FAS apoptotic inhibitory molecule 2 is a stress-induced intrinsic neuroprotective factor in the retina.

PubMed

Pawar, Mercy; Busov, Boris; Chandrasekhar, Aaruran; Yao, Jingyu; Zacks, David N; Besirli, Cagri G

2017-10-01

We report the neuroprotective role of FAS apoptotic inhibitory molecule 2 (FAIM2), an inhibitor of the FAS signaling pathway, during stress-induced photoreceptor apoptosis. Retinal detachment resulted in increased FAIM2 levels in photoreceptors with higher amounts detected at the tips of outer segments. Activation of FAS death receptor via FAS-ligand led to JNK-mediated FAIM2 phosphorylation, decreased proteasome-mediated degradation and increased association with the FAS receptor. Photoreceptor apoptosis was accelerated in Faim2 knockout mice following experimental retinal detachment. We show that FAIM2 is primarily involved in reducing stress-induced photoreceptor cell death but this effect was transient. FAIM2 was found to interact with both p53 and HSP90 following the activation of the FAS death pathway and FAIM2/HSP90 interaction was dependent on the phosphorylation of FAIM2. Lack of FAIM2 led to increased expression of proadeath genes Fas and Ripk1 in the retina under physiologic conditions. These results demonstrate that FAIM2 is an intrinsic neuroprotective factor activated by stress in photoreceptors and delays FAS-mediated photoreceptor apoptosis. Modulation of this pathway to increase FAIM2 expression may be a potential therapeutic option to prevent photoreceptor death.

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

PubMed

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Deleting the 14-3-3 protein Bmh1 extends life span in Saccharomyces cerevisiae by increasing stress response.

PubMed

Wang, Chen; Skinner, Craig; Easlon, Erin; Lin, Su-Ju

2009-12-01

Enhanced stress response has been suggested to promote longevity in many species. Calorie restriction (CR) and conserved nutrient-sensing target of rapamycin (TOR) and protein kinase A (PKA) pathways have also been suggested to extend life span by increasing stress response, which protects cells from age-dependent accumulation of oxidative damages. Here we show that deleting the yeast 14-3-3 protein, Bmh1, extends chronological life span (CLS) by activating the stress response. 14-3-3 proteins are highly conserved chaperone-like proteins that play important roles in many cellular processes. bmh1Delta-induced heat resistance and CLS extension require the general stress-response transcription factors Msn2, Msn4, and Rim15. The bmh1Delta mutant also displays a decreased reactive oxygen species level and increased heat-shock-element-driven transcription activity. We also show that BMH1 genetically interacts with CR and conserved nutrient-sensing TOR- and PKA-signaling pathways to regulate life span. Interestingly, the level of phosphorylated Ser238 on Bmh1 increases during chronological aging, which is delayed by CR or by reduced TOR activities. In addition, we demonstrate that PKA can directly phosphorylate Ser238 on Bmh1. The status of Bmh1 phosphorylation is therefore likely to play important roles in life-span regulation. Together, our studies suggest that phosphorylated Bmh1 may cause inhibitory effects on downstream longevity factors, including stress-response proteins. Deleting Bmh1 may eliminate the inhibitory effects of Bmh1 on these longevity factors and therefore extends life span.

Two Bistable Switches Govern M Phase Entry.

PubMed

Mochida, Satoru; Rata, Scott; Hino, Hirotsugu; Nagai, Takeharu; Novák, Béla

2016-12-19

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15 [1, 2]. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible [2-4]. Here, we surprisingly find that Cdk1 auto-activation is dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). We show that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, we found a sharp Cdk1 threshold for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. Our results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Serotonin receptor 1A–modulated phosphorylation of glycine receptor α3 controls breathing in mice

PubMed Central

Manzke, Till; Niebert, Marcus; Koch, Uwe R.; Caley, Alex; Vogelgesang, Steffen; Hülsmann, Swen; Ponimaskin, Evgeni; Müller, Ulrike; Smart, Trevor G.; Harvey, Robert J.; Richter, Diethelm W.

2010-01-01

Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A–specific (5-HTR1A–specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A–GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A–GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction. PMID:20978350

Lipopolysaccharide inhibits transforming growth factor-beta1-stimulated Smad6 expression by inducing phosphorylation of the linker region of Smad3 through a TLR4-IRAK1-ERK1/2 pathway.

PubMed

Kim, Eun-Ye; Kim, Byung-Chul

2011-03-09

Smad6, one of the inhibitory Smads, plays an important role in transforming growth factor-beta1 (TGF-β1)-mediated negative regulation of pro-inflammatory signaling. In this study, we found that bacterial endotoxin lipopolysaccharide (LPS) inhibits TGF-β1-induced expression of Smad6 in RAW264.7 cells. This repression was accompanied by increased Smad3 linker phosphorylation at Thr-179 and Ser-208 and was dependent on ERK1/2 activity via the TLR4-IRAK1-linked signaling cascade. The expression of a mutant Smad3, that lacks the phosphorylation sites in the linker regions, significantly reversed the inhibitory effect of LPS on TGF-β1-induced Smad6 expression and its anti-inflammatory capacity. Collectively, our findings show how LPS pro-inflammatory signal antagonizes the anti-inflammatory activity of TGF-β1. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Cross-talk between Rho-associated kinase and cyclic nucleotide-dependent kinase signaling pathways in the regulation of smooth muscle myosin light chain phosphatase.

PubMed

Grassie, Michael E; Sutherland, Cindy; Ulke-Lemée, Annegret; Chappellaz, Mona; Kiss, Enikö; Walsh, Michael P; MacDonald, Justin A

2012-10-19

Ca(2+) sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr(697) and/or Thr(855) (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser(696) prevents phosphorylation at Thr(697). However, the effects of Ser(854) and dual Ser(696)-Thr(697) and Ser(854)-Thr(855) phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser(696), Thr(697), Ser(854), and Thr(855)), Ser phosphorylation events (Ser(696)/Ser(854)) and dual Ser/Thr phosphorylation events (Ser(696)-Thr(697) and Ser(854)-Thr(855)). Dual phosphorylation at Ser(696)-Thr(697) and Ser(854)-Thr(855) by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr(697) and Thr(855) by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser(696), Thr(697), Ser(854), and Thr(855) in rat caudal artery, whereas U46619 induced Thr(697) and Thr(855) phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser(696)-Thr(697) and Ser(854)-Thr(855) inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.

Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM

PubMed Central

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T.

2017-01-01

SUMMARY Ataxia-Telangiectasia Mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. PMID:27939942

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

PubMed

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

2017-01-05

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway

PubMed Central

D’Ambrosio, Steven M.; Han, Chunhua; Pan, Li; Kinghorn, A. Douglas; Ding, Haiming

2011-01-01

Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003), was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compound 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF-induced activation of EGFR (Tyr1173). When compounds 1 and 2 were combined they synergistically inhibited c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation, and human oral cancer cell proliferation. The present data suggest that the potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins that target two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. PMID:21596018

Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway.

PubMed

D'Ambrosio, Steven M; Han, Chunhua; Pan, Li; Kinghorn, A Douglas; Ding, Haiming

2011-06-10

Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003) was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compounds 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF-induced activation of the EGFR (Tyr1173). When compounds 1 and 2 were combined they synergistically inhibited c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation, and human oral cancer cell proliferation. The present data suggest that the potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins that target two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Copyright © 2011 Elsevier Inc. All rights reserved.

p38 Mitogen-Activated Protein Kinase/Signal Transducer and Activator of Transcription-3 Pathway Signaling Regulates Expression of Inhibitory Molecules in T Cells Activated by HIV-1–Exposed Dendritic Cells

PubMed Central

Che, Karlhans Fru; Shankar, Esaki Muthu; Muthu, Sundaram; Zandi, Sasan; Sigvardsson, Mikael; Hinkula, Jorma; Messmer, Davorka; Larsson, Marie

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1–primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. PMID:22777388

A role for NRAGE in NF-κB activation through the non-canonical BMP pathway

PubMed Central

2010-01-01

Background Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-κB has yet to be explored. Results Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -α/β and subsequent transcriptional activation of the p65 subunit of NF-κB. Ablation of endogenous NRAGE by siRNA inhibited NF-κB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-κB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor. Conclusion Modulation of NRAGE expression revealed novel roles in regulating NF-κB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway. PMID:20100315

NT-3 promotes proprioceptive axon regeneration when combined with activation of the mTor intrinsic growth pathway but not with reduction of myelin extrinsic inhibitors

PubMed Central

Liu, Yingpeng; Kelamangalath, Lakshmi; Kim, Hyukmin; Han, Seung Baek; Tang, Xiaoqing; Zhai, Jinbin; Hong, Jee W; Lin, Shen; Son, Young-Jin; Smith, George M.

2016-01-01

Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons. PMID:27264357

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

PubMed

Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

2009-04-01

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

PubMed

Li, Hui; Yang, Duxiao; Ning, Shanglei; Xu, Yinghui; Yang, Fan; Yin, Rusha; Feng, Taihu; Han, Shouqing; Guo, Lu; Zhang, Pengju; Qu, Wenjie; Guo, Renbo; Song, Chen; Xiao, Peng; Zhou, Chengjun; Xu, Zhigang; Sun, Jin-Peng; Yu, Xiao

2018-01-01

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY 1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY 1196 -PAK1-T 423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways. © FASEB.

Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji

2013-06-14

Highlights: •H{sub 2}O{sub 2} inhibits TGF-β1-induced cell cycle arrest. •H{sub 2}O{sub 2} induces Smad3 linker phosphorylation through Akt-ERK1/2 pathway. •H{sub 2}O{sub 2}-mediated suppression of TGF-β signal requires Smad3 linker phosphorylation. •This is a first report about interplay between H{sub 2}O{sub 2} and growth inhibition pathway. -- Abstract: Hydrogen peroxide (H{sub 2}O{sub 2}) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H{sub 2}O{sub 2} are less understood. Here we report an important mechanism for antagonistic effectsmore » of H{sub 2}O{sub 2} on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H{sub 2}O{sub 2} (0.05–0.2 mM) completely blocked TGF-β1-mediated induction of p15{sup INK4B} expression and increase of its promoter activity. Interestingly, H{sub 2}O{sub 2} selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H{sub 2}O{sub 2} increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H{sub 2}O{sub 2} on TGF-β1-induced increase of p15{sup INK4B}-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H{sub 2}O{sub 2} as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H{sub 2}O{sub 2} antagonizes the cytostatic function of TGF-β1.« less

Regulation of lipogenesis by cyclin-dependent kinase 8–mediated control of SREBP-1

PubMed Central

Zhao, Xiaoping; Feng, Daorong; Wang, Qun; Abdulla, Arian; Xie, Xiao-Jun; Zhou, Jie; Sun, Yan; Yang, Ellen S.; Liu, Lu-Ping; Vaitheesvaran, Bhavapriya; Bridges, Lauren; Kurland, Irwin J.; Strich, Randy; Ni, Jian-Quan; Wang, Chenguang; Ericsson, Johan; Pessin, Jeffrey E.; Ji, Jun-Yuan; Yang, Fajun

2012-01-01

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding–induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis. PMID:22684109

Sarsaparilla (Smilax Glabra Rhizome) Extract Activates Redox-Dependent ATM/ATR Pathway to Inhibit Cancer Cell Growth by S Phase Arrest, Apoptosis, and Autophagy.

PubMed

She, Tiantian; Feng, Junnan; Lian, Shenyi; Li, Ruobing; Zhao, Chuanke; Song, Guoliang; Luo, Jie; Dawuti, Rouxianguli; Cai, Shaoqing; Qu, Like; Shou, Chengchao

2017-01-01

Sarsaparilla (Smilax Glabra Rhizome) exerts growth inhibitory effect on multiple cancer cells in vitro and in vivo, and redox-dependent persistent activation of ERK1/2 has been reported to underlie this effect. Here, we report an activation of ATM/ATR-dependent signaling pathway also as a mechanism for the cancer cell growth inhibition induced by the supernatant fraction of the water-soluble extract from sarsaparilla (SW). SW treatment (3.5 μg/μL) promoted the phosphorylations of ATM, ATR, and CHK1 in AGS and HT-29 cells. The ATM kinase inhibitor, KU55933, could reverse SW-induced ERK phosphorylation but not the reduced glutathione/oxidized glutathione (GSH/GSSG) imbalance in AGS cells. However, both the redox inhibitor glutathione (GSH) and ERK inhibitor U0126 antagonized SW-induced phosphorylations of ATM, ATR, and CHK1 in AGS cells. We further found KU55933 significantly antagonized SW-induced S phase arrest, apoptosis, autophagy and the resultant cell growth inhibition. Our results provide another molecular basis for the anticancer action of sarsaparilla.

Structure-Based Virtual Screening of Protein Tyrosine Phosphatase Inhibitors: Significance, Challenges, and Solutions.

PubMed

Reddy, Rallabandi Harikrishna; Kim, Hackyoung; Cha, Seungbin; Lee, Bongsoo; Kim, Young Jun

2017-05-28

Phosphorylation, a critical mechanism in biological systems, is estimated to be indispensable for about 30% of key biological activities, such as cell cycle progression, migration, and division. It is synergistically balanced by kinases and phosphatases, and any deviation from this balance leads to disease conditions. Pathway or biological activity-based abnormalities in phosphorylation and the type of involved phosphatase influence the outcome, and cause diverse diseases ranging from diabetes, rheumatoid arthritis, and numerous cancers. Protein tyrosine phosphatases (PTPs) are of prime importance in the process of dephosphorylation and catalyze several biological functions. Abnormal PTP activities are reported to result in several human diseases. Consequently, there is an increased demand for potential PTP inhibitory small molecules. Several strategies in structure-based drug designing techniques for potential inhibitory small molecules of PTPs have been explored along with traditional drug designing methods in order to overcome the hurdles in PTP inhibitor discovery. In this review, we discuss druggable PTPs and structure-based virtual screening efforts for successful PTP inhibitor design.

The design and delivery of a PKA inhibitory polypeptide to treat SCA1.

PubMed

Hearst, Scoty M; Shao, Qingmei; Lopez, Mariper; Raucher, Drazen; Vig, Parminder J S

2014-10-01

Spinocerebellar ataxia-1 (SCA1) is a neurodegenerative disease that primarily targets Purkinje cells (PCs) of the cerebellum. The exact mechanism of PC degeneration is unknown, however, it is widely believed that mutant ataxin-1 becomes toxic because of the phosphorylation of its serine 776 (S776) residue by cAMP-dependent protein kinase A (PKA). Therefore, to directly modulate mutant ATXN1 S776 phosphorylation and aggregation, we designed a therapeutic polypeptide to inhibit PKA. This polypeptide comprised of a thermally responsive elastin-like peptide (ELP) carrier, which increases peptide half-life, a PKA inhibitory peptide (PKI), and a cell-penetrating peptide (Synb1). We observed that our therapeutic polypeptide, Synb1-ELP-PKI, inhibited PKA activity at concentrations similar to the PKI peptide. Additionally, Synb1-ELP-PKI significantly suppressed mutant ATXN1 S776 phosphorylation and intranuclear inclusion formation in cell culture. Further, Synb1-ELP-PKI treatment improved SCA1 PC morphology in cerebellar slice cultures. Furthermore, the Synb1-ELP peptide carrier crossed the blood-brain barrier and localized to the cerebellum via the i.p. or intranasal route. Here, we show the intranasal delivery of ELP-based peptides to the brain as a novel delivery strategy. We also demonstrate that our therapeutic polypeptide has a great potential to target the neurotoxic S776 phosphorylation pathway in the SCA1 disease. © 2014 International Society for Neurochemistry.

Regulation of miR-21 expression in human melanoma via UV-ray-induced melanin pigmentation.

PubMed

Lin, Kuan-Yu; Chen, Chien-Min; Lu, Cheng-You; Cheng, Chun-Yuan; Wu, Yu-Hsin

2017-08-01

Excessive environmental ultraviolet (UV) radiation produces genetic mutations that can lead to skin cancer. This study was designed to assess the potential inhibitory activity of microRNA-21 (miR-21) on the UV irradiation-stimulated melanogenesis signal pathway in melanoma cells. The molecular mechanism of miR-21-induced inhibitory activity on UV-ray-stimulated melanogenesis-regulating proteins was examined in A375.S2 human melanoma and B16F10 mouse melanoma cells. UV irradiation for 30 min induced melanogenesis signal pathway by increasing melanin production and the number of A375.S2 cells. Similarly, UV radiation increased the expression of α-melanocyte-stimulating hormone (α-MSH) protein and decreased the melanogenesis-regulating signal, such as EGFR and Akt phosphorylation. Notably, miR-21 overexpression in UV-ray-stimulated A375.S2 cells decreased α-MSH expression and increased EGFR and Akt phosphorylation levels. Furthermore, miR-21 on UV-ray- induced melanogenesis was down-regulated by the Akt inhibitor and the EGFR inhibitor (Gefitinib). Results suggest that the suppressive activity of miR-21 on UV-ray-stimulated melanogenesis may involve the down-regulation of α-MSH and the activation in both of EGFR and Akt. © 2017 Wiley Periodicals, Inc.

Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

PubMed

Sapkota, Gopal; Knockaert, Marie; Alarcón, Claudio; Montalvo, Ermelinda; Brivanlou, Ali H; Massagué, Joan

2006-12-29

Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.

Metabolic regulation of oocyte cell death through the CaMKII-mediated phosphorylation of caspase-2.

PubMed

Nutt, Leta K; Margolis, Seth S; Jensen, Mette; Herman, Catherine E; Dunphy, William G; Rathmell, Jeffrey C; Kornbluth, Sally

2005-10-07

Vertebrate female reproduction is limited by the oocyte stockpiles acquired during embryonic development. These are gradually depleted over the organism's lifetime through the process of apoptosis. The timer that triggers this cell death is yet to be identified. We used the Xenopus egg/oocyte system to examine the hypothesis that nutrient stores can regulate oocyte viability. We show that pentose-phosphate-pathway generation of NADPH is critical for oocyte survival and that the target of this regulation is caspase-2, previously shown to be required for oocyte death in mice. Pentose-phosphate-pathway-mediated inhibition of cell death was due to the inhibitory phosphorylation of caspase-2 by calcium/calmodulin-dependent protein kinase II (CaMKII). These data suggest that exhaustion of oocyte nutrients, resulting in an inability to generate NADPH, may contribute to ooctye apoptosis. These data also provide unexpected links between oocyte metabolism, CaMKII, and caspase-2.

The involvement of ethylene in regulation of Arabidopsis gravitropism

NASA Astrophysics Data System (ADS)

Li, Ning; Zhu, Lin

Plant gravitropism is a directional response to gravity stimulus. This response involves a com-plex signaling network. Ethylene, a major plant hormone, has been found to modulate grav-itropism. The biosynthesis of ethylene is induced by the gravi-stimulus and the requirement for ethylene during gravitropism is tissue-dependent. While ethylene plays a modulating role in inflorescence stems, the light-grown hypocotyls of Arabidopsis requires ethylene to achieve a maximum gravicurvature. Because both inhibitory and stimulatory effects of ethylene on gravitropism have been overwhelmingly documented, there is a need to postulate a new theory to consolidate the apparently contradictory results. A dual-and-opposing effects (DOE) theory is therefore hypothesized to address how ethylene is involved in regulation of Arabidopsis grav-itropism, in which it is suggested that both stimulatory and inhibitory effects act on the same organ of a plant and co-exist at the same time in a mutually opposing manner. The final out-come of gravitropic response is determined by the dynamic display between the two opposing effects. A prolonged pretreatment of ethylene promotes the gravitropism in both inflorescence and light-grown hypocotyls, while a short ethylene pretreatment inhibits gravitropism. Gener-ally speaking, the inhibitory effect of ethylene is dominant over the expression of the stimula-tory effect in light-grown hypocotyls, whereas the stimulatory effect is dominant in inflorescence stem. Each effect is also positively correlated with concentrations of ethylene and in a time-dependent manner. The stimulatory effect occurs slowly but continues to react after the removal of ethylene, whereas the inhibitory effect takes place abruptly and diminishes shortly after its removal. Forward genetic screening based on the DOE phenotype of ethylene-treated Arabidop-sis has revealed a novel component in gravity signaling pathway: EGY1 (ethylene-dependent gravitropism-deficient and yellow green 1, Chen et al 2005; Guo et al 2008). To address the molecular mechanism by which ethylene regulates gravitropism, a cutting-edge phosphopro-teomics approach has been adopted to discover new components involved in ethylene signaling pathways (Li et al 2009). Two putative ethylene response transcription factors: EIL1 and ERF110, have been identified to contain ethylene-regulated phosphorylation sites, the phos-phorylation status of which are ethylene treatment-dependent but EIN2-independent, strongly suggestive of the existence of novel signaling components mediating an alternative ethylene signal pathway. Combination of the time-dependent ethylene treatments with the systematic profiling of protein phosphorylation using functional phosphoproteomics among Arabidopsis ethylene response mutants is able to provide more valuable information about the molecular mechanisms underlying ethylene and gravity signaling pathways. (This work is supported by grants: RPC07/08.SC16, 661408, 661207, N HKUST627/06, DAG04/05.SC08, HKUST6105/01M, and HKUST6413/06M)

Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Yun-Jeong; Cho, Hyun-Ji; Magae, Junji

2013-12-15

Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α throughmore » an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.« less

Berberine inhibits macrophage M1 polarization via AKT1/SOCS1/NF-κB signaling pathway to protect against DSS-induced colitis.

PubMed

Liu, Yunxin; Liu, Xiang; Hua, Weiwei; Wei, Qingyan; Fang, Xianjun; Zhao, Zheng; Ge, Chun; Liu, Chao; Chen, Chen; Tao, Yifu; Zhu, Yubing

2018-04-01

Berberine has been reported to have protective effects in colitis treatment. However, the detailed mechanisms remain unclear. Herein, we demonstrated that berberine could protect against dextran sulfate sodium (DSS)-induced colitis in mice by regulating macrophage polarization. In the colitis mouse model, berberine ameliorated DSS-induced colon shortening and colon tissue injury. Moreover, berberine-treated mice showed significant reduction in the disease activity index (DAI), pro-inflammatory cytokines expression and macrophages infiltration compared with the DSS-treated mice. Notably, berberine significantly reduced the percentage of M1 macrophages. In vitro analysis also confirmed the inhibitory effects of berberine on macrophages M1 polarization in RAW267.4 cells. Further investigation showed that berberine promoted AKT1 expression in mRNA and protein level. Silence of AKT1 abolished the inhibitory effect of berberine on macrophages M1 polarization. The berberine-induced AKT1 expression promoted suppressers of cytokine signaling (SOCS1) activation, which inhibited nuclear factor-kappa B (NF-κB) phosphorylation. In addition, we also found that berberine activated AKT1/SOCS1 signaling pathway but inhibited p65 phosphorylation in macrophages in vivo. Therefore, we concluded that berberine played a regulatory role in macrophages M1 polarization in DSS-induced colitis via AKT1/SOCS1/NF-κB signaling pathway. This unexpected property of berberine may provide a potential explanation for its protective effects in colitis treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

Role of Rho/ROCK and p38 MAP kinase pathways in transforming growth factor-beta-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo.

PubMed

Kamaraju, Anil K; Roberts, Anita B

2005-01-14

TGF-beta is a multifunctional cytokine known to exert its biological effects through a variety of signaling pathways of which Smad signaling is considered to be the main mediator. At present, the Smad-independent pathways, their interactions with each other, and their roles in TGF-beta-mediated growth inhibitory effects are not well understood. To address these questions, we have utilized a human breast cancer cell line MCF10CA1h and demonstrate that p38 MAP kinase and Rho/ROCK pathways together with Smad2 and Smad3 are necessary for TGF-beta-mediated growth inhibition of this cell line. We show that Smad2/3 are indispensable for TGF-beta-mediated growth inhibition, and that both p38 and Rho/ROCK pathways affect the linker region phosphorylation of Smad2/3. Further, by using Smad3 mutated at the putative phosphorylation sites in the linker region, we demonstrate that phosphorylation at Ser203 and Ser207 residues is required for the full transactivation potential of Smad3, and that these residues are targets of the p38 and Rho/ROCK pathways. We demonstrate that activation of the p38 MAP kinase pathway is necessary for the full transcriptional activation potential of Smad2/Smad3 by TGF-beta, whereas activity of Rho/ROCK is necessary for both down-regulation of c-Myc protein and up-regulation of p21waf1 protein, directly interfering with p21waf1 transcription. Our results not only implicate Rho/ROCK and p38 MAPK pathways as necessary for TGF-beta-mediated growth inhibition, but also demonstrate their individual contributions and the basis for their cooperation with each other.

Aliphatic acetogenin constituents of avocado fruits inhibit human oral cancer cell proliferation by targeting the EGFR/RAS/RAF/MEK/ERK1/2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Ambrosio, Steven M.; Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210; Han, Chunhua

2011-06-10

Highlights: {yields} The aliphatic acetogenins [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] (1) and [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate] (2) isolated from avocado fruit inhibit phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). {yields} Aliphatic acetogenin 2, but not 1, prevents EGF-induced activation of EGFR (Tyr1173). {yields} Combination of both aliphatic acetogenins synergistically inhibits c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation and human oral cancer cell proliferation. {yields} The potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins targeting two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. {yields} Providing a double hit on a critical cancer pathway such as EGFR/RAS/RAF/MEK/ERK1/2 by phytochemicals like thosemore » found in avocado fruit could lead to more effective approach toward cancer prevention. -- Abstract: Avocado (Persea americana) fruits are consumed as part of the human diet and extracts have shown growth inhibitory effects in various types of human cancer cells, although the effectiveness of individual components and their underlying mechanism are poorly understood. Using activity-guided fractionation of the flesh of avocado fruits, a chloroform-soluble extract (D003) was identified that exhibited high efficacy towards premalignant and malignant human oral cancer cell lines. From this extract, two aliphatic acetogenins of previously known structure were isolated, compounds 1 [(2S,4S)-2,4-dihydroxyheptadec-16-enyl acetate] and 2 [(2S,4S)-2,4-dihydroxyheptadec-16-ynyl acetate]. In this study, we show for the first time that the growth inhibitory efficacy of this chloroform extract is due to blocking the phosphorylation of EGFR (Tyr1173), c-RAF (Ser338), and ERK1/2 (Thr202/Tyr204) in the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway. Compounds 1 and 2 both inhibited phosphorylation of c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204). Compound 2, but not compound 1, prevented EGF-induced activation of the EGFR (Tyr1173). When compounds 1 and 2 were combined they synergistically inhibited c-RAF (Ser338) and ERK1/2 (Thr202/Tyr204) phosphorylation, and human oral cancer cell proliferation. The present data suggest that the potential anticancer activity of avocado fruits is due to a combination of specific aliphatic acetogenins that target two key components of the EGFR/RAS/RAF/MEK/ERK1/2 cancer pathway.« less

Nitric oxide, PKC-ε, and connexin43 are crucial for ischemic preconditioning-induced chemical gap junction uncoupling.

PubMed

Rong, Bing; Xie, Fei; Sun, Tao; Hao, Li; Lin, Ming-Jie; Zhong, Jing-Quan

2016-10-25

Ischemic preconditioning (IPC) maintains connexin43 (Cx43) phosphorylation and reduces chemical gap junction (GJ) coupling in cardiomyocytes to protect against ischemic damage. However, the signal transduction pathways underlying these effects are not fully understood. Here, we investigated whether nitric oxide (NO) and protein kinase C-ε (PKC-ε) contribute to IPC-induced cardioprotection by maintaining Cx43 phosphorylation and inhibiting chemical GJ coupling. IPC reduced ischemia-induced myocardial infarction and increased cardiomyocyte survival; phosphorylated Cx43, eNOS, and PKC-ε levels; and chemical GJ uncoupling. Administration of the NO donor SNAP mimicked the effects of IPC both in vivo and in vitro, maintaining Cx43 phosphorylation, promoting chemical GJ uncoupling, and reducing myocardial infarction. Preincubation with the NO synthase inhibitor L-NAME or PKC-ε translocation inhibitory peptide (PKC-ε-TIP) abolished these effects of IPC. Additionally, by inducing NO production, IPC induced translocation of PKC-ε, but not PKC-δ, from the cytosolic to the membrane fraction in primary cardiac myocytes. IPC-induced cardioprotection thus involves increased NO production, PKC-ε translocation, Cx43 phosphorylation, and chemical GJ uncoupling.

E-cadherin and, in its absence, N-cadherin promotes Nanog expression in mouse embryonic stem cells via STAT3 phosphorylation.

PubMed

Hawkins, Kate; Mohamet, Lisa; Ritson, Sarah; Merry, Catherine L R; Ward, Christopher M

2012-09-01

We have recently shown that loss of E-cadherin in mouse embryonic stem cells (mESCs) results in significant alterations to both the transcriptome and hierarchy of pluripotency-associated signaling pathways. Here, we show that E-cadherin promotes kruppel-like factor 4 (Klf4) and Nanog transcript and protein expression in mESCs via STAT3 phosphorylation and that β-catenin, and its binding region in E-cadherin, is required for this function. To further investigate the role of E-cadherin in leukemia inhibitory factor (LIF)-dependent pluripotency, E-cadherin null (Ecad(-/-)) mESCs were cultured in LIF/bone morphogenetic protein supplemented medium. Under these conditions, Ecad(-/-) mESCs exhibited partial restoration of cell-cell contact and STAT3 phosphorylation and upregulated Klf4, Nanog, and N-cadherin transcripts and protein. Abrogation of N-cadherin using an inhibitory peptide caused loss of phospho STAT3, Klf4, and Nanog in these cells, demonstrating that N-cadherin supports LIF-dependent pluripotency in this context. We therefore identify a novel molecular mechanism linking E- and N-cadherin to the core circuitry of pluripotency in mESCs. This mechanism may explain the recently documented role of E-cadherin in efficient induced pluripotent stem cell reprogramming. Copyright © 2012 AlphaMed Press.

Inhibitory Effect of Arctigenin from Fructus Arctii Extract on Melanin Synthesis via Repression of Tyrosinase Expression

PubMed Central

Park, Hwayong; Song, Kwang Hoon; Jung, Pil Mun; Kim, Ji-Eun; Kim, Mi Yoon; Ma, Jin Yeul

2013-01-01

To identify the active compound arctigenin in Fructus Arctii (dried seed of medicinal plant Arctium lappa) and to elucidate the inhibitory mechanism in melanogenesis, we analyzed melanin content and tyrosinase activity on B16BL6 murine melanoma and melan-A cell cultures. Water extracts of Fructus Arctii were shown to inhibit tyrosinase activity in vitro and melanin content in α-melanocyte stimulating hormone-stimulated cells to similar levels as the well-known kojic acid and arbutin, respectively. The active compound arctigenin of Fructus Arctii displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin content and tyrosinase activity in a dose-dependent manner. Melanogenic inhibitory activity was also identified in vivo with zebrafish embryo. To determine the mechanism of inhibition, the effects of arctigenin on tyrosinase gene expression and tyrosinase promoter activity were examined. Also in addition, in the signaling cascade, arctigenin dose dependently decreased the cAMP level and promoted the phosphorylation of extracellular signal-regulated kinase. This result suggests that arctigenin downregulates cAMP and the tyrosinase enzyme through its gene promoter and subsequently upregulates extracellular signal-regulated kinase activity by increasing phosphorylation in the melanogenesis signaling pathway, which leads to a lower melanin content. PMID:23781272

Pirfenidone inhibits transforming growth factor β1-induced extracellular matrix production in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Park, Il-Ho; Lee, Heung-Man

2015-01-01

Pirfenidone has been shown to have antifibrotic and anti-inflammatory effects in the lungs. The purpose of this study was to evaluate the inhibitory effects of pirfenidone on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and extracellular matrix accumulation. We also determined the molecular mechanisms of pirfenidone in nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from nasal polyps from eight patients who had chronic rhinosinusitis with nasal polyp. Pirfenidone was used to treat TGF-β1-induced NPDFs. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Fibroblast migration was evaluated with scratch assays. Expression levels of α-smooth muscle actin (SMA), fibronectin, and phosphorylated Smad2/3 were determined by Western blot and/or reverse transcription-polymerase chain reaction and immunofluorescent staining. Total collagen production was analyzed with the Sircol collagen assay and contractile activity was measured by a collagen gel contraction assay. Pirfenidone (0-2 mg/mL) has no significant cytotoxic effects in TGF-β1-induced NPDFs. Migration of NPDFs was significantly inhibited by pirfenidone treatment. The expression levels of α-SMA and fibronectin were significantly reduced in pirfenidone-treated NPDFs. Collagen contraction and production were also significantly decreased by pirfenidone treatment. Finally, pirfenidone significantly inhibited phosphorylation of the Smad2/3 pathway in TGF-β1-induced NPDFs. Pirfenidone has an inhibitory effect on TGF-β1-induced migration, myofibroblast differentiation (α-SMA), extracellular matrix accumulation, and collagen contraction by blocking the phosphorylation of Smad2/3 pathways in NPDFs. Thus, pirfenidone may inhibit TGF-β1-induced extracellular matrix by regulating Smad2/3.

Regulation of the Na(+)-K(+)-2Cl(-) cotransporter by cGMP/cGMP-dependent protein kinase I after furosemide administration.

PubMed

Limmer, Franziska; Schinner, Elisabeth; Castrop, Hayo; Vitzthum, Helga; Hofmann, Franz; Schlossmann, Jens

2015-10-01

Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well-established effects of nitric oxide on NKCC2 activity, cGMP is likely involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) and cGKIα-rescue mice with furosemide. cGKIα-rescue mice expressed cGKIα only under the control of the smooth muscle-specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα-rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in cGKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2. © 2015 FEBS.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mogi, Makio, E-mail: makio@dpc.aichi-gakuin.ac.jp; Kondo, Ayami

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor regulates bone mass by inhibiting osteoclastic bone resorption. mTOR, which is the mammalian target of rapamycin, is a kinase and central regulator of cell growth, proliferation, and survival. By using Rapamycin, we studied whether mTOR pathway is associated with OPG protein production in the mouse bone marrow-derived stromal cell line ST2. Rapamycin markedly increased the level of soluble OPG in ST2 cells. This antibiotic treatment resulted in the suppression of phosphorylation of mTOR. Rapamycin had no effects on the proliferation, differentiation, or apoptosis of the cells. Treatment with bone morphogenetic protein-4, which can induce OPG proteinmore » in ST2 cells, also resulted in a decrease in the density of the phospho-mTOR-band, suggesting that the suppression of the phospho-mTOR pathway is necessary for OPG production in ST2 cells. Thus, suitable suppression of mTOR phosphorylation is a necessary requirement for OPG production in bone marrow stromal cells.« less

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

PubMed Central

2010-01-01

Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin) may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin). Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs) phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and endothelial nitric oxide synthase (eNOS) expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP-eNOS/NO-cyclic GMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA2 cascade, and finally inhibition of platelet aggregation. PMID:20525309

Prelimbic cortex extracellular signal-regulated kinase 1/2 activation is required for memory retrieval of long-term inhibitory avoidance.

PubMed

Luo, Fei; Zheng, Jian; Sun, Xuan; Deng, Wei-Ke; Li, Bao Ming; Liu, Fang

2017-04-15

Neural mechanism underlying memory retrieval has been extensively studied in the hippocampus and amygdala. However, little is known about the role of medial prefrontal cortex in long-term memory retrieval. We evaluate this issue in one-trial step-through inhibitory avoidance (IA) paradigm. Our results showed that, 1) inactivation of mPFC by local infusion of GABA A -receptor agonist muscimol caused severe deficits in retrieval of 1-day and 7-day but had no effects on 2-h inhibitory avoidance memory; 2) the protein level of phosphorylated-ERK1/2 in mPFC were significantly increased following retrieval of 1-day and 7-day IA memory, so did the numbers of phosphorylated-ERK (pERK) and phosphorylated-CREB (pCREB) labeled neurons; 3) intra-mPFC infusion of ERK kinase inhibitor PD98095 significantly reduced phosphorylated ERK1/2 levels and phosphorylated-ERK1/2 and phosphorylated-CREB labeled cells, and severely impaired retrieval of 7-day IA memory when the drugs were administrated 30min prior to test. The present study provides evidence that retrieval of long-lasting memory for inhibitory avoidance requires mPFC and involves the ERK-CREB signaling cascade. Copyright © 2017 Elsevier B.V. All rights reserved.

Nitric oxide, PKC-ε, and connexin43 are crucial for ischemic preconditioning-induced chemical gap junction uncoupling

PubMed Central

Sun, Tao; Hao, Li; Lin, Ming-Jie; Zhong, Jing-Quan

2016-01-01

Ischemic preconditioning (IPC) maintains connexin43 (Cx43) phosphorylation and reduces chemical gap junction (GJ) coupling in cardiomyocytes to protect against ischemic damage. However, the signal transduction pathways underlying these effects are not fully understood. Here, we investigated whether nitric oxide (NO) and protein kinase C-ε (PKC-ε) contribute to IPC-induced cardioprotection by maintaining Cx43 phosphorylation and inhibiting chemical GJ coupling. IPC reduced ischemia-induced myocardial infarction and increased cardiomyocyte survival; phosphorylated Cx43, eNOS, and PKC-ε levels; and chemical GJ uncoupling. Administration of the NO donor SNAP mimicked the effects of IPC both in vivo and in vitro, maintaining Cx43 phosphorylation, promoting chemical GJ uncoupling, and reducing myocardial infarction. Preincubation with the NO synthase inhibitor L-NAME or PKC-ε translocation inhibitory peptide (PKC-ε-TIP) abolished these effects of IPC. Additionally, by inducing NO production, IPC induced translocation of PKC-ε, but not PKC-δ, from the cytosolic to the membrane fraction in primary cardiac myocytes. IPC-induced cardioprotection thus involves increased NO production, PKC-ε translocation, Cx43 phosphorylation, and chemical GJ uncoupling. PMID:27655723

Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.

PubMed

Adragna, Norma C; Ravilla, Nagendra B; Lauf, Peter K; Begum, Gulnaz; Khanna, Arjun R; Sun, Dandan; Kahle, Kristopher T

2015-01-01

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K(+) and Cl(-) efflux via activation of K(+) channels, volume-regulated anion channels (VRACs), and the K(+)-Cl(-) cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K(+) content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.

Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis

PubMed Central

Adragna, Norma C.; Ravilla, Nagendra B.; Lauf, Peter K.; Begum, Gulnaz; Khanna, Arjun R.; Sun, Dandan; Kahle, Kristopher T.

2015-01-01

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K+ and Cl− efflux via activation of K+ channels, volume-regulated anion channels (VRACs), and the K+-Cl− cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na+-K+-2Cl− cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K+ content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD. PMID:26217182

EPA blocks TNF-α-induced inhibition of sugar uptake in Caco-2 cells via GPR120 and AMPK.

PubMed

Castilla-Madrigal, Rosa; Barrenetxe, Jaione; Moreno-Aliaga, María J; Lostao, María Pilar

2018-03-01

The aim of the present work was to investigate in Caco-2 cells whether eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, could block the inhibitory effect of tumor necrosis factor-α (TNF-α) on sugar transport, and identify the intracellular signaling pathways involved. After pre-incubation of the Caco-2 cells with TNF-α and EPA for 1 hr, EPA prevented the inhibitory effect of the cytokine on α-methyl-d-glucose (αMG) uptake (15 min) and on SGLT1 expression at the brush border membrane, measured by Western blot. The ERK1/2 inhibitor PD98059 and the AMPK activator AICAR also prevented the inhibitory effect of TNF-α on both αMG uptake and SGLT1 expression. Interestingly, the AMPK inhibitor, Compound C, abolished the ability of EPA to prevent TNF-α-induced reduction of sugar uptake and transporter expression. The GPR120 antagonist, AH7614, also blocked the preventive effect of EPA on TNF-α-induced decrease of αMG uptake and AMPK phosphorylation. In summary, TNF-α inhibits αMG uptake by decreasing SGLT1 expression in the brush border membrane through the activation of ERK1/2 pathway. EPA prevents the inhibitory effect of TNF-α through the involvement of GPR120 and AMPK activation. © 2017 Wiley Periodicals, Inc.

Engagement of CD22 on B cells with the monoclonal antibody epratuzumab stimulates the phosphorylation of upstream inhibitory signals of the B cell receptor.

PubMed

Lumb, Simon; Fleischer, Sarah J; Wiedemann, Annika; Daridon, Capucine; Maloney, Alison; Shock, Anthony; Dörner, Thomas

2016-06-01

The binding of antigen to the B cell receptor (BCR) results in a cascade of signalling events that ultimately drive B cell activation. Uncontrolled B cell activation is regulated by negative feedback loops that involve inhibitory co-receptors such as CD22 and CD32B that exert their functions following phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). The CD22-targeted antibody epratuzumab has previously been shown to inhibit BCR-driven signalling events, but its effects on ITIM phosphorylation of CD22 and CD32B have not been properly evaluated. The present study therefore employed both immunoprecipitation and flow cytometry approaches to elucidate the effects of epratuzumab on direct phosphorylation of key tyrosine (Tyr) residues on both these proteins, using both transformed B cell lines and primary human B cells. Epratuzumab induced the phosphorylation of Tyr(822) on CD22 and enhanced its co-localisation with SHP-1. Additionally, in spite of high basal phosphorylation of other key ITIMs on CD22, in primary human B cells epratuzumab also enhanced phosphorylation of Tyr(807), a residue involved in the recruitment of Grb2. Such initiation events could explain the effects of epratuzumab on downstream signalling in B cells. Finally, we were able to demonstrate that epratuzumab stimulated the phosphorylation of Tyr(292) on the low affinity inhibitory Fc receptor CD32B which would further attenuate BCR-induced signalling. Together, these data demonstrate that engagement of CD22 with epratuzumab leads to the direct phosphorylation of key upstream inhibitory receptors of BCR signalling and may help to explain how this antibody modulates B cell function.

Curcumin inhibits urothelial tumor development by suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway.

PubMed

Tian, Binqiang; Zhao, Yingmei; Liang, Tao; Ye, Xuxiao; Li, Zuowei; Yan, Dongliang; Fu, Qiang; Li, Yonghui

2017-08-01

We have previously reported that curcumin inhibits urothelial tumor development in a rat bladder carcinogenesis model. In this study, we report that curcumin inhibits urothelial tumor development by suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway. Curcumin inhibits IGF2 expression at the transcriptional level and decreases the phosphorylation levels of IGF1R and IRS-1 in bladder cancer cells and N-methyl-N-nitrosourea (MNU)-induced urothelial tumor tissue. Ectopic expression of IGF2 and IGF1R, but not IGF1, in bladder cancer cells restored this process, suggesting that IGF2 is a target of curcumin. Moreover, introduction of constitutively active AKT1 abolished the inhibitory effect of curcumin on cell proliferation, migration, and restored the phosphorylation levels of 4E-BP1 and S6K1, suggesting that curcumin functions via suppressing IGF2-mediated AKT/mTOR signaling pathway. In summary, our results reveal that suppressing IGF2 and IGF2-mediated PI3K/AKT/mTOR signaling pathway is one of the mechanisms of action of curcumin. Our findings suggest a new therapeutic strategy against human bladder cancer caused by aberrant activation of IGF2, which are useful for translational application of curcumin.

Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR.

PubMed

Wang, Zheng; Wu, Xue; Liang, Yan-Ni; Wang, Li; Song, Zhong-Xing; Liu, Jian-Li; Tang, Zhi-Shu

2016-09-27

Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G₀/G₁ phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-mediated Inhibitory Signaling is Regulated by Sequential Phosphorylation Mediated by Distinct Nonreceptor Tyrosine Kinases: A Case Study Involving PECAM-1

PubMed Central

Tourdot, Benjamin E.; Brenner, Michelle K.; Keough, Kathleen C.; Holyst, Trudy; Newman, Peter J.; Newman, Debra K.

2013-01-01

The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing non-receptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton’s tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk, and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals. PMID:23418871

Targeting SHP-1, 2 and SHIP Pathways: A Novel Strategy for Cancer Treatment?

PubMed

Dempke, Wolfram C M; Uciechowski, Peter; Fenchel, Klaus; Chevassut, Timothy

2018-06-20

Well-balanced levels of tyrosine phosphorylation, maintained by the reversible and coordinated actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are critical for a wide range of cellular processes including growth, differentiation, metabolism, migration, and survival. Aberrant tyrosine phosphorylation, as a result of a perturbed balance between the activities of PTKs and PTPs, is linked to the pathogenesis of numerous human diseases, including cancer, suggesting that PTPs may be innovative molecular targets for cancer treatment. Two PTPs that have an important inhibitory role in haematopoietic cells are SHP-1 and SHP-2. SHP-1, 2 promote cell growth and act by both upregulating positive signaling pathways and by downregulating negative signaling pathways. SHIP is another inhibitory phosphatase that is specific for the inositol phospholipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). SHIP acts as a negative regulator of immune response by hydrolysing PIP3, and SHIP deficiency results in myeloproliferation and B-cell lymphoma in mice. The validation of SHP-1, 2 and SHIP as oncology targets has generated interest in the development of inhibitors as potential therapeutic agents for cancers; however, SHP-1, 2 and SHIP have proven to be an extremely difficult target for drug discovery, primarily due to the highly conserved and positively charged nature of their PTP active site, and many PTP inhibitors lack either appro-priate selectivity or membrane permeability. To overcome these caveats, novel techniques have been employed to synthesise new inhibitors that specifically attenuate the PTP-dependent signaling inside the cell and amongst them; some are already in clinical development which are discussed in this review. © 2018 S. Karger AG, Basel.

β-Adducin siRNA disruption of the spectrin-based cytoskeleton in differentiating keratinocytes prevented by calcium acting through calmodulin/epidermal growth factor receptor/cadherin pathway.

PubMed

Wu, Jianghong; Masci, Paul P; Chen, Chenfeng; Chen, Jiezhong; Lavin, Martin F; Zhao, Kong-Nan

2015-01-01

Here, we report that siRNA transfection of β-adducin significantly disrupted the spectrin-based cytoskeleton and cytoskeletal arrangements of both β-adducin and PKCδ by substantially inhibiting the expression of β-adducin, spectrin and PKCδ proteins in differentiating keratinocytes. However, extracellular Ca2+ treatment blocked the inhibitory effects of the β-adducin siRNA. Ca2+ also prevented the significant down-regulation of two differentiation markers involucrin and K1/10 and the distinct up-regulation of proliferation marker K14 in β-adducin siRNA transfected keratinocytes. In addition, β-adducin knockdown resulted in a substantial reduction of epidermal growth factor receptor (EGFR), cadherin and β-catenin and enhanced phosphorylation of EGFR on tyrosine 1173 and Ca2+ prevented these changes. Furthermore, Ca2+ blocked the inhibitory effects of β-adducin siRNA on the expression of calmodulin, phosphorylated-calmodulin (P-CaM((Tyr138))) and myristoylated alanine-rich C-kinase substrate (MARCKS) in keratinocytes. Co-immunoprecipitation studies further revealed that calmodulin, not MARCKS, strongly interacted with EGFR, cadherin and β-catenin. Our data suggest that Ca2+ plays an important role in regulating the expression and function of β-adducin to sustain normal organization of the spectrin-based cytoskeleton and the differentiation properties in keratinocytes through the calmodulin/EGFR/cadherin signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Diosgenin inhibits superoxide generation in FMLP-activated mouse neutrophils via multiple pathways.

PubMed

Lin, Y; Jia, R; Liu, Y; Gao, Y; Zeng, X; Kou, J; Yu, B

2014-12-01

Diosgenin possesses anti-inflammatory and anticancer properties. Activated neutrophils produce high concentrations of the superoxide anion which is involved in the pathophysiology of inflammation-related diseases and cancer. In the present study, the inhibitory effect and possible mechanisms of diosgenin on superoxide generation were investigated in mouse bone marrow neutrophils. Diosgenin potently and concentration-dependently inhibited the extracellular and intracellular superoxide anion generation in Formyl-Met-Leu-Phe (FMLP)- activated neutrophils, with IC50 values of 0.50 ± 0.08 μM and 0.66 ± 0.13 μM, respectively. Such inhibition was not mediated by scavenging the superoxide anion or by a cytotoxic effect. Diosgenin inhibited the phosphorylation of p47phox and membrane translocation of p47phox and p67phox, and thus blocking the assembly of nicotinamide adenine dinucleotide phosphate oxidase. Moreover, cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) expression were also effectively increased by diosgenin. It attenuated FMLP-induced increase of phosphorylation of cytosolic phospholipase A (cPLA2), p21-activated kinase (PAK), Akt, p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK). Our data indicate that diosgenin exhibits inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways. The results may explain the clinical implications of diosgenin in the treatment of inflammation-related disorders.

Irreversible dual inhibitory mode: the novel Btk inhibitor PLS-123 demonstrates promising anti-tumor activity in human B-cell lymphoma.

PubMed

Ding, Ning; Li, Xitao; Shi, Yunfei; Ping, Lingyan; Wu, Lina; Fu, Kai; Feng, Lixia; Zheng, Xiaohui; Song, Yuqin; Pan, Zhengying; Zhu, Jun

2015-06-20

The B-cell receptor (BCR) signaling pathway has gained significant attention as a therapeutic target in B-cell malignancies. Recently, several drugs that target the BCR signaling pathway, especially the Btk inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicates that pharmacological inhibition of BCR pathway holds promise in B-cell lymphoma treatment. Here we present a novel covalent irreversible Btk inhibitor PLS-123 with more potent anti-proliferative activity compared with ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and dual-action inhibitory mode of Btk activation. The phosphorylation of BCR downstream activating AKT/mTOR and MAPK signal pathways was also more significantly reduced after treatment with PLS-123 than ibrutinib. Gene expression profile analysis further suggested that the different selectivity profile of PLS-123 led to significant downregulation of oncogenic gene PTPN11 expression, which might also offer new opportunities beyond what ibrutinib has achieved. In addition, PLS-123 dose-dependently attenuated BCR- and chemokine-mediated lymphoma cell adhesion and migration. Taken together, Btk inhibitor PLS-123 suggested a new direction to pharmacologically modulate Btk function and develop novel therapeutic drug for B-cell lymphoma treatment.

Irreversible dual inhibitory mode: the novel Btk inhibitor PLS-123 demonstrates promising anti-tumor activity in human B-cell lymphoma

PubMed Central

Ding, Ning; Li, Xitao; Shi, Yunfei; Ping, Lingyan; Wu, Lina; Fu, Kai; Feng, Lixia; Zheng, Xiaohui; Song, Yuqin; Pan, Zhengying; Zhu, Jun

2015-01-01

The B-cell receptor (BCR) signaling pathway has gained significant attention as a therapeutic target in B-cell malignancies. Recently, several drugs that target the BCR signaling pathway, especially the Btk inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicates that pharmacological inhibition of BCR pathway holds promise in B-cell lymphoma treatment. Here we present a novel covalent irreversible Btk inhibitor PLS-123 with more potent anti-proliferative activity compared with ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and dual-action inhibitory mode of Btk activation. The phosphorylation of BCR downstream activating AKT/mTOR and MAPK signal pathways was also more significantly reduced after treatment with PLS-123 than ibrutinib. Gene expression profile analysis further suggested that the different selectivity profile of PLS-123 led to significant downregulation of oncogenic gene PTPN11 expression, which might also offer new opportunities beyond what ibrutinib has achieved. In addition, PLS-123 dose-dependently attenuated BCR- and chemokine-mediated lymphoma cell adhesion and migration. Taken together, Btk inhibitor PLS-123 suggested a new direction to pharmacologically modulate Btk function and develop novel therapeutic drug for B-cell lymphoma treatment. PMID:25944695

Neochlorogenic acid inhibits against LPS-activated inflammatory responses through up-regulation of Nrf2/HO-1 and involving AMPK pathway.

PubMed

Park, Sun Young; Jin, Mei Ling; Yi, Eun Hye; Kim, Yoon; Park, Geuntae

2018-06-08

Acute and chronic inflammatory diseases are associated with excessive inflammation due to the accumulation of pro-inflammatory mediators and cytokines produced by macrophages. In the present study, we investigated the anti-inflammatory properties of neochlorogenic acid (nCGA) from Lonicera japonica on lipopolysaccharide (LPS)-activated inflammation in macrophages and participation of the AMPK/Nrf2 pathway. nCGA pretreatment significantly reduced the production of nitric oxide, prostaglandin E 2 , TNF-α, reactive oxygen species, IL-1β, and IL-6 by LPS-activated macrophages. Moreover, both transcript and protein levels of inducible nitric oxide synthase and cyclooxygenase-2 were reduced by nCGA in LPS-activated macrophages. nCGA inhibited NF-κB activation by attenuating IKKα/β and IκBα phosphorylation in LPS-stimulated macrophages. Moreover, nCGA attenuated LPS-elevated JAK-1, STAT-1, and MAPK phosphorylation. We further evaluated the possible role of nCGA in the induction of AMPK/Nrf2 signal pathways required for the protein expression of HO-1 and NQO-1. nCGA induced AMPK activation via phosphorylation of LKB1 and CaMKII and by the inhibitory phosphorylation of GSK3β. It stimulated the overexpression of Nrf2/ARE-regulated downstream proteins, such as NQO-1 and HO-1. Furthermore, the anti-inflammatory effects of nCGA were attenuated in macrophages subjected to siRNAs specific for HO-1, NQO-1, Nrf2, and AMPK. Accordingly, these results indicate that nCGA, as an AMPK/Nrf2 signal activator, prevents excessive macrophage-mediated responses associated with acute and chronic inflammatory disorders. Copyright © 2018 Elsevier B.V. All rights reserved.

Suppressive effects of Lithospermum erythrorhizon extracts on lipopolysaccharide-induced activation of AP-1 and NF-kappaB via mitogen-activated protein kinase pathways in mouse macrophage cells.

PubMed

Han, Kyu Yeon; Kwon, Taek Hwan; Lee, Tae Hoon; Lee, Sung-Joon; Kim, Sung-Hoon; Kim, Jiyoung

2008-04-30

A variety of anti-inflammatory agents have been shown to exert chemopreventive activity via targeting of transcription factors such as NF-kappaB and AP-1. Lithospermum erythrorhizon (LE) has long been used in traditional oriental medicine. In this study, we demonstrated the inhibitory effects of LE extracts on lipopolysaccharide (LPS)-stimulated production of inflammatory cytokines. As an underlying mechanism of inhibition, LE extracts reduced LPS-induced transactivation of AP-1 as well as NF-kappaB in mouse macrophage cells. Electrophoretic mobility shift assays indicated that LE extracts inhibited the DNA binding activities of AP-1 and NF-kappaB. In addition, phosphorylation of IkappaB-alpha protein was suppressed by LE extracts. Moreover, LE extracts inhibited c-Jun N-terminal kinase and extracellular signal-regulated signaling pathways. Our results suggest that the anti-inflammatory activity of LE extracts may be mediated by the inhibition of signal transduction pathways that normally lead to the activation of AP-1and NF-kappaB. These inhibitory effects may be useful for chemoprevention of cancer or other chronic inflammatory diseases.

Identification of a novel Drosophila SMAD on the X chromosome.

PubMed

Henderson, K D; Andrew, D J

1998-11-09

TGF-beta signaling from the cell surface to the nucleus is mediated by the SMAD family of proteins, which have been grouped into three classes based upon sequence identity and function. Receptor-regulated, or pathway-restricted, SMADs (R-SMADs) are phosphorylated by ligand-specific serine/threonine kinase receptors. Phosphorylated R-SMADs oligomerize with the coactivating, or shared, SMAD (Co-SMAD) mediator and translocate to the nucleus where the complex directs transcription of downstream target genes. Inhibitory SMADs (I-SMADs) block receptor-mediated phosphorylation of R-SMADs. In Drosophila, one member of each class of SMAD has been reported: MAD, an R-SMAD, MEDEA, a Co-SMAD, and DAD, an I-SMAD. Here, we report the first identification of a novel Drosophila R-SMAD, which we have named Smox for Smad on X. We have localized the Smox gene to a specific interval on the X chromosome and shown that Smox is transcribed throughout development. Copyright 1998 Academic Press.

Osthole pretreatment alleviates TNBS-induced colitis in mice via both cAMP/PKA-dependent and independent pathways.

PubMed

Sun, Wu; Cai, Yun; Zhang, Xin-Xin; Chen, Hao; Lin, Yan-Die; Li, Hao

2017-08-01

Osthole, a natural coumarin found in traditional Chinese medicinal plants, has shown multiple biological activities. In the present study, we investigated the preventive effects of osthole on inflammatory bowel disease (IBD). Colitis was induced in mice by infusing TNBS into the colonic lumen. Before TNBS treatment, the mice received osthole (100 mg·kg -1 ·d -1 , ip) for 3 d. Pretreatment with osthole significantly ameliorated the clinical scores, colon length shortening, colonic histopathological changes and the expression of inflammatory mediators in TNBS-induced colitis. Pretreatment with osthole elevated serum cAMP levels; but treatment with the PKA inhibitor H89 (10 mg·kg -1 ·d -1 , ip) did not abolish the beneficial effects of osthole on TNBS-induced colitis. In mouse peritoneal macrophages, pretreatment with osthole (50 μmol/L) significantly attenuated the LPS-induced elevation of cytokines at the mRNA level; inhibition of PKA completely reversed the inhibitory effects of osthole on IL-1β, IL-6, COX2, and MCP-1 but not on TNFα. In Raw264.7 cells, the p38 inhibitor SB203580 markedly suppressed LPS-induced upregulation of the cytokines, whereas the PKA inhibitors H89 or KT5720 did not abolish the inhibitory effects of SB203580. Moreover, in LPS-stimulated mouse peritoneal macrophages, SB203580 strongly inhibited the restored expression of IL-1β, IL-6, COX2, and MCP-1, which was achieved by abolishing the suppressive effects of osthole with the PKA inhibitors. Western blot analysis showed that osthole significantly suppressed the phosphorylation of p38, which was induced by TNBS in mice or by LPS in Raw264.7 cells. Inhibition of PKA partially reversed the suppressive effects of osthole on p38 phosphorylation in LPS-stimulated cells. Collectively, our results suggest that osthole is effective in the prevention of TNBS-induced colitis by reducing the expression of inflammatory mediators and attenuating p38 phosphorylation via both cAMP/PKA-dependent and independent pathways, among which the cAMP/PKA-independent pathway plays a major role.

Tetramethylpyrazine attenuates TNF-α-induced iNOS expression in human endothelial cells: Involvement of Syk-mediated activation of PI3K-IKK-IκB signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Zhen; Li, Zhiliang; Chen, Song

2013-08-15

Endothelial cells produce nitric oxide (NO) by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NO synthase (iNOS). We explored the effect of tetramethylpyrazine (TMP), a compound derived from chuanxiong, on tumor necrosis factor (TNF)-α-induced iNOS in human umbilical vein endothelial cells (HUVECs) and explored the signal pathways involved by using RT-PCR and Western blot. TMP suppressed TNF-α-induced expression of iNOS by inhibiting IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation, which were required for NO gene transcription. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathwaymore » might be phosphoinositide-3-kinase (PI3K) dependent. Spleen tyrosine kinase (Syk) inhibitor piceatannol significantly inhibited NO production. Furthermore, piceatannol obviously suppressed TNF-α-induced IκB phosphorylation and the downstream NF-κB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/IKK/IκB-mediated signaling. TMP significantly inhibited TNF-α-induced phosphorylation of Syk and PI3K. Our data indicate that TMP might repress iNOS expression, at least in part, through its inhibitory effect of Syk-mediated PI3K phosphorylation in TNF-α-stimulated HUVECs. -- Highlights: •TMP suppressed TNF-α-induced expression of iNOS by inhibiting IKK/IκB/NF-κB pathway. •PI3K inhibitor wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression. •Syk inhibitor piceatannol repressed PI3K/IKK/IκB mediated NO production. •Syk is an upstream regulator in the activation of PI3K/IKK/IκB-mediated signaling. •TMP might repress iNOS expression through Syk-mediated PI3K pathway.« less

The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Wei; Department of cardiology, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150081; Guo, Ting

2011-05-01

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration.more » Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.« less

Interferon-γ alters downstream signaling originating from epidermal growth factor receptor in intestinal epithelial cells: functional consequences for ion transport.

PubMed

Paul, Gisela; Marchelletta, Ronald R; McCole, Declan F; Barrett, Kim E

2012-01-13

The epidermal growth factor receptor (EGFr) regulates many cellular functions, such as proliferation, apoptosis, and ion transport. Our aim was to investigate whether long term treatment with interferon-γ (IFN-γ) modulates EGF activation of downstream signaling pathways in intestinal epithelial cells and if this contributes to dysregulation of epithelial ion transport in inflammation. Polarized monolayers of T(84) and HT29/cl.19A colonocytes were preincubated with IFN-γ prior to stimulation with EGF. Basolateral potassium transport was studied in Ussing chambers. We also studied inflamed colonic mucosae from C57BL/6 mice treated with dextran sulfate sodium or mdr1a knock-out mice and controls. IFN-γ increased intestinal epithelial EGFr expression without increasing its phosphorylation. Conversely, IFN-γ caused a significant decrease in EGF-stimulated phosphorylation of specific EGFr tyrosine residues and activation of ERK but not Akt-1. In IFNγ-pretreated cells, the inhibitory effect of EGF on carbachol-stimulated K(+) channel activity was lost. In inflamed colonic tissues, EGFr expression was significantly increased, whereas ERK phosphorylation was reduced. Thus, although it up-regulates EGFr expression, IFN-γ causes defective EGFr activation in colonic epithelial cells via reduced phosphorylation of specific EGFr tyrosine residues. This probably accounts for altered downstream signaling consequences. These observations were corroborated in the setting of colitis. IFN-γ also abrogates the ability of EGF to inhibit carbachol-stimulated basolateral K(+) currents. Our data suggest that, in the setting of inflammation, the biological effect of EGF, including the inhibitory effect of EGF on Ca(2+)-dependent ion transport, is altered, perhaps contributing to diarrheal and other symptoms in vivo.

St. John's wort extract and hyperforin inhibit multiple phosphorylation steps of cytokine signaling and prevent inflammatory and apoptotic gene induction in pancreatic β cells.

PubMed

Novelli, Michela; Menegazzi, Marta; Beffy, Pascale; Porozov, Svetlana; Gregorelli, Alex; Giacopelli, Daniela; De Tata, Vincenzo; Masiello, Pellegrino

2016-12-01

The extract of the herbaceous plant St. John's wort (SJW) and its phloroglucinol component hyperforin (HPF) were previously shown to inhibit cytokine-induced STAT-1 and NF-κB activation and prevent damage in pancreatic β cells. To further clarify the mechanisms underlying their protective effects, we evaluated the phosphorylation state of various factors of cytokine signaling pathways and the expression of target genes involved in β-cell function, inflammatory response and apoptosis induction. In the INS-1E β-cell line, exposed to a cytokine mixture with/without SJW extract (2-5μg/ml) or HPF (1-5μM), protein phosphorylation was assessed by western blotting and expression of target genes by real-time quantitative PCR. SJW and HPF markedly inhibited, in a dose-dependent manner (from 60 to 100%), cytokine-induced activating phosphorylations of STAT-1, NF-κB p65 subunit and IKK (NF-κB inhibitory subunit IκBα kinase). MAPK and Akt pathways were also modulated by the vegetal compounds through hindrance of p38 MAPK, ERK1/2, JNK and Akt phosphorylations, each reduced by at least 65% up to 100% at the higher dose. Consistently, SJW and HPF a) abolished cytokine-induced mRNA expression of pro-inflammatory genes; b) avoided down-regulation of relevant β-cell functional/differentiation genes; c) corrected cytokine-driven imbalance between pro- and anti-apoptotic factors, by fully preventing up-regulation of pro-apoptotic genes and preserving expression or function of anti-apoptotic Bcl-2 family members; d) protected INS-1E cells against cytokine-induced apoptosis. In conclusion, SJW extract and HPF exert their protective effects through simultaneous inhibition of multiple phosphorylation steps along various cytokine signaling pathways and consequent restriction of inflammatory and apoptotic gene expression. Thus, they have a promising therapeutic potential for the prevention or limitation of immune-mediated β-cell dysfunction and damage leading to type 1 diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.

PubMed

Battistone, M A; Da Ros, V G; Salicioni, A M; Navarrete, F A; Krapf, D; Visconti, P E; Cuasnicú, P S

2013-09-01

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.

TNFα- and IKKβ-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKγ and NF-κB activation

PubMed Central

Bonif, Marianne; Meuwis, Marie-Alice; Close, Pierre; Benoit, Valérie; Heyninck, Karen; Chapelle, Jean-Paul; Bours, Vincent; Merville, Marie-Paule; Piette, Jacques; Beyaert, Rudi; Chariot, Alain

2005-01-01

Pro-inflammatory cytokines trigger signalling cascades leading to NF-κB (nuclear factor-κB)-dependent gene expression through IKK [IκB (inhibitory κB) kinase]-dependent phosphorylation and subsequent degradation of the IκB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-κB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKβ upon TNFα stimulation and that this modification negatively regulates TANK binding to NEMO (NF-κB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFα-mediated induction of a subset of NF-κB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFα by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKβ-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-κB activation. PMID:16336209

LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Huiling; Li, Ridong; Li, Li

In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{submore » 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.« less

Quercetin suppresses the chymotrypsin-like activity of proteasome via inhibition of MEK1/ERK1/2 signaling pathway in hepatocellular carcinoma HepG2 cells.

PubMed

Ding, Youming; Chen, Xiaoyan; Wang, Bin; Yu, Bin; Ge, Jianhui; Shi, Xiaokang

2018-05-01

The proteasomal system is a promising target for cancer treatment. Quercetin (Que), a flavonoid compound with antitumor ability, displays the inhibitory effect on proteasome activity. However, the underlying molecular mechanisms are ill defined. The present study found that Que treatment significantly reduced the chymotrypsin-like protease activity of proteasome whereas the trypsin- and caspase-like protease activities remained unchanged in HepG2 cancer cells, along with activation of p38 MAPK and JNK and reduction of ERK1/2 phosphorylation. Que-reduced proteasome activity could not be reverted by inhibition of p38 MAPK and JNK signaling pathway. In addition, MEK1 overexpression or knockdown upregulated or downregulated the chymotrypsin-like protease activity of proteasome, respectively. Both Que and MEK1/ERK1/2 inhibitor attenuated the expression levels of proteasome β subunits. These results indicate that Que-induced suppression of MEK1/ERK1/2 signaling and subsequent reduction of proteasome β subunits is responsible for its inhibitory impacts on proteasome activity.

Double-edged swords as cancer therapeutics: novel, orally active, small molecules simultaneously inhibit p53-MDM2 interaction and the NF-κB pathway.

PubMed

Zhuang, Chunlin; Miao, Zhenyuan; Wu, Yuelin; Guo, Zizhao; Li, Jin; Yao, Jianzhong; Xing, Chengguo; Sheng, Chunquan; Zhang, Wannian

2014-02-13

Simultaneous inactivation of p53 and hyperactivation of nuclear factor-κB (NF-κB) is a common occurrence in human cancer. Currently, antitumor agents are being designed to selectively activate p53 or inhibit NF-κB. However, there is no concerted effort yet to deliberately design inhibitors that can simultaneously do both. This paper provided a proof-of-concept study that p53-MDM2 interaction and NF-κB pathway can be simultaneously targeted by a small-molecule inhibitor. A series of pyrrolo[3,4-c]pyrazole derivatives were rationally designed and synthesized as the first-in-class inhibitors of p53-MDM2 interaction and NF-κB pathway. Most of the compounds were identified to possess nanomolar p53-MDM2 inhibitory activity. Compounds 5q and 5s suppressed NF-κB activation through inhibition of IκBα phosphorylation and elevation of the cytoplasmic levels of p65 and phosphorylated IKKα/β. Biochemical assay for the kinases also supported the fact that pyrrolo[3,4-c]pyrazole compounds directly targeted the NF-κB pathway. In addition, four compounds (5j, 5q, 5s, and 5u) effectively inhibited tumor growth in the A549 xenograft model. Further pharmacokinetic study revealed that compound 5q exhibited excellent oral bioavailability (72.9%).

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

PubMed

Bijak, Michal; Szelenberger, Rafal; Dziedzic, Angela; Saluk-Bijak, Joanna

2018-02-10

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet activation.

Hydrogen peroxide inhibits transforming growth factor-β1-induced cell cycle arrest by promoting Smad3 linker phosphorylation through activation of Akt-ERK1/2-linked signaling pathway.

PubMed

Choi, Jiyeon; Park, Seong Ji; Jo, Eun Ji; Lee, Hui-Young; Hong, Suntaek; Kim, Seong-Jin; Kim, Byung-Chul

2013-06-14

Hydrogen peroxide (H2O2) functions as a second messenger in growth factor receptor-mediated intracellular signaling cascade and is tumorigenic by virtue of its ability to promote cell proliferation; however, the mechanisms underlying the growth stimulatory action of H2O2 are less understood. Here we report an important mechanism for antagonistic effects of H2O2 on growth inhibitory response to transforming growth factor-β1 (TGF-β1). In Mv1Lu and HepG2 cells, pretreatment of H2O2 (0.05-0.2 mM) completely blocked TGF-β1-mediated induction of p15(INK4B) expression and increase of its promoter activity. Interestingly, H2O2 selectively suppressed the transcriptional activation potential of Smad3, not Smad2, in the absence of effects on TGF-β1-induced phosphorylation of the COOH-tail SSXS motif of Smad3 and its nuclear translocation. Mechanism studies showed that H2O2 increases the phosphorylation of Smad3 at the middle linker region in a concentration- and time-dependent manner and this effect is mediated by activation of extracellular signal-activated kinase 1/2 through Akt. Furthermore, expression of a mutant Smad3 in which linker phosphorylation sites were ablated significantly abrogated the inhibitory effects of H2O2 on TGF-β1-induced increase of p15(INK4B)-Luc reporter activity and blockade of cell cycle progression from G1 to S phase. These findings for the first time define H2O2 as a signaling molecule that modulate Smad3 linker phosphorylation and its transcriptional activity, thus providing a potential mechanism whereby H2O2 antagonizes the cytostatic function of TGF-β1. Copyright © 2013 Elsevier Inc. All rights reserved.

Flavonoids from sea buckthorn inhibit the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages through the MAPK and NF-κB pathways.

PubMed

Jiang, Fan; Guan, Haining; Liu, Danyi; Wu, Xi; Fan, Mingcheng; Han, Jianchun

2017-03-22

Sea buckthorn has long been used as a functional food to regulate cholesterol, relieve angina, and diminish inflammation. Flavonoids are one of the main active components in sea buckthorn. We investigated the effects of sea buckthorn flavonoid (SF) treatment on two pathways that mediate inflammation, the mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways, to explore the anti-inflammatory activity of SFs in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The LPS-induced over-production of nitric oxide (NO) and prostaglandin E2 (PGE 2 ) was inhibited by SFs through a mechanism related to the modulatory effects of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) genes. Additionally, SFs downregulated the production and mRNA expression of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β. Moreover, SFs inhibited the phosphorylation of the p38 and stress-activated protein kinase/jun amino-terminal kinase (SAPK/JNK) MAPK pathways, and they reduced the nuclear translocation of NF-κB to prevent its activation by blocking the phosphorylation and degradation of inhibitor protein of NF-κB α (IκB-α). Based on these findings, SFs may exert their inhibitory effects on inflammation by regulating the release of inflammatory mediators through the MAPK and NF-κB pathways. SFs highlight the potential benefits of using functional foods with anti-inflammatory actions to combat inflammatory diseases.

Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies.

PubMed

Ricciardi, Maria Rosaria; Scerpa, Maria Cristina; Bergamo, Paola; Ciuffreda, Ludovica; Petrucci, Maria Teresa; Chiaretti, Sabina; Tavolaro, Simona; Mascolo, Maria Grazia; Abrams, Stephen L; Steelman, Linda S; Tsao, Twee; Marchetti, Antonio; Konopleva, Marina; Del Bufalo, Donatella; Cognetti, Francesco; Foà, Robin; Andreeff, Michael; McCubrey, James A; Tafuri, Agostino; Milella, Michele

2012-10-01

In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70 % of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML.

Tyrosine phosphorylation of dihydrolipoamide dehydrogenase as a potential cadmium target and its inhibitory role in regulating mouse sperm motility.

PubMed

Li, Xinhong; Wang, Lirui; Li, Yuhua; Fu, Jieli; Zhen, Linqing; Yang, Qiangzhen; Li, Sisi; Zhang, Yukun

2016-05-16

Cadmium (Cd) is reported to reduce sperm motility and functions. However, the molecular mechanisms of Cd-induced toxicity remain largely unknown, presenting a major knowledge gap in research on reproductive toxicology. In the present study, we identified a candidate protein, dihydrolipoamide dehydrogenase (DLD), which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation in mouse sperm exposed to Cd both in vivo and in vitro. Immunoprecipitation assay demonstrated DLD was phosphorylated in tyrosine residues without altered expression after Cd treatment, which further confirmed our identified result. However, the tyrosine phosphorylation of DLD did not participate in mouse sperm capacitation and Bovine Serum Albumin (BSA) effectively prevented the tyrosine phosphorylation of DLD. Moreover, Cd-induced tyrosine phosphorylation of DLD lowered its dehydrogenase activity and meanwhile, Nicotinamide Adenine Dinucleotide Hydrogen (NADH) content, Adenosine Triphosphate (ATP) production and sperm motility were all inhibited by Cd. Interestingly, when the tyrosine phosphorylation of DLD was blocked by BSA, the decrease of DLD activity, NADH and ATP content as well as sperm motility was also suppressed simultaneously. These results suggested that Cd-induced tyrosine phosphorylation of DLD inhibited its activity and thus suppressed the tricarboxylic acid (TCA) cycle, which resulted in the reduction of NADH and hence the ATP production generated through oxidative phosphorylation (OPHOXS). Taken together, our results revealed that Cd induced DLD tyrosine phosphorylation, in response to regulate TCA metabolic pathway, which reduced ATP levels and these negative effects led to decreased sperm motility. This study provided new understanding of the mechanisms contributing to the harmful effects of Cd on the motility and function of spermatozoa. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling

PubMed Central

Chen, Qing; Su, Yi; Wesslowski, Janine; Hagemann, Anja I; Ramialison, Mirana; Wittbrodt, Joachim; Scholpp, Steffen; Davidson, Gary

2014-01-01

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6. Subject Categories Membrane & Intracellular Transport; Post-translational Modifications, Proteolysis & Proteomics PMID:25391905

Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis

PubMed Central

Wang, Zihao; Udeshi, Namrata D.; Slawson, Chad; Compton, Philip D.; Sakabe, Kaoru; Cheung, Win D.; Shabanowitz, Jeffrey; Hunt, Donald F.; Hart, Gerald W.

2010-01-01

Like phosphorylation, the addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways, but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division. PMID:20068230

Insulin Activates RSK (p90 Ribosomal S6 Kinase) to Trigger a New Negative Feedback Loop That Regulates Insulin Signaling for Glucose Metabolism*

PubMed Central

Smadja-Lamère, Nicolas; Shum, Michael; Déléris, Paul; Roux, Philippe P.; Abe, Jun-Ichi; Marette, André

2013-01-01

We previously demonstrated that the mTORC1/S6K1 pathway is activated by insulin and nutrient overload (e.g. amino acids (AA)), which leads to the inhibition of the PI3K/Akt pathway via the inhibitory serine phosphorylation of IRS-1, notably on serine 1101 (Ser-1101). However, even in the absence of AA, insulin can still promote IRS-1 Ser-1101 phosphorylation by other kinases that remain to be fully characterized. Here, we describe a new negative regulator of IRS-1, the p90 ribosomal S6 kinase (RSK). Computational analyses revealed that Ser-1101 within IRS-1 falls into the consensus motif of RSK. Moreover, recombinant RSK phosphorylated IRS-1 C-terminal fragment on Ser-1101, which was prevented by mutations of this site or when a kinase-inactive mutant of RSK was used. Using antibodies directed toward the phosphorylation sites located in the activation segment of RSK (Ser-221 or Ser-380), we found that insulin activates RSK in L6 myocytes in the absence of AA overload. Inhibition of RSK using either the pharmacological inhibitor BI-D1870 or after adenoviral expression of a dominant negative RSK1 mutant (RSK1-DN) showed that RSK selectively phosphorylates IRS-1 on Ser-1101. Accordingly, expression of the RSK1-DN mutant in L6 myocytes and FAO hepatic cells improved insulin action on glucose uptake and glucose production, respectively. Furthermore, RSK1 inhibition prevented insulin resistance in L6 myocytes chronically exposed to high glucose and high insulin. These results show that RSK is a novel regulator of insulin signaling and glucose metabolism and a potential mediator of insulin resistance, notably through the negative phosphorylation of IRS-1 on Ser-1101. PMID:24036112

Curcumin inhibits the invasion of lung cancer cells by modulating the PKCα/Nox-2/ROS/ATF-2/MMP-9 signaling pathway.

PubMed

Fan, Zhigang; Duan, Xiaoyi; Cai, Hui; Wang, Li; Li, Min; Qu, Jingkun; Li, Wanjun; Wang, Yongheng; Wang, Jiansheng

2015-08-01

Invasion and metastasis are the major causes of tumor-related mortality in lung cancer. It is believed that curcumin is an effective drug possessing anti-invasive and anti-metastatic activities in the treatment of cancer. However, the specific mechanisms remain unclear. In the present study, we investigated whether the PKCα/Nox-2/ATF-2/MMP-9 signaling pathway is involved in the invasive behavior of lung cancer and whether curcumin could inhibit invasion by modulating this pathway. The cytotoxic effect of curcumin was evaluated by MTT assay and the capacity of invasion was assessed by Transwell assay. siRNA and plasmid transfection techniques were used to study the function of targeted genes. Real-time PCR and western blot analysis were used to evaluate the expression levels of PKCα, Nox-2, MMP-9 and the phosphorylation of ATF-2. The results showed that curcumin inhibited the proliferation and invasion of A549 cells in a dose-dependent manner. Overexpression of MMP-9 enhanced the invasion of A549 cells. However, inhibition of MMP-9 by siRNA or curcumin suppressed cell invasion. Moreover, we also demonstrated the catalytic role of PKCα in expression of MMP-9 and cellular invasion in A549 cells, which was dependent on the expression of Nox-2 and phosphorylation of ATF-2. Finally, we also showed that curcumin dose-dependently reduced the expression of PKCα, P47phox, Nox-2 and phosphorylated ATF-2, as well as intracellular ROS generation, suggesting the inhibitory effect of curcumin on the activation of the PKCα/Nox-2/ROS/ATF-2 pathway. In conclusion, the PKCα/Nox-2/ROS/ATF-2/MMP-9 signaling pathway is activated in lung cancer A549 cells, which could be modulated by curcumin to inhibit cell invasiveness.

HSPA6 augments garlic extract-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; Implication for cell cycle dysregulation, signaling pathway alteration, and transcription factor-associated MMP-9 regulation

PubMed Central

Hwang, Byungdoo; Noh, Dae-Hwa; Park, Sung Lyea; Kim, Won Tae; Park, Sung-Soo; Kim, Wun-Jae; Moon, Sung-Kwon

2017-01-01

Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies. PMID:28187175

Anti-inflammatory effect of procyanidins from wild grape (Vitis amurensis) seeds in LPS-induced RAW 264.7 cells.

PubMed

Bak, Min-Ji; Truong, Van Long; Kang, Hey-Sook; Jun, Mira; Jeong, Woo-Sik

2013-01-01

In the present study, the anti-inflammatory effect and underlying mechanisms of wild grape seeds procyanidins (WGP) were examined using lipopolysaccharide- (LPS-) stimulated RAW 264.7 cells. We used nitric oxide (NO) and prostaglandin E2 (PGE2) and reactive oxygen species (ROS) assays to examine inhibitory effect of WGP and further investigated the mechanisms of WGP suppressed LPS-mediated genes and upstream expression by Western blot and confocal microscopy analysis. Our data indicate that WGP significantly reduced NO, PGE2, and ROS production and also inhibited the expression of proinflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions. Consistently, WGP significantly reduced LPS-stimulated expression of proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin- (IL-) 1 β . Moreover, WGP prevented nuclear translocation of nuclear factor- κ B (NF κ B) p65 subunit by reducing inhibitory κ B- α (I κ B α) and NF κ B phosphorylation. Furthermore, we found that WGP inhibited LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). Taken together, our results demonstrated that WGP exerts potent anti-inflammatory activity through the inhibition of iNOS and COX-2 by regulating NF κ B and p38 MAPK pathway.

Phosphorylated Akt/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and invasive ductal adenocarcinoma of the pancreas.

PubMed

Semba, Shuho; Moriya, Toshiyuki; Kimura, Wataru; Yamakawa, Mitsunori

2003-04-01

Akt/PKB promotes cell proliferation and rescues cells from apoptosis. To evaluate the role of Akt/PKB, a key molecule in phosphatidylinositol 3-OH kinase (PI3K) signaling, during the development of pancreatic duct neoplasias such as intraductal papillary-mucinous tumor (IPMT) and invasive ductal adenocarcinoma (IDAC) of the pancreas. In PK-45H pancreatic cancer cells, the growth-inhibitory and apoptosis-inducing effects of LY294002, a PI3K inhibitor, were detected in a concentration-dependent manner, followed by the reduction of phosphorylated Akt levels. Immunohistochemical analyses revealed that frequent overexpression of phosphorylated Akt (Ser473) was detected in 10 (63%) of 16 IPMTs and 14 (70%) of 20 IDACs. It is interesting that the incidence of Akt phosphorylation closely correlated with Ki-67 immunoreactivity and had an inverse association with the number of cases of apoptotic bodies in both IPMT and IDAC. Although there was no good correlation with other clinicopathologic parameters, the two patients with recurrent IPMT had high levels of phosphorylated Akt. Our findings suggest that activation of Akt plays an important role during the progression of these pancreatic duct neoplasias at the early stage. Furthermore, inhibition of the PI3K-Akt/PKB pathway may have therapeutic potential in the treatment of pancreatic duct tumors.

The regulation of smooth muscle contractility by zipper-interacting protein kinase.

PubMed

Ihara, Eikichi; MacDonald, Justin A

2007-01-01

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

Antibody to human α-fetoprotein inhibits cell growth of human hepatocellular carcinoma cells by resuscitating the PTEN molecule: in vitro experiments.

PubMed

Ohkawa, Kiyoshi; Asakura, Tadashi; Tsukada, Yutaka; Matsuura, Tomokazu

2017-06-01

It has been proposed that α-fetoprotein (AFP) is a new member of the intracellular signaling molecule family of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway via interaction with the phosphatase and tensin homolog (PTEN). In this study, the effects of anti-human AFP antibody on the functions of PTEN were examined using an AFP-producing human hepatoma cell line. The antibody caused significant inhibition of cell growth, compared to a normal IgG control, with the accumulation of intracellular immune complexes followed by significant reduction of cytosolic functional AFP. Decrease in the amount of AKT phosphorylated on serine (S) 473 indicated that PI3K/AKT signaling was suppressed in the cells. S380-phosphorylated PTEN increased markedly by the second day after antibody treatment, with slight but significant increase in the PTEN protein level. Since phosphorylation at S380 is critical for PTEN stability, the increase in S380-phosphorylated PTEN indicated maintenance of the number of PTEN molecules and the related potential to control PI3K/AKT signaling. p53 protein (P53) significantly, but slightly increased during antibody treatment, because PTEN expression increased the stability and function of P53 via both molecular interactions. P53 phosphorylated at S20 or at S392 dramatically increased, suggesting an increase in the stability, accumulation and activation of P53. Glucose transporter 1 (GLUT1) increased immediately after antibody treatment, pointing to a deficiency of glucose in the cells. Immunofluorescence cytology revealed that antibody-treatment re-distributed GLUT1 molecules throughout the cytoplasm with a reduction of their patchy localization on the cell surface. This suggested that translocation of GLUT1 depends on the PI3K/AKT pathway, in particular on PTEN expression. Antibody therapy targeted at AFP-producing tumor cells showed an inhibitory effect on the PI3K/AKT pathway via the liberation, restoration and functional stabilization of PTEN. PTEN simultaneously induced both P53 activation and intracellular translocation of GLUT1, since these are closely associated with PTEN.

Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and {beta}4 integrin function in MDA-MB-231 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, W.-J.; Chen, W.-K.; Wang, C.-J.

2008-01-15

Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration andmore » anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of {beta}4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin {beta}4 function including cell-matrix adhesion and cell-endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By histological and gross examination of mouse lung and real-time PCR analysis of human alu in host tissues, it showed that apigenin, wortmannin, as well as anti-{beta}4 antibody all inhibit HGF-promoted metastasis. These data support the inhibitory effect of apigenin on HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and integrin {beta}4 function.« less

Protective effect of Ginkgo biloba leaves extract, EGb761, on endotoxin-induced acute lung injury via a JNK- and Akt-dependent NFκB pathway.

PubMed

Lee, Chien-Ying; Yang, Jiann-Jou; Lee, Shiuan-Shinn; Chen, Chun-Jung; Huang, Yi-Chun; Huang, Kuang-Hua; Kuan, Yu-Hsiang

2014-07-09

Acute lung injury (ALI) is a clinical syndrome mainly caused by Gram-negative bacteria which is still in need of an effective therapeutic medicine. EGb761, an extract of Ginkgo biloba leaves, has several bioeffects including anti-inflammation, cardioprotection, neuroprotection, and free radical scavenging. Preadministration of EGb761 inhibited lipopolysaccharide (LPS)-induced histopathological changes and exchange of arterial blood gas. In addition, LPS-induced expression of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, macrophage inflammatory protein (MIP)-2, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were suppressed by EGb761. The activation of nuclear factor (NF)κB, a transcription factor of proinflammatory mediators, and phosphorylation of IκB, an inhibitor of NFκB, were also reduced by EGb761. Furthermore, we found the inhibitory concentration of EGb761 on phosphorylation of JNK and Akt was less than those of ERK and p38 MAPK. In conclusion, EGb761 is a potential protective agent for ALI, possibly via downregulating the JNK- and Akt-dependent NFκB activation pathway.

Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: the role of GSK-3beta in the maintenance of steady-state levels of insulin receptor signaling molecules and Na(v)1.7 sodium channel in adrenal chromaffin cells.

PubMed

Nemoto, Takayuki; Yanagita, Toshihiko; Kanai, Tasuku; Wada, Akihiko

2009-02-01

Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, where the majority of its substrates undergo inactivation/proteolysis by phosphorylation. Extracellular stimuli (e.g., insulin) catalyze inhibitory Ser(9)-phosphorylation of GSK-3beta, turning on signaling and causing other biological consequences otherwise constitutively suppressed by GSK-3beta. Regulated and dysregulated activities of GSK-3beta are pivotal to health, disease, and therapeutics (e.g., insulin resistance, neurodegeneration, tumorigenesis, inflammation); however, the underlying mechanisms of multifunctional GSK-3beta remain elusive. In cultured bovine adrenal chromaffin cells, 1) constitutive and negatively-regulated activities of GSK-3beta up- and down-regulated insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, and Akt levels via controlling proteasomal degradation and protein synthesis; 2) nicotinic receptor/protein kinase C-alpha (PKC-alpha)/extracellular signal-regulated kinase (ERK) pathway up-regulated IRS-1 and IRS-2 levels, enhancing insulin-induced the phosphoinositide 3-kinase (PI3K)/Akt/GSK-3beta pathway; 3) inhibition of calcineurin by cyclosporin A or FK506 down-regulated IRS-2 level, attenuating insulin-like growth factor-I (IGF-I)-induced ERK and GSK-3beta pathways; and 4) insulin, IGF-I or therapeutics (e.g., lithium) up-regulated the voltage-dependent Na(v)1.7 sodium channel.

The adaptor protein Crk controls activation and inhibition of natural killer cells.

PubMed

Liu, Dongfang; Peterson, Mary E; Long, Eric O

2012-04-20

Natural killer (NK) cell inhibitory receptors recruit tyrosine phosphatases to prevent activation, induce phosphorylation and dissociation of the small adaptor Crk from cytoskeleton scaffold complexes, and maintain NK cells in a state of responsiveness to subsequent activation events. How Crk contributes to inhibition is unknown. We imaged primary NK cells over lipid bilayers carrying IgG1 Fc to stimulate CD16 and human leukocyte antigen (HLA)-E to inhibit through receptor CD94-NKG2A. HLA-E alone induced Crk phosphorylation in NKG2A(+) NK cells. At activating synapses with Fc alone, Crk was required for the movement of Fc microclusters and their ability to trigger activation signals. At inhibitory synapses, HLA-E promoted central accumulation of both Fc and phosphorylated Crk and blocked the Fc-induced buildup of F-actin. We propose a unified model for inhibitory receptor function: Crk phosphorylation prevents essential Crk-dependent activation signals and blocks F-actin network formation, thereby reducing constraints on subsequent engagement of activation receptors. Copyright © 2012 Elsevier Inc. All rights reserved.

Involvement of Epidermal Growth Factor Receptor Signaling in Estrogen Inhibition of Oocyte Maturation Mediated Through the G Protein-Coupled Estrogen Receptor (Gper) in Zebrafish (Danio rerio)1

PubMed Central

Peyton, Candace; Thomas, Peter

2011-01-01

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression. PMID:21349822

Involvement of epidermal growth factor receptor signaling in estrogen inhibition of oocyte maturation mediated through the G protein-coupled estrogen receptor (Gper) in zebrafish (Danio rerio).

PubMed

Peyton, Candace; Thomas, Peter

2011-07-01

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression.

Arctigenin enhances chemosensitivity of cancer cells to cisplatin through inhibition of the STAT3 signaling pathway.

PubMed

Yao, Xiangyang; Zhu, Fenfen; Zhao, Zhihui; Liu, Chang; Luo, Lan; Yin, Zhimin

2011-10-01

Arctigenin is a dibenzylbutyrolactone lignan isolated from Bardanae fructus, Arctium lappa L, Saussureamedusa, Torreya nucifera, and Ipomea cairica. It has been reported to exhibit anti-inflammatory activities, which is mainly mediated through its inhibitory effect on nuclear transcription factor-kappaB (NF-κB). But the role of arctigenin in JAK-STAT3 signaling pathways is still unclear. In present study, we investigated the effect of arctigenin on signal transducer and activator of transcription 3 (STAT3) pathway and evaluated whether suppression of STAT3 activity by arctigenin could sensitize cancer cells to a chemotherapeutic drug cisplatin. Our results show that arctigenin significantly suppressed both constitutively activated and IL-6-induced STAT3 phosphorylation and subsequent nuclear translocation in cancer cells. Inhibition of STAT3 tyrosine phosphorylation was found to be achieved through suppression of Src, JAK1, and JAK2, while suppression of STAT3 serine phosphorylation was mediated by inhibition of ERK activation. Pervanadate reversed the arctigenin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, arctigenin can obviously induce the expression of the PTP SHP-2. Furthermore, the constitutive activation level of STAT3 was found to be correlated to the resistance of cancer cells to cisplatin-induced apoptosis. Arctigenin dramatically promoted cisplatin-induced cell death in cancer cells, indicating that arctigenin enhanced the sensitivity of cancer cells to cisplatin mainly via STAT3 suppression. These observations suggest a novel anticancer function of arctigenin and a potential therapeutic strategy of using arctigenin in combination with chemotherapeutic agents for cancer treatment. Copyright © 2011 Wiley-Liss, Inc.

Anti-inflammatory action of high molecular weight Mytilus edulis hydrolysates fraction in LPS-induced RAW264.7 macrophage via NF-κB and MAPK pathways.

PubMed

Kim, Young-Sang; Ahn, Chang-Bum; Je, Jae-Young

2016-07-01

Anti-inflammatory Mytilus edulis hydrolysates (MEHs) were prepared by peptic hydrolysis and MEH was further fractionated into three fractions based on molecular weight, namely >5kDa, 1-5kDa, and <1kDa. The >5kDa peptide fraction exerted the highest nitric oxide (NO) inhibitory activity and inhibited prostaglandin E2 (PGE2) secretion in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Pretreatment with the >5kDa peptide fraction markedly inhibited LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and gene expressions. Stimulation by LPS induced the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β), whereas co-treatment with the >5kDa peptide fraction suppressed pro-inflammatory cytokine production. The >5kDa peptide fraction inhibited the translocation of NF-κB (nuclear factor-kappa B) through the prevention of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation and also inhibited the MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

A taspine derivative supresses Caco-2 cell growth by competitively targeting EphrinB2 and regulating its pathway.

PubMed

Dai, Bingling; Wang, Wenjie; Ma, Yujiao; Liu, Rui; Zhang, Yanmin

2016-09-01

Colorectal cancer is a common gastrointestinal malignancy worldwide and it is a lethal and aggressive malignancy with a dismal prognosis. In the present study, we investigated the effect of taspine derivative 12k on human colorectal cancer targeted at EphrinB2 and its PDZ. The results indicated that 12k could bind to EphrinB2 and showed a higher suppressive effect on EphrinB2/HEK293 than on HEK293 cells. Caco‑2 cells were screened for high expression of EphrinB2. We found that 12k not only significantly decreased Caco‑2 cell viability and colony formation but impaired migration. Meanwhile, 12k effectively inhibited blood vessel formation in a tissue model of angiogenesis. Mechanistic studies revealed that 12k significantly reduced the phosphorylation of EphrinB2 and PDZ protein PICK1. Accordingly, it was associated with the downregulation by 12k of the PI3K/AKT/mTOR and MAPK signaling pathways which were downstream of VEGFR2, yet it had no effect on VEGFR3. Moreover, the expression of CD34, CD45 and HIF‑1α were downregulated in the Caco‑2 cells. In conclusion, our findings showed that 12k had an inhibitory effect on the growth of Caco-2 cells, and it functioned by interrupting the phosphorylation of EphrinB2 and its related signaling pathway.

Role of Spm-Cer-S1P signalling pathway in MMP-2 mediated U46619-induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin-3-gallate.

PubMed

Chowdhury, Animesh; Sarkar, Jaganmay; Chakraborti, Tapati; Chakraborti, Sajal

2015-10-01

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occurs, which contributes to pulmonary hypertension. Thromboxane A2 (TxA2) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin-3-gallate (EGCG) on the TxA2 mimetic, U46619-induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p(38)MAPK, NF-κB and MMP-2 significantly inhibit U46619-induced cell proliferation. EGCG markedly abrogate U46619-induced p(38)MAPK phosphorylation, NF-κB activation, proMMP-2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619-induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP-2 markedly abrogate U46619-induced SMase activity and S1P level. EGCG markedly inhibit U46619-induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline-Ceramide-Sphingosine-1-phosphate (Spm-Cer-S1P) signalling axis plays an important role in MMP-2 mediated U46619-induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP-2 activation by modulating p(38)MAPK-NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.

Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways

PubMed Central

Watanabe, Nobumoto; Arai, Harumi; Iwasaki, Jun-ichi; Shiina, Masaaki; Ogata, Kazuhiro; Hunter, Tony; Osada, Hiroyuki

2005-01-01

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCFβ-TrCP. The F-box protein β-TrCP (β-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to β-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved β-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in β-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to β-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of β-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second β-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis. PMID:16085715

[Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells].

PubMed

Horinouchi, Takahiro; Mazaki, Yuichi; Terada, Koji; Miwa, Soichi

2018-01-01

Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr 308 and Ser 473 , and [ 3 H]-labelled 2-deoxy-D-glucose ([ 3 H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [ 3 H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ET A R), inhibition of G q/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ET A R with ET-1 inhibits insulin-induced Akt phosphorylation and [ 3 H]2-DG uptake in a G q/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ET A R and GRK2 are potential targets for insulin resistance.

WIF1 re-expression in glioblastoma inhibits migration through attenuation of non-canonical WNT signaling by downregulating the lncRNA MALAT1.

PubMed

Vassallo, I; Zinn, P; Lai, M; Rajakannu, P; Hamou, M-F; Hegi, M E

2016-01-07

Glioblastoma is the most aggressive primary brain tumor in adults and due to the invasive nature cannot be completely removed. The WNT inhibitory factor 1 (WIF1), a secreted inhibitor of WNTs, is systematically downregulated in glioblastoma and acts as strong tumor suppressor. The aim of this study was the dissection of WIF1-associated tumor-suppressing effects mediated by canonical and non-canonical WNT signaling. We found that WIF1 besides inhibiting the canonical WNT pathway selectively downregulates the WNT/calcium pathway associated with significant reduction of p38-MAPK (p38-mitogen-activated protein kinase) phosphorylation. Knockdown of WNT5A, the only WNT ligand overexpressed in glioblastoma, phenocopied this inhibitory effect. WIF1 expression inhibited cell migration in vitro and in an orthotopic brain tumor model, in accordance with the known regulatory function of the WNT/Ca(2+) pathway on migration and invasion. In search of a mediator for this function differential gene expression profiles of WIF1-expressing cells were performed. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA and key positive regulator of invasion, emerged as the top downregulated gene. Indeed, knockdown of MALAT1 reduced migration in glioblastoma cells, without effect on proliferation. Hence, loss of WIF1 enhances the migratory potential of glioblastoma through WNT5A that activates the WNT/Ca(2+) pathway and MALAT1. These data suggest the involvement of canonical and non-canonical WNT pathways in glioblastoma promoting key features associated with this deadly disease, proliferation on one hand and invasion on the other. Successful targeting will require a dual strategy affecting both canonical and non-canonical WNT pathways.

Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3.

PubMed

Chen, Shuzhen; Cao, Wei; Yue, Ping; Hao, Chunhai; Khuri, Fadlo R; Sun, Shi-Yong

2011-10-01

Celecoxib is a COX-2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of cellular FLICE-inhibitory protein (c-FLIP), a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of glycogen synthase kinase-3 (GSK3), itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3, including the α and β forms, even in cell lines, where phosphorylated Akt levels were not increased. Phosphoinositide 3-kinase inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, protein kinase C (PKC) inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism that relied upon PKC and not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.

RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle

PubMed Central

Safdie, Gracia; Liewald, Jana F.; Kagan, Sarah; Battat, Emil; Gottschalk, Alexander; Treinin, Millet

2016-01-01

Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons. Here we show that phosphorylation of the ER-resident chaperone of nicotinic acetylcholine receptors, RIC-3, leads to increased muscle excitability. RIC-3 phosphorylation at Ser-164 depends on opposing functions of the phosphatase calcineurin (TAX-6), and of the casein kinase II homologue KIN-10. Effects of calcineurin down-regulation and of phosphorylated RIC-3 on muscle excitability are mediated by GABAA receptor inhibition. Thus RIC-3 phosphorylation enables effects of this chaperone on GABAA receptors in addition to nAChRs. This dual effect provides coordinated regulation of excitation and inhibition and enables fine-tuning of the excitation–inhibition balance. Moreover, regulation of inhibitory GABAA signaling by calcineurin, a calcium- and calmodulin-dependent phosphatase, enables homeostatic balancing of excitation and inhibition. PMID:27489343

Macrophage migration inhibitory factor limits activation-induced apoptosis of platelets via CXCR7-dependent Akt signaling.

PubMed

Chatterjee, Madhumita; Borst, Oliver; Walker, Britta; Fotinos, Anna; Vogel, Sebastian; Seizer, Peter; Mack, Andreas; Alampour-Rajabi, Setareh; Rath, Dominik; Geisler, Tobias; Lang, Florian; Langer, Harald F; Bernhagen, Jürgen; Gawaz, Meinrad

2014-11-07

Macrophage migration inhibitory factor (MIF) is released on platelet activation. Circulating MIF could potentially regulate platelets and thereby platelet-mediated inflammatory and regenerative mechanisms. However, the effect of MIF on platelets is unknown. The present study evaluated MIF in regulating platelet survival and thrombotic potential. MIF interacted with CXCR4-CXCR7 on platelets, defining CXCR7 as a hitherto unrecognized receptor for MIF on platelets. MIF internalized CXCR4, but unlike CXCL12 (SDF-1α), it did not phosphorylate Erk1/2 after CXCR4 ligation because of the lack of CD74 and failed in subsequent CXCR7 externalization. MIF did not alter the activation status of platelets. However, MIF rescued platelets from activation and BH3 mimetic ABT-737-induced apoptosis in vitro via CXCR7 and enhanced circulating platelet survival when administered in vivo. The antiapoptotic effect of MIF was absent in Cxcr7(-/-) murine embryonic cells but pronounced in CXCR7-transfected Madin-Darby canine kidney cells. This prosurvival effect was attributed to the MIF-CXCR7-initiated PI3K-Akt pathway. MIF induced CXCR7-Akt-dependent phosphorylation of BCL-2 antagonist of cell death (BAD) both in vitro and in vivo. Consequentially, MIF failed to rescue Akt(-/-) platelets from thrombin-induced apoptosis when challenged ex vivo, also in prolonging platelet survival and in inducing BAD phosphorylation among Akt(-/-) mice in vivo. MIF reduced thrombus formation under arterial flow conditions in vitro and retarded thrombotic occlusion after FeCl3-induced arterial injury in vivo, an effect mediated through CXCR7. MIF interaction with CXCR7 modulates platelet survival and thrombotic potential both in vitro and in vivo and thus could regulate thrombosis and inflammation. © 2014 American Heart Association, Inc.

New insights into the Vav1 activation cycle in lymphocytes.

PubMed

Barreira, María; Rodríguez-Fdez, Sonia; Bustelo, Xosé R

2018-05-01

Vav1 is a hematopoietic-specific Rho GDP/GTP exchange factor and signaling adaptor. Although these activities are known to be stimulated by direct Vav1 phosphorylation, little information still exists regarding the regulatory layers that influence the overall Vav1 activation cycle. Using a collection of cell models and activation-mimetic Vav1 mutants, we show here that the dephosphorylated state of Vav1 in nonstimulated T cells requires the presence of a noncatalytic, phospholipase Cγ1-Slp76-mediated inhibitory pathway. Upon T cell stimulation, Vav1 becomes rapidly phosphorylated via the engagement of Lck and, to a much lesser extent, other Src family kinases and Zap70. In this process, Lck, Zap70 and the adaptor protein Lat contribute differently to the dynamics and amplitude of the Vav1 phosphorylated pool. Consistent with a multiphosphosite activation mechanism, the optimal stimulation of Vav1 can only be recapitulated by the combination of several activation-mimetic phosphosite mutants. The analysis of these mutants has also unveiled the presence of different Vav1 signaling competent states that are influenced by phosphosites present in the N- and C-terminal domains of the protein. Copyright © 2018 Elsevier Inc. All rights reserved.

Induction of apoptosis by withaferin A in human leukemia U937 cells through down-regulation of Akt phosphorylation.

PubMed

Oh, Jung Hwa; Lee, Tae-Jin; Kim, Sang Hyun; Choi, Yung Hyun; Lee, Sang Han; Lee, Jin Man; Kim, Young-Ho; Park, Jong-Wook; Kwon, Taeg Kyu

2008-12-01

Withaferin A, a major chemical constituent of Withania somnifera, has been reported for its tumor cell growth inhibitory activity, antitumor effects, and impairing metastasis and angiogenesis. The mechanism by which withaferin A initiates apoptosis remains poorly understood. In the present report, we investigated the effect of withaferin A on the apoptotic pathway in U937 human promonocytic cells. We show that withaferin A induces apoptosis in association with the activation of caspase-3. JNK and Akt signal pathways play crucial roles in withaferin A-induced apoptosis in U937 cells. Furthermore, we have shown that overexpression of Bcl-2 and active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase-3, and PLC-gamma1 cleavage by withaferin A. Taken together, our results indicated that the JNK and Akt pathways and inhibition of NF-kappaB activity were key regulators of apoptosis in response to withaferin A in human leukemia U937 cells.

Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats

PubMed Central

Bernabeu, Ramon; Bevilaqua, Lia; Ardenghi, Patricia; Bromberg, Elke; Schmitz, Paulo; Bianchin, Marino; Izquierdo, Ivan; Medina, Jorge H.

1997-01-01

cAMP/cAMP-dependent protein kinase (PKA) signaling pathway has been recently proposed to participate in both the late phase of long term potentiation in the hippocampus and in the late, protein synthesis-dependent phase of memory formation. Here we report that a late memory consolidation phase of an inhibitory avoidance learning is regulated by an hippocampal cAMP signaling pathway that is activated, at least in part, by D1/D5 receptors. Bilateral infusion of SKF 38393 (7.5 μg/side), a D1/D5 receptor agonist, into the CA1 region of the dorsal hippocampus, enhanced retention of a step-down inhibitory avoidance when given 3 or 6 h, but not immediately (0 h) or 9 h, after training. In contrast, full retrograde amnesia was obtained when SCH 23390 (0.5 μg/side), a D1/D5 receptor antagonist, was infused into the hippocampus 3 or 6 h after training. Intrahippocampal infusion of 8Br-cAMP (1.25 μg/side), or forskolin (0.5 μg/side), an activator of adenylyl cyclase, enhanced memory when given 3 or 6 h after training. KT5720 (0.5 μg/side), a specific inhibitor of PKA, hindered memory consolidation when given immediately or 3 or 6 h posttraining. Rats submitted to the avoidance task showed learning-specific increases in hippocampal 3H-SCH 23390 binding and in the endogenous levels of cAMP 3 and 6 h after training. In addition, PKA activity and P-CREB (phosphorylated form of cAMP responsive element binding protein) immunoreactivity increased in the hippocampus immediately and 3 and 6 h after training. Together, these findings suggest that the late phase of memory consolidation of an inhibitory avoidance is modulated cAMP/PKA signaling pathways in the hippocampus. PMID:9192688

Involvement of ERK phosphorylation in brainstem neurons in modulation of swallowing reflex in rats

PubMed Central

Tsujimura, Takanori; Kondo, Masahiro; Kitagawa, Junichi; Tsuboi, Yoshiyuki; Saito, Kimiko; Tohara, Haruka; Ueda, Koichiro; Sessle, Barry J; Iwata, Koichi

2009-01-01

In order to evaluate the neuronal mechanisms underlying functional abnormalities of swallowing in orofacial pain patients, this study investigated the effects of noxious orofacial stimulation on the swallowing reflex, phosphorylated extracellular signal-regulated kinase (pERK) and γ-aminobutyric acid (GABA) immunohistochemical features in brainstem neurons, and also analysed the effects of brainstem lesioning and of microinjection of GABA receptor agonist or antagonist into the nucleus tractus solitarii (NTS) on the swallowing reflex in anaesthetized rats. The swallowing reflex elicited by topical administration of distilled water to the pharyngolaryngeal region was inhibited after capsaicin injection into the facial (whisker pad) skin or lingual muscle. The capsaicin-induced inhibitory effect on the swallowing reflex was itself depressed after the intrathecal administration of MAPK kinase (MEK) inhibitor. No change in the capsaicin-induced inhibitory effect was observed after trigeminal spinal subnucleus caudalis lesioning, but the inhibitory effect was diminished by paratrigeminal nucleus (Pa5) lesioning. Many pERK-like immunoreactive neurons in the NTS showed GABA immunoreactivity. The local microinjection of the GABAA receptor agonist muscimol into the NTS produced a significant reduction in swallowing reflex, and the capsaicin-induced depression of the swallowing reflex was abolished by microinjection of the GABAA receptor antagonist bicuculline into the NTS. The present findings suggest that facial skin–NTS, lingual muscle–NTS and lingual muscle–Pa5–NTS pathways are involved in the modulation of swallowing reflex by facial and lingual pain, respectively, and that the activation of GABAergic NTS neurons is involved in the inhibition of the swallowing reflex following noxious stimulation of facial and intraoral structures. PMID:19124539

Protein Phosphatase 2A Antagonizes ATM and ATR in a Cdk2- and Cdc7-Independent DNA Damage Checkpoint

PubMed Central

Petersen, Paris; Chou, Danny M.; You, Zhongsheng; Hunter, Tony; Walter, Johannes C.; Walter, Gernot

2006-01-01

We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts. PMID:16479016

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: II. Mutational analysis.

PubMed

Kitani, T; Okuno, S; Fujisawa, H

2001-10-01

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation.

PubMed

Liangpunsakul, Suthat; Wou, Sung-Eun; Zeng, Yan; Ross, Ruth A; Jayaram, Hiremagalur N; Crabb, David W

2008-12-01

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.

CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

PubMed

Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

2016-08-01

The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

Antiangiogenic properties of cafestol, a coffee diterpene, in human umbilical vein endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Shuaiyu; Korea Food Research Institute, 516 Baekhyun-dong, Bundang-gu, Songnam, Kyungki-do 463-746; Yoon, Yeo Cho

2012-05-11

Highlights: Black-Right-Pointing-Pointer Cafestol inhibits tube formation and migration of VEGF-stimulated HUVEC. Black-Right-Pointing-Pointer Cafestol inhibits phosphorylation of FAK and Akt. Black-Right-Pointing-Pointer Cafestol decreases NO production. -- Abstract: As angiogenesis plays important roles in tumor growth and metastasis, searching for antiangiogenic compounds is a promising tactic for treating cancers. Cafestol, a diterpene found mainly in unfiltered coffee, provides benefit through varied biological activity, including antitumorigenic, antioxidative, and anti-inflammatory effects. This study aimed to investigate the effects of cafestol on angiogenesis and to uncover the associated mechanism. We show that cafestol inhibits angiogenesis of human umbilical vascular endothelial cells. This inhibition affects themore » following specific steps of the angiogenic process: proliferation, migration, and tube formation. The inhibitory effects of cafestol are accompanied by decreasing phosphorylation of FAK and Akt and by a decrease in nitric oxide production. Overall, cafestol inhibits angiogenesis by affecting the angiogenic signaling pathway.« less

Glycogen synthase kinase-3 as drug target: from wallflower to center of attention.

PubMed

Van Wauwe, Jean; Haefner, Burkhard

2003-11-01

Some 20 years ago, glycogen synthase kinase-3 (GSK-3) was categorized as one of several protein kinases that could phosphorylate glycogen synthase and regulate the glucose metabolism pathway. Today, GSK-3 is being identified as a ubiquitous serine/threonine protein kinase that participates in a multitude of cellular processes, ranging from cell membrane-to-nucleus signaling, gene transcription, translation, cytoskeletal organization to cell cycle progression and survival. Two functional aspects make GSK-3 a peculiar kinase: its activity is constitutive and downregulated after cell activation by phosphorylation or interaction with inhibitory proteins, and the enzyme prefers substrates that are specifically prepared, that is prephosphorylated, by other kinases. Its pleiotropic but unique activities have made GSK-3 a much sought-after target for the treatment of prevalent human diseases such as type 2 diabetes and Alzheimer's disease. Recent drug discovery efforts have identified small-molecule, orally active inhibitors of GSK-3. This accomplishment may represent the first step toward the development of novel therapeutic agents.

Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila.

PubMed

Liu, Bo; Zheng, Yonggang; Yin, Feng; Yu, Jianzhong; Silverman, Neal; Pan, Duojia

2016-01-28

The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rehman, Kanwal; Chen, Zhe; Wang, Wen Wen

2012-09-15

Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involvedmore » in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs{sup III}) and its intermediate metabolites such as monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA{sup III} and DMA{sup III}) but not by iAs{sup III}. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA{sup III} directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA{sup III} strongly inhibited activity of PTP1B. ► DMA{sup III} directly bound to PTP1B, resulting in inhibition of enzyme activity.« less

Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

PubMed

Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

2016-01-01

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

Chlorogenic acid inhibits hypoxia-induced pulmonary artery smooth muscle cells proliferation via c-Src and Shc/Grb2/ERK2 signaling pathway.

PubMed

Li, Qun-Yi; Zhu, Ying-Feng; Zhang, Meng; Chen, Li; Zhang, Zhen; Du, Yong-Li; Ren, Guo-Qiang; Tang, Jian-Min; Zhong, Ming-Kang; Shi, Xiao-Jin

2015-03-15

Chlorogenic acid (CGA), abundant in coffee and particular fruits, can modulate hypertension and vascular dysfunction. Hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) proliferation has been tightly linked to vascular remodeling in pulmonary arterial hypertension (PAH). Thus, the present study was designed to investigate the effect of CGA on hypoxia-induced proliferation in cultured rat PASMCs. The data showed that CGA potently inhibited PASMCs proliferation and DNA synthesis induced by hypoxia. These inhibitory effects were associated with G1 cell cycle arrest and down-regulation of cell cycle proteins. Treatment with CGA reduced hypoxia-induced hypoxia inducible factor 1α (HIF-1α) expression and trans-activation. Furthermore, hypoxia-evoked c-Src phosphorylation was inhibited by CGA. In vitro ELISA-based tyrosine kinase assay indicated that CGA was a direct inhibitor of c-Src. Moreover, CGA attenuated physical co-association of c-Src/Shc/Grb2 and ERK2 phosphorylation in PASMCs. These results suggest that CGA inhibits hypoxia-induced proliferation in PASMCs via regulating c-Src-mediated signaling pathway. In vivo investigation showed that chronic CGA treatment inhibits monocrotaline-induced PAH in rats. These findings presented here highlight the possible therapeutic use of CGA in hypoxia-related PAH. Copyright © 2015 Elsevier B.V. All rights reserved.

Growth Inhibition by Bupivacaine Is Associated with Inactivation of Ribosomal Protein S6 Kinase 1

PubMed Central

Beigh, Mushtaq Ahmad; Showkat, Mehvish; Bashir, Basharat; Bashir, Asma; Hussain, Mahboob ul; Andrabi, Khurshid Iqbal

2014-01-01

Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. PMID:24605337

Apo-9′-Fucoxanthinone, Isolated from Sargassum muticum, Inhibits CpG-Induced Inflammatory Response by Attenuating the Mitogen-Activated Protein Kinase Pathway

PubMed Central

Chae, Doobyeong; Manzoor, Zahid; Kim, Sung Chun; Kim, Sohyun; Oh, Tae-Heon; Yoo, Eun-Sook; Kang, Hee-Kyoung; Hyun, Jin-Won; Lee, Nam Ho; Ko, Mi-Hee; Koh, Young-Sang

2013-01-01

Sargassum muticum (S. muticum) is a brown edible alga and widely distributed in Korea. This report was designed to evaluate the anti-inflammatory properties of apo-9′-fucoxanthinone (APO-9′) isolated from S. muticum on pro-inflammatory cytokine production. S. muticum extract (SME) exhibited significant inhibitory effects on pro-inflammatory cytokine production in bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). APO-9′ pre-treatment in the CpG DNA-stimulated BMDMs and BMDCs showed a strong dose-dependent inhibitory effect on interleukin (IL)-12 p40, IL-6 and tumor necrosis factor (TNF)-α production with IC50 values ranging from 5.31 to 13.79. It exhibited a strong inhibitory effect on the phosphorylation of ERK1/2 and on activator protein (AP)-1 reporter activity. APO-9′ pre-treatment exhibited significant inhibition of CpG DNA-induced production of inducible nitric oxide synthase. Taken together, these data suggest that SME and APO-9′ have a significant anti-inflammatory property and warrant further studies concerning the potentials of SME and APO-9′ for medicinal use. PMID:23985898

ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.

2010-08-13

Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish themore » identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.« less

Verteporfin inhibits papillary thyroid cancer cells proliferation and cell cycle through ERK1/2 signaling pathway

PubMed Central

Liao, Tian; Wei, Wen-Jun; Wen, Duo; Hu, Jia-Qian; Wang, Yu; Ma, Ben; Cao, Yi-Min; Xiang, Jun; Guan, Qing; Chen, Jia-Ying; Sun, Guo-Hua; Zhu, Yong-Xue; Li, Duan-Shu; Ji, Qing-Hai

2018-01-01

Verteporfin, a FDA approved second-generation photosensitizer, has been demonstrated to have anticancer activity in various tumors, but not including papillary thyroid cancer (PTC). In current pre-clinical pilot study, we investigate the effect of verteporfin on proliferation, apoptosis, cell cycle and tumor growth of PTC. Our results indicate verteporfin attenuates cell proliferation, arrests cell cycle in G2/S phase and induces apoptosis of PTC cells. Moreover, treatment of verteporfin dramatically suppresses tumor growth from PTC cells in xenograft mouse model. We further illustrate that exposure to MEK inhibitor U0126 inactivates phosphorylation of ERK1/2 and MEK in verteporfin-treated PTC cells. These data suggest verteporfin exhibits inhibitory effect on PTC cells proliferation and cell cycle partially via ERK1/2 signalling pathway, which strongly encourages the further application of verteporfin in the treatment against PTC. PMID:29721041

Autonomic dysfunction in patients with Brugada syndrome: further biochemical evidence of altered signaling pathways.

PubMed

Paul, Matthias; Meyborg, Matthias; Boknik, Peter; Gergs, Ulrich; Schmitz, Wilhelm; Breithardt, Günter; Wichter, Thomas; Neumann, Joachim

2011-09-01

In patients with Brugada syndrome (BrS), life-threatening ventricular tachyarrhythmias predominantly occur during vagal stimulation at rest or during sleep. Previous imaging studies displayed an impaired autonomic function in BrS patients. However, it remains unclear whether these alterations primarily stem from a reduction of synaptic release of norepinephrine (NE) or an enhanced presynaptic reuptake. Both conditions could lead to reduced NE concentrations in the synaptic cleft. Therefore, we analyzed key components of the sympathoadrenergic signaling pathways in patients with BrS. Endomyocardial biopsies were obtained from eight BrS patients (seven male; age 49 ± 15 years) and five controls (three male; age 43 ± 13 years; P = ns). The concentrations of NE, epinephrine (Epi), NE transport (NET) carrier protein, cyclic adenosine 5'monophosphate (cyclic adenosine monophosphate [cAMP]), inhibitory G-proteins (G(i1,2) α), troponin-I (TNI), and phosphorylated TNI were analyzed. Levels of NET, G(i1,2) α, TNI, Epi, and phosphorylated TNI were comparable between the groups. Compared to controls, patients with BrS showed reduced cAMP and NE concentrations. The current findings expand the concept of adrenergic dysfunction in BrS: the reduction of NE in BrS could lead to an impaired stimulation of β-adrenoceptors resulting in a reduction of cAMP and alterations of the subsequent signaling pathway with potential implication for arrhythmogenesis. ©2011, The Authors. Journal compilation ©2011 Wiley Periodicals, Inc.

Microenvironment mesenchymal cells protect ovarian cancer cell lines from apoptosis by inhibiting XIAP inactivation

PubMed Central

Castells, M; Milhas, D; Gandy, C; Thibault, B; Rafii, A; Delord, J-P; Couderc, B

2013-01-01

Epithelial ovarian carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. Carcinoma-associated mesenchymal stem cells (CA-MSC) have been shown to induce ovarian cancer chemoresistance through trogocytosis. Here we examined CA-MSC properties to protect ovarian cancer cells from carboplatin-induced apoptosis. Apoptosis was determined by Propidium Iodide and Annexin-V-FITC labelling and poly-ADP-ribose polymerase cleavage analysis. We showed a significant increase of inhibitory concentration 50 and a 30% decrease of carboplatin-induced apoptosis in ovarian cancer cells incubated in the presence of CA-MSC-conditioned medium (CM). A molecular analysis of apoptosis signalling pathway in response to carboplatin revealed that the presence of CA-MSC CM induced a 30% decrease of effector caspases-3 and -7 activation and proteolysis activity. CA-MSC secretions promoted Akt and X-linked inhibitor of apoptosis protein (XIAP; caspase inhibitor from inhibitor of apoptosis protein (IAP) family) phosphorylation. XIAP depletion by siRNA strategy permitted to restore apoptosis in ovarian cancer cells stimulated by CA-MSC CM. The factors secreted by CA-MSC are able to confer chemoresistance to carboplatin in ovarian cancer cells through the inhibition of effector caspases activation and apoptosis blockade. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and the phosphorylation of its downstream target XIAP underlined the implication of this signalling pathway in ovarian cancer chemoresistance. This study reveals the potentialities of targeting XIAP in ovarian cancer therapy. PMID:24176845

Wasabi-derived 6-(methylsulfinyl)hexyl isothiocyanate induces apoptosis in human breast cancer by possible involvement of the NF-κB pathways.

PubMed

Fuke, Yoko; Hishinuma, Madoka; Namikawa, Mayumi; Oishi, Yoshie; Matsuzaki, Takeshi

2014-01-01

6-(methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi (Wasabia japonica), which is a popular spice in Japan. 6-MSITC has been reported to inhibit the proliferation of breast cancer and melanoma cell lines. We inoculated 30 female Balb-nu/nu mice with MDA-MB-231 or -453 cells, and orally administered varying concentrations of 6-MSITC for 12 days following tumor growth. The tumor volumes and tumor weights from mice inoculated with MDA-MB-231 cells, and the tumor volumes of MDA-MB-453 cells were significantly inhibited by 6-MSITC on Days 9 and 11 after drug administration. DNA fragmentation, DNA ladder, and caspase 3/7 activity performed in vitro revealed that 6-MSITC induced apoptosis of MDA-MB-231, MDA-MB-453, and MCF-7 cells. Furthermore, nuclear factor-κB (NF-κB) expression in the nuclei and phosphorylation of inhibitor κBα (IκBα) was downregulated by 6-MSITC in a concentration-dependent manner; however, this activity was not observed in MCF-7 cells. Moreover, this downregulation of phosphorylated IκBα by 6-MSITC in MDA-MB-231 and -453 cells supports its inhibitory effects on NF-κB activity. The expression of phosphorylated AKT (pAKT) reduced by 6-MSITC was confirmed in MDA-MB-231 cells. Thus, we conclude that 6-MITC promotes apoptosis of breast cancer cells by inhibiting NF-kB and therefore releasing its control of the PI3K/AKT pathway.

Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K(2) is associated with p53 and independent of the intrinsic apoptotic pathway.

PubMed

Li, Lu; Qi, Zhiling; Qian, Jin; Bi, Fuyong; Lv, Jun; Xu, Lei; Zhang, Ling; Chen, Hongyu; Jia, Renbing

2010-09-01

Vitamin K(2) (VK(2)) can exert cell growth inhibitory effects in various human cancer cells. In this study, we investigated the cell growth inhibitory effects of VK(2) in hepatocellular carcinoma Smmc-7721 cells and the mechanisms involved. We found that VK(2)-inhibited cell proliferation in Smmc-7721 cells in a dose-dependent manner, and the IC50 of VK(2) in Smmc-7721 cells was 9.73 microM at 24 h. The data from flow cytometric analyses, DNA fragmentation assays, and caspase 3 activity assays revealed that apoptosis was the determining factor in VK(2) activity. Furthermore, a significant increase in p53 phosphorylation and protein level was exhibited in apoptotic cells treated with VK(2), although there were no changes in p53 mRNA expression. Bax expression was unaffected by VK(2) in Smmc-7721 cells. In addition, our study showed that caspase 3 was activated by caspase 8, not caspase 9, in Smmc-7721 cells treated with VK(2). In summary, these data suggested that VK(2) can inhibit the growth of Smmc-7721 cells by induction of apoptosis involving caspase 8 activation and p53. This apoptotic process was not mediated by the intrinsic apoptotic pathway.

Apoptosis induced by β,β-dimethylacrylshikonin is associated with Bcl-2 and NF-κB in human breast carcinoma MCF-7 cells

PubMed Central

XIONG, YAO; MA, XIU-YING; ZHANG, ZIRAN; SHAO, ZHEN-JUN; ZHANG, YUAN-YUAN; ZHOU, LI-MING

2013-01-01

β,β-dimethylacrylshikonin (DA) is a natural naphthoquinone derivative compound of Lithospermum erythrorhizon with various biological activities. The present study aimed to investigate the inhibitory effects and underlying mechanisms of DA in human breast carcinoma MCF-7 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that DA inhibited the proliferation of MCF-7 cells in a dose- and time-dependent manner. The half maximal inhibitory concentration of DA with regard to the proliferation of MCF-7 cells was 0.050±0.016 mM. The characteristics of cell apoptosis, including cell shrinkage, nuclear pyknosis and chromatin condensation, were all observed in DA-treated cells. DA decreased the expression levels of Bcl-2 and increased the expression of Bax and caspase-3 compared with those in the control. DA inhibited the activity of the nuclear factor (NF)-κB pathway, by downregulating the expression of the p65 subunit, and inhibited the Iκb phosphorylation. DA inhibits the proliferation of MCF-7 cells in vitro by inducing apoptosis through the downregulation of Bcl-2, upregulation of Bax and partial inactivation of the NF-κB pathway. PMID:24260077

IR/IGF1R signaling as potential target for treatment of high-grade osteosarcoma

PubMed Central

2013-01-01

Background High-grade osteosarcoma is an aggressive tumor most often developing in the long bones of adolescents, with a second peak in the 5th decade of life. Better knowledge on cellular signaling in this tumor may identify new possibilities for targeted treatment. Methods We performed gene set analysis on previously published genome-wide gene expression data of osteosarcoma cell lines (n=19) and pretreatment biopsies (n=84). We characterized overexpression of the insulin-like growth factor receptor (IGF1R) signaling pathways in human osteosarcoma as compared with osteoblasts and with the hypothesized progenitor cells of osteosarcoma – mesenchymal stem cells. This pathway plays a key role in the growth and development of bone. Since most profound differences in mRNA expression were found at and upstream of the receptor of this pathway, we set out to inhibit IR/IGF1R using OSI-906, a dual inhibitor for IR/IGF1R, on four osteosarcoma cell lines. Inhibitory effects of this drug were measured by Western blotting and cell proliferation assays. Results OSI-906 had a strong inhibitory effect on proliferation of 3 of 4 osteosarcoma cell lines, with IC50s below 100 nM at 72 hrs of treatment. Phosphorylation of IRS-1, a direct downstream target of IGF1R signaling, was inhibited in the responsive osteosarcoma cell lines. Conclusions This study provides an in vitro rationale for using IR/IGF1R inhibitors in preclinical studies of osteosarcoma. PMID:23688189

Mechanistic Target of Rapamycin Complex 1 (mTORC1) and mTORC2 as Key Signaling Intermediates in Mesenchymal Cell Activation*

PubMed Central

Walker, Natalie M.; Belloli, Elizabeth A.; Stuckey, Linda; Chan, Kevin M.; Lin, Jules; Lynch, William; Chang, Andrew; Mazzoni, Serina M.; Fingar, Diane C.; Lama, Vibha N.

2016-01-01

Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states. PMID:26755732

Novel compounds TAD-1822-7-F2 and F5 inhibited HeLa cells growth through the JAK/Stat signaling pathway.

PubMed

Yang, Tianfeng; Shi, Xianpeng; Kang, Yuan; Zhu, Man; Fan, Mengying; Zhang, Dongdong; Zhang, Yanmin

2018-07-01

Cervical carcinoma remains the second most common malignancy with a high mortality rate among women worldwide. TAD-1822-7-F2 (F2) and TAD-1822-7-F5 (F5) are novel compounds synthesized on the chemical structure of taspine derivatives, and show an effective suppression for HeLa cells. Our study aims to confirm the potential targets of F2 and F5, and investigate the underlying mechanism of the inhibitory effect on HeLa cells. In this study, Real Time Cell Analysis and crystal violet staining assay were conducted to investigate the effect of F2 and F5 on HeLa cells proliferation. And the analytical methods of surface plasmon resonance and quartz crystal microbalance were established and employed to study the interaction between F2 and F5 and potential target protein JAK2, suggesting that both compounds have strong interaction with the JAK2 protein. Western blot analysis, immunofluorescence staining study and PCR was conducted to investigate the molecules of JAK/Stat signaling pathway. Interestingly, F2 and F5 showed diverse regulation for signaling molecules because of their different chemical structure. F2 increased the expression of JAK2 and downregulated the level of P-JAK1 and P-JAK2, and decreased P-Stat3 (Ser727). While F5 could increase the expression of JAK2 and naturally decrease the phosphorylation of JAK1 and Tyk2, and decreased the expression of P-Stat6. Moreover, F2 and F5 showed the same downregulation on the P-Stat3 (Tyr705). Therefore, F2 and F5 could target the JAK2 protein and prevent the phosphorylation of JAKs to suppress the phosphorylation of the downstream effector Stats, which suggested that F2 and F5 have great potential to be the inhibitors of the JAK/Stat signaling pathway. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

[Inhibitory effect of exogenous insulin-like growth factor binding protein 7 on proliferation of human breast cancer cell line MDA-MB-453 and its mechanism].

PubMed

Yuan, Lei; Fan, Wen-Juan; Yang, Xu-Guang; Rao, Shu-Mei; Song, Jin-Ling; Song, Guo-Hua

2013-10-25

The present study was to investigate the effects of exogenous insulin-like growth factor binding protein 7 (IGFBP7) on the proliferation of human breast cancer cell line MDA-MB-453 and its possible mechanism. By means of MTT method in vitro, the results showed exogenous IGFBP7 inhibited the growth of MDA-MB-453 cells (IC50 of IGFBP7 = 8.49 μg/mL) in time- and concentration-dependent manner. SB203580, p38(MAPK) inhibitor, blocked the anti-proliferative effect of exogenous IGFBP7. The flow cytometry assay showed that exogenous IGFBP7 remarkably induced G0/G1 arrest in MDA-MB-453 cells. The Western blot showed that exogenous IGFBP7 promoted phosphorylation of p38(MAPK), up-regulated expression of p21(CIP1/WAF1), and inhibited phosphorylation of Rb. SB203580 restrained exogenous IGFBP7-induced regulation of p21(CIP1/WAF1) and p-Rb in MDA-MB-453 cells. In conclusion, the present study suggests that exogenous IGFBP7 could activate the p38(MAPK) signaling pathway, upregulate p21(CIP1/WAF1) expression, inhibit phosphorylation of Rb, and finally induce G0/G1 arrest in MDA-MB-453 cells.

Integrative annotation and knowledge discovery of kinase post-translational modifications and cancer-associated mutations through federated protein ontologies and resources.

PubMed

Huang, Liang-Chin; Ross, Karen E; Baffi, Timothy R; Drabkin, Harold; Kochut, Krzysztof J; Ruan, Zheng; D'Eustachio, Peter; McSkimming, Daniel; Arighi, Cecilia; Chen, Chuming; Natale, Darren A; Smith, Cynthia; Gaudet, Pascale; Newton, Alexandra C; Wu, Cathy; Kannan, Natarajan

2018-04-25

Many bioinformatics resources with unique perspectives on the protein landscape are currently available. However, generating new knowledge from these resources requires interoperable workflows that support cross-resource queries. In this study, we employ federated queries linking information from the Protein Kinase Ontology, iPTMnet, Protein Ontology, neXtProt, and the Mouse Genome Informatics to identify key knowledge gaps in the functional coverage of the human kinome and prioritize understudied kinases, cancer variants and post-translational modifications (PTMs) for functional studies. We identify 32 functional domains enriched in cancer variants and PTMs and generate mechanistic hypotheses on overlapping variant and PTM sites by aggregating information at the residue, protein, pathway and species level from these resources. We experimentally test the hypothesis that S768 phosphorylation in the C-helix of EGFR is inhibitory by showing that oncogenic variants altering S768 phosphorylation increase basal EGFR activity. In contrast, oncogenic variants altering conserved phosphorylation sites in the 'hydrophobic motif' of PKCβII (S660F and S660C) are loss-of-function in that they reduce kinase activity and enhance membrane translocation. Our studies provide a framework for integrative, consistent, and reproducible annotation of the cancer kinomes.

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation.

PubMed

Law, Nathan C; Hunzicker-Dunn, Mary E

2016-02-26

The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

PubMed

Peng, Z G; Yao, Y B; Yang, J; Tang, Y L; Huang, X

2015-05-12

This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.

Inhibitory Effect of Dried Pomegranate Concentration Powder on Melanogenesis in B16F10 Melanoma Cells; Involvement of p38 and PKA Signaling Pathways

PubMed Central

Kang, Su Jin; Choi, Beom Rak; Lee, Eun Kyoung; Kim, Seung Hee; Yi, Hae Yeon; Park, Hye Rim; Song, Chang Hyun; Lee, Young Joon; Ku, Sae Kwang

2015-01-01

Plants rich in antioxidant substances may be useful for preventing skin aging. Pomegranates, containing flavonoids and other polyphenolic compounds, are widely consumed due to their beneficial properties. We examined the underlying mechanisms of dried pomegranate concentrate powder (PCP) on melanin synthesis in B16F10 melanoma cells. The antioxidant effects of PCP were determined by measuring free radical scavenging capacity and transcript levels of antioxidant enzymes. To explore the inhibitory effects of PCP on melanin synthesis, we measured tyrosinase activity and melanin content in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. In addition, the levels of tyrosinase-related protein-1 (TRP-1), TRP-2, tyrosinase, and microphthalmia-associated transcription factor (MITF) expression were determined by Western blotting. Changes in the phosphorylation status of protein kinase A (PKA), cAMP response element-binding protein (CREB), mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase Akt, and glycogen kinase 3β (GSK3β) were also examined. The free radical scavenging activity of PCP increased in a dose-dependent manner. In PCP-treated B16F10 cells, transcript levels of glutathione peroxidase-1 (GPx-1) were increased compared with α-MSH-stimulated cells. In addition, PCP led to the down-regulation of phospho-p38, phospho-PKA, phospho-CREB, phospho-GSK3β, MITF, and TRP-1 compared with α-MSH-stimulated B16F10 cells. We believe this effect may be associated with PCP activity, which leads to the inhibition of melanin production and tyrosinase activity. These results suggest that PCP decreases tyrosinase activity and melanin production via inactivation of the p38 and PKA signaling pathways, and subsequently decreases phosphorylation of CREB, MITF, and melanogenic enzymes. These observations provided new insights on the molecular mechanisms of the skin-whitening property of PCP. PMID:26473849

Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

PubMed

Adamczewski, M; Paolini, R; Kinet, J P

1992-09-05

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.

Cotinine administration improves impaired cognition in the mouse model of Fragile X syndrome.

PubMed

Pardo, Marta; Beurel, Eleonore; Jope, Richard S

2017-02-01

Cotinine is the major metabolite of nicotine and has displayed some capacity for improving cognition in mouse models following chronic administration. We tested if acute cotinine treatment is capable of improving cognition in the mouse model of Fragile X syndrome, Fmr1 -/- knockout mice, and if this is related to inhibition by cotinine treatment of glycogen synthase kinase-3β (GSK3β), which is abnormally active in Fmr1 -/- mice. Acute cotinine treatment increased the inhibitory serine-phosphorylation of GSK3β and the activating phosphorylation of AKT, which can mediate serine-phosphorylation of GSK3β, in both wild-type and Fmr1 -/- mouse hippocampus. Acute cotinine treatment improved cognitive functions of Fmr1 -/- mice in coordinate and categorical spatial processing, novel object recognition, and temporal ordering. However, cotinine failed to restore impaired cognition in GSK3β knockin mice, in which a serine9-to-alanine9 mutation blocks the inhibitory serine phosphorylation of GSK3β, causing GSK3β to be hyperactive. These results indicate that acute cotinine treatment effectively repairs impairments of these four cognitive tasks in Fmr1 -/- mice, and suggest that this cognition-enhancing effect of cotinine is linked to its induction of inhibitory serine-phosphorylation of GSK3. Taken together, these results show that nicotinic receptor agonists can act as cognitive enhancers in a mouse model of Fragile X syndrome and highlight the potential role of inhibiting GSK3β in mediating the beneficial effects of cotinine on memory. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors*

PubMed Central

Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian

2010-01-01

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. PMID:20952396

The metabolic waste ammonium regulates mTORC2 and mTORC1 signaling

PubMed Central

Merhi, Ahmad; Delrée, Paul; Marini, Anna Maria

2017-01-01

Two structurally and functionally distinct mammalian TOR complexes control cell growth and metabolism in physiological and pathological contexts including cancer. Upregulated glutaminolysis is part of the metabolic reprogramming occurring in cancer, providing fuels for growth but also liberating ammonium, a potent neurotoxic waste product. Here, we identify ammonium as a novel dose-dependent signal mediating rapid mTORC2 activation and further regulating mTORC1. We show that ammonium induces rapid RICTOR-dependent phosphorylation of AKT-S473, a process requiring the PI3K pathway and further involving the Src-family kinase YES1, the FAK kinase and the ITGβ1 integrin. Release of calcium from the endoplasmic reticulum store triggers rapid mTORC2 activation, similar to ammonium-induced activation, the latter being conversely prevented by calcium chelation.Moreover, in analogy to growth factors, ammonium triggers the AKT-dependent phosphoinhibition of the TSC complex and of PRAS40, two negative regulators of mTORC1. Consistent with mTORC1 stimulation, ammonium induces the inhibitory phosphorylation of 4EBP1, a negative regulator of protein biogenesis. Ammonium however dually impacts on the phosphorylation of p70S6K1 triggering a transient AKT-independent decrease in the phosphorylation of this second mTORC1 readout. Finally, we reveal ammonium as a dose-dependent stimulator of proliferation. This study underscores an mTORC2 and mTORC1 response to the so-called ammonium waste. PMID:28303961

The metabolic waste ammonium regulates mTORC2 and mTORC1 signaling.

PubMed

Merhi, Ahmad; Delrée, Paul; Marini, Anna Maria

2017-03-17

Two structurally and functionally distinct mammalian TOR complexes control cell growth and metabolism in physiological and pathological contexts including cancer. Upregulated glutaminolysis is part of the metabolic reprogramming occurring in cancer, providing fuels for growth but also liberating ammonium, a potent neurotoxic waste product. Here, we identify ammonium as a novel dose-dependent signal mediating rapid mTORC2 activation and further regulating mTORC1. We show that ammonium induces rapid RICTOR-dependent phosphorylation of AKT-S473, a process requiring the PI3K pathway and further involving the Src-family kinase YES1, the FAK kinase and the ITGβ1 integrin. Release of calcium from the endoplasmic reticulum store triggers rapid mTORC2 activation, similar to ammonium-induced activation, the latter being conversely prevented by calcium chelation.Moreover, in analogy to growth factors, ammonium triggers the AKT-dependent phosphoinhibition of the TSC complex and of PRAS40, two negative regulators of mTORC1. Consistent with mTORC1 stimulation, ammonium induces the inhibitory phosphorylation of 4EBP1, a negative regulator of protein biogenesis. Ammonium however dually impacts on the phosphorylation of p70S6K1 triggering a transient AKT-independent decrease in the phosphorylation of this second mTORC1 readout. Finally, we reveal ammonium as a dose-dependent stimulator of proliferation. This study underscores an mTORC2 and mTORC1 response to the so-called ammonium waste.

Combination of two insulin-like growth factor-I receptor inhibitory antibodies targeting distinct epitopes leads to an enhanced antitumor response.

PubMed

Dong, Jianying; Demarest, Stephen J; Sereno, Arlene; Tamraz, Susan; Langley, Emma; Doern, Adam; Snipas, Tracey; Perron, Keli; Joseph, Ingrid; Glaser, Scott M; Ho, Steffan N; Reff, Mitchell E; Hariharan, Kandasamy

2010-09-01

The insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor tyrosine kinase that mediates cell survival signaling and supports tumor progression in multiple tumor types. We identified a spectrum of inhibitory IGF-IR antibodies with diverse binding epitopes and ligand-blocking properties. By binding distinct inhibitory epitopes, two of these antibodies, BIIB4 and BIIB5, block both IGF-I and IGF-II binding to IGF-IR using competitive and allosteric mechanisms, respectively. Here, we explored the inhibitory effects of combining BIIB4 and BIIB5. In biochemical assays, the combination of BIIB4 and BIIB5 improved both the potency and extent of IGF-I and IGF-II blockade compared with either antibody alone. In tumor cells, the combination of BIIB4 and BIIB5 accelerated IGF-IR downregulation and more efficiently inhibited IGF-IR activation as well as downstream signaling, particularly AKT phosphorylation. In several carcinoma cell lines, the antibody combination more effectively inhibited ligand-driven cell growth than either BIIB4 or BIIB5 alone. Notably, the enhanced tumor growth-inhibitory activity of the BIIB4 and BIIB5 combination was much more pronounced at high ligand concentrations, where the individual antibodies exhibited substantially reduced activity. Compared with single antibodies, the BIIB4 and BIIB5 combination also significantly further enhanced the antitumor activity of the epidermal growth factor receptor inhibitor erlotinib and the mTOR inhibitor rapamycin. Moreover, in osteosarcoma and hepatocellular carcinoma xenograft models, the BIIB4 and BIIB5 combination significantly reduced tumor growth to a greater degree than each single antibody. Taken together, our results suggest that targeting multiple distinct inhibitory epitopes on IGF-IR may be a more effective strategy of affecting the IGF-IR pathway in cancer.

Calcium-permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors Trigger Neuronal Nitric-oxide Synthase Activation to Promote Nerve Cell Death in an Src Kinase-dependent Fashion*

PubMed Central

Socodato, Renato; Santiago, Felipe N.; Portugal, Camila C.; Domingues, Ana F.; Santiago, Ana R.; Relvas, João B.; Ambrósio, António F.; Paes-de-Carvalho, Roberto

2012-01-01

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-dl-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death. PMID:22992730

Doxycycline inhibits leukemic cell migration via inhibition of matrix metalloproteinases and phosphorylation of focal adhesion kinase

PubMed Central

WANG, CHUNHUAI; XIANG, RU; ZHANG, XIANGZHONG; CHEN, YUNXIAN

2015-01-01

Doxycycline, a tetracycline-based antibiotic, has been reported to attenuate melanoma cell migration through inhibiting the focal adhesion kinase (FAK) signaling pathway. However, it remains to be elucidated whether doxycycline exerts this effect on leukemia cell migration. The present study aimed to examine the role of doxycycline in leukemia cell migration. The invasion capacities of the human leukemia cell lines KG1a (acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were evaluated using Matrigel® matrix-coated Transwell® chamber assays; leukemic cell lines treated with doxycycline (1 µg/ml) or anti-β1-integrin antibodies were added to the upper chamber, while untreated cells were included as controls. Reverse transcription quantitative polymerase chain reaction was performed in order to further understand the influence of doxycycline treatment on the expression of FAK and gelatinases in the KG1a and K562 leukemic cell lines. In addition, FAK protein expression and phosphorylation were determined using western blot analysis in order to investigate the mechanism by which doxycycline inhibited leukemic cell migration. The results revealed that doxycycline treatment significantly attenuated the migration of KG1a and K562 cells, which was demonstrated to be associated with inhibition of the expression and phosphorylation of FAK. In addition, doxycycline treatment inhibited matrix metalloproteinase (MMP)-2 and MMP-9 expression. Furthermore, incubation with blocking anti-β1-integrin antibodies had an analogous inhibitory effect on leukemic cell migration to that of doxycycline. In conclusion, the results of the present study suggested that doxycycline attenuated leukemic cell migration through inhibiting the FAK signaling pathway. Therefore, doxycycline may have potential for use as a novel strategy for the treatment of leukemia. PMID:26004127

Allosteric MEK1/2 Inhibitor Refametinib (BAY 86-9766) in Combination with Sorafenib Exhibits Antitumor Activity in Preclinical Murine and Rat Models of Hepatocellular Carcinoma12

PubMed Central

Schmieder, Roberta; Puehler, Florian; Neuhaus, Roland; Kissel, Maria; Adjei, Alex A; Miner, Jeffrey N; Mumberg, Dominik; Ziegelbauer, Karl; Scholz, Arne

2013-01-01

OBJECTIVE: The objectives of the study were to evaluate the allosteric mitogen-activated protein kinase kinase (MEK) inhibitor BAY 86-9766 in monotherapy and in combination with sorafenib in orthotopic and subcutaneous hepatocellular carcinoma (HCC) models with different underlying etiologies in two species. DESIGN: Antiproliferative potential of BAY 86-9766 and synergistic effects with sorafenib were studied in several HCC cell lines. Relevant pathway signaling was studied in MH3924a cells. For in vivo testing, the HCC cells were implanted subcutaneously or orthotopically. Survival and mode of action (MoA) were analyzed. RESULTS: BAY 86-9766 exhibited potent antiproliferative activity in HCC cell lines with half-maximal inhibitory concentration values ranging from 33 to 762 nM. BAY 86-9766 was strongly synergistic with sorafenib in suppressing tumor cell proliferation and inhibiting phosphorylation of the extracellular signal-regulated kinase (ERK). BAY 86-9766 prolonged survival in Hep3B xenografts, murine Hepa129 allografts, and MH3924A rat allografts. Additionally, tumor growth, ascites formation, and serum alpha-fetoprotein levels were reduced. Synergistic effects in combination with sorafenib were shown in Huh-7, Hep3B xenografts, and MH3924A allografts. On the signaling pathway level, the combination of BAY 86-9766 and sorafenib led to inhibition of the upregulatory feedback loop toward MEK phosphorylation observed after BAY 86-9766 monotreatment. With regard to the underlying MoA, inhibition of ERK phosphorylation, tumor cell proliferation, and microvessel density was observed in vivo. CONCLUSION: BAY 86-9766 shows potent single-agent antitumor activity and acts synergistically in combination with sorafenib in preclinical HCC models. These results support the ongoing clinical development of BAY 86-9766 and sorafenib in advanced HCC. PMID:24204195

The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway

NASA Technical Reports Server (NTRS)

Hu, B.; Zhou, X. Y.; Wang, X.; Zeng, Z. C.; Iliakis, G.; Wang, Y.

2001-01-01

Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.

Calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors trigger neuronal nitric-oxide synthase activation to promote nerve cell death in an Src kinase-dependent fashion.

PubMed

Socodato, Renato; Santiago, Felipe N; Portugal, Camila C; Domingues, Ana F; Santiago, Ana R; Relvas, João B; Ambrósio, António F; Paes-de-Carvalho, Roberto

2012-11-09

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.

Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases.

PubMed

Inamoto, Teruo; Azuma, Haruhito; Sakamoto, Takeshi; Kiyama, Satoshi; Ubai, Takanobu; Kotake, Yatsugu; Watanabe, Masahito; Katsuoka, Yoji

2007-10-01

Gamma-aminobutyric acid (GABA) was first discovered as an inhibitory neurotransmitter in the central nervous system (CNS) and has been reported to have a variety of functions, including regulation of cell division, cell differentiation and maturation, and to be involved in the development of certain cancers outside the CNS. In the present study, using the human renal cell carcinoma cell line Caki-2, we demonstrated that GABA stimulation significantly increased the expression of MMP-2 and -9 and subsequently increased the invasive activity of the cancer cells. Because MAPK signaling is one of the key regulators of MMP expression, we further evaluated MAPK signaling after stimulation with GABA. It was found that GABA stimulation promoted the phosphorylation of MAPKs, including ERK1/2, JNK, and p38. ERK1/2 phosphorylation was sustained for up to 12 h, while phosphorylation of JNK and p38 returned to the endogenous level by 30 min. It was noteworthy that the ras/raf/MEK/ERK pathway inhibitor PD98059 attenuated GABA-induced MMP-9 expression and that both PD98059 and MMP inhibitors attenuated the GABA-induced invasive activity of Caki-2 cells. Moreover, data obtained by depletion of the MEK/ERK pathway using interfering RNA transfection of Caki-2 cells clearly corroborated the above results, as both MMP-9 expression and GABA-induced invasive ability were decreased significantly. We also demonstrated that the GABA-induced increase in invasive ability via ERK1/2 up-regulation was mediated mainly through the GABA-B receptor. These results indicate that GABA stimulation promotes cancer cell invasion and that the effect is partly due to ERK1/2-dependent up-regulation of MMPs.

Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

PubMed

Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

2017-06-01

To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

The flower of Edgeworthia gardneri (wall.) Meisn. suppresses adipogenesis through modulation of the AMPK pathway in 3T3-L1 adipocytes.

PubMed

Gao, Die; Zhang, Yong-Lan; Yang, Feng-Qing; Li, Fan; Zhang, Qi-Hui; Xia, Zhi-Ning

2016-09-15

The flower of Edgeworthia gardneri (Wall.) Meisn., locally named "Lvluohua, ", has been widely used as Tibetan folk medicine for the treatment of metabolic diseases for a long time. To evaluate the anti-adipogenesis effect of ethyl acetate extract of the flower of E. gardneri (EEG extract) in 3T3-L1 adipocytes. Obesity-related parameters such as lipid accumulation and TG content were determined by Oil red O staining and enzymatic kit, respectively. Western blotting was used to determine the expressions of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBPα), phosphorylated adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). Moreover, main constituents of EEG extract were analyzed by high performance liquid chromatography (HPLC). EEG extract decreased the lipid and triglyceride (TG) accumulations during the differentiation process and down-regulated the adipogenesis-related transcriptional factors PPARγ and C/EBPα. EEG extract treatment increased AMPK and ACC phosphorylation. In addition, pretreatment with AMPK inhibitor, weakened the inhibitory effects of EEG extract on the expressions of PPARγand C/EBPα. HPLC analysis indicated that tiliroside was the main constituent in EEG extract. These results suggest that EEG extract may exert anti-adipogenic effects through modulation of the AMPK signaling pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Co-clustering of Fcgamma and B cell receptors induces dephosphorylation of the Grb2-associated binder 1 docking protein.

PubMed

Koncz, G; Tóth, G K; Bökönyi, G; Kéri, G; Pecht, I; Medgyesi, D; Gergely, J; Sármay, G

2001-07-01

The immunoreceptor tyrosine-based inhibitory motif (ITIM) of human type IIb Fcgamma receptor (FcgammaRIIb) is phosphorylated on its tyrosine upon co-clustering with the B cell receptor (BCR). The phosphorylated ITIM (p-ITIM) binds to the SH2 domains of polyphosphoinositol 5-phosphatase (SHIP) and the tyrosine phosphatase, SHP-2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcgammaRIIb in the activation of SHP-2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p-ITIM and the N-terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP-2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcgammaRII-SHIP complexes formed in the intact cells may also activate SHP-2. Grb2-associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine-phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP-2, SHIP and the p85 subunit of phosphatidyl inositol 3-kinase (PtdIns3-K) and may regulate their activity. Gab1 is a potential substrate of SHP-2, thus its binding to FcgammaRIIb may modify the Gab1-bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcgammaRIIb upon its co-clustering with BCR. Gab1 may recruit SH2 domain-containing molecules to the phosphorylated FcgammaRIIb. SHP-2, activated upon the binding to FcgammaRIIb-SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3-K and ultimately in the inhibition of downstream activation pathways in BCR/FcgammaRIIb co-aggregated cells.

Lopinavir Impairs Protein Synthesis and Induces eEF2 Phosphorylation via the Activation of AMP-Activated Protein Kinase

PubMed Central

Hong-Brown, Ly Q.; Brown, C. Randell; Huber, Danuta S.; Lang, Charles H.

2008-01-01

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K. PMID:18712774

Uncoupling of oxidative phosphorylation by curcumin: implication of its cellular mechanism of action.

PubMed

Lim, Han Wern; Lim, Hwee Ying; Wong, Kim Ping

2009-11-06

Curcumin is a phytochemical isolated from the rhizome of turmeric. Recent reports have shown curcumin to have antioxidant, anti-inflammatory and anti-tumor properties as well as affecting the 5'-AMP activated protein kinase (AMPK), mTOR and STAT-3 signaling pathways. We provide evidence that curcumin acts as an uncoupler. Well-established biochemical techniques were performed on isolated rat liver mitochondria in measuring oxygen consumption, F(0)F(1)-ATPase activity and ATP biosynthesis. Curcumin displays all the characteristics typical of classical uncouplers like fccP and 2,4-dinitrophenol. In addition, at concentrations higher than 50 microM, curcumin was found to inhibit mitochondrial respiration which is a characteristic feature of inhibitory uncouplers. As a protonophoric uncoupler and as an activator of F(0)F(1)-ATPase, curcumin causes a decrease in ATP biosynthesis in rat liver mitochondria. The resulting change in ATP:AMP could disrupt the phosphorylation status of the cell; this provides a possible mechanism for its activation of AMPK and its downstream mTOR and STAT-3 signaling.

Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways

PubMed Central

Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

2015-01-01

Abstract The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity. PMID:26061361

Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Corona, Giulia; Dipartimento di Biologia Sperimentale, Sez. Patologia Sperimentale, Universita degli Studi di Cagliari, 09042 Monserrato; Deiana, Monica

2007-10-26

We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 {mu}g/ml) the number of cells in the G2/M phase increased to 51.82 {+-} 2.69% relative to control cells (15.1 {+-} 2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 {mu}g/ml) to exert rapid inhibitionmore » of p38 (38.7 {+-} 4.7%) and CREB (28.6 {+-} 5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9 {+-} 9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development.« less

Uncoupling of oxidative phosphorylation by curcumin: Implication of its cellular mechanism of action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Han Wern; Lim, Hwee Ying; Wong, Kim Ping, E-mail: bchsitkp@nus.edu.sg

2009-11-06

Curcumin is a phytochemical isolated from the rhizome of turmeric. Recent reports have shown curcumin to have antioxidant, anti-inflammatory and anti-tumor properties as well as affecting the 5'-AMP activated protein kinase (AMPK), mTOR and STAT-3 signaling pathways. We provide evidence that curcumin acts as an uncoupler. Well-established biochemical techniques were performed on isolated rat liver mitochondria in measuring oxygen consumption, F{sub 0}F{sub 1}-ATPase activity and ATP biosynthesis. Curcumin displays all the characteristics typical of classical uncouplers like fccP and 2,4-dinitrophenol. In addition, at concentrations higher than 50 {mu}M, curcumin was found to inhibit mitochondrial respiration which is a characteristic featuremore » of inhibitory uncouplers. As a protonophoric uncoupler and as an activator of F{sub 0}F{sub 1}-ATPase, curcumin causes a decrease in ATP biosynthesis in rat liver mitochondria. The resulting change in ATP:AMP could disrupt the phosphorylation status of the cell; this provides a possible mechanism for its activation of AMPK and its downstream mTOR and STAT-3 signaling.« less

Optimization of N-benzyl-benzoxazol-2-ones as receptor antagonists of macrophage migration inhibitory factor (MIF)

PubMed Central

Hare, Alissa A.; Leng, Lin; Gandavadi, Sunilkumar; Du, Xin; Cournia, Zoe; Bucala, Richard; Jorgensen, William L.

2010-01-01

The cytokine MIF is involved in inflammation and cell proliferation via pathways initiated by its binding to the transmembrane receptor CD74. MIF also exhibits keto-enol tautomerase activity, believed to be vestigial in mammals. Starting from a 1-μM hit from virtual screening, substituted benzoxazol-2-ones have been discovered as antagonists with IC50 values as low as 7.5 nM in a tautomerase assay and 80 nM in a MIF-CD74 binding assay. Additional studies for one of the potent inhibitors demonstrated that it is not a covalent inhibitor of MIF and that it attenuates MIF-dependent ERK1/2 phosphorylation in human synovial fibroblasts. PMID:20728358

ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function

PubMed Central

Collins, Natalie B.; Wilson, James B.; Bush, Thomas; Thomashevski, Andrei; Roberts, Kate J.; Jones, Nigel J.

2009-01-01

Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage–specific event that is downstream of ATR and is functionally important in the FA pathway. PMID:19109555

Juvenile Hormone Prevents 20-Hydroxyecdysone-induced Metamorphosis by Regulating the Phosphorylation of a Newly Identified Broad Protein*

PubMed Central

Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-01-01

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5′-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. PMID:25096576

Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation*

PubMed Central

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-01-01

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically. PMID:23609448

Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.

PubMed

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-06-07

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage.

PubMed

Bluhm, Björn; Ehlen, Harald W A; Holzer, Tatjana; Georgieva, Veronika S; Heilig, Juliane; Pitzler, Lena; Etich, Julia; Bortecen, Toman; Frie, Christian; Probst, Kristina; Niehoff, Anja; Belluoccio, Daniele; Van den Bergen, Jocelyn; Brachvogel, Bent

2017-10-01

Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here, we characterize a miRNA that promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only Mek1 was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes Mek1 mRNA to raise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation of miR322 in mice linked the loss of miR-322 to decreased MEK1 levels and to increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development. © 2017. Published by The Company of Biologists Ltd.

Effect of quercetin on the production of nitric oxide in murine macrophages stimulated with lipopolysaccharide from Prevotella intermedia

PubMed Central

Cho, Yun-Jung

2013-01-01

Purpose Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action. Methods LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory κB (IκB)-α degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting. Results Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of IκB-α induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling. Conclusions Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-κB and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease. PMID:24040572

Effect of quercetin on the production of nitric oxide in murine macrophages stimulated with lipopolysaccharide from Prevotella intermedia.

PubMed

Cho, Yun-Jung; Kim, Sung-Jo

2013-08-01

Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action. LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory κB (IκB)-α degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting. Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of IκB-α induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling. Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-κB and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease.

Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-beta signaling.

PubMed

Wrighton, Katharine H; Willis, Danielle; Long, Jianyin; Liu, Fang; Lin, Xia; Feng, Xin-Hua

2006-12-15

Transforming growth factor-beta (TGF-beta) controls a diverse set of cellular processes, and its canonical signaling is mediated via TGF-beta-induced phosphorylation of receptor-activated Smads (2 and 3) at the C-terminal SXS motif. We recently discovered that PPM1A can dephosphorylate Smad2/3 at the C-terminal SXS motif, implicating a critical role for phosphatases in regulating TGF-beta signaling. Smad2/3 activity is also regulated by phosphorylation in the linker region (and N terminus) by a variety of intracellular kinases, making it a critical platform for cross-talk between TGF-beta and other signaling pathways. Using a functional genomic approach, we identified the small C-terminal domain phosphatase 1 (SCP1) as a specific phosphatase for Smad2/3 dephosphorylation in the linker and N terminus. A catalytically inactive SCP1 mutant (dnSCP1) had no effect on Smad2/3 phosphorylation in vitro or in vivo. Of the other FCP/SCP family members SCP2 and SCP3, but not FCP1, could also dephosphorylate Smad2/3 in the linker/N terminus. Depletion of SCP1/2/3 enhanced Smad2/3 linker phosphorylation. SCP1 increased TGF-beta-induced transcriptional activity in agreement with the idea that phosphorylation in the Smad2/3 linker must be removed for a full transcriptional response. SCP1 overexpression also counteracts the inhibitory effect of epidermal growth factor on TGF-beta-induced p15 expression. Taken together, this work identifies the first example of a Smad2/3 linker phosphatase(s) and reveals an important new substrate for SCPs.

Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

PubMed

Doytcheva, Petia; Bächler, Thomas; Tarasco, Erika; Marzolla, Vincenzo; Engeli, Michael; Pellegrini, Giovanni; Stivala, Simona; Rohrer, Lucia; Tona, Francesco; Camici, Giovanni G; Vanhoutte, Paul M; Matter, Christian M; Lutz, Thomas A; Lüscher, Thomas F; Osto, Elena

2017-11-14

Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits. After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability. Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Wee1 and Cdc25 are controlled by conserved PP2A-dependent mechanisms in fission yeast.

PubMed

Lucena, Rafael; Alcaide-Gavilán, Maria; Anastasia, Steph D; Kellogg, Douglas R

2017-03-04

Wee1 and Cdc25 are conserved regulators of mitosis. Wee1 is a kinase that delays mitosis via inhibitory phosphorylation of Cdk1, while Cdc25 is a phosphatase that promotes mitosis by removing the inhibitory phosphorylation. Although Wee1 and Cdc25 are conserved proteins, it has remained unclear whether their functions and regulation are conserved across diverse species. Here, we analyzed regulation of Wee1 and Cdc25 in fission yeast. Both proteins undergo dramatic cell cycle-dependent changes in phosphorylation that are dependent upon PP2A associated with the regulatory subunit Pab1. The mechanisms that control Wee1 and Cdc25 in fission yeast appear to share similarities to those in budding yeast and vertebrates, which suggests that there may be common mechanisms that control mitotic entry in all eukaryotic cells.

Wee1 and Cdc25 are controlled by conserved PP2A-dependent mechanisms in fission yeast

PubMed Central

2017-01-01

ABSTRACT Wee1 and Cdc25 are conserved regulators of mitosis. Wee1 is a kinase that delays mitosis via inhibitory phosphorylation of Cdk1, while Cdc25 is a phosphatase that promotes mitosis by removing the inhibitory phosphorylation. Although Wee1 and Cdc25 are conserved proteins, it has remained unclear whether their functions and regulation are conserved across diverse species. Here, we analyzed regulation of Wee1 and Cdc25 in fission yeast. Both proteins undergo dramatic cell cycle-dependent changes in phosphorylation that are dependent upon PP2A associated with the regulatory subunit Pab1. The mechanisms that control Wee1 and Cdc25 in fission yeast appear to share similarities to those in budding yeast and vertebrates, which suggests that there may be common mechanisms that control mitotic entry in all eukaryotic cells. PMID:28103117

Human immunodeficiency virus type 1 Vpr induces cell cycle G2 arrest through Srk1/MK2-mediated phosphorylation of Cdc25.

PubMed

Huard, Sylvain; Elder, Robert T; Liang, Dong; Li, Ge; Zhao, Richard Y

2008-03-01

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G(2) arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G(2) arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G(2)/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G(2) arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G(2) delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G(2)/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G(2) arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G(2) arrest at least in part through a Srk1/MK2-mediated mechanism.

Exploring the interactions of EGFR with phosphorylated Mig6 by molecular dynamics simulations and MM-PBSA calculations.

PubMed

Zhang, Yue; Zheng, Qing-Chuan

2018-06-14

Mig6, a negative regulator, directly binds to epidermal growth factor receptor (EGFR), including Mig6-segment1 and Mig6-segment2. Mig6 requires phosphorylation of Y394 on Mig6-segment2 in order to inhibit EGFR. Two phosphorylation pathways for Y394 have been previously reported and the first way may phosphorylate Y394 primed by Y395 phosphorylation. Besides, the binding mechanism of phosphorylated Mig6-segment2 with EGFR has not been elucidated clearly. Focused on EGFR complex with phosphorylated Mig6-segment2, molecular dynamics (MD) simulations were performed to explore the interactions of Mig6-segment2 with EGFR. Our results indicate a probable phosphorylation pathway on Y394 and some key residues of EGFR play important roles in binding to phosphorylated Mig6-segment2. In addition, a special L-shaped structure was found to be possibly associated with irreversible inhibition of EGFR by Mig6. Our work can give meaningful information to better understand the phosphorylation pathways for Y394 and the interactions of EGFR binding to phosphorylated Mig6-segment2. Copyright © 2018 Elsevier Ltd. All rights reserved.

BAD phosphorylation determines ovarian cancer chemo-sensitivity and patient survival

PubMed Central

Marchion, Douglas C.; Cottrill, Hope M.; Xiong, Yin; Chen, Ning; Bicaku, Elona; Fulp, William J.; Bansal, Nisha; Chon, Hye Sook; Stickles, Xiaomang B.; Kamath, Siddharth G.; Hakam, Ardeshir; Li, Lihua; Su, Dan; Moreno, Carolina; Judson, Patricia L.; Berchuck, Andrew; Wenham, Robert M.; Apte, Sachin M.; Gonzalez-Bosquet, Jesus; Bloom, Gregory C.; Eschrich, Steven A.; Sebti, Said; Chen, Dung-Tsa; Lancaster, Johnathan M.

2011-01-01

Purpose Despite initial sensitivity to chemotherapy, ovarian cancers (OVCA) often develop drug-resistance, which limits patient survival. Using specimens and/or genomic data from 289 patients and a panel of cancer cell lines, we explored genome-wide expression changes that underlie the evolution of OVCA chemo-resistance and characterized the BCL2 antagonist of cell death (BAD) apoptosis pathway as a determinant of chemo-sensitivity and patient survival. Experimental Design Serial OVCA cell cisplatin treatments were performed in parallel with measurements of genome-wide expression changes. Pathway analysis was performed on genes associated with increasing cisplatin-resistance (EC50). BAD-pathway expression and BAD-protein phosphorylation were evaluated in patient samples and cell lines as determinants of chemo-sensitivity and/or clinical outcome and as therapeutic targets. Results Induced in vitro OVCA cisplatin-resistance was associated with BAD-pathway expression (P < 0.001). In OVCA cell lines and primary specimens, BAD-protein phosphorylation was associated with platinum-resistance (n = 147, P < 0.0001) and also with overall patient survival (n = 134, P = 0.0007). Targeted modulation of BAD-phosphorylation levels influenced cisplatin sensitivity. A 47-gene BAD-pathway score was associated with in vitro phosphorylated-BAD levels and with survival in 142 patients with advanced-stage (III/IV) serous OVCA. Integration of BAD-phosphorylation or BAD-pathway score with OVCA surgical cytoreductive status was significantly associated with overall survival by log-rank test (P = 0.004 and <0.0001, respectively). Conclusion The BAD apoptosis pathway influences OVCA chemo-sensitivity and overall survival, likely via modulation of BAD-phosphorylation. The pathway has clinical relevance as a biomarker of therapeutic response, patient survival, and as a promising therapeutic target. PMID:21849418

Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

PubMed

Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

2016-12-21

Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

Modulation of GABAergic receptor binding by activation of calcium and calmodulin-dependent kinase II membrane phosphorylation.

PubMed

Churn, S B; DeLorenzo, R J

1998-10-26

gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Because of the important role that GABA plays in the CNS, alteration of GABAA receptor function would significantly affect neuronal excitability. Protein phosphorylation is a major mechanism for regulating receptor function in the brain and has been implicated in modulating GABAA receptor function. Therefore, this study was initiated to determine the role of calmodulin-dependent kinase II (CaM kinase II) membrane phosphorylation on GABAA receptor binding. Synaptosomal membrane fractions were tested for CaM kinase II activity towards endogenous substrates. In addition, muscimol binding was evaluated under equilibrium conditions in synaptosomal membrane fractions subjected to either basal (Mg2+ alone) or maximal CaM kinase II-dependent phosphorylation. Activation of endogenous CaM kinase II-dependent phosphorylation resulted in a significant enhancement of the apparent Bmax for muscimol binding without significantly altering the apparent binding affinity. The enhanced muscimol binding could be increased further by the addition of exogenous CaM kinase II to synaptosomal membrane fractions. Co-incubation with inhibitors of kinase activity during the phosphorylation reactions blocked the CaM kinase II-dependent increase in muscimol binding. The data support the hypothesis that activation of CaM kinase II-dependent phosphorylation caused an increased GABAA receptor binding and may play an important role in modulating the function of this inhibitory receptor/chloride ion channel complex. Copyright 1998 Elsevier Science B.V.

Role of Smad signaling in kidney disease.

PubMed

Zhang, Yanhua; Wang, Songyan; Liu, Shengmao; Li, Chunguang; Wang, Ji

2015-12-01

Smads are the key intermediates of canonical transforming growth factor-beta (TGF-β) signaling. These intermediates are divided into three distinct subgroups based on their role in TGF-β family signal transduction: Receptor-regulated Smads (R-Smads) 1, 2, 3, 5 and 8, common Smad4, and inhibitory Smads6 and 7. TGF-β signaling through Smad pathway involves phosphorylation, ubiquitination, sumoylation, acetylation, and protein-protein interactions with mitogen-activated protein kinases, PI3K-Akt/PKB, and Wnt/GSK-3. Several studies have suggested that upregulation or downregulation of TGF-β/Smad signaling pathways may be a pathogenic mechanism in the progression of chronic kidney disease. Smad2 and 3 are the two major downstream R-Smads in TGF-β-mediated renal fibrosis, while Smad7 also controls renal inflammation. In this review, we characterize the role of Smads in kidney disease, describe the molecular mechanisms, and discuss the potential of Smads as a therapeutic target in chronic kidney disease.

Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

PubMed

Shemon, Anne N; Heil, Gary L; Granovsky, Alexey E; Clark, Mathew M; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R; Koide, Shohei

2010-05-05

Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

PubMed

Cai, Mei-Juan; Liu, Wen; Pei, Xu-Yang; Li, Xiang-Ru; He, Hong-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

2014-09-19

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The integrated role of ACh, ERK and mTOR in the mechanisms of hippocampal inhibitory avoidance memory.

PubMed

Giovannini, Maria Grazia; Lana, Daniele; Pepeu, Giancarlo

2015-03-01

The purpose of this review is to summarize the present knowledge on the interplay among the cholinergic system, Extracellular signal-Regulated Kinase (ERK) and Mammalian Target of Rapamycin (mTOR) pathways in the development of short and long term memories during the acquisition and recall of the step-down inhibitory avoidance in the hippocampus. The step-down inhibitory avoidance is a form of associative learning that is acquired in a relatively simple one-trial test through several sensorial inputs. Inhibitory avoidance depends on the integrated activity of hippocampal CA1 and other brain areas. Recall can be performed at different times after acquisition, thus allowing for the study of both short and long term memory. Among the many neurotransmitter systems involved, the cholinergic neurons that originate in the basal forebrain and project to the hippocampus are of crucial importance in inhibitory avoidance processes. Acetylcholine released from cholinergic fibers during acquisition and/or recall of behavioural tasks activates muscarinic and nicotinic acetylcholine receptors and brings about a long-lasting potentiation of the postsynaptic membrane followed by downstream activation of intracellular pathway (ERK, among others) that create conditions favourable for neuronal plasticity. ERK appears to be salient not only in long term memory, but also in the molecular mechanisms underlying short term memory formation in the hippocampus. Since ERK can function as a biochemical coincidence detector in response to extracellular signals in neurons, the activation of ERK-dependent downstream effectors is determined, in part, by the duration of ERK phosphorylation itself. Long term memories require protein synthesis, that in the synapto-dendritic compartment represents a direct mechanism that can produce rapid changes in protein content in response to synaptic activity. mTOR in the brain regulates protein translation in response to neuronal activity, thereby modulating synaptic plasticity and long term memory formation. Some studies demonstrate a complex interplay among the cholinergic system, ERK and mTOR. It has been shown that co-activation of muscarinic acetylcholine receptors and β-adrenergic receptors facilitates the conversion of short term to long term synaptic plasticity through an ERK- and mTOR-dependent mechanism which requires translation initiation. It seems therefore that the complex interplay among the cholinergic system, ERK and mTOR is crucial in the development of new inhibitory avoidance memories in the hippocampus. Copyright © 2015 Elsevier Inc. All rights reserved.

An Investigation of Hierachical Protein Recruitment to the Inhibitory Platelet Receptor, G6B-b

PubMed Central

Coxon, Carmen H.; Sadler, Amanda J.; Huo, Jiandong; Campbell, R. Duncan

2012-01-01

Platelet activation is regulated by both positive and negative signals. G6B-b is an inhibitory platelet receptor with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). The molecular basis of inhibition by G6B-b is currently unknown but thought to involve the SH2 domain-containing tyrosine phosphatase SHP-1. Here we show that G6B-b also associates with SHP-2, as well as SHP-1, in human platelets. Using a number of biochemical approaches, we found these interactions to be direct and that the tandem SH2 domains of SHP-2 demonstrated a binding affinity for G6B-b 100-fold higher than that of SHP-1. It was also observed that while SHP-1 has an absolute requirement for phosphorylation at both motifs to bind, SHP-2 can associate with G6B-b when only one motif is phosphorylated, with the N-terminal SH2 domain and the ITIM being most important for the interaction. A number of other previously unreported SH2 domain-containing proteins, including Syk and PLCγ2, also demonstrated specificity for G6B-b phosphomotifs and may serve to explain the observation that G6B-b remains inhibitory in the absence of both SHP-1 and SHP-2. In addition, the presence of dual phosphorylated G6B-b in washed human platelets can reduce the EC50 for both CRP and collagen. PMID:23185356

Molecular mechanism of APC/C activation by mitotic phosphorylation.

PubMed

Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-05-12

In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

Roles for PI(3,5)P2 in nutrient sensing through TORC1

PubMed Central

Jin, Natsuko; Mao, Kai; Jin, Yui; Tevzadze, Gela; Kauffman, Emily J.; Park, Sujin; Bridges, Dave; Loewith, Robbie; Saltiel, Alan R.; Klionsky, Daniel J.; Weisman, Lois S.

2014-01-01

TORC1, a conserved protein kinase, regulates cell growth in response to nutrients. Localization of mammalian TORC1 to lysosomes is essential for TORC1 activation. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), an endosomal signaling lipid, is implicated in insulin-dependent stimulation of TORC1 activity in adipocytes. This raises the question of whether PI(3,5)P2 is an essential general regulator of TORC1. Moreover, the subcellular location where PI(3,5)P2 regulates TORC1 was not known. Here we report that PI(3,5)P2 is required for TORC1 activity in yeast and regulates TORC1 on the vacuole (lysosome). Furthermore, we show that the TORC1 substrate, Sch9 (a homologue of mammalian S6K), is recruited to the vacuole by direct interaction with PI(3,5)P2, where it is phosphorylated by TORC1. Of importance, we find that PI(3,5)P2 is required for multiple downstream pathways via TORC1-dependent phosphorylation of additional targets, including Atg13, the modification of which inhibits autophagy, and phosphorylation of Npr1, which releases its inhibitory function and allows nutrient-dependent endocytosis. These findings reveal PI(3,5)P2 as a general regulator of TORC1 and suggest that PI(3,5)P2 provides a platform for TORC1 signaling from lysosomes. PMID:24478451

Alpha-lipoic acid impairs body weight gain of young broiler chicks via modulating peripheral AMPK.

PubMed

Wang, Yufeng; Everaert, Nadia; Song, Zhigang; Decuypere, Eddy; Vermeulen, Daniel; Buyse, Johan

2017-09-01

In mammals, the AMP-activated protein kinase (AMPK) pathways in the central and peripheral tissues coordinately integrate inputs from multiple sources to regulate energy balance. The present study was aimed to explore the potential role of hepatic AMPK in the energy homeostasis of broiler chickens. Diets with 0, 0.05% or 0.1% alpha-lipoic acid (α-LA), a known AMPK inhibitor were provided to broiler chicks for 7days. As a result, α-LA supplementation decreased the relative growth rate of broiler chicks. Hepatic AMPKα2 mRNA levels were significantly upregulated by dietary α-LA, in concert with the increased phosphorylated AMPKα protein levels. In addition, hepatic FAS mRNA levels together with the malonyl-CoA to total CoA ester ratio were reduced by α-LA supplementation. Moreover, the hepatic phosphorylated glycogen synthase levels were increased resulting in a markedly decreased hepatic glycogen content. In conclusion, dietary α-LA supplementation decreased the in vivo hepatic glycogenesis and lipogenesis via stimulating hepatic AMPKα mRNA levels and the phosphorylated gene product. The stimulatory effect of α-LA on hepatic AMPK mRNA and pAMPKα protein levels together with our previous observations regarding its inhibitory effect on hypothalamic AMPK may have altered the energy balance and hence impaired body weight gain of broiler chicks. Copyright © 2017 Elsevier Inc. All rights reserved.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors

PubMed Central

Anton, Olga M.; Vielkind, Susina; Peterson, Mary E.; Tagaya, Yutaka; Long, Eric O.

2015-01-01

IL-15 bound to the IL-15 receptor α chain (IL-15Rα) is presented in trans to cells bearing the IL-2 receptor β and γc chains. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor–ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR–HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to down-regulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A+ cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Co-engagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis. PMID:26453750

Betulinic acid exerts anti-hepatitis C virus activity via the suppression of NF-κB- and MAPK-ERK1/2-mediated COX-2 expression.

PubMed

Lin, Chun-Kuang; Tseng, Chin-Kai; Chen, Kai-Hsun; Wu, Shih-Hsiung; Liaw, Chih-Chuang; Lee, Jin-Ching

2015-06-23

This study was designed to evaluate the effect of betulinic acid (BA), extracted from Avicennia marina, on the replication of hepatitis C virus (HCV) and to investigate the mechanism of this BA-mediated anti-HCV activity. HCV replicon and infectious systems were used to evaluate the anti-HCV activity of BA. Exogenous COX-2 or knock-down of COX-2 expression was used to investigate the role of COX-2 in the anti-HCV activity of BA. The effects of BA on the phosphorylation of NF-κB and on kinases in the MAPK signalling pathway were determined. The anti-HCV activity of BA in combination with other HCV inhibitors was also determined to assess its use as an anti-HCV supplement. BA inhibited HCV replication in both Ava5 replicon cells and in a cell culture-derived infectious HCV particle system. Treatment with a combination of BA and IFN-α, the protease inhibitor telaprevir or the NS5B polymerase inhibitor sofosbuvir resulted in the synergistic suppression of HCV RNA replication. Exogenous overexpression of COX-2 gradually attenuated the inhibitory effect of BA on HCV replication, suggesting that BA reduces HCV replication by suppressing the expression of COX-2. In particular, BA down-regulated HCV-induced COX-2 expression by reducing the phosphorylation of NF-κB and ERK1/2 of the MAPK signalling pathway. BA inhibits HCV replication by suppressing the NF-κB- and ERK1/2-mediated COX-2 pathway and may serve as a promising compound for drug development or as a potential supplement for use in the treatment of HCV-infected patients. © 2015 The British Pharmacological Society.

Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation.

PubMed

Foster, D Brian; Huang, Renjian; Hatch, Victoria; Craig, Roger; Graceffa, Philip; Lehman, William; Wang, C-L Albert

2004-12-17

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.

Two Naturally Occurring Terpenes, Dehydrocostuslactone and Costunolide, Decrease Intracellular GSH Content and Inhibit STAT3 Activation

PubMed Central

Butturini, Elena; Cavalieri, Elisabetta; Carcereri de Prati, Alessandra; Darra, Elena; Rigo, Antonella; Shoji, Kazuo; Murayama, Norie; Yamazaki, Hiroshi; Watanabe, Yasuo; Suzuki, Hisanori; Mariotto, Sofia

2011-01-01

The main purpose of the present study is to envisage the molecular mechanism of inhibitory action ofdehydrocostuslactone (DCE) andcostunolide (CS), two naturally occurring sesquiterpene lactones, towards the activation of signal transducer and activator of transcription 3 (STAT3). We report that, in human THP-1 cell line, they inhibit IL-6-elicited tyrosine phosphorylation of STAT3 and its DNA binding activity with EC50 of 10 µM with concomitantdown-regulation ofthe phosphorylation of the tyrosine Janus kinases JAK1, JAK2 and Tyk2. Furthermore, these compounds that contain an α-β-unsatured carbonyl moiety and function as potent Michael reaction acceptor, induce a rapid drop in intracellular glutathione (GSH) concentration by direct interaction with it, thereby triggering S-glutathionylation of STAT3. Dehydrocostunolide (HCS), the reduced form of CS lacking only the α-β-unsaturated carbonyl group, fails to exert any inhibitory action. Finally, the glutathione ethylene ester (GEE), the cell permeable GSH form, reverts the inhibitory action of DCE and CS on STAT3 tyrosine phosphorylation. We conclude that these two sesquiterpene lactones are able to induce redox-dependent post-translational modification of cysteine residues of STAT3 protein in order to regulate its function. PMID:21625597

Discovery of Tyk2 inhibitors via the virtual site-directed fragment-based drug design.

PubMed

Jang, Woo Dae; Kim, Jun-Tae; Son, Hoon Young; Park, Seung Yeon; Cho, Young Sik; Koo, Tae-sung; Lee, Hyuk; Kang, Nam Sook

2015-09-15

In this study, we synthesized compound 12 with potent Tyk2 inhibitory activity from FBDD study and carried out a cell-based assay for Tyk2/STAT3 signaling activation upon IFNα5 stimulation. Compound 12 completely suppressed the IFNα5-mediated Tyk2/STAT3 signaling pathway as well as the basal levels of pSTAT3. Stimulation with IFNα/β leads to the tyrosine phosphorylation of the JAK1 and Tyk2 receptor-associated kinases with subsequent STATs activation, transmitting signals from the cell surface receptor to the nucleus. In conclusion, the potency of compound 12 to interrupt the signal transmission of Tyk2/STAT3 appeared to be equivalent or superior to that of the reference compound. Copyright © 2015 Elsevier Ltd. All rights reserved.

Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase Cε-Dependent Mechanism in Neurons

PubMed Central

Björnström, Karin; Turina, Dean; Strid, Tobias; Sundqvist, Tommy; Eintrei, Christina

2014-01-01

Background The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction. Methods In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser729 phosphorylation were detected with Western blot. Results The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser729 phosphorylation, a crucial step in the activation of PKCε. Conclusions Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge. PMID:24828410

Ameliorative effects of Qingfei Tongluo formula on experimental mycoplasmal pneumonia in mice.

PubMed

Jiang, Yong-hong; Yu, Jian-er; Guo, Ai-hua; Li, Xiao; Lin, Yan; Jiang, Zhi-yan; Xiao, Zhen

2016-04-01

Mycoplasma pneumoniae pneumonia (MPP) is a common disease in children. Qingfei Tongluo formula (QTF) has been used for the treatment of MPP clinically, but the chemical constituents and mechanism involved remain unclear. This study aimed to analyze the main chemical constituents and to explore the possible mechanism of action associated with QTF treatment of MPP. Liquid chromatography-mass spectrometry was employed to identify the compounds contained in the QTF extract. A BALB/c mouse model of MP infection was established. After treatment with QTF (0.85 and 1.70 g/kg) for 3 days, hematoxylin and eosin staining was performed in lung tissues for histological examination. Inflammatory cytokines were detected by ELISA. Western blot analysis was used for detecting phosphorylated proteins involved in MAPK and nuclear factor (NF)-κB signaling pathways. In the mouse model, a large amount of pulmonary interstitial infiltration of lymphocytes and plasmacytes were seen as well as bronchus and vasodilation congestion. Following QTF treatment, inflammation was alleviated significantly compared with the model group. Inflammatory cytokines [interleukin (IL)-6, transforming growth factor-β1, IL-8, IL-1β and tumor necrosis factor-α] in bronchoalveolar lavage fluid were decreased dramatically. In addition, we found that QTF inhibited activation of phosphorylation of JNK, ERK and NF-κB. In conclusion, QTF alleviates MPP inflammation possibly via inhibitory activation of MAPK/NF-κB pathways, which can act as a new agent for MPP treatment.

Lnk adaptor suppresses radiation resistance and radiation-induced B-cell malignancies by inhibiting IL-11 signaling

PubMed Central

Louria-Hayon, Igal; Frelin, Catherine; Ruston, Julie; Gish, Gerald; Jin, Jing; Kofler, Michael M.; Lambert, Jean-Philippe; Adissu, Hibret A.; Milyavsky, Michael; Herrington, Robert; Minden, Mark D.; Dick, John E.; Gingras, Anne-Claude; Iscove, Norman N.; Pawson, Tony

2013-01-01

The Lnk (Sh2b3) adaptor protein dampens the response of hematopoietic stem cells and progenitors (HSPCs) to a variety of cytokines by inhibiting JAK2 signaling. As a consequence, Lnk−/− mice develop hematopoietic hyperplasia, which progresses to a phenotype resembling the nonacute phase of myeloproliferative neoplasm. In addition, Lnk mutations have been identified in human myeloproliferative neoplasms and acute leukemia. We find that Lnk suppresses the development of radiation-induced acute B-cell malignancies in mice. Lnk-deficient HSPCs recover more effectively from irradiation than their wild-type counterparts, and this resistance of Lnk−/− HSPCs to radiation underlies the subsequent emergence of leukemia. A search for the mechanism responsible for radiation resistance identified the cytokine IL-11 as being critical for the ability of Lnk−/− HSPCs to recover from irradiation and subsequently become leukemic. In IL-11 signaling, wild-type Lnk suppresses tyrosine phosphorylation of the Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 and its association with the growth factor receptor-bound protein 2, as well as activation of the Erk MAP kinase pathway. Indeed, Src homology region 2 domain-containing phosphatase-2 has a binding motif for the Lnk Src Homology 2 domain that is phosphorylated in response to IL-11 stimulation. IL-11 therefore drives a pathway that enhances HSPC radioresistance and radiation-induced B-cell malignancies, but is normally attenuated by the inhibitory adaptor Lnk. PMID:24297922

Regulation of FAK Ser-722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration.

PubMed

Bianchi, Mariarita; De Lucchini, Stefania; Marin, Oriano; Turner, David L; Hanks, Steven K; Villa-Moruzzi, Emma

2005-10-15

In addition to tyrosine sites, FAK (focal adhesion kinase) is phosphorylated on multiple serine residues. In the present study, the regulation of two of these sites, Ser-722 (S1) and Ser-911 (S4), was investigated. Phosphorylation of S1 (but not S4) decreased in resuspended cells, and recovered during spreading on fibronectin, indicating adhesion-dependent regulation. GSK3 (glycogen synthase kinase 3) inhibitors decreased S1 phosphorylation, and siRNA (short interfering RNA) silencing indicated further the involvement of GSK3beta. Furthermore, GSK3beta was found to become activated during cell spreading on fibronectin, and to physically associate with FAK. S1 phosphorylation was observed to decrease in wounded cell monolayers, while GSK3beta underwent inactivation and later was observed to increase to the original level within 24 h. Direct phosphorylation of S1, requiring pre-phosphorylation of Ser-726 in the +4 position, was demonstrated using purified GSK3 and a synthetic peptide containing FAK residues 714-730. An inhibitory role for S1 phosphorylation in FAK signalling was indicated by findings that both alanine substitution for S1 and dephosphorylation of S1 by PP1 (serine/threonine protein phosphatase type-1) resulted in an increase in FAK kinase activity; likewise, this role was also shown by cell treatment with the GSK3 inhibitor LiCl. The inhibitory role was confirmed by the finding that cells expressing FAK with alanine substitution for S1 displayed improved cell spreading and faster migration in wound-healing and trans-well assays. Finally, the finding that S1 phosphorylation increased in cells treated with the PP1 inhibitor okadaic acid indicated targeting of this site by PP1. These results indicate an additional mechanism for regulation of FAK activity during cell spreading and migration, involving Ser-722 phosphorylation modulated through the competing actions of GSK3beta and PP1.

Prostaglandin E2 mediates phosphorylation and down-regulation of the tuberous sclerosis-2 tumor suppressor (tuberin) in human endometrial adenocarcinoma cells via the Akt signaling pathway.

PubMed

Sales, Kurt J; Battersby, Sharon; Williams, Alistair R W; Anderson, Richard A; Jabbour, Henry N

2004-12-01

Prostaglandin (PG) E2 promotes tumor growth via interaction with its G protein-coupled receptors and activation of intracellular signaling. Tuberous sclerosis 2 (tuberin) is a tumor suppressor, which negatively regulates cell growth. Its phosphorylation results in its inactivation and targeted down- regulation, thus lifting the growth inhibition effects. This study investigated the expression and localization of tuberin in neoplastic and normal endometrium and the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Quantitative RT-PCR and Western blot analysis demonstrated reduced expression of tuberin in neoplastic tissue, compared with normal endometrial tissue. Tuberin expression was localized by immunohistochemistry to the glandular epithelial compartment in neoplastic and normal endometrium. We investigated the effect of PGE2 on phosphorylation of tuberin via the Akt pathway. Treatment of neoplastic and normal endometrium with 100 nm PGE2 enhanced phosphorylated tuberin immunoreactivity in the glandular epithelium. PGE2 also phosphorylated Akt and tuberin in Ishikawa endometrial adenocarcinoma cells, leading to a reduction in expression of total tuberin protein. Cotreatment of cells with wortmannin or LY294002 inhibited the PGE2-induced phosphorylation of Akt and tuberin. These data suggest that PGE2 signaling may promote endometrial tumorigenesis by inactivation of tuberin after its phosphorylation via the Akt signaling pathway.

Cytotoxicity of withaferin A in glioblastomas involves induction of an oxidative stress-mediated heat shock response while altering Akt/mTOR and MAPK signaling pathways

PubMed Central

Grogan, Patrick T.; Sleder, Kristina D.; Samadi, Abbas K.; Timmermann, Barbara N.; Cohen, Mark S.

2012-01-01

Withaferin A (WA), a steroidal lactone derived from the plant Vassobia breviflora, has been reported to have anti-proliferative, pro-apoptotic, and anti-angiogenic properties against cancer growth. In this study, we identified several key underlying mechanisms of anticancer action of WA in glioblastoma cells. WA was found to inhibit proliferation by inducing a dose-dependent G2/M cell cycle arrest and promoting cell death through both intrinsic and extrinsic apoptotic pathways. This was accompanied by an inhibitory shift in the Akt/mTOR signaling pathway which included diminished expression and/or phosphorylation of Akt, mTOR, p70 S6K, and p85 S6K with increased activation of AMPKα and the tumor suppressor tuberin/TSC2. Alterations in proteins of the MAPK pathway and cell surface receptors like EGFR, Her2/ErbB2, and c-Met were also observed. WA induced an N-acetyl-L-cysteinerepressible enhancement in cellular oxidative potential/stress with subsequent induction of a heat shock stress response primarily through HSP70, HSP32, and HSP27 upregulation and HSF1 downregulation. Taken together, we suggest that WA may represent a promising chemotherapeutic candidate in glioblastoma therapy warranting further translational evaluation. PMID:23129310

Wu-Tou Decoction in Rheumatoid Arthritis: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation

PubMed Central

Guo, Qingqing; Zheng, Kang; Fan, Danping; Zhao, Yukun; Li, Li; Bian, Yanqin; Qiu, Xuemei; Liu, Xue; Zhang, Ge; Ma, Chaoying; He, Xiaojuan; Lu, Aiping

2017-01-01

Purpose: This study aimed to explore underlying action mechanism of Wu-Tou decoction (WTD) in rheumatoid arthritis (RA) through network pharmacology prediction and experimental verification. Methods: Chemical compounds and human target proteins of WTD as well as RA-related human genes were obtained from TCM Database @ Taiwan, PubChem and GenBank, respectively. Subsequently, molecular networks and canonical pathways presumably involved in the treatment of WTD on RA were generated by ingenuity pathway analysis (IPA) software. Furthermore, experimental validation was carried out with MIP-1β-induced U937 cell model and collagen induced arthritis (CIA) rat model. Results: CCR5 signaling pathway in macrophages was shown to be the top one shared signaling pathway associated with both cell immune response and cytokine signaling. In addition, protein kinase C (PKC) δ and p38 in this pathway were treated as target proteins of WTD in RA. In vitro experiments indicated that WTD inhibited MIP-1β-induced production of TNF-α, MIP-1α, and RANTES as well as phosphorylation of CCR5, PKC δ, and p38 in U937 cells. WTD treatment maintained the inhibitory effects on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. In vivo experiments demonstrated that WTD ameliorated symptoms in CIA rats, decreased the levels of IL-1β, IL-2, IL-6, TNF-α, MIP-1α, MIP-2, RANTES, and IP-10 in serum of CIA rats, as well as mRNA levels of MIP-1α, MIP-2, RANTES, and IP-10 in ankle joints of CIA rats. Furthermore, WTD also lowered the phosphorylation levels of CCR5, PKC δ and p38 in both ankle joints and macrophages in ankle joints from CIA rats. Conclusion: It was demonstrated in this research that WTD played a role in inhibiting inflammatory response in RA which was closely connected with the modulation effect of WTD on CCR5 signaling pathway in macrophages. PMID:28515692

Davallia bilabiata inhibits TNF-α-induced adhesion molecules and chemokines by suppressing IKK/NF-kappa B pathway in vascular endothelial cells.

PubMed

Yang, Rong-Chi; Chang, Cheng-Chieh; Sheen, Jer-Ming; Wu, Hsiao-Ting; Pang, Jong-Hwei S; Huang, Sheng-Teng

2014-01-01

Davallia bilabiata (D. bilabiata) is also called GuSuiBu in Taiwan and is used as a substitute for Drynaria fortunei J. Sm. It is often used for trauma and bone repair. The inhibitory effect of D. bilabiata on inflammatory activity has not been reported. In the present study, we aimed to study the mechanism of anti-inflammation of D. bilabiata on the adhesion of leukocytes to vascular endothelial cells. The results showed that D. bilabiata, at concentrations without cytotoxic effect, inhibited the adhesion of monocytes (THP-1) to the TNF-α-stimulated human umbilical vascular endothelial cells (HUVECs). D. bilabiata suppressed the expression of the adhesion molecules ICAM, VCAM, and E-selectin at both the mRNA and protein level. In addition, both of the TNF-α-induced mRNA and protein expression of chemokines including fractalkine/CX3CL1, MCP-1 and RANTES as well as the level of secreted soluble fractalkine were decreased by D. bilabiata. We also verified that D. bilabiata inhibited the TNF-α-induced nuclear translocation of NF-κB through the inhibitory process on the TNF-α-activated phosphorylation of IKKα, IKKβ, IκB and NF-κB. All together, we concluded that the D. bilabiata affected the canonical pathway of TNF-α-induced NF-κB activation and down-regulated cell adhesion molecules and chemokine expression through inhibition of the NF-κB/IκBα/IKK signaling pathway. These findings strongly indicated that D. bilabiata might be a promising alternative/adjunct treatment for inflammatory diseases, such as rheumatoid arthritis and osteoarthritis.

Inhibitory effect 6-gingerol on adipogenesis through activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes.

PubMed

Li, Chunbo; Zhou, Lin

2015-12-25

6-Gingerol has been reported to inhibit adipogenesis and lipid content accumulation. However, the mechanism of its anti-adipogenic effect remains unclear. Our aim is to investigate the molecular mechanism of the anti-adipogenic effect of 6-gingerol. The lipid content in adipocytes was measured by Oil Red O staining and cell viability was analyzed by MTT assay. The extent of suppression of differentiation by 6-gingerol was characterized by measuring the triglyceride content and GPDH activity. The regulation of adipogenic markers and the components of the Wnt/β-catenin pathway were analyzed by real-time PCR and Western blotting. The nuclear location of β-catenin was identified using immunofluorescence assay. Small interfering RNA transfection was conducted to elucidate the crucial role of β-catenin in anti-adipogenic effect of 6-gingerol. Our results showed that 6-gingerol inhibited the adipogenesis and lowered the mRNA expression levels of transcription factors and the key lipogenic enzymes in 3T3-L1 cells. The effect of 6-gingerol on adipogenic differentiation was accompanied by stimulating the activation of the Wnt/β-catenin signaling. In addition, we found that 6-gingerol induced phosphorylations of glycogen synthase kinase-3β(GSK-3β), and promoted the nuclear accumulation of β-catenin. Importantly, the inhibitory effect of 6-gingerol on adipogenic differentiation was reversed after the siRNA knockdown of β-catenin was added. Our findings demonstrated that 6-gingerol inhibits the adipogenic differentiation of 3T3-L1 cells through activating the Wnt/β-catenin signaling pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation.

PubMed

Gou, Xiangbo; Wang, Wenying; Zou, Sihao; Qi, Yajuan; Xu, Yanfang

2018-03-01

The slowly activating delayed rectifier K + current (I Ks ) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates I Ks through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of I Ks by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record I Ks in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited I Ks in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1-2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human I Ks in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCβ) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on I Ks and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism underlying the modulation of the KCNQ1/KCNE1 channel. Copyright © 2018 Elsevier Ltd. All rights reserved.

β-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity*

PubMed Central

Sun, Jie-Jie; Lan, Jiang-Feng; Shi, Xiu-Zhen; Yang, Ming-Chong; Niu, Guo-Juan; Ding, Ding; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

2016-01-01

The Toll signaling pathway plays an important role in the innate immunity of Drosophila melanogaster and mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense against Staphylococcus aureus by regulating expression of antimicrobial peptides in shrimp. We then found that β-arrestins negatively regulate Toll signaling in two different ways. β-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of β-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. β-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser276 that impairs Dorsal transcriptional activity. Our study suggests that β-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity. PMID:26846853

Sophora subprosrate polysaccharide inhibited cytokine/chemokine secretion via suppression of histone acetylation modification and NF-κb activation in PCV2 infected swine alveolar macrophage.

PubMed

Yang, Jian; Tan, Hong-Lian; Gu, Li-Yuan; Song, Man-Ling; Wu, Yuan-Ying; Peng, Jian-Bo; Lan, Zong-Bao; Wei, Ying-Yi; Hu, Ting-Jun

2017-11-01

In the present study, effect of Sophora subprosrate polysaccharide on PCV2 infection-induced inflammation and histone acetylation modification in swine alveolar macrophage 3D4/2 cells was described for the first time. The relationship between histone acetylation modifications and inflammation response was investigated. The results showed that PCV2 infection induced inflammation by promoting the secretion of TNF-α, IL-1β, IL-6 and IL-10 in 3D4/2 cells. The production of TNF-α, IL-1β and IL-6 and their mRNA expression levels markedly decreased while the level and mRNA expression of IL-10 were elevated when the cells were treated with Sophora subprosrate polysaccharide. The SSP also decreased the activity of HATs, histone H3 acetylation (Ac-H3) and histone H4 acetylation (Ac-H4), p65 phosphorylation (P-p65) in the cells infected with PCV2 while HDACs activity was down-regulated, which involved in the inhibitory effect of SSP on histone acetylation and NF-κB signaling pathways activation. Down-regulation of HAT1 mRNA expression and up-regulation of HDAC1 mRNA expression further support the inhibitory effect of SSP on histone acetylation. In conclusion, Sophora subprosrate polysaccharide antagonized inflammatory responses induced by PCV2, via mechanisms involved in histone acetylation and NF-κB signaling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

[Inhibitory effect of apatinib on HCT-116 cells and its mechanism].

PubMed

Yin, Liang; Wang, Jin; Huang, Feng-Chang; Zhang, Yun-Fei; Xu, Ning; Wen, Zheng-Qi; Li, Wen-Liang; Dong, Jian

2017-03-20

To investigate the inhibitory effects of apatinib on colorectal carcinoma HCT-116 cells in vitro and the signaling pathways involved. The cytotoxicity of different concentrations (0, 0.5, 1, 1.5, and 2 µmol/L) of apatinib in HCT-116 cells was assessed by MTT assay, using capecitabine as the positive control. The apoptosis rate of apatinib-treated HCT-116 cells was detected using flow cytometry, and the expressions of Bcl-2, Bax, and caspase-3 were determined with quantitative real-time PCR and Western blotting. The effect of apatinib on the expressions of Akt, pAkt, Erk1/2 and pErk1/2 in HCT-116 cells was evaluated using Western blotting. Apatinib significantly inhibited the proliferation of HCT-116 cells in a concentration-dependent manner with an IC 50 value of 1.335 µmol/L. Flow cytometric analysis showed that apatinib significantly increased the apoptotic rate of HCT-116 cells dose-dependently. Apatinib induced the expression of the pro-apoptotic genes Bax and caspase-3 at both the mRNA and protein levels while inhibited the expression of the anti- apoptotic gene Bcl-2. The expressions of p-Akt and p-Erk1/2 were decreased in HCT-116 cells after apatinib treatment, but the total protein levels did not undergo obvious changes. Apatinib inhibits the proliferation and induces apoptosis of HCT-116 cells by suppressing the phosphorylation of Erk1/2 and Akt in the MAPK/Erk and PI3K/Akt signaling pathways.

Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pincus, David; Ryan, Christopher J.; Smith, Richard D.

2013-03-12

Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sitesmore » whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.« less

Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat.

PubMed

Liu, Fan; Wang, Zhiyao; Qiu, Yue; Wei, Min; Li, Chunyan; Xie, Yikuan; Shen, Le; Huang, Yuguang; Ma, Chao

2017-03-31

MyD88 is the adaptor protein of MyD88-dependent signaling pathway of TLRs and IL-1 receptor and regulates innate immune response. However, it was not clear whether and how MyD88 and related signaling pathways in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) are involved in neuropathic pain. Chronic constriction injury (CCI) was used to induce neuropathic pain in the rat. The expression of MyD88, TRIF, IBA1, and GFAP was detected with immunofluorescent staining and Western blot. The expression of interleukin-1 beta (IL-1β), high mobility group box 1 (HMGB1), NF-κB-p65, phosphorylated NF-κB-p65, ERK, phosphorylated ERK, and tumor necrosis factor-alpha (TNF-α) was detected with Western blot. Pain-related behavioral effects of MyD88 homodimerization inhibitory peptide (MIP) were accessed up to 3 weeks after intrathecal administration. Peripheral nerve injury significantly increased the protein level of MyD88 in the DRG and SDH, but had no effect on TRIF. MyD88 was found partly distributed in the nociceptive neurons in the DRGs and the astrocytes and microglia in the SDH. HMGB1 and IL-1β were also found upregulated in nociceptive pathways of CCI rats. Intrathecal application of MIP significantly alleviated mechanical and thermal hyperalgesia in the CCI rats and also reversed CCI-induced upregulation of MyD88 in both DRG and SDH. Further investigation revealed that suppression of MyD88 protein reduced the release of TNF-α and glial activation in the SDH in the CCI rats. MyD88-dependent TIR pathway in the DRG and SDH may play a role in CCI-induced neuropathic pain. MyD88 might serve as a potential therapeutic target for neuropathic pain.

Berberine improves airway inflammation and inhibits NF-κB signaling pathway in an ovalbumin-induced rat model of asthma.

PubMed

Li, Zhenghao; Zheng, Jie; Zhang, Ning; Li, Chengde

2016-12-01

Berberine has been reported for its various activities including anti-inflammatory effects and has been used in treating many diseases. However, its effects on airway inflammation in asthma have not been investigated. This study mainly aimed to detect its effects on the airway inflammation and the nuclear factor-κB (NF-κB) signaling pathway activity in a rat model of asthma. Asthma was induced by ovalbumin (OVA) sensitization and challenge. The asthmatic rats were respectively treated with vehicle PBS or berberine (100 mg/kg or 200 mg/kg) for 28 days. The control rats were treated with PBS. Inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted and the lung inflammation was scored. Levels of NF-κB p65 (mRNA and protein), phosphorylated NF-κB p65 (p-NF-κB p65), inhibitory κB alpha (IκBα) (mRNA and protein) and phosphorylated IκBα (p-IκBα), as well as NF-κB p65 DNA-binding activity, were measured to assess the activity of NF-κB signaling pathway. Levels of the downstream inflammatory mediators of NF-κB signaling, IL-1β, IL-4, IL-5, IL-6, IL-13 and IL-17 in BALF, were measured. Besides, the serum levels of OVA-specific immunoglobulin (Ig)E were measured. Results showed that OVA increased the number of inflammatory cells in BALF, elevated lung inflammation scores, enhanced the NF-κB signaling activity and promoted the production of IgE in rats. Berberine dose-dependently reversed the alterations induced by OVA in the asthmatic rats. The findings suggested a therapeutic potential of berberine on OVA- induced airway inflammation. The ameliorative effects on the OVA-induced airway inflammation might be associated with the inhibition of the NF-κB signaling pathway.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.

PubMed

Anton, Olga M; Vielkind, Susina; Peterson, Mary E; Tagaya, Yutaka; Long, Eric O

2015-11-15

IL-15 bound to the IL-15Rα-chain (IL-15Rα) is presented in trans to cells bearing the IL-2Rβ-chain and common γ-chain. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor-ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR-HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to downregulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A(+) cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Coengagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across, inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15-dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis. Copyright © 2015 by The American Association of Immunologists, Inc.

Olive Oil Phenolics Prevent Oxysterol‐Induced Proinflammatory Cytokine Secretion and Reactive Oxygen Species Production in Human Peripheral Blood Mononuclear Cells, Through Modulation of p38 and JNK Pathways

PubMed Central

Deiana, Monica; Spencer, Jeremy P. E.; Corona, Giulia

2017-01-01

Scope The aim of the present study was to investigate the ability of extra virgin olive oil (EVOO) polyphenols to counteract the proinflammatory effects induced by dietary and endogenous oxysterols in ex vivo immune cells. Methods and results Peripheral blood mononuclear cells (PBMCs), separated from the whole blood of healthy donors, were utilized and were stimulated with an oxysterols mixture, in the presence of physiologically relevant concentrations of the EVOO polyphenols, hydroxytyrosol, tyrosol, and homovanillic alcohol. Oxysterols significantly increased the production of proinflammatory cytokines, interleukin‐1β, regulated on activation, normal T‐cell expressed and secreted and macrophage migration inhibitory factor in ex vivo cultured PBMCs. Increased levels of reactive oxygen species (ROS) were also detected along with increased phosphorylation of the p38 and JNK. All phenolic compounds significantly reduced cytokine secretion induced by the oxysterols and inhibited ROS production and mitogen activated protein kinase phosphorylation. Conclusions These results suggest that extra virgin olive oil polyphenols modulate the immune response induced by dietary and endogenous cholesterol oxidation products in human immune cells and may hold benefit in controlling chronic immune and/or inflammatory processes. PMID:28815947

Functional evaluation of synthetic flavonoids and chalcones for potential antiviral and anticancer properties.

PubMed

Mateeva, Nelly; Eyunni, Suresh V K; Redda, Kinfe K; Ononuju, Ucheze; Hansberry, Tony D; Aikens, Cecilia; Nag, Anita

2017-06-01

Flavonoids, stilbenes, and chalcones are plant secondary metabolites that often possess diverse biological activities including anti-inflammatory, anti-cancer, and anti-viral activities. The wide range of bioactivities poses a challenge to identify their targets. Here, we studied a set of synthetically generated flavonoids and chalcones to evaluate for their biological activity, and compared similarly substituted flavonoids and chalcones. Substituted chalcones, but not flavonoids, showed inhibition of viral translation without significantly affecting viral replication in cells infected with hepatitis C virus (HCV). We suggest that the chalcones used in this study inhibit mammalian target of rapamycin (mTOR) pathway by ablating phosphorylation of ribosomal protein 6 (rps6), and also the kinase necessary for phosphorylating rps6 in Huh7.5 cells (pS6K1). In addition, selected chalcones showed inhibition of growth in Ishikawa, MCF7, and MDA-MB-231 cells resulting an IC 50 of 1-6µg/mL. When similarly substituted flavonoids were used against the same set of cancer cells, we did not observe any inhibitory effect. Together, we report that chalcones show potential for anti-viral and anti-cancer activities compared to similarly substituted flavonoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system.

PubMed

Miao, Linqing; Yang, Liu; Huang, Haoliang; Liang, Feisi; Ling, Chen; Hu, Yang

2016-03-30

Injured mature CNS axons do not regenerate in mammals. Deletion of PTEN, the negative regulator of PI3K, induces CNS axon regeneration through the activation of PI3K-mTOR signaling. We have conducted an extensive molecular dissection of the cross-regulating mechanisms in axon regeneration that involve the downstream effectors of PI3K, AKT and the two mTOR complexes (mTORC1 and mTORC2). We found that the predominant AKT isoform in CNS, AKT3, induces much more robust axon regeneration than AKT1 and that activation of mTORC1 and inhibition of GSK3β are two critical parallel pathways for AKT-induced axon regeneration. Surprisingly, phosphorylation of T308 and S473 of AKT play opposite roles in GSK3β phosphorylation and inhibition, by which mTORC2 and pAKT-S473 negatively regulate axon regeneration. Thus, our study revealed a complex neuron-intrinsic balancing mechanism involving AKT as the nodal point of PI3K, mTORC1/2 and GSK3β that coordinates both positive and negative cues to regulate adult CNS axon regeneration.

Ursolic acid inhibits proliferation and induces apoptosis of HT-29 colon cancer cells by inhibiting the EGFR/MAPK pathway*

PubMed Central

Shan, Jian-zhen; Xuan, Yan-yan; Zheng, Shu; Dong, Qi; Zhang, Su-zhan

2009-01-01

Objective: To investigate the effects of ursolic acid on the proliferation and apoptosis of human HT-29 colon cancer cells. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays were performed to evaluate the effects of ursolic acid on the growth and apoptosis of HT-29 cells. Western blot analysis was applied to investigate the inhibitory effects of ursolic acid on the phosphorylation of the epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), and the activity of B cell leukemia-2 (Bcl-2), B cell leukemia-xL (Bcl-xL), caspase-3, and caspase-9. Results: Ursolic acid inhibited the growth of HT-29 cells in dose- and time-dependent manners. The median inhibition concentration (IC50) values for 24, 48, and 72 h treatment were 26, 20, and 18 μmol/L, respectively. The apoptotic rates of 10, 20, and 40 μmol/L ursolic acid treatments for 24 h were 5.74%, 14.49%, and 33.05%, and for 48 h were 9%, 21.39%, and 40.49%, respectively. Ursolic acid suppressed the phosphorylation of EGFR, ERK1/2, p38 MAPK, and JNK, which is well correlated with its growth inhibitory effect. 10, 20, and 40 μmol/L ursolic acid significantly inhibited the proliferation of EGF-stimulated HT-29 cells (P<0.05). Cell proliferation was most significantly inhibited when treated with 10 and 20 μmol/L ursolic acid combined with 200 nmol/L AG 1478 or 10 μmol/L U0126 (P<0.01). Besides, it also down-regulated the expression of Bcl-2 and Bcl-xL and activated caspase-3 and caspase-9. Conclusion: Ursolic acid induces apoptosis in HT-29 cells by suppressing the EGFR/MAPK pathway, suggesting that it may be a potent agent for the treatment of colorectal cancer. PMID:19735099

Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4 or Rab11 pathway.

PubMed

Wang, Feifei; Chen, Xiaoqing; Zhang, Xiaoqing; Ma, Lan

2008-08-01

Agonist-induced phosphorylation, internalization, and intracellular trafficking of G protein-coupled receptors are critical in regulating both cellular responsiveness and signal transduction. The current study investigated the role of receptor phosphorylation state in regulation of agonist-induced internalization and intracellular trafficking of mu-opioid receptor (MOR). Our results showed that after agonist stimulation, the recycle of a mutant MOR that lacks the C-terminal residues after Asn(362) (MOR362T) was greatly decreased, whereas a C-terminal phosphorylation sites-mutated MOR (MOR3A), which is deficient in agonist-induced phosphorylation recycled back to the membrane at a level comparable to that of the wild-type receptor, however, interestingly at a slower rate. Inhibition of functions of either Rab4 or Rab11 by dominant-negative mutants and small interfering RNA both significantly impaired the recycling of the wild-type MOR, whereas the recycling of the phosphorylation-deficient mutant was only inhibited by the dominant-negative mutant and small interfering RNA of Rab11, suggesting that the recycling of nonphosphorylated MOR is exclusively via Rab11-mediated pathway. Furthermore, phosphorylated MOR was observed accumulated in Rab5- and Rab4-, but not Rab11-positive vesicles. Our data indicate that both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation, and the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively, which may ultimately lead to differential cellular responsiveness or downstream signaling.

Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands

PubMed Central

Granovsky, Alexey E.; Clark, Mathew M.; McElheny, Dan; Chimon, Alexander; Rosner, Marsha R.; Koide, Shohei

2010-01-01

Background Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. Methods/Findings In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. Conclusions/Significance This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential. PMID:20463977

Anti-inflammatory Effects of Cardamonin in Ovarian Cancer Cells Are Mediated via mTOR Suppression.

PubMed

Chen, Huajiao; Shi, Daohua; Niu, Peiguang; Zhu, Yanting; Zhou, Jintuo

2018-05-17

Cardamonin exhibits a variety of pharmacological activities including anti-inflammatory and antitumor, which are correlated with the inhibition of nuclear factor-kappaB and the mammalian target of rapamycin, respectively. However, whether the anti-inflammatory effects of cardamonin are mediated by the mammalian target of rapamycin remains unknown. In this study, ovarian cancer SKOV3 cells were cultured with lipopolysaccharide to induce inflammation, and the inhibitory effects and underlying molecular mechanisms of cardamonin were investigated using specific inhibitors of the mammalian target of rapamycin and the nuclear factor-kappaB pathway (rapamycin and pyrrolidine dithiocarbamate, respectively). Our results indicated that cardamonin inhibited the viability of normal and lipopolysaccharide-pretreated SKOV3 cells in a concentration-dependent manner. In accordance with rapamycin, the activation of the mammalian target of rapamycin and its downstream target, ribosomal protein S6 kinase 1, was inhibited by cardamonin, while pyrrolidine dithiocarbamate substantially blocked nuclear factor-kappaB activation and mildly inhibited the phosphorylation of the mammalian target of rapamycin and ribosomal protein S6 kinase 1. Pretreated with pyrrolidine dithiocarbamate, the effect of cardamonin on the mammalian target of rapamycin signalling was not affected, but the expression of inflammatory factors was further reduced. In cells pretreated with rapamycin, the inhibitory effects of cardamonin were completely suppressed with regards to the phosphorylation of the mammalian target of rapamycin, ribosomal protein S6 kinase 1, TNF- α , and interleukin-6, and nuclear factor-kappaB p65 protein expression was decreased. In conclusion, our findings indicate that the anti-inflammatory effects of cardamonin are correlated with mammalian target of rapamycin inhibition. Georg Thieme Verlag KG Stuttgart · New York.

Bisphenol A and Related Alkylphenols Exert Nongenomic Estrogenic Actions Through a G Protein-Coupled Estrogen Receptor 1 (Gper)/Epidermal Growth Factor Receptor (Egfr) Pathway to Inhibit Meiotic Maturation of Zebrafish Oocytes1

PubMed Central

Fitzgerald, Amanda C.; Peyton, Candace; Dong, Jing; Thomas, Peter

2015-01-01

Xenobiotic estrogens, such as bisphenol A (BPA), disrupt a wide variety of genomic estrogen actions, but their nongenomic estrogen actions remain poorly understood. We investigated nongenomic estrogenic effects of low concentrations of BPA and three related alkylphenols on the inhibition of zebrafish oocye maturation (OM) mediated through a G protein-coupled estrogen receptor 1 (Gper)-dependent epidermal growth factor receptor (Egfr) pathway. BPA (10–100 nM) treatment for 3 h mimicked the effects of estradiol-17beta (E2) and EGF, decreasing spontaneous maturation of defolliculated zebrafish oocytes, an effect not blocked by coincubation with actinomycin D, but blocked by coincubation with a Gper antibody. BPA displayed relatively high binding affinity (15.8% that of E2) for recombinant zebrafish Gper. The inhibitory effects of BPA were attenuated by inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase with ilomastat. Treatment with an inhibitor of Egfr transactivation, AG1478, and an inhibitor of the mitogen-activated protein kinase (MAPK) 3/1 pathway, U0126, increased spontaneous OM and blocked the inhibitory effects of BPA, E2, and the selective GPER agonist, G-1. Western blot analysis showed that BPA (10–200 nM) mimicked the stimulatory effects of E2 and EGF on Mapk3/1 phosphorylation. Tetrabromobisphenol A, 4-nonylphenol, and tetrachlorobisphenol A (5–100 nM) also inhibited OM, an effect blocked by cotreatment with AG1478, as well as with the GPER antagonist, G-15, and displayed similar binding affinities as BPA to zebrafish Gper. The results suggest that BPA and related alkylphenols disrupt zebrafish OM by a novel nongenomic estrogenic mechanism involving activation of the Gper/Egfr/Mapk3/1 pathway. PMID:26490843

Bisphenol A and Related Alkylphenols Exert Nongenomic Estrogenic Actions Through a G Protein-Coupled Estrogen Receptor 1 (Gper)/Epidermal Growth Factor Receptor (Egfr) Pathway to Inhibit Meiotic Maturation of Zebrafish Oocytes.

PubMed

Fitzgerald, Amanda C; Peyton, Candace; Dong, Jing; Thomas, Peter

2015-12-01

Xenobiotic estrogens, such as bisphenol A (BPA), disrupt a wide variety of genomic estrogen actions, but their nongenomic estrogen actions remain poorly understood. We investigated nongenomic estrogenic effects of low concentrations of BPA and three related alkylphenols on the inhibition of zebrafish oocye maturation (OM) mediated through a G protein-coupled estrogen receptor 1 (Gper)-dependent epidermal growth factor receptor (Egfr) pathway. BPA (10-100 nM) treatment for 3 h mimicked the effects of estradiol-17beta (E2) and EGF, decreasing spontaneous maturation of defolliculated zebrafish oocytes, an effect not blocked by coincubation with actinomycin D, but blocked by coincubation with a Gper antibody. BPA displayed relatively high binding affinity (15.8% that of E2) for recombinant zebrafish Gper. The inhibitory effects of BPA were attenuated by inhibition of upstream regulators of Egfr, intracellular tyrosine kinase (Src) with PP2, and matrix metalloproteinase with ilomastat. Treatment with an inhibitor of Egfr transactivation, AG1478, and an inhibitor of the mitogen-activated protein kinase (MAPK) 3/1 pathway, U0126, increased spontaneous OM and blocked the inhibitory effects of BPA, E2, and the selective GPER agonist, G-1. Western blot analysis showed that BPA (10-200 nM) mimicked the stimulatory effects of E2 and EGF on Mapk3/1 phosphorylation. Tetrabromobisphenol A, 4-nonylphenol, and tetrachlorobisphenol A (5-100 nM) also inhibited OM, an effect blocked by cotreatment with AG1478, as well as with the GPER antagonist, G-15, and displayed similar binding affinities as BPA to zebrafish Gper. The results suggest that BPA and related alkylphenols disrupt zebrafish OM by a novel nongenomic estrogenic mechanism involving activation of the Gper/Egfr/Mapk3/1 pathway. © 2015 by the Society for the Study of Reproduction, Inc.

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway.

PubMed

Yi, Lian; Shi, Tujin; Gritsenko, Marina A; X'avia Chan, Chi-Yuet; Fillmore, Thomas L; Hess, Becky M; Swensen, Adam C; Liu, Tao; Smith, Richard D; Wiley, H Steven; Qian, Wei-Jun

2018-04-17

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway as our model. A total of 43 phosphopeptides from the EGFR-MAPK pathway were selected for the study. The recovery and sensitivity of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO 2 ), combined with selected reaction monitoring (SRM)-MS were evaluated. The recovery of phosphopeptides by IMAC and TiO 2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1 to 100 μg starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 μg peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25 μg range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 10 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.

Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Lian; Shi, Tujin; Gritsenko, Marina A.

2018-03-27

Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichmentmore » methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.« less

The G2 block induced by DNA damage: a caffeine-resistant component independent of Cdc25C, MPM-2 phosphorylation, and H1 kinase activity.

PubMed

Barratt, R A; Kao, G; McKenna, W G; Kuang, J; Muschel, R J

1998-06-15

Treatment of cells with agents that cause DNA damage often results in a delay in G2. There is convincing evidence showing that inhibition of p34cdc2 kinase activation is involved in the DNA damage-induced G2 delay. In this study, we have demonstrated the existence of an additional pathway, independent of the p34cdc2 kinase activation pathway, that leads to a G2 arrest in etoposide-treated cells. Both the X-ray-induced and the etoposide-induced G2 arrest were associated with inhibition of the p34cdc2 H1 kinase activation pathway as judged by p34cdc2 H1 kinase activity and phosphorylation of cdc25C. Caffeine treatment restored these activities after either of the treatments. However, the etoposide-treated cells did not resume cycling, revealing the presence of an alternative pathway leading to a G2 arrest. To explore the possibility that this additional pathway involved phosphorylation of the MPM-2 epitope that is shared by a large family of mitotic phosphoproteins, we monitored the phosphorylation status of the MPM-2 epitope after DNA damage and after treatment with caffeine. Phosphorylation of the MPM-2 epitope was depressed in both X-ray and etoposide-treated cells, and the depression was reversed by caffeine in both cases. The results indicate that the pathway affecting MPM-2 epitope phosphorylation is involved in the G2 delay caused by DNA damage. However, it is not part of the caffeine-insensitive pathway leading to a G2 block seen in etoposide-treated cells.

Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells.

PubMed

Wang, Yufei; Whittall, Trevor; McGowan, Edward; Younson, Justine; Kelly, Charles; Bergmeier, Lesley A; Singh, Mahavir; Lehner, Thomas

2005-03-15

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407-426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457-496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457-496 but enhanced by p407-426.

Protein tyrosine phosphatase 1B inhibitory activity of lavandulyl flavonoids from roots of Sophora flavescens.

PubMed

Sasaki, Tatsunori; Li, Wei; Higai, Koji; Quang, Tran Hong; Kim, Young Ho; Koike, Kazuo

2014-05-01

Protein tyrosine phosphatase 1B is a non-transmembrane protein tyrosine phosphatase and major negative regulator in insulin signaling cascades, and much attention has been paid to protein tyrosine phosphatase 1B inhibitors as potential therapies for diabetes. The screening of a natural compound library led to the discovery of five lavandulyl flavonoids, which were isolated from the roots of Sophora flavescens, as novel PTP1B inhibitors: kuraridin (1), norkurarinone (2), kurarinone (3), 2'-methoxykurarinone (4), and kushenol T (5). The three most potent compounds, 1, 2, and 4 (IC50 < 30 µM), were demonstrated to be noncompetitive inhibitors of protein tyrosine phosphatase 1B based on a kinetic analysis, and they exhibited different inhibitory selectivities against four homologous protein tyrosine phosphatases (T cell protein tyrosine phosphatase, vaccinia H1-related phosphatase, and Src homology domain 2-containing protein tyrosine phosphatases 1 and 2). Compounds 1, 2, and 4 also exhibited cellular activity in the insulin signaling pathway by increasing the insulin-stimulated Akt phosphorylation level in human hepatocellular liver carcinoma HepG2 cells, suggesting their potential for new anti-insulin-resistant drug developments. Georg Thieme Verlag KG Stuttgart · New York.

Inhibitory Effect of the Ethanol Extract of a Rice Bran Mixture Comprising Angelica gigas, Cnidium officinale, Artemisia princeps, and Camellia sinensis on Brucella abortus Uptake by Professional and Nonprofessional Phagocytes.

PubMed

Hop, Huynh Tan; Arayan, Lauren Togonon; Reyes, Alisha Wehdnesday Bernardo; Huy, Tran Xuan Ngoc; Baek, Eun Jin; Min, WonGi; Lee, Hu Jang; Lee, Chun Hee; Rhee, Man Hee; Kim, Suk

2017-10-28

In this study, we evaluated the inhibitory effect of a rice bran mixture extract (RBE) on Brucella abortus pathogenesis in professional (RAW 264.7) and nonprofessional (HeLa) phagocytes. We fermented the rice bran mixture and then extracted it with 50% ethanol followed by gas chromatography-mass spectrometry to identify the components in RBE. Our results clearly showed that RBE caused a significant reduction in the adherence of B. abortus in both cell lines. Furthermore, analysis of phagocytic signaling proteins by western blot assay revealed that RBE pretreatment resulted in inhibition of phosphorylation of JNK, ERK, and p38, leading to decline of internalization compared with the controls. Additionally, the intensity of F-actin observed by fluorescence microscopy and FACS was remarkably reduced in RBE-pretreated cells compared with control cells. However, the intracellular replication of B. abortus within phagocytes was not affected by RBE. Taken together, these findings suggest that the phagocytic receptor blocking and suppressive effects of RBE on the MAPK-linked phagocytic signaling pathway could negatively affect the invasion of B. abortus into phagocytes.

A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

NASA Astrophysics Data System (ADS)

Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

2016-04-01

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

Role of Substance P in the Regulation of Glucose Metabolism via Insulin Signaling-Associated Pathways

PubMed Central

Bakirtzi, Kyriaki; Kokkotou, Efi; Stavrakis, Dimitris; Margolis, Kara Gross; Thomou, Thomas; Giorgadze, Nino; Kirkland, James L.

2011-01-01

Substance P (SP), encoded by the tachykinin 1 (Tac1) gene, is the most potent tachykinin ligand for the high-affinity neurokinin-1 receptor (NK-1R). We previously reported that NK-1R-deficient mice show less weight gain and reduced circulating levels of leptin and insulin in response to a high-fat diet (HFD) and demonstrated the presence of functional NK-1R in isolated human preadipocytes. Here we assessed the effects of SP on weight gain in response to HFD and determined glucose metabolism in Tac1-deficient (Tac1−/−) mice. The effect of SP on the expression of molecules that may predispose to reduced glucose uptake was also determined in isolated human mesenteric, omental, and sc preadipocytes. We show that although weight accumulation in response to HFD was similar between Tac1−/− mice and wild-type littermates, Tac1−/− mice demonstrated lower glucose and leptin and increased adiponectin blood levels and showed improved responses to insulin challenge after HFD. SP stimulated phosphorylation of c-Jun N-terminal kinase, protein kinase Cθ, mammalian target of rapamycin, and inhibitory serine insulin receptor substrate-1 phosphorylation in human preadipocytes in vitro. Preincubation of human mesenteric preadipocytes with the protein kinase Cθ pseudosubstrate inhibitor reduced insulin receptor substrate 1 phosphorylation in response to SP. Lastly, SP also induced insulin receptor substrate-1 phosphorylation in mature human sc adipocytes. Our results demonstrate an important role for SP in adipose tissue responses and obesity-associated pathologies. These novel SP effects on molecules that enhance insulin resistance at the adipocyte level may reflect an important role for this peptide in the pathophysiology of type 2 diabetes. PMID:22009727

Role of substance P in the regulation of glucose metabolism via insulin signaling-associated pathways.

PubMed

Karagiannides, Iordanes; Bakirtzi, Kyriaki; Kokkotou, Efi; Stavrakis, Dimitris; Margolis, Kara Gross; Thomou, Thomas; Giorgadze, Nino; Kirkland, James L; Pothoulakis, Charalabos

2011-12-01

Substance P (SP), encoded by the tachykinin 1 (Tac1) gene, is the most potent tachykinin ligand for the high-affinity neurokinin-1 receptor (NK-1R). We previously reported that NK-1R-deficient mice show less weight gain and reduced circulating levels of leptin and insulin in response to a high-fat diet (HFD) and demonstrated the presence of functional NK-1R in isolated human preadipocytes. Here we assessed the effects of SP on weight gain in response to HFD and determined glucose metabolism in Tac1-deficient (Tac1(-/-)) mice. The effect of SP on the expression of molecules that may predispose to reduced glucose uptake was also determined in isolated human mesenteric, omental, and sc preadipocytes. We show that although weight accumulation in response to HFD was similar between Tac1(-/-) mice and wild-type littermates, Tac1(-/-) mice demonstrated lower glucose and leptin and increased adiponectin blood levels and showed improved responses to insulin challenge after HFD. SP stimulated phosphorylation of c-Jun N-terminal kinase, protein kinase C, mammalian target of rapamycin, and inhibitory serine insulin receptor substrate-1 phosphorylation in human preadipocytes in vitro. Preincubation of human mesenteric preadipocytes with the protein kinase C pseudosubstrate inhibitor reduced insulin receptor substrate 1 phosphorylation in response to SP. Lastly, SP also induced insulin receptor substrate-1 phosphorylation in mature human sc adipocytes. Our results demonstrate an important role for SP in adipose tissue responses and obesity-associated pathologies. These novel SP effects on molecules that enhance insulin resistance at the adipocyte level may reflect an important role for this peptide in the pathophysiology of type 2 diabetes.

Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.

PubMed

von Willebrand, M; Williams, S; Tailor, P; Mustelin, T

1998-06-01

Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.

Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis

PubMed Central

Sury, Matthias D; Vorlet-Fawer, Lorianne; Agarinis, Claudia; Yousefi, Shida; Grandgirard, Denis; Leib, Stephen L; Christen, Stephan

2010-01-01

Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-triphosphate (PIP3) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP3 and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis. PMID:20875857

Interleukin-1β inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts.

PubMed

Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

2013-12-05

Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth. Published by Elsevier Ireland Ltd.

Friedelane-type triterpenoids as selective anti-inflammatory agents by regulation of differential signaling pathways in LPS-stimulated macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villar-Lorenzo, Andrea, E-mail: avillar@iib.uam.es

A series of 31 pentacyclic triterpenoids isolated from the root barks of Celastrus vulcanicola and Maytenus jelskii were tested for cytotoxicity and inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 18 (C18) and 25 (C25) exhibited significant inhibition of LPS-induced NO release at 50 and 25 μM concentrations, respectively, and decreased mRNAs of pro-inflammatory cytokines. At the molecular level, C18 neither inhibited LPS-mediated phosphorylation of mitogen activated protein kinases (MAPKs) nor nuclear translocation of nuclear factor kappa beta (NFκB). Instead, C18 enhanced and prolonged nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) andmore » increased the expression of its target genes including hemeoxigenase 1 (HO1). C25 efficiently inhibited LPS-mediated phosphorylation of JNK, p38 and ERK, without affecting NFκB or Nrf2 signaling pathways. Both compounds reduced LPS-mediated processing of caspase-1 and the cleavage of interleukin 1β (IL1β) proform, reflecting their ability to target the inflammasome. C25 also counteracted LPS effects on iNOS expression and pro-inflammatory cytokines mRNA levels in Bv-2 microglial cells. The anti-inflammatory effect of both compounds was also assessed in human macrophages. Our results suggest that triterpenoids C18 and C25 possess anti-inflammatory effects, which may be therapeutically relevant for diseases linked to inflammation. - Highlights: • Compounds 18 (C18) and 25 (C25) exert anti-inflammatory effects in macrophages. • C18 enhanced nuclear translocation of Nrf2 and increased HO1 expression. • C25 inhibited the phosphorylation of JNK, p38 and ERK, members of the MAPKs family. • C25 reduced LPS-mediated processing of caspase-1 and the cleavage of interleukin 1β. • C18 and C25 may be therapeutic agents for diseases linked to inflammation.« less

Interleukin-1β Inhibits Insulin Signaling and Prevents Insulin-Stimulated System A Amino Acid Transport in Primary Human Trophoblasts

PubMed Central

Aye, Irving L. M. H.; Jansson, Thomas; Powell, Theresa L.

2013-01-01

Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10 pg/ml) for 24 hours. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth. PMID:23891856

CS2164, a novel multi-target inhibitor against tumor angiogenesis, mitosis and chronic inflammation with anti-tumor potency.

PubMed

Zhou, You; Shan, Song; Li, Zhi-Bin; Xin, Li-Jun; Pan, De-Si; Yang, Qian-Jiao; Liu, Ying-Ping; Yue, Xu-Peng; Liu, Xiao-Rong; Gao, Ji-Zhou; Zhang, Jin-Wen; Ning, Zhi-Qiang; Lu, Xian-Ping

2017-03-01

Although inhibitors targeting tumor angiogenic pathway have provided improvement for clinical treatment in patients with various solid tumors, the still very limited anti-cancer efficacy and acquired drug resistance demand new agents that may offer better clinical benefits. In the effort to find a small molecule potentially targeting several key pathways for tumor development, we designed, discovered and evaluated a novel multi-kinase inhibitor, CS2164. CS2164 inhibited the angiogenesis-related kinases (VEGFR2, VEGFR1, VEGFR3, PDGFRα and c-Kit), mitosis-related kinase Aurora B and chronic inflammation-related kinase CSF-1R in a high potency manner with the IC 50 at a single-digit nanomolar range. Consequently, CS2164 displayed anti-angiogenic activities through suppression of VEGFR/PDGFR phosphorylation, inhibition of ligand-dependent cell proliferation and capillary tube formation, and prevention of vasculature formation in tumor tissues. CS2164 also showed induction of G2/M cell cycle arrest and suppression of cell proliferation in tumor tissues through the inhibition of Aurora B-mediated H3 phosphorylation. Furthermore, CS2164 demonstrated the inhibitory effect on CSF-1R phosphorylation that led to the suppression of ligand-stimulated monocyte-to-macrophage differentiation and reduced CSF-1R + cells in tumor tissues. The in vivo animal efficacy studies revealed that CS2164 induced remarkable regression or complete inhibition of tumor growth at well-tolerated oral doses in several human tumor xenograft models. Collectively, these results indicate that CS2164 is a highly selective multi-kinase inhibitor with potent anti-tumor activities against tumor angiogenesis, mitosis and chronic inflammation, which may provide the rationale for further clinical assessment of CS2164 as a therapeutic agent in the treatment of cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Rosmanol potently inhibits lipopolysaccharide-induced iNOS and COX-2 expression through downregulating MAPK, NF-kappaB, STAT3 and C/EBP signaling pathways.

PubMed

Lai, Ching-Shu; Lee, Jong Hun; Ho, Chi-Tang; Liu, Cheng Bin; Wang, Ju-Ming; Wang, Ying-Jan; Pan, Min-Hsiung

2009-11-25

Rosmanol is a natural polyphenol from the herb rosemary (Rosmarinus officinalis L.) with high antioxidant activity. In this study, we investigated the inhibitory effects of rosmanol on the induction of NO synthase (NOS) and COX-2 in RAW 264.7 cells induced by lipopolysaccharide (LPS). Rosmanol markedly inhibited LPS-stimulated iNOS and COX-2 protein and gene expression, as well as the downstream products, NO and PGE2. Treatment with rosmanol also reduced translocation of the nuclear factor-kappaB (NF-kappaB) subunits by prevention of the degradation and phosphorylation of inhibitor kappaB (IkappaB). Western blot analysis showed that rosmanol significantly inhibited translocation and phosphorylation of NF-kappaB, signal transducer and activator of transcription-3 (STAT3), and the protein expression of C/EBPbeta and C/EBPdelta. We also found that rosmanol suppressed LPS-induced phosphorylation of ERK1/2, p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Our results demonstrate that rosmanol downregulates inflammatory iNOS and COX-2 gene expression by inhibiting the activation of NF-kappaB and STAT3 through interfering with the activation of PI3K/Akt and MAPK signaling. Taken together, rosmanol might contribute to the potent anti-inflammatory effect of rosemary and may have potential to be developed into an effective anti-inflammatory agent.

Biosurfactant mannosyl-erythritol lipid inhibits secretion of inflammatory mediators from RBL-2H3 cells.

PubMed

Morita, Yosuke; Tadokoro, Satoshi; Sasai, Masao; Kitamoto, Dai; Hirashima, Naohide

2011-12-01

Biosurfactant mannosyl-erythritol lipids (MELs) are glycolipids produced by microbes that have various biological activities. It has been reported that MELs exhibit excellent surface-activity and also various bioactivities, such as induction of cell differentiation and apoptosis. However, little is known about their action related to drug discovery or drug seeds. We investigated the effects of MELs on the secretion of inflammatory mediators from mast cells that play a central role in allergic responses. Mast cells secrete three kinds of inflammatory mediators and we quantified these secreted mediators by photometer or ELISA. The action mechanisms of MELs were studied by Ca(2+)-sensitive fluorescence dye and Western blotting of phosphorylated proteins. MELs inhibited exocytotic release by antigen stimulation in a dose-dependent manner. We also found that MELs inhibited antigen-induced secretion of leukotriene C(4) and cytokine TNF-α (tumor necrosis factor-α). The inhibitory action of MELs on mediator secretion was mediated by inhibition of Ca(2+) increase, phosphorylation of MAP kinases and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) that serve as a molecular machinery for exocytotic membrane fusion. MELs have anti-inflammatory action inhibiting the secretion of inflammatory mediators from mast cells. MELs affects two of major intracellular signaling pathways including Ca(2+) increase and MAP kinases. MELs also inhibited the phosphorylation of SNARE proteins that is crucial for not only exocytosis but also intracellular vesicular trafficking. 2011 Elsevier B.V. All rights reserved.

Inhibitory Effects of Cytosolic Ca(2+) Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets.

PubMed

Kwon, Hyuk-Woo; Shin, Jung-Hae; Lee, Dong-Ha; Park, Hwa-Jin

2015-01-01

Intracellular Ca(2+) ([Ca(2+)] i ) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca(2+)-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca(2+)] i , which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI) (Ser(1756)) to inhibit [Ca(2+)] i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser(1756)) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), indicating inhibition of Ca(2+) influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser(1756)) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca(2+)] i , which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca(2+)-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

PKB/Akt phosphorylation of ERRγ contributes to insulin-mediated inhibition of hepatic gluconeogenesis.

PubMed

Kim, Don-Kyu; Kim, Yong-Hoon; Hynx, Debby; Wang, Yanning; Yang, Keum-Jin; Ryu, Dongryeol; Kim, Kyung Seok; Yoo, Eun-Kyung; Kim, Jeong-Sun; Koo, Seung-Hoi; Lee, In-Kyu; Chae, Ho-Zoon; Park, Jongsun; Lee, Chul-Ho; Biddinger, Sudha B; Hemmings, Brian A; Choi, Hueng-Sik

2014-12-01

Insulin resistance, a major contributor to the pathogenesis of type 2 diabetes, leads to increased hepatic glucose production (HGP) owing to an impaired ability of insulin to suppress hepatic gluconeogenesis. Nuclear receptor oestrogen-related receptor γ (ERRγ) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERRγ for the regulation of hepatic gluconeogenesis. We examined insulin-dependent phosphorylation and subcellular localisation of ERRγ in cultured cells and in the liver of C57/BL6, leptin receptor-deficient (db/db), liver-specific insulin receptor knockout (LIRKO) and protein kinase B (PKB) β-deficient (Pkbβ (-/-)) mice. To demonstrate the role of ERRγ in the inhibitory action of insulin on hepatic gluconeogenesis, we carried out an insulin tolerance test in C57/BL6 mice expressing wild-type or phosphorylation-deficient mutant ERRγ. We demonstrated that insulin suppressed the transcriptional activity of ERRγ by promoting PKB/Akt-mediated phosphorylation of ERRγ at S179 and by eliciting translocation of ERRγ from the nucleus to the cytoplasm through interaction with 14-3-3, impairing its ability to promote hepatic gluconeogenesis. In addition, db/db, LIRKO and Pkbβ (-/-) mice displayed enhanced ERRγ transcriptional activity due to a block in PKBβ-mediated ERRγ phosphorylation during refeeding. Finally, the phosphorylation-deficient mutant ERRγ S179A was resistant to the inhibitory action of insulin on HGP. These results suggest that ERRγ is a major contributor to insulin action in maintaining hepatic glucose homeostasis.

Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

PubMed Central

González-Terán, Bárbara; Cortés, José R.; Manieri, Elisa; Matesanz, Nuria; Verdugo, ρngeles; Rodríguez, María E.; González-Rodríguez, ρgueda; Valverde, ρngela; Martín, Pilar; Davis, Roger J.; Sabio, Guadalupe

2012-01-01

Bacterial LPS (endotoxin) has been implicated in the pathogenesis of acute liver disease through its induction of the proinflammatory cytokine TNF-α. TNF-α is a key determinant of the outcome in a well-established mouse model of acute liver failure during septic shock. One possible mechanism for regulating TNF-α expression is through the control of protein elongation during translation, which would allow rapid cell adaptation to physiological changes. However, the regulation of translational elongation is poorly understood. We found that expression of p38γ/δ MAPK proteins is required for the elongation of nascent TNF-α protein in macrophages. The MKK3/6-p38γ/δ pathway mediated an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase, which in turn promoted eEF2 activation (dephosphorylation) and subsequent TNF-α elongation. These results identify a new signaling pathway that regulates TNF-α production in LPS-induced liver damage and suggest potential cell-specific therapeutic targets for liver diseases in which TNF-α production is involved. PMID:23202732

18β-glycyrrhetinic acid inhibits migration and invasion of human gastric cancer cells via the ROS/PKC-α/ERK pathway.

PubMed

Cai, Hongke; Chen, Xi; Zhang, Jianbo; Wang, Jijian

2018-01-01

18β-glycyrrhetinic acid (18β-GA) is a bioactive component of licorice root which exerts pharmacological activities including anti-inflammatory, antiviral, anti-oxidative and anti-cancer effects. The current study further investigated the molecular mechanisms associated with the inhibitory effects of 18β-GA on tumor metastasis in human gastric cancer cells. The results indicated that 18β-GA significantly reduced invasion and migration activities and suppressed MMP-2 and 9 activities on SGC-7901cells in a dose-dependent manner. Further study showed 18β-GA upregulated E-cadherin expression but downregulated vimentin expression. The results also showed that 18β-GA inhibited ROS formation, PKC-α expression and the phosphorylation of ERK in a dose-dependent manner. In conclusion, this study revealed that 18β-GA inhibits migration and invasion via the ROS/PKC-α/ERK signaling pathway in gastric cancer cells. This suggests that 18β-GA has the potential to be used as an effective chemopreventive agent for the prevention of gastric cancer metastasis.

GPER-independent effects of estrogen in rat aortic vascular endothelial cells.

PubMed

Ding, Q; Hussain, Y; Chorazyczewski, J; Gros, R; Feldman, R D

2015-01-05

GPER (aka GPR30) has been identified as an important mechanism by which estrogen mediates its effects. Previous studies from our laboratories and those of others have demonstrated that GPER activation mediates a range of vascular contractile and growth regulatory responses. However, the importance of GPER in mediating the actions of estradiol (E2) in rat aortic endothelial cells is unclear. Therefore, we sought to determine the importance of GPER vs. the "classical" estrogen receptor (ER) in mediating the endothelial growth regulatory effects of E2. To do this we assessed the effect of E2 in regulating phosphoERK content and apoptotic rates in rat aortic endothelial cells and the role of GPER in mediating these effects. E2 mediated a concentration-dependent inhibition of both ERK phosphorylation and serum deprivation-induced apoptosis with a maximal effect at a concentration of 10 nM. Pretreatment with the ER antagonist ICI 182780 abolished E2-mediated inhibition of both ERK phosphorylation and apoptosis. In contrast, pretreatment with GPER antagonist G15 had no significant effect on E2-mediated inhibition of ERK phosphorylation or on apoptosis. Further, downregulation of GPER expression with a GPER shRNA adenovirus did not block E2-mediated inhibitory effects on ERK phosphorylation and apoptosis. In fact, these inhibitory effects of E2 were further enhanced by GPER downregulation. Downregulation of ERα expression reversed the E2-mediated inhibitory effects to stimulatory effects. E2's phosphoERK and apoptosis stimulatory effects seen with ERα downregulation are attenuated by pretreatment with G15. In conclusion, in rat aortic endothelial cells, E2-mediated endothelial effects are predominantly driven by ER and not by GPER. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

[Effects of electromagnetic radiation on RAF/MEK/ERK signaling pathway in rats hippocampus].

PubMed

Zuo, Hong-yan; Wang, De-wen; Peng, Rui-yun; Wang, Shui-ming; Gao, Ya-bing; Xu, Xin-ping; Ma, Jun-Jie

2009-05-01

To study the development of changes for signaling molecules related to Raf/MEK/ERK pathway in hippocampus of rats after electromagnetic radiation, and investigate the mechanisms of radiation injury. Rats were exposed to X-HPM, S-HPM and EMP radiation source respectively, and animal model of electromagnetic radiation was established. Western blot was used to detect the expression of Raf-1, phosphorylated Raf-1 and phospholylated ERK. The expression of Raf-1 down-regulated during 6 h-14 d after radiation, most significantly at 7 d, and recovered at 28 d. There was no significant difference between the radiation groups. The expression of phosphorylated Raf-1 and phosphorylated ERK both up-regulated at 6 h and 7 d after radiation, more significantly at 6 h, and the two microwave groups were more serious for phosphorylated ERK. During 6 h-14 d after S-HPM radiation, the expression of phosphorylated Raf-1 increased continuously, but phosphorylated ERK changed wavily, 6 h and 7 d were expression peak. Raf/MEK/ERK signaling pathway participates in the hippocampus injury induced by electromagnetic radiation. The excessive activation of ERK pathway may result in the apoptosis and death of neurons, which is the important mechanism of recognition disfunction caused by electromagnetic radiation.

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montes, Daniela K.; Brenet, Marianne; Muñoz, Vanessa C.

Highlights: •AVP induces mTOR phosphorylation in A-10 cells cultured in high glucose concentration. •The mTOR phosphorylation is mediated by the PI3K/Akt pathway activation. •The AVP-induced mTOR phosphorylation inhibited autophagy and stimulated cell proliferation. -- Abstract: Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. Themore » AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.« less

E-Cadherin Antagonizes Transforming Growth Factor β1 Gene Induction in Hepatic Stellate Cells by Inhibiting RhoA–Dependent Smad3 Phosphorylation

PubMed Central

Cho, Il Je; Kim, Young Woo; Han, Chang Yeob; Kim, Eun Hyun; Anderson, Richard A.; Lee, Young Sok; Lee, Chang Ho; Hwang, Se Jin; Kim, Sang Geon

2011-01-01

Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fibrosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECAD’s potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1-inducible or Smad3-inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Conclusion Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFβ1 and its target genes, and facilitates liver fibrosis. PMID:20890948

E-cadherin antagonizes transforming growth factor β1 gene induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation.

PubMed

Cho, Il Je; Kim, Young Woo; Han, Chang Yeob; Kim, Eun Hyun; Anderson, Richard A; Lee, Young Sok; Lee, Chang Ho; Hwang, Se Jin; Kim, Sang Geon

2010-12-01

Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fibrosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECAD's potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1-inducible or Smad3-inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFβ1 and its target genes, and facilitates liver fibrosis. Copyright © 2010 American Association for the Study of Liver Diseases.

The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway.

PubMed

Vasilcanu, Daiana; Girnita, Ada; Girnita, Leonard; Vasilcanu, Radu; Axelson, Magnus; Larsson, Olle

2004-10-14

The insulin-like growth factor-1 receptor (IGF-1R) is crucial for many functions in neoplastic cells, for example, antiapoptosis. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. PPP preferentially reduced phosphorylated Akt, as compared to phosphorylated Erk1/2, and caused apoptosis. Now, we aimed to investigate how PPP inhibits the IGF-1R tyrosine kinase (IGF-1RTK) and the PI3K/Akt apoptotic pathway. Using a baculovirus driven IGF-1RTK we found that PPP interfered with tyrosine phosphorylation in the activation loop of the kinase domain. Specifically, it blocked phosphorylation of tyrosine (Y) 1136, while sparing the two others (Y1131 and Y1135). To explore the impact of inhibition of Y1136 on Akt phosphorylation we transfected P6 cells (overexpressing IGF-1R) and malignant melanoma cells with different IGF-1R mutants, including Y1136F (tyrosine replaced by phenylalanine). Y1136F was found to strongly decrease IGF-1 stimulated phosphorylation of Akt. Conversely, Akt phosphorylation was weakly affected in the Y1131F transfectant. Taken together, our data suggest that the preferential inhibition of phosphorylated Akt, after PPP treatment, may be due to specific inhibition of Y1136. PPP was proven not to interfere directly with Akt or any of its downstream molecules in the apoptotic pathway.

Porcine reproductive and respiratory syndrome virus infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff.

PubMed

Li, Yang; Fang, Liurong; Zhou, Yanrong; Tao, Ran; Wang, Dang; Xiao, Shaobo

2018-06-13

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has caused tremendous economic losses in the global swine industry since it was discovered in the late 1980s. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread. Here, we demonstrate that PRRSV infection causes host translation suppression, which is strongly dependent on viral replication. By screening PRRSV-encoded nonstructural proteins (nsps), we found that nsp2 participates in the induction of host translation shutoff and that its transmembrane (TM) domain is required for this process. Nsp2-induced translation suppression is independent of protein degradation pathways and the phosphorylation of eukaryotic initiation factor 2α (eIF2α). However, the overexpression of nsp2 or its TM domain significantly attenuated the mammalian target of rapamycin (mTOR) signaling pathway, an alternative pathway for modulating host gene expression. PRRSV infection also attenuated the mTOR signaling pathway, and PRRSV-induced host translation shutoff could be partly reversed when the attenuated mTOR phosphorylation was reactivated by an activator of the mTOR pathway. PRRSV infection still negatively regulated the host translation when the effects of eIF2α phosphorylation were completely reversed. Taken together, our results demonstrate that PRRSV infection induces host translation shutoff and that nsp2 is associated with this process. Both eIF2α phosphorylation and the attenuation of the mTOR signaling pathway contribute to PRRSV-induced host translation arrest. IMPORTANCE Viruses are obligate parasites, and the production of progeny viruses relies strictly on the host translation machinery. Therefore, the efficient modulation of host mRNA translation benefits viral replication, spread, and evolution. In this study, we provide evidence that porcine reproductive and respiratory syndrome virus (PRRSV) infection induces host translation shutoff and that the viral nonstructural protein nsp2 is associated with this process. Many viruses induce host translation shutoff by phosphorylating eukaryotic initiation factor 2α (eIF2α). However, PRRSV nsp2 does not induce eIF2α phosphorylation but attenuates the mTOR signaling pathway, another pathway regulating the host cell translational machinery. We also found that PRRSV-induced host translation shutoff was partly reversed by dephosphorylating eIF2α or reactivating the mTOR pathway, indicating that PRRSV infection induces both eIF2α-phosphorylation-dependent and -independent host translation shutoff. Copyright © 2018 American Society for Microbiology.

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types. Images PMID:8388543

Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice

PubMed Central

Tomimatsu, Nozomi; Mukherjee, Bipasha; Hardebeck, Molly Catherine; Ilcheva, Mariya; Camacho, Cristel Vanessa; Harris, Janelle Louise; Porteus, Matthew; Llorente, Bertrand; Khanna, Kum Kum; Burma, Sandeep

2014-01-01

Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. While CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival, and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favor HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In sum, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice. PMID:24705021

Computer-Aided Drug Design Applied to Marine Drug Discovery: Meridianins as Alzheimer's Disease Therapeutic Agents.

PubMed

Llorach-Pares, Laura; Nonell-Canals, Alfons; Sanchez-Martinez, Melchor; Avila, Conxita

2017-11-27

Computer-aided drug discovery/design (CADD) techniques allow the identification of natural products that are capable of modulating protein functions in pathogenesis-related pathways, constituting one of the most promising lines followed in drug discovery. In this paper, we computationally evaluated and reported the inhibitory activity found in meridianins A-G, a group of marine indole alkaloids isolated from the marine tunicate Aplidium , against various protein kinases involved in Alzheimer's disease (AD), a neurodegenerative pathology characterized by the presence of neurofibrillary tangles (NFT). Balance splitting between tau kinase and phosphate activities caused tau hyperphosphorylation and, thereby, its aggregation and NTF formation. Inhibition of specific kinases involved in its phosphorylation pathway could be one of the key strategies to reverse tau hyperphosphorylation and would represent an approach to develop drugs to palliate AD symptoms. Meridianins bind to the adenosine triphosphate (ATP) binding site of certain protein kinases, acting as ATP competitive inhibitors. These compounds show very promising scaffolds to design new drugs against AD, which could act over tau protein kinases Glycogen synthetase kinase-3 Beta (GSK3β) and Casein kinase 1 delta (CK1δ, CK1D or KC1D), and dual specificity kinases as dual specificity tyrosine phosphorylation regulated kinase 1 (DYRK1A) and cdc2-like kinases (CLK1). This work is aimed to highlight the role of CADD techniques in marine drug discovery and to provide precise information regarding the binding mode and strength of meridianins against several protein kinases that could help in the future development of anti-AD drugs.

A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

PubMed

Belmont, Judson; Gu, Tao; Mudd, Ashley; Salomon, Arthur R

2017-08-04

Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca 2+ signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr 192 phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck's targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr 192 phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd.

Uncaria rhynchophylla inhibits the production of nitric oxide and interleukin-1β through blocking nuclear factor κB, Akt, and mitogen-activated protein kinase activation in macrophages.

PubMed

Kim, Ji-Hee; Bae, Chang Hwan; Park, Sun Young; Lee, Sang Joon; Kim, YoungHee

2010-10-01

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition.

PubMed

Thakur, Meghna; Seo, Eun Joo; Dever, Thomas E

2014-02-01

Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.

Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

PubMed

Shemon, Anne N; Eves, Eva M; Clark, Matthew C; Heil, Gary; Granovsky, Alexey; Zeng, Lingchun; Imamoto, Akira; Koide, Shohei; Rosner, Marsha Rich

2009-06-24

Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

Growth-inhibitory and antiangiogenic activity of the MEK inhibitor PD0325901 in malignant melanoma with or without BRAF mutations.

PubMed

Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

2009-08-01

The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC(50) in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G(1)-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27(KIP1)) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations12

PubMed Central

Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

2009-01-01

The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma. PMID:19649202

Dual mechanisms of NF-kappaB inhibition in carnosol-treated endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lian, K.-C.; Chuang, J.-J.; Hsieh, C.-W.

2010-05-15

The increased adhesion of monocytes to injured endothelial layers is a critical early event in atherogenesis. Under inflammatory conditions, there is increased expression of specific cell adhesion molecules on activated vascular endothelial cells, which increases monocyte adhesion. In our current study, we demonstrate a putative mechanism for the anti-inflammatory effects of carnosol, a diterpene derived from the herb rosemary. Our results show that both carnosol and rosemary essential oils inhibit the adhesion of TNFalpha-induced monocytes to endothelial cells and suppress the expression of ICAM-1 at the transcriptional level. Moreover, carnosol was found to exert its inhibitory effects by blocking themore » degradation of the inhibitory protein IkappaBalpha in short term pretreatments but not in 12 h pretreatments. Our data show that carnosol reduces IKK-beta phosphorylation in pretreatments of less than 3 h. In TNFalpha-treated ECs, NF-kappaB nuclear translocation and transcriptional activity was abolished by up to 12 h of carnosol pretreatment and this was blocked by Nrf-2 siRNA. The long-term inhibitory effects of carnosol thus appear to be mediated through its induction of Nrf-2-related genes. The inhibition of ICAM-1 expression and p65 translocation is reversed by HO-1 siRNA. Carnosol also upregulates the Nrf-2-related glutathione synthase gene and thereby increases the GSH levels after 9 h of exposure. Treating ECs with a GSH synthesis inhibitor, BSO, blocks the inhibitory effects of carnosol. In addition, carnosol increases p65 glutathionylation. Hence, our present findings indicate that carnosol suppresses TNFalpha-induced singling pathways through the inhibition of IKK-beta activity or the upregulation of HO-1 expression. The resulting GSH levels are dependent, however, on the length of the carnosol pretreatment period.« less

The mammalian target of rapamycin signaling pathway regulates myocyte enhancer factor-2C phosphorylation levels through integrin-linked kinase in goat skeletal muscle satellite cells.

PubMed

Wu, Haiqing; Ren, Yu; Pan, Wei; Dong, Zhenguo; Cang, Ming; Liu, Dongjun

2015-11-01

Mammalian target of rapamycin (mTOR) signaling pathway plays a key role in muscle development and is involved in multiple intracellular signaling pathways. Myocyte enhancer factor-2 (MEF2) regulates muscle cell proliferation and differentiation. However, how the mTOR signaling pathway regulates MEF2 activity remains unclear. We isolated goat skeletal muscle satellite cells (gSSCs) as model cells to explore mTOR signaling pathway regulation of MEF2C. We inhibited mTOR activity in gSSCs with PP242 and found that MEF2C phosphorylation was decreased and that muscle creatine kinase (MCK) expression was suppressed. Subsequently, we detected integrin-linked kinase (ILK) using MEF2C coimmunoprecipitation; ILK and MEF2C were colocalized in the gSSCs. We found that inhibiting mTOR activity increased ILK phosphorylation levels and that inhibiting ILK activity with Cpd 22 and knocking down ILK with small interfering RNA increased MEF2C phosphorylation and MCK expression. In the presence of Cpd 22, mTOR activity inhibition did not affect MEF2C phosphorylation. Moreover, ILK dephosphorylated MEF2C in vitro. These results suggest that the mTOR signaling pathway regulates MEF2C positively and regulates ILK negatively and that ILK regulates MEF2C negatively. It appears that the mTOR signaling pathway regulates MEF2C through ILK, further regulating the expression of muscle-related genes in gSSCs. © 2015 International Federation for Cell Biology.

Effects of HCM cTnI Mutation R145G on Troponin Structure and Modulation by PKA Phosphorylation Elucidated by Molecular Dynamics Simulations

PubMed Central

Lindert, Steffen; Cheng, Yuanhua; Kekenes-Huskey, Peter; Regnier, Michael; McCammon, J. Andrew

2015-01-01

Cardiac troponin (cTn) is a key molecule in the regulation of human cardiac muscle contraction. The N-terminal cardiac-specific peptide of the inhibitory subunit of troponin, cTnI (cTnI1-39), is a target for phosphorylation by protein kinase A (PKA) during β-adrenergic stimulation. We recently presented evidence indicating that this peptide interacts with the inhibitory peptide (cTnl137–147) when S23 and S24 are phosphorylated. The inhibitory peptide is also the target of the point mutation cTnI-R145G, which is associated with hypertrophic cardiomyopathy (HCM), a disease associated with sudden death in apparently healthy young adults. It has been shown that both phosphorylation and this mutation alter the cTnC-cTnI (C-I) interaction, which plays a crucial role in modulating contractile activation. However, little is known about the molecular-level events underlying this modulation. Here, we computationally investigated the effects of the cTnI-R145G mutation on the dynamics of cTn, cTnC Ca2+ handling, and the C-I interaction. Comparisons were made with the cTnI-R145G/S23D/S24D phosphomimic mutation, which has been used both experimentally and computationally to study the cTnI N-terminal specific effects of PKA phosphorylation. Additional comparisons between the phosphomimic mutations and the real phosphorylations were made. For this purpose, we ran triplicate 150 ns molecular dynamics simulations of cTnI-R145G Ca2+-bound cTnC1-161-cTnI1-172-cTnT236-285, cTnI-R145G/S23D/S24D Ca2+-bound cTnC1-161-cTnI1-172-cTnT236-285, and cTnI-R145G/PS23/PS24 Ca2+-bound cTnC1-161-cTnI1-172-cTnT236-285, respectively. We found that the cTnI-R145G mutation did not impact the overall dynamics of cTn, but stabilized crucial Ca2+-coordinating interactions. However, the phosphomimic mutations increased overall cTn fluctuations and destabilized Ca2+ coordination. Interestingly, cTnI-R145G blunted the intrasubunit interactions between the cTnI N-terminal extension and the cTnI inhibitory peptide, which have been suggested to play a crucial role in modulating troponin function during β-adrenergic stimulation. These findings offer a molecular-level explanation for how the HCM mutation cTnI-R145G reduces the modulation of cTn by phosphorylation of S23/S24 during β-adrenergic stimulation. PMID:25606687

Effects of HCM cTnI mutation R145G on troponin structure and modulation by PKA phosphorylation elucidated by molecular dynamics simulations.

PubMed

Lindert, Steffen; Cheng, Yuanhua; Kekenes-Huskey, Peter; Regnier, Michael; McCammon, J Andrew

2015-01-20

Cardiac troponin (cTn) is a key molecule in the regulation of human cardiac muscle contraction. The N-terminal cardiac-specific peptide of the inhibitory subunit of troponin, cTnI (cTnI(1-39)), is a target for phosphorylation by protein kinase A (PKA) during β-adrenergic stimulation. We recently presented evidence indicating that this peptide interacts with the inhibitory peptide (cTnl(137-147)) when S23 and S24 are phosphorylated. The inhibitory peptide is also the target of the point mutation cTnI-R145G, which is associated with hypertrophic cardiomyopathy (HCM), a disease associated with sudden death in apparently healthy young adults. It has been shown that both phosphorylation and this mutation alter the cTnC-cTnI (C-I) interaction, which plays a crucial role in modulating contractile activation. However, little is known about the molecular-level events underlying this modulation. Here, we computationally investigated the effects of the cTnI-R145G mutation on the dynamics of cTn, cTnC Ca(2+) handling, and the C-I interaction. Comparisons were made with the cTnI-R145G/S23D/S24D phosphomimic mutation, which has been used both experimentally and computationally to study the cTnI N-terminal specific effects of PKA phosphorylation. Additional comparisons between the phosphomimic mutations and the real phosphorylations were made. For this purpose, we ran triplicate 150 ns molecular dynamics simulations of cTnI-R145G Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), cTnI-R145G/S23D/S24D Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), and cTnI-R145G/PS23/PS24 Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), respectively. We found that the cTnI-R145G mutation did not impact the overall dynamics of cTn, but stabilized crucial Ca(2+)-coordinating interactions. However, the phosphomimic mutations increased overall cTn fluctuations and destabilized Ca(2+) coordination. Interestingly, cTnI-R145G blunted the intrasubunit interactions between the cTnI N-terminal extension and the cTnI inhibitory peptide, which have been suggested to play a crucial role in modulating troponin function during β-adrenergic stimulation. These findings offer a molecular-level explanation for how the HCM mutation cTnI-R145G reduces the modulation of cTn by phosphorylation of S23/S24 during β-adrenergic stimulation. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The inhibitory mechanism of Cordyceps sinensis on cigarette smoke extract-induced senescence in human bronchial epithelial cells.

PubMed

Liu, Ailing; Wu, Jinxiang; Li, Aijun; Bi, Wenxiang; Liu, Tian; Cao, Liuzhao; Liu, Yahui; Dong, Liang

2016-01-01

Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or stress. The bronchial epithelial cell is often injured by inhaled toxic substances, such as cigarette smoke. In the present study, we investigated whether exposure to cigarette smoke extract (CSE) induces senescence of bronchial epithelial cells; and Cordyceps sinensis mechanism of inhibition of CSE-induced cellular senescence. Human bronchial epithelial cells (16HBE cells) cultured in vitro were treated with CSE and/or C. sinensis. p16, p21, and senescence-associated-galactosidase activity were used to detect cellular senescence with immunofluorescence, quantitative polymerase chain reaction, and Western blotting. Reactive oxygen species (ROS), PI3K/AKT/mTOR and their phosphorylated proteins were examined to testify the activation of signaling pathway by ROS fluorescent staining and Western blotting. Then, inhibitors of ROS and PI3K were used to further confirm the function of this pathway. Cellular senescence was upregulated by CSE treatment, and C. sinensis can decrease CSE-induced cellular senescence. Activation of ROS/PI3K/AKT/mTOR signaling pathway was enhanced by CSE treatment, and decreased when C. sinensis was added. Blocking ROS/PI3K/AKT/mTOR signaling pathway can attenuate CSE-induced cellular senescence. CSE can induce cellular senescence in human bronchial epithelial cells, and ROS/PI3K/AKT/mTOR signaling pathway may play an important role in this process. C. sinensis can inhibit the CSE-induced senescence.

Communication Between the Calcium and cAMP Pathways Regulate the Expression of the TSH Receptor: TRPC2 in the Center of Action

PubMed Central

Löf, Christoffer; Sukumaran, Pramod; Viitanen, Tero; Vainio, Minna; Kemppainen, Kati; Pulli, Ilari; Näsman, Johnny; Kukkonen, Jyrki P.

2012-01-01

Transient receptor potential (TRP) cation channels are widely expressed and function in many physiologically important processes. Perturbations in the expression or mutations of the channels have implications for diseases. Many thyroid disorders, as excessive growth or disturbed thyroid hormone production, can be a result of dysregulated TSH signaling. In the present study, we found that of TRP canonicals (TRPCs), only TRPC2 was expressed in Fischer rat thyroid low-serum 5% cells (FRTL-5 cells). To investigate the physiological importance of the channel, we developed stable TRPC2 knockdown cells using short hairpin RNA (shTRPC2 cells). In these cells, the ATP-evoked entry of calcium was significantly decreased. This led to increased cAMP production, because inhibitory signals from calcium to adenylate cyclase 5/6 were decreased. Enhanced cAMP signaling projected to Ras-related protein 1-MAPK kinase 1 (MAPK/ERK kinase 1) pathway leading to phosphorylation of ERK1/2. The activated ERK1/2 pathway increased the expression of the TSH receptor. In contrast, secretion of thyroglobulin was decreased in shTRPC2 cells, due to improper folding and glycosylation of the protein. We show here a novel role for TRPC2 in regulating thyroid cell function. PMID:23015753

CCN5 attenuates profibrotic phenotypes of fibroblasts through the Smad6-CCN2 pathway: Potential role in epidural fibrosis.

PubMed

Xu, Honghai; Liu, Cong; Sun, Zhengming; Guo, Xiong; Zhang, Yuelin; Liu, Mengting; Li, Peng

2015-07-01

Epidural fibrosis is characterized by the development of dense and thick scar tissue adjacent to the dural mater and ranked as the major contributor for post-operative pain recurrence after laminectomy or discectomy. Recently, CCN5 exhibited an inhibitory effect on connective tissue growth factor (CTGF)/CCN2 (a critical regulator for fibrotic disease)‑mediated fibrogenesis. However, its function in epidural fibrosis and the underlying mechanisms involved remain to be determined. In this study, an obvious downregulation of CCN5 was observed in scar tissues from laminectomized rats, concomitant with a marked upregulation of CCN2, suggesting a potential negative regulatory role of CCN5 in fibrogenesis. Furthermore, CCN5 overexpression notably mitigated transforming growth factor‑β1-enhanced fibroblast viability and proliferation. Of note, CCN5 upregulation inhibited the switch of fibroblasts into myofibroblasts as its overexpression abrogated the expression of the myofibroblast marker, α-smooth muscle actin (α-SMA). CCN5 upregulation also reduced an increase in collagen type I, α1 (COL1A1) and total collagen concentrations. Additionally, CCN5 over-expression decreased CCN2 expression and increased Smad6 phosphorylation. Mechanism analysis revealed that blocking Smad6 signaling significantly ameliorated the inhibitory effect of CCN5 on the CCN2 levels, accompanied by the reduction in cell proliferation and collagen production. These results confirm that CCN5 exerts an anti-fibrotic function by regulating the Smad6-CCN2 pathway, thereby indicating a potential approach for ameliorating epidural fibrosis after laminectomy.

Regulation of PCNA Function by Tyrosine Phosphorylation in Prostate Cancer

DTIC Science & Technology

2012-10-01

PCNA in the prostate cell lines and examine the effects on cell proliferation and in modulating response to combination chemotherapy. As shown...Figure 3. PC-3 cells were specifically sensitive to the growth inhibitory effect of the Y211F peptide. Cells were plated and treated...lysates was also determined. Refer to Figure 1 for the effect on Y211 phosphorylation in PC-3 cells. Figure 7. Inhibition of Y211

Lithium protects against methamphetamine-induced neurotoxicity in PC12 cells via Akt/GSK3β/mTOR pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Jintao; Zhu, Dexiao; Zhang, Jing

Methamphetamine (MA) is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to MA causes psychosis and increases the risk of Parkinson's disease. Lithium (Li) is a known mood stabilizer and has neuroprotective effects. Previous studies suggest that MA exposure decreases the phosphorylation of Akt/GSK3β pathway in vivo, whereas Li facilitates the phosphorylation of Akt/GSK3β pathway. Moreover, GSK3β and mTOR are implicated in the locomotor sensitization induced by psychostimulants and mTOR plays a critical role in MA induced toxicity. However, the effect of MA on Akt/GSK3β/mTOR pathway has not been fully investigated in vitro. Here, we found that MA exposure significantly dephosphorylated Akt/GSK3β/mTOR pathwaymore » in PC12 cells. In addition, Li remarkably attenuated the dephosphorylation effect of MA exposure on Akt/GSK3β/mTOR pathway. Furthermore, Li showed obvious protective effects against MA toxicity and LY294002 (Akt inhibitor) suppressed the protective effects of Li. Together, MA exposure dephosphorylates Akt/GSK3β/mTOR pathway in vitro, while lithium protects against MA-induced neurotoxicity via phosphorylation of Akt/GSK3β/mTOR pathway. - Highlights: • Lithium protects against methamphetamine-induced neurotoxicity in vitro. • Methamphetamine exposure dephosphorylates Akt/GSK3β/mTOR pathway. • Lithium attenuates methamphetamine-induced toxicity via phosphorylating Akt/GSK3β/mTOR pathway.« less

Synaptically Driven Phosphorylation of Ribosomal Protein S6 Is Differentially Regulated at Active Synapses versus Dendrites and Cell Bodies by MAPK and PI3K/mTOR Signaling Pathways

ERIC Educational Resources Information Center

Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

2017-01-01

High-frequency stimulation of the medial perforant path triggers robust phosphorylation of ribosomal protein S6 (rpS6) in activated dendritic domains and granule cell bodies. Here we dissect the signaling pathways responsible for synaptically driven rpS6 phosphorylation in the dentate gyrus using pharmacological agents to inhibit PI3-kinase/mTOR…

Pin1 promotes transforming growth factor-beta-induced migration and invasion.

PubMed

Matsuura, Isao; Chiang, Keng-Nan; Lai, Chen-Yu; He, Dongming; Wang, Guannan; Ramkumar, Romila; Uchida, Takafumi; Ryo, Akihide; Lu, Kunping; Liu, Fang

2010-01-15

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

Non-Antibody Universal Detection of Protein Phosphorylation Using pIMAGO

PubMed Central

Iliuk, Anton B.; Tao, W. Andy

2015-01-01

With recent technical advances, important signaling pathways have continuously been discovered and dissected in many biological events. The vast majority of these signaling pathways involve reversible protein phosphorylation, and the dynamics of phosphorylation provides important mechanisms on how signaling networks function and interact. With a variety of research projects using lab models or clinical samples, a simple and reliable phosphorylation assay is highly desirable for routine detection of phosphorylation in sample mixtures. The protocols in this article describe the general procedure for a new non-antibody strategy for phosphorylation assay, termed pIMAGO (phospho-imaging). This novel design takes advantage of not only the unique properties of the soluble nanoparticles, but also of the multiple functionality of the molecule, allowing for highly selective, sensitive and quantitative assessment of protein phosphorylation without the use of either radioactive isotopes or limited phosphospecific antibodies. It also offers the capability for multiplexed detection of phosphorylation and total protein amount simultaneously. The described procedures allow for straightforward and routine detection and quantitation of general phosphorylation on any site of any protein in Western Blot and ELISA formats. PMID:25727060

Cardioprotective Effects of Transfusion of Late-Phase Preconditioned Plasma May Be Induced by Activating the Reperfusion Injury Salvage Kinase Pathway but Not the Survivor Activating Factor Enhancement Pathway in Rats.

PubMed

Zhao, Yang; Zheng, Zhi-Nan; Pi, Yan-Na; Liang, Xue; Jin, San-Qing

2017-01-01

A previous study in our laboratory demonstrated that transfusion of plasma collected at the late phase of remote ischemic preconditioning (RIPC) could reduce myocardial infarct size. Here, we tested whether the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways are involved in transferring protection. In a two-part study, donor rats ( n = 3) donated plasma 48 hours after RIPC (preconditioned plasma) or control (nonpreconditioned plasma). Normal (part 1) or ischemic (part 2) myocardia were collected from recipients ( n = 6) 24 hours after receiving normal saline, nonpreconditioned plasma, and preconditioned plasma or after further suffering ischemia reperfusion. Western blot was performed to analyze STAT3, Akt, and Erk1/2 phosphorylation in normal and ischemic myocardium (central area and border area). In normal myocardia, preconditioned plasma increased Akt and Erk1/2 phosphorylation significantly compared to nonpreconditioned plasma and normal saline; no STAT3 phosphorylation was detected. In ischemic myocardia, preconditioned plasma increased Akt and Erk1/2 phosphorylation significantly in both central and border areas compared to other fluids; no significant difference in STAT3 phosphorylation occurred among groups. Transfusion of preconditioned plasma collected at the late phase of RIPC could activate the RISK but not SAFE pathway, suggesting that RISK pathway may be involved in transferring protection.

The Glycerate and Phosphorylated Pathways of Serine Synthesis in Plants: The Branches of Plant Glycolysis Linking Carbon and Nitrogen Metabolism.

PubMed

Igamberdiev, Abir U; Kleczkowski, Leszek A

2018-01-01

Serine metabolism in plants has been studied mostly in relation to photorespiration where serine is formed from two molecules of glycine. However, two other pathways of serine formation operate in plants and represent the branches of glycolysis diverging at the level of 3-phosphoglyceric acid. One branch (the glycerate - serine pathway) is initiated in the cytosol and involves glycerate formation from 3-phosphoglycerate, while the other (the phosphorylated serine pathway) operates in plastids and forms phosphohydroxypyruvate as an intermediate. Serine formed in these pathways becomes a precursor of glycine, formate and glycolate accumulating in stress conditions. The pathways can be linked to GABA shunt via transamination reactions and via participation of the same reductase for both glyoxylate and succinic semialdehyde. In this review paper we present a hypothesis of the regulation of redox balance in stressed plant cells via participation of the reactions associated with glycerate and phosphorylated serine pathways. We consider these pathways as important processes linking carbon and nitrogen metabolism and maintaining cellular redox and energy levels in stress conditions.

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

PubMed

Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2004-09-23

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Fructose-enriched diet induces inflammation and reduces antioxidative defense in visceral adipose tissue of young female rats.

PubMed

Kovačević, Sanja; Nestorov, Jelena; Matić, Gordana; Elaković, Ivana

2017-02-01

The consumption of refined, fructose-enriched food continuously increases and has been linked to development of obesity, especially in young population. Low-grade inflammation and increased oxidative stress have been implicated in the pathogenesis of obesity-related disorders including type 2 diabetes. In this study, we examined alterations in inflammation and antioxidative defense system in the visceral adipose tissue (VAT) of fructose-fed young female rats, and related them to changes in adiposity and insulin sensitivity. We examined the effects of 9-week fructose-enriched diet applied immediately after weaning on nuclear factor κB (NF-κB) intracellular distribution, and on the expression of pro-inflammatory cytokines (IL-1β and TNFα) and key antioxidative enzymes in the VAT of female rats. Insulin signaling in the VAT was evaluated at the level of insulin receptor substrate-1 (IRS-1) protein and its inhibitory phosphorylation on Ser 307 . Fructose-fed rats had increased VAT mass along with increased NF-κB nuclear accumulation and elevated IL-1β, but not TNFα expression. The protein levels of antioxidative defense enzymes, mitochondrial manganese superoxide dismutase 2, and glutathione peroxidase, were reduced, while the protein content of IRS-1 and its inhibitory phosphorylation were not altered by fructose diet. The results suggest that fructose overconsumption-related alterations in pro-inflammatory markers and antioxidative capacity in the VAT of young female rats can be implicated in the development of adiposity, but do not affect inhibitory phosphorylation of IRS-1.

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

PubMed

Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

Mechanisms involved in p53 downregulation by leptin in trophoblastic cells.

PubMed

Toro, Ayelén Rayen; Pérez-Pérez, Antonio; Corrales Gutiérrez, Isabel; Sánchez-Margalet, Víctor; Varone, Cecilia Laura

2015-11-01

Leptin, a 16-kDa polypeptide hormone, is produced by the adipocyte and can also be synthesized by placenta. We previously demonstrated that leptin promotes proliferation and survival in placenta, in part mediated by the p53 pathway. In this work, we investigated the mechanisms involved in leptin down-regulation of p53 level. The human first trimester cytotrophoblastic Swan-71 cell line and human placental explants at term were used. In order to study the late phase of apoptosis, triggered by serum deprivation, experiments of DNA fragmentation were carried out. Exogenous leptin added to human placental explants, showed a decrease on DNA ladder formation and MAPK pathway is involved in this leptin effect. We also found that under serum deprivation condition, leptin decreases p53 levels and the inhibitory leptin effect is lost when cells were pretreated with 50 μM PD98059 or 10 μM LY29004; or were transfected with dominant negative mutants of intermediates of these pathways, suggesting that MAPK and PI3K signaling pathways are necessaries for leptin action. Additionally, leptin diminished Ser-46 p53 phosphorylation and this effect in placental explants was mediated by the activation of MAPK and PI3K pathways. Finally, in order to assess leptin effect on p53 half-life experiments with cycloheximide were performed and MDM-2 expression was analyzed. Leptin diminished p53 half-life and up-regulated MDM-2 expression. In summary, we provided evidence suggesting that leptin anti-apoptotic effect is mediated by MAPK and PI3K pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

An Unbiased Approach to Identifying Tau Kinases That Phosphorylate Tau at Sites Associated with Alzheimer Disease

PubMed Central

Cavallini, Annalisa; Brewerton, Suzanne; Bell, Amanda; Sargent, Samantha; Glover, Sarah; Hardy, Clare; Moore, Roger; Calley, John; Ramachandran, Devaki; Poidinger, Michael; Karran, Eric; Davies, Peter; Hutton, Michael; Szekeres, Philip; Bose, Suchira

2013-01-01

Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3β, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3β using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies. PMID:23798682

Phosphorylation of threonine 290 in the activation loop of Tpl2/Cot is necessary but not sufficient for kinase activity.

PubMed

Luciano, Brenda S; Hsu, Sang; Channavajhala, Padma L; Lin, Lih-Ling; Cuozzo, John W

2004-12-10

Cot/Tpl2/MAP3K8 is a serine/threonine kinase known to activate the ERK, p38, and JNK kinase pathways. Studies of Tpl2 knock-out mice reveal a clear defect in tumor necrosis factor-alpha production, although very little detail is known about its regulation and the signaling events involved. In the present study we demonstrated that phosphorylation of Cot was required for its maximal activity as phosphatase treatment of Cot decreased its kinase activity. The Cot sequence contains a conserved threonine at position 290 in the activation loop of the kinase domain. We found that mutation of this residue to alanine eliminated its ability to activate MEK/ERK and NF-kappaB pathways, whereas a phosphomimetic mutation to aspartic acid could rescue the ability to activate MEK. Thr-290 was also required for robust autophosphorylation of Cot. Antibody generated to phospho-Thr-290-Cot recognized both wild-type and kinase-dead Cot, suggesting that phosphorylation of Thr-290 did not occur through autophosphorylation but via another kinase. We showed that Cot was constitutively phosphorylated at Thr-290 in transfected human embryonic kidney 293T cells as well as human monocytes as this residue was phosphorylated in unstimulated and lipopolysaccharide-stimulated cells to the same degree. Treatment with herbimycin A inhibited Cot activity in the MEK/ERK pathway but did not inhibit phosphorylation at Thr-290. Together these results showed that phosphorylation of Cot at Thr-290 is necessary but not sufficient for full kinase activity in the MEK/ERK pathway.

Amplification and Demultiplexing in Insulin-regulated Akt Protein Kinase Pathway in Adipocytes*

PubMed Central

Tan, Shi-Xiong; Ng, Yvonne; Meoli, Christopher C.; Kumar, Ansu; Khoo, Poh-Sim; Fazakerley, Daniel J.; Junutula, Jagath R.; Vali, Shireen; James, David E.; Stöckli, Jacqueline

2012-01-01

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5–22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease. PMID:22207758

Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer

PubMed Central

Vahid, Sepideh; Thaper, Daksh; Gibson, Kate F.; Bishop, Jennifer L.; Zoubeidi, Amina

2016-01-01

Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers. PMID:27555231

Molecular chaperone Hsp27 regulates the Hippo tumor suppressor pathway in cancer.

PubMed

Vahid, Sepideh; Thaper, Daksh; Gibson, Kate F; Bishop, Jennifer L; Zoubeidi, Amina

2016-08-24

Heat shock protein 27 (Hsp27) is a molecular chaperone highly expressed in aggressive cancers, where it is involved in numerous pro-tumorigenic signaling pathways. Using functional genomics we identified for the first time that Hsp27 regulates the gene signature of transcriptional co-activators YAP and TAZ, which are negatively regulated by the Hippo Tumor Suppressor pathway. The Hippo pathway inactivates YAP by phosphorylating and increasing its cytoplasmic retention with the 14.3.3 proteins. Gain and loss of function experiments in prostate, breast and lung cancer cells showed that Hsp27 knockdown induced YAP phosphorylation and cytoplasmic localization while overexpression of Hsp27 displayed opposite results. Mechanistically, Hsp27 regulates the Hippo pathway by accelerating the proteasomal degradation of ubiquitinated MST1, the core Hippo kinase, resulting in reduced phosphorylation/activity of LATS1 and MOB1, its downstream effectors. Importantly, our in vitro results were supported by data from human tumors; clinically, high expression of Hsp27 in prostate tumors is correlated with increased expression of YAP gene signature and reduced phosphorylation of YAP in lung and invasive breast cancer clinical samples. This study reveals for the first time a link between Hsp27 and the Hippo cascade, providing a novel mechanism of deregulation of this tumor suppressor pathway across multiple cancers.

Intervention of Prostate Cancer by a Flavonoid Antioxidant Silymarin

DTIC Science & Technology

2001-04-01

effect of silymarin on PCA tumor growth in nude mice. Treatment of LNCaP and DUl45 human prostate carcinoma cells with silibinin (the pure form of...dimerization. These inhibitory effects of silibinin also corroborate with its inhibitory effect on both cellular and released expression of TGFalpha in...these two cell lines. In cytoplasmic signaling, silibinin showed strong decrease in ERKl/2 phosphorylation in both LNCaP and DUl45 cells and inhibition in

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-beta induced apoptosis.

PubMed

Conery, Andrew R; Cao, Yanna; Thompson, E Aubrey; Townsend, Courtney M; Ko, Tien C; Luo, Kunxin

2004-04-01

Transforming growth factor beta (TGF-beta) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-beta is specified. Smad proteins are critical mediators of TGF-beta signalling. After stimulation by TGF-beta, Smad2 and Smad3 become phosphorylated by the activated TGF-beta receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-beta target genes. Here we report that the sensitivity to TGF-beta induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-beta induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-beta. These findings identify an important determinant of sensitivity to TGF-beta-induced apoptosis that involves crosstalk between the TGF-beta and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.

Studying the Function of the Phosphorylated Pathway of Serine Biosynthesis in Arabidopsis thaliana.

PubMed

Krueger, Stephan; Benstein, Ruben M; Wulfert, Sabine; Anoman, Armand D; Flores-Tornero, María; Ros, Roc

2017-01-01

Photorespiration is an essential pathway in photosynthetic organisms and is particularly important to detoxify and recycle 2-phosphoglycolate (2-PG), a by-product of oxygenic photosynthesis. The enzymes that catalyze the reactions in the photorespiratory core cycle and closely associated pathways have been identified; however, open questions remain concerning the metabolic network in which photorespiration is embedded. The amino acid serine represents one of the major intermediates in the photorespiratory pathway and photorespiration is thought to be the major source of serine in plants. The restriction of photorespiration to autotrophic cells raises questions concerning the source of serine in heterotrophic tissues. Recently, the phosphorylated pathway of serine biosynthesis has been found to be extremely important for plant development and metabolism. In this protocol, we describe a detailed methodological workflow to analyze the generative and vegetative phenotypes of plants deficient in the phosphorylated pathway of serine biosynthesis, which together allow a better understanding of its function in plants.

The Phosphatidylinositol 3-Kinase/Akt Pathway Regulates Transforming Growth Factor-β Signaling by Destabilizing Ski and Inducing Smad7*

PubMed Central

Band, Arja M.; Björklund, Mia; Laiho, Marikki

2009-01-01

Ski is an oncoprotein that negatively regulates transforming growth factor (TGF)-β signaling. It acts as a transcriptional co-repressor by binding to TGF-β signaling molecules, Smads. Efficient TGF-β signaling is facilitated by rapid proteasome-mediated degradation of Ski by TGF-β. Here we report that Ski is phosphorylated by Akt/PKB kinase. Akt phosphorylates Ski on a highly conserved Akt motif at threonine 458 both in vitro and in vivo. The phosphorylation of Ski at threonine 458 is induced by Akt pathway activators including insulin, insulin-like growth factor-1, and hepatocyte growth factor. The phosphorylation of Ski causes its destabilization and reduces Ski-mediated inhibition of expression of another negative regulator of TGF-β, Smad7. Induction of Smad7 levels leads to inactivation of TGF-β receptors and TGF-β signaling cascade, as indicated by reduced induction of TGF-β target p15. Therefore, Akt modulates TGF-β signaling by temporarily adjusting the levels of two TGF-β pathway negative regulators, Ski and Smad7. These novel findings demonstrate that Akt pathway activation directly impacts TGF-β pathway. PMID:19875456

Discovery of novel, high potent, ABC type PTP1B inhibitors with TCPTP selectivity and cellular activity.

PubMed

Liu, Peihong; Du, Yongli; Song, Lianhua; Shen, Jingkang; Li, Qunyi

2016-08-08

Protein tyrosine phosphatase 1B (PTP1B) as a key negative regulator of both insulin and leptin receptor pathways has been an attractive therapeutic target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. With the goal of enhancing potency and selectivity of the PTP1B inhibitors, a series of methyl salicylate derivatives as ABC type PTP1B inhibitors (P1-P7) were discovered. More importantly, compound P6 exhibited high potent inhibitory activity (IC50 = 50 nM) for PTP1B with 15-fold selectivity over T-cell PTPase (TCPTP). Further studies on cellular activities revealed that compound P6 could enhance insulin-mediated insulin receptor β (IRβ) phosphorylation and insulin-stimulated glucose uptake. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Wild-type H- and N-Ras promote mutant K-Ras driven tumorigenesis by modulating the DNA damage response

PubMed Central

Grabocka, Elda; Pylayeva-Gupta, Yuliya; Jones, Mathew JK; Lubkov, Veronica; Yemanaberhan, Eyoel; Taylor, Laura; Jeng, Hao Hsuan; Bar-Sagi, Dafna

2014-01-01

SUMMARY Mutations in KRAS are prevalent in human cancers and universally predictive of resistance to anti-cancer therapeutics. Although it is widely accepted that acquisition of an activating mutation endows RAS genes with functional autonomy, recent studies suggest that the wild-type forms of Ras may contribute to mutant Ras-driven tumorigenesis. Here we show that downregulation of wild-type H-Ras or N-Ras in mutant K-Ras cancer cells leads to hyperactivation of the Erk/p90RSK and PI3K/Akt pathways, and consequently, the phosphorylation of Chk1 at an inhibitory site, Ser 280. The resulting inhibition of ATR/Chk1 signaling abrogates the activation of the G2 DNA damage checkpoint and confers specific sensitization of mutant K-Ras cancer cells to DNA damage chemotherapeutic agents in vitro and in vivo. PMID:24525237

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed

Husain, S; Abdel-Latif, A A

1999-08-15

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle.

Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

PubMed Central

Husain, S; Abdel-Latif, A A

1999-01-01

We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle. PMID:10432304

Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

PubMed Central

Denhardt, D T

1996-01-01

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways. PMID:8836113

Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

PubMed

Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin; Xie, Yan; Zhou, Xueping

2017-08-15

Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N. benthamiana ASYMMETRIC LEAVES 1. To our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as both a VSR and a symptom determinant and to provide a mechanistic explanation of how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses. Copyright © 2017 American Society for Microbiology.

Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant

PubMed Central

Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin

2017-01-01

ABSTRACT Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana. However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N. benthamiana ASYMMETRIC LEAVES 1. To our knowledge, this is the first report establishing an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as both a VSR and a symptom determinant and to provide a mechanistic explanation of how SNF1-related protein kinase 1 acts as a host defense factor. These findings expand the scope of phosphorylation-mediated defense mechanisms and contribute to further understanding of plant defense mechanisms against geminiviruses. PMID:28539450

Ligand-independent pathway that controls stability of interferon alpha receptor

PubMed Central

Liu, Jianghuai; Plotnikov, Alexander; Banerjee, Anamika; Kumar, K.G. Suresh; Ragimbeau, Josiane; Marijanovic, Zrinka; Baker, Darren P.; Pellegrini, Sandra; Fuchs, Serge Y.

2008-01-01

SUMMARY Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance. PMID:18166147

Bone Alkaline Phosphatase and Tartrate-Resistant Acid Phosphatase: Potential Co-regulators of Bone Mineralization.

PubMed

Halling Linder, Cecilia; Ek-Rylander, Barbro; Krumpel, Michael; Norgård, Maria; Narisawa, Sonoko; Millán, José Luis; Andersson, Göran; Magnusson, Per

2017-07-01

Phosphorylated osteopontin (OPN) inhibits hydroxyapatite crystal formation and growth, and bone alkaline phosphatase (BALP) promotes extracellular mineralization via the release of inorganic phosphate from the mineralization inhibitor inorganic pyrophosphate (PPi). Tartrate-resistant acid phosphatase (TRAP), produced by osteoclasts, osteoblasts, and osteocytes, exhibits potent phosphatase activity towards OPN; however, its potential capacity as a regulator of mineralization has not previously been addressed. We compared the efficiency of BALP and TRAP towards the endogenous substrates for BALP, i.e., PPi and pyridoxal 5'-phosphate (PLP), and their impact on mineralization in vitro via dephosphorylation of bovine milk OPN. TRAP showed higher phosphatase activity towards phosphorylated OPN and PPi compared to BALP, whereas the activity of TRAP and BALP towards PLP was comparable. Bovine milk OPN could be completely dephosphorylated by TRAP, liberating all its 28 phosphates, whereas BALP dephosphorylated at most 10 phosphates. OPN, dephosphorylated by either BALP or TRAP, showed a partially or completely attenuated phosphorylation-dependent inhibitory capacity, respectively, compared to native OPN on the formation of mineralized nodules. Thus, there are phosphorylations in OPN important for inhibition of mineralization that are removed by TRAP but not by BALP. In conclusion, our data indicate that both BALP and TRAP can alleviate the inhibitory effect of OPN on mineralization, suggesting a potential role for TRAP in skeletal mineralization. Further studies are warranted to explore the possible physiological relevance of TRAP in bone mineralization.

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed Central

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-01-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets. PMID:10947961

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines

PubMed Central

Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

2014-01-01

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways. PMID:25486532

Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

PubMed

Li, Chang-Ling; Yang, Jin-Gang; Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

2014-01-01

Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.

Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

PubMed Central

Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

2016-01-01

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. DOI: http://dx.doi.org/10.7554/eLife.15460.001 PMID:27304076

SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways.

PubMed

Chen, Shuliang; Bonifati, Serena; Qin, Zhihua; St Gelais, Corine; Kodigepalli, Karthik M; Barrett, Bradley S; Kim, Sun Hee; Antonucci, Jenna M; Ladner, Katherine J; Buzovetsky, Olga; Knecht, Kirsten M; Xiong, Yong; Yount, Jacob S; Guttridge, Denis C; Santiago, Mario L; Wu, Li

2018-04-17

Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from SAMHD1 knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.

AP39, a mitochondria‐targeting hydrogen sulfide (H2S) donor, protects against myocardial reperfusion injury independently of salvage kinase signalling

PubMed Central

Karwi, Qutuba G; Bornbaum, Julia; Boengler, Kerstin; Torregrossa, Roberta; Whiteman, Matthew; Wood, Mark E; Schulz, Rainer

2017-01-01

Background and Purpose H2S protects myocardium against ischaemia/reperfusion injury. This protection may involve the cytosolic reperfusion injury salvage kinase (RISK) pathway, but direct effects on mitochondrial function are possible. Here, we investigated the potential cardioprotective effect of a mitochondria‐specific H2S donor, AP39, at reperfusion against ischaemia/reperfusion injury. Experimental Approach Anaesthetized rats underwent myocardial ischaemia (30 min)/reperfusion (120 min) with randomization to receive interventions before reperfusion: vehicle, AP39 (0.01, 0.1, 1 μmol·kg−1), or control compounds AP219 and ADT‐OH (1 μmol·kg−1). LY294002, L‐NAME or ODQ were used to investigate the involvement of the RISK pathway. Myocardial samples harvested 5 min after reperfusion were analysed for RISK protein phosphorylation and isolated cardiac mitochondria were used to examine the direct mitochondrial effects of AP39. Key Results AP39, dose‐dependently, reduced infarct size. Inhibition of either PI3K/Akt, eNOS or sGC did not affect this effect of AP39. Western blot analysis confirmed that AP39 did not induce phosphorylation of Akt, eNOS, GSK‐3β or ERK1/2. In isolated subsarcolemmal and interfibrillar mitochondria, AP39 significantly attenuated mitochondrial ROS generation without affecting respiratory complexes I or II. Furthermore, AP39 inhibited mitochondrial permeability transition pore (PTP) opening and co‐incubation of mitochondria with AP39 and cyclosporine A induced an additive inhibitory effect on the PTP. Conclusion and Implications AP39 protects against reperfusion injury independently of the cytosolic RISK pathway. This cardioprotective effect could be mediated by inhibiting PTP via a cyclophilin D‐independent mechanism. Thus, selective delivery of H2S to mitochondria may be therapeutically applicable for employing the cardioprotective utility of H2S. PMID:27930802

Cell-free extracts of Propionibacterium acnes stimulate cytokine production through activation of p38 MAPK and Toll-like receptor in SZ95 sebocytes.

PubMed

Huang, Yu-Chun; Yang, Chao-Hsun; Li, Ting-Ting; Zouboulis, Christos C; Hsu, Han-Chi

2015-10-15

Propionibacterium acnes has been considered to influence the acne lesions. The present study intended to elucidate the underlying signaling pathways of P. acnes in human sebaceous gland cells relative to the generation of proinflammatory cytokines. Cell-free extracts of P. acnes under stationary growth phase were co-incubated with human immortalized SZ95 sebocytes. Then, cell-free P. acnes extracts-induced cytokine expression was evaluated by measuring mRNA and protein levels using quantitative RT-PCR and ELISA. Changes of phosphorylated cell signaling proteins and transcription factors were measured by Western blots and Milliplex assay. The interactive molecular mechanisms of P. acnes and sebocytes were examined through use of shRNA and the specific inhibitors of signaling pathways. Cell-free extracts of P. acnes significantly stimulated secretion of interleukin (IL)-8 and IL-6 in SZ95 sebocytes. The degradation of IκB-α and increased phosphorylation of IκB-α, p38 mitogen activated protein kinase (MAPK), CREB, and STAT3 were demonstrated. Quantitative RT-PCR measurements revealed that gene expression of IL-8 and Toll-like receptor 2 (TLR2) was enhanced by cell-free extracts of P. acnes. In addition, the NF-κB inhibitor BMS345541, p38 MAPK inhibitor SB203580, or anti-TLR2 neutralizing antibody prevented cell-free P. acnes extracts-induced secretion of IL-8. Knockdown of TLR2 using shRNA exerted similar inhibitory effects on IL-8 expression. Moreover, inhibition of STAT3 activity by STA-21 enhanced P. acnes-mediated secretion of IL-8. Cell-free extracts of P. acnes are capable to activate NF-κB and p38 MAPK pathways and up-regulate secretion of IL-8 through TLR2-dependent signaling in human SZ95 sebocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Coprinus comatus Cap Inhibits Adipocyte Differentiation via Regulation of PPARγ and Akt Signaling Pathway

PubMed Central

Jang, Sun-Hee; Kang, Suk Nam; Jeon, Beong-Sam; Ko, Yeoung-Gyu; Kim, Hong-Duck; Won, Chung-Kil; Kim, Gon-Sup; Cho, Jae-Hyeon

2014-01-01

This study assessed the effects of Coprinus comatus cap (CCC) on adipogenesis in 3T3-L1 adipocytes and the effects of CCC on the development of diet-induced obesity in rats. Here, we showed that the CCC has an inhibitory effect on the adipocyte differentiation of 3T3-L1 cells, resulting in a significant decrease in lipid accumulation through the downregulation of several adipocyte specific-transcription factors, including CCAAT/enhancer binding protein β, C/EBPδ, and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, treatment with CCC during adipocyte differentiation induced a significant down-regulation of PPARγ and adipogenic target genes, including adipocyte protein 2, lipoprotein lipase, and adiponectin. Interestingly, the CCC treatment of the 3T3-L1 adipocytes suppressed the insulin-stimulated Akt and GSK3β phosphorylation, and these effects were stronger in the presence of an inhibitor of Akt phosphorylation, LY294002, suggesting that CCC inhibited adipocyte differentiation through the down-regulation of Akt signaling. In the animal study, CCC administration significantly reduced the body weight and adipose tissue weight of rats fed a high fat diet (HFD) and attenuated lipid accumulation in the adipose tissues of the HFD-induced obese rats. The size of the adipocyte in the epididymal fat of the CCC fed rats was significantly smaller than in the HFD rats. CCC treatment significantly reduced the total cholesterol and triglyceride levels in the serum of HFD rats. These results strongly indicated that the CCC-mediated decrease in body weight was due to a reduction in adipose tissue mass. The expression level of PPARγ and phospho-Akt was significantly lower in the CCC-treated HFD rats than that in the HFD obesity rats. These results suggested that CCC inhibited adipocyte differentiation by the down-regulation of major transcription factor involved in the adipogenesis pathway including PPARγ through the regulation of the Akt pathway in 3T3-L1 cells and HFD adipose tissue. PMID:25181477

Arctigenin exerts anti-colitis efficacy through inhibiting the differentiation of Th1 and Th17 cells via an mTORC1-dependent pathway.

PubMed

Wu, Xin; Dou, Yannong; Yang, Yan; Bian, Difei; Luo, Jinque; Tong, Bei; Xia, Yufeng; Dai, Yue

2015-08-15

Arctigenin, the main effective constituent of Arctium lappa L. fruit, has previously been proven to dramatically attenuate dextran sulfate sodium (DSS)-induced colitis in mice, a frequently used animal model of inflammatory bowel disease (IBD). As Th1 and Th17 cells play a crucial role in the pathogenesis of IBD, the present study addressed whether and how arctigenin exerted anti-colitis efficacy by interfering with the differentiation and activation of Th1/Th17 cells. In vitro, arctigenin was shown to markedly inhibit the differentiation of Th17 cells from naïve T cells, and moderately inhibit the differentiation of Th1 cells, which was accompanied by lowered phosphorylation of STAT3 and STAT4, respectively. In contrast, arctigenin was lack of marked effect on the differentiation of either Th2 or regulatory T cells. Furthermore, arctigenin was shown to suppress the mammalian target of rapamycin complex 1 (mTORC1) pathway in T cells as demonstrated by down-regulated phosphorylation of the downstream target genes p70S6K and RPS6, and it functioned independent of two well-known upstream kinases PI3K/AKT and ERK. Arctigenin was also able to inhibit the activity of mTORC1 by dissociating raptor from mTOR. Interestingly, the inhibitory effect of arctigenin on T cell differentiation disappeared under a status of mTORC1 overactivation via knockdown of tuberous sclerosis complex 2 (TSC2, a negative regulator of mTORC1) or pretreatment of leucine (an agonist of mTOR). In DSS-induced mice, the inhibition of Th1/Th17 responses and anti-colitis effect of arctigenin were abrogated by leucine treatment. In conclusion, arctigenin ameliorates colitis through down-regulating the differentiation of Th1 and Th17 cells via mTORC1 pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

A genetic and developmental pathway from STAT3 to the OCT4–NANOG circuit is essential for maintenance of ICM lineages in vivo

PubMed Central

Do, Dang Vinh; Ueda, Jun; Messerschmidt, Daniel M.; Lorthongpanich, Chanchao; Zhou, Yi; Feng, Bo; Guo, Guoji; Lin, Peiyu J.; Hossain, Md Zakir; Zhang, Wenjun; Moh, Akira; Wu, Qiang; Robson, Paul; Ng, Huck Hui; Poellinger, Lorenz; Knowles, Barbara B.; Solter, Davor; Fu, Xin-Yuan

2013-01-01

Although it is known that OCT4–NANOG are required for maintenance of pluripotent cells in vitro, the upstream signals that regulate this circuit during early development in vivo have not been identified. Here we demonstrate, for the first time, signal transducers and activators of transcription 3 (STAT3)-dependent regulation of the OCT4–NANOG circuitry necessary to maintain the pluripotent inner cell mass (ICM), the source of in vitro-derived embryonic stem cells (ESCs). We show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the four-cell and later stage embryos. Using leukemia inhibitory factor (Lif)-null embryos, we found that STAT3 phosphorylation is dependent on LIF in four-cell stage embryos. In blastocysts, interleukin 6 (IL-6) acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by janus kinase 1 (JAK1), a LIF- and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm formation. Indeed, STAT3 directly binds to the Oct4 and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem cells. These results provide a novel genetic model of cell fate determination operating through STAT3 in the preimplantation embryo and pluripotent stem cells in vivo. PMID:23788624

Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells.

PubMed

Sands, William A; Woolson, Hayley D; Milne, Gillian R; Rutherford, Claire; Palmer, Timothy M

2006-09-01

Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway.

PubMed

Feng, Xiuyan; Zhang, Yiqian; Shao, Ningjun; Wang, Yanhui; Zhuang, Zhizhi; Wu, Ping; Lee, Matthew J; Liu, Yingli; Wang, Xiaonan; Zhuang, Jieqiu; Delpire, Eric; Gu, Dingying; Cai, Hui

2015-05-15

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs.

Aldosterone modulates thiazide-sensitive sodium chloride cotransporter abundance via DUSP6-mediated ERK1/2 signaling pathway

PubMed Central

Feng, Xiuyan; Zhang, Yiqian; Shao, Ningjun; Wang, Yanhui; Zhuang, Zhizhi; Wu, Ping; Lee, Matthew J.; Liu, Yingli; Wang, Xiaonan; Zhuang, Jieqiu; Delpire, Eric; Gu, Dingying

2015-01-01

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs. PMID:25761881

Phosphorylation of human INO80 is involved in DNA damage tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kato, Dai; Waki, Mayumi; Umezawa, Masaki

Highlights: Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced PCNA ubiquitination. Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. Black-Right-Pointing-Pointer Western blot analyses showed phosphorylated hINO80 C-terminus. Black-Right-Pointing-Pointer Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in themore » DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.« less

Modulation of dendritic cell and T cell cross-talk during aging: The potential role of checkpoint inhibitory molecules.

PubMed

Gardner, Joanne K; Mamotte, Cyril D S; Jackaman, Connie; Nelson, Delia J

2017-09-01

Dendritic cells (DCs) undergo continuous changes throughout life, and there is evidence that elderly DCs have a reduced capacity to stimulate T cells, which may contribute to impaired anti-tumour immune responses in elderly people with cancer. Changes in checkpoint inhibitory molecules/pathways during aging may be one mechanism that impairs the ability of elderly DCs to activate T cells. However, little is currently known regarding the combined effects of aging and cancer on DC and T cell inhibitory molecules/pathways. In this review, we discuss our current understanding of the influence of aging and cancer on key DC and T cell inhibitory molecules/pathways, the potential underlying cellular and molecular mechanisms contributing to their modulation, and the possibility of therapeutically targeting inhibitory molecules in elderly cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibitory effect on HT-29 colon cancer cells of a water-soluble polysaccharide obtained from highland barley.

PubMed

Cheng, Dai; Zhang, Xinyu; Meng, Meng; Han, Lirong; Li, Caijiao; Hou, Lihua; Qi, Wentao; Wang, Chunling

2016-11-01

A water-soluble polysaccharide (BP-1) was obtained from highland barley (Hordeum vulgare L.) by hot water extraction and purification of sepharose column chromatography. BP-1 had an average molecular weight of about 6.7×10 4 Da and was composed of glucose (Glc), xylose (Xyl), arabinose (Ara) and rhamnose (Rha) with a relative molar ratio of 8.82:1.92:1.50:1.00. It was found that BP-1 inhibited proliferation of human colon cancer cells (HT-29) in a time- and dose-dependent manner with half maximal inhibitory concentration at 48h of 48.18μg/mL. Western blotting results showed that BP-1 enhanced the phosphorylation of c-Jun N-terminal kinase (JNK), processes associated with the reactive oxygen species (ROS) formation and inhibited nuclear factor-κB (NF-κB) translocation from cytoplasm into nucleus. Meanwhile, the BP-1-induced apoptosis was related to the regulation of apoptosis-associated proteins, such as B-cell lymphoma-2 (Bcl-2), release of cytochrome C from mitochondria to cytoplasm and activation of caspase-8 and caspase-9. These results suggest that BP-1-induced HT-29 apoptosis through ROS-JNK and NF-κB-mediated caspase pathways. Copyright © 2016. Published by Elsevier B.V.

Naringin induces autophagy-mediated growth inhibition by downregulating the PI3K/Akt/mTOR cascade via activation of MAPK pathways in AGS cancer cells.

PubMed

Raha, Suchismita; Yumnam, Silvia; Hong, Gyeong Eun; Lee, Ho Jeong; Saralamma, Venu Venkatarame Gowda; Park, Hyeon-Soo; Heo, Jeong Doo; Lee, Sang Joon; Kim, Eun Hee; Kim, Jin-A; Kim, Gon Sup

2015-09-01

Naringin, one of the major bioflavonoid of Citrus, has been demonstrated as potential anticancer agent. However, the underlying anticancer mechanism still needs to be explored further. This study investigated the inhibitory effect of Naringin on human AGS cancer cells. AGS cell proliferation was inhibited by Naringin in a dose- and time-dependent manner. Naringin did not induce apoptotic cell death, determined by no DNA fragmentation and the reduced Bax/Bcl-xL ratio. Growth inhibitory role of Naringin was observed by western blot analysis demonstrating downregulation of PI3K/Akt/mTOR cascade with an upregulated p21CIPI/WAFI. Formation of cytoplasmic vacuoles and autophagosomes were observed in Naringin-treated AGS cells, further confirmed by the activation of autophagic proteins Beclin 1 and LC3B with a significant phosphorylation of mitogen activated protein kinases (MAPKs). Collectively, our observed results determined that anti-proliferative activity of Naringin in AGS cancer cells is due to suppression of PI3K/Akt/mTOR cascade via induction of autophagy with activated MAPKs. Thus, the present finding suggests that Naringin induced autophagy- mediated growth inhibition shows potential as an alternative therapeutic agent for human gastric carcinoma.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id; Department of Mathematic Faculty of MIPA Universitas Gadjah Mada; Kusumo, F. A.

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present amore » mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.« less

Proteome and behavioral alterations in phosphorylation-deficient mutant Collapsin Response Mediator Protein2 knock-in mice.

PubMed

Nakamura, Haruko; Takahashi-Jitsuki, Aoi; Makihara, Hiroko; Asano, Tetsuya; Kimura, Yayoi; Nakabayashi, Jun; Yamashita, Naoya; Kawamoto, Yuko; Nakamura, Fumio; Ohshima, Toshio; Hirano, Hisashi; Tanaka, Fumiaki; Goshima, Yoshio

2018-05-11

CRMP2, alternatively designated as DPYSL2, was the first CRMP family member to be identified as an intracellular molecule mediating the signaling of the axon guidance molecule Semaphorin 3A (Sema3A). In Sema3A signaling, cyclin-dependent kinase 5 (Cdk5) primarily phosphorylates CRMP2 at Ser522. Glycogen synthase kinase-3β (GSK-3β) subsequently phosphorylates the residues of Thr509 and Thr514 of CRMP2. Previous studies showed that CRMP2 is involved in pathogenesis of neurological disorders such as Alzheimer's disease. In Alzheimer's disease, hyper-phosphorylated forms of CRMP2 are accumulated in the paired helical filaments. To get insight into the possible involvement of the phosphorylation of CRMP2 in pathogenesis of neurological disorders, we previously created CRMP2 S522A knock-in (crmp2 ki/ki ) mice and demonstrated that the phosphorylation of CRMP2 at Ser522 is involved in normal dendrite patterning in cortical neurons. However, the behavioral impact and in vivo signaling network of the CRMP2 phosphorylation are not fully understood. In this study, we performed behavioral and proteomics analysis of crmp2 ki/ki mice. The crmp2 ki/ki mice appeared healthy and showed no obvious differences in physical characteristics compared to wild-type mice, but they showed impaired emotional behavior, reduced sociality, and low sensitivity to pain stimulation. Through mass-spectrometry-based proteomic analysis, we found that 59 proteins were increased and 77 proteins were decreased in the prefrontal cortex of crmp2 ki/ki mice. Notably, CRMP3, CRMP4, and CRMP5, the other CRMP family proteins, were increased in crmp2 ki/ki mice. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses identified 14 pathways in increased total proteins and 13 pathways in decreased total proteins which are associated with the pathogenesis of Parkinson's, Alzheimer's, and Huntington's diseases. We also detected 20 pathways in increased phosphopeptides and 16 pathways in decreased phosphopeptides including "inflammatory mediator regulation of TRP channels" in crmp2 ki/ki mice. Our study suggests that the phosphorylation of CRMP2 at Ser522 is involved in the signaling pathways that may be related to neuropsychiatric and neurodegenerative diseases and pain. Copyright © 2018. Published by Elsevier Ltd.

Actin-binding and cell proliferation activities of angiomotin family members are regulated by Hippo pathway-mediated phosphorylation.

PubMed

Chan, Siew Wee; Lim, Chun Jye; Guo, Fusheng; Tan, Ivan; Leung, Thomas; Hong, Wanjin

2013-12-27

Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.

Salmonella Biofilm Development Depends on the Phosphorylation Status of RcsB

PubMed Central

Latasa, Cristina; García, Begoña; Echeverz, Maite; Toledo-Arana, Alejandro; Valle, Jaione; Campoy, Susana; García-del Portillo, Francisco; Solano, Cristina

2012-01-01

The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that Salmonella biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in Salmonella enterica serovar Typhimurium. PMID:22582278

CDK5-mediated tau accumulation triggers methamphetamine-induced neuronal apoptosis via endoplasmic reticulum-associated degradation pathway.

PubMed

Xiao, Ning; Zhang, Fu; Zhu, Bofeng; Liu, Chao; Lin, Zhoumeng; Wang, Huijun; Xie, Wei-Bing

2018-08-01

Overexposure to methamphetamine (METH) causes apoptosis in a number of cell types, particularly neuronal cells. However, the underlying mechanisms of METH-induced neuronal apoptosis remain to be elucidated. Accumulation of microtubule-associated protein Tau can lead to activation of multiple neurotoxic pathways, which is closely correlated with neuronal apoptosis. The aim of this study was to determine the role of Tau in METH-induced neuronal apoptosis. We determined the expression of two phosphorylated Tau proteins (serine 396 and threonine 231) in the human neuroblastoma SH-SY5Y cells and in the hippocampus of Sprague-Dawley rats treated with vehicle or METH using western blotting, immunohistochemical staining and immunofluorescence staining. We also measured the expression levels of the phosphorylated Tau protein, ubiquitination proteins, the intermediate products of proteasome degradation pathway, CD3-δ (a substrate of proteasome degradation pathway), endoplasmic reticulum stress signal molecule phosphorylated PERK (pPERK), and endoplasmic reticulum stress-specific apoptotic signal molecule caspase-12 in SH-SY5Y cells and in rats after inhibiting the expression of an upstream regulatory factor of phosphorylated Tau protein (CDK5) using siRNA or virus transfection. The results showed that exposure to METH significantly up-regulated the expression of phosphorylated Tau protein in vivo and in vitro and silencing the expression of CDK5 inhibited the up-regulation of phosphorylated Tau induced by METH exposure. METH exposure also significantly increased the expression of ubiquitination protein and CD3-δ and these effects were blocked by CDK5 silencing. In addition, METH exposure significantly elevated the levels of phosphorylated PERK and caspase-12 and these effects were suppressed after CDK5 silencing, which indicates that blockade of CDK5 expression can mitigate METH-induced neuronal apoptosis. These results suggest that METH can impair the endoplasmic reticulum-associated degradation (ERAD) pathway and induce neuronal apoptosis through endoplasmic reticulum stress, which is mainly mediated by abnormal CDK5-regulated Tau phosphorylation. Copyright © 2018 Elsevier B.V. All rights reserved.

Insulin-like Growth Factor 1 Rescues R28 Retinal Neurons from Apoptotic Death through ERK-mediated BimEL Phosphorylation Independent of Akt

PubMed Central

Kong, Dejuan; Gong, Lijie; Arnold, Edith; Shanmugam, Sumathi; Fort, Patrice E.; Gardner, Thomas W.; Abcouwer, Steven F.

2016-01-01

Insulin-like growth factor 1 (IGF-1) can provide long-term neurotrophic support by activation of Akt, inhibition of FoxO nuclear localization and suppression of Bim gene transcription in multiple neuronal systems. However, MEK/ERK activation can also promote neuron survival through phosphorylation of BimEL. We explored the contribution of the PI3K/Akt/FoxO and MEK/ERK/BimEL pathways in IGF-1 stimulated survival after serum deprivation (SD) of R28 cells differentiated to model retinal neurons. IGF-1 caused rapid activation of Akt leading to FoxO1/3-T32/T24 phosphorylation, and prevented FoxO1/3 nuclear translocation and Bim mRNA upregulation in response to SD. IGF-1 also caused MAPK/MEK pathway activation as indicated by ERK1/2-T202/Y204 and Bim-S65 phosphorylation. Overexpression of FoxO1 increased Bim mRNA expression and amplified the apoptotic response to SD without shifting the serum response curve. Inhibition of Akt activation with LY294002 or by Rictor knockdown did not block the protective effect of IGF-1, while inhibition of MEK activity with PD98059 prevented Bim phosphorylation and blocked IGF-1 protection. In addition, knockdown of Bim expression was protective during SD, while co-silencing of FoxO1 and Fox03 expression had little effect. Thus, the PI3K/Akt/FoxO pathway was not essential for protection from SD-induced apoptosis by IGF-1 in R28 cells. Instead, IGF-1 protection was dependent on activation of the MEK/ERK pathway leading to BimEL phosphorylation, which is known to prevent Bax/Bak oligomerization and activation of the intrinsic mitochondrial apoptosis pathway. These studies demonstrate the requirement of the MEK/ERK pathway in a model of retinal neuron cell survival and highlight the cell specificity for IGF-1 signaling in this response. PMID:27511131

Cyclic AMP Enhances TGFβ Responses of Breast Cancer Cells by Upregulating TGFβ Receptor I Expression

PubMed Central

Oerlecke, Ilka; Bauer, Elke; Dittmer, Angela; Leyh, Benjamin; Dittmer, Jürgen

2013-01-01

Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein) and TβRI (TGFβ receptor 1), were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB) nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation. As a consequence, cancer cell functions such as proliferation are affected. PMID:23349840

Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells

PubMed Central

Chen, Jie; Wang, Hong; Xu, Wei-Ping; Wei, Si-Si; Li, Hui Joyce; Mei, Yun-Qing; Li, Yi-Gang; Wang, Yue-Peng

2016-01-01

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent. PMID:27276708

Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells.

PubMed

Chen, Jie; Wang, Hong; Xu, Wei-Ping; Wei, Si-Si; Li, Hui Joyce; Mei, Yun-Qing; Li, Yi-Gang; Wang, Yue-Peng

2016-08-30

CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent.

Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain

PubMed Central

Li, Mei-yi; Fong, Peter; Zhang, Ji-guo; Zhang, Can-wen; Gong, Ke-rui; Yang, Ming-feng; Niu, Jing-zhong; Ji, Xun-ming; Lv, Guo-wei

2015-01-01

Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC) and extracellular signal-regulated kinase1/2 (ERK1/2) in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6–S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling pathway. Thus, our results suggest a novel mechanism of gabapentin-mediated analgesia for visceral inflammatory pain through a PKC-ERK1/2 signaling pathway that may be a future therapeutic target for the treatment of visceral inflammatory pain. PMID:26512901

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells.

PubMed

Iijima, Yoshihiro; Laser, Martin; Shiraishi, Hirokazu; Willey, Christopher D; Sundaravadivel, Balasubramanian; Xu, Lin; McDermott, Paul J; Kuppuswamy, Dhandapani

2002-06-21

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.

ROS mediates interferon gamma induced phosphorylation of Src, through the Raf/ERK pathway, in MCF-7 human breast cancer cell line.

PubMed

Zibara, Kazem; Zeidan, Asad; Bjeije, Hassan; Kassem, Nouhad; Badran, Bassam; El-Zein, Nabil

2017-03-01

Interferon gamma (IFN-ɣ) is a pleiotropic cytokine which plays dual contrasting roles in cancer. Although IFN-ɣ has been clinically used to treat various malignancies, it was recently shown to have protumorigenic activities. Reactive oxygen species (ROS) are overproduced in cancer cells, mainly due to NADPH oxidase activity, which results into several changes in signaling pathways. In this study, we examined IFN-ɣ effect on the phosphorylation levels of key signaling proteins, through ROS production, in the human breast cancer cell line MCF-7. After treatment by IFN-ɣ, results showed a significant increase in the phosphorylation of STAT1, Src, raf, AKT, ERK1/2 and p38 signaling molecules, in a time specific manner. Src and Raf were found to be involved in early stages of IFN-ɣ signaling since their phosphorylation increased very rapidly. Selective inhibition of Src-family kinases resulted in an immediate significant decrease in the phosphorylation status of Raf and ERK1/2, but not p38 and AKT. On the other hand, IFN-ɣ resulted in ROS generation, through H 2 O 2 production, whereas pre-treatment with the ROS inhibitor NAC caused ROS inhibition and a significant decrease in the phosphorylation levels of AKT, ERK1/2, p38 and STAT1. Moreover, pretreatment with a selective NOX1 inhibitor resulted in a significant decrease of AKT phosphorylation. Finally, no direct relationship was found between ROS production and calcium mobilization. In summary, IFN-ɣ signaling in MCF-7 cell line is ROS-dependent and follows the Src/Raf/ERK pathway whereas its signaling through the AKT pathway is highly dependent on NOX1.

TGF-β signaling is often attenuated during hepatotumorigenesis, but is retained for the malignancy of hepatocellular carcinoma cells.

PubMed

Mu, Xiaoxin; Lin, Shu; Yang, Junhua; Chen, Chen; Chen, Yun; Herzig, Maryanne C; Washburn, Kenneth; Halff, Glenn A; Walter, Christi A; Sun, Beicheng; Sun, Lu-Zhe

2013-01-01

The role of transforming growth factor-beta (TGF-β) signaling in hepatocarcinogenesis remains controversial. We aimed to reveal TGF-β signaling status in human and murine tissues of hepatocellular carcinoma (HCC) and the mechanisms that mediate TGF-β's role in regulating HCC malignancy. Here, TGF-β pathway component expression and activation in human and murine HCC tissues were measured with quantitative RT-PCR and Western blotting assays. The role of TGF-β receptor and Smad signaling in the growth and survival of several HCC cell lines was determined with several in vitro and in vivo approaches. We found that TGF-β receptor II (TβRII) expression was downregulated in two different HCC patient cohorts. Consistently, Smad3 phosphorylation was also downregulated in HCC tissues in comparison to that in adjacent normal tissues. Interestingly, many HCC cell lines were sensitive to TGF-β and growth-inhibited by exogenous TGF-β. However, stable knockdown of TβRII inhibited cell growth on plastic and in soft agar, and induced apoptosis resulting in suppressed subcutaneous tumor growth and metastatic potential in vivo. Furthermore, knockdown of Smad4 also led to a significant inhibition of growth on plastic and in soft agar with concomitant increase of apoptosis, PTEN expression, and reduced nuclear accumulation of linker region-phosphorylated Smad3. Taken together, TGF-β signaling pathway plays a dichotomous role in hepatocellular carcinogenesis. It appears to suppress HCC development, but is retained for HCC cell survival and malignancy. Furthermore, Smad4 can mediate both growth inhibitory activity induced by exogenous TGF-β and the survival activity induced by autocrine TGF-β revealing a delicate selection of the two opposing activities of TGF-β during HCC evolution.

Ability of Bruton's Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but not B-Cell Receptor Signaling.

PubMed

Bender, Andrew T; Gardberg, Anna; Pereira, Albertina; Johnson, Theresa; Wu, Yin; Grenningloh, Roland; Head, Jared; Morandi, Federica; Haselmayer, Philipp; Liu-Bujalski, Lesley

2017-03-01

Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Artemisinin Protects Retinal Neuronal Cells against Oxidative Stress and Restores Rat Retinal Physiological Function from Light Exposed Damage.

PubMed

Yan, Fengxia; Wang, Haitao; Gao, Yang; Xu, Jiangping; Zheng, Wenhua

2017-08-16

Oxidative stress plays a key role in the pathogenesis of age-related macular degeneration (AMD), a leading cause of severe visual loss and blindness in the aging population which lacks any effective treatments currently. In this study, artemisinin, a well-known antimalarial drug was found to suppress hydrogen peroxide (H 2 O 2 )-induced cell death in retinal neuronal RGC-5 cells. Artemisinin, in the therapeutically relevant dosage, concentration-dependently attenuated the accumulation of intracellular reactive oxygen species (ROS), increased mitochondrial membrane potential and decreased cell apoptosis in RGC-5 cells induced by H 2 O 2 . Western blot analysis showed that artemisinin upregulated the phosphorylation of p38 and extracellular signal-regulated kinases1/2 (ERK1/2) and reversed the inhibitory effect of H 2 O 2 on the phosphorylation of these two kinases. Moreover, protective effect of artemisinin was blocked by the p38 kinase inhibitor PD169316 or ERK1/2 kinase pathway inhibitor PD98059, respectively. In contrast, c-Jun N-terminal kinase inhibitor and rapamycin had no effect in the protective effect of artemisinin. Taken together, these results demonstrated that artemisinin promoted the survival of RGC-5 cells from H 2 O 2 toxicity via the activation of the p38 and ERK1/2 pathways. Interestingly, intravitreous injection of artimisinin, concentration-dependently reversed light exposed-damage (a dry AMD animal model) of rat retinal physiological function detected by flash electroretinogram. These results indicate that artemisinin can protect retinal neuronal functions from H 2 O 2 -induced damage in vitro and in vivo and suggest the potential application of artemisinin as a new drug in the treatment of retinal disorders like AMD.

Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality

PubMed Central

Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

2015-01-01

Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

Tectorigenin sensitizes paclitaxel-resistant human ovarian cancer cells through downregulation of the Akt and NFκB pathway.

PubMed

Yang, Yeong-In; Lee, Kyung-Tae; Park, Hee-Juhn; Kim, Tae Jin; Choi, Youn Seok; Shih, Ie-Ming; Choi, Jung-Hye

2012-12-01

Paclitaxel (Taxol) is currently used as the front-line chemotherapeutic agent for several cancers including ovarian carcinoma; however, the drug frequently induces drug resistance through multiple mechanisms. The new strategy of using natural compounds in combination therapies is highly attractive because those compounds may enhance the efficacy of chemotherapy. In this study, we found that tectorigenin, an isoflavonoid isolated from flower of Pueraria thunbergiana, enhanced the growth-inhibitory effect of paclitaxel in paclitaxel-resistant ovarian cancer cells (MPSC1(TR), A2780(TR) and SKOV3(TR)) as well as their naive counterparts. The combination of tectorigenin with paclitaxel resulted in a synergistic apoptosis compared with either agent alone through activation of caspases-3, -8 and -9. Treatment with tectorigenin inhibited the nuclear translocation of NFκB and the expression of NFκB-dependent genes such as FLIP, XIAP, Bcl-2, Bcl-xL and COX-2, which are known to be associated with chemoresistance. In addition, the tectorigenin-paclitaxel combination inhibited the phosphorylation of IκB and IKK and the activation of Akt in paclitaxel-resistant cancer cells. Moreover, tectorigenin-paclitaxel-induced cell growth inhibition was enhanced by pretreatment with the Akt inhibitor LY294002 or overexpression of the dominant negative Akt (Akt-DN), but reduced by overexpression of constitutively activated Akt (Akt-Myr). Furthermore, we found that Akt-Myr, at least in part, reversed tectorigenin-paclitaxel-induced nuclear translocation of NFκB and the phosphorylation of IκB and IKK. These data suggest that tectorigenin could sensitize paclitaxel-resistant human ovarian cancer cells through inactivation of the Akt/IKK/IκB/NFκB signaling pathway, and promise a new intervention to chemosensitize paclitaxel-induced cytotoxicity in ovarian cancer.

α-Methyl artoflavanocoumarin from Juniperus chinensis exerts anti-diabetic effects by inhibiting PTP1B and activating the PI3K/Akt signaling pathway in insulin-resistant HepG2 cells.

PubMed

Jung, Hee Jin; Seong, Su Hui; Ali, Md Yousof; Min, Byung-Sun; Jung, Hyun Ah; Choi, Jae Sue

2017-12-01

Diabetes mellitus is one of the greatest global health issues and much research effort continues to be directed toward identifying novel therapeutic agents. Insulin resistance is a challenging integrally related topic and molecules capable of overcoming it are of considerable therapeutic interest in the context of type 2 diabetes mellitus (T2DM). Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling transduction and is regarded a novel therapeutic target in T2DM. Here, we investigated the inhibitory effect of α-methyl artoflavanocoumarin (MAFC), a natural flavanocoumarin isolated from Juniperus chinensis, on PTP1B in insulin-resistant HepG2 cells. MAFC was found to potently inhibit PTP1B with an IC 50 of 25.27 ± 0.14 µM, and a kinetics study revealed MAFC is a mixed type PTP1B inhibitor with a K i value of 13.84 µM. Molecular docking simulations demonstrated MAFC can bind to catalytic and allosteric sites of PTP1B. Furthermore, MAFC significantly increased glucose uptake and decreased the expression of PTP1B in insulin-resistant HepG2 cells, down-regulated the phosphorylation of insulin receptor substrate (IRS)-1 (Ser307), and dose-dependently enhanced the protein levels of IRS-1, phosphorylated phosphoinositide 3-kinase (PI3K), Akt, and ERK1. These results suggest that MAFC from J. chinensis has therapeutic potential in T2DM by inhibiting PTP1B and activating insulin signaling pathways.

Decursin inhibits retinal neovascularization via suppression of VEGFR-2 activation.

PubMed

Kim, Jeong Hun; Kim, Jin Hyoung; Lee, You Mie; Ahn, Eun-Mi; Kim, Kyu-Won; Yu, Young Suk

2009-09-12

Pathologic angiogenesis in the retina leads to the catastrophic loss of vision. Retinopathy of prematurity (ROP), a vasoproliferative retinopathy, is a leading cause of blindness in children. We evaluated the inhibitory effect of decursin on retinal neovascularization. Anti-angiogenic activity of decursin was evaluated by vascular endothelial growth factor (VEGF)-induced proliferation, migration, and in vitro tube formation assay of human retinal microvascular endothelial cells (HRMECs). We also used western blot analysis to assess inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) phosphorylation by decursin. After intravitreal injection of decursin in a mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting in cross-sections. The toxicity of decursin was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HRMECs as well as histologic and immunohistochemistry examination for glial fibrillary acidic protein in the retina. Decursin significantly inhibited VEGF-induced proliferation, migration, and the formation of capillary-like networks of retinal endothelial cells in a dose-dependent manner. Decursin inhibited VEGF-induced phosphorylation of VEGFR-2, blocking the VEGFR-2 signaling pathway. When intravitreously injected, decursin dramatically suppressed retinal neovascularization in a mouse model of ROP. Even in a high concentration, decursin never induced any structural or inflammatory changes to cells in retinal or vitreous layers. Moreover, the upregulation of glial fibrillary acidic protein expression was not detected in Mueller cells. Our data suggest that decursin may be a potent anti-angiogenic agent targeting the VEGFR-2 signaling pathway, which significantly inhibits retinal neovascularization without retinal toxicity and may be applicable in various other vasoproliferative retinopathies as well.

Decursin inhibits retinal neovascularization via suppression of VEGFR-2 activation

PubMed Central

Kim, Jeong Hun; Kim, Jin Hyoung; Lee, You Mie; Ahn, Eun-Mi; Kim, Kyu-Won

2009-01-01

Purpose Pathologic angiogenesis in the retina leads to the catastrophic loss of vision. Retinopathy of prematurity (ROP), a vasoproliferative retinopathy, is a leading cause of blindness in children. We evaluated the inhibitory effect of decursin on retinal neovascularization. Methods Anti-angiogenic activity of decursin was evaluated by vascular endothelial growth factor (VEGF)-induced proliferation, migration, and in vitro tube formation assay of human retinal microvascular endothelial cells (HRMECs). We also used western blot analysis to assess inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) phosphorylation by decursin. After intravitreal injection of decursin in a mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting in cross-sections. The toxicity of decursin was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HRMECs as well as histologic and immunohistochemistry examination for glial fibrillary acidic protein in the retina. Results Decursin significantly inhibited VEGF-induced proliferation, migration, and the formation of capillary-like networks of retinal endothelial cells in a dose-dependent manner. Decursin inhibited VEGF-induced phosphorylation of VEGFR-2, blocking the VEGFR-2 signaling pathway. When intravitreously injected, decursin dramatically suppressed retinal neovascularization in a mouse model of ROP. Even in a high concentration, decursin never induced any structural or inflammatory changes to cells in retinal or vitreous layers. Moreover, the upregulation of glial fibrillary acidic protein expression was not detected in Mueller cells. Conclusions Our data suggest that decursin may be a potent anti-angiogenic agent targeting the VEGFR-2 signaling pathway, which significantly inhibits retinal neovascularization without retinal toxicity and may be applicable in various other vasoproliferative retinopathies as well. PMID:19756180

Epidermal Growth Factor Receptor activation promotes ADA3 acetylation through the AKT-p300 pathway

PubMed Central

Srivastava, Shashank; Mohibi, Shakur; Mirza, Sameer; Band, Hamid; Band, Vimla

2017-01-01

ABSTRACT The ADA3 (Alteration/Deficiency in Activation 3) protein is an essential adaptor component of several Lysine Acetyltransferase (KAT) complexes involved in chromatin modifications. Previously, we and others have demonstrated a crucial role of ADA3 in cell cycle progression and in maintenance of genomic stability. Recently, we have shown that acetylation of ADA3 is key to its role in cell cycle progression. Here, we demonstrate that AKT activation downstream of Epidermal Growth Factor Receptor (EGFR) family proteins stimulation leads to phosphorylation of p300, which in turn promotes the acetylation of ADA3. Inhibition of upstream receptor tyrosine kinases (RTKs), HER1 (EGFR)/HER2 by lapatinib and the accompanying reduction of phospho-AKT levels led to a decrease in p300 phosphorylation and ADA3 protein levels. The p300/PCAF inhibitor garcinol also destabilized the ADA3 protein in a proteasome-dependent manner and an ADA3 mutant with K→R mutations exhibited a marked increase in half-life, consistent with opposite role of acetylation and ubiquitination of ADA3 on shared lysine residues. ADA3 knockdown led to cell cycle inhibitory effects, as well as apoptosis similar to those induced by lapatinib treatment of HER2+ breast cancer cells, as seen by accumulation of CDK inhibitor p27, reduction in mitotic marker pH3(S10), and a decrease in the S-phase marker PCNA, as well as the appearance of cleaved PARP. Taken together our results reveal a novel RTK-AKT-p300-ADA3 signaling pathway involved in growth factor-induced cell cycle progression. PMID:28759294

Ingestion of onion soup high in quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in man: a pilot study.

PubMed

Hubbard, Gary P; Wolffram, Siegfried; de Vos, Ric; Bovy, Arnaud; Gibbins, Jonathan M; Lovegrove, Julie A

2006-09-01

Epidemiological data suggest that those who consume a diet rich in quercetin-containing foods may have a reduced risk of CVD. Furthermore, in vitro and ex vivo studies have observed the inhibition of collagen-induced platelet activation by quercetin. The aim of the present study was to investigate the possible inhibitory effects of quercetin ingestion from a dietary source on collagen-stimulated platelet aggregation and signalling. A double-blind randomised cross-over pilot study was undertaken. Subjects ingested a soup containing either a high or a low amount of quercetin. Plasma quercetin concentrations and platelet aggregation and signalling were assessed after soup ingestion. The high-quercetin soup contained 69 mg total quercetin compared with the low-quercetin soup containing 5 mg total quercetin. Plasma quercetin concentrations were significantly higher after high-quercetin soup ingestion than after low-quercetin soup ingestion and peaked at 2.59 (sem 0.42) mumol/l. Collagen-stimulated (0.5 mug/ml) platelet aggregation was inhibited after ingestion of the high-quercetin soup in a time-dependent manner. Collagen-stimulated tyrosine phosphorylation of a key component of the collagen-signalling pathway via glycoprotein VI, Syk, was significantly inhibited by ingestion of the high-quercetin soup. The inhibition of Syk tyrosine phosphorylation was correlated with the area under the curve for the high-quercetin plasma profile. In conclusion, the ingestion of quercetin from a dietary source of onion soup could inhibit some aspects of collagen-stimulated platelet aggregation and signalling ex vivo. This further substantiates the epidemiological data suggesting that those who preferentially consume high amounts of quercetin-containing foods have a reduced risk of thrombosis and potential CVD risk.

The Neuroprotective Effect of Klotho is Mediated via Regulation of Members of the Redox System*

PubMed Central

Zeldich, Ella; Chen, Ci-Di; Colvin, Teresa A.; Bove-Fenderson, Erin A.; Liang, Jennifer; Tucker Zhou, Tracey B.; Harris, David A.; Abraham, Carmela R.

2014-01-01

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid β (oAβ)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAβ, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAβ, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect. PMID:25037225

Serine-204 in the Linker Region of Smad3 Mediates the Collagen-I Response to TGF-β in a Cell Phenotype-Specific Manner

PubMed Central

Browne, James A.; Liu, Xiaoying; Schnaper, H. William; Hayashida, Tomoko

2013-01-01

Regulation of TGF-β1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-β-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-β are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-β-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-β stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake. Renal epithelial cells (HKC) respond differently from MEF or mesangial cells; blocking ERK neither changed TGF-β-stimulated S204 phosphorylation nor prevented Smad3-mediated COL1A2 promoter activity in HKC. Furthermore, re-expression of wild type-Smad3 or the S204A-Smad3 mutant in Smad3-knockdown HKC reconstituted Smad3-mediated COL1A2 promoter activity. Collectively, these data suggest that Serine-204 phosphorylation in the Smad3LR is a critical event by which ERK enhances Smad3-mediated COL1A2 promoter activity in mesenchymal cells. PMID:24080014

Serine-204 in the linker region of Smad3 mediates the collagen-I response to TGF-β in a cell phenotype-specific manner.

PubMed

Browne, J A; Liu, X; Schnaper, H W; Hayashida, T

2013-11-15

Regulation of TGF-β1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-β-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-β are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-β-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-β stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake. Renal epithelial cells (HKC) respond differently from MEF or mesangial cells; blocking ERK neither changed TGF-β-stimulated S204 phosphorylation nor prevented Smad3-mediated COL1A2 promoter activity in HKC. Furthermore, re-expression of wild type-Smad3 or the S204A-Smad3 mutant in Smad3-knockdown HKC reconstituted Smad3-mediated COL1A2 promoter activity. Collectively, these data suggest that Serine-204 phosphorylation in the Smad3LR is a critical event by which ERK enhances Smad3-mediated COL1A2 promoter activity in mesenchymal cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

PubMed

Ku, H; Meier, K E

2000-04-14

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

Rubus idaeus extract suppresses migration and invasion of human oral cancer by inhibiting MMP-2 through modulation of the Erk1/2 signaling pathway.

PubMed

Huang, Yi-Wen; Chuang, Chun-Yi; Hsieh, Yih-Shou; Chen, Pei-Ni; Yang, Shun-Fa; Shih-Hsuan-Lin; Chen, Yang-Yu; Lin, Chiao-Wen; Chang, Yu-Chao

2017-03-01

Raspberries (Rubus idaeus L.) have been extensively studies worldwide because of their beneficial effects on health. Recently reports indicate that crude extracts of Rubus idaeus (RIE) have antioxidant and anticancer ability. The aim of this study was to evaluate the mechanism of its antimetastatic ability in oral cancer cells. In this study, SCC-9 and SAS oral cancer cells were subjected to a treatment with RIE and then analyzed the effect of RIE on migration and invasion. The addition of RIE inhibited the migration and invasion ability of oral cancer cells. Real time PCR, western blot and zymography analysis demonstrated that mRNA, protein expression and enzyme activity of matrix metalloproteinases-2 (MMP-2) were down-regulated by RIE. Moreover, the phosphorylation of Focal adhesion kinase (FAK), src, and extracellular signal-regulated kinase (ERK) were inhibited after RIE treatment. In conclusion, these results demonstrated that RIE exerted an inhibitory effect of migration and invasion in oral cancer cells and alter metastasis by suppression of MMP-2 expression through FAK/Scr/ERK signaling pathway. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1037-1046, 2017. © 2016 Wiley Periodicals, Inc.

Antagonistic effects of IL-17 and D-resolvins on endothelial Del-1 expression through a GSK-3β-C/EBPβ pathway.

PubMed

Maekawa, Tomoki; Hosur, Kavita; Abe, Toshiharu; Kantarci, Alpdogan; Ziogas, Athanasios; Wang, Baomei; Van Dyke, Thomas E; Chavakis, Triantafyllos; Hajishengallis, George

2015-09-16

Del-1 is an endothelial cell-secreted anti-inflammatory protein. In humans and mice, Del-1 expression is inversely related to that of IL-17, which inhibits Del-1 through hitherto unidentified mechanism(s). Here we show that IL-17 downregulates human endothelial cell expression of Del-1 by targeting a critical transcription factor, C/EBPβ. Specifically, IL-17 causes GSK-3β-dependent phosphorylation of C/EBPβ, which is associated with diminished C/EBPβ binding to the Del-1 promoter and suppressed Del-1 expression. This inhibitory action of IL-17 can be reversed at the GSK-3β level by PI3K/Akt signalling induced by D-resolvins. The biological relevance of this regulatory network is confirmed in a mouse model of inflammatory periodontitis. Intriguingly, resolvin-D1 (RvD1) confers protection against IL-17-driven periodontal bone loss in a Del-1-dependent manner, indicating an RvD1-Del-1 axis against IL-17-induced pathological inflammation. The dissection of signalling pathways regulating Del-1 expression provides potential targets to treat inflammatory diseases associated with diminished Del-1 expression, such as periodontitis and multiple sclerosis.

In Silico Modeling of Crabtree Effect.

PubMed

Ghosh, Debraj; De, Rajat K

2017-01-01

Glycolytic activity during Crabtree effect is similar to that in tumor cells. Research regarding Crabtree effect is very much crucial. The mechanism of metabolic activities in glycolysis pathway and oxidative phosphorylation pathway in regards to Crabtree effect in Saccharomyces cerevisiae was studied in this paper. We also explored the effects of hexose phosphates in the activities of respiratory chain complexes (III and IV) in inhibition of respiration. Besides, the enhancement of fermentation in response to excess glucose concentration was studied. We discussed the significance of Crabtree effect in mammalian cancer in terms of Crabtree effect in a Crabtree positive organism, as it is similar to cancer metabolism in mammalian cells. We developed an in silico model of Crabtree effect. A comparative study was performed with laboratory experiments regarding inhibitory role of fructose 1,6-bisphosphate on metabolic respiration. The model was simulated for different concentration levels of glucose and hexose phosphates using COPASI and SNOOPY tools. We have shown that a hike in glucose concentration increases ethanol concentration and leads glycolytic activity towards fermentation. This phenomenon occurs during Crabtree effect. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders

PubMed Central

Gueller, Saskia; Chumakova, Katya; Kawamata, Norihiko; Liu, Liqin; Koeffler, H. Phillip

2007-01-01

Recently, activating myeloproliferative leukemia virus oncogene (MPL) mutations, MPLW515L/K, were described in myeloproliferative disorder (MPD) patients. MPLW515L leads to activation of downstream signaling pathways and cytokine-independent proliferation in hematopoietic cells. The adaptor protein Lnk is a negative regulator of several cytokine receptors, including MPL. We show that overexpression of Lnk in Ba/F3-MPLW515L cells inhibits cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhances proliferation. Lnk blocks the activation of Jak2, Stat3, Erk, and Akt in these cells. Furthermore, MPLW515L-expressing cells are more susceptible to Lnk inhibitory functions than their MPL wild-type (MPLWT)–expressing counterparts. Lnk associates with activated MPLWT and MPLW515L and colocalizes with the receptors at the plasma membrane. The SH2 domain of Lnk is essential for its binding and for its down-regulation of MPLWT and MPLW515L. Lnk itself is tyrosine-phosphorylated following thrombopoietin stimulation. Further elucidating the cellular pathways that attenuate MPLW515L will provide insight into the pathogenesis of MPD and could help develop specific therapeutic approaches. PMID:17693582

Adaptor protein Lnk negatively regulates the mutant MPL, MPLW515L associated with myeloproliferative disorders.

PubMed

Gery, Sigal; Gueller, Saskia; Chumakova, Katya; Kawamata, Norihiko; Liu, Liqin; Koeffler, H Phillip

2007-11-01

Recently, activating myeloproliferative leukemia virus oncogene (MPL) mutations, MPLW515L/K, were described in myeloproliferative disorder (MPD) patients. MPLW515L leads to activation of downstream signaling pathways and cytokine-independent proliferation in hematopoietic cells. The adaptor protein Lnk is a negative regulator of several cytokine receptors, including MPL. We show that overexpression of Lnk in Ba/F3-MPLW515L cells inhibits cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhances proliferation. Lnk blocks the activation of Jak2, Stat3, Erk, and Akt in these cells. Furthermore, MPLW515L-expressing cells are more susceptible to Lnk inhibitory functions than their MPL wild-type (MPLWT)-expressing counterparts. Lnk associates with activated MPLWT and MPLW515L and colocalizes with the receptors at the plasma membrane. The SH2 domain of Lnk is essential for its binding and for its down-regulation of MPLWT and MPLW515L. Lnk itself is tyrosine-phosphorylated following thrombopoietin stimulation. Further elucidating the cellular pathways that attenuate MPLW515L will provide insight into the pathogenesis of MPD and could help develop specific therapeutic approaches.

Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses.

PubMed

Twu, Olivia; Dessí, Daniele; Vu, Anh; Mercer, Frances; Stevens, Grant C; de Miguel, Natalia; Rappelli, Paola; Cocco, Anna Rita; Clubb, Robert T; Fiori, Pier Luigi; Johnson, Patricia J

2014-06-03

The human-infective parasite Trichomonas vaginalis causes the most prevalent nonviral sexually transmitted infection worldwide. Infections in men may result in colonization of the prostate and are correlated with increased risk of aggressive prostate cancer. We have found that T. vaginalis secretes a protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), that is 47% similar to human macrophage migration inhibitory factor (HuMIF), a proinflammatory cytokine. Because HuMIF is reported to be elevated in prostate cancer and inflammation plays an important role in the initiation and progression of cancers, we have explored a role for TvMIF in prostate cancer. Here, we show that TvMIF has tautomerase activity, inhibits macrophage migration, and is proinflammatory. We also demonstrate that TvMIF binds the human CD74 MIF receptor with high affinity, comparable to that of HuMIF, which triggers activation of ERK, Akt, and Bcl-2-associated death promoter phosphorylation at a physiologically relevant concentration (1 ng/mL, 80 pM). TvMIF increases the in vitro growth and invasion through Matrigel of benign and prostate cancer cells. Sera from patients infected with T. vaginalis are reactive to TvMIF, especially in males. The presence of anti-TvMIF antibodies indicates that TvMIF is released by the parasite and elicits host immune responses during infection. Together, these data indicate that chronic T. vaginalis infections may result in TvMIF-driven inflammation and cell proliferation, thus triggering pathways that contribute to the promotion and progression of prostate cancer.

Raf Kinase Inhibitory Protein Protects Cells against Locostatin-Mediated Inhibition of Migration

PubMed Central

Shemon, Anne N.; Eves, Eva M.; Clark, Matthew C.; Heil, Gary; Granovsky, Alexey; Zeng, Lingchun; Imamoto, Akira

2009-01-01

Background Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. Methods/Findings We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP−/−) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP−/− MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. Conclusions/Significance These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells. PMID:19551145

The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

PubMed

Dodd, Dana A; Worth, Randall G; Rosen, Michael K; Grinstein, Sergio; van Oers, Nicolai S C; Hansen, Eric J

2014-05-20

Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for phagocytic activity. Haemophilus ducreyi, a sexually transmitted pathogen, secretes a 4,153-amino-acid (aa) protein (LspA1) that effectively inhibits FcγR-mediated phagocytic activity. In this study, we show that a 294-aa domain within this bacterial protein binds to C-terminal Src kinase (Csk) and stimulates its catalytic activity, resulting in a significant attenuation of Src kinase activity and consequent inhibition of phagocytosis. The ability to inhibit phagocytosis via Csk is not unique to H. ducreyi, because we found that the Helicobacter pylori CagA protein also inhibits phagocytosis in a Csk-dependent manner. Harnessing Csk to subvert the FcγR-mediated phagocytic pathway represents a new bacterial effector mechanism for circumventing the innate immune response. Copyright © 2014 Dodd et al.

Identification of Ser153 in ICL2 of the gonadotropin-releasing hormone (GnRH) receptor as a phosphorylation-independent site for inhibition of Gq coupling.

PubMed

Shacham, Sharon; Cheifetz, Maya N; Fridkin, Mati; Pawson, Adam J; Millar, Robert P; Naor, Zvi

2005-08-12

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized alphaT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser153 with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser151, Ser153, and Thr142) or only Ser153 was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser153) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in alphaT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser153 operates through a phosphorylation-independent mechanism.

Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

PubMed

Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

2016-01-05

Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

mTOR pathway and Ca2+ stores mobilization in aged smooth muscle cells

PubMed Central

Martín-Cano, Francisco E; Camello-Almaraz, Cristina; Hernandez, David; Pozo, Maria J; Camello, Pedro J

2013-01-01

Aging is considered to be driven by the so called senescence pathways, especially the mTOR route, although there is almost no information on its activity in aged tissues. Aging also induces Ca2+ signal alterations, but information regarding the mechanisms for these changes is almost inexistent. We investigated the possible involvement of the mTOR pathway in the age-dependent changes on Ca2+ stores mobilization in colonic smooth muscle cells of young (4 month old) and aged (24 month old) guinea pigs. mTORC1 activity was enhanced in aged smooth muscle, as revealed by phosphorylation of mTOR and its direct substrates S6K1 and 4E-BP1. Mobilization of intracellular Ca2+ stores through IP3R or RyR channels was impaired in aged cells, and it was facilitated by mTOR and by FKBP12, as indicated by the inhibitory effects of KU0063794 (a direct mTOR inhibitor), rapamycin (a FKBP12-mediated mTOR inhibitor) and FK506 (an FKBP12 binding immunosuppressant). Aging suppressed the facilitation of the Ca2+ mobilization by FKBP12 but not by mTOR, without changing the total expression of FKBP12 protein. In conclusion, or study shows that in smooth muscle aging enhances the constitutive activity of mTORC1 pathway and impairs Ca2+ stores mobilization by suppression of the FKBP12-induced facilitation of Ca2+ release. PMID:23661091

Prebiotic synthesis of phosphoenol pyruvate by α-phosphorylation-controlled triose glycolysis

NASA Astrophysics Data System (ADS)

Coggins, Adam J.; Powner, Matthew W.

2017-04-01

Phosphoenol pyruvate is the highest-energy phosphate found in living organisms and is one of the most versatile molecules in metabolism. Consequently, it is an essential intermediate in a wide variety of biochemical pathways, including carbon fixation, the shikimate pathway, substrate-level phosphorylation, gluconeogenesis and glycolysis. Triose glycolysis (generation of ATP from glyceraldehyde 3-phosphate via phosphoenol pyruvate) is among the most central and highly conserved pathways in metabolism. Here, we demonstrate the efficient and robust synthesis of phosphoenol pyruvate from prebiotic nucleotide precursors, glycolaldehyde and glyceraldehyde. Furthermore, phosphoenol pyruvate is derived within an α-phosphorylation controlled reaction network that gives access to glyceric acid 2-phosphate, glyceric acid 3-phosphate, phosphoserine and pyruvate. Our results demonstrate that the key components of a core metabolic pathway central to energy transduction and amino acid, sugar, nucleotide and lipid biosyntheses can be reconstituted in high yield under mild, prebiotically plausible conditions.

Double-Stranded RNA Induces Biphasic STAT1 Phosphorylation by both Type I Interferon (IFN)-Dependent and Type I IFN-Independent Pathways

PubMed Central

Dempoya, Junichi; Imaizumi, Tadaatsu; Hayakari, Ryo; Xing, Fei; Yoshida, Hidemi; Okumura, Ken; Satoh, Kei

2012-01-01

Upon viral infection, pattern recognition receptors sense viral nucleic acids, leading to the production of type I interferons (IFNs), which initiate antiviral activities. Type I IFNs bind to their cognate receptor, IFNAR, resulting in the activation of signal-transducing activators of transcription 1 (STAT1). Thus, it has long been thought that double-stranded RNA (dsRNA)-induced STAT1 phosphorylation is mediated by the transactivation of type I IFN signaling. Foreign RNA, such as viral RNA, in cells is sensed by the cytoplasmic sensors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). In this study, we explored the molecular mechanism responsible for STAT1 phosphorylation in response to the sensing of dsRNA by cytosolic RNA sensors. Polyinosinic-poly(C) [poly(I:C)], a synthetic dsRNA that is sensed by both RIG-I and MDA-5, induces STAT1 phosphorylation. We found that the poly(I:C)-induced initial phosphorylation of STAT1 is dependent on the RIG-I pathway and that MDA-5 is not involved in STAT1 phosphorylation. Furthermore, pretreatment of the cells with neutralizing antibody targeting the IFN receptor suppressed the initial STAT1 phosphorylation in response to poly(I:C), suggesting that this initial phosphorylation event is predominantly type I IFN dependent. In contrast, neither the known RIG-I pathway nor type I IFN is involved in the late phosphorylation of STAT1. In addition, poly(I:C) stimulated STAT1 phosphorylation in type I IFN receptor-deficient U5A cells with delayed kinetics. Collectively, our study provides evidence of a comprehensive regulatory mechanism in which dsRNA induces STAT1 phosphorylation, indicating the importance of STAT1 in maintaining very tight regulation of the innate immune system. PMID:22973045

Emodin Inhibition of Influenza A Virus Replication and Influenza Viral Pneumonia via the Nrf2, TLR4, p38/JNK and NF-kappaB Pathways.

PubMed

Dai, Jian-Ping; Wang, Qian-Wen; Su, Yun; Gu, Li-Ming; Zhao, Ying; Chen, Xiao-Xua; Chen, Cheng; Li, Wei-Zhong; Wang, Ge-Fei; Li, Kang-Sheng

2017-10-18

Lasting activations of toll-like receptors (TLRs), MAPK and NF-κB pathways can support influenza A virus (IAV) infection and promote pneumonia. In this study, we have investigated the effect and mechanism of action of emodin on IAV infection using qRT-PCR, western blotting, ELISA, Nrf2 luciferase reporter, siRNA and plaque inhibition assays. The results showed that emodin could significantly inhibit IAV (ST169, H1N1) replication, reduce IAV-induced expressions of TLR2/3/4/7, MyD88 and TRAF6, decrease IAV-induced phosphorylations of p38/JNK MAPK and nuclear translocation of NF-κB p65. Emodin also activated the Nrf2 pathway, decreased ROS levels, increased GSH levelss and GSH/GSSG ratio, and upregulated the activities of SOD, GR, CAT and GSH-Px after IAV infection. Suppression of Nrf2 via siRNA markedly blocked the inhibitory effects of emodin on IAV-induced activations of TLR4, p38/JNK, and NF-κB pathways and on IAV-induced production of IL-1β, IL-6 and expression of IAV M2 protein. Emodin also dramatically increased the survival rate of mice, reduced lung edema, pulmonary viral titer and inflammatory cytokines, and improved lung histopathological changes. In conclusion, emodin can inhibit IAV replication and influenza viral pneumonia, at least in part, by activating Nrf2 signaling and inhibiting IAV-induced activations of the TLR4, p38/JNK MAPK and NF-κB pathways.

The inhibitory mechanism of Cordyceps sinensis on cigarette smoke extract-induced senescence in human bronchial epithelial cells

PubMed Central

Liu, Ailing; Wu, Jinxiang; Li, Aijun; Bi, Wenxiang; Liu, Tian; Cao, Liuzhao; Liu, Yahui; Dong, Liang

2016-01-01

Objectives Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or stress. The bronchial epithelial cell is often injured by inhaled toxic substances, such as cigarette smoke. In the present study, we investigated whether exposure to cigarette smoke extract (CSE) induces senescence of bronchial epithelial cells; and Cordyceps sinensis mechanism of inhibition of CSE-induced cellular senescence. Methods Human bronchial epithelial cells (16HBE cells) cultured in vitro were treated with CSE and/or C. sinensis. p16, p21, and senescence-associated-galactosidase activity were used to detect cellular senescence with immunofluorescence, quantitative polymerase chain reaction, and Western blotting. Reactive oxygen species (ROS), PI3K/AKT/mTOR and their phosphorylated proteins were examined to testify the activation of signaling pathway by ROS fluorescent staining and Western blotting. Then, inhibitors of ROS and PI3K were used to further confirm the function of this pathway. Results Cellular senescence was upregulated by CSE treatment, and C. sinensis can decrease CSE-induced cellular senescence. Activation of ROS/PI3K/AKT/mTOR signaling pathway was enhanced by CSE treatment, and decreased when C. sinensis was added. Blocking ROS/PI3K/AKT/mTOR signaling pathway can attenuate CSE-induced cellular senescence. Conclusion CSE can induce cellular senescence in human bronchial epithelial cells, and ROS/PI3K/AKT/mTOR signaling pathway may play an important role in this process. C. sinensis can inhibit the CSE-induced senescence. PMID:27555762

A coordinated phosphorylation cascade initiated by p38MAPK/MSK1 directs RARα to target promoters

PubMed Central

Bruck, Nathalie; Vitoux, Dominique; Ferry, Christine; Duong, Vanessa; Bauer, Annie; de Thé, Hughes; Rochette-Egly, Cécile

2009-01-01

The nuclear retinoic acid (RA) receptor alpha (RARα) is a transcriptional transregulator that controls the expression of specific gene subsets through binding at response elements and dynamic interactions with coregulators, which are coordinated by the ligand. Here, we highlighted a novel paradigm in which the transcription of RARα target genes is controlled by phosphorylation cascades initiated by the rapid RA activation of the p38MAPK/MSK1 pathway. We demonstrate that MSK1 phosphorylates RARα at S369 located in the ligand-binding domain, allowing the binding of TFIIH and thereby phosphorylation of the N-terminal domain at S77 by cdk7/cyclin H. MSK1 also phosphorylates histone H3 at S10. Finally, the phosphorylation cascade initiated by MSK1 controls the recruitment of RARα/TFIIH complexes to response elements and subsequently RARα target gene activation. Cancer cells characterized by a deregulated p38MAPK/MSK1 pathway, do not respond to RA, outlining the essential contribution of the RA-triggered phosphorylation cascade in RA signalling. PMID:19078967

Gene expression profile during testicular development in patients with SRY-negative 46,XX testicular disorder of sex development.

PubMed

Mizuno, Kentaro; Kojima, Yoshiyuki; Kamisawa, Hideyuki; Moritoki, Yoshinobu; Nishio, Hidenori; Kohri, Kenjiro; Hayashi, Yutaro

2013-12-01

To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes. We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)-based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed. We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression. On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes. Copyright © 2013 Elsevier Inc. All rights reserved.

Anthelmintic drug ivermectin inhibits angiogenesis, growth and survival of glioblastoma through inducing mitochondrial dysfunction and oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Yingying; Fang, Shanshan; Sun, Qiushi

Glioblastoma is one of the most vascular brain tumour and highly resistant to current therapy. Targeting both glioblastoma cells and angiogenesis may present an effective therapeutic strategy for glioblastoma. In our work, we show that an anthelmintic drug, ivermectin, is active against glioblastoma cells in vitro and in vivo, and also targets angiogenesis. Ivermectin significantly inhibits growth and anchorage-independent colony formation in U87 and T98G glioblastoma cells. It induces apoptosis in these cells through a caspase-dependent manner. Ivermectin significantly suppresses the growth of two independent glioblastoma xenograft mouse models. In addition, ivermectin effectively targets angiogenesis through inhibiting capillary network formation, proliferation andmore » survival in human brain microvascular endothelial cell (HBMEC). Mechanistically, ivermectin decreases mitochondrial respiration, membrane potential, ATP levels and increases mitochondrial superoxide in U87, T98G and HBMEC cells exposed to ivermectin. The inhibitory effects of ivermectin are significantly reversed in mitochondria-deficient cells or cells treated with antioxidants, further confirming that ivermectin acts through mitochondrial respiration inhibition and induction of oxidative stress. Importantly, we show that ivermectin suppresses phosphorylation of Akt, mTOR and ribosomal S6 in glioblastoma and HBMEC cells, suggesting its inhibitory role in deactivating Akt/mTOR pathway. Altogether, our work demonstrates that ivermectin is a useful addition to the treatment armamentarium for glioblastoma. Our work also highlights the therapeutic value of targeting mitochondrial metabolism in glioblastoma. - Highlights: • Ivermectin is effective in glioblastoma cells in vitro and in vivo. • Ivermectin inhibits angiogenesis. • Ivermectin induces mitochondrial dysfunction and oxidative stress. • Ivermectin deactivates Akt/mTOR signaling pathway.« less

Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

PubMed Central

2011-01-01

Background Pigmentation is one of the essential defense mechanisms against oxidative stress or UV irradiation; however, abnormal hyperpigmentation in human skin may pose a serious aesthetic problem. C-phycocyanin (Cpc) is a phycobiliprotein from spirulina and functions as an antioxidant and a light harvesting protein. Though it is known that spirulina has been used to reduce hyperpigmentation, little literature addresses the antimelanogenic mechanism of Cpc. Herein, we investigated the rationale for the Cpc-induced inhibitory mechanism on melanin synthesis in B16F10 melanoma cells. Methods Cpc-induced inhibitory effects on melanin synthesis and tyrosinase expression were evaluated. The activity of MAPK pathways-associated molecules such as MAPK/ERK and p38 MAPK, were also examined to explore Cpc-induced antimelanogenic mechanisms. Additionally, the intracellular localization of Cpc was investigated by confocal microscopic analysis to observe the migration of Cpc. Results Cpc significantly (P < 0.05) reduced both tyrosinase activity and melanin production in a dose-dependent manner. This phycobiliprotein elevated the abundance of intracellular cAMP leading to the promotion of downstream ERK1/2 phosphorylation and the subsequent MITF (the transcription factor of tyrosinase) degradation. Further, Cpc also suppressed the activation of p38 causing the consequent disturbed activation of CREB (the transcription factor of MITF). As a result, Cpc negatively regulated tyrosinase gene expression resulting in the suppression of melanin synthesis. Moreover, the entry of Cpc into B16F10 cells was revealed by confocal immunofluorescence localization and immunoblot analysis. Conclusions Cpc exerted dual antimelanogenic mechanisms by upregulation of MAPK/ERK-dependent degradation of MITF and downregulation of p38 MAPK-regulated CREB activation to modulate melanin formation. Cpc may have potential applications in biomedicine, food, and cosmetic industries. PMID:21988805

Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling.

PubMed

Diniz, Luan Pereira; Tortelli, Vanessa; Garcia, Matheus Nunes; Araújo, Ana Paula Bérgamo; Melo, Helen M; Silva, Gisele S Seixas da; Felice, Fernanda G De; Alves-Leon, Soniza Vieira; Souza, Jorge Marcondes de; Romão, Luciana Ferreira; Castro, Newton Gonçalves; Gomes, Flávia Carvalho Alcantara

2014-12-01

The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-β1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-β pathway. TGF-β1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-β1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-β1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-β1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission. © 2014 Wiley Periodicals, Inc.

Tyrosine Phosphorylation in Toll-Like Receptor Signaling

PubMed Central

Chattopadhyay, Saurabh; Sen, Ganes C.

2014-01-01

There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses.

PubMed

Nguyen, Quynh-Anh; Horn, Meryl E; Nicoll, Roger A

2016-11-02

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively.

Phosphorylation of eukaryotic elongation factor 2 (eEF2) by cyclin A-cyclin-dependent kinase 2 regulates its inhibition by eEF2 kinase.

PubMed

Hizli, Asli A; Chi, Yong; Swanger, Jherek; Carter, John H; Liao, Yi; Welcker, Markus; Ryazanov, Alexey G; Clurman, Bruce E

2013-02-01

Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A-cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.

Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) by Cyclin A–Cyclin-Dependent Kinase 2 Regulates Its Inhibition by eEF2 Kinase

PubMed Central

Hizli, Asli A.; Chi, Yong; Swanger, Jherek; Carter, John H.; Liao, Yi; Welcker, Markus; Ryazanov, Alexey G.

2013-01-01

Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A–cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity. PMID:23184662

How quantitative measures unravel design principles in multi-stage phosphorylation cascades.

PubMed

Frey, Simone; Millat, Thomas; Hohmann, Stefan; Wolkenhauer, Olaf

2008-09-07

We investigate design principles of linear multi-stage phosphorylation cascades by using quantitative measures for signaling time, signal duration and signal amplitude. We compare alternative pathway structures by varying the number of phosphorylations and the length of the cascade. We show that a model for a weakly activated pathway does not reflect the biological context well, unless it is restricted to certain parameter combinations. Focusing therefore on a more general model, we compare alternative structures with respect to a multivariate optimization criterion. We test the hypothesis that the structure of a linear multi-stage phosphorylation cascade is the result of an optimization process aiming for a fast response, defined by the minimum of the product of signaling time and signal duration. It is then shown that certain pathway structures minimize this criterion. Several popular models of MAPK cascades form the basis of our study. These models represent different levels of approximation, which we compare and discuss with respect to the quantitative measures.

Phosphorylation of CaMKII in the rat dorsal raphe nucleus plays an important role in sleep-wake regulation.

PubMed

Cui, Su-Ying; Li, Sheng-Jie; Cui, Xiang-Yu; Zhang, Xue-Qiong; Yu, Bin; Sheng, Zhao-Fu; Huang, Yuan-Li; Cao, Qing; Xu, Ya-Ping; Lin, Zhi-Ge; Yang, Guang; Song, Jin-Zhi; Ding, Hui; Wang, Zi-Jun; Zhang, Yong-He

2016-02-01

The Ca(2+) modulation in the dorsal raphe nucleus (DRN) plays an important role in sleep-wake regulation. Calmodulin-dependent kinase II (CaMKII) is an important signal-transducing molecule that is activated by Ca(2+) . This study investigated the effects of intracellular Ca(2+) /CaMKII signaling in the DRN on sleep-wake states in rats. Maximum and minimum CaMKII phosphorylation was detected at Zeitgeber time 21 (ZT 21; wakefulness state) and ZT 3 (sleep state), respectively, across the light-dark rhythm in the DRN in rats. Six-hour sleep deprivation significantly reduced CaMKII phosphorylation in the DRN. Microinjection of the CAMKII activation inhibitor KN-93 (5 or 10 nmol) into the DRN suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REM sleep (NREMS). Application of a high dose of KN-93 (10 nmol) increased slow-wave sleep (SWS) time, SWS bouts, the mean duration of SWS, the percentage of SWS relative to total sleep, and delta power density during NREMS. Microinjection of CaCl2 (50 nmol) in the DRN increased CaMKII phosphorylation and decreased NREMS, SWS, and REMS. KN-93 abolished the inhibitory effects of CaCl2 on NREMS, SWS, and REMS. These data indicate a novel wake-promoting and sleep-suppressing role for the Ca(2+) /CaMKII signaling pathway in DRN neurons. We propose that the intracellular Ca(2+) /CaMKII signaling in the dorsal raphe nucleus (DRN) plays wake-promoting and sleep-suppressing role in rats. Intra-DRN application of KN-93 (CaMKII activation inhibitor) suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Intra-DRN application of CaCl2 attenuated REMS and NREMS. We think these findings should provide a novel cellular and molecular mechanism of sleep-wake regulation. © 2015 International Society for Neurochemistry.

Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase.

PubMed

Chang, Chao C; Lu, Wan J; Chiang, Cheng W; Jayakumar, Thanasekaran; Ong, Eng T; Hsiao, George; Fong, Tsorng H; Chou, Duen S; Sheu, Joen R

2010-12-01

Sesamol is a potent phenolic antioxidant which possesses antimutagenic, antihepatotoxic and antiaging properties. Platelet activation is relevant to a variety of acute thrombotic events and coronary heart diseases. There have been few studies on the effect of sesamol on platelets. Therefore, the aim of this study was to systematically examine the detailed mechanisms of sesamol in preventing platelet activation in vitro and in vivo. Sesamol (2.5-5 μM) exhibited more potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists. Sesamol inhibited collagen-stimulated platelet activation accompanied by [Ca(2+)](i) mobilization, thromboxane A(2) (TxA(2)) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) phosphorylation in washed platelets. Sesamol markedly increased cAMP and cGMP levels, endothelial nitric oxide synthase (eNOS) expression and NO release, as well as vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the sesamol-mediated inhibitory effects on platelet aggregation and p38 MAPK phosphorylation, and sesamol-mediated stimulatory effects on VASP and eNOS phosphorylation, and NO release. Sesamol also reduced hydroxyl radical (OH(●)) formation in platelets. In an in vivo study, sesamol (5 mg/kg) significantly prolonged platelet plug formation in mice. The most important findings of this study demonstrate for the first time that sesamol possesses potent antiplatelet activity, which may involve activation of the cAMP-eNOS/NO-cGMP pathway, resulting in inhibition of the PLCγ2-PKC-p38 MAPK-TxA(2) cascade, and, finally, inhibition of platelet aggregation. Sesamol treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders. Copyright © 2010 Elsevier Inc. All rights reserved.

Mangiferin ameliorates Porphyromonas gingivalis-induced experimental periodontitis by inhibiting phosphorylation of nuclear factor-κB and Janus kinase 1-signal transducer and activator of transcription signaling pathways.

PubMed

Li, H; Wang, Q; Ding, Y; Bao, C; Li, W

2017-02-01

Mangiferin is a natural polyphenol compound with anti-inflammatory properties. However, there have been few reports on the effect of mangiferin on periodontitis. Here, we investigated the anti-inflammatory effects of this compound on experimental periodontitis and the underlying mechanisms. Mice were inoculated with Porphyromonas gingivalis to induce periodontitis, and treated with mangiferin orally (50 mg/kg bodyweight, once a day) for 8 wk. Then, the alveolar bone loss was examined using a scanning electronic microscope. Expression of tumor necrosis factor-α (TNF-α) and the phosphorylation levels of nuclear factor-κB (NF-κB) and Janus kinase 1-signal transducer and activator of adhesion (JAK1-STAT) pathways in the gingival epithelium were detected using western blot analysis and immunohistochemical staining. The results showed that mice with periodontitis exhibited greater alveolar bone loss, stronger expression of TNF-α and higher phosphorylation levels of NF-κB and JAK1-STAT1/3 pathways in gingival epithelia, compared with control mice with no periodontitis. Moreover, treatment with mangiferin could significantly inhibit alveolar bone loss, TNF-α production and phosphorylation of NF-κB and JAK1-STAT1/3 pathways in gingival epithelia. Mangiferin has anti-inflammatory effects on periodontitis, which is associated with its ability to down-regulate the phosphorylation of NF-κB and JAK1-STAT1/3 pathways in gingival epithelia. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K.

PubMed

Cheng, Z; Surichan, S; Ruparelia, K; Arroo, R; Boarder, M R

2011-04-01

The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4'-hydroxy-5,6,7,8-tetramethoxyflavone (4'-OH-TMF) in this effect. We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [(3)H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade. Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4'-OH-TMF. Low micromolar concentrations of tangeretin or 4'-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4'-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR. Tangeretin and 4'-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4'-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Single agent BMS-911543 Jak2 inhibitor has distinct inhibitory effects on STAT5 signaling in genetically engineered mice with pancreatic cancer

PubMed Central

Mace, Thomas A.; Shakya, Reena; Elnaggar, Omar; Wilson, Kristin; Komar, Hannah M.; Yang, Jennifer; Pitarresi, Jason R.; Young, Gregory S.; Ostrowski, Michael C.; Ludwig, Thomas; Bekaii-Saab, Tanios; Bloomston, Mark; Lesinski, Gregory B.

2015-01-01

The Jak/STAT pathway is activated in human pancreatic ductal adenocarcinoma (PDAC) and cooperates with mutant Kras to drive initiation and progression of PDAC in murine models. We hypothesized that the small-molecule Jak2 inhibitor (BMS-911543) would elicit anti-tumor activity against PDAC and decrease immune suppressive features of the disease. We used an aggressive genetically engineered PDAC model with mutant KrasG12D, tp53R270H, and Brca1 alleles (KPC-Brca1 mice). Mice with confirmed tumor burden were treated orally with vehicle or 30 mg/kg BMS-911543 daily for 14 days. Histologic analysis of pancreata from treated mice revealed fewer foci of adenocarcinoma and significantly decreased Ki67+ cells versus controls. In vivo administration of BMS-911543 significantly reduced pSTAT5 and FoxP3 positive cells within the pancreas, but did not alter STAT3 phosphorylation. Continuous dosing of KPC-Brca1 mice with BMS-911543 resulted in a median survival of 108 days, as compared to a median survival of 87 days in vehicle treated animals, a 23% increase (p = 0.055). In vitro experiments demonstrated that PDAC cell lines were poorly sensitive to BMS-911543, requiring high micromolar concentrations to achieve targeted inhibition of Jak/STAT signaling. Similarly, BMS-911543 had little in vitro effect on the viability of both murine and human PDAC-derived stellate cell lines. However, BMS-911543 potently inhibited phosphorylation of pSTAT3 and pSTAT5 at low micromolar doses in human PBMC and reduced in vitro differentiation of Foxp3+ T regulatory cells. These results indicate that single agent Jak2i deserves further study in preclinical models of PDAC and has distinct inhibitory effects on STAT5 mediated signaling. PMID:26575024

Active Components of Ginger Potentiate β-Agonist–Induced Relaxation of Airway Smooth Muscle by Modulating Cytoskeletal Regulatory Proteins

PubMed Central

Zhang, Yi; Xu, Carrie; Wakita, Ryo; Emala, Charles W.

2014-01-01

β-Agonists are the first-line therapy to alleviate asthma symptoms by acutely relaxing the airway. Purified components of ginger relax airway smooth muscle (ASM), but the mechanisms are unclear. By elucidating these mechanisms, we can explore the use of phytotherapeutics in combination with traditional asthma therapies. The objectives of this study were to: (1) determine if 6-gingerol, 8-gingerol, or 6-shogaol potentiate β-agonist–induced ASM relaxation; and (2) define the mechanism(s) of action responsible for this potentiation. Human ASM was contracted in organ baths. Tissues were relaxed dose dependently with β-agonist, isoproterenol, in the presence of vehicle, 6-gingerol, 8-gingerol, or 6-shogaol (100 μM). Primary human ASM cells were used for cellular experiments. Purified phosphodiesterase (PDE) 4D or phospholipase C β enzyme was used to assess inhibitory activity of ginger components using fluorescent assays. A G-LISA assay was used to determine the effects of ginger constituents on Ras homolog gene family member A activation. Significant potentiation of isoproterenol-induced relaxation was observed with each of the ginger constituents. 6-Shogaol showed the largest shift in isoproterenol half-maximal effective concentration. 6-Gingerol, 8-gingerol, or 6-shogaol significantly inhibited PDE4D, whereas 8-gingerol and 6-shogaol also inhibited phospholipase C β activity. 6-Shogaol alone inhibited Ras homolog gene family member A activation. In human ASM cells, these constituents decreased phosphorylation of 17-kD protein kinase C–potentiated inhibitory protein of type 1 protein phosphatase and 8-gingerol decreased myosin light chain phosphorylation. Isolated components of ginger potentiate β-agonist–induced relaxation in human ASM. This potentiation involves PDE4D inhibition and cytoskeletal regulatory proteins. Together with β-agonists, 6-gingerol, 8-gingerol, or 6-shogaol may augment existing asthma therapy, resulting in relief of symptoms through complementary intracellular pathways. PMID:23962082

Pinocembrin attenuates lipopolysaccharide-induced inflammatory responses in Labeo rohita macrophages via the suppression of the NF-κB signalling pathway.

PubMed

Giri, Sib Sankar; Sen, Shib Sankar; Sukumaran, Venkatachalam; Park, Se Chang

2016-09-01

Pinocembrin is a flavonoid that has been reported to exhibit various pharmacological and biological activities including antimicrobial, antioxidant, and anti-inflammatory. To explore the anti-inflammatory activity of pinocembrin in a fish cell line, we investigated its ability to regulate the inflammatory mediators elevated by lipopolysaccharide (LPS) in Labeo rohita head-kidney (HK) macrophages. HK macrophages of L. rohita were treated with LPS (1 μg mL(-1)) in the presence or absence of pinocembrin. We examined the inhibitory effect of pinocembrin on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production. The inhibitory effect of pinocembrin on nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was investigated by RT-PCR and western blot. The effect of pinocembrin on pro-inflammatory cytokines (tumour necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β)) and anti-inflammatory cytokine IL-10 was investigated by ELISA and RT-PCR. The phosphorylation of three mitogen activated protein kinases (MAPKs) ERK, JNK, and p38 was analysed by western blot. Pinocembrin inhibited LPS-induced productions of NO and PGE2, and also markedly inhibited TNF-α, IL-1β, iNOS, and COX-2 production in a concentration-dependent manner. In addition, TNF-α and IL-1β mRNA expression levels decreased significantly, while IL-10 mRNA expression increased (P < 0.05) with pinocembrin pre-treatment. RT-PCR and western blot analysis showed that pinocembrin decreased both the mRNA and protein expression levels of LPS-induced iNOS and COX-2 in HK macrophages. Pinocembrin suppressed the phosphorylation of MAPK in LPS-stimulated HK macrophages. Further, pinocembrin significantly inhibited LPS-induced NF-κB transcriptional activity via the attenuation of IκBα degradation. Taken together, pinocembrin reduced the levels of pro-inflammatory mediators, such as iNOS, COX-2, TNF-α, and IL-1β, by inhibiting NF-κB activation via the suppression of ERK and p38 phosphorylation, and by attenuating the degradation of IκBα. These results suggest that pinocembrin is a potential novel candidate for the treatment of inflammatory conditions in L. rohita macrophages. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

PubMed

Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

2009-04-10

Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity.

A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1

PubMed Central

Bar-Yosef, Hagit; Gildor, Tsvia; Ramírez-Zavala, Bernardo; Schmauch, Christian; Weissman, Ziva; Pinsky, Mariel; Naddaf, Rawi; Morschhäuser, Joachim; Arkowitz, Robert A.; Kornitzer, Daniel

2018-01-01

The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation. PMID:29473018

A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1.

PubMed

Bar-Yosef, Hagit; Gildor, Tsvia; Ramírez-Zavala, Bernardo; Schmauch, Christian; Weissman, Ziva; Pinsky, Mariel; Naddaf, Rawi; Morschhäuser, Joachim; Arkowitz, Robert A; Kornitzer, Daniel

2018-01-01

The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

Picropodophyllin inhibits the growth of Ewing's sarcoma cells through the insulin‑like growth factor‑1 receptor/Akt signaling pathway.

PubMed

Wu, Yong-Tao; Wang, Bao-Jun; Miao, Sheng-Wu; Gao, Jian-Jun

2015-11-01

Ewing's sarcoma (ES) is the second most common type of pediatric bone tumor, and is associated with a poor prognosis. Picropodophyllin (PPP), a novel selective inhibitor of insulin‑like growth factor‑1 receptor (IGF‑1R), is able to strongly inhibit various types of cancers. However, the effect of IGF‑1R on ES remains unclear. Following treatment with various concentrations of PPP for various times, cell viability was determined using an MTT assay. In addition, cell proliferation and apoptosis was investigated separately by bromodeoxyuridine staining and flow cytometry, respectively. The PPP‑associated signaling pathway was also investigated. The results of the present study suggested that PPP inhibited cell proliferation and viability of A673 and SK‑ES‑1 human Ewing's sarcoma cells in a dose- and time‑dependent manner. In addition, cell apoptosis rates were increased following treatment with PPP. Further investigation of the underlying mechanism revealed that PPP inhibited Akt phosphorylation. Fumonisin B1, an Akt‑specific activator, reversed the inhibitory effects of PPP on cell growth. Furthermore, the results suggested that PPP decreased the expression levels of IGF‑1R, a common activator of Akt signaling. PPP inhibited the growth of human Ewing's sarcoma cells by targeting the IGF‑1R/Akt signaling pathway. Therefore, PPP may prove useful in the development of an effective strategy for the treatment of Ewing's sarcoma.

Non-thermal plasma inhibits human cervical cancer HeLa cells invasiveness by suppressing the MAPK pathway and decreasing matrix metalloproteinase-9 expression

NASA Astrophysics Data System (ADS)

Li, Wei; Yu, K. N.; Bao, Lingzhi; Shen, Jie; Cheng, Cheng; Han, Wei

2016-01-01

Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. However, the mechanism underlying its biological effects remains unclear. In this study, we investigated the inhibitory effect of NTP on the invasion of HeLa cells, and explored the possible mechanism. Our results showed that NTP exposure for 20 or 40 s significantly suppressed the migration and invasion of HeLa cells on the basis of matrigel invasion assay and wound healing assay, respectively. Moreover, NTP reduced the activity and protein expression of the matrix metalloproteinase (MMP)-9 enzyme. Western blot analysis indicated that NTP exposure effectively decreased phosphorylation level of both ERK1/2 and JNK, but not p38 MAPK. Furthermore, treatment with MAPK signal pathway inhibitors or NTP all exhibited significant depression of HeLa cells migration and MMP-9 expression. The result showed that NTP synergistically suppressed migration and MMP-9 expression in the presence of ERK1/2 inhibitor and JNK inhibitor, but not p38 MAPK inhibitor. Taken together, these findings suggested that NTP exposure inhibited the migration and invasion of HeLa cells via down-regulating MMP-9 expression in ERK1/2 and JNK signaling pathways dependent manner. These findings provide hints to the potential clinical research and therapy of NTP on cervical cancer metastasis.

The antiproliferative and apoptotic effects of apigenin on glioblastoma cells.

PubMed

Stump, Trevor A; Santee, Brittany N; Williams, Lauren P; Kunze, Rachel A; Heinze, Chelsae E; Huseman, Eric D; Gryka, Rebecca J; Simpson, Denise S; Amos, Samson

2017-07-01

Glioblastoma (GBM) is highly proliferative, infiltrative, malignant and the most deadly form of brain tumour. The epidermal growth factor receptor (EGFR) is overexpressed, amplified and mutated in GBM and has been shown to play key and important roles in the proliferation, growth and survival of this tumour. The goal of our study was to investigate the antiproliferative, apoptotic and molecular effects of apigenin in GBM. Proliferation and viability tests were carried out using the trypan blue exclusion, MTT and lactate dehydrogenase (LDH) assays. Flow cytometry was used to examine the effects of apigenin on the cell cycle check-points. In addition, we determined the effects of apigenin on EGFR-mediated signalling pathways by Western blot analyses. Our results showed that apigenin reduced cell viability and proliferation in a dose- and time-dependent manner while increasing cytotoxicity in GBM cells. Treatment with apigenin-induced is poly ADP-ribose polymerase (PARP) cleavage and caused cell cycle arrest at the G2M checkpoint. Furthermore, our data revealed that apigenin inhibited EGFR-mediated phosphorylation of mitogen-activated protein kinase (MAPK), AKT and mammalian target of rapamycin (mTOR) signalling pathways and attenuated the expression of Bcl-xL. Our results demonstrated that apigenin has potent inhibitory effects on pathways involved in GBM proliferation and survival and could potentially be used as a therapeutic agent for GBM. © 2017 Royal Pharmaceutical Society.

VEGF attenuated increase of outward delayed-rectifier potassium currents in hippocampal neurons induced by focal ischemia via PI3-K pathway.

PubMed

Wu, K W; Yang, P; Li, S S; Liu, C W; Sun, F Y

2015-07-09

We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents (Kv) and outward delayed-rectifier potassium currents (IK), but not outward transient potassium currents (IA), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced IK increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of IK via activation of the PI3-K signaling pathway. Published by Elsevier Ltd.

Bioenergetics of Mycobacterium: An Emerging Landscape for Drug Discovery

PubMed Central

Iqbal, Iram Khan; Bajeli, Sapna; Akela, Ajit Kumar

2018-01-01

Mycobacterium tuberculosis (Mtb) exhibits remarkable metabolic flexibility that enables it to survive a plethora of host environments during its life cycle. With the advent of bedaquiline for treatment of multidrug-resistant tuberculosis, oxidative phosphorylation has been validated as an important target and a vulnerable component of mycobacterial metabolism. Exploiting the dependence of Mtb on oxidative phosphorylation for energy production, several components of this pathway have been targeted for the development of new antimycobacterial agents. This includes targeting NADH dehydrogenase by phenothiazine derivatives, menaquinone biosynthesis by DG70 and other compounds, terminal oxidase by imidazopyridine amides and ATP synthase by diarylquinolines. Importantly, oxidative phosphorylation also plays a critical role in the survival of persisters. Thus, inhibitors of oxidative phosphorylation can synergize with frontline TB drugs to shorten the course of treatment. In this review, we discuss the oxidative phosphorylation pathway and development of its inhibitors in detail. PMID:29473841

Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Zhengyu; Department of Neurology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437; Yang, Qi

2014-03-28

Highlights: • TK promotes EGFR phosphorylation in SH-SY5Y cells. • TK activates ERK1/2 and p38 phosphorylation in SH-SY5Y cells. • TK mediates SH-SY5Y cell proliferation via EGFR and ERK1/2 pathway. - Abstract: Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depletedmore » of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ciomperlik, Jessica J.; Basta, Holly A.; Palmenberg, Ann C., E-mail: acpalmen@wisc.edu

Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler's murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts.more » The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions. - Highlights: • Nup98, but not Nup50 becomes phosphorylated by cardiovirus Leader protein-dependent mechanisms. • At least four independent nucleocytoplasmic trafficking pathways are inhibited by this process. • Nups must be presented in a nuclear pore context for Leader-directed phosphorylation. • Leader, by itself, does not cause activation of cellular kinases.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2more » activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular fractions from control, MVAL or propiconazole-treated cells revealed increased Ras protein in the cytoplasmic fraction of L-744,832-treated cells, while propiconazole or MVAL reversed these effects. Western blot analysis indicated that phosphorylation of Erk1/2, a protein downstream of Ras, was increased by propiconazole. These data indicate that propiconazole increases cell proliferation by increasing the levels of cholesterol biosynthesis intermediates presumably through a negative feedback mechanism within the pathway, a result of CYP51 inhibition. This feedback mechanism increases Erk1/2 signaling through mevalonate-mediated Ras activation. These results provide an explanation for the observed effects of propiconazole on hepatic cholesterol pathways and on the increased hepatic cell proliferation induced by propiconazole in mice. Highlights: ► Propiconazole increases cell proliferation in AML12 mouse hepatocytes. ► Propiconazole increases Ras farnesylation and alters Ras membrane localization. ► Propiconazole increases Erk1/2 phosphorylation. ► Dysregulation of the cholesterol biosynthesis pathway can explain these results. ► These results can explain similar effects induced by propiconazole in mice.« less

Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

PubMed

Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

2017-11-15

A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min -1 , together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in itself. Copyright © 2017 Elsevier B.V. All rights reserved.

Wnt/β-catenin signaling: new (and old) players and new insights

PubMed Central

Huang, He; He, Xi

2008-01-01

Wnt/β-catenin signaling has central roles in embryogenesis and human diseases including cancer. A central scheme of the Wnt pathway is to stabilize the transcription coactivator β-catenin by preventing its phosphorylation-dependent degradation. Significant progress has been made towards the understanding of this critical regulatory pathway, including the protein complex that promotes β-catenin phosphorylation-degradation, and the mechanism by which the extracellular Wnt ligand engages cell surface receptors to inhibit β-catenin phosphorylation-degradation. Here we review some recent discoveries in these two areas, and highlight some critical questions that remain to be resolved. PMID:18339531

Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells.

PubMed

Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

2016-01-01

Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways.

Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells

PubMed Central

Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

2016-01-01

Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways. PMID:27143997

Activation of AMPK reduces the co-transporter activity of NKCC1.

PubMed

Fraser, Scott A; Davies, Matthew; Katerelos, Marina; Gleich, Kurt; Choy, Suet-Wan; Steel, Rohan; Galic, Sandra; Mount, Peter F; Kemp, Bruce E; Power, David A

2014-01-01

The co-transporter activity of Na(+)-K(+)-2Cl(-) 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1(T212/T217) phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1-293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217.

Analysis of genes involved in the PI3K/Akt pathway in radiation- and MNU-induced rat mammary carcinomas

PubMed Central

Showler, Kaye; Nishimura, Mayumi; Imaoka, Tatsuhiko; Nishimura, Yukiko; Morioka, Takamitsu; Blyth, Benjamin J.; Kokubo, Toshiaki; Takabatake, Masaru; Fukuda, Maki; Moriyama, Hitomi; Kakinuma, Shizuko; Fukushi, Masahiro

2017-01-01

Abstract The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis. PMID:27738081

Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

PubMed

Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

2017-07-01

TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Defective insulin signaling pathway and increased glycogen synthase kinase-3 activity in the brain of diabetic mice: parallels with Alzheimer's disease and correction by insulin.

PubMed

Jolivalt, C G; Lee, C A; Beiswenger, K K; Smith, J L; Orlov, M; Torrance, M A; Masliah, E

2008-11-15

We have evaluated the effect of peripheral insulin deficiency on brain insulin pathway activity in a mouse model of type 1 diabetes, the parallels with Alzheimer's disease (AD), and the effect of treatment with insulin. Nine weeks of insulin-deficient diabetes significantly impaired the learning capacity of mice, significantly reduced insulin-degrading enzyme protein expression, and significantly reduced phosphorylation of the insulin-receptor and AKT. Phosphorylation of glycogen synthase kinase-3 (GSK3) was also significantly decreased, indicating increased GSK3 activity. This evidence of reduced insulin signaling was associated with a concomitant increase in tau phosphorylation and amyloid beta protein levels. Changes in phosphorylation levels of insulin receptor, GSK3, and tau were not observed in the brain of db/db mice, a model of type 2 diabetes, after a similar duration (8 weeks) of diabetes. Treatment with insulin from onset of diabetes partially restored the phosphorylation of insulin receptor and of GSK3, partially reduced the level of phosphorylated tau in the brain, and partially improved learning ability in insulin-deficient diabetic mice. Our data indicate that mice with systemic insulin deficiency display evidence of reduced insulin signaling pathway activity in the brain that is associated with biochemical and behavioral features of AD and that it can be corrected by insulin treatment.

Various stressors rapidly activate the p38-MAPK signaling pathway in Mytilus galloprovincialis (Lam.).

PubMed

Gaitanaki, Catherine; Kefaloyianni, Erene; Marmari, Athina; Beis, Isidoros

2004-05-01

The stimulation of p38-MAPK signal transduction pathway by various stressful stimuli was investigated in the marine bivalve M. galloprovincialis. Oxidative stress (5 microM H2O2) induced a biphasic pattern of p38-MAPK phosphorylation with maximal values attained at 15 min (8.1-fold) and 1 h (8.0-fold) of treatment respectively. Furthermore, 1 microM SB203580 abolished the p38-MAPK phosphorylation induced by oxidative stress. Aerial exposure also induced a biphasic pattern of p38-MAPK phosphorylation, with maximal values attained at 1 h (6.8-fold) and 8 h (4.9-fold) respectively. Re-oxygenation following a 15 min of aerial exposure resulted in the progressive dephosphorylation of the kinase. Treatment with 0.5 M sorbitol (in normal seawater) induced the rapid kinase phosphorylation (9.2-fold) and this effect was reversible. Seawater salinities varying between 100-60% had no effect, whereas a salinity of 50% induced a significant p38-MAPK phosphorylation. Furthermore, hypertonicity (120% seawater) resulted in a moderate kinase phosphorylation. All the above results demonstrate for the first time in a marine invertebrate imposed to environmental and other forms of stress as an intact, living organism, that the p38-MAPK pathway is specifically activated by various stressful stimuli which this animal can often face and sustain in vivo.

Selective Effects of PD-1 on Akt and Ras Pathways Regulate Molecular Components of the Cell Cycle and Inhibit T Cell Proliferation

PubMed Central

Patsoukis, Nikolaos; Brown, Julia; Petkova, Victoria; Liu, Fang; Li, Lequn; Boussiotis, Vassiliki A.

2017-01-01

The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4+ T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCFSkp2 degrades p27kip1, an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G1 phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G1 phase inhibitor p15INK4 and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase–Akt and Ras–mitogen-activated and extracellular signal–regulated kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle. PMID:22740686

Inhibitory effects of CP on the growth of human gastric adenocarcinoma BGC-823 tumours in nude mice.

PubMed

Wang, Hai-Jun; Liu, Yu; Zhou, Bao-Jun; Zhang, Zhan-Xue; Li, Ai-Ying; An, Ran; Yue, Bin; Fan, Li-Qiao; Li, Yong

2018-05-01

Objective To investigate the potential antitumour effects of [2-(6-amino-purine-9-yl)-1-hydroxy-phosphine acyl ethyl] phosphonic acid (CP) against gastric adenocarcinoma. Methods Human BGC-823 xenotransplants were established in nude mice. Animals were randomly divided into control and CP groups, which were administered NaHCO 3 vehicle alone or CP dissolved in NaHCO 3 (200 µg/kg body weight) daily, respectively. Tumour volume was measured weekly for 6 weeks. Resected tumours were assayed for proliferative activity with anti-Ki-67 or anti-proliferating cell nuclear antigen (PCNA) antibodies. Cell apoptosis was examined using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays and with caspase-3 immunostaining. Proteins were measured by Western blotting. Results There was a significant reduction in tumour volume and a reduced percentage of Ki-67-positive or PCNA-positive cells in the CP group compared with the control group. The percentage of TUNEL-positive or caspase 3-positive cells significantly increased following CP treatment compared with the control group. Tumours from the CP group had higher levels of phosphorylated-extracellular signal-regulated kinase (p-ERK) and phosphorylated-AKT (p-AKT) compared with control tumours. Conclusion CP treatment inhibited tumour growth and induced tumour cell apoptosis in a nude mouse model of BGC-823 gastric adenocarcinoma. Activation of the AKT and ERK signalling pathways may mediate this antitumour activity.

FK-3000 isolated from Stephania delavayi Diels. inhibits MDA-MB-231 cell proliferation by decreasing NF-κB phosphorylation and COX-2 expression.

PubMed

Xu, Hong De; Cho, Soon-Chang; Bang, Mi-Ae; Bae, Chun-Sik; Choi, Yeonshik; Li, Yong-Chun; Lim, Seung-Kil; Shim, Jaegal; Park, Dae-Hun

2015-01-01

The World Health Organization (WHO) has reported that cancer is one of the most prevalent diseases and a leading cause of death worldwide. Many anticancer drug development studies have been pursued over the last few decades and several viable drugs have been discovered, such as paclitaxel, topotecan and irinotecan. Previously, our research group uncovered the cytocidal and cytostatic effects of the plant Stephania delavayi Diels. In this study, we determined the active chemical to be 6,7-di-O-acetylsinococuline (FK-3000). The FK-3000 half maximal inhibitory concentration (IC50) in MDA-MB-231 breast carcinoma cells at 48 h was 0.52 µg/ml and it induced apoptosis in a dose- and time-dependent manner. FK-3000 suppressed NF-κB nuclear translocation, decreased NF-κB phosphorylation, and decreased COX-2 protein expression. MDA-MB-231 xenografted mice were treated with FK-3000, Taxol, or their combination for 21 days. The tumor size was smallest in the co-treatment group, indicating that FK-3000 may have a synergistic effect with Taxol. FK-3000 treatment showed no adverse effects on blood cell counts, serum protein levels, or pathology. These studies demonstrate that FK-3000, isolated from S. delavayi Diels., is a promising, pathway-specific anticancer agent that exhibits low toxicity.

Echinocystic acid inhibits RANKL-induced osteoclastogenesis by regulating NF-κB and ERK signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Jian-hui, E-mail: jianhui_yangxa@163.com; Li, Bing; Wu, Qiong

Receptor activator of nuclear factor-κB ligand (RANKL) is a key factor in the differentiation and activation of osteoclasts. Echinocystic acid (EA), a pentacyclic triterpene isolated from the fruits of Gleditsia sinensis Lam, was reported to prevent reduction of bone mass and strength and improve the cancellous bone structure and biochemical properties in ovariectomy rats. However, the molecular mechanism of EA on the osteoclast formation has not been reported. The purpose of this study was to investigate the effects and mechanism of EA on RANKL-induced osteoclastogenesis. Our results showed that EA inhibited the formation of osteoclast, as well as the expressionmore » of osteoclastogenesis-related marker proteins in bone marrow macrophages (BMMs). At molecular levels, EA inhibited RANKL-induced NF-κB activation and ERK phosphorylation in BMMs. In conclusion, the present study demonstrated that EA can suppress osteoclastogenesis in vitro. Moreover, we clarified that these inhibitory effects of EA occur through suppression of NF-κB and ERK activation. Therefore, EA may be a potential agent in the treatment of osteoclast-related diseases such as osteoporosis. - Highlights: • EA inhibited the formation of osteoclast in BMMs. • EA inhibits the expression of osteoclastogenesis-related marker proteins in BMMs. • EA inhibits RANKL-induced NF-κB activation in BMMs. • EA inhibits RANKL-induced ERK phosphorylation in BMMs.« less

Methionine sulfoximine-treatment and carbon starvation elicit Snf1-independent phosphorylation of the transcription activator Gln3 in Saccharomyces cerevisiae

PubMed Central

Tate, Jennifer J.; Rai, Rajendra; Cooper, Terrance G.

2008-01-01

SUMMARY Tor proteins are global regulators situated at the top of a signal transduction pathway conserved from yeast to humans. Specific inhibition of the two S. cerevisiae Tor proteins by rapamycin alters many cellular processes and the expression of hundreds of genes. Among the regulated genes are those whose expression is activated by the GATA-family transcription activator, Gln3. The extent of Gln3 phosphorylation has been thought to determine its intracellular localization, with phosphorylated and dephosphorylated forms accumulating in the cytoplasm and nucleus, respectively. Data presented here demonstrate that rapamycin and the glutamine synthetase inhibitor, methionine sulfoximine (MSX), although eliciting the same outcomes with respect to Gln3-Myc13 nuclear accumulation and NCR-sensitive transcription, generate diametrically opposite effects on Gln3-Myc13 phosphorylation. MSX increases Gln3-Myc13 phosphorylation while rapamycin decreases it. Gln3-Myc13 phosphorylation levels are regulated by at least three mechanisms: (i) one, observed during carbon starvation, depends on Snf1 kinase, (ii) another, observed during both carbon-starvation and MSX-treatment, is Snf1-independent, and (iii) the last is rapamycin-induced dephosphorylation. MSX and rapamycin act additively on Gln3-Myc13 phosphorylation, but MSX clearly predominates. These results suggest that MSX- and rapamycin-inhibited proteins are more likely to function in separate regulatory pathways than they are to function tandemly in a single pathway as previously thought. Further, Gln3 phosphorylation/dephosphorylation, that we and others have detected thus far, is not a demonstrably required step in achieving Gln3 nuclear localization and NCR-sensitive transcription in response to MSX- or rapamycin-treatment. PMID:15911613

Excitatory and inhibitory STDP jointly tune feedforward neural circuits to selectively propagate correlated spiking activity

PubMed Central

Kleberg, Florence I.; Fukai, Tomoki; Gilson, Matthieu

2014-01-01

Spike-timing-dependent plasticity (STDP) has been well established between excitatory neurons and several computational functions have been proposed in various neural systems. Despite some recent efforts, however, there is a significant lack of functional understanding of inhibitory STDP (iSTDP) and its interplay with excitatory STDP (eSTDP). Here, we demonstrate by analytical and numerical methods that iSTDP contributes crucially to the balance of excitatory and inhibitory weights for the selection of a specific signaling pathway among other pathways in a feedforward circuit. This pathway selection is based on the high sensitivity of STDP to correlations in spike times, which complements a recent proposal for the role of iSTDP in firing-rate based selection. Our model predicts that asymmetric anti-Hebbian iSTDP exceeds asymmetric Hebbian iSTDP for supporting pathway-specific balance, which we show is useful for propagating transient neuronal responses. Furthermore, we demonstrate how STDPs at excitatory–excitatory, excitatory–inhibitory, and inhibitory–excitatory synapses cooperate to improve the pathway selection. We propose that iSTDP is crucial for shaping the network structure that achieves efficient processing of synchronous spikes. PMID:24847242

The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

PubMed Central

Craig, Julie; Mikhailenko, Irina; Noyes, Nathaniel; Migliorini, Mary; Strickland, Dudley K.

2013-01-01

Background The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule. Methodology/Principal Findings To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis. Conclusions/Significance Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events. PMID:23922991

Insulin-like growth factor 1 rescues R28 retinal neurons from apoptotic death through ERK-mediated BimEL phosphorylation independent of Akt.

PubMed

Kong, Dejuan; Gong, Lijie; Arnold, Edith; Shanmugam, Sumathi; Fort, Patrice E; Gardner, Thomas W; Abcouwer, Steven F

2016-10-01

Insulin-like growth factor 1 (IGF-1) can provide long-term neurotrophic support by activation of Akt, inhibition of FoxO nuclear localization and suppression of Bim gene transcription in multiple neuronal systems. However, MEK/ERK activation can also promote neuron survival through phosphorylation of BimEL. We explored the contribution of the PI3K/Akt/FoxO and MEK/ERK/BimEL pathways in IGF-1 stimulated survival after serum deprivation (SD) of R28 cells differentiated to model retinal neurons. IGF-1 caused rapid activation of Akt leading to FoxO1/3-T32/T24 phosphorylation, and prevented FoxO1/3 nuclear translocation and Bim mRNA upregulation in response to SD. IGF-1 also caused MAPK/MEK pathway activation as indicated by ERK1/2-T202/Y204 and Bim-S65 phosphorylation. Overexpression of FoxO1 increased Bim mRNA expression and amplified the apoptotic response to SD without shifting the serum response curve. Inhibition of Akt activation with LY294002 or by Rictor knockdown did not block the protective effect of IGF-1, while inhibition of MEK activity with PD98059 prevented Bim phosphorylation and blocked IGF-1 protection. In addition, knockdown of Bim expression was protective during SD, while co-silencing of FoxO1 and Fox03 expression had little effect. Thus, the PI3K/Akt/FoxO pathway was not essential for protection from SD-induced apoptosis by IGF-1 in R28 cells. Instead, IGF-1 protection was dependent on activation of the MEK/ERK pathway leading to BimEL phosphorylation, which is known to prevent Bax/Bak oligomerization and activation of the intrinsic mitochondrial apoptosis pathway. These studies demonstrate the requirement of the MEK/ERK pathway in a model of retinal neuron cell survival and highlight the cell specificity for IGF-1 signaling in this response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Saussurea lappa extract suppresses TPA-induced cell invasion via inhibition of NF-κB-dependent MMP-9 expression in MCF-7 breast cancer cells

PubMed Central

2014-01-01

Background Saussurea lappa (SL) has been used as a traditional herbal medicine to treat abdominal pain and tenesmus, and has been suggested to possess various biological activities, including anti-tumor, anti-ulcer, anti-inflammatory, anti-viral, and cardiotonic activities. The effect of SL on breast cancer metastasis, however, is unknown. Cell migration and invasion are crucial in neoplastic metastasis. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is a major component in cancer cell invasion. Methods Cell viability was examined by MTT assay, whereas cell motility was measured by invasion assay. Western blot, Real-time PCR, and Zymography assays were used to investigate the inhibitory effects of ESL on matrix metalloproteinase-9 (MMP-9) expression level in MCF-7 cells. EMSA confirmed the inhibitory effects of ESL on DNA binding of NF- κB in MCF-7 cells. Results Cells threated with various concentrations of Saussurea lappa (ESL) for 24 h. Concentrations of 2 or 4 μM did not lead to a significant change in cell viability or morphology. Therefore, subsequent experiments utilized the optimal non-toxic concentration (2 or 4 μM) of ESL. In this study, we investigated the inhibitory effect of ethanol extract of ESL on MMP-9 expression and cell invasion in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MCF-7 cells. ESL inhibited the TPA-induced transcriptional activation of nuclear factor-kappa B (NF-κB). However, this result obtained that ESL did not block the TPA-induced phosphorylation of the kinases: p38, ERK, and JNK. Therefore, ELS-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of NF-kB pathway in MCF-7 cells. Conclusions These results indicate that ELS-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of NF-kB pathway in MCF-7 cells. Thus, ESL has potential for controlling breast cancer invasiveness in vitro. PMID:24885456

Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells.

PubMed

Gorelik, Gabriela J; Yarlagadda, Sushma; Patel, Dipak R; Richardson, Bruce C

2012-09-01

CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T(505) in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T(505) phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells. CD4+ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation. Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T(505) phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA-stimulated T(505) phosphorylation, similar to PKCδ in peroxynitrite-treated cells. Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus. Copyright © 2012 by the American College of Rheumatology.

Herpes simplex virus triggers activation of calcium-signaling pathways

PubMed Central

Cheshenko, Natalia; Del Rosario, Brian; Woda, Craig; Marcellino, Daniel; Satlin, Lisa M.; Herold, Betsy C.

2003-01-01

The cellular pathways required for herpes simplex virus (HSV) invasion have not been defined. To test the hypothesis that HSV entry triggers activation of Ca2+-signaling pathways, the effects on intracellular calcium concentration ([Ca2+]i) after exposure of cells to HSV were examined. Exposure to virus results in a rapid and transient increase in [Ca2+]i. Pretreatment of cells with pharmacological agents that block release of inositol 1,4,5-triphosphate (IP3)–sensitive endoplasmic reticulum stores abrogates the response. Moreover, treatment of cells with these pharmacological agents inhibits HSV infection and prevents focal adhesion kinase (FAK) phosphorylation, which occurs within 5 min after viral infection. Viruses deleted in glycoprotein L or glycoprotein D, which bind but do not penetrate, fail to induce a [Ca2+]i response or trigger FAK phosphorylation. Together, these results support a model for HSV infection that requires activation of IP3-responsive Ca2+-signaling pathways and that is associated with FAK phosphorylation. Defining the pathway of viral invasion may lead to new targets for anti-viral therapy. PMID:14568989

A new MCM modification cycle regulates DNA replication initiation

PubMed Central

Wei, Lei; Zhao, Xiaolan

2016-01-01

The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1 and activated in S phase to initiate replication. During this transition, the only known chemical change on MCM is the gain of multi-site phosphorylation that promotes cofactor recruitment. As replication initiation is intimately linked to multiple biological cues, additional changes on MCM can provide further regulatory points. Here, we describe a yeast MCM sumoylation cycle that negatively regulates replication. MCM subunits undergo sumoylation upon loading at origins in G1 prior to MCM phosphorylation. MCM sumoylation levels then decline as MCM phosphorylation levels rise, suggesting an inhibitory role in replication. Indeed, increasing MCM sumoylation impairs replication initiation through promoting the recruitment of a phosphatase that reduces MCM phosphorylation and activation. MCM sumoylation thus counterbalances kinase-based regulation to ensure accurate control of replication initiation. PMID:26854664

A new MCM modification cycle regulates DNA replication initiation.

PubMed

Wei, Lei; Zhao, Xiaolan

2016-03-01

The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1, and it initiates replication after being activated in S phase. During this transition, the only known chemical change to MCM is the gain of multisite phosphorylation that promotes cofactor recruitment. Because replication initiation is intimately linked to multiple biological cues, additional changes to MCM can provide further regulatory points. Here, we describe a yeast MCM SUMOylation cycle that regulates replication. MCM subunits undergo SUMOylation upon loading at origins in G1 before MCM phosphorylation. MCM SUMOylation levels then decline as MCM phosphorylation levels rise, thus suggesting an inhibitory role of MCM SUMOylation during replication. Indeed, increasing MCM SUMOylation impairs replication initiation, partly through promoting the recruitment of a phosphatase that decreases MCM phosphorylation and activation. We propose that MCM SUMOylation counterbalances kinase-based regulation, thus ensuring accurate control of replication initiation.

The leukocyte receptor CD84 inhibits Fc epsilon RI-mediated signaling through homophilic interaction in transfected RBL-2H3 cells.

PubMed

Oliver-Vila, Irene; Saborit-Villarroya, Ifigènia; Engel, Pablo; Martin, Margarita

2008-04-01

Signaling through the high-affinity receptor for immunoglobulin E (Fc epsilon RI) results in the coordinated activation of tyrosine kinases, thus leading to calcium mobilization, degranulation, and leukotriene and cytokine synthesis. Here, we show that CD84, a member of the CD150 family of leukocyte receptors, inhibits Fc epsilon RI-mediated mast cell degranulation in CD84-transfected rat basophilic leukaemia-2H3 mast cell line cells (RBL-2H3) through homophilic interaction. There was no reduction in overall protein phosphorylation following IgE triggering in CD84 RBL-2H3 cells. Indeed, phosphorylation of Dok-1 and c-Cbl increased in CD84 RBL-2H3, suggesting that inhibition is mediated by these molecules. MAP kinase phosphorylation (ERK1/2, JNK and p38) and cytokine synthesis were impaired in CD84 RBL-2H3. This inhibitory mechanism was independent of SAP and SHP-2 recruitment. Interestingly, CD84 mutants in tyrosines (Y279F and DeltaY324) reversed this inhibitory profile. These data suggest that CD84 may play a role in modulating Fc epsilon RI-mediated signaling in mast cells. Thus, CD84 could play a protective role against undesired allergic and inflammatory responses.

Matrix metalloproteinase-1 inhibitory activities of Morinda citrifolia seed extract and its constituents in UVA-irradiated human dermal fibroblasts.

PubMed

Masuda, Megumi; Murata, Kazuya; Naruto, Shunsuke; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

2012-01-01

The objective of this study was to examine whether a 50% ethanolic extract (MCS-ext) of the seeds of Morinda citrifolia (noni) and its constituents have matrix metalloproteinase-1 (MMP-1) inhibitory activity in UVA-irradiated normal human dermal fibroblasts (NHDFs). The MCS-ext (10 μg/mL) inhibited MMP-1 secretion from UVA-irradiated NHDFs, without cytotoxic effects, at 48 h after UV exposure. The ethyl acetate-soluble fraction of MCS-ext was the most potent inhibitor of MMP-1 secretion. Among the constituents of the fraction, a lignan, 3,3'-bisdemethylpinoresinol (1), inhibited the MMP-1 secretion at a concentration of 0.3 μM without cytotoxic effects. Furthermore, 1 (0.3 μM) reduced the level of intracellular MMP-1 expression. Other constituents, namely americanin A (2), quercetin (3) and ursolic acid (4), were inactive. To elucidate inhibition mechanisms of MMP-1 expression and secretion, the effect of 1 on mitogen-activated protein kinases (MAPKs) phosphorylation was examined. Western blot analysis revealed that 1 (0.3 μM) reduced the phosphorylations of p38 and c-Jun-N-terminal kinase (JNK). These results suggested that 1 suppresses intracellular MMP-1 expression, and consequent secretion from UVA-irradiated NHDFs, by down-regulation of MAPKs phosphorylation.

Dynamic Light Scattering Analysis of the Effect of Phosphorylated Osteopontin Peptides on Mineral Formation

NASA Astrophysics Data System (ADS)

Mozaffari, Maryam; Goiko, Maria; de Bruyn, John; Goldberg, Harvey

2015-03-01

Biomineralization is the process by which living organisms synthesize minerals. Osteopontin (OPN), a mineral-associated protein, has been shown to be a potent inhibitor of mineral formation, a process that is dependent on phosphorylation. To gain a better understanding of the mechanism of inhibition, dynamic light scattering (DLS) was used to monitor the initial stages of nucleation, providing information about the size and relative concentration of the growing crystals as a function of time. DLS was used to investigate the effect of phosphorylated (P3, pOPAR) and non-phosphorylated (P0, OPAR) OPN peptides on the formation and growth of hydroxyapatite (HA) crystals from supersaturated solutions of calcium and phosphate ions. The non-phosphorylated P0 had a limited effect on HA nucleation and growth, while its thrice-phosphorylated isoform, P3, was a potent inhibitor of HA nucleation. The aspartic acid-rich OPAR was found to moderately inhibit nucleation but not growth, while its singly-phosphorylated isoform, pOPAR, inhibited HA nucleation more effectively, with some effect on HA crystal growth. The order of the inhibitory potential of these peptides was pOPAR>OPAR>P3>P0. This work confirms that highly acidic and phosphorylated peptides can inhibit the nucleation of HA more effectively.

Effects of a brief high-fat diet and acute exercise on the mTORC1 and IKK/NF-κB pathways in rat skeletal muscle

PubMed Central

Castorena, Carlos M.; Arias, Edward B.; Sharma, Naveen; Cartee, Gregory D.

2016-01-01

One exercise session can improve subsequent insulin-stimulated glucose uptake by skeletal muscle in healthy and insulin-resistant individuals. Our first aim was to determine whether a brief (2 weeks) high-fat diet (HFD) that caused muscle insulin resistance would activate the mammalian target of rapamycin complex 1 (mTORC1) and/or inhibitor of κB kinase/nuclear factor κB (IKK/NF-κB) pathways, which are potentially linked to induction of insulin resistance. Our second aim was to determine whether acute exercise that improved insulin-stimulated glucose uptake by muscles would attenuate activation of these pathways. We compared HFD-fed rats with rats fed a low-fat diet (LFD). Some animals from each diet group were sedentary and others were studied 3 h postexercise, when insulin-stimulated glucose uptake was increased. The results did not provide evidence that brief HFD activated either the mTORC1 (including phosphorylation of mTORSer2448, TSC2Ser939, p70S6KThr412, and RPS6Ser235/236) or the IKK/NF-κB (including abundance of IκBα or phosphorylation of NF-κBSer536, IKKα/βSer177/181, and IκBSer32) pathway in insulin-resistant muscles. Exercise did not oppose the activation of either pathway, as evidenced by no attenuation of phosphorylation of key proteins in the IKK/NF-κB pathway (NF-κBSer536, IKKα/βSer177/181, and IκBSer32), unaltered IκBα abundance, and no attenuation of phosphorylation of key proteins in the mTORC1 pathway (mTORSer2448, TSC2Ser939, and RPS6Ser235/236). Instead, exercise induced greater phosphorylation of 2 proteins of the mTORC1 pathway (PRAS40Thr246 and p70S6KThr412) in insulin-stimulated muscles, regardless of diet. Insulin resistance induced by a brief HFD was not attributable to greater activation of the mTORC1 or the IKK/NF-κB pathway in muscle, and exercise-induced improvement in insulin sensitivity was not attributable to attenuated activation of these pathways in muscle. PMID:25706655

Caffeine inhibition of glycogen phosphorylase from Mytilus galloprovincialis mantle tissue.

PubMed

San Juan Serrano, F; Sánchez López, J L; García Martín, L O

1995-09-01

A different caffeine inhibition of both phosphorylated and unphosphorylated forms of glycogen phosphorylase from Mytilus mantle has been demonstrated. Caffeine increases the allosteric constant of phosphorylase b 30-fold, acting as an allosteric inhibitor (nH = 2) of mixed type with respect to inorganic phosphate (Pi) and AMP, and of single competitive type with respect to glycogen. The Mytilus phosphorylated form is also caffeine inhibited through competitive inhibition in relation to Pi and glycogen. In this case, the inhibitor does not modify the allosteric constant (near 2), neither does it display allosteric effects (nH = 1). The results demonstrate the notable modification of the nucleotide site promoted by the phosphorylation process and the existence of a functional inhibitory nucleoside site in Mytilus phosphorylase.

[Effect of endotoxin pretreatment-induced glycogen synthase kinase-3 inhibition on glycogen metabolism in rat liver and the mechanism].

PubMed

Chen, Xiaole; Gong, Jianping; Xu, Faliang

2014-02-01

To investigate the changes in the functional activity of glycogen synthase kinase-3 (GSK-3) in the hepatic tissue after endotoxin (lipopolysaccharide, LPS) tolerance and explore the effects of LPS-induced GSK-3 inhibition on glycogen metabolism in the liver. Male SD rats were randomly divided into normal control, endotoxin pretreatment and GSK-3 inhibitor (lithium chloride) groups with corresponding pretreatments prior to a large dose of LPS challenge (10 mg/kg) to induce liver injury. Glycogen deposition and content in the hepatic tissue was detected using periodic acid-Schiff (PAS) staining and a glycogen quantification kit, respectively. Western blotting was performed for semi-quantitative analysis of protein level and inhibitory phosphorylation of GSK-3, and a Coomassie brilliant blue G-250-based colorimetric assay was used to detect calpain activity in the liver. Glycogen content in the liver decreased significantly after LPS challenge in all the 3 groups (P<0.05) but showed no significant difference among the groups (P>0.05). Both LPS and lithium chloride pretreatments caused a significant increase of liver glycogen content (P<0.05). LPS pretreatment induced inhibitory phosphorylation of GSK-3β (P<0.05) and partial cleavage of GSK-3α but did not affect the expression of GSK-3 protein (P>0.05). Large-dose LPS challenge significantly increased the activity of calpain in the liver tissue (P<0.05) to a comparable level in the 3 groups (P>0.05). Endotoxin pretreatment induces inhibitory phosphorylation of GSK-3β and partial cleavage of GSK-3α and promotes the deposition of liver glycogen but does not affect the activity of calpain, which may contribute to an increased glycogen reserve for energy supply in the event of large-dose LPS challenge.

The In Vivo Activity of Ime1, the Key Transcriptional Activator of Meiosis-Specific Genes in Saccharomyces cerevisiae, Is Inhibited by the Cyclic AMP/Protein Kinase A Signal Pathway through the Glycogen Synthase Kinase 3-β Homolog Rim11

PubMed Central

Rubin-Bejerano, Ifat; Sagee, Shira; Friedman, Osnat; Pnueli, Lilach; Kassir, Yona

2004-01-01

Phosphorylation is the main mode by which signals are transmitted to key regulators of developmental pathways. The glycogen synthase kinase 3 family plays pivotal roles in the development and well-being of all eukaryotic organisms. Similarly, the budding yeast homolog Rim11 is essential for the exit of diploid cells from the cell cycle and for entry into the meiotic developmental pathway. In this report we show that in vivo, in cells grown in a medium promoting vegetative growth with acetate as the sole carbon source (SA medium), Rim11 phosphorylates Ime1, the master transcriptional activator required for entry into the meiotic cycle and for the transcription of early meiosis-specific genes. We demonstrate that in the presence of glucose, the kinase activity of Rim11 is inhibited. This inhibition could be due to phosphorylation on Ser-5, Ser-8, and/or Ser-12 because in the rim11S5AS8AS12A mutant, Ime1 is incorrectly phosphorylated in the presence of glucose and cells undergo sporulation. We further show that this nutrient signal is transmitted to Rim11 and consequently to Ime1 by the cyclic AMP/protein kinase A signal transduction pathway. Ime1 is phosphorylated in SA medium on at least two residues, Tyr-359 and Ser-302 and/or Ser-306. Ser-302 and Ser-306 are part of a consensus site for the mammalian homolog of Rim11, glycogen synthase kinase 3-β. Phosphorylation on Tyr-359 but not Ser-302 or Ser-306 is essential for the transcription of early meiosis-specific genes and sporulation. We show that Tyr-359 is phosphorylated by Rim11. PMID:15282298

A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration.

PubMed

Ziegler, Susanne; Eiseler, Tim; Scholz, Rolf-Peter; Beck, Alexander; Link, Gisela; Hausser, Angelika

2011-03-01

The multifunctional signal adapter protein Ras and Rab interactor 1 (RIN1) is a Ras effector protein involved in the regulation of epithelial cell processes such as cell migration and endocytosis. RIN1 signals via two downstream pathways, namely the activation of Rab5 and Abl family kinases. Protein kinase D (PKD) phosphorylates RIN1 at serine 351 in vitro, thereby regulating interaction with 14-3-3 proteins. Here, we report the identification of serine 292 in RIN1 as an in vivo PKD phosphorylation site. PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry. We demonstrate that phosphorylation at serine 292 controls RIN1-mediated inhibition of cell migration by modulating the activation of Abl kinases. We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD. Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.

Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

PubMed Central

Zhou, Yue; Yamada, Naoki; Tanaka, Tomohiro; Hori, Takashi; Yokoyama, Satoru; Hayakawa, Yoshihiro; Yano, Seiji; Fukuoka, Junya; Koizumi, Keiichi; Saiki, Ikuo; Sakurai, Hiroaki

2015-01-01

Crosstalk between inflammatory signalling pathways and receptor tyrosine kinases has been revealed as an indicator of cancer malignant progression. In the present study, we focus on EphA2 receptor tyrosine kinase, which is overexpressed in many human cancers. It has been reported that ligand-independent phosphorylation of EphA2 at Ser-897 is induced by Akt. We show that inflammatory cytokines promote RSK-, not Akt-, dependent phosphorylation of EphA2 at Ser-897. In addition, the RSK–EphA2 signalling pathway controls cell migration and invasion of metastatic breast cancer cells. Moreover, Ser-897-phosphorylated EphA2 co-localizes with phosphorylated active form of RSK in various human tumour specimens, and this double positivity is related to poor survival in lung cancer patients, especially those with a smoking history. Taken together, these results indicate that the phosphorylation of EphA2 at Ser-897 is controlled by RSK and the RSK–EphA2 axis might contribute to cell motility and promote tumour malignant progression. PMID:26158630

Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2.

PubMed

Zhou, Yue; Yamada, Naoki; Tanaka, Tomohiro; Hori, Takashi; Yokoyama, Satoru; Hayakawa, Yoshihiro; Yano, Seiji; Fukuoka, Junya; Koizumi, Keiichi; Saiki, Ikuo; Sakurai, Hiroaki

2015-07-09

Crosstalk between inflammatory signalling pathways and receptor tyrosine kinases has been revealed as an indicator of cancer malignant progression. In the present study, we focus on EphA2 receptor tyrosine kinase, which is overexpressed in many human cancers. It has been reported that ligand-independent phosphorylation of EphA2 at Ser-897 is induced by Akt. We show that inflammatory cytokines promote RSK-, not Akt-, dependent phosphorylation of EphA2 at Ser-897. In addition, the RSK-EphA2 signalling pathway controls cell migration and invasion of metastatic breast cancer cells. Moreover, Ser-897-phosphorylated EphA2 co-localizes with phosphorylated active form of RSK in various human tumour specimens, and this double positivity is related to poor survival in lung cancer patients, especially those with a smoking history. Taken together, these results indicate that the phosphorylation of EphA2 at Ser-897 is controlled by RSK and the RSK-EphA2 axis might contribute to cell motility and promote tumour malignant progression.

The Methoxyflavonoid Isosakuranetin Suppresses UV-B-Induced Matrix Metalloproteinase-1 Expression and Collagen Degradation Relevant for Skin Photoaging.

PubMed

Jung, Hana; Lee, Eunjoo H; Lee, Tae Hoon; Cho, Man-Ho

2016-09-01

Solar ultraviolet (UV) radiation is a main extrinsic factor for skin aging. Chronic exposure of the skin to UV radiation causes the induction of matrix metalloproteinases (MMPs), such as MMP-1, and consequently results in alterations of the extracellular matrix (ECM) and skin photoaging. Flavonoids are considered as potent anti-photoaging agents due to their UV-absorbing and antioxidant properties and inhibitory activity against UV-mediated MMP induction. To identify anti-photoaging agents, in the present study we examined the preventative effect of methoxyflavonoids, such as sakuranetin, isosakuranetin, homoeriodictyol, genkwanin, chrysoeriol and syringetin, on UV-B-induced skin photo-damage. Of the examined methoxyflavonoids, pretreatment with isosakuranetin strongly suppressed the UV-B-mediated induction of MMP-1 in human keratinocytes in a concentration-dependent manner. Isosakuranetin inhibited UV-B-induced phosphorylation of mitogen-activated protein kinase (MAPK) signaling components, ERK1/2, JNK1/2 and p38 proteins. This result suggests that the ERK1/2 kinase pathways likely contribute to the inhibitory effects of isosakuranetin on UV-induced MMP-1 production in human keratinocytes. Isosakuranetin also prevented UV-B-induced degradation of type-1 collagen in human dermal fibroblast cells. Taken together, our findings suggest that isosakuranetin has the potential for development as a protective agent for skin photoaging through the inhibition of UV-induced MMP-1 production and collagen degradation.

Ectopic expression of the Drosophila Cdk1 inhibitory kinases, Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development.

PubMed Central

Price, Donald M; Jin, Zhigang; Rabinovitch, Simon; Campbell, Shelagh D

2002-01-01

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis. PMID:12072468

The anti-HER3 antibody in combination with trastuzumab exerts synergistic antitumor activity in HER2-positive gastric cancer.

PubMed

Wang, Qiwei; Zhang, Xiaotian; Shen, Enyun; Gao, Jing; Cao, Fengqi; Wang, Xiaojuan; Li, Yilin; Tian, Tiantian; Wang, Jingyuan; Chen, Zuhua; Wang, Jiayuan; Shen, Lin

2016-09-28

The anti-HER2 monoclonal antibody trastuzumab is central to the treatment of HER2-positive gastric cancer (GC); however, its responses are limited. HER3 seems to be the preferred dimerization partner with HER2 and is emerging as a key target for complete blockade of downstream pathways and better clinical response. In this study, we report that novel anti-HER3 antibodies (1A5-3D4) that can neutralize multiple modes of HER3 activation, combined with trastuzumab, exhibited synergistic inhibitory effect on the cell proliferation in HER2-positive GC cell lines. Follow-up studies revealed that the combination treatment significantly inhibited phosphorylation of HER3 as well as AKT and ERK signals. In vivo experiments further showed that the anti-tumor effect of trastuzumab was enhanced by its combination with 1A5-3D4 in NCI-N87 xenograft and patient derived xenografts (PDX). Particularly in an HER2-negative whereas neuregulin1 (a ligand of HER3) positive PDX, the combination was also superior to monotherapy. 1A5-3D4 in combination with trastuzumab exhibits a synergistic inhibitory effect on tumor activity, suggesting that targeting both HER2 and HER3 resulted in an improved treatment effects on HER2-positive GC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

PubMed

Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

2018-05-01

Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts.

PubMed

Furukawa, Fukiko; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yoshida, Katsunori; Sugano, Yasushi; Yamagata, Hideo; Matsushita, Masanori; Seki, Toshihito; Inagaki, Yutaka; Nishizawa, Mikio; Fujisawa, Junichi; Inoue, Kyoichi

2003-10-01

Hepatic stellate cells (HSCs) spontaneously transdifferentiate into myofibroblast (MFB)-phenotype on plastic dishes. This response recapitulates the features of activation in vivo. Transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by MFBs. In quiescent HSCs, TGF-beta signaling involves TGF-beta type I receptor (TbetaRI)-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. The middle linker regions of Smad2 and Smad3 also are phosphorylated by mitogen-activated protein kinase (MAPK). This study elucidates the change of Smad3-mediated signals during the transdifferentiation process. By using antibodies highly specific to the phosphorylated C-terminal region and the phosphorylated linker region of Smad3, we found that TGF-beta-dependent Smad3 phosphorylation at the C-terminal region decreased, but that the phosphorylation at the linker region increased in the process of transdifferentiation. TGF-beta activated the p38 MAPK pathway, further leading to Smad3 phosphorylation at the linker region in the cultured MFBs, irrespective of Smad2. The phosphorylation promoted hetero-complex formation and nuclear translocation of Smad3 and Smad4. Once combined with TbetaRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to plasminogen activator inhibitor-type I promoter could enhance the transcription. In addition, Smad3 phosphorylation mediated by the activated TbetaRI was impaired severely in MFBs during chronic liver injury, whereas Smad3 phosphorylation at the linker region was remarkably induced by p38 MAPK pathway. In conclusion, p38 MAPK-dependent Smad3 phosphorylation promoted extracellular matrix production in MFBs both in vitro and in vivo.

TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Feifei; Jiang, Yinan; Zheng, Qiping

Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involvedmore » in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.« less

(S)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

PubMed Central

Zheng, Weiwei; Yang, Bei; Pi, Jingbo; He, Gengsheng; Qu, Weidong

2012-01-01

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5′-triphosphate (ATP) levels, 3′-5′-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH. PMID:22916194

(S)-α-chlorohydrin inhibits protein tyrosine phosphorylation through blocking cyclic AMP - protein kinase A pathway in spermatozoa.

PubMed

Zhang, Hao; Yu, Huan; Wang, Xia; Zheng, Weiwei; Yang, Bei; Pi, Jingbo; He, Gengsheng; Qu, Weidong

2012-01-01

α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5'-triphosphate (ATP) levels, 3'-5'-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH.

Propofol mediates signal transducer and activator of transcription 3 activation and crosstalk with phosphoinositide 3-kinase/AKT.

PubMed

Shravah, Jayant; Wang, Baohua; Pavlovic, Marijana; Kumar, Ujendra; Chen, David Dy; Luo, Honglin; Ansley, David M

2014-01-01

We previously demonstrated that propofol, an intravenous anesthetic with anti-oxidative properties, activated the phosphoinositide 3-kinase (PI3K)/AKT pathway to increase the expression of B cell lymphoma (Bcl)-2 and, therefore the anti-apoptotic potential on cardiomyocytes. Here, we wanted to determine if propofol can also activate the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway, another branch of cardioprotective signaling. The cellular response of nuclear factor kappa B (NFκB) and STAT3 was also evaluated. Cardiac H9c2 cells were treated by propofol alone or in combination with pretreatment by inhibitors for JAK2/STAT3 or PI3K/AKT pathway. STAT3 and AKT phosphorylation, and STAT3 translocation were measured by western blotting and immunofluorescence staining, respectively. Propofol treatment significantly increased STAT3 phosphorylation at both tyrosine 705 and serine 727 residues. Sustained early phosphorylation of STAT3 was observed with 25~75 μM propofol at 10 and 30 min. Nuclear translocation of STAT3 was seen at 4 h after treatment with 50 μM propofol. In cultured H9c2 cells, we further demonstrated that propofol-induced STAT3 phosphorylation was reduced by pretreatment with PI3K/AKT pathway inhibitors wortmannin or API-2. Conversely, pretreatment with JAK2/STAT3 pathway inhibitor AG490 or stattic inhibited propofol-induced AKT phosphorylation. In addition, propofol induced NFκB p65 subunit perinuclear translocation. Inhibition or knockdown of STAT3 was associated with increased levels of the NFκB p65 subunit. Our results suggest that propofol induces an adaptive response by dual activation and crosstalk of cytoprotective PI3K/AKT and JAK2/STAT3 pathways. Rationale to apply propofol clinically as a preemptive cardioprotectant during cardiac surgery is supported by our findings.

Inhibition of protein phosphorylation in MIA pancreatic cancer cells: Confluence of metabolic and signaling pathways

PubMed Central

Zhang, Hengwei; Cao, Rui; Lee, Wai-Nang Paul; Deng, Caishu; Zhao, Yingchun; Lappe, Joan; Recker, Robert; Yen, Yun; Wang, Qi; Tsai, Ming-Ying; Go, Vay Liang; Xiao, Gary Guishan

2010-01-01

Oxythiamine (OT), a transketolase inhibitor, is known to inhibit pancreatic cancer cell proliferation. In this study, we investigated the effect of inhibition of the transketolase pathway on signaling pathways in MIA PaCa cancer cells using in-house proteomic techniques. We hypothesized that OT alter protein phosphorylation thus affecting cell cycle arrest and cell proliferation. MIA PaCa-2 cells were cultured in media containing an algal 15N amino acid mixture at 50% enrichment, with and without OT, to determine protein expression and synthesis. Analysis of cell lysates using two-dimensional gel electrophoresis matrix assisted laser desorption and ionization time-of-flight and time-of-flight mass spectrometry (2-DE-MALDI-TOF/TOF MS) identified 12 phosphor proteins that were significantly suppressed by OT treatment. Many of these proteins are involved in regulation of cycle activities and apoptosis. Among the proteins identified, expression of the phosphor heat shock protein 27 (Hsp27) was dramatically inhibited by OT treatment while the level of its total protein remained unchanged. Hsp27 expression and phoshporylation is known to be associated with drug resistance and cancer cell survival. The changes in phosphorylation of key proteins of cancer proliferation and survival suggest that protein phosphorylation is the confluence of the effects of OT on metabolic and signaling pathways. PMID:20035555

Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis*S⃞

PubMed Central

Ha, Chang Hoon; Wang, Weiye; Jhun, Bong Sook; Wong, Chelsea; Hausser, Angelika; Pfizenmaier, Klaus; McKinsey, Timothy A.; Olson, Eric N.; Jin, Zheng-Gen

2008-01-01

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cγ-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis. PMID:18332134

HSF1 stress response pathway regulates autophagy receptor SQSTM1/p62-associated proteostasis.

PubMed

Watanabe, Yoshihisa; Tsujimura, Atsushi; Taguchi, Katsutoshi; Tanaka, Masaki

2017-01-02

Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis.

Decreased Phosphorylated Protein Kinase B (Akt) in Individuals with Autism Associated with High Epidermal Growth Factor Receptor (EGFR) and Low Gamma-Aminobutyric Acid (GABA).

PubMed

Russo, Anthony J

2015-01-01

Dysregulation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway could contribute to the pathogenesis of autism spectrum disorders. In this study, phosphorylated Akt concentration was measured in 37 autistic children and 12, gender and age similar neurotypical, controls using an enzyme-linked immunosorbent assay. Akt levels were compared to biomarkers known to be associated with epidermal growth factor receptor (EGFR) and c-Met (hepatocyte growth factor (HGF) receptor) pathways and severity levels of 19 autism-related symptoms. We found phosphorylated Akt levels significantly lower in autistic children and low Akt levels correlated with high EGFR and HGF and low gamma-aminobutyric acid, but not other biomarkers. Low Akt levels also correlated significantly with increased severity of receptive language, conversational language, hypotonia, rocking and pacing, and stimming, These results suggest a relationship between decreased phosphorylated Akt and selected symptom severity in autistic children and support the suggestion that the AKT pathways may be associated with the etiology of autism.

HSF1 stress response pathway regulates autophagy receptor SQSTM1/p62-associated proteostasis

PubMed Central

Watanabe, Yoshihisa; Tsujimura, Atsushi; Taguchi, Katsutoshi; Tanaka, Masaki

2017-01-01

ABSTRACT Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis. PMID:27846364

Thromboxane A2-induced bi-directional regulation of cerebral arterial tone.

PubMed

Neppl, Ronald L; Lubomirov, Lubomir T; Momotani, Ko; Pfitzer, Gabriele; Eto, Masumi; Somlyo, Avril V

2009-03-06

Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.

Hepatocyte-protective effect of nectandrin B, a nutmeg lignan, against oxidative stress: Role of Nrf2 activation through ERK phosphorylation and AMPK-dependent inhibition of GSK-3β

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Jae-Sook; Kim, Eun-Kyung; Choi, Yong-Won

Oxidative stress can contribute to the development and progression of liver diseases, such as drug-induced or alcoholic liver injury, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. Nectandrin B is a bioactive lignan isolated from nutmeg extract. To date, little information is available about its pharmacological activities in the liver. This study investigated the hepatocyte-protective effect of nectandrin B against tert-butylhydroperoxide-induced oxidative injury and the underlying molecular mechanism. The cell viability assay revealed that nectandrin B prevents apoptosis stimulated by tert-butylhydroperoxide in both HepG2 cells and primary mouse hepatocytes. Nectandrin B also attenuated ROS production and restored the depleted glutathione level.more » Real-time PCR and immunoblot analyses showed that the expression of glutamate-cysteine ligase, an enzyme responsible for the glutathione biosynthesis, was induced by nectandrin B, indicating its indirect antioxidative effect. The NF-E2-related factor-2 (Nrf2) regulates gene expression of an array of antioxidant enzymes in hepatocytes. Nectandrin B stimulated Nrf2 activation as evidenced by its enhanced nuclear accumulation and increased antioxidant response element (ARE)-luciferase activity. Intriguingly, the hepatocyte-protective effect of nectandrin B against oxidative damage was completely abrogated by Nrf2 knockdown using Nrf2 specific siRNA. Nectandrin B promoted ERK activation, but inactivated GSK-3β through the AMPK-mediated inhibitory phosphorylation. The enforced overexpression of dominant-negative mutant of MEK1 or AMPKα, or wild-type GSK-3β inhibited the increase in the NQO1-ARE-luciferase activity stimulated by nectandrin B, suggesting that both ERK and AMPK-GSK-3β signalings are involved in the activation of Nrf2/ARE pathway by nectandrin B. Consistent with this, cytoprotection and restoration of glutathione level by nectandrin B was also blocked by the overexpression of dominant-negative MEK1 or wild-type GSK-3β. Finally, our data demonstrate that nectandrin B has the ability to protect hepatocytes against oxidative injury through the activation of Nrf2/ARE pathway mediated by ERK phosphorylation and AMPK-dependent inactivation of GSK-3β. - Highlight: • Nectandrin B, a nutmeg lignan protects hepatocytes against oxidative damage. • Nectandrin B exerts an indirect antioxidative effect via the activation of Nrf2. • Both ERK and GSK-3β pathways contribute to the activation of Nrf2 and hepatocyte-protection by nectandrin B. • Nectandrin B can inactivate GSK-3β through the activation of AMPK in HepG2 cells.« less

PTP1B Inhibitory and Anti-Inflammatory Effects of Secondary Metabolites Isolated from the Marine-Derived Fungus Penicillium sp. JF-55

PubMed Central

Lee, Dong-Sung; Jang, Jae-Hyuk; Ko, Wonmin; Kim, Kyoung-Su; Sohn, Jae Hak; Kang, Myeong-Suk; Ahn, Jong Seog; Kim, Youn-Chul; Oh, Hyuncheol

2013-01-01

Protein tyrosine phosphatase 1B (PTP1B) plays a major role in the negative regulation of insulin signaling, and is thus considered as an attractive therapeutic target for the treatment of diabetes. Bioassay-guided investigation of the methylethylketone extract of marine-derived fungus Penicillium sp. JF-55 cultures afforded a new PTP1B inhibitory styrylpyrone-type metabolite named penstyrylpyrone (1), and two known metabolites, anhydrofulvic acid (2) and citromycetin (3). Compounds 1 and 2 inhibited PTP1B activity in a dose-dependent manner, and kinetic analyses of PTP1B inhibition suggested that these compounds inhibited PTP1B activity in a competitive manner. In an effort to gain more biological potential of the isolated compounds, the anti-inflammatory effects of compounds 1–3 were also evaluated. Among the tested compounds, only compound 1 inhibited the production of NO and PGE2, due to the inhibition of the expression of iNOS and COX-2. Penstyrylpyrone (1) also reduced TNF-α and IL-1β production, and these anti-inflammatory effects were shown to be correlated with the suppression of the phosphorylation and degradation of IκB-α, NF-κB nuclear translocation, and NF-κB DNA binding activity. In addition, using inhibitor tin protoporphyrin (SnPP), an inhibitor of HO-1, it was verified that the inhibitory effects of penstyrylpyrone (1) on the pro-inflammatory mediators and NF-κB DNA binding activity were associated with the HO-1 expression. Therefore, these results suggest that penstyrylpyrone (1) suppresses PTP1B activity, as well as the production of pro-inflammatory mediators via NF-κB pathway, through expression of anti-inflammatory HO-1. PMID:23612372

Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway.

PubMed

Yuan, Gangjun; Chen, Xin; Liu, Zhuowei; Wei, Wensu; Shu, Qinghai; Abou-Hamdan, Hussein; Jiang, Lijuan; Li, Xiangdong; Chen, Rixin; Désaubry, Laurent; Zhou, Fangjian; Xie, Dan

2018-02-07

Prohibitin 1 (PHB) is a potential target for the treatment of urothelial carcinoma of the bladder (UCB). FL3 is a newly synthesized agent that inhibits cancer cell proliferation by targeting the PHB protein; however, the effect of FL3 in UCB cells remains unexplored. FL3 was identified to be a potent inhibitor of UCB cell viability using CCK-8 (cell counting kit-8) assay. Then a series of in vitro and in vivo experiments were conducted to further demonstrate the inhibitory effect of FL3 on UCB cell proliferation and to determine the underlying mechanisms. FL3 inhibited UCB cell proliferation and growth both in vitro and in vivo. By targeting the PHB protein, FL3 inhibited the interaction of Akt and PHB as well as Akt-mediated PHB phosphorylation, which consequently decreases the localization of PHB in the mitochondria. In addition, FL3 treatment resulted in cell cycle arrest in the G2/M phase, and this inhibitory effect of FL3 could be mimicked by knockdown of PHB. Through the microarray analysis of mRNA expression after FL3 treatment and knockdown of PHB, we found that the mRNA expression of the growth arrest and DNA damage-inducible alpha (GADD45α) gene were significantly upregulated. When knocked down the expression of GADD45α, the inhibitory effect of FL3 on cell cycle was rescued, suggesting that FL3-induced cell cycle inhibition is GADD45α dependent. Our data provide that FL3 inhibits the interaction of Akt and PHB, which in turn activates the GADD45α-dependent cell cycle inhibition in the G2/M phase.

Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses

PubMed Central

Nguyen, Quynh-Anh; Horn, Meryl E; Nicoll, Roger A

2016-01-01

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively. DOI: http://dx.doi.org/10.7554/eLife.19236.001 PMID:27805570

Extracellular matrix collagen alters cell proliferation and cell cycle progression of human uterine leiomyoma smooth muscle cells.

PubMed

Koohestani, Faezeh; Braundmeier, Andrea G; Mahdian, Arash; Seo, Jane; Bi, JiaJia; Nowak, Romana A

2013-01-01

Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.

Extracellular Matrix Collagen Alters Cell Proliferation and Cell Cycle Progression of Human Uterine Leiomyoma Smooth Muscle Cells

PubMed Central

Koohestani, Faezeh; Braundmeier, Andrea G.; Mahdian, Arash; Seo, Jane; Bi, JiaJia; Nowak, Romana A.

2013-01-01

Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs. PMID:24040420

Evidence for Two Modes of Synergistic Induction of Apoptosis by Mapatumumab and Oxaliplatin in Combination with Hyperthermia in Human Colon Cancer Cells

PubMed Central

Song, Xinxin; Kim, Seog-Young; Lee, Yong J.

2013-01-01

Colorectal cancer is the third leading cause of cancer-related mortality in the world-- the main cause of death from colorectal cancer is hepatic metastases, which can be treated with isolated hepatic perfusion (IHP). Searching for the most clinically relevant approaches for treating colorectal metastatic disease by isolated hepatic perfusion (IHP), we developed the application of oxaliplatin concomitantly with hyperthermia and humanized death receptor 4 (DR4) antibody mapatumumab (Mapa), and investigated the molecular mechanisms of this multimodality treatment in human colon cancer cell lines CX-1 and HCT116 as well as human colon cancer stem cells Tu-12, Tu-21 and Tu-22. We showed here, in this study, that the synergistic effect of the multimodality treatment-induced apoptosis was caspase dependent and activated death signaling via both the extrinsic apoptotic pathway and the intrinsic pathway. Death signaling was activated by c-Jun N-terminal kinase (JNK) signaling which led to Bcl-xL phosphorylation at serine 62, decreasing the anti-apoptotic activity of Bcl-xL, which contributed to the intrinsic pathway. The downregulation of cellular FLICE inhibitory protein long isoform (c-FLIPL) in the extrinsic pathway was accomplished through ubiquitination at lysine residue (K) 195 and protein synthesis inhibition. Overexpression of c-FLIPL mutant (K195R) and Bcl-xL mutant (S62A) completely abrogated the synergistic effect. The successful outcome of this study supports the application of multimodality strategy to patients with colorectal hepatic metastases who fail to respond to standard chemoradiotherapy that predominantly targets the mitochondrial apoptotic pathway. PMID:24013390

Multiple Transduction Pathways Mediate Thyrotropin Receptor Signaling in Preosteoblast-Like Cells

PubMed Central

Boutin, Alisa; Neumann, Susanne

2016-01-01

It has been shown that the TSH receptor (TSHR) couples to a number of different signaling pathways, although the Gs-cAMP pathway has been considered primary. Here, we measured the effects of TSH on bone marker mRNA and protein expression in preosteoblast-like U2OS cells stably expressing TSHRs. We determined which signaling cascades are involved in the regulation of IL-11, osteopontin (OPN), and alkaline phosphatase (ALPL). We demonstrated that TSH-induced up-regulation of IL-11 is primarily mediated via the Gs pathway as IL-11 was up-regulated by forskolin (FSK), an adenylyl cyclase activator, and inhibited by protein kinase A inhibitor H-89 and by silencing of Gαs by small interfering RNA. OPN levels were not affected by FSK, but its up-regulation was inhibited by TSHR/Gi-uncoupling by pertussis toxin. Pertussis toxin decreased p38 MAPK kinase phosphorylation, and a p38 inhibitor and small interfering RNA knockdown of p38α inhibited OPN induction by TSH. Up-regulation of ALPL expression required high doses of TSH (EC50 = 395nM), whereas low doses (EC50 = 19nM) were inhibitory. FSK-stimulated cAMP production decreased basal ALPL expression, whereas protein kinase A inhibition by H-89 and silencing of Gαs increased basal levels of ALPL. Knockdown of Gαq/11 and a protein kinase C inhibitor decreased TSH-stimulated up-regulation of ALPL, whereas a protein kinase C activator increased ALPL levels. A MAPK inhibitor and silencing of ERK1/2 inhibited TSH-stimulated ALPL expression. We conclude that TSH regulates expression of different bone markers via distinct signaling pathways. PMID:26950201

Decoding the phosphorylation code in Hedgehog signal transduction

PubMed Central

Chen, Yongbin; Jiang, Jin

2013-01-01

Hedgehog (Hh) signaling plays pivotal roles in embryonic development and adult tissue homeostasis, and its deregulation leads to numerous human disorders including cancer. Binding of Hh to Patched (Ptc), a twelve-transmembrane protein, alleviates its inhibition of Smoothened (Smo), a seven-transmembrane protein related to G-protein-coupled receptors (GPCRs), leading to Smo phosphorylation and activation. Smo acts through intracellular signaling complexes to convert the latent transcription factor Cubitus interruptus (Ci)/Gli from a truncated repressor to a full-length activator, leading to derepression/activation of Hh target genes. Increasing evidence suggests that phosphorylation participates in almost every step in the signal relay from Smo to Ci/Gli, and that differential phosphorylation of several key pathway components may be crucial for translating the Hh morphogen gradient into graded pathway activities. In this review, we focus on the multifaceted roles that phosphorylation plays in Hh signal transduction, and discuss the conservation and difference between Drosophila and mammalian Hh signaling mechanisms. PMID:23337587

Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways.

PubMed

Shambharkar, Prashant B; Blonska, Marzenna; Pappu, Bhanu P; Li, Hongxiu; You, Yun; Sakurai, Hiroaki; Darnay, Bryant G; Hara, Hiromitsu; Penninger, Josef; Lin, Xin

2007-04-04

The IkappaB kinase (IKK) complex serves as the master regulator for the activation of NF-kappaB by various stimuli. It contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKalpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKalpha/beta activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKalpha/beta phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKalpha/beta and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.

γH2AX foci formation in the absence of DNA damage: mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway.

PubMed

Tu, Wen-Zhi; Li, Bing; Huang, Bo; Wang, Yu; Liu, Xiao-Dan; Guan, Hua; Zhang, Shi-Meng; Tang, Yan; Rang, Wei-Qing; Zhou, Ping-Kun

2013-11-01

Phosphorylated H2AX is considered to be a biomarker for DNA double-strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA-PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation-mimic CHK2-T68D abrogated the attenuation of mitotic γH2AX induced by DNA-PKcs inactivation. Thus, the DNA-PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du Jianhai; Guan Tongju; Zhang Hui

2008-04-04

Extracellular signal-regulated kinase (ERK) is one of the key protein kinases that regulate the growth and proliferation in cardiac fibroblasts (CFs). As an energy sensor of cellular metabolism, AMP-activated protein kinase (AMPK) is found recently to be involved in myocardial remodeling. In this study, we investigated the crosstalk between ERK and AMPK in the growth and proliferation of CFs. In neonatal rat cardiac fibroblasts (NRCFs), we found that serum significantly inhibited basal AMPK phosphorylation between 10 min and 24 h and also partially inhibited AMPK phosphorylation by AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR). Furthermore, ERK inhibitor could greatly reverse the inhibition ofmore » AMPK by serum. Conversely, activation of AMPK by AICAR also showed a significant inhibition of basal and serum-induced ERK phosphorylation but it showed a delayed and steadfast inhibition which appeared after 60 min and lasted until 12 h. Moreover, inhibition of ERK could repress the activation of p70S6K, an important kinase in cardiac proliferation, and AICAR could also inhibit p70S6K phosphorylation. In addition, under both serum and serum-free medium, AICAR significantly inhibited the DNA synthesis and cell numbers, and reduced cells at S phase. In conclusion, AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts, which involved inhibitory interactions between ERK and AMPK. This is the first report that AMPK could be a target of ERK in growth factors-induced proliferation, which may give a new mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts.« less

A link between mitotic entry and membrane growth suggests a novel model for cell size control

PubMed Central

Anastasia, Steph D.; Nguyen, Duy Linh; Thai, Vu; Meloy, Melissa; MacDonough, Tracy

2012-01-01

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2ACdc55). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function. PMID:22451696

In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

PubMed

Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

2014-04-01

A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBα Degradation.

PubMed

Zhao, Xuan; Hirota, Tsuyoshi; Han, Xuemei; Cho, Han; Chong, Ling-Wa; Lamia, Katja; Liu, Sihao; Atkins, Annette R; Banayo, Ester; Liddle, Christopher; Yu, Ruth T; Yates, John R; Kay, Steve A; Downes, Michael; Evans, Ronald M

2016-06-16

Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition. Copyright © 2016 Elsevier Inc. All rights reserved.

A link between mitotic entry and membrane growth suggests a novel model for cell size control.

PubMed

Anastasia, Steph D; Nguyen, Duy Linh; Thai, Vu; Meloy, Melissa; MacDonough, Tracy; Kellogg, Douglas R

2012-04-02

Addition of new membrane to the cell surface by membrane trafficking is necessary for cell growth. In this paper, we report that blocking membrane traffic causes a mitotic checkpoint arrest via Wee1-dependent inhibitory phosphorylation of Cdk1. Checkpoint signals are relayed by the Rho1 GTPase, protein kinase C (Pkc1), and a specific form of protein phosphatase 2A (PP2A(Cdc55)). Signaling via this pathway is dependent on membrane traffic and appears to increase gradually during polar bud growth. We hypothesize that delivery of vesicles to the site of bud growth generates a signal that is proportional to the extent of polarized membrane growth and that the strength of the signal is read by downstream components to determine when sufficient growth has occurred for initiation of mitosis. Growth-dependent signaling could explain how membrane growth is integrated with cell cycle progression. It could also control both cell size and morphogenesis, thereby reconciling divergent models for mitotic checkpoint function.

Gallic Acid Attenuates Platelet Activation and Platelet-Leukocyte Aggregation: Involving Pathways of Akt and GSK3β

PubMed Central

Chang, Shih-Sheng; Lee, Viola S. Y.; Tseng, Yu-Lun; Chang, Kuan-Cheng; Chen, Kuen-Bao; Chen, Yuh-Lien; Li, Chi-Yuan

2012-01-01

Platelet activation and its interaction with leukocytes play an important role in atherothrombosis. Cardiovascular diseases resulted from atherothrombosis remain the major causes of death worldwide. Gallic acid, a major constituent of red wine and tea, has been believed to have properties of cardiovascular protection, which is likely to be related to its antioxidant effects. Nonetheless, there were few and inconsistent data regarding the effects of gallic acid on platelet function. Therefore, we designed this in vitro study to determine whether gallic acid could inhibit platelet activation and the possible mechanisms. From our results, gallic acid could concentration-dependently inhibit platelet aggregation, P-selectin expression, and platelet-leukocyte aggregation. Gallic acid prevented the elevation of intracellular calcium and attenuated phosphorylation of PKCα/p38 MAPK and Akt/GSK3β on platelets stimulated by the stimulants ADP or U46619. This is the first mechanistic explanation for the inhibitory effects on platelets from gallic acid. PMID:22811749

Parallel comparative proteomics and phosphoproteomics reveal that cattle myostatin regulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway

PubMed Central

Yang, Shuping; Li, Xin; Liu, Xinfeng; Ding, Xiangbin; Xin, Xiangbo; Jin, Congfei; Zhang, Sheng; Li, Guangpeng; Guo, Hong

2018-01-01

MSTN-encoded myostatin is a negative regulator of skeletal muscle development. Here, we utilized the gluteus tissues from MSTN gene editing and wild type Luxi beef cattle which are native breed of cattle in China, performed tandem mass tag (TMT) -based comparative proteomics and phosphoproteomics analyses to investigate the regulatory mechanism of MSTN related to cellular metabolism and signaling pathway in muscle development. Out of 1,315 proteins, 69 differentially expressed proteins (DEPs) were found in global proteomics analysis. Meanwhile, 149 differentially changed phosphopeptides corresponding to 76 unique phosphorylated proteins (DEPPs) were detected from 2,600 identified phosphopeptides in 702 phosphorylated proteins. Bioinformatics analyses suggested that majority of DEPs and DEPPs were closely related to glycolysis, glycogenolysis, and muscle contractile fibre processes. The global discovery results were validated by Multiple Reaction Monitoring (MRM)-based targeted peptide quantitation analysis, western blotting, and muscle glycogen content measurement. Our data revealed that increase in abundance of key enzymes and phosphorylation on their regulatory sites appears responsible for the enhanced glycogenolysis and glycolysis in MSTN−/−. The elevated glycogenolysis was assocaited with an enhanced phosphorylation of Ser1018 in PHKA1, and Ser641/Ser645 in GYS1, which were regulated by upstream phosphorylated AKT-GSK3β pathway and highly consistent with the lower glycogen content in gluteus of MSTN−/−. Collectively, this study provides new insights into the regulatory mechanisms of MSTN involved in energy metabolism and muscle growth. PMID:29541418

Central insulin signaling is attenuated by long-term insulin exposure via insulin receptor substrate-1 serine phosphorylation, proteasomal degradation, and lysosomal insulin receptor degradation.

PubMed

Mayer, Christopher M; Belsham, Denise D

2010-01-01

Central insulin signaling is critical for the prevention of insulin resistance. Hyperinsulinemia contributes to insulin resistance, but it is not yet clear whether neurons are subject to cellular insulin resistance. We used an immortalized, hypothalamic, clonal cell line, mHypoE-46, which exemplifies neuronal function and expresses the components of the insulin signaling pathway, to determine how hyperinsulinemia modifies neuronal function. Western blot analysis indicated that prolonged insulin treatment of mHypoE-46 cells attenuated insulin signaling through phospho-Akt. To understand the mechanisms involved, time-course analysis was performed. Insulin exposure for 4 and 8 h phosphorylated Akt and p70-S6 kinase (S6K1), whereas 8 and 24 h treatment decreased insulin receptor (IR) and IR substrate 1 (IRS-1) protein levels. Insulin phosphorylation of S6K1 correlated with IRS-1 ser1101 phosphorylation and the mTOR-S6K1 pathway inhibitor rapamycin prevented IRS-1 serine phosphorylation. The proteasomal inhibitor epoxomicin and the lysosomal pathway inhibitor 3-methyladenine prevented the degradation of IRS-1 and IR by insulin, respectively, and pretreatment with rapamycin, epoxomicin, or 3-methyladenine prevented attenuation of insulin signaling by long-term insulin exposure. Thus, a sustained elevation of insulin levels diminishes neuronal insulin signaling through mTOR-S6K1-mediated IRS-1 serine phosphorylation, proteasomal degradation of IRS-1 and lysosomal degradation of the IR.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

PubMed Central

Ravi, R K; Weber, E; McMahon, M; Williams, J R; Baylin, S; Mal, A; Harter, M L; Dillehay, L E; Claudio, P P; Giordano, A; Nelkin, B D; Mabry, M

1998-01-01

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors. PMID:9421477

Lateral Orbitofrontal Cortical Modulation on the Medial Prefrontal Cortex-Amygdala Pathway: Differential Regulation of Intra-Amygdala GABAA and GABAB Receptors.

PubMed

Chang, Chun-Hui

2017-07-01

The basolateral complex of the amygdala receives inputs from neocortical areas, including the medial prefrontal cortex and lateral orbitofrontal cortex. Earlier studies have shown that lateral orbitofrontal cortex activation exerts an inhibitory gating on medial prefrontal cortex-amygdala information flow. Here we examined the individual role of GABAA and GABAB receptors in this process. In vivo extracellular single-unit recordings were done in anesthetized rats. We searched amygdala neurons that fire in response to medial prefrontal cortex activation, tested lateral orbitofrontal cortex gating at different delays (lateral orbitofrontal cortex-medial prefrontal cortex delays: 25, 50, 100, 250, 500, and 1000 milliseconds), and examined differential contribution of GABAA and GABAB receptors with iontophoresis. Relative to baseline, lateral orbitofrontal cortex stimulation exerted an inhibitory modulatory gating on the medial prefrontal cortex-amygdala pathway and was effective up to a long delay of 500 ms (long-delay latencies at 100, 250, and 500 milliseconds). Moreover, blockade of intra-amygdala GABAA receptors with bicuculline abolished the lateral orbitofrontal cortex inhibitory gating at both short- (25 milliseconds) and long-delay (100 milliseconds) intervals, while blockade of GABAB receptors with saclofen reversed the inhibitory gating at long delay (100 milliseconds) only. Among the majority of the neurons examined (8 of 9), inactivation of either GABAA or GABAB receptors during baseline did not change evoked probability per se, suggesting that local feed-forward inhibitory mechanism is pathway specific. Our results suggest that the effect of lateral orbitofrontal cortex inhibitory modulatory gating was effective up to 500 milliseconds and that intra-amygdala GABAA and GABAB receptors differentially modulate the short- and long-delay lateral orbitofrontal cortex inhibitory gating on the medial prefrontal cortex-amygdala pathway. © The Author 2017. Published by Oxford University Press on behalf of CINP.

Targeting arachidonic acid pathway by natural products for cancer prevention and therapy.

PubMed

Yarla, Nagendra Sastry; Bishayee, Anupam; Sethi, Gautam; Reddanna, Pallu; Kalle, Arunasree M; Dhananjaya, Bhadrapura Lakkappa; Dowluru, Kaladhar S V G K; Chintala, Ramakrishna; Duddukuri, Govinda Rao

2016-10-01

Arachidonic acid (AA) pathway, a metabolic process, plays a key role in carcinogenesis. Hence, AA pathway metabolic enzymes phospholipase A 2 s (PLA 2 s), cyclooxygenases (COXs) and lipoxygenases (LOXs) and their metabolic products, such as prostaglandins and leukotrienes, have been considered novel preventive and therapeutic targets in cancer. Bioactive natural products are a good source for development of novel cancer preventive and therapeutic drugs, which have been widely used in clinical practice due to their safety profiles. AA pathway inhibitory natural products have been developed as chemopreventive and therapeutic agents against several cancers. Curcumin, resveratrol, apigenin, anthocyans, berberine, ellagic acid, eugenol, fisetin, ursolic acid, [6]-gingerol, guggulsteone, lycopene and genistein are well known cancer chemopreventive agents which act by targeting multiple pathways, including COX-2. Nordihydroguaiaretic acid and baicalein can be chemopreventive molecules against various cancers by inhibiting LOXs. Several PLA 2 s inhibitory natural products have been identified with chemopreventive and therapeutic potentials against various cancers. In this review, we critically discuss the possible utility of natural products as preventive and therapeutic agents against various oncologic diseases, including prostate, pancreatic, lung, skin, gastric, oral, blood, head and neck, colorectal, liver, cervical and breast cancers, by targeting AA pathway. Further, the current status of clinical studies evaluating AA pathway inhibitory natural products in cancer is reviewed. In addition, various emerging issues, including bioavailability, toxicity and explorability of combination therapy, for the development of AA pathway inhibitory natural products as chemopreventive and therapeutic agents against human malignancy are also discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Parallel prefrontal pathways reach distinct excitatory and inhibitory systems in memory-related rhinal cortices.

PubMed

Bunce, Jamie G; Zikopoulos, Basilis; Feinberg, Marcia; Barbas, Helen

2013-12-15

To investigate how prefrontal cortices impinge on medial temporal cortices we labeled pathways from the anterior cingulate cortex (ACC) and posterior orbitofrontal cortex (pOFC) in rhesus monkeys to compare their relationship with excitatory and inhibitory systems in rhinal cortices. The ACC pathway terminated mostly in areas 28 and 35 with a high proportion of large terminals, whereas the pOFC pathway terminated mostly through small terminals in area 36 and sparsely in areas 28 and 35. Both pathways terminated in all layers. Simultaneous labeling of pathways and distinct neurochemical classes of inhibitory neurons, followed by analyses of appositions of presynaptic and postsynaptic fluorescent signal, or synapses, showed overall predominant association with spines of putative excitatory neurons, but also significant interactions with presumed inhibitory neurons labeled for calretinin, calbindin, or parvalbumin. In the upper layers of areas 28 and 35 the ACC pathway was associated with dendrites of neurons labeled with calretinin, which are thought to disinhibit neighboring excitatory neurons, suggesting facilitated hippocampal access. In contrast, in area 36 pOFC axons were associated with dendrites of calbindin neurons, which are poised to reduce noise and enhance signal. In the deep layers, both pathways innervated mostly dendrites of parvalbumin neurons, which strongly inhibit neighboring excitatory neurons, suggesting gating of hippocampal output to other cortices. These findings suggest that the ACC, associated with attention and context, and the pOFC, associated with emotional valuation, have distinct contributions to memory in rhinal cortices, in processes that are disrupted in psychiatric diseases. Copyright © 2013 Wiley Periodicals, Inc.

Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

NASA Astrophysics Data System (ADS)

He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

2015-07-01

Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS.

EPA Science Inventory

We have previously demonstrated that the PCB mixture, Aroclor 1254 (A1254), increases the phosphorylated form of CREB (pCREB), the cAMP-responsive element binding protein. This transcription factor is important in nervous system development and plasticity. Phosphorylation
of C...

GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

PubMed Central

Toh, Wei Hong; Chia, Pei Zhi Cheryl; Hossain, Mohammed Iqbal; Gleeson, Paul A.

2018-01-01

The diversion of the membrane-bound β-site amyloid precursor protein–(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production. PMID:29142073

ABI3, a component of the WAVE2 complex, is potentially regulated by PI3K/AKT pathway

PubMed Central

Moraes, Lais; Zanchin, Nilson I.T.; Cerutti, Janete M.

2017-01-01

We previously reported that ABI3 expression is lost in follicular thyroid carcinomas and its restoration significantly inhibited cell growth, invasiveness, migration, and reduced tumor growth in vivo. The mechanistic basis by which ABI3 exerts its tumor suppressive effects is not fully understood. In this study, we show that ABI3 is a phosphoprotein. Using proteomic array analysis, we showed that ABI3 modulated distinct cancer-related pathways in thyroid cancer cells. The KEA analysis found that PI3K substrates were enriched and forced expression of ABI3 markedly decreased the phosphorylation of AKT and the downstream-targeted protein pGSK3β. We next used immunoprecipitation combined with mass spectrometry to identify ABI3-interacting proteins that may be involved in modulating/integrating signaling pathways. We identified 37 ABI3 partners, including several components of the canonical WAVE regulatory complex (WRC) such as WAVE2/CYF1P1/NAP1, suggesting that ABI3 function might be regulated through WRC. Both, pharmacological inhibition of the PI3K/AKT pathway and mutation at residue S342 of ABI3, which is predicted to be phosphorylated by AKT, provided evidences that the non-phosphorylated form of ABI3 is preferentially present in the WRC protein complex. Collectively, our findings suggest that ABI3 might be a downstream mediator of the PI3K/AKT pathway that might disrupt WRC via ABI3 phosphorylation. PMID:28978070

ABI3, a component of the WAVE2 complex, is potentially regulated by PI3K/AKT pathway.

PubMed

Moraes, Lais; Zanchin, Nilson I T; Cerutti, Janete M

2017-09-15

We previously reported that ABI3 expression is lost in follicular thyroid carcinomas and its restoration significantly inhibited cell growth, invasiveness, migration, and reduced tumor growth in vivo . The mechanistic basis by which ABI3 exerts its tumor suppressive effects is not fully understood. In this study, we show that ABI3 is a phosphoprotein. Using proteomic array analysis, we showed that ABI3 modulated distinct cancer-related pathways in thyroid cancer cells. The KEA analysis found that PI3K substrates were enriched and forced expression of ABI3 markedly decreased the phosphorylation of AKT and the downstream-targeted protein pGSK3β. We next used immunoprecipitation combined with mass spectrometry to identify ABI3-interacting proteins that may be involved in modulating/integrating signaling pathways. We identified 37 ABI3 partners, including several components of the canonical WAVE regulatory complex (WRC) such as WAVE2/CYF1P1/NAP1, suggesting that ABI3 function might be regulated through WRC. Both, pharmacological inhibition of the PI3K/AKT pathway and mutation at residue S342 of ABI3, which is predicted to be phosphorylated by AKT, provided evidences that the non-phosphorylated form of ABI3 is preferentially present in the WRC protein complex. Collectively, our findings suggest that ABI3 might be a downstream mediator of the PI3K/AKT pathway that might disrupt WRC via ABI3 phosphorylation.

Isoform-specific and Protein Kinase C-mediated Regulation of CTP:Phosphoethanolamine Cytidylyltransferase Phosphorylation*

PubMed Central

Pavlovic, Zvezdan; Zhu, Lin; Pereira, Leanne; Singh, Ratnesh Kumar; Cornell, Rosemary B.; Bakovic, Marica

2014-01-01

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50–90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation. PMID:24519946

Methylmercury causes neuronal cell death through the suppression of the TrkA pathway: In vitro and in vivo effects of TrkA pathway activators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujimura, Masatake, E-mail: fujimura@nimd.go.jp; Usuki, Fusako

Methylmercury (MeHg) is an environmental toxin which induces cell death specific for the nervous systems. Here we show that MeHg causes neuronal cell death through the suppression of the tropomyosin receptor kinase A (TrkA) pathway, and that compounds activating the TrkA pathway prevent MeHg-induced nerve damage in vitro and in vivo. We first investigated the mechanism of MeHg-induced neurotoxicity in differentiating neurons using PC12 cells. Exposure to 100 nM MeHg for 1 day induced apoptosis in differentiating PC12 cells. Further, MeHg-induced apoptosis was preceded by inhibition of neurite extension, as determined by ELISA analyses of the neurite-specific protein neurofilament tripletmore » H protein (NF-H). To determine the mechanism of MeHg-induced apoptosis, we evaluated the effects of MeHg on the TrkA pathway, which is known to regulate neuronal differentiation and viability. Western blot analysis demonstrated that, like the TrkA phosphorylation inhibitor K252a, MeHg inhibited phosphorylation of TrkA and its downstream effectors. Furthermore, GM1 ganglioside and its analog MCC-257, which enhance TrkA phosphorylation, overcame the effect of MeHg in neurons, supporting the involvement of the TrkA pathway in MeHg-induced nerve damage. Finally, we demonstrated that MCC-257 rescued the clinical sign and pathological changes in MeHg-exposed rats. These findings indicate that MeHg-induced apoptosis in neuron is triggered by inhibition of the TrkA pathway, and that GM1 ganglioside and MCC-257 effectively prevent MeHg-induced nerve damage. - Highlights: • Exposure to 100 nM MeHg for 1 day induced apoptosis in differentiating PC12 cells. • Inhibition of neurite extension was involved in MeHg-induced apoptosis. • Like the TrkA phosphorylation inhibitor, MeHg inhibited phosphorylation of TrkA. • GM1 ganglioside and its analog effectively prevented MeHg-induced nerve damage.« less

Rho-associated coiled-coil kinase (ROCK) protein controls microtubule dynamics in a novel signaling pathway that regulates cell migration.

PubMed

Schofield, Alice V; Steel, Rohan; Bernard, Ora

2012-12-21

The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.

PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela

2012-04-15

The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKTmore » Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.« less

Quantitation of Met tyrosine phosphorylation using MRM-MS.

PubMed

Meng, Zhaojing; Srivastava, Apurva K; Zhou, Ming; Veenstra, Timothy

2013-01-01

Phosphorylation has long been accepted as a key cellular regulator of cell signaling pathways. The recent development of multiple-reaction monitoring mass spectrometry (MRM-MS) provides a useful tool for measuring the absolute quantity of phosphorylation occupancy at pivotal sites within signaling proteins, even when the phosphorylation sites are in close proximity. Here, we described a targeted quantitation approach to measure the absolute phosphorylation occupancy at Y1234 and Y1235 of Met. The approach is utilized to obtain absolute occupancy of the two phosphorylation sites in the full-length recombinant Met. It is further applied to quantitate the phosphorylation state of these two sites in SNU-5 cells treated with a Met inhibitor.

Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and transcription.

PubMed

Lanucara, Francesco; Lam, Connie; Mann, Jelena; Monie, Tom P; Colombo, Stefano A P; Holman, Stephen W; Boyd, James; Dange, Manohar C; Mann, Derek A; White, Michael R H; Eyers, Claire E

2016-07-01

The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple 'one modification, one effect' system. © 2016 The Authors.

Acquired BRAF V600E Mutation as Resistant Mechanism after Treatment with Osimertinib.

PubMed

Ho, Chao-Chi; Liao, Wei-Yu; Lin, Chih-An; Shih, Jin-Yuan; Yu, Chong-Jen; Chih-Hsin Yang, James

2017-03-01

AZD9291 (osimertinib) is designed for acquired T790M mutation after first- and second-generation EGFR) tyrosine kinase inhibitors have been used. Some of the resistance mechanisms that present after osimertinib treatment, including a newly acquired EGFR C797S mutation, have been identified. It is unclear, however, whether the bypass pathway is also a mechanism of resistance in patients after osimertinib treatment. Cells from malignant pleural effusion were collected and cultured at the time of progression in a patient being treated with osimertinib. Tumor genotyping was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. EGFR, AKT, MEK, and ERK phosphorylation were determined. An anchorage-dependent colony formation assay was used for drug sensitivity. An acquired mutation, BRAF V600E, was found in the patient at the time of progression while being treated with osimertinib. Cells grown from malignant pleural effusion were sensitive to BRAF V600E inhibitor and were more vulnerable to a combination treatment with osimertinib. A potential mechanism of acquired resistance to osimertinib in patients with T790M is through the BRAF pathway. Simultaneous blockade of the BRAF and EGFR had a significant inhibitory effect. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Direct binding of arsenic trioxide to AMPK and generation of inhibitory effects on acute myeloid leukemia precursors

PubMed Central

Beauchamp, Elspeth M.; Kosciuczuk, Ewa M.; Serrano, Ruth; Nanavati, Dhaval; Swindell, Elden P.; Viollet, Benoit; O'Halloran, Thomas V.; Altman, Jessica K.; Platanias, Leonidas C.

2014-01-01

Arsenic trioxide (As2O3) exhibits potent antineoplastic effects and is used extensively in clinical oncology for the treatment of a subset of patients with acute myeloid leukemia (AML). Although As2O3 is known to regulate activation of several signaling cascades, the key events, accounting for its anti-leukemic properties, remain to be defined. We provide evidence that arsenic can directly bind to cysteine 299 in AMPKα and inhibit its activity. This inhibition of AMPK by arsenic is required in part for its cytotoxic effects on primitive leukemic progenitors from patients with AML, while concomitant treatment with an AMPK activator antagonizes in vivo the arsenic-induced antileukemic effects in a xenograft AML mouse model. A consequence of AMPK inhibition is activation of the mTOR pathway as a negative regulatory feedback loop. However, when AMPK expression is lost, arsenic-dependent activation of the kinase RSK downstream of MAPK activity compensates the generation of regulatory feedback signals through phosphorylation of downstream mTOR targets. Thus, therapeutic regimens with arsenic trioxide will need to include inhibitors of both the mTOR and RSK pathways in combination to prevent engagement of negative feedback loops and maximize antineoplastic responses. PMID:25344585

Kyungheechunggan-Tang-01, a New Herbal Medication, Suppresses LPS-Induced Inflammatory Responses through JAK/STAT Signaling Pathway in RAW 264.7 Macrophages

PubMed Central

Han, Hee-Soo; Shin, Ji-Sun; Inn, Kyung-Soo; Lee, Jang-Hoon; Park, Geonha

2017-01-01

Medicinal plants have been used as alternative therapeutic tools to alleviate inflammatory diseases. The objective of this study was to evaluate anti-inflammatory properties of Kyungheechunggan-tang- (KCT-) 01, KCT-02, and Injinchunggan-tang (IJCGT) as newly developed decoctions containing 3–11 herbs in LPS-induced macrophages. KCT-01 showed the most potent inhibitory effects on LPS-induced NO, PGE2, TNF-α, and IL-6 production among those three herbal formulas. In addition, KCT-01 significantly inhibited LPS-induced iNOS and COX-2 at protein levels and expression of iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Molecular data revealed that KCT-01 attenuated the activation of JAK/STAT signaling cascade without affecting NF-κB or AP-1 activation. In ear inflammation induced by croton oil, KCT-01 significantly reduced edema, MPO activity, expression levels of iNOS and COX-2, and STAT3 phosphorylation in ear tissues. Taken together, our findings suggest that KCT-01 can downregulate the expression of proinflammatory genes by inhibiting JAK/STAT signaling pathway under inflammatory conditions. This study provides useful data for further exploration and application of KCT-01 as a potential anti-inflammatory medicine. PMID:29348772

Oxidation inhibits PTH receptor signaling and trafficking

PubMed Central

Ardura, Juan A.; Alonso, Verónica; Esbrit, Pedro; Friedman, Peter A.

2017-01-01

Reactive Oxygen Species (ROS) increase during aging, potentially affecting many tissues including brain, heart, and bone. ROS alter signaling pathways and constitute potential therapeutic targets to limit oxidative damaging effects in aging-associated diseases. Parathyroid hormone receptors (PTHR) are widely expressed and PTH is the only anabolic therapy for osteoporosis. The effects of oxidative stress on PTHR signaling and trafficking have not been elucidated. Here, we used Fluorescence Resonance Energy Transfer (FRET)-based cAMP, ERK, and calcium fluorescent biosensors to analyze the effects of ROS on PTHR signaling and trafficking by live-cell imaging. PTHR internalization and recycling were measured in HEK-293 cells stably transfected with HA-PTHR. PTH increased cAMP production, ERK phosphorylation, and elevated intracellular calcium. Pre-incubation with H2O2 reduced all PTH-dependent signaling pathways. These inhibitory effects were not a result of PTH oxidation since PTH incubated with H2O2 triggered similar responses. PTH promoted internalization and recycling of the PTHR. Both events were significantly reduced by H2O2 pre-incubation. These findings highlight the role of oxidation on PTHR signaling and trafficking, and suggest the relevance of ROS as a putative target in diseases associated with oxidative stress such as age-related osteoporosis. PMID:27908723

Context-dependent modulation of Pol II CTD phosphatase SSUP-72 regulates alternative polyadenylation in neuronal development

PubMed Central

Chen, Fei; Zhou, Yu; Qi, Yingchuan B.; Khivansara, Vishal; Li, Hairi; Chun, Sang Young; Kim, John K.; Fu, Xiang-Dong; Jin, Yishi

2015-01-01

Alternative polyadenylation (APA) is widespread in neuronal development and activity-mediated neural plasticity. However, the underlying molecular mechanisms are largely unknown. We used systematic genetic studies and genome-wide surveys of the transcriptional landscape to identify a context-dependent regulatory pathway controlling APA in the Caenorhabditis elegans nervous system. Loss of function in ssup-72, a Ser5 phosphatase for the RNA polymerase II (Pol II) C-terminal domain (CTD), dampens transcription termination at a strong intronic polyadenylation site (PAS) in unc-44/ankyrin yet promotes termination at the weak intronic PAS of the MAP kinase dlk-1. A nuclear protein, SYDN-1, which regulates neuronal development, antagonizes the function of SSUP-72 and several nuclear polyadenylation factors. This regulatory pathway allows the production of a neuron-specific isoform of unc-44 and an inhibitory isoform of dlk-1. Dysregulation of the unc-44 and dlk-1 mRNA isoforms in sydn-1 mutants impairs neuronal development. Deleting the intronic PAS of unc-44 results in increased pre-mRNA processing of neuronal ankyrin and suppresses sydn-1 mutants. These results reveal a mechanism by which regulation of CTD phosphorylation controls coding region APA in the nervous system. PMID:26588990

PDK1 inhibitor GSK2334470 synergizes with proteasome inhibitor MG‑132 in multiple myeloma cells by inhibiting full AKT activity and increasing nuclear accumulation of the PTEN protein.

PubMed

Zhang, Jin; Yang, Chunmei; Zhou, Fengping; Chen, Xiaohui

2018-06-01

Phosphoinositide‑dependent kinase 1 (PDK1) is generally active in multiple myeloma (MM) and higher expression than other hematopoietic cells, which is associated with the drug resistance and the disease progression. Previous studies have demonstrated that PDK1 can be targeted therapeutically in MM. In the present study, we examined the combination effect of GSK2334470 (GSK‑470), a novel and highly specific inhibitor of PDK1, with proteasome inhibitor MG‑132 in MM cell lines. GSK‑470 monotherapy significantly inhibited growth of MM cell lines and induced apoptosis that was associated with the activation of both the intrinsic mitochondrial pathway and the extrinsic death receptor pathway. Moreover, GSK‑470 demonstrated synergistic growth inhibitory effects with MG‑132. Notably, treatment with these inhibitors resulted in an almost complete inhibition of phosphorylation of mammalian target of rapamycin on Ser2448 and Ser2481 and full activation of AKT. The combination therapy also caused an upregulation of PTEN and an increased nuclear accumulation of PTEN protein. Collectively, our results provide the rationale for novel combination treatment with PDK1 inhibitor and proteasome inhibitors to improve outcomes in patients with MM.

A parapoxviral virion protein inhibits NF-κB signaling early in infection

PubMed Central

Khatiwada, Sushil; Delhon, Gustavo; Nagendraprabhu, Ponnuraj; Chaulagain, Sabal; Luo, Shuhong; Diel, Diego G.; Flores, Eduardo F.

2017-01-01

Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection. PMID:28787456

APPL1-mediated activation of STAT3 contributes to inhibitory effect of adiponectin on hepatic gluconeogenesis.

PubMed

Ding, Youming; Zhang, Deling; Wang, Bin; Zhang, Yemin; Wang, Lei; Chen, Xiaoyan; Li, Mingxin; Tang, Zhao; Wang, Changhua

2016-09-15

Adiponectin has been shown to suppress hepatic gluconeogenesis. However, the signaling pathways underlying its action remain ill-defined. The purpose of this study was to examine the potential role of APPL1 in mediating anti-gluconeogenic ability of adiponectin. Primary hepatocytes were isolated from male C57BL/6 mice. Western blot and RT-PCR were performed to detect protein expression and mRNA level, respectively. The protein-protein association was determined by immunoprecipitation and GST pull-down assay. We found that APPL1 protein levels were negatively associated with expressions of proteins and mRNAs of gluconeogenesis enzymes under stimulation with adiponectin. In addition, adiponectin-stimulated STAT3 phosphorylation and acetylation were positively regulated by APPL1 and negative regulated by SirT1. Pharmacological and genetic inhibition of STAT3 mitigated impact of adiponectin on hepatic gluconeogenesis. Furthermore, adiponectin administration facilitated the binding of APPL1 to SirT1 and suppressed the association of SirT1 with STAT3. Taken together, our study showed that APPL1-SirT1-STAT3 pathway mediated adiponectin signaling in primary hepatocytes. This new finding provides a novel mechanism by which adiponectin suppresses hepatic gluconeogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

NSC 95397 Suppresses Proliferation and Induces Apoptosis in Colon Cancer Cells through MKP-1 and the ERK1/2 Pathway.

PubMed

Dubey, Navneet Kumar; Peng, Bou-Yue; Lin, Chien-Min; Wang, Peter D; Wang, Joseph R; Chan, Chun-Hao; Wei, Hong-Jian; Deng, Win-Ping

2018-05-31

NSC 95397, a quinone-based small molecule compound, has been identified as an inhibitor for dual-specificity phosphatases, including mitogen-activated protein kinase phosphatase-1 (MKP-1). MKP-1 is known to inactivate mitogen-activated protein kinases by dephosphorylating both of their threonine and tyrosine residues. Moreover, owing to their participation in tumorigenesis and drug resistance in colon cancer cells, MKP-1 is an attractive therapeutic target for colon cancer treatment. We therefore investigated the inhibitory activity of NSC 95397 against three colon cancer cell lines including SW480, SW620, and DLD-1, and their underlying mechanisms. The results demonstrated that NSC 95397 reduced cell viability and anchorage-independent growth of all the three colon cancer cell lines through inhibited proliferation and induced apoptosis via regulating cell-cycle-related proteins, including p21, cyclin-dependent kinases, and caspases. Besides, by using mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126, we provided mechanistic evidence that the antineoplastic effects of NSC 95397 were achieved via inhibiting MKP-1 activity followed by ERK1/2 phosphorylation. Conclusively, our results indicated that NSC 95397 might serve as an effective therapeutic intervention for colon cancer through regulating MKP-1 and ERK1/2 pathway.

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events.

PubMed

Timár, J; Tóth, S; Tóvári, J; Paku, S; Raz, A

1999-01-01

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1-5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCalpha to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal.

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

PubMed

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling.

PubMed

Namkoong, Seung; Kim, Chun-Ki; Cho, Young-Lai; Kim, Ji-Hee; Lee, Hansoo; Ha, Kwon-Soo; Choe, Jongseon; Kim, Pyeung-Hyeun; Won, Moo-Ho; Kwon, Young-Geun; Shim, Eun Bo; Kim, Young-Myeong

2009-06-01

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI.Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation,but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERKactivation and PI3K/Akt/eNOS/NO signaling.

Extensive Crosstalk between O-GlcNAcylation and Phosphorylation Regulates Akt Signaling

PubMed Central

Sun, Danni; Xin, Xianliang; Pan, Qiuming; Peng, Shuying; Liang, Zhongjie; Luo, Cheng; Yang, Yiming; Jiang, Hualiang; Huang, Min; Chai, Wengang; Ding, Jian; Geng, Meiyu

2012-01-01

O-linked N-acetylglucosamine glycosylations (O-GlcNAc) and O-linked phosphorylations (O-phosphate), as two important types of post-translational modifications, often occur on the same protein and bear a reciprocal relationship. In addition to the well documented phosphorylations that control Akt activity, Akt also undergoes O-GlcNAcylation, but the interplay between these two modifications and the biological significance remain unclear, largely due to the technique challenges. Here, we applied a two-step analytic approach composed of the O-GlcNAc immunoenrichment and subsequent O-phosphate immunodetection. Such an easy method enabled us to visualize endogenous glycosylated and phosphorylated Akt subpopulations in parallel and observed the inhibitory effect of Akt O-GlcNAcylations on its phosphorylation. Further studies utilizing mass spectrometry and mutagenesis approaches showed that O-GlcNAcylations at Thr 305 and Thr 312 inhibited Akt phosphorylation at Thr 308 via disrupting the interaction between Akt and PDK1. The impaired Akt activation in turn resulted in the compromised biological functions of Akt, as evidenced by suppressed cell proliferation and migration capabilities. Together, this study revealed an extensive crosstalk between O-GlcNAcylations and phosphorylations of Akt and demonstrated O-GlcNAcylation as a new regulatory modification for Akt signaling. PMID:22629392

Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

PubMed

Walther, Stefanie; Pluteanu, Florentina; Renz, Susanne; Nikonova, Yulia; Maxwell, Joshua T; Yang, Li-Zhen; Schmidt, Kurt; Edwards, Joshua N; Wakula, Paulina; Groschner, Klaus; Maier, Lars S; Spiess, Joachim; Blatter, Lothar A; Pieske, Burkert; Kockskämper, Jens

2014-09-01

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling. Copyright © 2014 the American Physiological Society.

UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53{sup Ser-15} phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader

2009-06-12

Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser{sup 15}. Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not preventmore » Dubca cell apoptosis, suggesting that p53{sup Ser-15} phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.« less

MAT2B promotes adipogenesis by modulating SAMe levels and activating AKT/ERK pathway during porcine intramuscular preadipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Cunzhen; Chen, Xiaochang; Wu, Wenjing

Intramuscular fat (IMF) has been demonstrated as one of the crucial factors of livestock meat quality. The MAT2B protein with MAT2α catalyzes the formation of methyl donor S- adenosylmethionine (SAMe) to mediate cell metabolism including proliferation and apoptosis. However, the regulatory effect of MAT2B on IMF deposition is still unclear. In this study, the effect of MAT2B on adipogenesis and its potential mechanism during porcine intramuscular preadipocyte differentiation was studied. The results showed that overexpression of MAT2B promoted adipogenesis and significantly up-regulated the mRNA and protein levels of adipogenic marker genes including FASN, PPARγ and aP2, consistently, knockdown of MAT2Bmore » inhibited lipid accumulation and down-regulated the mRNA and protein levels of the above genes. Furthermore, flow cytometry and EdU-labeling assay indicated that MAT2B regulate adipogenesis was partly due to influence intracellular SAMe levels and further affect cell clonal expansion. Also, increased expression of MAT2B activated the phosphorylations of AKT and ERK1/2, whereas knockdown of MAT2B blocked AKT signaling and repressed the phosphorylation of ERK1/2. Moreover, the inhibitory effect of LY294002 (a specific PI3K inhibitor) on the activities of AKT and ERK1/2 was partially recovered by overexpression of MAT2B in porcine intramuscular adipocytes. Finally, Co-IP experiments showed that MAT2B can directly interact with AKT. Taken together, our findings suggested that MAT2B acted as a positive regulator through modifying SAMe levels as well as activating AKT/ERK signaling pathway to promote porcine intramuscular adipocyte differentiation. - Highlights: • MAT2B up-regulates the expression of adipogenic marker genes and promotes porcine intramuscular preadipocyte differentiation. • MAT2B influences intracellular SAMe levels and further affects cell clonal expansion. • MAT2B interacts with AKT and activates AKT/ERK signaling pathway.« less

The protective effects of PCPA against monocrotaline-induced pulmonary arterial hypertension are mediated through the downregulation of NFAT-1 and NF-κB.

PubMed

Bai, Yang; Li, Zhong-Xia; Wang, Huai-Liang; Lian, Guo-Chao; Wang, Yun

2017-07-01

Inflammation and remodeling play a role in the pathogenesis of pulmonary arterial hypertension (PAH). Nuclear factor-κB (NF-κB) and nuclear factor of activated T cells-1 (NFAT-1) participate in inflammation and remodeling in a number of diseases. As a tryptophan hydroxylase inhibitor, 4-chloro-DL-phenylalanine (PCPA) had been reported to exert anti-inflammatory and remodeling effects. Therefore, we hypothesized that PCPA may attenuate monocrotaline (MCT)-induced PAH through the NFAT-1 and NF-κB signaling pathways. In order to confirm our hypothesis, we divided 68 Sprague-Dawley male rats into 4 groups as follows: the control, MCT, MCT + P1 and MCT + P2 groups. MCT was administered at a dose of 60 mg/kg once via intraperitoneal injection. PCPA was administered via intraperitoneal injection at a dose of 50 or 100 mg/kg once daily for 21 consecutive days. We then measured the hemodynamic index and morphological analysis was carried out on the lung tissues. Western blot analysis and immunohistochemistry were used to examine the levels of NFAT-1 and NF-κB p-65. The expression levels of phosphorylated inhibitor of NF-κB kinase (p-IKK), IKK, phosphorylated extracellular signal‑regulated kinase (p-ERK), ERK, intercellular adhesion molecule-1 (ICAM-1) and inter-leukin-6 (IL-6) were examined by western blot analysis. MCT was found to significantly induce PAH, with inflammation and remodeling of the lung tissues. This was associatd with an increased expression of NFAT-1, p-IKK, p-ERK and nuclear p65. PCPA significantly attenuated MCT-induced inflammation and arterial remodeling, and decreased the expression of NFAT-1, as well as that of relevant proteins of the NF-κB signaling pathway. The above-mentioned findings suggest that the inhibitory effects of PCPA on MCT-induced inflammation and arterial remodeling are related to the downregulation of the NFAT-1 and NF-κB signaling pathways in rats with PAH.

Thymoquinone inhibits TNF-α-induced inflammation and cell adhesion in rheumatoid arthritis synovial fibroblasts by ASK1 regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Umar, Sadiq; Hedaya, Omar; Singh, Anil K.

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1–5 μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p < 0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effectmore » on NF-κB pathway, in RA-FLS (p < 0.05; n = 4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p < 0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA. - Highlights: • Evolving evidence suggests that ASK1 plays a central role in rheumatic arthritis (RA). • TNF-α activates ASK1, which regulate downstream signaling through JNK/p38 activation in RA-FLS. • ASK1 may be used as a potential therapeutic target in RA. • Thymoquinone was able to selectively inhibit TNF-α-induced phosphorylation of ASK1 in RA-FLS. • Thymoquinone might serve as a potential small-molecule inhibitor in RA.« less

An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

PubMed Central

Heibeck, Tyler H.; Ding, Shi-Jian; Opresko, Lee K.; Zhao, Rui; Schepmoes, Athena A.; Yang, Feng; Tolmachev, Aleksey V.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Wiley, H. Steven; Qian, Wei-Jun

2010-01-01

Protein tyrosine phosphorylation represents a central regulatory mechanism in cell signaling. Here we present an extensive survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell line by applying anti-phosphotyrosine peptide immunoaffinity purification coupled with high sensitivity capillary liquid chromatography tandem mass spectrometry. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and acute stimulation with epidermal growth factor (EGF). The estimated false discovery rate was 1.0% as determined by searching against a scrambled database. Comparison of these data with existing literature showed significant agreement for previously reported sites. However, we observed 281 sites that were not previously reported for HMEC cultures and 29 of which have not been reported for any human cell or tissue system. The analysis showed that the majority of highly phosphorylated proteins were relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed, raising the possibility of more important functional roles for such highly phosphorylated pTyr sites. By mapping to major signaling networks, such as the EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which provides interesting targets for future hypothesis-driven and targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems. PMID:19534553

Evidence for a role of Collapsin response mediator protein-2 in signaling pathways that regulate the proliferation of non-neuronal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tahimic, Candice Ginn T.; Tomimatsu, Nozomi; Nishigaki, Ryuichi

Collapsin response mediator protein-2 or Crmp-2 plays a critical role in the establishment of neuronal polarity. In this study, we present evidence that apart from its functions in neurodevelopment, Crmp-2 is also involved in pathways that regulate the proliferation of non-neuronal cells through its phosphorylation by regulatory proteins. We show that Crmp-2 undergoes dynamic phosphorylation changes in response to contact inhibition-induced quiescence and that hyperphosphorylation of Crmp-2 occurs in a tumor. We further suggest that de-regulation of Crmp-2 phosphorylation levels at certain amino acid residues may lead to aberrant cell proliferation and consequently, tumorigenesis.

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

Cancer.gov

The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

Analysis of genes involved in the PI3K/Akt pathway in radiation- and MNU-induced rat mammary carcinomas.

PubMed

Showler, Kaye; Nishimura, Mayumi; Daino, Kazuhiro; Imaoka, Tatsuhiko; Nishimura, Yukiko; Morioka, Takamitsu; Blyth, Benjamin J; Kokubo, Toshiaki; Takabatake, Masaru; Fukuda, Maki; Moriyama, Hitomi; Kakinuma, Shizuko; Fukushi, Masahiro; Shimada, Yoshiya

2017-03-01

The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis.

PubMed

Yang, Jung-Bo; Quan, Juan-Hua; Kim, Ye-Eun; Rhee, Yun-Ee; Kang, Byung-Hyun; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min; Lee, Young-Ha

2015-08-01

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.

SKI knockdown inhibits human melanoma tumor growth in vivo.

PubMed

Chen, Dahu; Lin, Qiushi; Box, Neil; Roop, Dennis; Ishii, Shunsuke; Matsuzaki, Koichi; Fan, Tao; Hornyak, Thomas J; Reed, Jon A; Stavnezer, Ed; Timchenko, Nikolai A; Medrano, Estela E

2009-12-01

The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

Oxytocin in the regulation of social behaviours in medial amygdala-lesioned mice via the inhibition of the extracellular signal-regulated kinase signalling pathway.

PubMed

Wang, Yu; Zhao, Shanshan; Wu, Zhe; Feng, Yu; Zhao, Chuansheng; Zhang, Chaodong

2015-05-01

The neuropeptide oxytocin (OXT) has been implicated in the pathophysiology of behavioural deficits among patients with autism spectrum disorder (ASD). However, the molecular mechanisms underlying its role in ASD remain unclear. In the present study, a murine model with ASD-like phenotypes was induced by intra-medial amygdala injection of N-methyl-d-aspartate, and it was used to investigate the role of OXT in behaviour regulation. Behavioural tests were performed to verify the ASD-like phenotypes of N-methyl-d-aspartate-treated mice, and the results showed that mice with bilateral medial amygdala lesions presented significant behavioural deficits, including impaired learning and memory and increased anxiety and depression. We also observed a notably decreased level of OXT in both the plasma and the hypothalamus of medial amygdala-lesioned mice, and the extracellular signal-regulated kinase (ERK) was activated. Further studies demonstrated that the administration of OXT alleviated ASD-like symptoms and significantly inhibited phosphorylation of ERK; the inhibitory effect was similar to that of U0126, an ERK signalling inhibitor. In addition, OXT administration modulated the expression of downstream proteins of the ERK signalling pathway, such as cyclic adenosine monophosphate response element binding and c-fos. Taken together, our data indicate that OXT plays an important role in ameliorating behavioural deficits in an ASD-like mouse model, which was mediated by inhibiting the ERK signalling pathway and its downstream proteins. © 2015 Wiley Publishing Asia Pty Ltd.

Proteomic interactions in the mouse vitreous-retina complex.

PubMed

Skeie, Jessica M; Mahajan, Vinit B

2013-01-01

Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina. Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software. We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor. Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.

Nardosinone protects H9c2 cardiac cells from angiotensin II-induced hypertrophy.

PubMed

Du, Meng; Huang, Kun; Gao, Lu; Yang, Liu; Wang, Wen-Shuo; Wang, Bo; Huang, Kai; Huang, Dan

2013-12-01

Pathological cardiac hypertrophy induced by angiotensin II (AngII) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngII-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosinone (25, 50, 100, and 200 μmol/L) or AngII (1 μmol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngII. The mRNA expression of ANP, BNP and β-MHC was obviously elevated in AngII-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 μmol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang II-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways.

(Z)3,4,5,4‧-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level

NASA Astrophysics Data System (ADS)

Fan, Xing-Xing; Yao, Xiao-Jun; Xu, Su Wei; Wong, Vincent Kam-Wai; He, Jian-Xing; Ding, Jian; Xue, Wei-Wei; Mujtaba, Tahira; Michelangeli, Francesco; Huang, Min; Huang, Jun; Xiao, Da-Kai; Jiang, Ze-Bo; Zhou, Yan-Ling; Kin-Ting Kam, Richard; Liu, Liang; Lai-Han Leung, Elaine

2015-11-01

Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4‧-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients.

Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

PubMed

Kumar, Raj; Calhoun, William J

2008-12-01

Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and related transcription factors. This review provides currently available information regarding the role of GR phosphorylation in its action, and highlights the possible underlying mechanisms of action.

IGFBP-1 hyperphosphorylation in response to leucine deprivation is mediated by the AAR pathway

PubMed Central

Malkani, Niyati; Jansson, Thomas; Gupta, Madhulika B.

2017-01-01

Insulin-like growth factor-1 (IGF-I) is the key regulator of fetal growth. IGF-I bioavailability is markedly diminished by IGF binding protein-1 (IGFBP-1) phosphorylation. Leucine deprivation strongly induces IGFBP-1hyperphosphorylation, and plays an important role in fetal growth restriction (FGR). FGR is characterized by decreased amino acid availability, which activates the amino acid response (AAR) and inhibits the mechanistic target of rapamycin (mTOR) pathway. We investigated the role of AAR and mTOR in mediating IGFBP-1 secretion and phosphorylation in HepG2 cells in leucine deprivation. mTOR inhibition (rapamycin or raptor+rictor siRNA), or activation (DEPTOR siRNA) demonstrated a role of mTOR in leucine deprivation-induced IGFBP-1 secretion but not phosphorylation. When the AAR was blocked (U0126, or ERK/GCN2 siRNA), both IGFBP-1 secretion and phosphorylation (Ser101/Ser119/Ser169) due to leucine deprivation were prevented. CK2 inhibition by TBB also attenuated IGFBP-1 phosphorylation in leucine deprivation. These results suggest that the AAR and mTOR independently regulate IGFBP-1 secretion and phosphorylation in leucine deprivation. PMID:25957086

Identification of differential pathways in papillary thyroid carcinoma utilizing pathway co-expression analysis.

PubMed

Qiu, Wei-Hai; Chen, Gui-Yan; Cui, Lu; Zhang, Ting-Ming; Wei, Feng; Yang, Yong

2016-01-01

To identify differential pathways between papillary thyroid carcinoma (PTC) patients and normal controls utilizing a novel method which combined pathway with co-expression network. The proposed method included three steps. In the first step, we conducted pretreatments for background pathways and gained representative pathways in PTC. Subsequently, a co-expression network for representative pathways was constructed using empirical Bayes (EB) approach to assign a weight value for each pathway. Finally, random model was extracted to set the thresholds of identifying differential pathways. We obtained 1267 representative pathways and their weight values based on the co-expressed pathway network, and then by meeting the criterion (Weight > 0.0296), 87 differential pathways in total across PTC patients and normal controls were identified. The top three ranked differential pathways were CREB phosphorylation, attachment of GPI anchor to urokinase plasminogen activator receptor (uPAR) and loss of function of SMAD2/3 in cancer. In conclusion, we successfully identified differential pathways (such as CREB phosphorylation, attachment of GPI anchor to uPAR and post-translational modification: synthesis of GPI-anchored proteins) for PTC using the proposed pathway co-expression method, and these pathways might be potential biomarkers for target therapy and detection of PTC.

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

PubMed

Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

2005-04-01

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

PI3K/Akt Signaling Pathway Activates the WNK-OSR1/SPAK-NCC Phosphorylation Cascade in Hyperinsulinemic db/db Mice

PubMed Central

Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

2013-01-01

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt-sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently-identified WNK kinase-OSR1/SPAK kinases-NCC transporter phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the PI3K/Akt signaling cascade in the kidney in response to hyperinsulinemia. A PI3K inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific PI3K inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the PI3K/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions such as the metabolic syndrome. PMID:22949526

Profiling the HER3/PI3K Pathway in Breast Tumors Using Proximity-Directed Assays Identifies Correlations between Protein Complexes and Phosphoproteins

PubMed Central

Mukherjee, Ali; Badal, Youssouf; Nguyen, Xuan-Thao; Miller, Johanna; Chenna, Ahmed; Tahir, Hasan; Newton, Alicia; Parry, Gordon; Williams, Stephen

2011-01-01

Background The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples. Methodology and Principal Findings Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation. Significance This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models. PMID:21297994

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors.

PubMed

Kobayashi, Yuhko; Murata, Michiharu; Minami, Hideyuki; Yamamoto, Shuhei; Kagaya, Yasuaki; Hobo, Tokunori; Yamamoto, Akiko; Hattori, Tsukaho

2005-12-01

The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.

Phosphorylated c-MPL tyrosine 591 regulates thrombopoietin-induced signaling

PubMed Central

Sangkhae, Veena; Saur, Sebastian Jonas; Kaushansky, Alexis; Kaushansky, Kenneth; Hitchcock, Ian Stuart

2018-01-01

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation and differentiation through binding to and stimulation of the cell surface receptor c-MPL. Activating mutations in c-MPL constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-MPL that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study we focused on c-MPL tyrosine 591 (Y591). We found Y591 of wild-type c-MPL to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using an SH2/PTB domain binding microarray, we identified novel c-MPL binding partners for phosphorylated Y591, including Src homology phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through siRNA treatment of Ba/F3-MPL cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by SYK. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development. PMID:24607955

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation.

PubMed

Li, Ping; Liu, Ping; Peng, Yan; Zhang, Zhuo-Hang; Li, Xiao-Ming; Xiong, Ren-Ping; Chen, Xing; Zhao, Yan; Ning, Ya-Lei; Yang, Nan; Zhang, Bo; Zhou, Yuan-Guo

2018-06-27

Increasing evidence has suggested that bidirectional regulation of cell proliferation is one important effect of TGF-β1 in wound healing. Increased c-Ski expression plays a role in promoting fibroblast proliferation at low TGF-β1 concentrations, but the mechanism by which low TGF-β1 concentrations regulate c-Ski levels remains unclear. In this study, the proliferation of rat primary fibroblasts was assessed with an ELISA BrdU kit. The mRNA and protein expression and phosphorylation levels of corresponding factors were measured by RT-qPCR, immunohistochemistry or Western blotting. We first found that low TGF-β1 concentrations not only promoted c-Ski mRNA and protein expression in rat primary fibroblasts but also increased the phosphorylation levels of Extracellular Signal-Regulated Kinases (ERK) and cAMP response element binding (CREB) protein. An ERK kinase (mitogen-activated protein kinase kinase, MEK) inhibitor significantly inhibited ERK1/2 phosphorylation levels, markedly reducing c-Ski expression and CREB phosphorylation levels and abrogating the growth-promoting effect of low TGF-β1 concentrations. At the same time, Smad2/3 phosphorylation levels were not significantly changed. Taken together, these results suggest that the increased cell proliferation induced by low TGF-β1 concentrations mediates c-Ski expression potentially through the ERK/CREB pathway rather than through the classic TGF-β1/Smad pathway.