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Sample records for extracellular signal-regulated protein

  1. Synbindin in Extracellular Signal-Regulated Protein Kinase Spatial Regulation and Gastric Cancer Aggressiveness

    PubMed Central

    2013-01-01

    Background The molecular mechanisms that control the aggressiveness of gastric cancer (GC) remain poorly defined. Here we show that synbindin contributes to the aggressiveness of GC by activating extracellular signal-regulated protein kinase (ERK) signaling on the Golgi apparatus. Methods Expression of synbindin was examined in normal gastric mucosa (n = 44), intestinal metaplastic gastric mucosa (n = 66), and GC tissues (n=52), and the biological effects of synbindin on tumor growth and ERK signaling were detected in cultured cells, nude mice, and human tissue samples. The interaction between synbindin and mitogen-activated protein kinase kinase (MEK1)/ERK was determined by immunofluorescence and fluorescence resonance energy transfer assays. The transactivation of synbindin by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was detected using luciferase reporter assay and chromatin immunoprecipitation. Results High expression of synbindin was associated with larger tumor size (120.8 vs 44.8cm3; P = .01), advanced tumor node metastasis (TNM) stage (P = .003), and shorter patient survival (hazard ratio = 1.51; 95% confidence interval [CI] = 1.01 to 2.27; P = .046). Synbindin promotes cell proliferation and invasion by activating ERK2 on the Golgi apparatus, and synbindin is directly transactivated by NF-κB. Synbindin expression level was statistically significantly higher in human GCs with activated ERK2 than those with low ERK2 activity (intensity score of 11.5, 95% CI = 10.4 to 12.4 vs intensity score of 4.6, 95% CI 3.9 to 5.3; P < .001). Targeting synbindin in xenograft tumors decreased ERK2 phosphorylation and statistically significantly reduced tumor volume (451.2mm3, 95% CI = 328.3 to 574.1 vs 726.1mm3, 95% CI = 544.2 to 908.2; P = .01). Conclusions Synbindin contributes to malignant phenotypes of GC by activating ERK on the Golgi, and synbindin is a potential biomarker and therapeutic target for GC. PMID:24104608

  2. Resting extracellular signal-regulated protein kinase 1/2 expression following a continuum of chronic resistance exercise training paradigms.

    PubMed

    Galpin, Andrew J; Fry, Andrew C; Nicoll, Justin X; Moore, Christopher A; Schilling, Brian K; Thomason, Donald B

    2016-01-01

    Extracellular signal-regulated protein kinase 1/2 (ERK1/2) moderates skeletal muscle growth; however, chronic responses of this protein to unique resistance exercise (RE) paradigms are yet to be explored. The purpose of this investigation was to describe the long-term response of ERK1/2 following circuit weight training (CWT), recreationally weight training (WT), powerlifting (PL) and weightlifting (WL). Independent t-tests were used to determine differences in trained groups compared to sedentary controls. Total ERK1/2 content was lower in PL and WL compared to their controls (p ≤ 0.05). Specific trained groups displayed large (WL: pERK/total-ERK; d = 1.25) and moderate (CWT: total ERK1/2; d = 0.54) effect sizes for altered kinase expression compared to controls. The results indicate ERK1/2 expression is down-regulated after chronic RE in well-trained weightlifters and powerlifters. Lower expression of this protein may be a method in which anabolism is tightly regulated after many years of high-intensity RE. PMID:27396416

  3. Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement, Attachment, Pairing and Egg Release in Schistosoma mansoni

    PubMed Central

    Ressurreição, Margarida; De Saram, Paulu; Kirk, Ruth S.; Rollinson, David; Emery, Aidan M.; Page, Nigel M.; Davies, Angela J.; Walker, Anthony J.

    2014-01-01

    Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance. PMID:24921927

  4. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    SciTech Connect

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  5. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes

    PubMed Central

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-01-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  6. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes.

    PubMed

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-08-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  7. Effect of training on activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in rat soleus muscle.

    PubMed

    Lee, Jong Sam; Bruce, Clinton R; Spurrell, Brian E; Hawley, John A

    2002-08-01

    1. The effect of a chronic programme of either low- or moderate-to-high-intensity treadmill running on the activation of the extracellular-signal regulated protein kinase (ERK1/2) and the p38 mitogen-activated protein kinase (MAPK) pathways was determined in rat muscle. 2. Sprague-Dawley rats were assigned to one of three groups: (i) sedentary (NT; n = 8); (ii) low-intensity training (8 m/min; LIT; n = 16); and (iii) moderate-to-high-intensity training (28 m/min; HIT; n = 16). The training regimens were planned so that animals covered the same distance and had similar glycogen utilization for both LIT and HIT exercise sessions. 3. A single bout of LIT or HIT following 8 weeks of training led to a twofold increase in the phosphorylation of ERK1/2 (P = 0.048) and a two- to threefold increase in p38 MAPK (P = 0.005). Extracellular signal-regulated kinase 1/2 phosphorylation in muscle sampled 48 h after the last exercise bout was similar to sedentary values, while p38 MAPK phosphorylation was 70-80% lower than sedentary. One bout of LIT or HIT increased total ERK1/2 and p38 MAPK expression, with the magnitude of this increase being independent of prior exercise intensity or duration. Extracellular signal- regulated kinase 1/2 expression was increased three- to fourfold in muscle sampled 48 h after the last exercise bout irrespective of the prior training programme (P = 0.027), but p38 MAPK expression was approximately 90% lower than sedentary values. 4. In conclusion, exercise-training of different intensities/ durations results in selective postexercise activation of intracellular signalling pathways, which may be one mechanism regulating specific adaptations induced by diverse training programmes. PMID:12099995

  8. Extracellular signal-regulated protein kinases 1 and 2 activation by addictive drugs: a signal toward pathological adaptation.

    PubMed

    Pascoli, Vincent; Cahill, Emma; Bellivier, Frank; Caboche, Jocelyne; Vanhoutte, Peter

    2014-12-15

    Addiction is a chronic and relapsing psychiatric disorder that is thought to occur in vulnerable individuals. Synaptic plasticity evoked by drugs of abuse in the so-called neuronal circuits of reward has been proposed to underlie behavioral adaptations that characterize addiction. By increasing dopamine in the striatum, addictive drugs alter the balance of dopamine and glutamate signals converging onto striatal medium-sized spiny neurons (MSNs) and activate intracellular events involved in long-term behavioral alterations. Our laboratory contributed to the identification of salient molecular changes induced by administration of addictive drugs to rodents. We pioneered the observation that a common feature of addictive drugs is to activate, by a double tyrosine/threonine phosphorylation, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the striatum, which control a plethora of substrates, some of them being critically involved in cocaine-mediated molecular and behavioral adaptations. Herein, we review how the interplay between dopamine and glutamate signaling controls cocaine-induced ERK1/2 activation in MSNs. We emphasize the key role of N-methyl-D-aspartate receptor potentiation by D1 receptor to trigger ERK1/2 activation and its subsequent nuclear translocation where it modulates both epigenetic and genetic processes engaged by cocaine. We discuss how cocaine-induced long-term synaptic and structural plasticity of MSNs, as well as behavioral adaptations, are influenced by ERK1/2-controlled targets. We conclude that a better knowledge of molecular mechanisms underlying ERK1/2 activation by drugs of abuse and/or its role in long-term neuronal plasticity in the striatum may provide a new route for therapeutic treatment in addiction. PMID:24844603

  9. The Vaccinia Virus O1 Protein Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase 1/2 and Promotes Viral Virulence

    PubMed Central

    Lukassen, Susanne; Späth, Michaela; Wolferstätter, Michael; Babel, Eveline; Brinkmann, Kay; Wielert, Ursula; Chaplin, Paul; Suter, Mark

    2012-01-01

    Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo. PMID:22171261

  10. Albumin stimulates p44/p42 extracellular-signal-regulated mitogen-activated protein kinase in opossum kidney proximal tubular cells.

    PubMed

    Dixon, R; Brunskill, N J

    2000-03-01

    The presence of protein in the urine of patients with renal disease is an adverse prognostic feature. It has therefore been suggested that proteinuria per se may be responsible for the development of renal tubulo-interstitial scarring and fibrosis, and disturbances in tubular cell growth and proliferation. We have used the opossum kidney proximal tubular cell line to investigate the effects of albumin on cell growth. The effect of albumin on cell proliferation was investigated by cell counting and measurement of [(3)H]thymidine incorporation. We studied the effect of recombinant human albumin on the activity of p44/p42 extracellular-signal-regulated mitogen-activated protein kinase (MAP kinase ) using an in vitro kinase assay, and immunoblotting with antibodies against active extracellular-signal-regulated kinase (ERK). The effects of the ERK inhibitor PD98059 were also examined. Recombinant human albumin was found to stimulate proliferation of opossum kidney cells in a dose-dependent manner, with maximal stimulation at a concentration of 1 mg/ml. In addition, recombinant human albumin activated ERK in a time-dependent (maximal after 5 min) and dose-dependent (maximal at 1 mg/ml) fashion. These effects on cell proliferation and ERK activity were inhibited by PD98059, and were not reproduced by ovalbumin or mannitol. The data therefore indicate that albumin is able to stimulate growth and proliferation of proximal tubular cells that is dependent on the ERK family of MAP kinases. The potential importance of this pathway in the development of renal disease is discussed. PMID:10677388

  11. Na/H Exchange Regulatory Factor 1, a Novel AKT-associating Protein, Regulates Extracellular Signal-regulated Kinase Signaling through a B-Raf–Mediated Pathway

    PubMed Central

    Wang, Bin; Yang, Yanmei

    2008-01-01

    Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5′-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf. PMID:18272783

  12. Dopamine D1 Receptors Regulate Protein Synthesis-Dependent Long-Term Recognition Memory via Extracellular Signal-Regulated Kinase 1/2 in the Prefrontal Cortex

    ERIC Educational Resources Information Center

    Nagai, Taku; Takuma, Kazuhiro; Kamei, Hiroyuki; Ito, Yukio; Nakamichi, Noritaka; Ibi, Daisuke; Nakanishi, Yutaka; Murai, Masaaki; Mizoguchi, Hiroyuki; Nabeshima, Toshitaka; Yamada, Kiyofumi

    2007-01-01

    Several lines of evidence suggest that extracellular signal-regulated kinase1/2 (ERK1/2) and dopaminergic system is involved in learning and memory. However, it remains to be determined if the dopaminergic system and ERK1/2 pathway contribute to cognitive function in the prefrontal cortex (PFC). The amount of phosphorylated ERK1/2 was increased in…

  13. MEK Kinase 2 and the Adaptor Protein Lad Regulate Extracellular Signal-Regulated Kinase 5 Activation by Epidermal Growth Factor via Src

    PubMed Central

    Sun, Weiyong; Wei, Xudong; Kesavan, Kamala; Garrington, Timothy P.; Fan, Ruihua; Mei, Junjie; Anderson, Steven M.; Gelfand, Erwin W.; Johnson, Gary L.

    2003-01-01

    Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex. PMID:12640115

  14. Pre-LTP requires extracellular signal-regulated kinase in the ACC

    PubMed Central

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush

    2016-01-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  15. Pre-LTP requires extracellular signal-regulated kinase in the ACC.

    PubMed

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush; Zhuo, Min

    2016-02-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  16. Dual-specificity Phosphatase 1 Deficiency Induces Endometrioid Adenocarcinoma Progression via Activation of Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Pathway

    PubMed Central

    Yang, Yuan; Zhou, Jing-Yi; Zhao, Li-Jun; Gao, Bao-Rong; Wan, Xiao-Ping; Wang, Jian-Liu

    2016-01-01

    Background: Previously, we reported that dual-specificity phosphatase 1 (DUSP1) was differentially expressed in endometrioid adenocarcinoma (EEA). However, the role of DUSP1 in EEA progression and the relationship between DUSP1 and medroxyprogesterone (MPA) are still unclear. Methods: The expression of DUSP1 in EEA specimens was detected by immunohistochemical analysis. The effect of DUSP1 on cell proliferation was analyzed by Cell Counting Kit 8 and colony formation assay, and cell migration was analyzed by transwell assay. MPA-induced DUSP1 expression in EEA cells was measured by Western blot. Results: DUSP1 expression was deficient in advanced International Federation of Gynecology and Obstetrics stage, high-grade and myometrial invasive EEA. In EEA cell lines (Hec1A, Hec1B, RL952, and Ishikawa), the DUSP1 expression was substantially higher in Ishikawa cells than in other cell lines (P < 0.05). Knockdown of DUSP1 promoted Ishikawa cells proliferation, migration, and activation of mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/Erk) pathway. MPA-induced DUSP1 expression and inhibited MAPK/Erk pathway in Ishikawa cells. Conclusions: Our data suggest that DUSP1 deficiency promotes EEA progression via MAPK/Erk pathway, which may be reversed by MPA, suggesting that DUSP1 may serve as a potential therapeutic target for the treatment of EEA. PMID:27174322

  17. The Novel Anticancer Drug Hydroxytriolein Inhibits Lung Cancer Cell Proliferation via a Protein Kinase Cα- and Extracellular Signal-Regulated Kinase 1/2-Dependent Mechanism.

    PubMed

    Guardiola-Serrano, Francisca; Beteta-Göbel, Roberto; Rodríguez-Lorca, Raquel; Ibarguren, Maitane; López, David J; Terés, Silvia; Alvarez, Rafael; Alonso-Sande, María; Busquets, Xavier; Escribá, Pablo V

    2015-08-01

    Membrane lipid therapy is a novel approach to rationally design or discover therapeutic molecules that target membrane lipids. This strategy has been used to design synthetic fatty acid analogs that are currently under study in clinical trials for the treatment of cancer. In this context, and with the aim of controlling tumor cell growth, we have designed and synthesized a hydroxylated analog of triolein, hydroxytriolein (HTO). Both triolein and HTO regulate the biophysical properties of model membranes, and they inhibit the growth of non-small-cell lung cancer (NSCLC) cell lines in vitro. The molecular mechanism underlying the antiproliferative effect of HTO involves regulation of the lipid membrane structure, protein kinase C-α and extracellular signal-regulated kinase activation, the production of reactive oxygen species, and autophagy. In vivo studies on a mouse model of NSCLC showed that HTO, but not triolein, impairs tumor growth, which could be associated with the relative resistance of HTO to enzymatic degradation. The data presented explain in part why olive oil (whose main component is the triacylglycerol triolein) is preventive but not therapeutic, and they demonstrate a potent effect of HTO against cancer. HTO shows a good safety profile, it can be administered orally, and it does not induce nontumor cell (fibroblast) death in vitro or side effects in mice, reflecting its specificity for cancer cells. For these reasons, HTO is a good candidate as a drug to combat cancer that acts by regulating lipid structure and function in the cancer cell membrane. PMID:26065701

  18. Chronic activation of extracellular-signal-regulated protein kinases by phenylephrine is required to elicit a hypertrophic response in cardiac myocytes.

    PubMed Central

    Barron, Anthony J; Finn, Stephen G; Fuller, Stephen J

    2003-01-01

    Extracellular-signal-regulated protein kinases (ERKs) are activated rapidly and transiently in response to phenylephrine (PE) and endothelin-1 (ET-1) in cardiac myocytes, but whether this is linked to the subsequent development of the hypertrophic phenotype remains equivocal. To investigate this, we examined the dependence of the hypertrophic response on the length of exposure to PE in neonatal myocyte cultures. In addition to the initial transient activation of ERKs (maximum at 5-10 min), PE (10 microM) induced a second, more prolonged peak of activity several hours later. The activity of a transfected atrial natriuretic factor-luciferase reporter gene was increased 10- to 24-fold by PE. This response was inhibited by the alpha(1)-antagonist prazosin (100 nM) and by U0126 (10 microM) and PD184352 (1 microM), inhibitors of ERK activation, irrespective of whether these were added before or up to 24 h after the addition of PE. Prazosin had no effect on ET-1 (50 nM)-stimulated atrial natriuretic factor-luciferase activity. Protein synthesis was enhanced by 35+/-6% by PE, and this was blocked by prazosin added 1 h after the addition of PE, but decreased only by half when added 8 h after PE. Similarly, PE (48 h) increased myocyte area by 49% and this was prevented by prazosin added 1 h after PE, but decreased only by half when added at 24 h. These results demonstrate that prolonged exposure to PE is required to elicit alterations in gene expression, protein synthesis and cell size, characteristic of hypertrophied myocytes, and they confirm that the initial peak of ERK activity is insufficient to trigger hypertrophic responses. PMID:12513686

  19. Extracellular signal-regulated kinases modulate capacitation of human spermatozoa.

    PubMed

    Luconi, M; Barni, T; Vannelli, G B; Krausz, C; Marra, F; Benedetti, P A; Evangelista, V; Francavilla, S; Properzi, G; Forti, G; Baldi, E

    1998-06-01

    Recent evidence indicates the presence of p21 Ras and of a protein with characteristics similar to mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), in mammalian spermatozoa, suggesting the occurrence of the Ras/ERK cascade in these cells. In the present study we investigated the subcellular localization of ERKs and their biological functions in human spermatozoa. Immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy demonstrated localization of ERKs in the postacrosomal region of spermatozoa. After stimulation of acrosome reaction with the calcium ionophore A23187 and progesterone, ERKs were mostly localized at the level of the equatorial region, indicating redistribution of these proteins in acrosome-reacted spermatozoa. Two proteins of 42 and 44 kDa that are tyrosine phosphorylated in a time-dependent manner during in vitro capacitation were identified as p42 (ERK-2) and p44 (ERK-1) by means of specific antibodies. The increase in tyrosine phosphorylation of these proteins during capacitation was accompanied by increased kinase activity, as determined by the ability of ERK-1 and ERK-2 to phosphorylate the substrate myelin basic protein. The role of this activity in the occurrence of sperm capacitation was also investigated by using PD098059, an inhibitor of the MAPK cascade. The presence of this compound during in vitro capacitation inhibits ERK activation and significantly reduces the ability of spermatozoa to undergo the acrosome reaction in response to progesterone. Since only capacitated spermatozoa are able to respond to progesterone, these data strongly indicate that ERKs are involved in the regulation of capacitation. In summary, our data demonstrate the presence of functional ERKs in human spermatozoa and indicate that these enzymes are involved in activation of these cells during capacitation, providing new insight in clarifying the molecular mechanisms and the

  20. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

    PubMed Central

    Reszka, A A; Bulinski, J C; Krebs, E G; Fischer, E H

    1997-01-01

    The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs. Images PMID:9243503

  1. Osthole-mediated cell differentiation through bone morphogenetic protein-2/p38 and extracellular signal-regulated kinase 1/2 pathway in human osteoblast cells.

    PubMed

    Kuo, Po-Lin; Hsu, Ya-Ling; Chang, Cheng-Hsiung; Chang, Jiunn-Kae

    2005-09-01

    The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients. Osthole (7-methoxy-8-isopentenoxycoumarin) is a coumarin derivative present in many medicinal plants. By means of alkaline phosphatase (ALP) activity, osteocalcin, osteopontin, and type I collagen, enzyme-linked immunosorbent assay, we have shown that osthole exhibits a significant induction of differentiation in two human osteoblast-like cell lines, MG-63 and hFOB. Induction of differentiation by osthole was associated with increased bone morphogenetic protein (BMP)-2 production and the activations of SMAD1/5/8 and p38 and extracellular signal-regulated kinase (ERK) 1/2 kinases. Addition of purified BMP-2 protein did not increase the up-regulation of ALP activity and osteocalcin by osthole, whereas the BMP-2 antagonist noggin blocked both osthole and BMP-2-mediated ALP activity enhancement, indicating that BMP-2 production is required in osthole-mediated osteoblast maturation. Pretreatment of osteoblast cells with noggin abrogated p38 activation but only partially decreased ERK1/2 activation, suggesting that BMP-2 signaling is required in p38 activation and is partially involved in ERK1/2 activation in osthole-treated osteoblast cells. Cotreatment of p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] or p38 small interfering RNA (siRNA) expression inhibited osthole-mediated activation of ALP but only slightly affected osteocalcin production. In contrast, the production of osteocalcin induced by osthole was inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) or by expression of an ERK2 siRNA. These data suggest that BMP-2/p38 pathway links to the early phase, whereas ERK1/2 pathway is associated with the later phase in osthole-mediated differentiation of osteoblast cells. In this study, we demonstrate that osthole is a promising agent for treating osteoporosis

  2. Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.

    2010-12-01

    The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

  3. Role of adenosine A2A receptor in cerebral ischemia reperfusion injury: Signaling to phosphorylated extracellular signal-regulated protein kinase (pERK1/2).

    PubMed

    Mohamed, R A; Agha, A M; Abdel-Rahman, A A; Nassar, N N

    2016-02-01

    Following brain ischemia reperfusion (IR), the dramatic increase in adenosine activates A2AR to induce further neuronal damage. Noteworthy, A2A antagonists have proven efficacious in halting IR injury, however, the detailed downstream signaling remains elusive. To this end, the present study aimed to investigate the possible involvement of phospho-extracellular signal-regulated kinase (pERK1/2) pathway in mediating protection afforded by the central A2A blockade. Male Wistar rats (250-270 g) subjected to bilateral carotid occlusion for 45 min followed by a 24-h reperfusion period showed increased infarct size corroborating histopathological damage, memory impairment and motor incoordination as well as increased locomotor activity. Those events were mitigated by the unilateral intrahippocampal administration of the selective A2A antagonist SCH58261 via a decrease in pERK1/2 downstream from diacyl glycerol (DAG) signaling. Consequent to pERK1/2 inhibition, reduced hippocampal microglial activation, glial tumor necrosis factor-alpha (TNF-α) and brain-derived neurotropic factor (BDNF) expression, glutamate (Glu), inducible nitric oxide synthase (iNOS) and thiobarbituric acid reactive substances (TBARS) were evident in animals receiving SCH58261. Additionally, the anti-inflammatory cytokine interleukin-10 (IL-10) increased following nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). Taken all together, these events suppressed apoptotic pathways via a reduction in cytochrome c (Cyt. c) as well as caspase-3 supporting a crucial role for pERK1/2 inhibition in consequent reduction of inflammatory and excitotoxic cascades as well as correction of the redox imbalance. PMID:26642806

  4. Extracellular signal-regulated kinases in pain of peripheral origin.

    PubMed

    White, John P M; Cibelli, Mario; Fidalgo, Antonio Rei; Nagy, Istvan

    2011-01-10

    Activation of members of the family of enzymes known as extracellular signal-regulated kinases (ERKs) is now known to be involved in the development and/or maintenance of the pain associated with many inflammatory conditions, such as herniated spinal disc pain, chronic inflammatory articular pain, and the pain associated with bladder inflammation. Moreover, ERKs are implicated in the development of neuropathic pain signs in animals which are subjected to the lumbar 5 spinal nerve ligation model and the chronic constriction injury model of neuropathic pain. The position has now been reached where all scientists working on pain subjects ought to be aware of the importance of ERKs, if only because certain of these enzymes are increasingly employed as experimental markers of nociceptive processing. Here, we introduce the reader, first, to the intracellular context in which these enzymes function. Thereafter, we consider the involvement of ERKs in mediating nociceptive signalling to the brain resulting from noxious stimuli at the periphery which will be interpreted by the brain as pain of peripheral origin. PMID:20950608

  5. Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase C(alpha), src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.

    PubMed

    Canet-Aviles, Rosa-Maria; Anderton, Mark; Hooper, Nigel M; Turner, Anthony J; Vaughan, Peter F T

    2002-06-15

    The signalling pathways by which muscarine and epidermal growth factor (EGF) regulate the secretion of the alpha-secretase cleavage product (sAPPalpha) of the amyloid precursor protein (APP) were examined in the human neuroblastoma SH-SY5Y. Using specific inhibitors it was found that over 80% of sAPPalpha secretion, enhanced by muscarine, occurred via the extracellular signal-regulated kinase (ERK1/2) member of the mitogen-activated protein kinase (MAPK) family and was dependent on protein kinase Calpha (PKCalpha) and a member of the Src family of non-receptor tyrosine kinases (Src-TK). In contrast the stimulation of sAPPalpha secretion by EGF was not affected by inhibitors of PKC nor Src-TK but was dependent on ERK1/2. In addition muscarine-enhanced sAPPalpha secretion and ERK1/2 activation were inhibited 60 and 80%, respectively, by micromolar concentrations of the phosphatidylinositol 3 kinase (PI-3K) inhibitor wortmannin. In comparison wortmannin decreased EGF stimulation of sAPPalpha secretion and ERK 1/2 activation by approximately 40%. Unexpectedly, U73122, an inhibitor of phosphoinositide-specific phospholipase C, did not inhibit muscarine enhancement of sAPPalpha secretion. These data are discussed in relation to a pathway for the enhancement of sAPPalpha secretion by muscarine which involves the activation of a Src-TK by G-protein beta/gamma-subunits leading to activation of PKCalpha, and ERK1/2 by a mechanism not involving phospholipase C. PMID:12191495

  6. Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.

    PubMed Central

    Wilson, N J; Moss, S T; Csar, X F; Ward, A C; Hamilton, J A

    1999-01-01

    Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A. PMID:10215588

  7. Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase*

    PubMed Central

    Cottom, Joshua; Salvador, Lisa M.; Maizels, Evelyn T.; Reierstad, Scott; Park, Youngkyu; Carr, Daniel W.; Davare, Monika A.; Hell, Johannes W.; Palmer, Stephen S.; Dent, Paul; Kawakatsu, Hisaaki; Ogata, Masato; Hunzicker-Dunn, Mary

    2006-01-01

    In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca2+ channel blocker, and chelation of extracellular Ca2+. These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase. PMID:12493768

  8. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways.

    PubMed

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P; Taub, Dennis D

    2014-12-20

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levels and impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  9. Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

    PubMed Central

    Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

    2014-01-01

    Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

  10. Angiotensin II regulates ACE and ACE2 in neurons through p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 signaling

    PubMed Central

    Xiao, Liang; Haack, Karla K. V.

    2013-01-01

    Brain ANG II plays an important role in modulating sympathetic function and homeostasis. The generation and degradation of ANG II are carried out, to a large extent, through the angiotensin-converting enzyme (ACE) and ACE2, respectively. In disease states, such as hypertension and chronic heart failure, central expression of ACE is upregulated and ACE2 is decreased in central sympathoregulatory neurons. In this study, we determined the expression of ACE and ACE2 in response to ANG II in a neuronal cell culture and the subsequent signaling mechanism(s) involved. A mouse catecholaminergic neuronal cell line (CATH.a) was treated with ANG II (30, 100, and 300 nM) for 24 h, and protein expression was determined by Western blot analysis. ANG II induced a significant dose-dependent increase in ACE and decrease in ACE2 mRNA and protein expression in CATH.a neurons. This effect was abolished by pretreatment of the cells with the p38 MAPK inhibitor SB-203580 (10 μM) 30 min before administration of ANG II or the ERK1/2 inhibitor U-0126 (10 μM). These data suggest that ANG II increases ACE and attenuates ACE2 expression in neurons via the ANG II type 1 receptor, p38 MAPK, and ERK1/2 signaling pathways. PMID:23535237

  11. Angiotensin II type 2 receptor-interacting protein 3a suppresses proliferation, migration and invasion in tongue squamous cell carcinoma via the extracellular signal-regulated kinase-Snai2 pathway

    PubMed Central

    ZHAO, TINGTING; HE, QIANTING; LIU, ZHONGHUA; DING, XUEQIANG; ZHOU, XIAOFENG; WANG, ANXUN

    2016-01-01

    Our previous studies demonstrated that the downregulation of microtubule-associated tumor suppressor 1/angiotensin II type 2 receptor-interacting protein (MTUS1/ATIP) is associated with poor differentiation and prognosis in tongue squamous cell carcinoma (TSCC), and that ATIP1 exerts an antiproliferative effect on TSCC. The aim of the present study was to further investigate the anticancer effect of MTUS1/ATIP3a in TSCC. It was observed that UM1 cells (a TSCC cell line with high migration and invasion ability) exhibited lower expression of ATIP3a compared with UM2 cells (a TSCC cell line with lower migration and invasion ability). Restoration of ATIP3a expression in UM1 cells exerted antiproliferative effects and inhibited migration and invasion, whereas knockdown of ATIP3a promoted proliferation, migration and invasion in UM2 cells. Restoration of ATIP3a expression inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and the expression of Snai2 and vimentin in UM1 cells, whereas knockdown of ATIP3a promoted the phosphorylation of ERK1/2 and the expression of Snai2 and vimentin in UM2 cells. Therefore, MTUS1/ATIP3a was found to suppress the proliferation, migration and invasion of TSCC cells via the ERK1/2-Snai2 pathway. PMID:26870214

  12. Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

    SciTech Connect

    Kumari, Gita; Mahalingam, S.

    2009-10-01

    Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

  13. Post-exercise impact of ingested whey protein hydrolysate on gene expression profiles in rat skeletal muscle: activation of extracellular signal-regulated kinase 1/2 and hypoxia-inducible factor-1α.

    PubMed

    Kanda, Atsushi; Ishijima, Tomoko; Shinozaki, Fumika; Nakayama, Kyosuke; Fukasawa, Tomoyuki; Nakai, Yuji; Abe, Keiko; Kawahata, Keiko; Ikegami, Shuji

    2014-06-28

    We have previously shown that whey protein hydrolysate (WPH) causes a greater increase in muscle protein synthesis than does a mixture of amino acids that is identical in amino acid composition. The present study was conducted to investigate the effect of WPH on gene expression. Male Sprague-Dawley rats subjected to a 2 h swimming exercise were administered either a carbohydrate-amino acid diet or a carbohydrate-WPH diet immediately after exercise. At 1 h after exercise, epitrochlearis muscle mRNA was sampled and subjected to DNA microarray analysis. We found that ingestion of WPH altered 189 genes after considering the false discovery rate. Among the up-regulated genes, eight Gene Ontology (GO) terms were enriched, which included key elements such as Cd24, Ccl2, Ccl7 and Cxcl1 involved in muscle repair after exercise. In contrast, nine GO terms were enriched in gene sets that were down-regulated by the ingestion of WPH, and these GO terms fell into two clusters, 'regulation of ATPase activity' and 'immune response'. Furthermore, we found that WPH activated two upstream proteins, extracellular signal-regulated kinase 1/2 (ERK1/2) and hypoxia-inducible factor-1α (HIF-1α), which might act as key factors for regulating gene expression. These results suggest that ingestion of WPH, compared with ingestion of a mixture of amino acids with an identical amino acid composition, induces greater changes in the post-exercise gene expression profile via activation of the proteins ERK1/2 and HIF-1α. PMID:24598469

  14. Suppression of Mitochondrial Biogenesis through Toll-Like Receptor 4–Dependent Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling in Endotoxin-Induced Acute Kidney Injury

    PubMed Central

    Smith, Joshua A.; Stallons, L. Jay; Collier, Justin B.; Chavin, Kenneth D.

    2015-01-01

    Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1β) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis. PMID:25503387

  15. Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNF{alpha} in human epidermal keratinocytes (HaCaT)

    SciTech Connect

    Onda, Kenji . E-mail: knjond@ps.toyaku.ac.jp; Nagashima, Masahiro; Kawakubo, Yo; Inoue, Shota; Hirano, Toshihiko; Oka, Kitaro

    2006-12-08

    Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) {alpha} reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNF{alpha} was not accompanied by changes in mRNA expressions of GR isoforms ({alpha} or {beta}). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNF{alpha}. Additionally, we observed that TNF{alpha} reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNF{alpha}-mediated GC insensitivity. Our data suggest that overexpression of TNF{alpha} leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.

  16. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  17. p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps

    PubMed Central

    Wu, Zhenguo; Woodring, Pamela J.; Bhakta, Kunjan S.; Tamura, Kumiko; Wen, Fang; Feramisco, James R.; Karin, Michael; Wang, Jean Y. J.; Puri, Pier Lorenzo

    2000-01-01

    The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle differentiation. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun–N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of p38 prevents the differentiation program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle differentiation even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts derived from p38α−/− embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic growth) in myotubes. PMID

  18. Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

    PubMed Central

    Dine, Julien; Ducourneau, Vincent R R; Fénelon, Valérie S; Fossat, Pascal; Amadio, Aurélie; Eder, Matthias; Israel, Jean-Marc; Oliet, Stéphane H R; Voisin, Daniel L

    2014-01-01

    Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of

  19. Extracellular signal-regulated kinase (ERK) activation is required for porcine epidemic diarrhea virus replication.

    PubMed

    Kim, Youngnam; Lee, Changhee

    2015-10-01

    Porcine epidemic diarrhea virus (PEDV) is a highly enteropathogenic coronavirus of swine that causes acute enteritis with high mortality in nursery piglets. To date, the cellular factors involved in PEDV replication have not been well defined. The extracellular signal-regulated kinase (ERK) that serves as a critical component of cellular signal transduction pathways to modulate a variety of cellular functions has been shown to regulate several viral infections. In the present study, we found that PEDV activates ERK1/2 early in infection independently of viral replication. The PEDV-induced ERK1/2 activation resulted in the phosphorylation of its downstream substrate Elk-1 in infected cells. Treatment with ERK inhibitors or ERK1/2 knockdown significantly suppressed viral progeny production. Inhibition of ERK activation also diminished viral protein expression and genomic and subgenomic RNA transcription. These findings indicate that the ERK signaling pathway plays an important role in the PEDV life cycle and beneficially contributes to viral infection. PMID:26115165

  20. Learned stressor resistance requires extracellular signal-regulated kinase in the prefrontal cortex

    PubMed Central

    Christianson, John P.; Flyer-Adams, Johanna G.; Drugan, Robert C.; Amat, Jose; Daut, Rachel A.; Foilb, Allison R.; Watkins, Linda R.; Maier, Steven F.

    2014-01-01

    Behaviorally controllable stressors confer protection from the neurochemical and behavioral consequences of future uncontrollable stressors, a phenomenon termed “behavioral immunization”. Recent data implicate protein synthesis within the ventromedial prefrontal cortex (mPFC) as critical to behavioral immunization. Adult, male Sprague-Dawley rats were exposed to a series of controllable tailshocks and 1 week later to uncontrollable tailshocks, followed 24 h later by social exploration and shuttlebox escape tests. To test the involvement of N-methyl-D-aspartate receptors (NMDARs) and the extracellular signal-regulated kinase (ERK) cascade in behavioral immunization, either D-AP5 or the MEK inhibitor U0126 was injected to the prelimbic (PL) or infralimbic (IL) mPFC prior to controllable stress exposure. Phosphorylated ERK and P70S6K, regulators of transcription and translation, were quantified by Western blot or immunohistochemistry after controllable or uncontrollable tailshocks. Prior controllable stress prevented the social exploration and shuttlebox performance deficits caused by the later uncontrollable stressor, and this effect was blocked by injections of D-AP5 into mPFC. A significant increase in phosphorylated ERK1 and ERK2, but not P70S6K, occurred within the PL and IL in rats exposed to controllable stress, but not to uncontrollable stress. However, U0126 only prevented behavioral immunization when injected to the PL. We provide evidence that NMDAR and ERK dependent signaling within the PL region is required for behavioral immunization, a learned form of stressor resistance. PMID:25324750

  1. Interleukin-1β Increases Gap Junctional Communication among Synovial Fibroblasts via the Extracellular Signal Regulated Kinase Pathway

    PubMed Central

    Niger, Corinne; Howell, Floyd D.; Stains, Joseph P.

    2009-01-01

    Background information The gap junction protein, connexin43, has been implicated in the etiology of osteoarthritis. Work from others has revealed that the size and number of gap junctions increases in synovial biopsies from patients with osteoarthritis. Further, pharmacologic inhibition of connexin43 function has been shown to reduce interleukin-1β induced metalloproteinase production by synovial fibroblasts in vitro. Results In this study, we examine the link between interleukin-1β and connexin43 function. We demonstrate that treatment of a rabbit synovial fibroblast cell line with interleukin-1β markedly increases connexin43 protein in a dose- and time-dependent manner. The impact on connexin43 protein levels appears to occur post-transcriptionally as mRNA levels are unaffected by interleukin-1β administration. Additionally, we show by fluorescence microscopy that interleukin-1β alters the cellular distribution of connexin43 to cell-cell junctions and is concomitant to a striking increase in gap junction communication. Further, we demonstrate that the increase in connexin43 protein and the associated change in protein localization and gap junction communication following interleukin-1β treatment are dependent upon activation of the extracellular signal regulated kinase signaling cascade. Conclusions These data show that interleukin-1β acts through the extracellular signal regulated kinase signaling cascade to alter the expression and function of connexin43 in synovial fibroblasts. PMID:19656083

  2. Cadmium activates extracellular signal-regulated kinase 5 in HK-2 human renal proximal tubular cells

    SciTech Connect

    Kondo, Mio; Inamura, Hisako; Matsumura, Ken-ichi; Matsuoka, Masato

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cadmium exposure induces ERK5 phosphorylation in HK-2 renal proximal tubular cells. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced ERK5 but not ERK1/2 phosphorylation. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced CREB and c-Fos phosphorylation. Black-Right-Pointing-Pointer ERK5 activation by cadmium exposure may play an anti-apoptotic role in HK-2 cells. -- Abstract: We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the phosphorylation and functionality of extracellular signal-regulated kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, in HK-2 human renal proximal tubular cells. Following exposure to CdCl{sub 2}, ERK5 phosphorylation increased markedly, but the level of total ERK5 was unchanged. ERK5 phosphorylation following CdCl{sub 2} exposure was rapid and transient, similar to the time course of ERK1/2 phosphorylation. Treatment of HK-2 cells with the MAPK/ERK kinase 5 inhibitor, BIX02189, suppressed CdCl{sub 2}-induced ERK5 but not ERK1/2 phosphorylation. The CdCl{sub 2}-induced increase of phosphorylated cAMP response element-binding protein (CREB) and activating transcription factor-1 (ATF-1), as well as the accumulation of mobility-shifted c-Fos protein, were suppressed by BIX02189 treatment. Furthermore, BIX02189 treatment enhanced cleavage of poly(ADP-ribose) polymerase and increased the level of cytoplasmic nucleosomes in HK-2 cells exposed to CdCl{sub 2}. These findings suggest that ERK5 pathway activation by CdCl{sub 2} exposure might induce the phosphorylation of cell survival-transcription factors, such as CREB, ATF-1, and c-Fos, and may exert a partial anti-apoptotic role in HK-2 cells.

  3. Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

    SciTech Connect

    Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

    2009-12-01

    Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

  4. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

    NASA Technical Reports Server (NTRS)

    Park, H.; Go, Y. M.; Darji, R.; Choi, J. W.; Lisanti, M. P.; Maland, M. C.; Jo, H.

    2000-01-01

    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

  5. Phosphorylation of human immunodeficiency virus type 1 capsid protein at serine 16, required for peptidyl-prolyl isomerase-dependent uncoating, is mediated by virion-incorporated extracellular signal-regulated kinase 2.

    PubMed

    Dochi, Takeo; Nakano, Takashi; Inoue, Mutsumi; Takamune, Nobutoki; Shoji, Shozo; Sano, Kouichi; Misumi, Shogo

    2014-05-01

    We reported previously that Pin1 facilitates human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid core through the phosphorylated Ser(16)-Pro(17) motif. However, the specific kinase responsible for Ser(16) phosphorylation has remained unknown. Here, we showed that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser(16). The characterization of immature virions produced by exposing chronically HIV-1LAV-1-infected CEM/LAV-1 cells to 10 µM saquinavir indicated that Ser(16) is phosphorylated after the initiation of Pr55(Gag) processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser(16) residue in the Ser(16)-Pro(17) motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser(16) phosphorylation level inside virions, and virus partially defective in Ser(16) phosphorylation showed impaired reverse transcription and attenuated replication owing to attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser(16) phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed impaired reverse transcription and attenuated HIV-1 replication. Taken together, these findings provide insights into the as-yet-obscure processes in Pin1-dependent HIV-1 uncoating. PMID:24509437

  6. Extracellular signal-regulated kinase-2 within the ventral tegmental area regulates responses to stress.

    PubMed

    Iñiguez, Sergio D; Vialou, Vincent; Warren, Brandon L; Cao, Jun-Li; Alcantara, Lyonna F; Davis, Lindsey C; Manojlovic, Zarko; Neve, Rachael L; Russo, Scott J; Han, Ming-Hu; Nestler, Eric J; Bolaños-Guzmán, Carlos A

    2010-06-01

    Neurotrophic factors and their signaling pathways have been implicated in the neurobiological adaptations in response to stress and the regulation of mood-related behaviors. A candidate signaling molecule implicated in mediating these cellular responses is the extracellular signal-regulated kinase (ERK1/2), although its functional role in mood regulation remains to be fully elucidated. Here we show that acute (1 d) or chronic (4 weeks) exposure to unpredictable stress increases phosphorylation of ERK1/2 and of two downstream targets (ribosomal S6 kinase and mitogen- and stress-activated protein kinase 1) within the ventral tegmental area (VTA), an important substrate for motivated behavior and mood regulation. Using herpes simplex virus-mediated gene transfer to assess the functional significance of this ERK induction, we show that overexpressing ERK2 within the VTA increases susceptibility to stress as measured in the forced swim test, responses to unconditioned nociceptive stimuli, and elevated plus maze in Sprague Dawley male rats, and in the tail suspension test and chronic social defeat stress procedure in C57BL/6 male mice. In contrast, blocking ERK2 activity in the VTA produces stress-resistant behavioral responses in these same assays and also blocks a chronic stress-induced reduction in sucrose preference. The effects induced by ERK2 blockade were accompanied by decreases in the firing frequency of VTA dopamine neurons, an important electrophysiological hallmark of resilient-like behavior. Together, these results strongly implicate a role for ERK2 signaling in the VTA as a key modulator of responsiveness to stress and mood-related behaviors. PMID:20519540

  7. Slow Inhibition and Conformation Selective Properties of Extracellular Signal-Regulated Kinase 1 and 2 Inhibitors

    PubMed Central

    Rudolph, Johannes; Xiao, Yao; Pardi, Arthur; Ahn, Natalie G.

    2016-01-01

    The mitogen-activated protein (MAP) kinase pathway is a target for anticancer therapy, validated using inhibitors of B-Raf and MAP kinase kinase (MKK) 1 and 2. Clinical outcomes show a high frequency of acquired resistance in patient tumors, involving upregulation of activity of the MAP kinase, extracellular signal-regulated kinase (ERK) 1 and 2. Thus, inhibitors for ERK1/2 are potentially important for targeted therapeutics against cancer. The structures and potencies of different ERK inhibitors have been published, but their kinetic mechanisms have not been characterized. Here we perform enzyme kinetic studies on six representative ERK inhibitors, with potencies varying from 100 pM to 20 μM. Compounds with significant biological activity (IC50 < 100 nM) that inhibit in the subnanomolar range (Vertex-11e and SCH772984) display slow-onset inhibition and represent the first inhibitors of ERK2 known to demonstrate slow dissociation rate constants (values of 0.2 and 1.1 h−1, respectively). Furthermore, we demonstrate using kinetic competition assays that Vertex-11e binds with differing affinities to ERK2 in its inactive, unphosphorylated and active, phosphorylated forms. Finally, two-dimensional heteronuclear multiple-quantum correlation nuclear magnetic resonance experiments reveal that distinct conformational states are formed in complexes of Vertex-11e with inactive and active ERK2. Importantly, two conformers interconvert in equilibrium in the active ERK2 apoenzyme, but Vertex-11e strongly shifts the equilibrium completely to one conformer. Thus, a high-affinity, slow dissociation inhibitor stabilizes different enzyme conformations depending on the activity state of ERK2 and reveals properties of conformational selection toward the active kinase. PMID:25350931

  8. Extracellular-signal-regulated kinase 5 modulates the antioxidant response by transcriptionally controlling Sirtuin 1 expression in leukemic cells.

    PubMed

    Lopez-Royuela, Nuria; Rathore, Moeez G; Allende-Vega, Nerea; Annicotte, Jean-Sébastien; Fajas, Lluis; Ramachandran, Bindu; Gulick, Tod; Villalba, Martin

    2014-08-01

    Cancer cell metabolism differs from that of non-transformed cells in the same tissue. This specific metabolism gives tumor cells growing advantages besides the effect in increasing anabolism. One of these advantages is immune evasion mediated by a lower expression of the mayor histocompatibility complex class I molecules. The extracellular-signal-regulated kinase-5 regulates both mayor histocompatibility complex class I expression and metabolic activity. However, the mechanisms underlying are largely unknown. We show here that extracellular-signal-regulated kinase-5 regulates the transcription of the NADH(+)-dependent histone deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin 1) in leukemic Jurkat T cells. This involves the activation of the transcription factor myocyte enhancer factor-2 and its binding to the sirt1 promoter. In addition, extracellular-signal-regulated kinase-5 is required for T cell receptor-induced and oxidative stress-induced full Sirtuin 1 expression. Extracellular-signal-regulated kinase-5 induces the expression of promoters containing the antioxidant response elements through a Sirtuin 1-dependent pathway. On the other hand, down modulation of extracellular-signal-regulated kinase-5 expression impairs the anti-oxidant response. Notably, the extracellular-signal-regulated kinase-5 inhibitor BIX02189 induces apoptosis in acute myeloid leukemia tumor cells without affecting T cells from healthy donors. Our results unveil a new pathway that modulates metabolism in tumor cells. This pathway represents a promising therapeutic target in cancers with deep metabolic layouts such as acute myeloid leukemia. PMID:24880091

  9. Calcium-independent activation of extracellular signal-regulated kinases 1 and 2 by cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Sumpio, B. E.

    1998-01-01

    We have previously demonstrated that cyclic strain induces extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation in endothelial cells (EC). The aim of this study was to investigate the effect of Ca2+ on the activation of ERK1/2. Bovine aortic EC were pretreated with a chelator of extracellular Ca2+, ethylaneglycol-bis(aminoethylether)-tetra-acetate (EGTA), a depleter of Ca2+ pools, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone (BHQ), or a Ca2+ channel blocker, GdCl3, and subjected to an average 10 % strain at a rate of 60 cycles/min for 10 min. BHQ and GdCl3 did not inhibit the strain-induced ERK1/2 activation. Chelation of normal extracellular Ca2+ (1.8 mM) medium with EGTA (3 mM) acutely stimulated baseline phosphorylation and activation of ERK1/2, thereby obscuring any strain-induced activation of ERK1/2. However, in EC preincubated for 24 hours in Ca2+-free medium, elevated baseline phosphorylation was minimally activated by EGTA (200 microM) such that cyclic strain stimulated ERK1/2 in the presence or absence of BHQ. These results suggest a Ca2+ independence of the ERK1/2 signaling pathway by cyclic strain. Copyright 1998 Academic Press.

  10. Nicotine Shifts the Temporal Activation of Hippocampal Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2 to Enhance Long-Term, but not Short-term, Hippocampus-Dependent Memory

    PubMed Central

    Gould, Thomas J.; Wilkinson, Derek S.; Yildirim, Emre; Poole, Rachel L. F.; Leach, Prescott T.; Simmons, Steven J.

    2014-01-01

    Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular regulated signaling kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4 hours but not 2 hours post-training, delineating time points for STM (2 hours) and LTM (4 hours and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 hours but not 2 hours post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories. PMID:24457151

  11. Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module*

    PubMed Central

    Glatz, Gábor; Gógl, Gergő; Alexa, Anita; Reményi, Attila

    2013-01-01

    Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins. PMID:23382384

  12. Sex differences in fear extinction and involvements of extracellular signal-regulated kinase (ERK).

    PubMed

    Matsuda, Shingo; Matsuzawa, Daisuke; Ishii, Daisuke; Tomizawa, Haruna; Sutoh, Chihiro; Shimizu, Eiji

    2015-09-01

    Stress-related disorders, such as post-traumatic stress disorder (PTSD) and panic disorders, are disproportionately prevalent in females. However, the biological mechanism underlying these sex differences in the prevalence rate remains unclear. In the present study, we examined sex differences in fear memory, fear extinction, and spontaneous recovery of fear. We investigated the presence of sex differences in recent and remote fear memory in mice using contextual fear conditioning, as well as sex differences in spontaneous recovery of fear memory using a consecutive fear extinction paradigm. We examined the number of fear extinction days required to prevent spontaneous recovery of fear in either sex. We investigated whether ovariectomy affected fear extinction and spontaneous recovery. We also measured the activation of extracellular signal-regulated kinase (ERK) 1 and 2 in the dorsal hippocampus and the medial prefrontal cortex following fear extinction sessions. In our results, we found no sex difference in recent or remote fear memory. However, females required more fear extinction sessions compared to males to prevent spontaneous recovery. Within-extinction freezing also differed between males and females. Moreover, females required more extinction sessions than males to increase ERK2 phosphorylation in the dorsal hippocampus. Our data suggest that contextual fear extinction was unstable in females compared to males and that such sex differences may be related to the ERK2 phosphorylation in the hippocampus. PMID:26079214

  13. Albumin-induced apoptosis of glomerular parietal epithelial cells is modulated by extracellular signal-regulated kinase 1/2

    PubMed Central

    Ohse, Takamoto; Krofft, Ron D.; Wu, Jimmy S.; Eddy, Allison A.; Pippin, Jeffrey W.; Shankland, Stuart J.

    2012-01-01

    Background. The biological role(s) of glomerular parietal epithelial cells (PECs) is not fully understood in health or disease. Given its location, PECs are constantly exposed to low levels of filtered albumin, which is increased in nephrotic states. We tested the hypothesis that PECs internalize albumin and increased uptake results in apoptosis. Methods. Confocal microscopy of immunofluorescent staining and immunohistochemistry were used to demonstrate albumin internalization in PECs and to quantitate albumin uptake in normal mice and rats as well as experimental models of membranous nephropathy, minimal change disease/focal segmental glomerulosclerosis and protein overload nephropathy. Fluorescence-activated cell sorting analysis was performed on immortalized cultured PECs exposed to fluorescein isothiocyanate (FITC)-labeled albumin in the presence of an endosomal inhibitor or vehicle. Apoptosis was measured by Hoechst staining in cultured PECs exposed to bovine serum albumin. Levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2) were restored by retroviral infection of mitogen-activated protein kinase (MEK) 1/2 and reduced by U0126 in PECs exposed to high albumin levels in culture and apoptosis measured by Hoechst staining. Results. PECs internalized albumin normally, and this was markedly increased in all of the experimental disease models (P < 0.05 versus controls). Cultured immortalized PECs also internalize FITC-labeled albumin, which was reduced by endosomal inhibition. A consequence of increased albumin internalization was PEC apoptosis in vitro and in vivo. Candidate signaling pathways underlying these events were examined. Data showed markedly reduced levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in PECs exposed to high albumin levels in nephropathy and in culture. A role for ERK1/2 in limiting albumin-induced apoptosis was shown by restoring p-ERK1/2 by retroviral infection, which reduced

  14. The extracellular signal-regulated kinase mitogen-activated protein kinase/ribosomal S6 protein kinase 1 cascade phosphorylates cAMP response element-binding protein to induce MUC5B gene expression via D-prostanoid receptor signaling.

    PubMed

    Choi, Yeon Ho; Lee, Sang-Nam; Aoyagi, Hiroki; Yamasaki, Yasundo; Yoo, Jung-Yoon; Park, Boryung; Shin, Dong Min; Yoon, Ho-Geun; Yoon, Joo-Heon

    2011-09-30

    Mucus hypersecretion is a prominent feature of respiratory diseases, and MUC5B is a major airway mucin. Mucin gene expression can be affected by inflammatory mediators, including prostaglandin (PG) D(2,) an inflammatory mediator synthesized by hematopoietic PGD synthase (H-PGDS). PGD(2) binds to either D-prostanoid receptor (DP1) or chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). We investigated the mechanisms by which PGD(2) induces MUC5B gene expression in airway epithelial cells. Western blot analysis showed that H-PGDS was highly expressed in nasal polyps. Similar results were obtained for PGD(2) expression. In addition, we could clearly detect the expressions of both H-PGDS and DP1 in nasal epithelial cells but not CRTH2. We demonstrated that PGD(2) increased MUC5B gene expression in normal human nasal epithelial cells as well as in NCI-H292 cells in vitro. S5751, a DP1 antagonist, inhibited PGD(2)-induced MUC5B expression, whereas a CRTH2 antagonist (OC0459) did not. These data suggest that PGD(2) induced MUC5B expression via DP1. Pretreatment with extracellular signal-regulated kinase (ERK) inhibitor (PD98059) blocked both PGD(2)-induced ERK mitogen-activated protein kinase (MAPK) activation and MUC5B expression. Proximity ligation assays showed direct interaction between RSK1 and cAMP response element-binding protein (CREB). Stimulation with PGD(2) caused an increase in intracellular cAMP levels, whereas intracellular Ca(2+) did not have such an effect. PGD(2)-induced MUC5B mRNA levels were regulated by CREB via direct interaction with two cAMP-response element sites (-921/-914 and -900/-893). Finally, we demonstrated that PGD(2) can induce MUC5B overproduction via ERK MAPK/RSK1/CREB signaling and that DP1 receptor may have suppressive effects in controlling MUC5B overproduction in the airway. PMID:21832046

  15. Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy.

    PubMed

    Yue, T L; Gu, J L; Wang, C; Reith, A D; Lee, J C; Mirabile, R C; Kreutz, R; Wang, Y; Maleeff, B; Parsons, A A; Ohlstein, E H

    2000-12-01

    The extracellular signal-regulated kinase (ERK) pathway is activated by hypertrophic stimuli in cardiomyocytes. However, whether ERK plays an essential role or is implicated in all major components of cardiac hypertrophy remains controversial. Using a selective MEK inhibitor, U0126, and a selective Raf inhibitor, SB-386023, to block the ERK signaling pathway at two different levels and adenovirus-mediated transfection of dominant-negative Raf, we studied the role of ERK signaling in response of cultured rat cardiomyocytes to hypertrophic agonists, endothelin-1 (ET-1), and phenylephrine (PE). U0126 and SB-386023 blocked ET-1 and PE-induced ERK but not p38 and JNK activation in cardiomyocytes. Both compounds inhibited ET-1 and PE-induced protein synthesis and increased cell size, sarcomeric reorganization, and expression of beta-myosin heavy chain in myocytes with IC(50) values of 1-2 microm. Furthermore, both inhibitors significantly reduced ET-1- and PE-induced expression of atrial natriuretic factor. In cardiomyocytes transfected with a dominant-negative Raf, ET-1- and PE-induced increase in cell size, sarcomeric reorganization, and atrial natriuretic factor production were remarkably attenuated compared with the cells infected with an adenovirus-expressing green fluorescence protein. Taken together, our data strongly support the notion that the ERK signal pathway plays an essential role in ET-1- and PE-induced cardiomyocyte hypertrophy. PMID:10984495

  16. Epidermal-growth-factor receptor and metalloproteinases mediate thromboxane A2-dependent extracellular-signal-regulated kinase activation.

    PubMed Central

    Gallet, Carole; Blaie, Stéphanie; Lévy-Toledano, Sylviane; Habib, Aïda

    2003-01-01

    The signalling pathways that link G-protein-coupled receptors to mitogen-activated protein kinases involve receptor and non-receptor tyrosine kinases and protein kinase C (PKC). We explored the pathways that are implicated in the thromboxane (TX) A(2)-dependent activation of extracellular-signal-regulated protein kinase (ERK) and the role of the two TX receptor (TP) isoforms, TP alpha and TP beta. ERK activation by IBOP, a TX analogue, was dependent on epidermal-growth-factor receptor (EGFR) in TP alpha- or TP beta-transfected cells and in human aortic smooth muscle cells (hASMCs), since AG1478, a selective inhibitor of tyrosine phosphorylation of the EGFR, strongly blocked ERK and EGFR phosphorylation. In addition, EGFR transactivation leading to ERK activation involved matrix metalloproteinases (MMPs), since BB2516, an inhibitor of MMP, decreased ERK and EGFR phosphorylation in TP alpha- or TP beta-transfected cells. Moreover, we showed that both isoforms activate ERK phosphorylation in an Src-kinase-dependent manner, whereas PKC was mainly implicated in ERK activation and EGFR phosphorylation by TP beta. In hASMCs, we showed that ERK activation depended on both pertussis-sensitive and -insensitive G alpha-proteins. We demonstrated further that EGFRs, PKC, Src kinase and MMPs are involved in ERK activation by TX. The results of the present study highlight a role for MMPs and PKC in EGFR transactivation triggered by the TPs and demonstrate this mechanism for the first time in primary cells, i.e. hASMCs. PMID:12534349

  17. Neuronal extracellular signal-regulated kinase (ERK) activity as marker and mediator of alcohol and opioid dependence

    PubMed Central

    Zamora-Martinez, Eva R.; Edwards, Scott

    2014-01-01

    Early pioneering work in the field of biochemistry identified phosphorylation as a crucial post-translational modification of proteins with the ability to both indicate and arbitrate complex physiological processes. More recent investigations have functionally linked phosphorylation of extracellular signal-regulated kinase (ERK) to a variety of neurophysiological mechanisms ranging from acute neurotransmitter action to long-term gene expression. ERK phosphorylation serves as an intracellular bridging mechanism that facilitates neuronal communication and plasticity. Drugs of abuse, including alcohol and opioids, act as artificial yet powerful rewards that impinge upon natural reinforcement processes critical for survival. The graded progression from initial exposure to addiction (or substance dependence) is believed to result from drug- and drug context-induced adaptations in neuronal signaling processes across brain reward and stress circuits following excessive drug use. In this regard, commonly abused drugs as well as drug-associated experiences are capable of modifying the phosphorylation of ERK within central reinforcement systems. In addition, chronic drug and alcohol exposure may drive ERK-regulated epigenetic and structural alterations that underlie a long-term propensity for escalating drug use. Under the influence of such a neurobiological vulnerability, encountering drug-associated cues and contexts can produce subsequent alterations in ERK signaling that drive relapse to drug and alcohol seeking. Current studies are determining precisely which molecular and regional ERK phosphorylation-associated events contribute to the addiction process, as well as which neuroadaptations need to be targeted in order to return dependent individuals to a healthy state. PMID:24653683

  18. LDL immune complexes stimulate LDL receptor expression in U937 histiocytes via extracellular signal-regulated kinase and AP-1.

    PubMed

    Fu, Yuchang; Huang, Yan; Bandyopadhyay, Sumita; Virella, Gabriel; Lopes-Virella, Maria F

    2003-07-01

    We have previously shown that LDL-containing immune complexes (LDL-ICs) induce up-regulation of LDL receptor (LDLR) expression in human macrophages. The present study further investigated the molecular mechanisms leading to LDLR up-regulation by LDL-ICs as well as the signaling pathways involved. Results showed that treatment of U937 histiocytes with LDL-ICs did not increase the precursors and the cleaved forms of sterol-regulatory element binding proteins (SREBPs) 1a and 2, suggesting that SREBPs may not be involved in LDLR up-regulation by LDL-ICs. Promoter deletion and mutation studies showed that the AP-1 binding sites were essential for LDL-IC-stimulated LDLR expression. Electrophoretic mobility shift assays further demonstrated that LDL-ICs stimulated transcription factor AP-1 activity. Studies assessing the signaling pathways involved in LDLR up-regulation by LDL-ICs showed that the up-regulation of LDLR was extracellular signal-regulated kinase (ERK) dependent. In conclusion, the present study shows that LDL-ICs up-regulate LDLR expression via the ERK signaling pathway and the AP-1 motif-dependent transcriptional activation. PMID:12730303

  19. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells.

    PubMed

    Voss, Kelsey; Amaya, Moushimi; Mueller, Claudius; Roberts, Brian; Kehn-Hall, Kylene; Bailey, Charles; Petricoin, Emanuel; Narayanan, Aarthi

    2014-11-01

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. PMID:25261871

  20. Porcine circovirus type 2 replication is impaired by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

    SciTech Connect

    Wei Li; Liu Jue

    2009-03-30

    Postweaning multisystemic wasting syndrome, which is primarily caused by porcine circovirus type 2 (PCV2), is an emerging and important swine disease. We have recently shown that PCV2 induces nuclear factor kappa B activation and its activation is required for active replication, but the other cellular factors involved in PCV2 replication are not well defined. The extracellular signal-regulated kinase (ERK) which served as an important component of cellular signal transduction pathways has been shown to regulate many viral infections. In this report, we show that PCV2 activates ERK1/2 in PCV2-infected PK15 cells dependent on viral replication. The PCV2-induced ERK1/2 leads to phosphorylation of the ternary complex factor Elk-1, which kinetically paralleled ERK1/2 activation. Inhibition of ERK activation with U0126, a specific MEK1/2 inhibitor, significantly reduced viral progeny release. Investigations into the mechanism of ERK1/2 regulation revealed that inhibition of ERK activation leads to decreased viral transcription and lower virus protein expression. These data indicate that the ERK signaling pathway is involved in PCV2 infection and beneficial to PCV2 replication in the cultured cells.

  1. Activation of the Extracellular Signal-Regulated Kinase Signaling Is Critical for Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Differentiation

    PubMed Central

    Li, Chen-Shuang; Zheng, Zhong; Su, Xiao-Xia; Wang, Fei; Ling, Michelle; Zou, Min; Zhou, Hong

    2016-01-01

    Human umbilical cord mesenchymal stem cells (hUCMSCs) are recognized as candidate progenitor cells for bone regeneration. However, the mechanism of hUCMSC osteogenesis remains unclear. In this study, we revealed that mitogen-activated protein kinases (MAPKs) signaling is involved in hUCMSC osteogenic differentiation in vitro. Particularly, the activation of c-Jun N-terminal kinases (JNK) and p38 signaling pathways maintained a consistent level in hUCMSCs through the entire 21-day osteogenic differentiation period. At the same time, the activation of extracellular signal-regulated kinases (ERK) signaling significantly increased from day 5, peaked at day 9, and declined thereafter. Moreover, gene profiling of osteogenic markers, alkaline phosphatase (ALP) activity measurement, and alizarin red staining demonstrated that the application of U0126, a specific inhibitor for ERK activation, completely prohibited hUCMSC osteogenic differentiation. However, when U0126 was removed from the culture at day 9, ERK activation and osteogenic differentiation of hUCMSCs were partially recovered. Together, these findings demonstrate that the activation of ERK signaling is essential for hUCMSC osteogenic differentiation, which points out the significance of ERK signaling pathway to regulate the osteogenic differentiation of hUCMSCs as an alternative cell source for bone tissue engineering. PMID:26989682

  2. Chrysin inhibits human airway smooth muscle cells proliferation through the extracellular signal-regulated kinase 1/2 signaling pathway.

    PubMed

    Yao, Jing; Zhang, Yun-Shi; Feng, Gan-Zhu; Du, Qiang

    2015-11-01

    Asthma is a chronic airway inflammatory disease characterized by an increased mass of airway smooth muscle (ASM). Chrysin (5,7-dihydroxyflavone), a natural flavonoid, has been shown to exert multiple biological activities, including anti-inflammatory, anti-proliferative and anti-oxidant effects, as well as the potency to ameliorate asthma in animal models. The objective of the present study was to identify the underlying mechanism of the therapeutic effects of chrysin. The impact of chrysin on basal and platelet-derived growth factor (PDGF)-induced proliferation and apoptosis of human airway smooth muscle cells (HASMCs) was investigated. Furthermore, the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway was evaluated in HASMCs. The results revealed that chrysin significantly inhibited basal as well as PDGF-induced HASMC proliferation, most likely through the suppression of ERK1/2 phosphorylation. However, chrysin did not significantly reduce PDGF-induced apoptosis of HASMCs. The present study indicated that chrysin may be a promising medication for controlling airway remodeling and clinical manifestations of asthma. PMID:26502995

  3. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

    SciTech Connect

    Voss, Kelsey; Amaya, Moushimi; Mueller, Claudius; Roberts, Brian; Kehn-Hall, Kylene; Bailey, Charles; Petricoin, Emanuel; Narayanan, Aarthi

    2014-11-15

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

  4. Regulation of Autophagic Activation by Rta of Epstein-Barr Virus via the Extracellular Signal-Regulated Kinase Pathway

    PubMed Central

    Chen, Lee-Wen; Wang, Wen-Hung; Chang, Pey-Jium; Chiu, Ya-Fang; Hung, Chen-Chia; Lin, Ying-Ju; Liou, Jieh-Yuan; Tsai, Wan-Ju; Hung, Chia-Ling

    2014-01-01

    ABSTRACT Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development. PMID:25122800

  5. Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY.

    PubMed

    Gógl, Gergő; Alexa, Anita; Kiss, Bence; Katona, Gergely; Kovács, Mihály; Bodor, Andrea; Reményi, Attila; Nyitray, László

    2016-01-01

    Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca(2+)-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca(2+)-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca(2+)-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a "fuzzy" complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas. PMID:26527685

  6. Effects of Chronic Sleep Deprivation on the Extracellular Signal-Regulated Kinase Pathway in the Temporomandibular Joint of Rats

    PubMed Central

    Wang, Peihuan; Wu, Longmei; Zhu, Guoxiong; Zhao, Huaqiang

    2014-01-01

    Objectives To examine the possible involvement and regulatory mechanisms of extracellular signal-regulated kinase (ERK) pathway in the temporomandibular joint (TMJ) of rats subjected to chronic sleep deprivation (CSD). Methods Rats were subjected to CSD using the modified multiple platform method (MMPM). The serum levels of corticosterone (CORT) and adrenocorticotropic hormone (ACTH) were tested and histomorphology and ultrastructure of the TMJ were observed. The ERK and phospho-ERK (p-ERK) expression levels were detected by Western blot analysis, and the MMP-1, MMP-3, and MMP-13 expression levels were detected by real-time quantitative polymerase chain reaction (PCR) and Western blotting. Results The elevated serum CORT and ACTH levels confirmed that the rats were under CSD stress. Hematoxylin and eosin (HE) staining and scanning electron microscopy (SEM) showed pathological alterations in the TMJ following CSD; furthermore, the p-ERK was activated and the mRNA and protein expression levels of MMP-1, MMP-3, and MMP-13 were upregulated after CSD. In the rats administered with the selective ERK inhibitor U0126, decreased tissue destruction was observed. Phospho-ERK activation was visibly blocked and the MMP-1, MMP-3, and MMP-13 mRNA and protein levels were lower than the corresponding levels in the CSD without U0126 group. Conclusion These findings indicate that CSD activates the ERK pathway and upregulates the MMP-1, MMP-3, and MMP-13 mRNA and protein levels in the TMJ of rats. Thus, CSD induces ERK pathway activation and causes pathological alterations in the TMJ. ERK may be associated with TMJ destruction by promoting the expression of MMPs. PMID:25226519

  7. Cell division in the unicellular microalga Dunaliella viridis depends on phosphorylation of extracellular signal-regulated kinases (ERKs).

    PubMed

    Jiménez, Carlos; Cossío, Belén R; Rivard, Christopher J; Berl, Tomás; Capasso, Juan M

    2007-01-01

    In mammalian cells, MAPKs are involved in both stress response (JNK and p38 pathways) and cell proliferation and differentiation [extracellular signal-regulated kinase (ERK)] through protein kinase cascades. Exposure of Dunaliella viridis cell cultures to PD98059, a very specific inhibitor of the ERK signalling pathway, resulted in a total arrest of cell proliferation and a complete dephosphorylation of ERK. As shown by flow cytometry analysis of propidium iodide-stained cells, PD98059 stopped mitosis at the G(2) phase after the S phase has been completed. Multiple physiological parameters such as cell motility and reducing power generation (NADPH) clearly indicate that the treated cells are wholly viable. Exposure of D. viridis to environmental stresses that impair cell division, such as hyperosmotic shock, nitrogen starvation, or sublethal UV irradiation, caused a marked decrease in the phospho-ERK levels as detected by western blot. Two 400 bp polynucleotides from D. viridis with high homologies to published sequences of ERK1 and ERK2 were cloned, sequenced, and submitted to GenBank. Northern blot analysis revealed two mRNA bands of approximately 1.9 kb, consistent with the expected size of ERK proteins ( approximately 40 kDa). Sequence analysis showed that they contained several mitogen-activated protein kinase (MAPK) conserved domains, including II, III, VIb, VII, and the double phosphorylation motif. Interestingly, in D. viridis, this motif was T*DY* instead of the canonic T*EY*. Based on this finding, ERK plant sequences can be divided into two groups, one termed the T*DY* branch and the other termed the T*EY* branch. The molecular and functional data presented here suggest that ERK is a very ancient signalling pathway and that it was already present in the last common ancestor of all eukaryotic cells. PMID:17220513

  8. Extracellular Signal-regulated Kinase (ERK)-dependent Phosphorylation of Y-Box-binding Protein 1 (YB-1) Enhances Gene Expression in Granulosa Cells in Response to Follicle-stimulating Hormone (FSH).

    PubMed

    Donaubauer, Elyse M; Hunzicker-Dunn, Mary E

    2016-06-01

    Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells (GCs). Stimulation of GCs by FSH leads to their proliferation and differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for follicular maturation. Herein, we investigated the role of Y-box-binding protein-1 (YB-1) within GCs. YB-1 is a nucleic acid binding protein that regulates transcription and translation. Our results show that FSH promotes an increase in the phosphorylation of YB-1 on Ser(102) within 15 min that is maintained at significantly increased levels until ∼8 h post treatment. FSH-stimulated phosphorylation of YB-1(Ser(102)) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKA inhibitor (PKI), the MEK inhibitor PD98059, or the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Thus, phosphorylation of YB-1 on Ser(102) is PKA-, ERK-, and RSK-2-dependent. However, pretreatment of GCs with the protein phosphatase 1 (PP1) inhibitor tautomycin increased phosphorylation of YB-1(Ser(102)) in the absence of FSH; FSH did not further increase YB-1(Ser(102)) phosphorylation. This result suggests that the major effect of RSK-2 is to inhibit PP1 rather than to directly phosphorylate YB-1 on Ser(102) YB-1 coimmunoprecipitated with PP1β catalytic subunit and RSK-2. Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant prevented the induction by FSH of Egfr, Cyp19a1, Inha, Lhcgr, Cyp11a1, Hsd17b1, and Pappa mRNAs and estradiol-17β production. Collectively, our results reveal that phosphorylation of YB-1 on Ser(102) via the ERK/RSK-2 signaling pathway is necessary for FSH-mediated expression of target genes required for maturation of follicles to a preovulatory phenotype. PMID:27080258

  9. Shift from extracellular signal-regulated kinase to AKT/cAMP response element-binding protein pathway increases survival-motor-neuron expression in spinal-muscular-atrophy-like mice and patient cells.

    PubMed

    Branchu, Julien; Biondi, Olivier; Chali, Farah; Collin, Thibault; Leroy, Felix; Mamchaoui, Kamel; Makoukji, Joelle; Pariset, Claude; Lopes, Philippe; Massaad, Charbel; Chanoine, Christophe; Charbonnier, Frédéric

    2013-03-01

    Spinal muscular atrophy (SMA), a recessive neurodegenerative disease, is characterized by the selective loss of spinal motor neurons. No available therapy exists for SMA, which represents one of the leading genetic causes of death in childhood. SMA is caused by a mutation of the survival-of-motor-neuron 1 (SMN1) gene, leading to a quantitative defect in the survival-motor-neuron (SMN) protein expression. All patients retain one or more copies of the SMN2 gene, which modulates the disease severity by producing a small amount of stable SMN protein. We reported recently that NMDA receptor activation, directly in the spinal cord, significantly enhanced the transcription rate of the SMN2 genes in a mouse model of very severe SMA (referred as type 1) by a mechanism that involved AKT/CREB pathway activation. Here, we provide the first compelling evidence for a competition between the MEK/ERK/Elk-1 and the phosphatidylinositol 3-kinase/AKT/CREB signaling pathways for SMN2 gene regulation in the spinal cord of type 1 SMA-like mice. The inhibition of the MEK/ERK/Elk-1 pathway promotes the AKT/CREB pathway activation, leading to (1) an enhanced SMN expression in the spinal cord of SMA-like mice and in human SMA myotubes and (2) a 2.8-fold lifespan extension in SMA-like mice. Furthermore, we identified a crosstalk between ERK and AKT signaling pathways that involves the calcium-dependent modulation of CaMKII activity. Together, all these data open new perspectives to the therapeutic strategy for SMA patients. PMID:23467345

  10. Keratins Modulate c-Flip/Extracellular Signal-Regulated Kinase 1 and 2 Antiapoptotic Signaling in Simple Epithelial Cells

    PubMed Central

    Gilbert, Stéphane; Loranger, Anne; Marceau, Normand

    2004-01-01

    Among the large family of intermediate filament proteins, the keratin 8 and 18 (K8/K18) pair constitutes a hallmark for all simple epithelial cells, such as hepatocytes and mammary cells. Functional studies with different cell models have suggested that K8/K18 are involved in simple epithelial cell resistance to several forms of stress that may lead to cell death. We have reported recently that K8/K18-deprived hepatocytes from K8-null mice are more sensitive to Fas-mediated apoptosis. Here we show that upon Fas, tumor necrosis factor alpha receptor, or tumor necrosis factor alpha-related apoptosis-inducing ligand receptor stimulation, an inhibition of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation sensitizes wild-type but not K8-null mouse hepatocytes to apoptosis and that a much weaker ERK1/2 activation occurs in K8-null hepatocytes. In turn, this impaired ERK1/2 activation in K8-null hepatocytes is associated with a drastic reduction in c-Flip protein, an event that also holds in a K8-null mouse mammary cell line. c-Flip, along with Raf-1, is part of a K8/K18-immunoisolated complex from wild-type hepatocytes, and Fas stimulation leads to further c-Flip and Raf-1 recruitment in the complex. This points to a new regulatory role of simple epithelium keratins in the c-Flip/ERK1/2 antiapoptotic signaling pathway. PMID:15282307

  11. Pterygium epithelium abnormal differentiation related to activation of extracellular signal-regulated kinase signaling pathway in vitro

    PubMed Central

    Peng, Juan; Sha, Xiang-Yin; Liu, Yi; Yang, Rui-Ming; Wen, Ye

    2015-01-01

    AIM To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro. METHODS The expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis. RESULTS The expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression. CONCLUSION We suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation. PMID:26682158

  12. Morelloflavone, a biflavonoid, inhibits tumor angiogenesis by targeting rho GTPases and extracellular signal-regulated kinase signaling pathways.

    PubMed

    Pang, Xiufeng; Yi, Tingfang; Yi, Zhengfang; Cho, Sung Gook; Qu, Weijing; Pinkaew, Decha; Fujise, Ken; Liu, Mingyao

    2009-01-15

    Morelloflavone, a biflavonoid extracted from Garcinia dulcis, has shown antioxidative, antiviral, and anti-inflammatory properties. However, the function and the mechanism of this compound in cancer treatment and tumor angiogenesis have not been elucidated to date. In this study, we postulated that morelloflavone might have the ability to inhibit angiogenesis, the pivotal step in tumor growth, invasiveness, and metastasis. We showed that morelloflavone could inhibit vascular endothelial growth factor (VEGF)-induced cell proliferation, migration, invasion, and capillary-like tube formation of primary cultured human umbilical vascular endothelial cells in a dose-dependent manner. Morelloflavone effectively inhibited microvessel sprouting of endothelial cells in the mouse aortic ring assay and the formation of new blood microvessels induced by VEGF in the mouse Matrigel plug assay. Furthermore, morelloflavone inhibited tumor growth and tumor angiogenesis of prostate cancer cells (PC-3) in xenograft mouse tumor model in vivo, suggesting that morelloflavone inhibited tumorigenesis by targeting angiogenesis. To understand the underlying mechanism of morelloflavone on the inhibitory effect of tumor growth and angiogenesis, we showed that morelloflavone could inhibit the activation of both RhoA and Rac1 GTPases but have little effect on the activation of Cdc42 GTPase. Additionally, morelloflavone inhibited the phosphorylation and activation of Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway kinases without affecting VEGF receptor 2 activity. Together, our results indicate that morelloflavone exerts antiangiogenic action by targeting the activation of Rho-GTPases and ERK signaling pathways. These findings are the first to reveal the novel functions of morelloflavone in tumor angiogenesis and its molecular basis for the anticancer action. PMID:19147565

  13. Dopamine D2 receptor activates extracellular signal-regulated kinase through the specific region in the third cytoplasmic loop.

    PubMed

    Takeuchi, Yusuke; Fukunaga, Kohji

    2004-06-01

    To investigate whether the third cytoplasmic loop and the C-terminal cytoplasmic tail of dopamine D(2) receptor (D2R) are involved in extracellular signal-regulated kinase (ERK) activation and subsequent regulation of transcription factors, we established NG108-15 cells stably expressing D2LR and D2SR deleted 40 amino acid residues in the third cytoplasmic loop (NGD2LR-3rd-dele and NGD2SR-3rd-dele) or the C-terminal cytoplasmic tail (NGD2LR-C-dele and NGD2SR-C-dele) and evaluated these receptors' functions using luciferase reporter gene assay. Immunocytochemical studies showed similar intracellular distributions of D2LR-3rd-dele and D2SR-3rd-dele to D2LR and D2SR, respectively. Quinpirole-induced inhibition of forskolin-induced cyclic AMP responsive element (CRE) activation was not affected by the deletion of 40 amino acid residues. However, nuclear factor-kappa B (NF-kappaB) activation by D2R-3rd-dele was largely attenuated compared to that by D2R. Similarly, ERK or serum-responsive element (SRE) activation by quinpirole treatment was totally abolished in NGD2R-3rd-dele cells. Moreover, D2R-C-dele was diffusely distributed or clustered in the cell bodies and lost the receptor functions. Taken together, the 40 amino acid residues in the third cytoplasmic loop are essential for the ERK activation but not for inhibition of adenylyl cyclase through Gi/o proteins. In addition, the C-terminal cytoplasmic tail is essential for membrane association of D2Rs to elicit the receptor functions. PMID:15189353

  14. Ribosomal protein S6 kinase 1 signaling regulates mammalian lifespan

    PubMed Central

    Selman, Colin; Tullet, Jennifer M.A.; Wieser, Daniela; Irvine, Elaine; Lingard, Steven J.; Choudhury, Agharul I.; Claret, Marc; Al-Qassab, Hind; Carmignac, Danielle; Ramadani, Faruk; Woods, Angela; Robinson, Iain C.A.; Schuster, Eugene; Batterham, Rachel L.; Kozma, Sara C.; Thomas, George; Carling, David; Okkenhaug, Klaus; Thornton, Janet M.; Partridge, Linda; Gems, David; Withers, Dominic J.

    2016-01-01

    Caloric restriction (CR) protects against aging and disease but the mechanisms by which this affects mammalian lifespan are unclear. We show in mice that deletion of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway component ribosomal S6 protein kinase 1 (S6K1) led to increased lifespan and resistance to age-related pathologies such as bone, immune and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian lifespan, and suggest therapeutic manipulation of S6K1 and AMPK might mimic CR and provide broad protection against diseases of aging. PMID:19797661

  15. Extracellular signal-regulated kinase 1/2 signalling in SLE T cells is influenced by oestrogen and disease activity.

    PubMed

    Gorjestani, S; Rider, V; Kimler, B F; Greenwell, C; Abdou, N I

    2008-06-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women of reproductive age. The disease is characterized by exaggerated T-cell activity and abnormal T-cell signalling. The mitogen-activated protein kinase (MAPK) pathway is involved in the maintenance of T-cell tolerance that fails in patients with SLE. Oestrogen is a female sex hormone that binds to nuclear receptors and alters the rate of gene transcription. Oestrogen can also act through the plasma membrane and rapidly stimulate second messengers including calcium flux and kinase activation. In this study, we investigated whether oestrogen influences the activation of MAPK signalling through the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in activated SLE T cells. SLE and control T cells were cultured in serum-free medium without and with oestradiol (10(-7) M) for 18 h. The T cells were activated with phorbol 12 myristate 13-acetate and ionomycin for various time points (0-60 min), and the amount of phosphorylated ERK1/2 was measured by immunoblotting. There were no differences in ERK1/2 phosphorylation between SLE and control T cells at 5 and 15 min after the activation stimulus. However, comparison between the amount of phosphorylated ERK1/2 in SLE T cells from the same patients cultured without and with oestradiol showed a significant oestrogen-dependent suppression (P=0.48) of ERK1/2 in patients with inactive/mild systemic lupus erythematosus disease activity index (SLEDAI) (0-2) compared with patients with moderate (4-6) or active (8-12) SLEDAI scores. These results suggest that the suppression of MAPK through ERK1/2 phosphorylation is sensitive to oestradiol in patients with inactive or mild disease, but the sensitivity is not maintained when disease activity increases. Furthermore, studies are now necessary to understand the mechanisms by which oestrogen influences MAPK activation in SLE T cells. PMID:18539708

  16. Constitutive hypophosphorylation of extracellular signal-regulated kinases-1/2 and down-regulation of c-Jun in human gastric adenocarcinoma

    SciTech Connect

    Wu, William Ka Kei; Sung, Joseph Joe Yiu; Yu Le; Li Zhijie; Chu, Kent Man; Cho, C.H.

    2008-08-22

    Hyperphosphorylation of extracellular signal-regulated protein kinases-1/2 (ERK1/2) is known to promote cancer cell proliferation. We therefore investigated the constitutive phosphorylation levels of ERK1/2 and the expression of its downstream targets c-Fos, c-Jun, and cyclooxygenase-2 (COX-2) in biopsied human gastric cancer tissues. Results showed that ERK1/2 phosphorylation and c-Jun expression were significantly lowered in gastric cancer compared with the non-cancer adjacent tissues. The expression of c-Fos, however, was not altered while COX-2 was significantly up-regulated. To conclude, we demonstrate that hypophosphorylation of ERK1/2 may occur in gastric cancer. Such discovery may have implication in the application of pathway-directed therapy for this malignant disease.

  17. Bone Morphogenetic Protein Signaling Regulates Gastric Epithelial Cell Development and Proliferation in Mice

    PubMed Central

    Shinohara, Masahiko; Mao, Maria; Keeley, Theresa M.; El–Zaatari, Mohamad; Lee, Hyuk–Joon; Eaton, Kathryn A.; Samuelson, Linda C.; Merchant, Juanita L.; Goldenring, James R.; Todisco, Andrea

    2010-01-01

    BACKGROUND & AIMS We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach. METHODS The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses. RESULTS In the stomachs of the transgenic mice, phosphorylation of Smad1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and griffonia simplicifolialectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2. CONCLUSIONS Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis. PMID:20826155

  18. Extracellular Signal-Regulated Kinase 7 (ERK7), a Novel ERK with a C-Terminal Domain That Regulates Its Activity, Its Cellular Localization, and Cell Growth

    PubMed Central

    Abe, Mark K.; Kuo, Wen-Liang; Hershenson, Marc B.; Rosner, Marsha Rich

    1999-01-01

    Mitogen-activated protein (MAP) kinases play distinct roles in a variety of cellular signaling pathways and are regulated through multiple mechanisms. In this study, a novel 61-kDa member of the MAP kinase family, termed extracellular signal-regulated kinase 7 (ERK7), has been cloned and characterized. Although it has the signature TEY activation motif of ERK1 and ERK2, ERK7 is not activated by extracellular stimuli that typically activate ERK1 and ERK2 or by common activators of c-Jun N-terminal kinase (JNK) and p38 kinase. Instead, ERK7 has appreciable constitutive activity in serum-starved cells that is dependent on the presence of its C-terminal domain. Interestingly, the C-terminal tail, not the kinase domain, of ERK7 regulates its nuclear localization and inhibition of growth. Taken together, these results elucidate a novel type of MAP kinase whereby interactions via its C-terminal tail, rather than extracellular signal-mediated activation cascades, regulate its activity, localization, and function. PMID:9891064

  19. Discovery and structure activity relationship study of novel indazole amide inhibitors for extracellular signal-regulated kinase1/2 (ERK1/2).

    PubMed

    Li, Lei; Liu, Feifei; Jin, Nan; Tang, Shuai; Chen, Zhuxi; Yang, Xiaotong; Ding, Jian; Geng, Meiyu; Jiang, Lei; Huang, Min; Cao, Jianhua

    2016-06-01

    The discovery and optimization of a series of indazole amide based extracellular signal-regulated kinase inhibitors via structure/knowledge based drug design and kinase screen is reported. The optimized compounds demonstrate potent inhibition of ERK1/2 enzyme activity, growth of BRAF mutant HT29 cells and ERK signaling in HT29 cells. PMID:27106711

  20. Extracellular signal regulated kinase and GEF-H1 mediate depolarization-induced Rho activation and paracellular permeability increase

    PubMed Central

    Waheed, Faiza; Speight, Pam; Kawai, Glenn; Dan, Qinghong; Kapus, András; Szászi, Katalin

    2011-01-01

    Plasma membrane depolarization activates the Rho/Rho kinase (ROK) pathway and thereby enhances myosin light chain (MLC) phosphorylation, which in turn is thought to be a key regulator of paracellular permeability. However, the upstream mechanisms that couple depolarization to Rho activation and permeability changes are unknown. Here we show that three different depolarizing stimuli (high extracellular [K+], the lipophilic cation tetraphenylphosphonium or L-alanine, which is taken up by electrogenic Na+-cotransport) all provoke robust phosphorylation of Extracellular Signal Regulated Kinase (ERK) in LLC-PK1 and MDCK cells. Importantly, inhibition of ERK prevented the depolarization-induced activation of Rho. Searching for the underlying mechanism, we have identified GEF-H1 as the ERK-regulated critical exchange factor, responsible for the depolarization-induced Rho activation. This conclusion is based on our findings that a) depolarization activated GEF-H1, but not p115RhoGEF; b) siRNA-mediated GEF-H1 silencing eliminated the activation of the Rho pathway; c) ERK inhibition prevented the activation of GEF-H1. Moreover, we found that the Na+/K+ pump inhibitor ouabain also caused ERK, GEF-H1 and Rho activation, partially due to its depolarizing effect. Regarding functional consequences of this newly identified pathway, we found that depolarization increased paracellular permeability in LLC-PK1 and MDCK cells, and this effect was mitigated by inhibiting myosin using blebbistatin or a dominant negative (phosphorylation-incompetent) MLC. Taken together, we propose, that the ERK/GEF-H1/Rho/ROK/pMLC pathway could be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and regulate paracellular transport in the tubular epithelium. PMID:20237148

  1. Drug-induced alterations in the extracellular signal-regulated kinase (ERK) signalling pathway: implications for reinforcement and reinstatement.

    PubMed

    Zhai, Haifeng; Li, Yanqin; Wang, Xi; Lu, Lin

    2008-02-01

    Drug addiction, characterized by high rates of relapse, is recognized as a kind of neuroadaptive disorder. Since the extracellular signal-regulated kinase (ERK) pathway is critical to neuroplasticity in the adult brain, understanding the role this pathway plays is important for understanding the molecular mechanism underlying drug addiction and relapse. Here, we review previous literatures that focus on the effects of exposure to cocaine, amphetamine, Delta(9)-tetrahydrocannabinol (THC), nicotine, morphine, and alcohol on ERK signaling in the mesocorticolimbic dopamine system; these alterations of ERK signaling have been thought to contribute to the drug's rewarding effects and to the long-term maladaptation induced by drug abuse. We then discuss the possible upstreams of the ERK signaling pathway activated by exposure of drugs of abuse and the environmental cues previously paired with drugs. Finally, we argue that since ERK activation is a key molecular process in reinstatement of conditioned place preference and drug self-administration, the pharmacological manipulation of the ERK pathway is a potential treatment strategy for drug addiction. PMID:18041576

  2. Extracellular signal-regulated kinase 5 in the cerebrospinal fluid-contacting nucleus contributes to morphine physical dependence in rats.

    PubMed

    Wang, Chun-Guang; Ding, Yan-Ling; Zheng, Tian-Fang; Wei, Jing-Qiu; Liu, He; Chen, Yu-Feng; Wang, Jia-You; Zhang, Li-Cai

    2013-05-01

    The cerebrospinal fluid-contacting nucleus (CSF-CN) may influence actual composition of the CSF for non-synaptic signal transmission via releasing or absorbing bioactive substances, which distributes and localizes in the ventral periaqueductal central gray of the brainstem. Previous studies demonstrated that CSF-CN was involved in neuropathic pain and morphine dependence. Thus, to identify whether extracellular signal-regulated kinase 5 (ERK5) distributed in the CSF-CN and its function on the formation and development of morphine physical dependence, morphine withdrawal-like behavioral test and immunofluorescent technique were used in this research. Morphine was subcutaneously injected by an intermittent and escalating procedure to induce physical dependence, which was measured by withdrawal symptoms. In this study, we found that horseradish peroxidase-conjugated toxin subunit B/p-ERK5 double-labeled neurons expressed in the CSF-CN of normal rats. ERK5 signaling pathway was remarkably activated by naloxone-precipitated withdrawal in the CSF-CN. Moreover, selective attenuation of p-ERK5 expression in the CSF-CN by lateral ventricle injection of BIX02188 could significantly relieve morphine withdrawal symptom. These findings confirmed that the activation of p-ERK5 in the CSF-CN might contribute to morphine physical dependence. PMID:23143893

  3. The inactivation of extracellular signal-regulated kinase by glucagon-like peptide-1 contributes to neuroprotection against oxidative stress.

    PubMed

    Nakajima, Shingo; Numakawa, Tadahiro; Adachi, Naoki; Yoon, Hyung Shin; Odaka, Haruki; Ooshima, Yoshiko; Kunugi, Hiroshi

    2016-03-11

    Glucagon-like peptide-1 (GLP-1), an insulinotropic peptide secreted from enteroendocrine cells, has been known to have a neuroprotective effect. However, it is not fully understood the intracellular mediator of GLP-1 signaling in neuronal cells. In the present study, we examined the change in intracellular signaling of cortical neurons after GLP-1 application and luminal glucose stimulation in vitro and in vivo. GLP-1 receptor was highly expressed in cultured cortical neurons and brain tissues including the prefrontal cortex and hippocampus. The activation of GLP-1 receptor (5min) significantly decreased levels of phosphorylated extracellular signal-regulated kinase (pERK), which is involved in neuronal cell survival and death, in cultured cortical neurons. Oral glucose administration also rapidly reduced pERK levels in the prefrontal cortex, while intraperitoneal glucose injection did not show such an effect. Further, GLP-1 attenuated hydrogen peroxide-induced cell death and hyperactivity of ERK in cultured cortical neurons. It is possible that increased GLP-1 by luminal glucose stimulation affects cortical system including the maintenance of neuronal cell survival. PMID:26827720

  4. The chemopreventive agent myoinositol inhibits Akt and extracellular signal-regulated kinase in bronchial lesions from heavy smokers.

    PubMed

    Han, Wei; Gills, Joell J; Memmott, Regan M; Lam, Stephen; Dennis, Phillip A

    2009-04-01

    Myoinositol is an isomer of glucose that has chemopreventive activity in animal models of cancer. In a recent phase I clinical trial, myoinositol administration correlated with a statistically significant regression of preexisting bronchial dysplastic lesions in heavy smokers. To shed light on the potential mechanisms involved, activation of Akt and extracellular signal-regulated kinase (ERK), two kinases that control cellular proliferation and survival, was assessed in 206 paired bronchial biopsies from 21 patients who participated in this clinical trial. Before myoinositol treatment, strongly positive staining for activation of Akt was detected in 27% of hyperplastic/metaplastic lesions and 58% of dysplastic lesions (P = 0.05, chi(2) test). There was also a trend toward increased activation of ERK (28% in regions of hyperplasia/metaplasia to 42% of dysplastic lesions). Following myoinositol treatment, significant decreases in Akt and ERK phosphorylation were observed in dysplastic (P < 0.01 and 0.05, respectively) but not hyperplastic/metaplastic lesions (P > 0.05). In vitro, myoinositol decreased endogenous and tobacco carcinogen-induced activation of Akt and ERK in immortalized human bronchial epithelial cells, which decreased cell proliferation and induced a G(1)-S cell cycle arrest. These results show that the phenotypic progression of premalignant bronchial lesions from smokers correlates with increased activation of Akt and ERK and that these kinases are targets of myoinositol. Moreover, they suggest that myoinositol might cause regression of bronchial dysplastic lesions through inhibition of active Akt and ERK. PMID:19336734

  5. Activation of extracellular signal-regulated kinases Erk-1 and Erk-2 by cell swelling in H4IIE hepatoma cells.

    PubMed Central

    Schliess, F; Schreiber, R; Häussinger, D

    1995-01-01

    Hepatic metabolism and gene expression are among the factors controlled by the cellular hydration state, which changes within minutes in response to aniso-osmotic environments, cumulative substrate uptake, oxidative stress and under the influence of hormones such as insulin. The signalling events coupling cell-volume changes to altered cell function were studied in H4IIE rat hepatoma cells. Hypo-osmotic cell swelling resulted within 1 min in a tyrosine kinase-mediated activation of the extracellular signal-regulated protein kinases Erk-1 and Erk-2, which was independent of protein kinase C and cytosolic calcium. Activation of mitogen-activated protein kinases was followed by an increased phosphorylation of c-Jun, which may explain our recently reported finding of an about 5-fold increase in c-jun mRNA level in response to cell swelling. Pretreatment of cells with pertussis or cholera toxin abolished the swelling-induced activation of Erk-1 and Erk-2, suggesting the involvement of G-proteins. Thus, a signal-transduction pathway resembling growth factor signalling is activated already by osmotic water shifts across the plasma membrane, thereby providing a new perspective for adaption of cell function to alterations of the environment. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7619047

  6. Fluid shear stress regulates metalloproteinase-1 and 2 in human periodontal ligament cells: involvement of extracellular signal-regulated kinase (ERK) and P38 signaling pathways.

    PubMed

    Zheng, Lisha; Huang, Yan; Song, Wei; Gong, Xianghui; Liu, Meili; Jia, Xiaolin; Zhou, Gang; Chen, Luoping; Li, Ang; Fan, Yubo

    2012-09-21

    Matrix metalloproteinase (MMP)-1, 2, with their endogenous inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1, 2 are critical for extracellular matrix remodeling in human periodontal ligament (PDL) and their expression are sensitive to mechanical stresses. Shear stress as the main type of mechanical stress in tooth movement is involved in matrix turnover. However, how shear stress regulates MMPs and TIMPs system is still unclear. In this study, we investigated the effect of fluid shear stress on expression of MMP-1, 2 and TIMP-1, 2 in human PDL cells and the possible roles of mitogen-activated protein kinases in this process. Three levels of fluid shear stresses (6, 9 and 12 dyn/cm(2)) were loaded on PDL cells for 2, 4, 8 and 12h. The results indicated that fluid shear stress rearranged cytoskeleton in PDL cells. Fluid shear stress increased expression of MMP-1, 2, TIMP-1 and suppressed TIMP-2 expression. MAP kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were activated rapidly by fluid shear stress. The ERK inhibitor blocked fluid shear stress induced MMP-1 expression and P38 inhibitor reduced fluid shear stress stimulated MMP-2 expression. Our study suggested that fluid shear stress involved in PDL remodeling via regulating MMP-1, 2 and TIMP-1, 2 expression. ERK regulated fluid shear stress induced MMP-1 expression and P38 play a role in fluid shear stress induced MMP-2 upregulation. PMID:22863019

  7. Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes.

    PubMed

    Sundgren, Nathan C; Giraud, George D; Schultz, Jess M; Lasarev, Michael R; Stork, Philip J S; Thornburg, Kent L

    2003-12-01

    Growth of the fetal heart involves cardiomyocyte enlargement, division, and maturation. Insulin-like growth factor-1 (IGF-1) is implicated in many aspects of growth and is likely to be important in developmental heart growth. IGF-1 stimulates the IGF-1 receptor (IGF1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). We hypothesized that IGF-1 stimulates cardiomyocyte proliferation and enlargement through stimulation of the ERK cascade and stimulates cardiomyocyte differentiation through the PI3K cascade. In vivo administration of Long R3 IGF-1 (LR3 IGF-1) did not stimulate cardiomyocyte hypertrophy but led to a decreased percentage of cells that were binucleated in vivo. In culture, LR3 IGF-1 increased myocyte bromodeoxyuridine (BrdU) uptake by three- to five-fold. The blockade of either ERK or PI3K signaling (by UO-126 or LY-294002, respectively) completely abolished BrdU uptake stimulated by LR3 IGF-1. LR3 IGF-1 did not increase footprint area, but as expected, phenylephrine stimulated an increase in binucleated cardiomyocyte size. We conclude that 1) IGF-1 through IGF1R stimulates cardiomyocyte division in vivo; hyperplastic growth is the most likely explanation of IGF-1 stimulated heart growth in vivo; 2) IGF-1 through IGF1R does not stimulate binucleation in vitro or in vivo; 3) IGF-1 through IGF1R does not stimulate hypertrophy either in vivo or in vitro; and 4) IGF-1 through IGF1R requires both ERK and PI3K signaling for proliferation of near-term fetal sheep cardiomyocytes in vitro. PMID:12947030

  8. Extracellular Signal-Regulated Kinases Modulate DNA Damage Response - A Contributing Factor to Using MEK Inhibitors in Cancer Therapy

    PubMed Central

    Wei, F; Yan, J; Tang, D

    2011-01-01

    The Raf-MEK-ERK pathway is commonly activated in human cancers, largely attributable to the extracellular signal-regulated kinases (ERKs) being a common downstream target of growth factor receptors, Ras, and Raf. Elevation of these up-stream signals occurs frequently in a variety of malignancies and ERK kinases play critical roles in promoting cell proliferation. Therefore, inhibition of MEK-mediated ERK activation is very appealing in cancer therapy. Consequently, numerous MEK inhibitors have been developed over the years. However, clinical trials have yet to produce overwhelming support for using MEK inhibitors in cancer therapy. Although complex reasons may have contributed to this outcome, an alternative possibility is that the MEK-ERK pathway may not solely provide proliferation signals to malignancies, the central scientific rationale in developing MEK inhibitors for cancer therapy. Recent developments may support this alternative possibility. Accumulating evidence now demonstrated that the MEK-ERK pathway contributes to the proper execution of cellular DNA damage response (DDR), a major pathway of tumor suppression. During DDR, the MEK-ERK pathway is commonly activated, which facilitates the proper activation of DDR checkpoints to prevent cell division. Inhibition of MEK-mediated ERK activation, therefore, compromises checkpoint activation. As a result, cells may continue to proliferate in the presence of DNA lesions, leading to the accumulation of mutations and thereby promoting tumorigenesis. Alternatively, reduction in checkpoint activation may prevent efficient repair of DNA damages, which may cause apoptosis or cell catastrophe, thereby enhancing chemotherapy’s efficacy. This review summarizes our current understanding of the participation of the ERK kinases in DDR. PMID:22087839

  9. Extracellular signal-regulated kinase activation and endothelin-1 production in human endothelial cells exposed to vibration

    PubMed Central

    White, Charles R; Haidekker, Mark A; Stevens, Hazel Y; Frangos, John A

    2004-01-01

    Hand–arm vibration syndrome is a vascular disease of occupational origin and a form of secondary Raynaud's phenomenon. Chronic exposure to hand-held vibrating tools may cause endothelial injury. This study investigates the biomechanical forces involved in the transduction of fluid vibration in the endothelium. Human endothelial cells were exposed to direct vibration and rapid low-volume fluid oscillation. Rapid low-volume fluid oscillation was used to simulate the effects of vibration by generating defined temporal gradients in fluid shear stress across an endothelial monolayer. Extracellular signal-regulated kinase (ERK1/2) phosphorylation and endothelin-1 (ET-1) release were monitored as specific biochemical markers for temporal gradients and endothelial response, respectively. Both vibrational methods were found to phosphorylate ERK1/2 in a similar pattern. At a fixed frequency of fluid oscillation where the duration of each pulse cycle remained constant, ERK1/2 phosphorylation increased with the increasing magnitude of the applied temporal gradient. However, when the frequency of flow oscillation was increased (thus decreasing the duration of each pulse cycle), ERK1/2 phosphorylation was attenuated across all temporal gradient flow profiles. Fluid oscillation significantly stimulated ET-1 release compared to steady flow, and endothelin-1 was also attenuated with the increase in oscillation frequency. Taken together, these results show that both the absolute magnitude of the temporal gradient and the frequency/duration of each pulse cycle play a role in the biomechanical transduction of fluid vibrational forces in endothelial cells. Furthermore, this study reports for the first time a link between the ERK1/2 signal transduction pathway and transmission of vibrational forces in the endothelium. PMID:14724194

  10. Dynamic seizure-related changes in extracellular signal-regulated kinase activation in a mouse model of temporal lobe epilepsy

    PubMed Central

    Houser, Carolyn R.; Huang, Christine S.; Peng, Zechun

    2008-01-01

    Extracellular signal-regulated kinase (ERK) is highly sensitive to regulation by neuronal activity and is critically involved in several forms of synaptic plasticity. These features suggested that alterations in ERK signaling might occur in epilepsy. Previous studies have described increased ERK phosphorylation immediately after the induction of severe seizures, but patterns of ERK activation in epileptic animals during the chronic period have not been determined. Thus, the localization and abundance of phosphorylated ERK (pERK) were examined in a pilocarpine model of recurrent seizures in C57BL/6 mice during the seizure-free period and at short intervals after spontaneous seizures. Immunolabeling of pERK in control animals revealed an abundance of distinctly-labeled neurons within the hippocampal formation. However, in pilocarpine-treated mice during the seizure-free period, the numbers of pERK-labeled neurons were substantially decreased throughout much of the hippocampal formation. Double labeling with a general neuronal marker suggested that the decrease in pERK-labeled neurons was not due primarily to cell loss. The decreased ERK phosphorylation in seizure-prone animals was interpreted as a compensatory response to increased neuronal excitability within the network. Nevertheless, striking increases in pERK labeling occurred at the time of spontaneous seizures and were evident in large populations of neurons at very short intervals (as early as 2 min) after detection of a behavioral seizure. These findings suggest that increased pERK labeling could be one of the earliest immunohistochemical indicators of neurons that are activated at the time of a spontaneous seizure. PMID:18675888

  11. Agonist-induced activation of histamine H3 receptor signals to extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent mechanisms.

    PubMed

    Lai, Xiangru; Ye, Lingyan; Liao, Yuan; Jin, Lili; Ma, Qiang; Lu, Bing; Sun, Yi; Shi, Ying; Zhou, Naiming

    2016-04-01

    The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here

  12. Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons

    PubMed Central

    Cohen, Matthew R.; Johnson, William M.; Pilat, Jennifer M.; Kiselar, Janna; DeFrancesco-Lisowitz, Alicia; Zigmond, Richard E.

    2015-01-01

    Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca2+-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca2+-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca2+ signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca2+ signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function. PMID:26416880

  13. Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons.

    PubMed

    Cohen, Matthew R; Johnson, William M; Pilat, Jennifer M; Kiselar, Janna; DeFrancesco-Lisowitz, Alicia; Zigmond, Richard E; Moiseenkova-Bell, Vera Y

    2015-12-01

    Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca(2+)-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca(2+)-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca(2+) signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca(2+) signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function. PMID:26416880

  14. Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Cao, Pianpian; Zhu, Jian-kang; Tao, W. Andy

    2014-01-01

    Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. PMID:25022875

  15. Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants.

    PubMed

    Lee, Young H; Marquez, Ana P; Mungunsukh, Ognoon; Day, Regina M

    2010-12-01

    Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner. PMID:20926686

  16. Differential expression of extracellular-signal-regulated kinase 5 (ERK5) in normal and degenerated human nucleus pulposus tissues and cells

    SciTech Connect

    Liang, Weiguo; Fang, Dejian; Ye, Dongping; Zou, Longqiang; Shen, Yan; Dai, Libing; Xu, Jiake

    2014-07-11

    Highlights: • ERK5 involved in NP cells. • ERK5 involved in NP tissue. • It was important modulator. - Abstract: Extracellular-signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and regulates a wide variety of cellular processes such as proliferation, differentiation, necrosis, apoptosis and degeneration. However, the expression of ERK5 and its role in degenerated human nucleus pulposus (NP) is hitherto unknown. In this study, we observed the differential expression of ERK5 in normal and degenerated human nucleus pulposus tissues by using immunohistochemical staining and Western blot. Treatment of NP cells with Pro-inflammatory cytokine, TNF-α decreased ERK5 gene expression as well as NP marker gene expression; including the type II collagen and aggrecan. Suppression of ERK5 gene expression in NP cells by ERK5 siRNA resulted in decreased gene expression of type II collagen and aggrecan. Furthermore, inhibition of ERK5 activation by BIX02188 (5 μM) decreased the gene expression of type II collagen and aggrecan in NP cells. Our results document the expression of ERK5 in degenerated nucleus pulposus tissues, and suggest a potential involvement of ERK5 in human degenerated nucleus pulposus.

  17. Mitochondrial ATP production is necessary for activation of the extracellular-signal-regulated kinases during ischaemia/reperfusion in rat myocyte-derived H9c2 cells.

    PubMed Central

    Abas, L; Bogoyevitch, M A; Guppy, M

    2000-01-01

    To search for the stimuli involved in activating the mitogen-activated protein kinases (MAPKs) during ischaemia and reperfusion, we simulated the event in a system in vitro conducive to continuous and non-invasive measurements of several major perturbations that occur at the time: O(2) tension, mitochondrial respiration and energy status. Using H9c2 cells (a clonal line derived from rat heart), we found that activation of the extracellular signal-regulated MAPKs (ERKs) on reoxygenation was abolished if the mitochondria were inhibited prior to and during reoxygenation. Re-introduction of O(2) per se is therefore not sufficient to activate the ERKs. Recovery and maintenance of cellular ATP levels by mitochondrial respiration is necessary, although ATP recovery alone is not sufficient. ERK activation by H(2)O(2), but not phorbol esters, was also sensitive to mitochondrial inhibition. Thus, reoxygenation and H(2)O(2)-mediated oxidative stress share a mechanism of ERK activation that is ATP- or mitochondrion-dependent, and this common feature suggests that the reoxygenation response is mediated by reactive oxygen species. A correlation between ERK activity and ATP levels was also found during the anoxic phase of ischaemia, an effect that was not due to substrate limitation for the kinases. Our results reveal the importance of cellular metabolism in ERK activation, and introduce ATP as a novel participant in the mechanisms underlying the ERK cascade. PMID:10861219

  18. Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

    SciTech Connect

    Jin, Yonghui; Kato, Tomohisa; Furu, Moritoshi; Nasu, Akira; Kajita, Yoichiro; Mitsui, Hiroto; Ueda, Michiko; Aoyama, Tomoki; Nakayama, Tomitaka; Nakamura, Takashi; Toguchida, Junya

    2010-01-15

    Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO{sub 2}) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO{sub 2}) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.

  19. Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

    PubMed Central

    Seaberg, Bonnie; Henslee, Gabrielle; Wang, Shuo; Paez-Colasante, Ximena; Landreth, Gary E.

    2015-01-01

    The Ras–extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice. PMID:25605336

  20. Role of N-methyl-D-aspartate receptors in the neuroprotective activation of extracellular signal-regulated kinase 1/2 by cisplatin.

    PubMed

    Gozdz, Agata; Habas, Agata; Jaworski, Jacek; Zielinska, Magdalena; Albrecht, Jan; Chlystun, Marcin; Jalili, Ahmad; Hetman, Michal

    2003-10-31

    Neurons are exposed to damaging stimuli that can trigger cell death and subsequently cause serious neurological disorders. Therefore, it is important to define defense mechanisms that can be activated in response to damage to reduce neuronal loss. Here we report that cisplatin (CPDD), a neurotoxic anticancer drug that damages DNA, triggered apoptosis and activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in cultured rat cortical neurons. Inhibition of ERK1/2 activation using either pharmacological inhibitors or a dominant-negative mutant of the ERK1/2 activator, mitogen-activated protein kinase kinase 1, increased the toxicity of CPDD. Interestingly, N-methyl-d-aspartate (NMDA) receptor (NMDAR) antagonists reduced the ERK1/2 activation and exacerbated apoptosis in CPDD-treated neurons. Pre-treatment with CPDD increased ERK1/2 activation triggered by exogenous NMDA, suggesting that CPDD augmented NMDAR responsiveness. CPDD-enhanced response of NMDAR and CPDD-mediated ERK1/2 activation were both decreased by inhibition of poly(ADP-ribose) polymerase (PARP). Interestingly, PARP activation did not produce ATP depletion, suggesting involvement of a non-energetic mechanism in NMDAR regulation by PARP. Finally, CPDD toxicity was reduced by brain-derived neurotrophic factor, and this protection required ERK1/2. In summary, our data identify a novel compensatory circuit in central nervous system neurons that couples the DNA injury, through PARP and NMDAR, to the defensive ERK1/2 activation. PMID:12930843

  1. Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine

    PubMed Central

    Gutierrez-Mecinas, Maria; Polgár, Erika; Todd, Andrew J

    2016-01-01

    Background Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. Results Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. Conclusions Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway. PMID:27270268

  2. Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.

    PubMed Central

    Cohen, D M

    1996-01-01

    Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8855340

  3. Hippocampal Extracellular Signal-Regulated Kinase Signaling has a Role in Passive Avoidance Memory Retrieval Induced by GABAA Receptor Modulation in Mice

    PubMed Central

    Kim, Dong Hyun; Kim, Jong Min; Park, Se Jin; Lee, Seungheon; Shin, Chan Young; Cheong, Jae Hoon; Ryu, Jong Hoon

    2012-01-01

    Available evidence strongly suggests that the γ-aminobutyric acid type A (GABAA) receptor has a crucial role in memory retrieval. However, the signaling mechanisms underlying the role of GABAA receptor modulation in memory retrieval are unclear. We conducted one-trial passive avoidance task with pre-retention trial drug administration in the hippocampus to test the effects of GABAA receptor modulation on memory retrieval. We further tested the co-involvement of signaling molecules: extracellular signal-regulated kinase (ERK), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and cAMP responsive element-binding protein (CREB). First, we observed that the phosphorylation of hippocampal ERK was required for memory retrieval during the task. Accordingly, to investigate whether GABAA receptor activation or inhibition induces ERK phosphorylation during memory retrieval, drugs that target the GABAA receptor were administered into the hippocampus before the retention trial. Muscimol, a GABAA receptor agonist, and diazepam, an agonist to benzodiazepine-binding site of GABAA receptor, blocked retention trial-induced ERK phosphorylation and impaired memory retrieval. Furthermore, co-treatment with sub-effective dose of U0126, a mitogen-activated protein kinase inhibitor, blocked the upregulation of ERK phosphorylation and impaired memory retrieval, and bicuculline methiodide (BMI), a GABAA receptor antagonist, increased ERK phosphorylation induced by the retention trial and facilitated memory retrieval. Finally, the effects of BMI were blocked by the co-application of a sub-effective dose of U0126. These results suggest that GABAA receptor-mediated memory retrieval is closely related to ERK activity. PMID:22169949

  4. Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

    PubMed Central

    Lee-Kwon, W; Park, D; Bernier, M

    1998-01-01

    Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase. PMID:9531502

  5. Up-regulation of interleukin-8 by novel small cytoplasmic molecules of nontypeable Haemophilus influenzae via p38 and extracellular signal-regulated kinase pathways.

    PubMed

    Wang, Beinan; Cleary, P Patrick; Xu, Haidong; Li, Jian-Dong

    2003-10-01

    Nontypeable Haemophilus influenzae (NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases. Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of < or =3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention. PMID:14500470

  6. Homogeneous Time-Resolved Fluorescence-Based Assay to Monitor Extracellular Signal-Regulated Kinase Signaling in a High-Throughput Format

    PubMed Central

    Ayoub, Mohammed Akli; Trebaux, Julien; Vallaghe, Julie; Charrier-Savournin, Fabienne; Al-Hosaini, Khaled; Gonzalez Moya, Arturo; Pin, Jean-Philippe; Pfleger, Kevin D. G.; Trinquet, Eric

    2014-01-01

    The extracellular signal-regulated kinases (ERKs) are key components of multiple important cell signaling pathways regulating diverse biological responses. This signaling is characterized by phosphorylation cascades leading to ERK1/2 activation and promoted by various cell surface receptors including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). We report the development of a new cell-based Phospho-ERK1/2 assay (designated Phospho-ERK), which is a sandwich proximity-based assay using the homogeneous time-resolved fluorescence technology. We have validated the assay on endogenously expressed ERK1/2 activated by the epidermal growth factor as a prototypical RTK, as well as various GPCRs belonging to different classes and coupling to different heterotrimeric G proteins. The assay was successfully miniaturized in 384-well plates using various cell lines endogenously, transiently, or stably expressing the different receptors. The validation was performed for agonists, antagonists, and inhibitors in dose–response as well as kinetic analysis, and the signaling and pharmacological properties of the different receptors were reproduced. Furthermore, the determination of a Z′-factor value of 0.7 indicates the potential of the Phospho-ERK assay for high-throughput screening of compounds that may modulate ERK1/2 signaling. Finally, our study is of great interest in the current context of investigating ERK1/2 signaling with respect to the emerging concepts of biased ligands, G protein-dependent/independent ERK1/2 activation, and functional transactivation between GPCRs and RTKs, illustrating the importance of considering the ERK1/2 pathway in cell signaling PMID:25002860

  7. Inhibition of doxorubicin-induced autophagy in hepatocellular carcinoma Hep3B cells by sorafenib--the role of extracellular signal-regulated kinase counteraction.

    PubMed

    Manov, Irena; Pollak, Yulia; Broneshter, Rinata; Iancu, Theodore C

    2011-09-01

    A multikinase inhibitor of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK/ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co-treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1/2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl-2 family. ERK1/2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX-induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib-induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK/ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX-induced autophagy and thus promote cancer cell survival. The use of MEK/ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects. PMID:21790999

  8. Antimelanogenic effect of ginsenoside Rg3 through extracellular signal-regulated kinase-mediated inhibition of microphthalmia-associated transcription factor

    PubMed Central

    Lee, Seung Jae; Lee, Woo Jin; Chang, Sung Eun; Lee, Ga-Young

    2015-01-01

    Background Panax ginseng has been used to prolong longevity and is believed to be useful for improving skin complexion. Ginsenosides are the most active components isolated from ginseng, and ginsenoside Rg3 (G-Rg3) in particular has been demonstrated to possess antioxidative, antitumorigenic, and anti-inflammatory properties. The aim of this study was to examine the ability of G-Rg3 to inhibit melanogenesis. Methods The effects of G-Rg3 on melanin contents and the protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related protein 1 (TRP1) were evaluated. Melanogenesis-regulating signaling molecules such as Akt and extracellular signal-regulated kinase (ERK) were also examined to explore G-Rg3-induced antimelanogenic mechanisms. Results G-Rg3 was found to significantly inhibit the synthesis of melanin in normal human epidermal melanocytes and B16F10 cells in a dose-dependent manner. The activity of cellular tyrosinase and the expression of MITF, tyrosinase, and TRP1 were all reduced, whereas ERK was strongly activated. PD98059 (a specific inhibitor of ERK) attenuated the G-Rg3-induced inhibition of melanin synthesis and tyrosinase activity. Conclusion Taken together, these results showed that G-Rg3 induces the activation of ERK, which accounts for its antimelanogenic effects. G-Rg3 may be a promising safe skin-whitening agent, adding to the long list of uses of P. ginseng for the enhancement of skin beauty. PMID:26199555

  9. Ambroxol inhalation ameliorates LPS-induced airway inflammation and mucus secretion through the extracellular signal-regulated kinase 1/2 signaling pathway.

    PubMed

    Zhang, Shui-Juan; Jiang, Juan-Xia; Ren, Qian-Qian; Jia, Yong-Liang; Shen, Jian; Shen, Hui-Juan; Lin, Xi-Xi; Lu, Hong; Xie, Qiang-Min

    2016-03-15

    Ambroxol, a metabolite of bromhexine, is shown to exert several pharmacological activities, including secretolytic, anti-inflammatory and antioxidant actions. Oral and intravenous administration of ambroxol is useful for the airway inflammatory diseases. However, little is known about its potential in inhalation therapy for lipopolysaccharide (LPS)-induced mucous hypersecretion and inflammatory response. In the present study, we compared the pharmacological effects of ambroxol by inhalation with intravenous administration and preliminarily explored its mechanism of action. Our results demonstrated that ambroxol administered by inhalation inhibited MUC5AC expression, reduced glycosaminoglycan levels, enhanced the function of mucociliary clearance and promoted sputum excretion, suggesting that ambroxol increases expectoration of sputum by reducing its viscosity. Moreover, ambroxol significantly alleviated LPS-induced the influx of inflammatory cells and the extracellular signal-regulated kinase 1/2 (Erk 1/2) expression in lung tissues, and inhibited increases in the mRNA expression of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, CCL-2 (monocyte chemotactic protein-1), KC (keratinocyte cell protein) and interleukin (IL)-1β in lung tissues. The secretolytic and anti-inflammatory effects of inhaled ambroxol at a dose of 7.5mg/ml was comparable to that of ambroxol at 20mg/ml i.v. and dexamethasone at 0.5mg/kg i.p. In addition, we found that ambroxol dose-dependently inhibited LPS-induced increases in the mRNA expression of MUC5AC, TNF-α, and IL-1β in human bronchial epithelial cell (NCI-H292) by inhibiting the Erk signaling pathway. These results demonstrate the beneficial effects of ambroxol in inhalation therapy for the airway inflammatory diseases. PMID:26872986

  10. Effect of D-cycloserine in conjunction with fear extinction training on extracellular signal-regulated kinase activation in the medial prefrontal cortex and amygdala in rat.

    PubMed

    Gupta, Subhash C; Hillman, Brandon G; Prakash, Anand; Ugale, Rajesh R; Stairs, Dustin J; Dravid, Shashank M

    2013-06-01

    D-cycloserine (DCS) is currently under clinical trials for a number of neuropsychiatric conditions and has been found to augment fear extinction in rodents and exposure therapy in humans. However, the molecular mechanism of DCS action in these multiple modalities remains unclear. Here, we describe the effect of DCS administration, alone or in conjunction with extinction training, on neuronal activity (c-fos) and neuronal plasticity [phospho-extracellular signal-regulated kinase (pERK)] markers using immunohistochemistry. We found that intraperitoneal administration of DCS in untrained young rats (24-28 days old) increased c-fos- and pERK-stained neurons in both the prelimbic and infralimbic division of the medial prefrontal cortex (mPFC) and reduced pERK levels in the lateral nucleus of the central amygdala. Moreover, DCS administration significantly increased GluA1, GluN1, GluN2A, and GluN2B expression in the mPFC. In a separate set of animals, we found that DCS facilitated fear extinction and increased pERK levels in the infralimbic prefrontal cortex, prelimbic prefrontal cortex intercalated cells and lateral nucleus of the central amygdala, compared with saline control. In the synaptoneurosomal preparation, we found that extinction training increased iGluR protein expression in the mPFC, compared with context animals. No significant difference in protein expression was observed between extinction-saline and extinction-DCS groups in the mPFC. In contrast, in the amygdala DCS, the conjunction with extinction training led to an increase in iGluR subunit expression, compared with the extinction-saline group. Our data suggest that the efficacy of DCS in neuropsychiatric disorders may be partly due to its ability to affect neuronal activity and signaling in the mPFC and amygdala subnuclei. PMID:23551217

  11. Methyl gallate isolated from Spondias pinnata exhibits anticancer activity against human glioblastoma by induction of apoptosis and sustained extracellular signal-regulated kinase 1/2 activation

    PubMed Central

    Chaudhuri, Dipankar; Ghate, Nikhil Baban; Singh, Sudhir Shankar; Mandal, Nripendranath

    2015-01-01

    Background: Spondias pinnata has been reported for its efficient anticancer effects, but the studies were mostly focused on its extract. Objective: Since its bioactive compounds are largely unknown, this study was designed to characterize the lead components present in it and their anticancer activity against human glioblastoma cell line (U87). Materials and Methods: Major compounds from the ethyl acetate fraction were isolated by column chromatography and their anticancer potentials against U87 cells were evaluated. Furthermore, flow cytometric and immunoblotting analyses were performed to demonstrate the mechanism of apoptosis inducing activity of methyl gallate (MG) against U87 cell line. Results: Four major compounds were isolated from the ethyl acetate fraction. Amongst these, two compounds showed promising activities and with the help of different spectroscopic methods they were identified as gallic acid and MG. Flow cytometric studies revealed that MG-induced apoptosis in U87 cells dose-dependently; the same was confirmed by activation of caspases through cleavage of endogenous substrate poly (adenosine diphosphate-ribose) polymerase. MG treatment also induced the expression of p53 and B-cell lymphoma-2-associated X and cleavage of BH3 interacting-domain with a concomitant decrease in B-cell lymphoma-2 expression. Moreover, MG-induced sustained phosphorylation of extracellular signal-regulated kinase (ERK1/2) in U87 cells with no change in the phosphorylation of other mitogen-activated protein kinases (c-Jun N-terminal of stress-activated protein kinases, p38). Conclusion: MG is a potent antioxidant and it induces sustained ERK1/2 activation and apoptosis in human glioblastoma U87, and provide a rationale for evaluation of MG for other brain carcinoma cell lines for the advancement of glioblastoma therapy. PMID:25829764

  12. Spatial Phosphoprotein Profiling Reveals a Compartmentalized Extracellular Signal-regulated Kinase Switch Governing Neurite Growth and Retraction

    SciTech Connect

    Wang, Yingchun; Yang, Feng; Fu, Yi; Huang, Xiahe; Wang, Wei; Jiang, Xining; Gritsenko, Marina A.; Zhao, Rui; Monroe, Matthew E.; Pertz, Olivier C.; Purvine, Samuel O.; Orton, Daniel J.; Jacobs, Jon M.; Camp, David G.; Smith, Richard D.; Klemke, Richard L.

    2011-05-20

    Abstract - Brain development and spinal cord regeneration require neurite sprouting and growth cone navigation in response to extension and collapsing factors present in the extracellular environment. These external guidance cues control neurite growth cone extension and retraction processes through intracellular protein phosphorylation of numerous cytoskeletal, adhesion, and polarity complex signaling proteins. However, the complex kinase/substrate signaling networks that mediate neuritogenesis have not been investigated. Here, we compare the neurite phosphoproteome under growth and retraction conditions using neurite purification methodology combined with mass spectrometry. More than 4000 non-redundant phosphorylation sites from 1883 proteins have been annotated and mapped to signaling pathways that control kinase/phosphatase networks, cytoskeleton remodeling, and axon/dendrite specification. Comprehensive informatics and functional studies revealed a compartmentalized ERK activation/deactivation cytoskeletal switch that governs neurite growth and retraction, respectively. Our findings provide the first system-wide analysis of the phosphoprotein signaling networks that enable neurite growth and retraction and reveal an important molecular switch that governs neuritogenesis.

  13. A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP

    PubMed Central

    Woodard, Crystal; Liao, Gangling; Goodwin, C. Rory; Hu, Jianfei; Xie, Zhi; dos Reis, Thaila F.; Newman, Rob; Rho, Heesool; Qian, Jiang

    2015-01-01

    ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCE KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human

  14. Heme oxygenase-1 ameliorates kidney ischemia-reperfusion injury in mice through extracellular signal-regulated kinase 1/2-enhanced tubular epithelium proliferation.

    PubMed

    Chen, Hsin-Hung; Lu, Pei-Jung; Chen, Bo-Ron; Hsiao, Michael; Ho, Wen-Yu; Tseng, Ching-Jiunn

    2015-10-01

    Heme oxygenase (HO)-1 confers transient resistance against oxidative damage, including renal ischemia-reperfusion injury (IRI). We investigated the potential protective effect of HO-1 induction in a mouse model of renal IRI induced by bilateral clamping of the kidney arteries. The mice were randomly assigned to five groups to receive an intraperitoneal injection of PBS, hemin (an HO-1 inducer, 100μmol/kg), hemin+ZnPP (an HO-1 inhibitor, 5mg/kg), hemin+PD98059 (a MEK-ERK inhibitor, 10mg/kg) or a sham operation. All of the groups except for the sham-operated group underwent 25min of ischemia and 24 to 72h of reperfusion. Renal function and tubular damage were assessed in the mice that received hemin or the vehicle treatment prior to IRI. The renal injury score and HO-1 protein levels were evaluated via H&E and immunohistochemistry staining. Hemin-preconditioned mice exhibited preserved renal cell function (BUN: 40±2mg/dl, creatinine: 0.53±0.06mg/dl), and the tubular injury score at 72h (1.65±0.12) indicated that tubular damage was prevented. Induction of HO-1 induced the phosphorylation of extracellular signal-regulated kinases (ERK) 1/2. However, these effects were abolished with ZnPP treatment. Kidney function (BUN: 176±49mg/dl, creatinine: 1.54±0.39mg/dl) increased, and the tubular injury score (3.73±0.09) indicated that tubular damage also increased with ZnPP treatment. HO-1-induced tubular epithelial proliferation was attenuated by PD98059. Our findings suggest that HO-1 preconditioning promotes ERK1/2 phosphorylation and enhances tubular recovery, which subsequently prevents further renal injury. PMID:26232688

  15. Contribution of phospholipase D in endothelin-1-mediated extracellular signal-regulated kinase activation and proliferation in rat uterine leiomyoma cells.

    PubMed

    Robin, Philippe; Chouayekh, Sondes; Bole-Feysot, Christine; Leiber, Denis; Tanfin, Zahra

    2005-01-01

    Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth. PMID:15355882

  16. Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates tyrosine hydroxylase through extracellular signal-regulated kinase in rat striatum.

    PubMed

    Yu, Hyun Sook; Kim, Se Hyun; Park, Hong Geun; Kim, Yong Sik; Ahn, Yong Min

    2011-11-01

    Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000μM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000μM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000μM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model. PMID:21871514

  17. Activation of Extracellular Signal-Regulated Kinase in the Anterior Cingulate Cortex Contributes to the Induction and Expression of Affective Pain

    PubMed Central

    Cao, Hong; Gao, Yong-Jing; Ren, Wen-Hua; Li, Ting-Ting; Duan, Kai-Zheng; Cui, Yi-Hui; Cao, Xiao-Hua; Zhao, Zhi-Qi; Ji, Ru-Rong; Zhang, Yu-Qiu

    2009-01-01

    The anterior cingulate cortex (ACC) is implicated in the affective response to noxious stimuli. However, little is known about the molecular mechanisms involved. The present study demonstrated that extracellular signal-regulated kinase (ERK) activation in the ACC plays a crucial role in pain-related negative emotion. Intraplantar formalin injection produced a transient ERK activation in laminae V–VI and a persistent ERK activation in laminae II–III of the rostral ACC (rACC) bilaterally. Using formalin-induced conditioned place avoidance (F-CPA) in rats, which is believed to reflect the pain-related negative emotion, we found that blockade of ERK activation in the rACC with MEK inhibitors prevented the induction of F-CPA. Interestingly, this blockade did not affect formalin-induced two-phase spontaneous nociceptive responses and CPA acquisition induced by electric foot-shock or U69,593, an innocuous aversive agent. Upstream, NMDA receptor, adenylyl cyclase (AC) and PKA activators activated ERK in rACC slices. Consistently, intra-rACC microinjection of AC or PKA inhibitors prevented F-CPA induction. Downstream, phosphorylation of cAMP response element binding protein (CREB) was induced in the rACC by formalin injection and by NMDA, AC and PKA activators in brain slices, which was suppressed by MEK inhibitors. Furthermore, ERK also contributed to the expression of pain-related negative emotion. Thus, when rats were re-exposed to the conditioning context for retrieval of pain experience, ERK and CREB were re-activated in the rACC, and inhibiting ERK activation blocked the expression of F-CPA. All together, our results demonstrate that ERK activation in the rACC is required for the induction and expression of pain-related negative affect. PMID:19279268

  18. Establishment of Extracellular Signal-Regulated Kinase 1/2 Bistability and Sustained Activation through Sprouty 2 and Its Relevance for Epithelial Function▿

    PubMed Central

    Liu, Weimin; Tundwal, Kavita; Liang, Qiaoling; Goplen, Nicholas; Rozario, Sadee; Quayum, Nayeem; Gorska, Magdalena; Wenzel, Sally; Balzar, Silvana; Alam, Rafeul

    2010-01-01

    Our objective was to establish an experimental model of a self-sustained and bistable extracellular signal-regulated kinase 1/2 (ERK1/2) signaling process. A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 4 h. Repeated stimulation leads to sustained activation of ERK1/2 but not Jun N-terminal protein kinase (JNK), p38, or STAT6. The ERK1/2 activation lasts for 3 to 7 days and depends upon a positive-feedback mechanism involving Sprouty 2. Overexpression of Sprouty 2 induces, and its genetic deletion abrogates, ERK1/2 bistability. Sprouty 2 directly activates Fyn kinase, which then induces ERK1/2 activation. A genome-wide microarray analysis shows that the bistable phospho-ERK1/2 (pERK1/2) does not induce a high level of gene transcription. This is due to its nuclear exclusion and compartmentalization to Rab5+ endosomes. Cells with sustained endosomal pERK1/2 manifest resistance against growth factor withdrawal-induced cell death. They are primed for heightened cytokine production. Epithelial cells from cases of human asthma and from a mouse model of chronic asthma manifest increased pERK1/2, which is associated with Rab5+ endosomes. The increase in pERK1/2 was associated with a simultaneous increase in Sprouty 2 expression in these tissues. Thus, we have developed a cellular model of sustained ERK1/2 activation, which may provide a mechanistic understanding of self-sustained biological processes in chronic illnesses such as asthma. PMID:20123980

  19. Establishment of extracellular signal-regulated kinase 1/2 bistability and sustained activation through Sprouty 2 and its relevance for epithelial function.

    PubMed

    Liu, Weimin; Tundwal, Kavita; Liang, Qiaoling; Goplen, Nicholas; Rozario, Sadee; Quayum, Nayeem; Gorska, Magdalena; Wenzel, Sally; Balzar, Silvana; Alam, Rafeul

    2010-04-01

    Our objective was to establish an experimental model of a self-sustained and bistable extracellular signal-regulated kinase 1/2 (ERK1/2) signaling process. A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 4 h. Repeated stimulation leads to sustained activation of ERK1/2 but not Jun N-terminal protein kinase (JNK), p38, or STAT6. The ERK1/2 activation lasts for 3 to 7 days and depends upon a positive-feedback mechanism involving Sprouty 2. Overexpression of Sprouty 2 induces, and its genetic deletion abrogates, ERK1/2 bistability. Sprouty 2 directly activates Fyn kinase, which then induces ERK1/2 activation. A genome-wide microarray analysis shows that the bistable phospho-ERK1/2 (pERK1/2) does not induce a high level of gene transcription. This is due to its nuclear exclusion and compartmentalization to Rab5+ endosomes. Cells with sustained endosomal pERK1/2 manifest resistance against growth factor withdrawal-induced cell death. They are primed for heightened cytokine production. Epithelial cells from cases of human asthma and from a mouse model of chronic asthma manifest increased pERK1/2, which is associated with Rab5+ endosomes. The increase in pERK1/2 was associated with a simultaneous increase in Sprouty 2 expression in these tissues. Thus, we have developed a cellular model of sustained ERK1/2 activation, which may provide a mechanistic understanding of self-sustained biological processes in chronic illnesses such as asthma. PMID:20123980

  20. Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways.

    PubMed

    Bischoff, David S; Zhu, Jian-Hua; Makhijani, Nalini S; Yamaguchi, Dean T

    2008-07-01

    Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate. PMID:18275043

  1. Characterization of p96h2bk: immunoreaction with an anti-Erk(extracellular-signal-regulated kinase) peptide antibody and activity in Xenopus oocytes and eggs.

    PubMed Central

    Chen, D H; Chen, C T; Zhang, Y; Liu, M A; Campos-Gonzalez, R; Pan, B T

    1998-01-01

    We have shown previously that oncogenic Ras induces cell cycle arrest in activated Xenopus egg extracts [Pan, Chen and Lin (1994) J. Biol. Chem. 269, 5968-5975]. The cell cycle arrest correlates with the stimulation of a protein kinase activity that phosphorylates histone H2b in vitro (designated p96(h2bk)) [Chen and Pan (1994) J. Biol. Chem. 269, 28034-28043]. We report here that p96(h2bk) is likely to be p96(ram), a protein of approx. 96 kDa that immunoreacts with a monoclonal antibody (Mk-1) raised against a synthetic peptide derived from a sequence highly conserved in Erk1/Erk2 (where Erk is extracellular-signal-regulated kinase). This is supported by two lines of evidence. First, activation/inactivation of p96(h2bk) correlates with upward/downward bandshifts of p96(ram) in polyacrylamide gels. Secondly, both p96(h2bk) and p96(ram) can be immunoprecipitated by antibody Mk-1. We also studied the activity of p96(h2bk)/p96(ram) in Xenopus oocytes and eggs. p96(h2bk)/p96(ram) was inactive in stage 6 oocytes, was active in unfertilized eggs, and became inactive again in eggs after fertilization. Since stage 6 oocytes are at G2-phase of the cell cycle, unfertilized eggs arrest at M-phase and eggs exit M-phase arrest after fertilization, the results thus indicate that p96(h2bk)/p96(ram) activity is cell cycle dependent. Moreover, microinjection of oncogenic Ras into fertilized eggs at the one-cell stage arrests the embryos at the two-cell stage, and this induced arrest is correlated with an inappropriate activation of p96(h2bk)/p96(ram). The data are consistent with the concept that inappropriate activation of p96(h2bk)/p96(ram) plays a role in the cell cycle arrest induced by oncogenic Ras. PMID:9742211

  2. Extracellular secretion of recombinant proteins

    SciTech Connect

    Linger, Jeffrey G.; Darzins, Aldis

    2014-07-22

    Nucleic acids encoding secretion signals, expression vectors containing the nucleic acids, and host cells containing the expression vectors are disclosed. Also disclosed are polypeptides that contain the secretion signals and methods of producing polypeptides, including methods of directing the extracellular secretion of the polypeptides. Exemplary embodiments include cellulase proteins fused to secretion signals, methods to produce and isolate these polypeptides, and methods to degrade lignocellulosic biomass.

  3. Sigma-1 receptor stimulation protects retinal ganglion cells from ischemia-like insult through the activation of extracellular-signal-regulated kinases 1/2.

    PubMed

    Mueller, Brett H; Park, Yong; Ma, Hai-Ying; Dibas, Adnan; Ellis, Dorette Z; Clark, Abbot F; Yorio, Thomas

    2014-11-01

    Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3-P7 Sprague-Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. RGCs were cultured for 7 days before subjecting the cells to an OGD insult (0.5% oxygen in glucose-free medium) for 6 h. During the OGD, RGCs were treated with pentazocine (σ-1 receptor agonist) with or without BD 1047 (σ-1 receptor antagonist). In other experiments, primary RGCs were treated with pentazocine in the presence or absence of an MEK1/2 inhibitor, PD098059. Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined by immunoblotting and immunofluorescence staining. RGCs subjected to OGD for 6 h induced 50% cell death in primary RGCs (p < 0.001) and inhibited pERK1/2 expression by 65% (p < 0.001). Cell death was attenuated when RGCs were treated with pentazocine under OGD (p < 0.001) and pERK1/2 expression was increased by 1.6 fold (p < 0.05) compared to OGD treated RGCs without pentazocine treatment. The co-treatment of PD098059 (MEK1/2 inhibitor) with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. Activation of the σ-1 receptor is a neuroprotective target that can protect RGCs from an ischemia-like insult. These results also established a direct relationship between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. These findings suggest that activation of

  4. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    PubMed

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  5. Increased Phosphorylation of extracellular signal-regulated kinase in trigeminal nociceptive neurons following propofol administration in rats

    PubMed Central

    Shoda, Emi; Kitagawa, Junichi; Suzuki, Ikuko; Nitta-Kubota, Ieko; Miyamoto, Makiko; Tsuboi, Yoshiyuki; Kondo, Masahiro; Masuda, Yuji; Oi, Yoshiyuki; Ren, Ke; Iwata, Koichi

    2009-01-01

    Although propofol (PRO) is widely used in clinic as a hypnotic agent, the underlying mechanisms of its action on pain pathways is still unknown. Sprague-Dawley rats were assigned to receive PRO or pentobarbital (PEN) and were divided into two groups as LIGHT and DEEP hypnotic levels based on the EEG analysis. Rats in each hypnotic level received capsaicin injection into the face and phosphorylated extracellular regulated-kinase (pERK) immunohistochemistry were performed in subnucleus caudalis (Vc) and upper cervical spinal cord. A large number of pERK-like immunoreactive (LI) cells was observed in the trigeminal spinal subnuclei interpolaris and caudalis transition zone (Vi/Vc), middle Vc and transition zone between Vc and upper cervical spinal cord (Vc/C2) in the rats with PEN or PRO administration following capsaicin injection into the whisker pad region. The number of pERK-LI cells in Vi/Vc, middle Vc and Vc/C2 was significantly larger in rats with PRO injection than those with PEN injection. The number of pERK-LI cells was increased following an increase in the dose of PRO but not in PEN. The pERK-LI cells were dominantly distributed in the Vi/Vc, middle Vc and Vc/C2 after the bolus injections of PRO. The expression of pERK-LI cells was depressed after the intravenous lidocaine application before PRO injection. The present findings suggested that PRO induced an enhancement of the activity of trigeminal nociceptive pathways through nociceptors innervating the venous structure, as indicated by a lidocaine-sensitive increase in pERK. This may explain deep pain around the injection regions during intravenous bolus injection of PRO. Perspective: The effect of propofol administration on ERK phosphorylation in the subregions of the spinal trigeminal complex and upper cervical spinal cord neurons were precisely analyzed in rats with PRO injection. A large number of pERK-LI cells was observed following intravenous PRO administration, suggesting an enhancement of

  6. Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3

    SciTech Connect

    Sun Maoyun; Wei Yuanyan; Yao Luyang; Xie Jianhui; Chen Xiaoning; Wang Hanzhou; Jiang Jianhai; Gu Jianxin . E-mail: jxgu@shmu.edu.cn

    2006-02-03

    Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation.

  7. The tumor suppressor p53 inhibits Net, an effector of Ras/extracellular signal-regulated kinase signaling.

    PubMed

    Nakade, Koji; Zheng, Hong; Ganguli, Gitali; Buchwalter, Gilles; Gross, Christian; Wasylyk, Bohdan

    2004-02-01

    The tumor suppressor function of p53 is linked to its ability to repress gene expression, but the mechanisms of specific gene repression are poorly understood. We report that wild-type p53 inhibits an effector of the Ras oncogene/mitogen-activated protein (MAP) kinase pathway, the transcription factor Net. Tumor-associated mutant p53s are less efficient inhibitors. p53 inhibits by preventing phosphorylation of Net by MAP kinases. Loss of p53 in vivo leads to increased Net phosphorylation in response to wound healing and UV irradiation of skin. Our results show that p53 can repress specific gene expression by inhibiting Net, a factor implicated in cell cycle entry. PMID:14729959

  8. Sodium-Calcium Exchanger 1 Regulates Epithelial Cell Migration via Calcium-dependent Extracellular Signal-regulated Kinase Signaling*

    PubMed Central

    Balasubramaniam, Sona Lakshme; Gopalakrishnapillai, Anilkumar; Gangadharan, Vimal; Duncan, Randall L.; Barwe, Sonali P.

    2015-01-01

    Na+/Ca2+ exchanger-1 (NCX1) is a major calcium extrusion mechanism in renal epithelial cells enabling the efflux of one Ca2+ ion and the influx of three Na+ ions. The gradient for this exchange activity is provided by Na,K-ATPase, a hetero-oligomer consisting of a catalytic α-subunit and a regulatory β-subunit (Na,K-β) that also functions as a motility and tumor suppressor. We showed earlier that mice with heart-specific ablation (KO) of Na,K-β had a specific reduction in NCX1 protein and were ouabain-insensitive. Here, we demonstrate that Na,K-β associates with NCX1 and regulates its localization to the cell surface. Madin-Darby canine kidney cells with Na,K-β knockdown have reduced NCX1 protein and function accompanied by 2.1-fold increase in free intracellular calcium and a corresponding increase in the rate of cell migration. Increased intracellular calcium up-regulated ERK1/2 via calmodulin-dependent activation of PI3K. Both myosin light chain kinase and Rho-associated kinase acted as mediators of ERK1/2-dependent migration. Restoring NCX1 expression in β-KD cells reduced migration rate and ERK1/2 activation, suggesting that NCX1 functions downstream of Na,K-β in regulating cell migration. In parallel, inhibition of NCX1 by KB-R7943 in Madin-Darby canine kidney cells, LLC-PK1, and human primary renal epithelial cells (HREpiC) increased ERK1/2 activation and cell migration. This increased migration was associated with high myosin light chain phosphorylation by PI3K/ERK-dependent mechanism in HREpiC cells. These data confirm the role of NCX1 activity in regulating renal epithelial cell migration. PMID:25770213

  9. Quercitrin treatment protects endothelial progenitor cells from oxidative damage via inducing autophagy through extracellular signal-regulated kinase.

    PubMed

    Zhi, Kangkang; Li, Maoquan; Bai, Jun; Wu, Yongfa; Zhou, Sili; Zhang, Xiaoping; Qu, Lefeng

    2016-07-01

    Atherosclerosis is a disease resulting from impaired endothelial function, often caused by oxidant injury or inflammation. Endothelial progenitor cells (EPCs) play a critical role in repairing damaged endothelium and protecting against atherosclerosis. Quercitrin, a plant-derived flavonoid compound, displays antioxidant and anti-inflammatory activities. In this study, we showed that quercitrin treatment reduced the apoptosis of EPCs caused by oxidized low-density lipoprotein (ox-LDL) in a dose-dependent manner. Quercitrin improved tube formation, migration and adhesion of ox-LDL-treated EPCs. To determine the effect of quercitrin in vivo, EPCs treated with or without ox-LDL and quercitrin were locally injected into the ischemic hind limb muscle of nude mice. Those injected with EPCs treated with ox-LDL and quercitrin showed significantly increased local accumulation of EPCs, blood flow recovery and capillary density compared with the control and ox-LDL only groups. Furthermore, we showed that quercitrin enhanced autophagy and upregulated mitogen-activated protein kinase and ERK phosphorylation in a dose-dependent manner in vitro. Autophagy inhibitors, chloroquine and 3-methyladenine, abrogated quercitrin-enhanced autophagy caused by ox-LDL as evidenced by decreased numbers of branch points, migratory cells and adherent cells, and increased numbers of apoptotic cells. The ERK inhibitor PD98059 abrogated quercitrin-enhanced autophagy, as identified by decreased autophagosome formation and downregulated ERK phosphorylation. The inhibition of ERK did not affect the expression of Rac1, but enhanced phosphorylation of Akt. Quercitrin treatment also increased the expression of E-cadherin, and PD98059 abrogated the upregulation of E-cadherin induced by quercitrin. Our findings suggested that autophagy is a protective mechanism in EPCs exposed to oxidative damage. Quercitrin can promote autophagy through the activation of ERK and the ERK signaling pathway is therefore

  10. Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

    SciTech Connect

    Lu, Zhengyu; Yang, Qi; Cui, Mei; Liu, Yanping; Wang, Tao; Zhao, Hong; Dong, Qiang

    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 depleted 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.

  11. Emblica officinalis exerts wound healing action through up-regulation of collagen and extracellular signal-regulated kinases (ERK1/2).

    PubMed

    Sumitra, Miriyala; Manikandan, Panchatcharam; Gayathri, Vinaya Subramani; Mahendran, Panchatcharam; Suguna, Lonchin

    2009-01-01

    During wound healing, the wound site is rich in oxidants, such as hydrogen peroxide, mostly contributed by neutrophils and macrophages. Ascorbic acid and tannins of low molecular weight, namely emblicanin A (2,3-di-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-2-keto-glucono-delta-lactone) and emblicanin B (2,3,4,6-bis-(S)-hexahydroxydiphenoyl-2-keto-glucono-delta-lactone) present in Emblica officinalis (emblica), have been shown to exhibit a very strong antioxidant action. We proposed that addition of these antioxidants to the wound microenvironment would support the repair process. The present investigation was undertaken to determine the efficacy of emblica on dermal wound healing in vivo. Full-thickness excision wounds were made on the back of the rat and topical application of emblica accelerated wound contraction and closure. Emblica increased cellular proliferation and cross-linking of collagen at the wound site, as evidenced by an increase in the activity of extracellular signal-regulated kinase 1/2, along with an increase in DNA, type III collagen, acid-soluble collagen, aldehyde content, shrinkage temperature and tensile strength. Higher levels of tissue ascorbic acid, alpha-tocopherol, reduced glutathione, superoxide dismutase, catalase, and glutathione peroxidase support the fact that emblica application promotes antioxidant activity at the wound site. In summary, this study provides firm evidence to support that topical application of emblica represents a feasible and productive approach to support dermal wound healing. PMID:19152656

  12. Regulation of dopamine D2 receptor-mediated extracellular signal-regulated kinase signaling and spine formation by GABAA receptors in hippocampal neurons.

    PubMed

    Yoon, Dong-Hoon; Yoon, Sehyoun; Kim, Donghoon; Kim, Hyun; Baik, Ja-Hyun

    2015-01-23

    Dopamine (DA) signaling via DA receptors is known to control hippocampal activity that contributes to learning, memory, and synaptic plasticity. In primary hippocampal neuronal culture, we observed that dopamine D2 receptors (D2R) co-localized with certain subtypes of GABAA receptors, namely α1, β3, and γ2 subunits, as revealed by double immunofluorocytochemical analysis. Treatment with the D2R agonist, quinpirole, was shown to elicit an increase in phosphorylation of extracellular signal-regulated kinase (ERK) in hippocampal neurons. This phosphorylation was inhibited by pretreatment with the GABAA receptor agonist, muscimol. Furthermore, treatment of hippocampal neurons with quinpirole increased the dendritic spine density and this regulation was totally blocked by pretreatment with a MAP kinase kinase (MEK) inhibitor (PD98059), D2R antagonist (haloperidol), or by the GABAA receptor agonist, muscimol. These results suggest that D2R-mediated ERK phosphorylation can control spine formation and that the GABAA receptor negatively regulates the D2R-induced spine formation through ERK signaling in hippocampal neurons, thus indicating a potential role of D2R in the control of hippocampal neuronal excitability. PMID:25483619

  13. Retinoic Acid Modulates Interferon-γ Production by Hepatic Natural Killer T Cells via Phosphatase 2A and the Extracellular Signal-Regulated Kinase Pathway

    PubMed Central

    Chang, Heng-Kwei

    2015-01-01

    Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-γ production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-γ production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-γ production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-γ expression by activated NKT cells. PMID:25343668

  14. BRAF, KIT and NRAS mutations and expression of c-KIT, phosphorylated extracellular signal-regulated kinase and phosphorylated AKT in Japanese melanoma patients.

    PubMed

    Oyama, Satomi; Funasaka, Yoko; Watanabe, Atsushi; Takizawa, Toshihiro; Kawana, Seiji; Saeki, Hidehisa

    2015-05-01

    To clarify the status of gene mutation and activation of growth signal in melanoma of Japanese patients in vivo, we analyzed the mutation of BRAF exon 15, NRAS exon 2, and KIT exons 9, 11, 13, 17 and 18 in melanoma cells obtained by laser capture microdissection, and performed direct sequencing in 20 cases of acral lentiginous melanoma (ALM) and 17 cases of superficial spreading melanoma (SSM). In the study of the mutation of BRAF, pyrosequencing was also done. To examine the cell proliferation signaling, immunohistochemistry for phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT (phosphorylated AKT) and c-KIT was done. The mutation of BRAF p.V600E was detected in 13 cases of ALM (65.0%) and 12 cases of SSM (70.6%). No NRAS mutation was found in all cases. The mutation in exons 9, 11, and 18 of KIT was detected in nine cases. The mutation of BRAF and KIT showed no correlation with clinical stage, lymph node metastasis, tumor thickness, ulceration and histology. pERK and pAKT was observed in small population of melanoma cells and there was no correlation with gene mutation. Our results indicate that the mutations of BRAF and KIT exist in Japanese melanoma patients, however, the cell growth signaling may be regulated by not only these mutated genes, but by other unknown regulatory factors, which may affect the prognosis of melanoma. PMID:25766129

  15. Sensitization of the transient receptor potential vanilloid type 1 ion channel by isoflurane or sevoflurane does not result in extracellular signal-regulated kinase 1/2 activation in rat spinal dorsal horn neurons.

    PubMed

    White, J P M; Cibelli, M; Fidalgo, A R; Paule, C C; Anderson, P J; Jenes, A; Rice, A S C; Nagy, I

    2010-03-17

    Clinically relevant concentrations of isoflurane or sevoflurane sensitize transient receptor potential vanilloid type 1 to several of its activators, including capsaicin. It has, moreover, been suggested these volatile general anaesthetics may augment nociceptive signalling arising from surgical procedures and thereby contribute to post-operative pain. To investigate this suggestion, we have studied intraplantar capsaicin injection-induced phosphorylation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons (which is a recognized marker of spinal nociceptive processing) in rat during isoflurane or sevoflurane anaesthesia after 60 min under anaesthesia. Control animals were anaesthetized with pentobarbital (which of itself does not activate extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons). Unilateral intraplantar capsaicin injection in control animals evoked extracellular signal-regulated kinase 1/2 phosphorylation in a group of neurons in lamina I and lamina II of the ipsilateral spinal dorsal horn in a somatotopically appropriate area. In contrast, both anaesthetic gases (given for 60 min and without subsequent capsaicin injection) induced extracellular signal-regulated kinase 1/2 activation in a different group of mainly lamina I neurons bilaterally. The total number of spinal dorsal horn neurons labelled on the ipliateral side following capsaicin injection into the isoflurane-, or sevoflurane-, anaesthetized animals was significantly less than that produced by capsaicin alone. Further, capsaicin injection into isoflurane-, or sevoflurane-, anaesthetized animals reduced extracellular signal-regulated kinase 1/2 phosphorylation induced by the gases alone on both sides. These findings do not support the suggestion that isoflurane-, or sevoflurane-, induced sensitization of transient receptor potential vanilloid type 1 by capsaicin, or other agonist, is translated into induction of spinal nociceptive processing and

  16. Dopamine- and cAMP-regulated Phosphoprotein of 32-kDa (DARPP-32)-dependent Activation of Extracellular Signal-regulated Kinase (ERK) and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Experimental Parkinsonism*

    PubMed Central

    Santini, Emanuela; Feyder, Michael; Gangarossa, Giuseppe; Bateup, Helen S.; Greengard, Paul; Fisone, Gilberto

    2012-01-01

    Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia. PMID:22753408

  17. Dopamine- and cAMP-regulated phosphoprotein of 32-kDa (DARPP-32)-dependent activation of extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) signaling in experimental parkinsonism.

    PubMed

    Santini, Emanuela; Feyder, Michael; Gangarossa, Giuseppe; Bateup, Helen S; Greengard, Paul; Fisone, Gilberto

    2012-08-10

    Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia. PMID:22753408

  18. S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC via extracellular signal-regulated kinase and nuclear factor-κB pathways.

    PubMed

    Kang, Jin Hyun; Hwang, Sae Mi; Chung, Il Yup

    2015-01-01

    Airway mucus hyperproduction is a common feature of chronic airway diseases such as severe asthma, chronic obstructive pulmonary disease and cystic fibrosis, which are closely associated with neutrophilic airway inflammation. S100A8, S100A9 and S100A12 are highly abundant proteins released by neutrophils and have been identified as important biomarkers in many inflammatory diseases. Herein, we report a new role for S100A8, S100A9 and S100A12 for producing MUC5AC, a major mucin protein in the respiratory tract. All three S100 proteins induced MUC5AC mRNA and the protein in normal human bronchial epithelial cells as well as NCI-H292 lung carcinoma cells in a dose-dependent manner. A Toll-like receptor 4 (TLR4) inhibitor almost completely abolished MUC5AC expression by all three S100 proteins, while neutralization of the receptor for advanced glycation end-products (RAGE) inhibited only S100A12-mediated production of MUC5AC. The S100 protein-mediated production of MUC5AC was inhibited by the pharmacological agents that block prominent signalling molecules for MUC5AC expression, such as mitogen-activated protein kinases, nuclear factor-κB (NF-κB) and epidermal growth factor receptor. S100A8, S100A9 and S100A12 equally elicited both phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear translocation of NF-κB/degradation of cytosolic IκB with similar kinetics through TLR4. In contrast, S100A12 preferentially activated the ERK pathway rather than the NF-κB pathway through RAGE. Collectively, these data reveal the capacity of these three S100 proteins to induce MUC5AC production in airway epithelial cells, suggesting that they all serve as key mediators linking neutrophil-dominant airway inflammation to mucin hyperproduction. PMID:24975020

  19. Extracellular signal-regulated kinase mediates gonadotropin subunit gene expression and LH release responses to endogenous gonadotropin-releasing hormones in goldfish.

    PubMed

    Klausen, Christian; Booth, Morgan; Habibi, Hamid R; Chang, John P

    2008-08-01

    The possible involvement of extracellular signal-regulated kinase (ERK) in mediating the stimulatory actions of two endogenous goldfish gonadotropin-releasing hormones (salmon (s)GnRH and chicken (c)GnRH-II) on gonadotropin synthesis and secretion was examined. Western blot analysis revealed the presence of ERK and phosphorylated (p)ERK in goldfish brain, pituitary, liver, ovary, testis and muscle tissue extracts, as well as extracts of dispersed goldfish pituitary cells and HeLa cells. Interestingly, a third ERK-like immunoreactive band of higher molecular mass was detected in goldfish tissue and pituitary cell extracts in addition to the ERK1-p44- and ERK2-p42-like immunoreactive bands. Incubation of primary cultures of goldfish pituitary cells with either a PKC-activating 4beta-phorbol ester (TPA) or a synthetic diacylglycerol, but not a 4alpha-phorbol ester, elevated the ratio of pERK/total (t)ERK for all three ERK isoforms. The stimulatory effects of TPA were attenuated by the PKC inhibitor GF109203X and the MEK inhibitor PD98059. sGnRH and cGnRH-II also elevated the ratio of pERK/tERK for all three ERK isoforms, in a time-, dose- and PD98059-dependent manner. In addition, treatment with PD98059 reduced the sGnRH-, cGnRH-II- and TPA-induced increases in gonadotropin subunit mRNA levels in Northern blot studies and sGnRH- and cGnRH-II-elicited LH release in cell column perifusion studies with goldfish pituitary cells. These results indicate that GnRH and PKC can activate ERK through MEK in goldfish pituitary cells. More importantly, the present study suggests that GnRH-induced gonadotropin subunit gene expression and LH release involve MEK/ERK signaling in goldfish. PMID:18558406

  20. Severe burn injury induces a characteristic activation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons.

    PubMed

    White, John P M; Ko, Chin Wing; Fidalgo, Antonio Rei; Cibelli, Mario; Paule, Cleoper C; Anderson, Peter J; Cruz, Celia; Gomba, Szabolcs; Matesz, Klara; Veress, Gabor; Avelino, Antonio; Nagy, Istvan

    2011-08-01

    We have studied scalding-type burn injury-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the spinal dorsal horn, which is a recognised marker for spinal nociceptive processing. At 5min after severe scalding injury to mouse hind-paw, a substantial number of phosphorylated ERK1/2 (pERK1/2) immunopositive neurons were found in the ipsilateral dorsal horn. At 1h post-injury, the number of pERK1/2-labelled neurons remained substantially the same. However, at 3h post-injury, a further increase in the number of labelled neurons was found on the ipsilateral side, while a remarkable increase in the number of labelled neurons on the contralateral side resulted in there being no significant difference between the extent of the labelling on both sides. By 6h post-injury, the number of labelled neurons was reduced on both sides without there being significant difference between the two sides. A similar pattern of severe scalding injury-induced activation of ERK1/2 in spinal dorsal horn neurons over the same time-course was found in mice which lacked the transient receptor potential type 1 receptor (TRPV1) except that the extent to which ERK1/2 was activated in the ipsilateral dorsal horn at 5 min post-injury was significantly greater in wild-type animals when compared to TRPV1 null animals. This difference in activation of ERK1/2 in spinal dorsal horn neurons was abolished within 1h after injury, demonstrating that TRPV1 is not essential for the maintenance of ongoing spinal nociceptive processing in inflammatory pain conditions in mouse resulting from at least certain types of severe burn injury. PMID:21371920

  1. Xenin-induced feeding suppression is not mediated through the activation of central extracellular signal-regulated kinase signaling in mice.

    PubMed

    Kim, Eun Ran; Lew, Pei San; Spirkina, Alexandra; Mizuno, Tooru M

    2016-10-01

    Xenin is a gut hormone that reduces food intake by partly acting through the hypothalamus via neurotensin receptor 1 (Ntsr1). However, specific signaling pathways that mediate xenin-induced feeding suppression are not fully understood. Activation of Ntsr1 leads to the activation of the extracellular signal-regulated kinase (ERK). Hypothalamic ERK participates in the regulation of food intake by mediating the effect of hormonal signals. Therefore, we hypothesized that the anorectic effect of xenin is mediated by hypothalamic ERK signaling. To address this hypothesis, we compared levels of phosphorylation of ERK1/2 (pERK1/2) in the hypothalamus of both control and xenin-treated mice. The effect of xenin on ERK1/2 phosphorylation was also examined in mouse hypothalamic neuronal cell lines with or without Ntsr1. We also examined the effect of the blockade of central ERK signaling on xenin-induced feeding suppression in mice. The intraperitoneal (i.p.) injection of xenin caused a significant increase in the number of pERK1/2-immunoreactive cells in the hypothalamic arcuate nucleus. The majority of pERK1/2-positive cells expressed neuronal nuclei (NeuN), a marker for neurons. Xenin treatment increased pERK1/2 levels in one cell line expressing Ntsr1 but not another line without Ntsr1 expression. Both i.p. and intracerebroventricular (i.c.v.) injections of xenin reduced food intake in mice. The i.c.v. pre-treatment with U0126, a selective inhibitor of ERK1/2 upstream kinases, did not affect xenin-induced reduction in food intake. These findings suggest that although xenin activates ERK signaling in subpopulations of hypothalamic neurons, xenin does not require the activation of hypothalamic ERK signaling pathway to elicit feeding suppression. PMID:27316340

  2. Transcriptional repression of Mad-Max complex by human umbilical cord blood stem cells downregulates extracellular signal-regulated kinase in glioblastoma.

    PubMed

    Velpula, Kiran Kumar; Dasari, Venkata Ramesh; Tsung, Andrew J; Dinh, Dzung H; Rao, Jasti S

    2012-07-01

    Previously, we have shown that human umbilical cord blood stem cell (hUCBSC) treatment downregulate cyclin D1 in glioma cells. To study the cell cycle progression and investigate the upstream molecules regulating cyclin D1 expression, we analyzed the involvement of extracellular signal-regulated kinase (ERK) and its functionality after treatment with hUCBSC. We observed downregulation of pERK after hUCBSC treatment at both transcriptional and translational levels. Increased translocation of ERK from cytoplasm to the nucleus was observed in glioma cells, whereas hUCBSC cocultures with glioma cells showed suppressed nuclear translocation. This finding suggests that hUCBSC regulates ERK by suppressing its phosphorylation at phospho-Thr(202)/Tyr(204) retarding pERK nuclear translocation. ERK promoter analysis has shown c-Myc binding sites, indicative of possible transcriptional interactions that regulate cyclin D1 and ERK expression levels. Treatment of U251 and 5310 glioma cells with U0126, a MEK/ERK inhibitor receded pERK and c-Myc levels. In another experiment, U251 and 5310 cells treated with 10074-G5, c-Myc/Max inhibitor displayed reduction in pERK and c-Myc levels suggestive of a positive feedback loop between ERK/c-Myc/Max molecules. In the present study, we show that glioma cells exhibit abundant c-Myc expression and increased c-Myc/Max activity. In contrast, the glioma cells cocultured with hUCBSC demonstrated high Mad1 expression that competitively binds to Max to repress the c-Myc/Max mediated gene transcription. Our studies thus elucidate the potential role of hUCBSC in controlling glioma cell cycle progression and invasion by limiting Max binding to c-Myc, thus regulating the expression of glioma cell cycle and invasion associated molecules such as ERK, integrins via increased levels of Mad1 expression. PMID:21933022

  3. Transcriptional Repression of Mad-Max Complex by Human Umbilical Cord Blood Stem Cells Downregulates Extracellular Signal-Regulated Kinase in Glioblastoma

    PubMed Central

    Velpula, Kiran Kumar; Dasari, Venkata Ramesh; Tsung, Andrew J.; Dinh, Dzung H.

    2012-01-01

    Previously, we have shown that human umbilical cord blood stem cell (hUCBSC) treatment downregulate cyclin D1 in glioma cells. To study the cell cycle progression and investigate the upstream molecules regulating cyclin D1 expression, we analyzed the involvement of extracellular signal-regulated kinase (ERK) and its functionality after treatment with hUCBSC. We observed downregulation of pERK after hUCBSC treatment at both transcriptional and translational levels. Increased translocation of ERK from cytoplasm to the nucleus was observed in glioma cells, whereas hUCBSC cocultures with glioma cells showed suppressed nuclear translocation. This finding suggests that hUCBSC regulates ERK by suppressing its phosphorylation at phospho-Thr202/Tyr204 retarding pERK nuclear translocation. ERK promoter analysis has shown c-Myc binding sites, indicative of possible transcriptional interactions that regulate cyclin D1 and ERK expression levels. Treatment of U251 and 5310 glioma cells with U0126, a MEK/ERK inhibitor receded pERK and c-Myc levels. In another experiment, U251 and 5310 cells treated with 10074-G5, c-Myc/Max inhibitor displayed reduction in pERK and c-Myc levels suggestive of a positive feedback loop between ERK/c-Myc/Max molecules. In the present study, we show that glioma cells exhibit abundant c-Myc expression and increased c-Myc/Max activity. In contrast, the glioma cells cocultured with hUCBSC demonstrated high Mad1 expression that competitively binds to Max to repress the c-Myc/Max mediated gene transcription. Our studies thus elucidate the potential role of hUCBSC in controlling glioma cell cycle progression and invasion by limiting Max binding to c-Myc, thus regulating the expression of glioma cell cycle and invasion associated molecules such as ERK, integrins via increased levels of Mad1 expression. PMID:21933022

  4. MicroRNAs 206 and 21 Cooperate To Promote RAS–Extracellular Signal-Regulated Kinase Signaling by Suppressing the Translation of RASA1 and SPRED1

    PubMed Central

    Sharma, Sriganesh B.; Lin, Chen-Chung; Farrugia, Mark K.; McLaughlin, Sarah L.; Ellis, Emily J.; Brundage, Kathleen M.; Salkeni, Mohamad A.

    2014-01-01

    Despite the low prevalence of activating point mutation of RAS or RAF genes, the RAS–extracellular signal-regulated kinase (ERK) pathway is implicated in breast cancer pathogenesis. Indeed, in triple-negative breast cancer (TNBC), there is recurrent genetic alteration of pathway components. Using short hairpin RNA (shRNA) methods, we observed that the zinc finger transcription factor Krüppel-like factor 4 (KLF4) can promote RAS-ERK signaling in TNBC cells. Endogenous KLF4 bound to the promoter regions and promoted the expression of two microRNAs (miRs), miR-206 and miR-21 (i.e., miR-206/21). Antisense-mediated knockdown (anti-miR) revealed that miR-206/21 coordinately promote RAS-ERK signaling and the corresponding cell phenotypes by inhibiting translation of the pathway suppressors RASA1 and SPRED1. In TNBC cells, including cells with mutation of RAS, the suppression of either RASA1 or SPRED1 increased the levels of GTP-bound, wild-type RAS and activated ERK 1/2. Unlike the control cells, treatment of RASA1- or SPRED1-suppressed cells with anti-miR-206/21 had little or no impact on the level of activated ERK 1/2 or on cell proliferation and failed to suppress tumor initiation. These results identify RASA1 and SPRED1 mRNAs as latent RAS-ERK pathway suppressors that can be upregulated in tumor cells by anti-miR treatment. Consequently, KLF4-regulated miRs are important for the maintenance of RAS-ERK pathway activity in TNBC cells. PMID:25202123

  5. Corticotrigeminal Projections from the Insular Cortex to the Trigeminal Caudal Subnucleus Regulate Orofacial Pain after Nerve Injury via Extracellular Signal-Regulated Kinase Activation in Insular Cortex Neurons

    PubMed Central

    Wang, Jian; Li, Zhi-Hua; Feng, Ban; Zhang, Ting; Zhang, Han; Li, Hui; Chen, Tao; Cui, Jing; Zang, Wei-Dong; Li, Yun-Qing

    2015-01-01

    Cortical neuroplasticity alterations are implicated in the pathophysiology of chronic orofacial pain. However, the relationship between critical cortex excitability and orofacial pain maintenance has not been fully elucidated. We recently demonstrated a top-down corticospinal descending pain modulation pathway from the anterior cingulate cortex (ACC) to the spinal dorsal horn that could directly regulate nociceptive transmission. Thus, we aimed to investigate possible corticotrigeminal connections that directly influence orofacial nociception in rats. Infraorbital nerve chronic constriction injury (IoN-CCI) induced significant orofacial nociceptive behaviors as well as pain-related negative emotions such as anxiety/depression in rats. By combining retrograde and anterograde tract tracing, we found powerful evidence that the trigeminal caudal subnucleus (Vc), especially the superficial laminae (I/II), received direct descending projections from granular and dysgranular parts of the insular cortex (IC). Extracellular signal-regulated kinase (ERK), an important signaling molecule involved in neuroplasticity, was significantly activated in the IC following IoN-CCI. Moreover, in IC slices from IoN-CCI rats, U0126, an inhibitor of ERK activation, decreased both the amplitude and the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and reduced the paired-pulse ratio (PPR) of Vc-projecting neurons. Additionally, U0126 also reduced the number of action potentials in the Vc-projecting neurons. Finally, intra-IC infusion of U0126 obviously decreased Fos expression in the Vc, accompanied by the alleviation of both nociceptive behavior and negative emotions. Thus, the corticotrigeminal descending pathway from the IC to the Vc could directly regulate orofacial pain, and ERK deactivation in the IC could effectively alleviate neuropathic pain as well as pain-related negative emotions in IoN-CCI rats, probably through this top–down pathway. These findings may

  6. Tissue Strain Reorganizes Collagen With a Switchlike Response That Regulates Neuronal Extracellular Signal-Regulated Kinase Phosphorylation In Vitro: Implications for Ligamentous Injury and Mechanotransduction.

    PubMed

    Zhang, Sijia; Cao, Xuan; Stablow, Alec M; Shenoy, Vivek B; Winkelstein, Beth A

    2016-02-01

    Excessive loading of ligaments can activate the neural afferents that innervate the collagenous tissue, leading to a host of pathologies including pain. An integrated experimental and modeling approach was used to define the responses of neurons and the surrounding collagen fibers to the ligamentous matrix loading and to begin to understand how macroscopic deformation is translated to neuronal loading and signaling. A neuron-collagen construct (NCC) developed to mimic innervation of collagenous tissue underwent tension to strains simulating nonpainful (8%) or painful ligament loading (16%). Both neuronal phosphorylation of extracellular signal-regulated kinase (ERK), which is related to neuroplasticity (R2 ≥ 0.041; p ≤ 0.0171) and neuronal aspect ratio (AR) (R2 ≥ 0.250; p < 0.0001), were significantly correlated with tissue-level strains. As NCC strains increased during a slowly applied loading (1%/s), a "switchlike" fiber realignment response was detected with collagen reorganization occurring only above a transition point of 11.3% strain. A finite-element based discrete fiber network (DFN) model predicted that at bulk strains above the transition point, heterogeneous fiber strains were both tensile and compressive and increased, with strains in some fibers along the loading direction exceeding the applied bulk strain. The transition point identified for changes in collagen fiber realignment was consistent with the measured strain threshold (11.7% with a 95% confidence interval of 10.2-13.4%) for elevating ERK phosphorylation after loading. As with collagen fiber realignment, the greatest degree of neuronal reorientation toward the loading direction was observed at the NCC distraction corresponding to painful loading. Because activation of neuronal ERK occurred only at strains that produced evident collagen fiber realignment, findings suggest that tissue strain-induced changes in the micromechanical environment, especially altered local

  7. A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons.

    PubMed

    Hayashi, Yukari; Nishimune, Hiroshi; Hozumi, Katsuto; Saga, Yumiko; Harada, Akihiro; Yuzaki, Michisuke; Iwatsubo, Takeshi; Kopan, Raphael; Tomita, Taisuke

    2016-01-01

    Notch signaling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not γ-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but γ-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons. PMID:27040987

  8. A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons

    PubMed Central

    Hayashi, Yukari; Nishimune, Hiroshi; Hozumi, Katsuto; Saga, Yumiko; Harada, Akihiro; Yuzaki, Michisuke; Iwatsubo, Takeshi; Kopan, Raphael; Tomita, Taisuke

    2016-01-01

    Notch signaling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Although recent studies implicate that Notch regulates synaptic plasticity or cognitive performance, the molecular mechanism how Notch works in mature neurons remains uncertain. Here we demonstrate that a novel Notch signaling is involved in expression of synaptic proteins in postmitotic neurons. Levels of several synaptic vesicle proteins including synaptophysin 1 and VGLUT1 were increased when neurons were cocultured with Notch ligands-expressing NIH3T3 cells. Neuron-specific deletion of Notch genes decreased these proteins, suggesting that Notch signaling maintains the expression of synaptic vesicle proteins in a cell-autonomous manner. Unexpectedly, cGMP-dependent protein kinase (PKG) inhibitor, but not γ-secretase inhibitor, abolished the elevation of synaptic vesicle proteins, suggesting that generation of Notch intracellular domain is dispensable for this function. These data uncover a ligand-dependent, but γ-secretase-independent, non-canonical Notch signaling involved in presynaptic protein expression in postmitotic neurons. PMID:27040987

  9. PKC-dependent extracellular signal-regulated kinase 1/2 pathway is involved in the inhibition of Ib on AngiotensinII-induced proliferation of vascular smooth muscle cells

    SciTech Connect

    Wang Yu; Yan Tianhua; Wang Qiujuan Wang Wei; Xu Jinyi; Wu Xiaoming; Ji Hui

    2008-10-10

    AngiotensinII (AngII) induces vascular smooth muscle cell (VSMC) proliferation, which plays an important role in the development and progression of hypertension. AngII-induced cellular events have been implicated, in part, in the activation of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we investigated the effect of Ib, a novel nonpeptide AngII receptor type 1 (AT{sub 1}) antagonist, on the activation of PKC and ERK1/2 in VSMC proliferation induced by AngII. MTT, and [{sup 3}H]thymidine incorporation assay showed that AngII-induced VSMC proliferation was inhibited significantly by Ib. The specific binding of [{sup 125}I]AngII to AT{sub 1} receptors was blocked by Ib in a concentration-dependent manner with IC{sub 50} value of 0.96 nM. PKC activity assay and Western blot analysis demonstrated that Ib significantly inhibited the activation of PKC and phosphorylation of ERK1/2 induced by AngII, respectively. Furthermore, AngII-induced ERK1/2 activation was obviously blocked by GF109203X, a PKC inhibitor. These findings suggest that the suppression of Ib on AngII-induced VSMC proliferation may be attributed to its inhibitory effect on PKC-dependent ERK1/2 pathway.

  10. Allosteric and Biased G Protein-Coupled Receptor Signaling Regulation: Potentials for New Therapeutics

    PubMed Central

    Khoury, Etienne; Clément, Stéphanie; Laporte, Stéphane A.

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that participate in many aspects of the endocrine function and are important targets for drug development. They transduce signals mainly, but not exclusively, via hetero-trimeric G proteins, leading to a diversity of intracellular signaling cascades. Ligands binding at the hormone orthosteric sites of receptors have been classified as agonists, antagonists, and/or inverse agonists based on their ability to mainly modulate G protein signaling. Accumulating evidence also indicates that such ligands, alone or in combination with other ones such as those acting outside the orthosteric hormone binding sites (e.g., allosteric modulators), have the ability to selectively engage subsets of signaling responses as compared to the natural endogenous ligands. Such modes of functioning have been variously referred to as “functional selectivity” or “ligand-biased signaling.” In this review, we provide an overview of the current knowledge regarding GPCR-biased signaling and their functional regulation with a focus on the evolving concept that receptor domains can also be targeted to allosterically bias signaling, and discuss the usefulness of such modes of regulation for the design of more efficient therapeutics. PMID:24847311

  11. Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes

    PubMed Central

    Cailotto, Frederic; Bianchi, Arnaud; Sebillaud, Sylvie; Venkatesan, Narayanan; Moulin, David; Jouzeau, Jean-Yves; Netter, Patrick

    2007-01-01

    ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-β1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-β1. Chondrocytes were exposed to 10 ng/mL of TGF-β1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-β1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-β1. TGF-β1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-β1-induced ePPi generation. Induction of Ank by TGF-β1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-β1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCδ inhibitor). These data suggest a regulatory role for calcium in TGF-β1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-β1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed

  12. C-terminus of heat shock protein 70– interacting protein facilitates degradation of apoptosis signal-regulating kinase 1 and inhibits apoptosis signal-regulating kinase 1– dependent apoptosis

    PubMed Central

    Hwang, Jae Ryoung; Zhang, Chunlian; Patterson, Cam

    2005-01-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that is regulated under conditions of cellular stress. ASK1 phosphorylates c-Jun N-terminal kinase (JNK) and elicits an apoptotic response. ASK1 activity is regulated at multiple levels, 1 of which is through inhibition by cytosolic chaperones of the heat shock protein (Hsp) 70 family. Among the proteins that determine Hsp70 function, CHIP (C-terminus of Hsp70-interacting protein) is a cochaperone and ubiquitin ligase that interacts with Hsp70 through an amino-terminal tetratricopeptide repeat (TPR) domain. Prominent among the cellular functions mediated by CHIP is protection against physiologic stress. Because ASK1 is known to contain a TPR-acceptor site, we examined the role of CHIP in regulating ASK1 function. CHIP interacted with ASK1 in a TPR-dependent fashion and induced ubiquitylation and proteasome-dependent degradation of ASK1. Targeting of ASK1 by CHIP inhibited JNK activation in response to oxidative challenge and reduced ASK1-dependent apoptosis, whereas short interfering RNA (siRNA)-dependent depletion of CHIP enhanced JNK activation. Consistent with its ability to reduce cytoplasmic ASK1 levels, CHIP triggered the translocation of ASK1 partner protein death-associated protein (Daxx) into the nucleus, where it is known to activate an antiapoptotic response. These results indicate that CHIP regulates ASK1 activity by inducing its ubiquitylation and degradation, which, together with its effects on Daxx localization, provides a mechanism for the antiapoptotic effects of CHIP observed in the face of cellular and physiologic stress. PMID:16038411

  13. Dynamic Trk and G Protein Signalings Regulate Dopaminergic Neurodifferentiation in Human Trophoblast Stem Cells

    PubMed Central

    Lee, Tony Tung-Yin; Tsai, Cheng-Fang; Chen, Hung-Sheng; Lai, Feng-Jie; Yokoyama, Kazunari K.; Hsieh, Tsung-Hsun; Wu, Ruey-Meei; Lee, Jau-nan

    2015-01-01

    Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson’s disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic

  14. Dynamic Trk and G Protein Signalings Regulate Dopaminergic Neurodifferentiation in Human Trophoblast Stem Cells.

    PubMed

    Tsai, Eing-Mei; Wang, Yu-Chih; Lee, Tony Tung-Yin; Tsai, Cheng-Fang; Chen, Hung-Sheng; Lai, Feng-Jie; Yokoyama, Kazunari K; Hsieh, Tsung-Hsun; Wu, Ruey-Meei; Lee, Jau-Nan

    2015-01-01

    Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson's disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic

  15. Social defeat stress promotes tumor growth and angiogenesis by upregulating vascular endothelial growth factor/extracellular signal-regulated kinase/matrix metalloproteinase signaling in a mouse model of lung carcinoma.

    PubMed

    Wu, Xiao; Liu, Bao-Jun; Ji, Shumeng; Wu, Jing-Feng; Xu, Chang-Qing; Du, Yi-Jie; You, Xiao-Fang; Li, Bei; Le, Jing-Jing; Xu, Hai-Lin; Duan, Xiao-Hong; Dong, Jing-Cheng

    2015-07-01

    Numerous epidemiological and experimental animal studies have indicated that chronic psychological stress may promote tumor development. However, the underlying molecular mechanisms by which chronic stress promotes tumorigenesis remain to be fully elucidated and animal models have not yet been well established. In the present study, an established mouse model of repeated social defeat stress (RSDS), was generated and used to investigate the effect of stress on tumor growth and metastasis. C57BL/6 mice were exposed to RSDS for 10 days, followed by subcutaneousl inoculation with Lewis lung carcinoma cells for seven days. The tumor weight and volume as well as the number of the lung metastatic nodules were then determined. Vascular endothelial growth factor (VEGF) serum levels were measured using ELISAs. In addition, expression levels of VEGF receptor (VEGFR) and L1 cell adhesion molecule (L1CAM) messenger (m)RNA were confirmed using reverse transcription quantitative polymerase chain reaction. Furthermore, protein expression levels of phosphorlyated extracellular signal-regulated kinase (pERK), matrix metalloproteinase (MMP)-2 and MMP-9 were examined using western blot analysis. The results showed that RSDS significantly increased the weight and the volume of the primary tumor as well as the number of the lung metastatic nodules. Serum VEGF levels were significantly higher in the tumor-stress group compared with those of the unstressed tumor mice. In addition, tumors in stressed animals demonstrated markedly enhanced expression of VEGFR-2 and L1CAM mRNA as well as pERK, MMP-2 and MMP-9 protein expression. In conclusion, these results suggested that RSDS contributed to lung cancer progression, angiogenesis and metastasis, which was partially associated with increased VEGF secretion and therefore the activation of the ERK signaling pathway, resulting in the induction of MMP-2 and MMP-9 protein expression. PMID:25824133

  16. Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

    SciTech Connect

    Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C.; Ballestas, Mary E.; Elmets, Craig A.; Robbins, David J.; Matalon, Sadis; Deshane, Jessy S.; Afaq, Farrukh; Bickers, David R.; Athar, Mohammad

    2013-11-01

    Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.

  17. Retinoic acid signaling regulates sonic hedgehog and bone morphogenetic protein signalings during genital tubercle development.

    PubMed

    Liu, Liqing; Suzuki, Kentaro; Nakagata, Naomi; Mihara, Kenichiro; Matsumaru, Daisuke; Ogino, Yukiko; Yashiro, Kenta; Hamada, Hiroshi; Liu, Zhonghua; Evans, Sylvia M; Mendelsohn, Cathy; Yamada, Gen

    2012-02-01

    Retinoic acid (RA) plays pivotal roles in organogenesis, and both excessive and reduced amounts of RA cause developmental abnormalities. Reproductive organs are susceptible to teratogen toxigenicity, and the genital tubercle (GT) is one such representative organ. The physiological function of endogenous RA signaling and the mechanisms of RA-induced teratogenicity are poorly understood during the GT development. The objective of this study is to understand the developmental and teratogenic roles of RA during GT development by analyzing genetically modified mouse models. We found dynamic patterns of gene expression for the RA-synthesizing enzyme, Raldh2, and for the RA-catabolizing enzyme, Cyp26b1, during GT development. Rarb, an indicator gene for RA signaling, starts its expression in the prospective corpus cavernosum penis and in the urethral plate epithelium (UE), which plays central roles during GT development. Excessive RA signaling in Cyp26b1(-/-) mutants leads to abnormal extents of cell proliferation and differentiation during GT development, and also upregulates expression of growth factor signalings. They include Sonic hedgehog (Shh) signaling and Bone morphogenetic protein (Bmp) signaling, which are expressed in the UE and its bilateral mesenchyme. RA signaling positively regulatesShh and Bmp4 expression during GT development as testified also by the experiment of RA administration and analyses of loss-of-function of RA signaling mutants. Thus, RA signaling is involved in the developmental cascade necessary for UE formation and GT development. PMID:22127979

  18. DNA-hypomethylating agent, 5'-azacytidine, induces cyclooxygenase-2 expression via the PI3-kinase/Akt and extracellular signal-regulated kinase-1/2 pathways in human HT1080 fibrosarcoma cells.

    PubMed

    Yu, Seon-Mi; Kim, Song-Ja

    2015-10-01

    The cytosine analogue 5'-azacytidine (5'-aza) induces DNA hypomethylation by inhibiting DNA methyltransferase. In clinical trials, 5'-aza is widely used in epigenetic anticancer treatments. Accumulated evidence shows that cyclooxygenase-2 (COX-2) is overexpressed in various cancers, indicating that it may play a critical role in carcinogenesis. However, few studies have been performed to explore the molecular mechanism underlying the increased COX-2 expression. Therefore, we tested the hypothesis that 5'-aza regulates COX-2 expression and prostaglandin E2 (PGE2) production. The human fibrosarcoma cell line HT1080, was treated with various concentrations of 5'-aza for different time periods. Protein expressions of COX-2, DNA (cytosine-5)-methyltransferase 1 (DNMT1), pAkt, Akt, extracellular signal-regulated kinase (ERK), and phosphorylated ERK (pERK) were determined using western blot analysis, and COX-2 mRNA expression was determined using RT-PCR. PGE2 production was evaluated using the PGE2 assay kit. The localization and expression of COX-2 were determined using immunofluorescence staining. Treatment with 5'-aza induces protein and mRNA expression of COX-2. We also observed that 5'-aza-induced COX-2 expression and PGE2 production were inhibited by S-adenosylmethionine (SAM), a methyl donor. Treatment with 5'-aza phosphorylates PI3-kinase/Akt and ERK-1/2; inhibition of these pathways by LY294002, an inhibitor of PI3-kinase/Akt, or PD98059, an inhibitor of ERK-1/2, respectively, prevents 5'-aza-induced COX-2 expression and PGE2 production. Overall, these observations indicate that the hypomethylating agent 5'-aza modulates COX-2 expression via the PI3-kinase/Akt and ERK-1/2 pathways in human HT1080 fibrosarcoma cells. PMID:26238650

  19. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  20. Overexpression of KAI1 induces autophagy and increases MiaPaCa-2 cell survival through the phosphorylation of extracellular signal-regulated kinases

    SciTech Connect

    Wu, Chun-Yan; Yan, Jun; Yang, Yue-Feng; Xiao, Feng-Jun; Li, Qing-Fang; Zhang, Qun-Wei; Wang, Li-Sheng; Guo, Xiao-Zhong; Wang, Hua

    2011-01-21

    Research highlights: {yields} We first investigate the effects of KAI1 on autophagy in MiaPaCa-2 cells. {yields} Our findings demonstrate that KAI1 induces autophagy, which in turn inhibits KAI1-induced apoptosis. {yields} This study also supplies a possible novel therapeutic method for the treatment of pancreatic cancer using autophagy inhibitors. -- Abstract: KAI1, a metastasis-suppressor gene belonging to the tetraspanin family, is known to inhibit cancer metastasis without affecting the primary tumorigenicity by inhibiting the epidermal growth factor (EGF) signaling pathway. Recent studies have shown that hypoxic conditions of solid tumors induce high-level autophagy and KAI1 expression. However, the relationship between autophagy and KAI1 remains unclear. By using transmission electron microscopy, confocal microscopy, and Western blotting, we found that KAI1 can induce autophagy in a dose- and time-dependent manner in the human pancreatic cell line MiaPaCa-2. KAI1-induced autophagy was confirmed by the expression of autophagy-related proteins LC3 and Beclin 1. KAI1 induces autophagy through phosphorylation of extracellular signal-related kinases rather than that of AKT. KAI1-induced autophagy protects MiaPaCa-2 cells from apoptosis and proliferation inhibition partially through the downregulation of poly [adenosine diphosphate (ADP)-ribose] polymerase (PARP) cleavage and caspase-3 activation.

  1. Suppression of nuclear factor erythroid 2-related factor 2 via extracellular signal-regulated kinase contributes to bleomycin-induced oxidative stress and fibrogenesis.

    PubMed

    Liu, Rui; Chen, Hongli; Bai, Hua; Zhang, Wei; Wang, Xin; Qin, Xujun; Zhang, Xiaodi; Li, Wenli; Liang, Xin; Hai, Chunxu

    2013-06-20

    Pulmonary fibrosis is a serious and irreversible lung injury with obscure etiologic mechanisms and no effective treatment to date. This study explored a crucial link between oxidative stress and pulmonary fibrogenesis, focusing on nuclear factor erythroid 2-related factor 2 (Nrf2), a core transcription factor in antioxidative regulation systems. Treatment of C57 BL/6 mice with bleomycin increased fibroblast viability and collagen production and significantly downregulated Nrf2. In addition, prominent oxidative stress was indicated by changes in superoxide dismutase, catalase activity, and glutathione and thiobarbituric acid-reactive substance levels. In a cell-based model, bleomycin suppressed Nrf2 activation via extracellular signal-related kinase phosphorylation, enhancing intracellular reactive oxygen species in lung fibroblasts and stimulating abnormal cell proliferation and collagen secretion. To confirm this novel mechanism of bleomycin-induced fibrogenesis, we attempted to upregulate Nrf2 and related antioxidant proteins in bleomycin-treated fibroblasts using a putative Nrf2 activator, caffeic acid phenethyl ester, and the results showed that bleomycin-induced fibroblast proliferation and collagen content were attenuated through improved redox balance. Collectively, these results disclose a potential regulatory mechanism in pulmonary fibrosis that will aid the development of new therapies. PMID:23570914

  2. Dexmedetomidine Dose-Dependently Attenuates Ropivacaine-Induced Seizures and Negative Emotions Via Inhibiting Phosphorylation of Amygdala Extracellular Signal-Regulated Kinase in Mice.

    PubMed

    Zhai, Ming-Zhu; Wu, Huang-Hui; Yin, Jun-Bin; Cui, Yuan-Yuan; Mei, Xiao-Peng; Zhang, Han; Zhu, Xia; Shen, Xue-Feng; Kaye, Alan David; Chen, Guo-Zhong

    2016-05-01

    Ropivacaine (Ropi), one of the newest and safest amino amide local anesthetics, is linked to toxicity, including the potential for seizures, changes in behavior, and even cardiovascular collapse. Dexmedetomidine (Dex), an α2-adrenergic receptor agonist, has been widely used in anesthesia and critical care practice. To date, the underlying mechanisms of the effects of Dex premedication on Ropi-induced toxicity have not been clearly identified. In the current study, we investigated the effects of increasing doses of Dex premedication on 50 % convulsive dose (CD50) of Ropi. With increasing doses of intraperitoneal (i.p.) Dex 10 min prior to each i.p. RopiCD50, the latency and duration of seizure activity were recorded. Open-field (OF) and elevated plus maze (EPM) test were used to measure negative behavioral emotions such as depression and anxiety. Immunohistochemistry and Western blot were utilized to investigate phosphorylation-extracellular regulated protein kinases (p-ERK) expression in the basolateral amygdala (BLA) on 2 h and in the central amygdala (CeA) on 24 h after convulsion in mice. The results of our investigation demonstrated that Dex dose-dependently increased RopiCD50, prolonged the latency and shortened the duration of each RopiCD50-induced seizure, improved the negative emotions revealed by both OF and EPM test, and inhibited p-ERK expression in the BLA and the CeA. PMID:26099305

  3. Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells.

    PubMed

    Joo, Donghyun; Woo, Jong Soo; Cho, Kwang-Hyun; Han, Seung Hyun; Min, Tae Sun; Yang, Deok-Chun; Yun, Cheol-Heui

    2016-04-01

    Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225]. PMID:26879318

  4. Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells

    PubMed Central

    Joo, Donghyun; Woo, Jong Soo; Cho, Kwang-Hyun; Han, Seung Hyun; Min, Tae Sun; Yang, Deok-Chun; Yun, Cheol-Heui

    2016-01-01

    Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225] PMID:26879318

  5. Mechanical signals regulate and activate SNAIL1 protein to control the fibrogenic response of cancer-associated fibroblasts.

    PubMed

    Zhang, Kun; Grither, Whitney R; Van Hove, Samantha; Biswas, Hirak; Ponik, Suzanne M; Eliceiri, Kevin W; Keely, Patricia J; Longmore, Gregory D

    2016-05-15

    Increased deposition of collagen in extracellular matrix (ECM) leads to increased tissue stiffness and occurs in breast tumors. When present, this increases tumor invasion and metastasis. Precisely how this deposition is regulated and maintained in tumors is unclear. Much has been learnt about mechanical signal transduction in cells, but transcriptional responses and the pathophysiological consequences are just becoming appreciated. Here, we show that the SNAIL1 (also known as SNAI1) protein level increases and accumulates in nuclei of breast tumor cells and cancer-associated fibroblasts (CAFs) following exposure to stiff ECM in culture and in vivo SNAIL1 is required for the fibrogenic response of CAFs when exposed to a stiff matrix. ECM stiffness induces ROCK activity, which stabilizes SNAIL1 protein indirectly by increasing intracellular tension, integrin clustering and integrin signaling to ERK2 (also known as MAPK1). Increased ERK2 activity leads to nuclear accumulation of SNAIL1, and, thus, avoidance of cytosolic proteasome degradation. SNAIL1 also influences the level and activity of YAP1 in CAFs exposed to a stiff matrix. This work describes a mechanism whereby increased tumor fibrosis can perpetuate activation of CAFs to sustain tumor fibrosis and promote tumor metastasis through regulation of SNAIL1 protein level and activity. PMID:27076520

  6. A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

    PubMed

    Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

    2003-02-28

    Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals. PMID:12446706

  7. Differential Modulation of Brainstem Phosphatidylinositol 3-Kinase/Akt and Extracellular Signal-Regulated Kinase 1/2 Signaling Underlies WIN55,212-2 Centrally Mediated Pressor Response in Conscious Rats

    PubMed Central

    Ibrahim, Badr Mostafa

    2012-01-01

    Our recent study demonstrated that central cannabinoid receptor 1 (CB1R) activation caused dose-related pressor response in conscious rats, and reported studies implicated the brainstem phosphatidylinositol 3-kinase (PI3K)/Akt-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in blood pressure control. Therefore, in this study, we tested the hypothesis that the modulation of brainstem PI3K/Akt-ERK1/2 signaling plays a critical role in the central CB1R-mediated pressor response. In conscious freely moving rats, the pressor response elicited by intracisternal (i.c.) (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt (WIN55,212-2) (15 μg) was associated with significant increases in ERK1/2 phosphorylation in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS). In contrast, Akt phosphorylation was significantly reduced in the same neuronal pools. Pretreatment with the selective CB1R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (30 μg i.c.) attenuated the neurochemical responses elicited by central CB1R activation. Furthermore, pretreatment with the ERK/mitogen-activated protein kinase kinase inhibitor 2′-amino-3′-methoxyflavone (PD98059) (5 μg i.c.) abrogated WIN55,212-2-evoked increases in blood pressure and neuronal ERK1/2 phosphorylation but not the reduction in Akt phosphorylation. On the other hand, prior PI3K inhibition with wortmannin (0.4 μg i.c.) exacerbated the WIN55,212-2 (7.5 and 15 μg i.c.) dose-related increases in blood pressure and ERK1/2 phosphorylation in the RVLM. The present neurochemical and integrative studies yield new insight into the critical role of two brainstem kinases, PI3K and ERK1/2, in the pressor response elicited by central CB1R activation in conscious rats. PMID:21946192

  8. Lactoferrin inhibits dexamethasone-induced chondrocyte impairment from osteoarthritic cartilage through up-regulation of extracellular signal-regulated kinase 1/2 and suppression of FASL, FAS, and Caspase 3

    SciTech Connect

    Tu, Yihui; Xue, Huaming; Francis, Wendy; Davies, Andrew P.; Pallister, Ian; Kanamarlapudi, Venkateswarlu; Xia, Zhidao

    2013-11-08

    Highlights: •Dex exerts dose-dependant inhibition of HACs viability and induction of apoptosis. •Dex-induced impairment of chondrocytes was attenuated by rhLF. •ERK and FASL/FAS signaling are involved in the effects of rhLF. •OA patients with glucocorticoid-induced cartilage damage may benefit from treatment with rhLF. -- Abstract: Dexamethasone (Dex) is commonly used for osteoarthritis (OA) with excellent anti-inflammatory and analgesic effect. However, Dex also has many side effects following repeated use over prolonged periods mainly through increasing apoptosis and inhibiting proliferation. Lactoferrin (LF) exerts significantly anabolic effect on many cells and little is known about its effect on OA chondrocytes. Therefore, the aim of this study is to investigate whether LF can inhibit Dex-induced OA chondrocytes apoptosis and explore its possible molecular mechanism involved in. MTT assay was used to determine the optimal concentration of Dex and recombinant human LF (rhLF) on chondrocytes at different time and dose points. Chondrocytes were then stimulated with Dex in the absence or presence of optimal concentration of rhLF. Cell proliferation and viability were evaluated using MTT and LIVE/DEAD assay, respectively. Cell apoptosis was evaluated by multi-parameter apoptosis assay kit using both confocal microscopy and flow cytometry, respectively. The expression of extracellular signal-regulated kinase (ERK), FAS, FASL, and Caspase-3 (CASP3) at the mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and immunocytochemistry, respectively. The optimal concentration of Dex (25 μg/ml) and rhLF (200 μg/ml) were chosen for the following experiments. rhLF significantly reversed the detrimental effect of Dex on chondrocytes proliferation, viability, and apoptosis. In addition, rhLF significantly prevented Dex-induced down-regulation of ERK and up-regulation of FAS, FASL, and CASP3. These findings demonstrated that rhLF acts as

  9. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer*

    PubMed Central

    Young, Christian D.; Zimmerman, Lisa J.; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B.; Gatza, Michael L.; Morrison, Meghan M.; Moore, Preston D.; Whitwell, Corbin A.; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E.; Silva, Grace O.; Patel, Premal; Brantley-Sieders, Dana M.; Levin, Maren; Horiates, Marina; Palma, Norma A.; Wang, Kai; Stephens, Philip J.; Perou, Charles M.; Weaver, Alissa M.; O'Shaughnessy, Joyce A.; Chang, Jenny C.; Park, Ben Ho; Liebler, Daniel C.; Cook, Rebecca S.; Arteaga, Carlos L.

    2015-01-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. PMID:25953087

  10. Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

    PubMed

    Young, Christian D; Zimmerman, Lisa J; Hoshino, Daisuke; Formisano, Luigi; Hanker, Ariella B; Gatza, Michael L; Morrison, Meghan M; Moore, Preston D; Whitwell, Corbin A; Dave, Bhuvanesh; Stricker, Thomas; Bhola, Neil E; Silva, Grace O; Patel, Premal; Brantley-Sieders, Dana M; Levin, Maren; Horiates, Marina; Palma, Norma A; Wang, Kai; Stephens, Philip J; Perou, Charles M; Weaver, Alissa M; O'Shaughnessy, Joyce A; Chang, Jenny C; Park, Ben Ho; Liebler, Daniel C; Cook, Rebecca S; Arteaga, Carlos L

    2015-07-01

    Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR. PMID:25953087

  11. Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.

    PubMed

    Asthagiri, A R; Nelson, C M; Horwitz, A F; Lauffenburger, D A

    1999-09-17

    Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. We begin to address this by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum fibronectin density. Plating cells on fibronectin-coated surfaces initiated a transient, biphasic ERK2 response, the magnitude and kinetics of which depended on integrin-ligand binding properties. Whereas ERK2 activity initially increased with a rate proportional to integrin-ligand bond number for low fibronectin density, the desensitization rate was independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation followed a superficially disparate time course. However, analysis of the early kinetics of the two signals revealed them to be correlated. The initial rates of FAK and ERK2 signal generation exhibited similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to differences in feedback regulation of these signals. Whereas FAK phosphorylation increased to a steady-state level as new integrin-ligand bond formation continued during cell spreading, ERK2 activity was decoupled from the integrin-ligand stimulus and decayed back to a basal level. Accordingly, we propose different functional metrics for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the integral of the pulse response for

  12. Ligand-biased activation of extracellular signal-regulated kinase 1/2 leads to differences in opioid induced antinociception and tolerance.

    PubMed

    Bobeck, Erin N; Ingram, Susan L; Hermes, Sam M; Aicher, Sue A; Morgan, Michael M

    2016-02-01

    Opioids produce antinociception by activation of G protein signaling linked to the mu-opioid receptor (MOPr). However, opioid binding to the MOPr also activates β-arrestin signaling. Opioids such as DAMGO and fentanyl differ in their relative efficacy for activation of these signaling cascades, but the behavioral consequences of this differential signaling are not known. The purpose of this study was to evaluate the behavioral significance of G protein and internalization dependent signaling within ventrolateral periaqueductal gray (vlPAG). Antinociception induced by microinjecting DAMGO into the vlPAG was attenuated by blocking Gαi/o protein signaling with administration of pertussis toxin (PTX), preventing internalization with administration of dynamin dominant-negative inhibitory peptide (dyn-DN) or direct inhibition of ERK1/2 with administration of the MEK inhibitor, U0126. In contrast, the antinociceptive effect of microinjecting fentanyl into the vlPAG was not altered by administration of PTX or U0126, and was enhanced by administration of dyn-DN. Microinjection of DAMGO, but not fentanyl, into the vlPAG induced phosphorylation of ERK1/2, which was blocked by inhibiting receptor internalization with administration of dyn-DN, but not by inhibition of Gαi/o proteins. ERK1/2 inhibition also prevented the development and expression of tolerance to repeated DAMGO microinjections, but had no effect on fentanyl tolerance. These data reveal that ERK1/2 activation following MOPr internalization contributes to the antinociceptive effect of some (e.g., DAMGO), but not all opioids (e.g., fentanyl) despite the known similarities for these agonists to induce β-arrestin recruitment and internalization. PMID:26497105

  13. Extracellular signal-regulated kinase mediates expression of arginase II but not inducible nitric-oxide synthase in lipopolysaccharide-stimulated macrophages.

    PubMed

    Jin, Yi; Liu, Yusen; Nelin, Leif D

    2015-01-23

    The mitogen-activated protein kinases (MAPK) have been shown to participate in iNOS induction following lipopolysaccharide (LPS) stimulation, while the role of MAPKs in the regulation of arginase remains unclear. We hypothesized that different MAPK family members are involved in iNOS and arginase expression following LPS stimulation. LPS-stimulated RAW 264.7 cells exhibited increased protein and mRNA levels for iNOS, arginase I, and arginase II; although the induction of arginase II was more robust than that for arginase I. A p38 inhibitor completely prevented iNOS expression while it only attenuated arginase II induction. In contrast, a MEK1/2 inhibitor (ERK pathway) completely abolished arginase II expression while actually enhancing iNOS induction in LPS-stimulated cells. Arginase II promoter activity was increased by ∼4-fold following LPS-stimulation, which was prevented by the ERK pathway inhibitor. Arginase II promoter activity was unaffected by a p38 inhibitor or JNK pathway interference. Transfection with a construct expressing a constitutively active RAS mutant increased LPS-induced arginase II promoter activity, while transfection with a vector expressing a dominant negative ERK2 mutant or a vector expressing MKP-3 inhibited the arginase II promoter activity. LPS-stimulated nitric oxide (NO) production was increased following siRNA-mediated knockdown of arginase II and decreased when arginase II was overexpressed. Our results demonstrate that while both the ERK and p38 pathways regulate arginase II induction in LPS-stimulated macrophages, iNOS induction by LPS is dependent on p38 activation. These results suggest that differential inhibition of the MAPK pathway may be a potential therapeutic strategy to regulate macrophage phenotype. PMID:25451938

  14. The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells

    PubMed Central

    Hong, Seung-Keun; Kim, Jin-Hwan; Lin, Ming-Fong; Park, Jong-In

    2011-01-01

    Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16INK4A and p21CIP1, but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation. PMID:21871886

  15. Agonist-mediated activation of Bombyx mori diapause hormone receptor signals to extracellular signal-regulated kinases 1 and 2 through Gq-PLC-PKC-dependent cascade.

    PubMed

    Jiang, Xue; Yang, Jingwen; Shen, Zhangfei; Chen, Yajie; Shi, Liangen; Zhou, Naiming

    2016-08-01

    Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca(2+) and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca(2+) chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause. PMID:27318251

  16. Radiosensitization and downregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) upon inhibition of mitogen/extracellular signal-regulated kinase (MEK) in malignant melanoma cells

    PubMed Central

    Eder, Stefan; Lamkowski, Andreas; Priller, Markus; Port, Matthias; Steinestel, Konrad

    2015-01-01

    Background Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an important cofactor in the p53-mediated DNA damage response pathway upon ionizing radiation (IR) and exerts anti-apoptotic effects also independent of p53 pathway activation. Furthermore, hnRNP K is overexpressed in various neoplasms including malignant melanoma (MM). Here, we investigate the role of hnRNP K in the radioresistance of MM cells. Methods and results Our results show cytoplasmic expression of hnRNP K in human MM surgical specimens, but not in benign nevi, and a quick dose- and time-dependent upregulation in response to IR accompanied by cytoplasmic redistribution of the protein in the IPC-298 cellular tumor model carrying an activating NRAS mutation (p.Q61L). SiRNA-based knockdown of hnRNP K induced a delayed decline in γH2AX/53BP1-positive DNA repair foci upon IR. Pharmacological interference with MAPK signaling abrogated ERK phosphorylation, diminished cellular hnRNP K levels, impaired γH2AX/53BP1-foci repair and proliferative capability and increased apoptosis comparable to the observed hnRNP K knockdown phenotype in IPC-298 cells. Conclusion Our results indicate that pharmacological interference with MAPK signaling increases vulnerability of NRAS-mutant malignant melanoma cells to ionizing radiation along with downregulation of endogenous hnRNP K and point towards a possible use for combined MEK inhibition and localized radiation therapy of MM in the NRAS-mutant setting where BRAF inhibitors offer no clinical benefit. PMID:26136337

  17. Autophagy-related Gene 7 (ATG7) and Reactive Oxygen Species/Extracellular Signal-regulated Kinase Regulate Tetrandrine-induced Autophagy in Human Hepatocellular Carcinoma*

    PubMed Central

    Gong, Ke; Chen, Chao; Zhan, Yao; Chen, Yan; Huang, Zebo; Li, Wenhua

    2012-01-01

    Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the broadly used Chinese medicinal herb Stephaniae tetrandrae, exhibits potent antitumor effects and has the potential to be used as a cancer chemotherapeutic agent. We previously reported that high concentrations of tetrandrine induce apoptosis in liver cancer cells. Here, we found that in human hepatocellular carcinoma (HCC) cells, a low dose of tetrandrine (5 μm) induced the expression of LC3-II, resulted in the formation of acidic autophagolysosome vacuoles (AVOs), and caused a punctate fluorescence pattern with the GFP-LC3 protein, which all are markers for cellular autophagy. Tetrandrine induced the production of intracellular reactive oxygen species (ROS), and treatment with ROS scavengers significantly abrogated the tetrandrine-induced autophagy. These results suggest that the generation of ROS plays an important role in promoting tetrandrine-induced autophagy. Tetrandrine-induced mitochondrial dysfunction resulted in ROS accumulation and autophagy. ROS generation activated the ERK MAP kinase, and the ERK signaling pathway at least partially contributed to tetrandrine-induced autophagy in HCC cells. Moreover, we found that tetrandrine transcriptionally regulated the expression of autophagy related gene 7 (ATG7), which promoted tetrandrine-induced autophagy. In addition to in vitro studies, similar results were also observed in vivo, where tetrandrine caused the accumulation of ROS and induced cell autophagy in a tumor xenograft model. Interestingly, tetrandrine treatment also induced autophagy in a ROS-dependent manner in C. elegans muscle cells. Therefore, these findings suggest that tetrandrine is a potent autophagy agonist and may be a promising clinical chemotherapeutic agent. PMID:22927446

  18. Magnificent seven: roles of G protein-coupled receptors in extracellular sensing in fungi

    PubMed Central

    Xue, Chaoyang; Hsueh, Yen-Ping; Heitman, Joseph

    2010-01-01

    G protein-coupled receptors (GPCRs) represent the largest family of transmembrane receptors and are responsible for transducing extracellular signals into intracellular responses that involve complex intracellular-signaling networks. This review highlights recent research advances in fungal GPCRs, including classification, extracellular sensing, and G protein-signaling regulation. The involvement of GPCRs in pheromone and nutrient sensing has been studied extensively over the past decade. Following recent advances in fungal genome sequencing projects, a panoply of GPCR candidates has been revealed and some have been documented to play key roles sensing diverse extracellular signals, such as pheromones, sugars, amino acids, nitrogen sources, and even photons. Identification and deorphanization of additional putative GPCRs may require the development of new research tools. Here, we compare research on GPCRs in fungi with information derived from mammalian systems to provide a useful road map on how to better understand ligand–GPCR–G protein interactions in general. We also emphasize the utility of yeast as a discovery tool for systemic studies of GPCRs from other organisms. PMID:18811658

  19. Developmental changes in insulin- and amino acid-induced mTOR signalling regulate muscle protein synthesis in neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enhanced efficiency, with which dietary protein is used for growth in the neonate, is due to the ability of neonatal muscle to markedly increase protein synthesis in response to feeding (Davis "et al.", 1996). The stimulation of protein synthesis by feeding in neonatal muscle is independently m...

  20. Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders.

    PubMed

    Li, Min; Quan, Chao; Toth, Rachel; Campbell, David G; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

    2015-12-11

    Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser(858) of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease. PMID:26499801

  1. Toll-Like Receptor 2-Dependent Extracellular Signal-Regulated Kinase Signaling in Mycobacterium tuberculosis-Infected Macrophages Drives Anti-Inflammatory Responses and Inhibits Th1 Polarization of Responding T Cells

    PubMed Central

    Richardson, Edward T.; Shukla, Supriya; Sweet, David R.; Wearsch, Pamela A.; Tsichlis, Philip N.; Boom, W. Henry

    2015-01-01

    Mycobacterium tuberculosis survives within macrophages and employs immune evasion mechanisms to persist in the host. Protective T helper type 1 (Th1) responses are induced, and the immune response in most individuals is sufficient to restrict M. tuberculosis to latent infection, but most infections are not completely resolved. As T cells and macrophages respond, a balance is established between protective Th1-associated and other proinflammatory cytokines, such as interleukin-12 (IL-12), interferon gamma (IFN-γ), and tumor necrosis factor alpha, and anti-inflammatory cytokines, such as IL-10. The mechanisms by which M. tuberculosis modulates host responses to promote its survival remain unclear. In these studies, we demonstrate that M. tuberculosis induction of IL-10, suppression of IL-12, and inhibition of class II major histocompatibility complex (MHC-II) molecules in infected macrophages are all driven by Toll-like receptor 2 (TLR2)-dependent activation of the extracellular signal-regulated kinases (ERK). Elimination of ERK signaling downstream of TLR2 by pharmacologic inhibition with U0126 or genetic deletion of Tpl2 blocks IL-10 secretion and enhances IL-12 p70 secretion. We demonstrate that M. tuberculosis regulation of these pathways in macrophages affects T cell responses to infected macrophages. Thus, genetic blockade of the ERK pathway in Tpl2−/− macrophages enhances Th1 polarization and IFN-γ production by antigen-specific CD4+ T cells responding to M. tuberculosis infection. These data indicate that M. tuberculosis and its potent TLR2 ligands activate ERK signaling in macrophages to promote anti-inflammatory macrophage responses and blunt Th1 responses against the pathogen. PMID:25776754

  2. β2-Glycoprotein I Inhibits Vascular Endothelial Growth Factor-Induced Angiogenesis by Suppressing the Phosphorylation of Extracellular Signal-Regulated Kinase 1/2, Akt, and Endothelial Nitric Oxide Synthase.

    PubMed

    Chiu, Wen-Chin; Chiou, Tzeon-Jye; Chung, Meng-Ju; Chiang, An-Na

    2016-01-01

    Angiogenesis is the process of new blood vessel formation, and it plays a key role in various physiological and pathological conditions. The β2-glycoprotein I (β2-GPI) is a plasma glycoprotein with multiple biological functions, some of which remain to be elucidated. This study aimed to identify the contribution of 2-GPI on the angiogenesis induced by vascular endothelial growth factor (VEGF), a pro-angiogenic factor that may regulate endothelial remodeling, and its underlying mechanism. Our results revealed that β2-GPI dose-dependently decreased the VEGF-induced increase in endothelial cell proliferation, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bromodeoxyuridine (BrdU) incorporation assays. Furthermore, incubation with both β2-GPI and deglycosylated β2-GPI inhibited the VEGF-induced tube formation. Our results suggest that the carbohydrate residues of β2-GPI do not participate in the function of anti-angiogenesis. Using in vivo Matrigel plug and angioreactor assays, we show that β2-GPI remarkably inhibited the VEGF-induced angiogenesis at a physiological concentration. Moreover, β2-GPI inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and endothelial nitric oxide synthase (eNOS). In summary, our in vitro and in vivo data reveal for the first time that β2-GPI inhibits the VEGF-induced angiogenesis and highlights the potential for β2-GPI in anti-angiogenic therapy. PMID:27579889

  3. An N-terminal nuclear export signal regulates trafficking and aggregation of Huntingtin (Htt) protein exon 1.

    PubMed

    Zheng, Zhiqiang; Li, Aimin; Holmes, Brandon B; Marasa, Jayne C; Diamond, Marc I

    2013-03-01

    Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity. PMID:23319588

  4. Glutathione S-transferase class μ regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries.

    PubMed

    Bhattacharya, Poulomi; Madden, Jill A; Sen, Nivedita; Hoyer, Patricia B; Keating, Aileen F

    2013-02-15

    4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2-8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. PMID:23274565

  5. Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

    SciTech Connect

    Bhattacharya, Poulomi; Madden, Jill A.; Sen, Nivedita; Hoyer, Patricia B.; Keating, Aileen F.

    2013-02-15

    4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2–8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P < 0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P < 0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P < 0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P < 0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. - Highlights: ► GSTM protein increases in response to ovarian VCD exposure. ► VCD increases Ask1 mRNA at the onset of follicle loss. ► Ovarian GSTM binds more ASK1 protein during VCD-induced ovotoxicity. ► PI3K regulates ovarian GSTM protein.

  6. Extracellular proteins limit the dispersal of biogenic nanoparticles

    USGS Publications Warehouse

    Moreau, J.W.; Weber, P.K.; Martin, M.C.; Gilbert, B.; Hutcheon, I.D.; Banfield, J.F.

    2007-01-01

    High-spatial-resolution secondary ion microprobe spectrometry, synchrotron radiation-based Fourier-transform infrared spectroscopy, and polyacrylamide gel analysis demonstrated the intimate association of proteins with spheroidal aggregates of biogenic zinc sulfide nanocrystals, an example of extracellular biomineralization. Experiments involving synthetic zinc sulfide nanoparticles and representative amino acids indicated a driving role for cysteine in rapid nanoparticle aggregation. These findings suggest that microbially derived extracellular proteins can limit the dispersal of nanoparticulate metal-bearing phases, such as the mineral products of bioremediation, that may otherwise be transported away from their source by subsurface fluid flow.

  7. Extracellular proteins from the Phakopsora pachyrhizi spore wall

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phakopsora pachyrhizi is the causal agent of Asian soybean rust, a disease of leguminous plants that has recently become established in the U.S. We previously applied two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry to identify a set of extracellular proteins that were washed...

  8. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. PMID:25789606

  9. Conformal Nanopatterning of Extracellular Matrix Proteins onto Topographically Complex Surfaces

    PubMed Central

    Sun, Yan; Jallerat, Quentin; Szymanski, John M.

    2015-01-01

    We report a method for conformal nanopatterning of extracellular matrix proteins onto engineered surfaces independent of underlying microtopography. This enables fibronectin, laminin, and other proteins to be applied to biomaterial surfaces in complex geometries inaccessible using traditional soft lithography techniques. Engineering combinatorial surfaces that integrate topographical and biochemical micropatterns enhances control of the biotic-abiotic interface, used here to understand cardiomyocyte response to competing physical and chemical cues in the microenvironment. PMID:25506720

  10. EXTRACELLULAR HEAT SHOCK PROTEINS: A NEW LOCATION, A NEW FUNCTION

    PubMed Central

    De Maio, Antonio; Vazquez, Daniel

    2015-01-01

    The expression of heat shock proteins (hsp) is a basic and well conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Since these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that hsp can also be present outside cells where they appear to display a function different than the well understood chaperone role. Extracellular hsp act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Since the majority of hsp do not possess a secretory peptide signal, they are likely be exported by a non-classical secretory pathway. Different mechanisms have been proposed to explain the export of hsp, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular hsp appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular hsp suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular hsp. PMID:23807250

  11. Extracellular Proteins: Novel Key Components of Metal Resistance in Cyanobacteria?

    PubMed Central

    Giner-Lamia, Joaquín; Pereira, Sara B.; Bovea-Marco, Miquel; Futschik, Matthias E.; Tamagnini, Paula; Oliveira, Paulo

    2016-01-01

    Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities, and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias toward the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria.

  12. Genetic analysis of extracellular proteins of Serratia marcescens.

    PubMed Central

    Hines, D A; Saurugger, P N; Ihler, G M; Benedik, M J

    1988-01-01

    Serratia marcescens, a gram-negative enteric bacterium, is capable of secreting a number of proteins extracellularly. The types of activity found in the growth media include proteases, chitinases, a nuclease, and a lipase. Genetic studies have been undertaken to investigate the mechanisms used for the extracellular secretion of these exoproteins by S. marcescens. Many independent mutations affecting the extracellular enzymes were isolated after chemical and transposon mutagenesis. Using indicator media, we have identified loci involved in the production or excretion of extracellular protease, nuclease, or chitinase by S. marcescens. None of the mutations represented general extracellular-excretion mutants; in no case was the production or excretion of multiple exoproteins affected. A variety of loci were identified, including regulatory mutations affecting nuclease and chitinase expression. A number of phenotypically different protease mutants arose. Some of them may represent different gene products required for the production and excretion of the major metalloprotease, a process more complex than that for the other S. marcescens exoproteins characterized to date. PMID:2842305

  13. Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

    PubMed

    Blake, James F; Burkard, Michael; Chan, Jocelyn; Chen, Huifen; Chou, Kang-Jye; Diaz, Dolores; Dudley, Danette A; Gaudino, John J; Gould, Stephen E; Grina, Jonas; Hunsaker, Thomas; Liu, Lichuan; Martinson, Matthew; Moreno, David; Mueller, Lars; Orr, Christine; Pacheco, Patricia; Qin, Ann; Rasor, Kevin; Ren, Li; Robarge, Kirk; Shahidi-Latham, Sheerin; Stults, Jeffrey; Sullivan, Francis; Wang, Weiru; Yin, Jianping; Zhou, Aihe; Belvin, Marcia; Merchant, Mark; Moffat, John; Schwarz, Jacob B

    2016-06-23

    The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity. PMID:27227380

  14. Extracellular Proteins Limit the Dispersal of BiogenicNanoparticles

    SciTech Connect

    Moreau, John W.; Weber, Peter K.; Martin, Michael C.; Gilbert,Benjamin; Hutcheon, Ian D.; Banfield, Jillian F.

    2007-04-27

    High spatial-resolution secondaryion microprobespectrometry, synchrotron radiation Fourier-transform infraredspectroscopy and polyacrylamide gel analysis demonstrate the intimateassociation of proteins with spheroidal aggregates of biogenic zincsulfide nanocrystals, an example of extracellular biomineralization.Experiments involving synthetic ZnS nanoparticles and representativeamino acids indicate a driving role for cysteine in rapid nanoparticleaggregation. These findings suggest that microbially-derivedextracellular proteins can limit dispersal of nanoparticulatemetal-bearing phases, such as the mineral products of bioremediation,that may otherwise be transported away from their source by subsurfacefluid flow.

  15. Extracellular Protease Digestion to Evaluate Membrane Protein Cell Surface Localization

    PubMed Central

    Besingi, Richard N.; Clark, Patricia L.

    2016-01-01

    Membrane proteins play crucial roles in signaling and as anchors for cell surface display. Proper secretion of a membrane protein can be evaluated by its susceptibility to digestion by an extracellular protease, but this requires a crucial control to confirm membrane integrity during digestion. This protocol describes how to use this approach to determine how efficiently a protein is secreted to the outer surface of Gram-negative bacteria. Its success relies upon careful selection of an appropriate intracellular reporter protein that will remain undigested if the membrane barrier remains intact, but is rapidly digested when cells are lysed prior to evaluation. Reporter proteins that are resistant to proteases (e.g. maltose-binding protein) do not return accurate results; in contrast, proteins that are more readily digested (e.g. SurA) serve as more sensitive reporters of membrane integrity, yielding more accurate measurements of membrane protein localization. Similar considerations apply when evaluating membrane protein localization in other contexts, including eukaryotic cells and organelle membranes. Evaluating membrane protein localization using this approach requires only standard biochemistry laboratory equipment for cell lysis, gel electrophoresis and western blotting. After expression of the protein of interest, this procedure can be completed in 4 h. PMID:26584447

  16. Heat treatment of peach fruit: modifications in the extracellular compartment and identification of novel extracellular proteins.

    PubMed

    Bustamante, Claudia A; Budde, Claudio O; Borsani, Julia; Lombardo, Verónica A; Lauxmann, Martin A; Andreo, Carlos S; Lara, María V; Drincovich, María F

    2012-11-01

    Ripening of peach (Prunus persica L. Batsch) fruit is accompanied by dramatic cell wall changes that lead to softening. Post-harvest heat treatment is effective in delaying softening and preventing some chilling injury symptoms that this fruit exhibits after storage at low temperatures. In the present work, the levels of twelve transcripts encoding proteins involved in cell wall metabolism, as well as the differential extracellular proteome, were examined after a post-harvest heat treatment (HT; 39 °C for 3 days) of "Dixiland" peach fruit. A typical softening behaviour, in correlation with an increase in 1-aminocyclopropane-1-carboxylic acid oxidase-1 (PpACO1), was observed for peach maintained at 20 °C for 3 days (R3). Six transcripts encoding proteins involved in cell wall metabolism significantly increased in R3 with respect to peach at harvest, while six showed no modification or even decreased. In contrast, after HT, fruit maintained their firmness, exhibiting low PpACO1 level and significant lower levels of the twelve cell wall-modifying genes than in R3. Differential proteomic analysis of apoplastic proteins during softening and after HT revealed a significant decrease of DUF642 proteins after HT; as well as an increase of glyceraldehyde-3-phosphate dehydrogenase (GAPC) after softening. The presence of GAPC in the peach extracellular matrix was further confirmed by in situ immunolocalization and transient expression in tomato fruit. Though further studies are required to establish the function of DUF642 and GAPC in the apoplast, this study contributes to a deeper understanding of the events during peach softening and after HT with a focus on this key compartment. PMID:22902552

  17. Extracellular Proteins: Novel Key Components of Metal Resistance in Cyanobacteria?

    PubMed

    Giner-Lamia, Joaquín; Pereira, Sara B; Bovea-Marco, Miquel; Futschik, Matthias E; Tamagnini, Paula; Oliveira, Paulo

    2016-01-01

    Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities, and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias toward the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria. PMID:27375598

  18. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  19. Vascular wall extracellular matrix proteins and vascular diseases

    PubMed Central

    Xu, Junyan; Shi, Guo-Ping

    2014-01-01

    Extracellular matrix proteins form the basic structure of blood vessels. Along with providing basic structural support to blood vessels, matrix proteins interact with different sets of vascular cells via cell surface integrin or non-integrin receptors. Such interactions induce vascular cell de novo synthesis of new matrix proteins during blood vessel development or remodeling. Under pathological conditions, vascular matrix proteins undergo proteolytic processing, yielding bioactive fragments to influence vascular wall matrix remodeling. Vascular cells also produce alternatively spliced variants that induce vascular cell production of different matrix proteins to interrupt matrix homeostasis, leading to increased blood vessel stiffness; vascular cell migration, proliferation, or death; or vascular wall leakage and rupture. Destruction of vascular matrix proteins leads to vascular cell or blood-borne leukocyte accumulation, proliferation, and neointima formation within the vascular wall; blood vessels prone to uncontrolled enlargement during blood flow diastole; tortuous vein development; and neovascularization from existing pathological tissue microvessels. Here we summarize discoveries related to blood vessel matrix proteins within the past decade from basic and clinical studies in humans and animals — from expression to cross-linking, assembly, and degradation under physiological and vascular pathological conditions, including atherosclerosis, aortic aneurysms, varicose veins, and hypertension. PMID:25045854

  20. Egg extracellular coat proteins: from fish to mammals.

    PubMed

    Litscher, E S; Wassarman, P M

    2007-03-01

    The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEalpha (approximately 58 kDa), VEbeta (approximately 54 kDa), and VEgamma (approximately 47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (approximately 200 kDa), ZP2 (approximately 120 kDa), and ZP3 (approximately 83 kDa). Overall, trout VE and mouse ZP proteins share approximately 25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEalpha, VEbeta, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEalpha and VEbeta are very similar in sequence (approximately 65% sequence identity) and are related to ZP1 and ZP2, whereas VEgamma is related to ZP3 (approximately 25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under hormonal control and transported in the bloodstream to growing oocytes in the ovary. The trout VE is assembled from VEalpha/gamma and VEbeta/gamma heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite approximately 400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C

  1. Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

    PubMed

    Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

    2013-11-01

    Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

  2. Expression of genes encoding extracellular matrix proteins: A macroarray study

    PubMed Central

    FUTYMA, KONRAD; MIOTŁA, PAWEŁ; RÓŻYŃSKA, KRYSTYNA; ZDUNEK, MAŁGORZATA; SEMCZUK, ANDRZEJ; RECHBERGER, TOMASZ; WOJCIEROWSKI, JACEK

    2014-01-01

    Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs. PMID:25231141

  3. Gene evolution and functions of extracellular matrix proteins in teeth.

    PubMed

    Yoshizaki, Keigo; Yamada, Yoshihiko

    2013-03-01

    The extracellular matrix (ECM) not only provides physical support for tissues, but it is also critical for tissue development, homeostasis and disease. Over 300 ECM molecules have been defined as comprising the "core matrisome" in mammals through the analysis of whole genome sequences. During tooth development, the structure and functions of the ECM dynamically change. In the early stages, basement membranes (BMs) separate two cell layers of the dental epithelium and the mesenchyme. Later in the differentiation stages, the BM layer is replaced with the enamel matrix and the dentin matrix, which are secreted by ameloblasts and odontoblasts, respectively. The enamel matrix genes and the dentin matrix genes are each clustered in two closed regions located on human chromosome 4 (mouse chromosome 5), except for the gene coded for amelogenin, the major enamel matrix protein, which is located on the sex chromosomes. These genes for enamel and dentin matrix proteins are derived from a common ancestral gene, but as a result of evolution, they diverged in terms of their specific functions. These matrix proteins play important roles in cell adhesion, polarity, and differentiation and mineralization of enamel and dentin matrices. Mutations of these genes cause diseases such as odontogenesis imperfect (OI) and amelogenesis imperfect (AI). In this review, we discuss the recently defined terms matrisome and matrixome for ECMs, as well as focus on genes and functions of enamel and dentin matrix proteins. PMID:23539364

  4. Glycoprotein nonmetastatic melanoma protein B extracellular fragment shows neuroprotective effects and activates the PI3K/Akt and MEK/ERK pathways via the Na+/K+-ATPase

    PubMed Central

    Ono, Yoko; Tsuruma, Kazuhiro; Takata, Masafumi; Shimazawa, Masamitsu; Hara, Hideaki

    2016-01-01

    Glycoprotein nonmetastatic melanoma protein B (GPNMB) plays important roles in various types of cancer and amyotrophic lateral sclerosis (ALS). The details of GPNMB function and its interacting protein have not been clarified. Therefore, to identify GPNMB binding partners on the cell membrane, we used membrane protein library/BLOTCHIP-MS technology, which enables us to analyze all cell membrane proteins as binding partners of the GPNMB extracellular fragment. As a result of a comprehensive search, we identified the alpha subunits of Na+/K+-ATPase (NKA) as a possible binding partner. We confirmed the interaction between the GPNMB extracellular fragment and NKA by immunoprecipitation and immunostaining in NSC-34 cells. Indeed, endogenous GPNMB extracellular fragment bound to and colocalized with NKA alpha subunits. Furthermore, exogenous GPNMB extracellular fragment, i.e., human recombinant GPNMB, also bound to and colocalized with NKA alpha subunits. Additionally, we found that the GPNMB extracellular fragment had neuroprotective effects and activated the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathways via NKA. These findings indicated that NKA may act as a novel “receptor” for the GPNMB extracellular fragment, offering additional molecular targets for the treatment of GPNMB-related diseases, including various types of cancer and ALS. PMID:26988030

  5. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  6. Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins.

    PubMed

    Capossela, S; Schläfli, P; Bertolo, A; Janner, T; Stadler, B M; Pötzel, T; Baur, M; Stoyanov, J V

    2014-01-01

    Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases. PMID:24706108

  7. Extracellular matrix protein expression is brain region dependent.

    PubMed

    Dauth, Stephanie; Grevesse, Thomas; Pantazopoulos, Harry; Campbell, Patrick H; Maoz, Ben M; Berretta, Sabina; Parker, Kevin Kit

    2016-05-01

    In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region-specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin-R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin-R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin-R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309-1336, 2016. © 2016 Wiley Periodicals, Inc. PMID:26780384

  8. Expression of extracellular matrix proteins in adenomatoid odontogenic tumor.

    PubMed

    Modolo, Filipe; Biz, Michelle Tillmann; Martins, Marília Trierveiller; Machado de Sousa, Suzana Orsini; de Araújo, Ney Soares

    2010-03-01

    Altered expression of extracellular matrix (ECM) components has been reported in several pathologies; however, few ECM proteins have been evaluated in adenomatoid odontogenic tumor (AOT). The aim of this study was to analyze the expression and distribution of the ECM proteoglycans: biglycan and decorin; and glycoproteins: osteonectin, osteopontin, bone sialoprotein and osteocalcin in the AOT. Three-micrometer sections from paraffin-embedded specimens were evaluated employing a streptavidin-biotin immunohistochemical method with the antibodies against the proteins previously cited. Only the osteonectin was expressed in the epithelial cells. The eosinophilic amorphous material and the connective tissue showed expression of all components studied. The calcification foci expressed only osteopontin. In conclusion, the low expression of the components studied in neoplastic epithelial cells suggests that the epithelial cells act probably as stimulators of the expression by the stroma, which in turn can act as agonist or antagonist of the tumor growth. These results suggest that the components studied probably have a key role in the biological behavior of the AOT. PMID:20070486

  9. Trovafloxacin-induced Replication Stress Sensitizes HepG2 Cells to Tumor Necrosis Factor-alpha-induced Cytotoxicity Mediated by Extracellular Signal-regulated Kinase and Ataxia Telangiectasia and Rad3-related

    PubMed Central

    Beggs, Kevin M.; Maiuri, Ashley R.; Fullerton, Aaron M.; Poulsen, Kyle L.; Breier, Anna B.; Ganey, Patricia E.; Roth, Robert A.

    2015-01-01

    Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress. PMID:25748550

  10. Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis

    PubMed Central

    Genereux, Joseph C; Qu, Song; Zhou, Minghai; Ryno, Lisa M; Wang, Shiyu; Shoulders, Matthew D; Kaufman, Randal J; Lasmézas, Corinne I; Kelly, Jeffery W; Wiseman, R Luke

    2015-01-01

    The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis. PMID:25361606

  11. Regulation of Lhb and Egr1 Gene Expression by GNRH Pulses in Rat Pituitaries Is Both c-Jun N-Terminal Kinase (JNK)- and Extracellular Signal-Regulated Kinase (ERK)-Dependent1

    PubMed Central

    Burger, Laura L.; Haisenleder, Daniel J.; Aylor, Kevin W.; Marshall, John C.

    2009-01-01

    Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1–24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription. PMID:19710510

  12. Baculovirus display for discovery of low-affinity extracellular receptor-ligand interactions using protein microarrays.

    PubMed

    Tom, Irene; Estevez, Alberto; Bowman, Krista; Gonzalez, Lino C

    2015-06-15

    When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors. PMID:25797350

  13. Measurement and modification of the expression level of the chaperone protein and signaling regulator GRP78/BiP in mammalian cells.

    PubMed

    Chen, Wan-Ting; Lee, Amy S

    2011-01-01

    GRP78/BiP is a major endoplasmic reticulum (ER) chaperone protein essential for protein quality control in the ER as well as a central regulator of unfolded protein response (UPR). The induction of GRP78 is well established as a marker for ER stress. Recently, mouse models targeting the Grp78 allele indicate that GRP78 has critical roles in cancer progression, drug resistance, angiogenesis, neurological diseases, and diabetes. The discovery of a cytosolic GRP78 isoform and cell surface GRP78 adds new insights to its function beyond the ER compartment in regulating growth factor signaling and cell viability. Here, we summarize and update several approaches for the detection and quantitation of total GRP78, cytosolic GRP78 isoform, and cell surface GRP78, and the use of small interfering RNA to knockdown GRP78 expression. These techniques can be applied to culture cells as well as tissues. PMID:21266253

  14. Large-scale screening for novel low-affinity extracellular protein interactions

    PubMed Central

    Bushell, K. Mark; Söllner, Christian; Schuster-Boeckler, Benjamin; Bateman, Alex; Wright, Gavin J.

    2008-01-01

    Extracellular protein–protein interactions are essential for both intercellular communication and cohesion within multicellular organisms. Approximately a fifth of human genes encode membrane-tethered or secreted proteins, but they are largely absent from recent large-scale protein interaction datasets, making current interaction networks biased and incomplete. This discrepancy is due to the unsuitability of popular high-throughput methods to detect extracellular interactions because of the biochemical intractability of membrane proteins and their interactions. For example, cell surface proteins contain insoluble hydrophobic transmembrane regions, and their extracellular interactions are often highly transient, having half-lives of less than a second. To detect transient extracellular interactions on a large scale, we developed AVEXIS (avidity-based extracellular interaction screen), a high-throughput assay that overcomes these technical issues and can detect very transient interactions (half-lives ≤ 0.1 sec) with a low false-positive rate. We used it to systematically screen for receptor–ligand pairs within the zebrafish immunoglobulin superfamily and identified novel ligands for both well-known and orphan receptors. Genes encoding receptor–ligand pairs were often clustered phylogenetically and expressed in the same or adjacent tissues, immediately implying their involvement in similar biological processes. Using AVEXIS, we have determined the first systematic low–affinity extracellular protein interaction network, supported by independent biological data. This technique will now allow large-scale extracellular protein interaction mapping in a broad range of experimental contexts. PMID:18296487

  15. Notch signaling regulates the phosphorylation of Akt and survival of lipopolysaccharide-activated macrophages via regulator of G protein signaling 19 (RGS19)

    PubMed Central

    Sangphech, Naunpun; Osborne, Barbara A.; Palaga, Tanapat

    2014-01-01

    Macrophages play critical roles in innate immune defense by sensing microbes using pattern-recognition receptors. Lipopolysaccharide (LPS) stimulates macrophages via TLR, which leads to activation of downstream signaling cascades. In this study, we investigated the roles of a conserved signaling pathway, Notch signaling, in regulating the downstream signaling cascades of the LPS/TLR4 pathways in macrophages. Using a phosphoproteomic approach and a gamma-secretase inhibitor (GSI) to suppress the processing and activation of Notch signaling, we identified regulator of G protein signaling 19 (RGS19) as a target protein whose phosphorylation was affected by GSI treatment. RGS19 is a guanosine triphosphatase (GTPase)-activating protein that functions to negatively regulate G protein-coupled receptors via Gαi/Gαq-linked signaling. Stimulation of RAW264.7 cells with LPS increased the level of the phosphorylated form of RGS19, while LPS stimulation in the presence of GSI decreased its level. GSI treatment did not alter the mRNA level of rgs19. Treatment with GSI or silencing of rgs19 in macrophages impaired the phosphorylation of Akt Thr308 upon LPS stimulation. Furthermore, targeted deletion of a DNA-binding protein and binding partner of the Notch receptor, RBP-Jκ/CSL, in macrophages resulted in delayed and decreased Akt phosphorylation. Because the PI3K/Akt pathway regulates cell survival in various cell types, the cell cycle and cell death were assayed upon GSI treatment, phosphatidylinositol 3 kinase (PI3K) inhibitor treatment or silencing of rgs19. GSI treatment resulted in decreased cell populations in the G1 and S phases, while it increased the cell population of cell death. Similarly, silencing of rgs19 resulted in a decreased cell population in the G1 phase and an increased cell population in the subG1 phase. Inhibition of Akt phosphorylation by PI3K inhibitor in LPS-stimulated macrophages increased cell population in G1 phase, suggesting a possible cell cycle

  16. The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor.

    PubMed

    Ihara, Yuichiro; Kihara, Yasuyuki; Hamano, Fumie; Yanagida, Keisuke; Morishita, Yasuyuki; Kunita, Akiko; Yamori, Takao; Fukayama, Masashi; Aburatani, Hiroyuki; Shimizu, Takao; Ishii, Satoshi

    2010-10-01

    Tumors often are associated with a low extracellular pH, which induces a variety of cellular events. However, the mechanisms by which tumor cells recognize and react to the acidic environment have not been fully elucidated. T-cell death-associated gene 8 (TDAG8) is an extracellular pH-sensing G protein-coupled receptor that is overexpressed in various tumors and tumor cell lines. In this report, we show that TDAG8 on the surface of tumor cells facilitates tumor development by sensing the acidic environment. Overexpression of TDAG8 in mouse Lewis lung carcinoma (LLC) cells enhanced tumor development in animal models and rendered LLC cells resistant to acidic culture conditions by increasing activation of protein kinase A and extracellular signal-regulated kinase in vitro. Moreover, shRNA-mediated knockdown of endogenous TDAG8 in NCI-H460 human non-small cell lung cancer cells reduced cell survival in an acidic environment in vitro as well as tumor development in vivo. Microarray analyses of tumor-containing lung tissues of mice injected with TDAG8-expressing LLC cells revealed up-regulation of genes related to cell growth and glycolysis. These results support the hypothesis that TDAG8 enhances tumor development by promoting adaptation to the acidic environment to enhance cell survival/proliferation. TDAG8 may represent a therapeutic target for arresting tumor growth. PMID:20855608

  17. Regulation of the death-associated protein kinase 1 expression and autophagy via ATF6 requires apoptosis signal-regulating kinase 1.

    PubMed

    Gade, Padmaja; Manjegowda, Srikanta B; Nallar, Shreeram C; Maachani, Uday B; Cross, Alan S; Kalvakolanu, Dhananjaya V

    2014-11-01

    The death-associated protein kinase 1 (DAPK1) is an important regulator of cell death and autophagy. Recently, we have identified that ATF6, an endoplasmic reticulum-resident transcription factor, in association with the transcription factor CEBP-β, regulates the gamma interferon (IFN-γ)-induced expression of Dapk1 (P. Gade et al., Proc. Natl. Acad. Sci. U. S. A. 109:10316-10321, 2012, doi.org/10.1073/pnas.1119273109). IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β were essential for the formation of a novel transcriptional complex that regulates DAPK1. Here, we report that IFN-γ activates the ASK1-MKK3/MKK6-p38 mitogen-activated protein kinase (MAPK) pathway for controlling the activity of ATF6. The terminal enzyme in this pathway, p38 MAPK, phosphorylates a critical threonine residue in ATF6 upstream of its DNA binding domain. ATF6 mutants defective for p38 MAPK phosphorylation fail to undergo proteolytic processing in the Golgi apparatus and drive IFN-γ-induced gene expression and autophagy. We also show that mice lacking Ask1 are highly susceptible to lethal bacterial infection owing to defective autophagy. Together, these results identify a novel host defense pathway controlled by IFN-γ signaling. PMID:25135476

  18. Multiple Protein Interactions Involving Proposed Extracellular Loop Domains of the Tight Junction Protein Occludin

    PubMed Central

    Nusrat, Asma; Brown, G. Thomas; Tom, Jeffrey; Drake, Alex; Bui, Tam T.T.; Quan, Cliff; Mrsny, Randall J.

    2005-01-01

    Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101–121 and O-B:210–228, respectively, to examine potential molecular interactions between these two regions of occludin and other TJ proteins. A superficial biophysical assessment of A:101–121 and O-B:210–228 showed them to have dissimilar solution conformation characteristics. Although O-A:101–121 failed to strongly interact with protein components of the human epithelial intestinal cell line T84, O-B:210–228 selectively associated with occludin, claudin-one and the junctional adhesion molecule (JAM)-A. Further, the presence of O-B:210–228, but not O-A:101–121, impeded the recovery of functional TJ structures. A scrambled peptide sequences of O-B:210–228 failed to influence TJ assembly. These studies demonstrate distinct properties for these two extracellular segments of the occludin protein and provide an improved understanding of how specific domains of occludin may interact with proteins present at TJ structures. PMID:15659655

  19. A Cysteine-Rich Extracellular Protein Containing a PA14 Domain Mediates Quorum Sensing in Dictyostelium discoideum

    PubMed Central

    Kolbinger, Alexandra; Gao, Tong; Brock, Debbie; Ammann, Robin; Kisters, Axel; Kellermann, Joseph; Hatton, Diane; Gomer, Richard H.; Wetterauer, Birgit

    2005-01-01

    Much remains to be understood about quorum-sensing factors that allow cells to sense their local density. Dictyostelium discoideum is a simple eukaryote that grows as single-celled amoebae and switches to multicellular development when food becomes limited. As the growing cells reach a high density, they begin expressing discoidin genes. The cells secrete an unknown factor, and at high cell densities the concomitant high levels of the factor induce discoidin expression. We report here the enrichment of discoidin-inducing complex (DIC), an ∼400-kDa protein complex that induces discoidin expression during growth and development. Two proteins in the DIC preparation, DicA1 and DicB, were identified by sequencing proteolytic digests. DicA1 and DicB were expressed in Escherichia coli and tested for their ability to induce discoidin during growth and development. Recombinant DicB was unable to induce discoidin expression, while recombinant DicA1 was able to induce discoidin expression. This suggests that DicA1 is an active component of DIC and indicates that posttranslational modification is dispensable for activity. DicA1 mRNA is expressed in vegetative and developing cells. The mature secreted form of DicA1 has a molecular mass of 80 kDa and has a 24-amino-acid cysteine-rich repeat that is similar to repeats in Dictyostelium proteins, such as the extracellular matrix protein ecmB/PstA, the prespore cell-inducing factor PSI, and the cyclic AMP phosphodiesterase inhibitor PDI. Together, the data suggest that DicA1 is a component of a secreted quorum-sensing signal regulating discoidin gene expression during Dictyostelium growth and development. PMID:15947191

  20. Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR.

    PubMed

    Müller, A; Benoist, P; Diem, H G; Schwencke, J

    1991-12-01

    To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest. PMID:15101385

  1. Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the Receptor of Advanced Glycation End Products/Raf Protein/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase/Extracellular Signal-regulated Kinase (RAGE/Raf/MEK/ERK) Pathway.

    PubMed

    Meng, Hong-Zheng; Zhang, Wei-Lin; Liu, Fei; Yang, Mao-Wei

    2015-11-20

    The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells; cell proliferation was stimulated after exposure to low dose AGE-BSA, but cell apoptosis was stimulated after exposure to high dose AGE-BSA. The low dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf/MEK/ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short term effects of low dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy, and regulated the proliferation and function of hFOB1.19 cells. PMID:26472922

  2. Extracellular Monomeric Tau Protein Is Sufficient to Initiate the Spread of Tau Protein Pathology*

    PubMed Central

    Michel, Claire H.; Kumar, Satish; Pinotsi, Dorothea; Tunnacliffe, Alan; St. George-Hyslop, Peter; Mandelkow, Eckhard; Mandelkow, Eva-Maria; Kaminski, Clemens F.; Kaminski Schierle, Gabriele S.

    2014-01-01

    Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau. PMID:24235150

  3. [Effect of fibronectin on the synthesis of extracellular matrix proteins in periodontal ligament cells].

    PubMed

    Wan, L; Wu, Z; Zhou, Y

    1996-11-01

    Immunofluorescence staining method and fluorescence spectrophotometry were used to study the synthesis of extracellular matrix proteins in periodontal ligament cells (PDL cells) when exogenous fibronectin (FN) existed. The results showed that the right amount of exogenous FN (0.044 mumol/l) could increase the amount of type I collagen and type III collagen in PDL cells (P < 0.01), inhibit the synthesis of FN itself (P < 0.01). It suggested that exogenous FN can effect the synthesis of extracellular matrix proteins so as to promote a new connective tissue attachment formation. PMID:9592289

  4. Proteomics unravels extracellular vesicles as carriers of classical cytoplasmic proteins in Candida albicans.

    PubMed

    Gil-Bona, Ana; Llama-Palacios, Arancha; Parra, Claudia Marcela; Vivanco, Fernando; Nombela, César; Monteoliva, Lucía; Gil, Concha

    2015-01-01

    The commensal fungus Candida albicans secretes a considerable number of proteins and, as in different fungal pathogens, extracellular vesicles (EVs) have also been observed. Our report contains the first proteomic analysis of EVs in C. albicans and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture supernatants from C. albicans were separated into EVs and EV-free supernatant and analyzed by LC-MS/MS. A total of 96 proteins were identified including 75 and 61 proteins in EVs and EV-free supernatant, respectively. Out of these, 40 proteins were found in secretome by proteomic analysis for the first time. The soluble proteins were enriched in cell wall and secreted pathogenesis related proteins. Interestingly, more than 90% of these EV-free supernatant proteins were classical secretory proteins with predicted N-terminal signal peptide, whereas all the leaderless proteins involved in metabolism, including some moonlighting proteins, or in the exocytosis and endocytosis process were exclusively cargo of the EVs. We propose a model of the different mechanisms used by C. albicans secreted proteins to reach the extracellular medium. Furthermore, we tested the potential of the Bgl2 protein, identified in vesicles and EV-free supernatant, to protect against a systemic candidiasis in a murine model. PMID:25367658

  5. Flocculation behaviour of hematite-kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides.

    PubMed

    Poorni, S; Natarajan, K A

    2014-02-01

    Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells. PMID:24185193

  6. Methods for Studying Wnt Protein Modifications/Inactivations by Extracellular Enzymes, Tiki and Notum.

    PubMed

    Zhang, Xinjun; He, Xi

    2016-01-01

    Wnt proteins are modified and inactivated by two extracellular enzymatic antagonists, Tiki and Notum. Tiki proteins act as membrane-tethered metalloproteases to cleave a fragment from the amino terminus of Wnt proteins. Notum is a Wnt deacylase that removes the lipid modification that is essential for Wnt activities. Here, we provide detailed procedures for preparing enzymatic active Tiki and Notum proteins and the in vitro enzymatic reactions. We also describe a metabolic labeling and click chemistry method for detection of Wnt protein acylation. PMID:27590149

  7. An extracellular interactome of Immunoglobulin and LRR proteins reveals receptor-ligand networks

    PubMed Central

    Özkan, Engin; Carrillo, Robert A.; Eastman, Catharine L.; Weiszmann, Richard; Waghray, Deepa; Johnson, Karl G.; Zinn, Kai; Celniker, Susan E.; Garcia, K. Christopher

    2013-01-01

    Extracellular domains of cell-surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein “interactome” datasets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on the Immunoglobulin Superfamily (IgSF), Fibronectin type-III (FnIII) and Leucine-rich repeat (LRR) families of Drosophila, a set of 202 proteins, many of which are known to be important in neuronal and developmental functions. Out of 20503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We ‘deorphanized’ the 20-member subfamily of defective in proboscis IgSF proteins, showing that they selectively interact with an 11-member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common ‘orphan’ LRR protein. We also observed new interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs. PMID:23827685

  8. MODULATION OF EASTERN OYSTER HEMOCYTE ACTIVITIES BY PERKINSUS MARINUS EXTRACELLULAR PROTEINS

    EPA Science Inventory

    The oyster pathogen Perkinsus marinusproduces many extracellular proteins (ECP) in vitro. Analysis of this ECP revealed a battery of hydrolytic enzymes. Some of these enzymes are known to modulate the activity of host defense cells. Although information on the effects of P. marin...

  9. Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain

    PubMed Central

    Banappagari, Sashikanth; McCall, Alecia; Fontenot, Krystal; Vicente, M. Graca H.; Gujar, Amit; Satyanarayanajois, Seetharama

    2013-01-01

    Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be modulated by small molecule ligands and, hence, these protein-protein interactions play a key role in biochemical reactions related to control of cell growth. A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of EGFRs was conjugated with BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene). Conjugation of BODIPY to the peptidomimetic was investigated by different approaches. The conjugate was characterized for its ability to bind to HER2 overexpressing SKBR-3 and BT-474 cells. Furthermore, cellular uptake of conjugate of BODIPY was studied in the presence of membrane tracker and Lyso tracker using confocal microscopy. Our results suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the conjugate. Inhibition of protein-protein interaction and downstream signaling effect of compound 5-1 was also studied by proximity ligation assay and western blot analysis. Results suggested that compound 5-1 inhibits protein-protein interactions of HER2-HER3 and phosphorylation of HER2 in a time-dependent manner. PMID:23688700

  10. Prediction of lipid-binding regions in cytoplasmic and extracellular loops of membrane proteins as exemplified by protein translocation membrane proteins.

    PubMed

    Keller, Rob C A

    2013-01-01

    The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation. PMID:22961045

  11. Glycation of extracellular matrix proteins impairs migration of immune cells.

    PubMed

    Haucke, Elisa; Navarrete-Santos, Alexander; Simm, Andreas; Silber, Rolf-Edgar; Hofmann, Britt

    2014-01-01

    The immune response during aging and diabetes is disturbed and may be due to the altered migration of immune cells in an aged tissue. Our study should prove the hypothesis that age and diabetes-related advanced glycation end products (AGEs) have an impact on the migration and adhesion of human T-cells. To achieve our purpose, we used in vitro AGE-modified proteins (soluble albumin and fibronectin [FN]), as well as human collagen obtained from bypass graft. A Boyden chamber was used to study cell migration. Migrated Jurkat T-cells were analyzed by flow cytometry and cell adhesion by crystal violet staining. Actin polymerization was determined by phalloidin-Alexa-fluor 488-labeled antibody and fluorescence microscopy. We found that significantly fewer cells (50%, p = 0.003) migrated through methylglyoxal modified FN. The attachment to FN in the presence of AGE-bovine serum albumin (BSA) was also reduced (p < 0.05). In ex vivo experiments, isolated collagen from human vein graft material negatively affected the migration of the cells depending on the grade of AGE modification of the collagen. Collagen with a low AGE level reduced the cell migration by 30%, and collagen with a high AGE level by 60%. Interaction of the cells with an AGE-modified matrix, but not with soluble AGEs like BSA-AGE per se, was responsible for a disturbed migration. The reduced migration was accompanied by an impaired actin polymerization. We conclude that AGEs-modified matrix protein inhibits cell migration and adhesion of Jurkat T-cells. PMID:24635174

  12. Metal affinity enrichment increases the range and depth of proteome identification for extracellular microbial proteins

    SciTech Connect

    Wheeler, Korin; Erickson, Brian K; Mueller, Ryan; Singer, Steven; Verberkmoes, Nathan C; Hwang, Mona; Thelen, Michael P.; Hettich, Robert {Bob} L

    2012-01-01

    Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added 20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as hypothetical were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.

  13. Simvastatin and atorvastatin facilitates amyloid β-protein degradation in extracellular spaces by increasing neprilysin secretion from astrocytes through activation of MAPK/Erk1/2 pathways.

    PubMed

    Yamamoto, Naoki; Fujii, Yoko; Kasahara, Rika; Tanida, Mamoru; Ohora, Kentaro; Ono, Yoko; Suzuki, Kenji; Sobue, Kazuya

    2016-06-01

    One of the major neuropathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid β-protein (Aβ) in the brain. Aβ accumulation seems to arise from an imbalance between Aβ production and clearance. Neprilysin (NEP) and insulin-degrading enzyme (IDE) are the important Aβ-degrading enzymes in the brain, and deficits in their expression may promote Aβ deposition in patients with sporadic late-onset AD. Statins, which are used clinically for reducing cholesterol levels, can exert beneficial effects on AD. Therefore, we examined whether various statins are associated with Aβ degradation by inducing NEP and IDE expression, and then evaluating the relation between activation of intracellular signaling transduction, inhibition of cholesterol production, and morphological changes to astrocytes. Treating cultured rat astrocytes with simvastatin and atorvastatin significantly decreased the expression of NEP but not IDE in a concentration- and time-dependent manner. The decrease in NEP expression was a result of activation of extracellular signal-regulated kinase (ERK) but not the reduction of cholesterol synthesis pathway. This NEP reduction was achieved by the release to the extracellular space of cultured astrocytes. Furthermore, the cultured medium prepared from simvastatin- and atorvastatin-treated astrocytes significantly induced the degradation of exogenous Aβ. These results suggest that simvastatin and atorvastatin induce the increase of Aβ degradation of NEP on the extracellular of astrocytes by inducing ERK-mediated pathway activity and that these reagents regulate the differential mechanisms between the secretion of NEP, the induction of cholesterol reduction, and the morphological changes in the cultured astrocytes. GLIA 2016;64:952-962. PMID:26875818

  14. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation

    PubMed Central

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. DOI: http://dx.doi.org/10.7554/eLife.15550.001 PMID:27435961

  15. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

    PubMed

    Plate, Lars; Cooley, Christina B; Chen, John J; Paxman, Ryan J; Gallagher, Ciara M; Madoux, Franck; Genereux, Joseph C; Dobbs, Wesley; Garza, Dan; Spicer, Timothy P; Scampavia, Louis; Brown, Steven J; Rosen, Hugh; Powers, Evan T; Walter, Peter; Hodder, Peter; Wiseman, R Luke; Kelly, Jeffery W

    2016-01-01

    Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases. PMID:27435961

  16. N-terminal specific conjugation of extracellular matrix proteins to 2-pyridinecarboxaldehyde functionalized polyacrylamide hydrogels.

    PubMed

    Lee, Jessica P; Kassianidou, Elena; MacDonald, James I; Francis, Matthew B; Kumar, Sanjay

    2016-09-01

    Polyacrylamide hydrogels have been used extensively to study cell responses to the mechanical and biochemical properties of extracellular matrix substrates. A key step in fabricating these substrates is the conjugation of cell adhesion proteins to the polyacrylamide surfaces, which typically involves nonspecifically anchoring these proteins via side-chain functional groups. This can result in a loss of presentation control and altered bioactivity. Here, we describe a new functionalization strategy in which we anchor full-length extracellular matrix proteins to polyacrylamide substrates using 2-pyridinecarboxaldehyde, which can be co-polymerized into polyacrylamide gels and used to immobilize proteins by their N-termini. This one-step reaction proceeds under mild aqueous conditions and does not require additional reagents. We demonstrate that these substrates can readily conjugate to various extracellular matrix proteins, as well as promote cell adhesion and spreading. Notably, this chemistry supports the assembly and cellular remodeling of large collagen fibers, which is not observed using conventional side-chain amine-conjugation chemistry. PMID:27348850

  17. Isolation of High-Purity Extracellular Vesicles by Extracting Proteins Using Aqueous Two-Phase System

    PubMed Central

    Kim, Jongmin; Shin, Hyunwoo; Kim, Jiyoon; Kim, Junho; Park, Jaesung

    2015-01-01

    We present a simple and rapid method to isolate extracellular vesicles (EVs) by using a polyethylene glycol/dextran aqueous two-phase system (ATPS). This system isolated more than ~75% of melanoma-derived EVs from a mixture of EVs and serum proteins. To increase the purity of EVs, a batch procedure was combined as additional steps to remove protein contaminants, and removed more than ~95% of the protein contaminants. We also performed RT-PCR and western blotting to verify the diagnostic applicability of the isolated EVs, and detected mRNA derived from melanoma cells and CD81 in isolated EVs. PMID:26090684

  18. Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites.

    PubMed

    Lasonder, Edwin; Green, Judith L; Grainger, Munira; Langsley, Gordon; Holder, Anthony A

    2015-08-01

    Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 (http://proteomecentral.proteomexchange.org/dataset/PXD001684). The observed differential phosphorylation between extra and intraerythrocytic life-cycle stages was confirmed using both phospho-site and phospho-motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium-dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2-like elF2-kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation-induced translational arrest as intracellular schizonts transform into extracellular merozoites. PMID:25886026

  19. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    PubMed

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding. PMID:25351253

  20. Extracellular peptidase hunting for improvement of protein production in plant cells and roots

    PubMed Central

    Lallemand, Jérôme; Bouché, Frédéric; Desiron, Carole; Stautemas, Jennifer; de Lemos Esteves, Frédéric; Périlleux, Claire; Tocquin, Pierre

    2015-01-01

    Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing. PMID:25705212

  1. Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain.

    PubMed

    Ouidir, Tassadit; Jarnier, Frédérique; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2014-09-01

    Protein phosphorylation on serine, threonine, and tyrosine is known to be involved in a wide variety of cellular processes, signal transduction, and bacterial virulence. We characterized, for the first time, the extracellular phosphoproteins of the Pseudomonas aeruginosa PA14 strain. We identified 28 phosphoproteins (59 phosphosites) including enzymes, with various phosphorylation sites, known as potent secreted virulence factors in P. aeruginosa. The high phosphorylation level of these virulence factors might reflect a relationship between Ser/Thr/Tyr phosphorylation and virulence. PMID:24965220

  2. Toxicity of extracellular proteins from Diplodia seriata and Neofusicoccum parvum involved in grapevine Botryosphaeria dieback.

    PubMed

    Bénard-Gellon, M; Farine, S; Goddard, M L; Schmitt, M; Stempien, E; Pensec, F; Laloue, H; Mazet-Kieffer, F; Fontaine, F; Larignon, P; Chong, J; Tarnus, C; Bertsch, C

    2015-03-01

    Botryosphaeria dieback, esca and Eutypa dieback are three economic major grapevine trunk diseases that cause severe yield reduction in vineyards worldwide. The frequency of disease symptoms has increased considerably over the past decade, and no efficient treatment is currently available to control these diseases. The different fungi associated with grapevine trunk diseases mainly induce necrotic wood and characteristic foliar symptoms. In this context, fungi virulence factors and host invasion are not well understood. We hypothesise that extracellular proteins produced by Diplodia seriata and Neofusicoccum parvum, two causal agents associated with Botryosphaeria dieback, are virulence factors responsible for the pathogenicity. In our previous work, we demonstrated that the total extracellular compounds produced by N. parvum induced more necrosis on Chardonnay calli and triggered a different defence gene expression pattern than those produced by D. seriata. Furthermore, this aggressiveness was not clearly correlated with the production of mellein, a characteristic phytotoxin of Botryosphaeriaceae, in our in vitro calli model. To characterise other potential virulence factors and to understand the mechanisms of host invasion by the fungus, we evaluated the profile, quantity and the impact of extracellular proteins produced by these fungi on Vitis vinifera calli necrosis and defence gene expression. Our results reveal that, under the same conditions, N. parvum produces more extracellular proteins and in higher concentrations than D. seriata. With Vitis vinifera cv. Chardonnay cells, we showed that equivalent concentrations of proteins secreted by N. parvum were more aggressive than those of D. seriata in producing necrosis and that they clearly induced more grapevine defence genes. PMID:25323623

  3. Extracellular Release and Signaling by Heat Shock Protein 27: Role in Modifying Vascular Inflammation

    PubMed Central

    Batulan, Zarah; Pulakazhi Venu, Vivek Krishna; Li, Yumei; Koumbadinga, Geremy; Alvarez-Olmedo, Daiana Gisela; Shi, Chunhua; O’Brien, Edward R.

    2016-01-01

    Heat shock protein 27 (HSP27) is traditionally viewed as an intracellular chaperone protein with anti-apoptotic properties. However, recent data indicate that a number of heat shock proteins, including HSP27, are also found in the extracellular space where they may signal via membrane receptors to alter gene transcription and cellular function. Therefore, there is increasing interest in better understanding how HSP27 is released from cells, its levels and composition in the extracellular space, and the cognate cell membrane receptors involved in effecting cell signaling. In this paper, the knowledge to date, as well as some emerging paradigms about the extracellular function of HSP27 is presented. Of particular interest is the role of HSP27 in attenuating atherogenesis by modifying lipid uptake and inflammation in the plaque. Moreover, the abundance of HSP27 in serum is an emerging new biomarker for ischemic events. Finally, HSP27 replacement therapy may represent a novel therapeutic opportunity for chronic inflammatory disorders, such as atherosclerosis. PMID:27507972

  4. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated.

    PubMed

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J; Vander Wall, Todd A; Groom, Joseph; Himmel, Michael E; Westpheling, Janet

    2015-01-01

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure

  5. Homologous Expression of the Caldicellulosiruptor bescii CelA Reveals that the Extracellular Protein Is Glycosylated

    PubMed Central

    Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-01-01

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. It will also allow the study of glycosylation of CelA itself and its role in the structure

  6. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated

    DOE PAGESBeta

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-03-23

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm formore » cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. Finally, it will also allow the study of glycosylation of CelA itself and its role

  7. Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

    PubMed Central

    Zhang, Weipeng; Sun, Jin; Ding, Wei; Lin, Jinshui; Tian, Renmao; Lu, Liang; Liu, Xiaofen; Shen, Xihui; Qian, Pei-Yuan

    2015-01-01

    Though the essential role of extracellular matrix in biofilm development has been extensively documented, the function of matrix-associated proteins is elusive. Determining the dynamics of matrix-associated proteins would be a useful way to reveal their functions in biofilm development. Therefore, we applied iTRAQ-based quantitative proteomics to evaluate matrix-associated proteins isolated from different phases of Pseudomonas aeruginosa ATCC27853 biofilms. Among the identified 389 proteins, 54 changed their abundance significantly. The increased abundance of stress resistance and nutrient metabolism-related proteins over the period of biofilm development was consistent with the hypothesis that biofilm matrix forms micro-environments in which cells are optimally organized to resist stress and use available nutrients. Secreted proteins, including novel putative effectors of the type III secretion system were identified, suggesting that the dynamics of pathogenesis-related proteins in the matrix are associated with biofilm development. Interestingly, there was a good correlation between the abundance changes of matrix-associated proteins and their expression. Further analysis revealed complex interactions among these modulated proteins, and the mutation of selected proteins attenuated biofilm development. Collectively, this work presents the first dynamic picture of matrix-associated proteins during biofilm development, and provides evidences that the matrix-associated proteins may form an integral and well regulated system that contributes to stress resistance, nutrient acquisition, pathogenesis and the stability of the biofilm. PMID:26029669

  8. The dynamic architecture of photoreceptor ribbon synapses: Cytoskeletal, extracellular matrix, and intramembrane proteins

    PubMed Central

    MERCER, AARON J.; THORESON, WALLACE B.

    2012-01-01

    Rod and cone photoreceptors possess ribbon synapses that assist in the transmission of graded light responses to second-order bipolar and horizontal cells of the vertebrate retina. Proper functioning of the synapse requires the juxtaposition of presynaptic release sites immediately adjacent to postsynaptic receptors. In this review, we focus on the synaptic, cytoskeletal, and extracellular matrix proteins that help to organize photoreceptor ribbon synapses in the outer plexiform layer. We examine the proteins that foster the clustering of release proteins, calcium channels, and synaptic vesicles in the presynaptic terminals of photoreceptors adjacent to their postsynaptic contacts. Although many proteins interact with one another in the presynaptic terminal and synaptic cleft, these protein–protein interactions do not create a static and immutable structure. Instead, photoreceptor ribbon synapses are remarkably dynamic, exhibiting structural changes on both rapid and slow time scales. PMID:22192503

  9. Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

    PubMed Central

    Son, Myunghan; Moon, Yuseok; Oh, Mi Jin; Han, Sang Bin; Park, Ki Hyun; Kim, Jung-Gon

    2012-01-01

    Pseudomonas fluorescens, a widespread Gram-negative bacterium, is an ideal protein manufacturing factory (PMF) because of its safety, robust growth, and high protein production. P. fluorescens possesses a type I secretion system (T1SS), which mediates secretion of a thermostable lipase (TliA) and a protease (PrtA) through its ATP-binding cassette (ABC) transporter. Recombinant proteins in P. fluorescens are attached to the C-terminal signal region of TliA for transport as fusion proteins to the extracellular medium. However, intrinsic TliA from the P. fluorescens genome interferes with detection of the recombinant protein and the secreted recombinant protein is hydrolyzed, due to intrinsic PrtA, resulting in decreased efficiency of the PMF. In this research, the lipase and protease genes of P. fluorescens SIK W1 were deleted using the targeted gene knockout method. Deletion mutant P. fluorescens ΔtliA ΔprtA secreted fusion proteins without TliA or protein degradation. Using wild-type P. fluorescens as an expression host, degradation of the recombinant protein varied depending on the type of culture media and aeration; however, degradation did not occur with the P. fluorescens ΔtliA ΔprtA double mutant irrespective of growth conditions. By homologous expression of tliA and the ABC transporter in a plasmid, TliA secreted from P. fluorescens ΔprtA and P. fluorescens ΔtliA ΔprtA cells was found to be intact, whereas that secreted from the wild-type P. fluorescens and P. fluorescens ΔtliA cells was found to be hydrolyzed. Our results demonstrate that the P. fluorescens ΔtliA ΔprtA deletion mutant is a promising T1SS-mediated PMF that enhances production and detection of recombinant proteins in extracellular media. PMID:23042178

  10. Staphylococcus aureus extracellular adherence protein contributes to biofilm formation in the presence of serum

    PubMed Central

    Thompson, Karl M.; Abraham, Nabil; Jefferson, Kimberly K.

    2010-01-01

    Staphylococcus aureus extracellular adherence protein (EAP) is secreted, but it can redock on the bacterial cell surface via neutral phosphatase (Nptase). EAP binds to certain blood proteins and to itself, and through these affinities, it contributes to adherence and aggregation. It has been demonstrated previously that EAP expression is iron regulated and it contributes to biofilm formation under iron-deplete conditions. In this study, we found that EAP and Nptase also play a role in biofilm formation under iron-replete conditions in the presence of human serum. PMID:20199571

  11. Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

    SciTech Connect

    Kim, Se-Hee; Schmitt, Christopher E.; Woolls, Melissa J.; Holland, Melinda B.; Kim, Jun-Dae; Jin, Suk-Won

    2013-01-25

    Highlights: ► VEGF-A signaling regulates the segregation of axial vessels. ► VEGF-A signaling is mediated by PKC and ERK in this process. ► Ectopic activation of ERK is sufficient to rescue defects in vessel segregation. -- Abstract: Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A) signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3 K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process.

  12. Novel Phakopsora pachyrhizi Extracellular Proteins Are Ideal Targets for Immunological Diagnostic Assays

    PubMed Central

    McMahon, Michael B.; Edwards, H. Herb; Boerma, Britney L.; Lewis Ivey, Melanie L.; Miller, Sally A.; Dorrance, Anne E.

    2012-01-01

    Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to spread across the southeast and midsouth regions of the United States, necessitating the use of fungicides by producers. Our objective in this research was to identify ASR proteins expressed early during infection for the development of immunodiagnostic assays. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for Phakopsora extracellular protein). Two highly expressed protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets for antibody development, after we detected PHEPs in plants as early as 3 days postinfection (dpi). Monoclonal antibodies (MAbs; 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants and for specificity against a set of common soybean pathogens. These antibodies should prove applicable in immunodiagnostic assays to detect infected soybeans and to identify ASR spores from sentinel surveillance plots. PMID:22447596

  13. Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction.

    PubMed

    Singhal, Neha; Martin, Paul T

    2011-11-01

    The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission. PMID:21766463

  14. Cell density modulates growth, extracellular matrix, and protein synthesis of cultured rat mesangial cells.

    PubMed

    Wolthuis, A; Boes, A; Grond, J

    1993-10-01

    Mesangial cell (MC) hyperplasia and accumulation of extracellular matrix are hallmarks of chronic glomerular disease. The present in vitro study examined the effects of cell density on growth, extracellular matrix formation, and protein synthesis of cultured rat MCs. A negative linear relationship was found between initial plating density and DNA synthesis per cell after 24 hours incubation in medium with 10% fetal calf serum (range: 1 x 10(3) to 7 x 10(5) MCs/2cm2, r = 0.996, P < 0.001). Enzyme-linked immunosorbent assay of the amount of fibronectin in the conditioned medium after 72 hours showed a negative relationship with increasing cell density. In contrast, the amount of cell-associated fibronectin increased to maximal values in confluent cultures, and no further increase was seen at supraconfluency. The relative collagen synthesis in the conditioned medium and cell layer--assessed by collagenase digestion after 5 hours [3H]proline pulse labeling--showed a similar pattern. Secreted collagen decreased with increasing cell density from 3.4% to 0.2% of total protein synthesis. In contrast, cell-associated collagen increased from 1.1% to 11.8% of newly synthesized protein until confluency followed by a decrease to 4.2% at supraconfluency. Specific immunoprecipitation of collagen types I, III, and IV revealed a significant (twofold) increase in collagen I synthesis per cell at confluency. Collagen III and IV synthesis was not affected by cell density. Specific protein expression in both the medium and cell layer were analyzed by two-dimensional polyacrylamide gel electrophoresis (150 to 20 kd, pI 5.0 to 7.0) after 20 hours steady-state metabolic labeling with [35S]methionine. Supraconfluent MCs displayed overexpression of 10, underexpression of four, new expression of five, and changed mobility of three different intracellular proteins. Of interest was the overexpression of two proteins (89 kd, pI 5.31 and 72 kd, pI 5.32) that were identified by immunoblotting as

  15. A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix*

    PubMed Central

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-01-01

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBankTM data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate. PMID:20150428

  16. A protein involved in the assembly of an extracellular calcium storage matrix.

    PubMed

    Glazer, Lilah; Shechter, Assaf; Tom, Moshe; Yudkovski, Yana; Weil, Simy; Aflalo, Eliahu David; Pamuru, Ramachandra Reddy; Khalaila, Isam; Bentov, Shmuel; Berman, Amir; Sagi, Amir

    2010-04-23

    Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate. PMID:20150428

  17. Proteomics of rice and Cochliobolus miyabeanus fungal interaction: insight into proteins at intracellular and extracellular spaces.

    PubMed

    Kim, Jin Yeong; Wu, Jingni; Kwon, Soon Jae; Oh, Haram; Lee, So Eui; Kim, Sang Gon; Wang, Yiming; Agrawal, Ganesh Kumar; Rakwal, Randeep; Kang, Kyu Young; Ahn, Il-Pyung; Kim, Beom-Gi; Kim, Sun Tae

    2014-10-01

    Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice-C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG-fractionation of total proteins coupled with MS (MALDI-TOF/TOF and nESI-LC-MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS-PAGE in combination with nESI-LC-MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice-C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects. PMID:25047395

  18. Anthrax lethal toxin impairs CD1d-mediated antigen presentation by targeting the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway.

    PubMed

    Khan, Masood A; Gallo, Richard M; Brutkiewicz, Randy R

    2010-05-01

    Lethal toxin (LT) is a critical virulence factor of Bacillus anthracis and an important means by which this bacterium evades the host's immune system. In this study, we demonstrate that CD1d-expressing cells treated with LT have reduced CD1d-mediated antigen presentation. We earlier showed an important role for the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2) in the regulation of CD1d-mediated antigen presentation, and we report here that LT impairs antigen presentation by CD1d in an ERK1/2-dependent manner. Similarly, LT and the ERK1/2 pathway-specific inhibitor U0126 caused a decrease in major histocompatibility complex (MHC) class II-mediated antigen presentation. Confocal microscopy analyses revealed altered intracellular distribution of CD1d and LAMP-1 in LT-treated cells, similar to the case for ERK1/2-inhibited cells. These results suggest that Bacillus anthracis has the ability to evade the host's innate immune system by reducing CD1d-mediated antigen presentation through targeting the ERK1/2 pathway. PMID:20194602

  19. Allosteric Modulation of G Protein Coupled Receptors by Cytoplasmic, Transmembrane and Extracellular Ligands

    PubMed Central

    Yanamala, Naveena; Klein-Seetharaman, Judith

    2010-01-01

    G protein coupled receptors (GPCRs) bind diverse classes of ligands, and depending on the receptor, these may bind in their transmembrane or the extracellular domains, demonstrating the principal ability of GPCRs to bind ligand in either domains. Most recently, it was also observed that small molecule ligands can bind in the cytoplasmic domain, and modulate binding and response to extracellular or transmembrane ligands. Thus, all three domains in GPCRs are potential sites for allosteric ligands, and whether a ligand is allosteric or orthosteric depends on the receptor. Here, we will review the evidence supporting the presence of putative binding pockets in all three domains of GPCRs and discuss possible pathways of communication between these pockets. PMID:24009470

  20. Single proteins that serve linked functions in intracellular and extracellular microenvironments

    SciTech Connect

    Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei; Bissell, Mina J.

    2009-06-03

    Maintenance of organ homeostasis and control of appropriate response to environmental alterations requires intimate coordination of cellular function and tissue organization. An important component of this coordination may be provided by proteins that can serve distinct, but linked, functions on both sides of the plasma membrane. Here we present a novel hypothesis in which non-classical secretion can provide a mechanism through which single proteins can integrate complex tissue functions. Single genes can exert a complex, dynamic influence through a number of different processes that act to multiply the function of the gene product(s). Alternative splicing can create many different transcripts that encode proteins of diverse, even antagonistic, function from a single gene. Posttranslational modifications can alter the stability, activity, localization, and even basic function of proteins. A protein can exist in different subcellular localizations. More recently, it has become clear that single proteins can function both inside and outside the cell. These proteins often lack defined secretory signal sequences, and transit the plasma membrane by mechanisms separate from the classical ER/Golgi secretory process. When examples of such proteins are examined individually, the multifunctionality and lack of a signal sequence are puzzling - why should a protein with a well known function in one context function in such a distinct fashion in another? We propose that one reason for a single protein to perform intracellular and extracellular roles is to coordinate organization and maintenance of a global tissue function. Here, we describe in detail three specific examples of proteins that act in this fashion, outlining their specific functions in the extracellular space and in the intracellular space, and we discuss how these functions may be linked. We present epimorphin/syntaxin-2, which may coordinate morphogenesis of secretory organs (as epimorphin) with control of

  1. Comparative Proteomic Analysis of Extracellular Proteins of Clostridium perfringens Type A and Type C Strains▿ †

    PubMed Central

    Sengupta, Nabonita; Alam, Syed Imteyaz; Kumar, Bhoj; Kumar, Ravi Bhushan; Gautam, Vandana; Kumar, Subodh; Singh, Lokendra

    2010-01-01

    Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Forty-eight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-γ) and interleukin 2 (IL-2), suggesting a Th1 type immune response. PMID:20605988

  2. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    PubMed Central

    Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

  3. Machine learning approaches for discrimination of Extracellular Matrix proteins using hybrid feature space.

    PubMed

    Ali, Farman; Hayat, Maqsood

    2016-08-21

    Extracellular Matrix (ECM) proteins are the vital type of proteins that are secreted by resident cells. ECM proteins perform several significant functions including adhesion, differentiation, cell migration and proliferation. In addition, ECM proteins regulate angiogenesis process, embryonic development, tumor growth and gene expression. Due to tremendous biological significance of the ECM proteins and rapidly increases of protein sequences in databases, it is indispensable to introduce a new high throughput computation model that can accurately identify ECM proteins. Various traditional models have been developed, but they are laborious and tedious. In this work, an effective and high throughput computational classification model is proposed for discrimination of ECM proteins. In this model, protein sequences are formulated using amino acid composition, pseudo amino acid composition (PseAAC) and di-peptide composition (DPC) techniques. Further, various combination of feature extraction techniques are fused to form hybrid feature spaces. Several classifiers were employed. Among these classifiers, K-Nearest Neighbor obtained outstanding performance in combination with the hybrid feature space of PseAAC and DPC. The obtained accuracy of our proposed model is 96.76%, which the highest success rate has been reported in the literature so far. PMID:27179459

  4. Odontoblast β-catenin signaling regulates fenestration of mouse Hertwig's epithelial root sheath.

    PubMed

    Zhang, Ran; Teng, Yan; Zhu, Liang; Lin, JingTing; Yang, Xiao; Yang, Guan; Li, TieJun

    2015-09-01

    The interaction between Hertwig's epithelial root sheath (HERS) and the adjacent mesenchyme is vitally important in mouse tooth root development. We previously generated odontoblast-specific Ctnnb1 (encoding β-catenin) deletion mice, and demonstrated that odontoblast β-catenin signaling regulates odontoblast proliferation and differentiation. However, the role of odontoblast β-catenin signaling in regulation of HERS behavior has not been fully investigated. Here, using the same odontoblast- specific Ctnnb1 deletion mice, we found that ablation of β-catenin signaling in odontoblasts led to aberrant HERS formation. Mechanistically, odontoblast-specific Ctnnb1 deletion resulted in elevated bone morphogenetic protein 7 (Bmp7) expression and reduced expression of noggin and follistatin, both of which encode extracellular inhibitors of BMPs. Furthermore, the levels of phosphorylated Smad1/5/8 were increased in HERS cells. In vitro tissue culture confirmed that BMP7 treatment disrupted the HERS structure. Taken together, we demonstrated that odontoblast β-catenin signaling may act through regulation of BMP signaling to maintain the integrity of HERS cells. PMID:26208822

  5. Potential Autoepitope within the Extracellular Region of Contactin-Associated Protein-like 2 in Mice

    PubMed Central

    Obregon, Demian F.; Zhu, Yuyan; Bailey, Antoinette R.; Portis, Samantha M.; Hou, Huayan; Zeng, Jin; Stock, Saundra L.; Murphy, Tanya K.; Bengtson, Michael A.; Tan, Jun

    2013-01-01

    Aims Implicated in autoimmune encephalitis, neuromyotonia and genetic forms of autism, here we report that contactin-associated protein-like 2 (CNTNAP2) contains a potential autoepitope within the extracellular region. Methodology CNTNAP2 sequence-similar regions (CSSRs) from human pathogens were identified. Sera from autistic and control children were obtained and analyzed for the presence of antibodies able to bind CSSRs. One such candidate CSSR was evaluated for evidence of autoimmune responses to CNTNAP2 in a mouse model of acute infection. Results Autistic and control children sera contained antibodies able to discrete regions of CNTNAP2. In a murine model of acute infection, a CSSR derived from the N-terminal extracellular region of CNTNAP2 resulted in anti-CNTNAP2 antibody production, proinflammatory cytokine elevation, cerebellar and cortical white matter T-cell infiltration as well as motor dysfunction. Conclusion Taken together, these data suggest that CNTNAP2 contains a potential autoepitope within the extracellular region. PMID:24466509

  6. Extracellular matrix interacts with interferon {alpha} protein: Retention and display of cytotoxicity

    SciTech Connect

    Yoshida, Kimiko; Kondoh, Atsushi; Narumi, Kenta; Yoshida, Teruhiko; Aoki, Kazunori

    2008-11-14

    We have been investigating the efficacy of an intratumoral interferon (IFN)-{alpha} gene transfer against solid cancers, and found that when the gene is transduced into the subcutaneous tumors, IFN-{alpha} concentration is markedly increased in the injected tumor but not in the serum. To explain this effective confinement of IFN-{alpha} to target tissues, we hypothesized that the extracellular matrix in the tumors interacts with IFN-{alpha}. In this study, a solid-phase-binding assay and immunoprecipitation demonstrated that the IFN-{alpha} binds directly to matrix proteins. Immunohistochemical staining showed a co-localization of IFN-{alpha} with pericellular fibronectin. In addition, matrix-bound IFN-{alpha} protein transduced intracellular signaling and potentiated its cytotoxic activity, suggesting that the retention of IFN-{alpha} protein on extracellular matrix is likely to play a role in its in vivo biological activity. The data suggest a therapeutic advantage of the intratumoral IFN-{alpha} gene transfer over the conventional parenteral therapy both in the safety and efficacy.

  7. Extracellular trafficking of a wheat cold-responsive protein, WLT10.

    PubMed

    Ohno, Ryoko; Takumi, Shigeo

    2015-02-01

    A cold-responsive wheat gene, WLT10, encodes a member of the cereal-specific low temperature-responsive/cold-responsive protein family, which contains a hydrophobic N-terminal 20 amino acid sequence that corresponds to signal peptides associated with extracellular trafficking. To verify the subcellular localization of WLT10 and the function of its putative signal peptide, we constructed three chimeric genes in which either the WLT10 signal peptide, a signal peptide with only 6 additional amino acids, or the full-length WLT10 polypeptide was fused to the N-terminus of green fluorescent protein (GFP). These fusion constructs were transiently introduced into onion epidermal cells by particle bombardment. GFP signals were observed not only in the extracellular space (ECS) but also in the endoplasmic reticulum (ER) and Golgi apparatus. The time course of GFP signal localization suggests the movement of WLT10 through the ER/Golgi pathway and into the ECS. Thus, WLT10 is a cold-responsive secreted protein, and its N-terminal 20 amino acid region is important for transport to the ECS. PMID:25462969

  8. An extracellular lipid transfer protein is relocalized intracellularly during seed germination.

    PubMed

    Pagnussat, Luciana; Burbach, Christian; Baluska, Frantisek; de la Canal, Laura

    2012-11-01

    Plant lipid transfer proteins (LTPs) constitute a family of small proteins recognized as being extracellular. In agreement with this notion, several lines of evidence have shown the apoplastic localization of HaAP10, a LTP from Helianthus annuus dry seeds. However, HaAP10 was recently detected intracellularly in imbibing seeds. To clarify its distribution, immunolocalization experiments were performed during the course of germination and confirmed its intracellular localization upon early seed imbibition. Further assays using a hydrophobic dye, FM4-64, inhibitors of vesicular traffic, and immunolocalization of the pectin rhamnogalacturonan-II, allowed the conclusion that endocytosis is activated as soon as seed imbibition starts. Furthermore, this study demonstrated that HaAP10 is endocytosed throughout imbibition. Biochemical and cellular approaches indicate that the intracellular fraction of this LTP appears associated with oil bodies and some evidence also suggest its presence in glyoxysomes. So, HaAP10 is apoplastic in dry seeds and upon imbibition is rapidly internalized and relocalized to organelles involved in lipid metabolism. The results suggest that HaAP10 may be acting as a fatty acid shuttle between the oil body and the glyoxysome during seed germination. This concept is consistent with the initial proposition that LTPs participate in the intracellular transfer of lipids which was further denied based on their apparent extracellular localization. This report reveals for the first time the relocalization of a lipid transfer protein and opens new perspectives on its role. PMID:23162115

  9. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

    PubMed Central

    Li, Jinghuan; Lee, Yi; Johansson, Henrik J.; Mäger, Imre; Vader, Pieter; Nordin, Joel Z.; Wiklander, Oscar P. B.; Lehtiö, Janne; Wood, Matthew J. A.; Andaloussi, Samir EL

    2015-01-01

    Extracellular vesicles (EVs) play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs. PMID:26022510

  10. Response of Inflammatory Mediators, Extracellular Matrix Proteins and Stem and Progenitor Cells to Emphysema.

    PubMed

    Skurikhin, E G; Pakhomova, A V; Krupin, V A; Pershina, O V; Pan, E S; Ermolaeva, L A; Vaizova, O E; Rybalkina, O Yu; Dygai, A M

    2016-08-01

    Inflammation, extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, and fibronectin), stem and progenitor cells (multipotent mesenchymal stromal cells, Clara cells, angiogenesis, precursors, endothelial and epithelial cells) were studied in female C57Bl/6 mice with experimental elastase-induced emphysema. Diffuse emphysema reduced the number of endothelial (CD45(-)CD31(+)CD34(+)) and epithelial (CD45(-)CD117(+)CD49f(+)) cells, induced microcirculation disturbances, and decreased the area occupied by the connective tissue. Emphysematous changes in the lungs were accompanied by infiltration of the alveolar septa with macrophages and lymphocytes, increase in the serum and lung concentrations of transforming growth factor-β, IL-1β, IL-2, IL-5, IL-10, and IL-13, and lung concentration of IL-17. In the lungs, inflammation was associated with marked increase in the number of multipotent mesenchymal stromal cells CD90(+)CD73(+)CD106(+)CD44(+)) and Clara cells (CD45(-)CD34(-)CD31(-)Sca1(+)) and overexpression of extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, fibronectin) and Clara cells protein. On the other hand, elastase reduced the number of angiogenic precursor cells (CD45(-)CD117(+)Flk1(+)). PMID:27591877

  11. Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM

    PubMed Central

    Johnson, Brant; Selle, Kurt; O’Flaherty, Sarah; Goh, Yong Jun

    2013-01-01

    Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers. PMID:24002751

  12. Identification of extracellular surface-layer associated proteins in Lactobacillus acidophilus NCFM.

    PubMed

    Johnson, Brant; Selle, Kurt; O'Flaherty, Sarah; Goh, Yong Jun; Klaenhammer, Todd

    2013-11-01

    Bacterial surface (S-) layers are crystalline arrays of self-assembling, proteinaceous subunits called S-layer proteins (Slps), with molecular masses ranging from 40 to 200 kDa. The S-layer-forming bacterium Lactobacillus acidophilus NCFM expresses three major Slps: SlpA (46 kDa), SlpB (47 kDa) and SlpX (51 kDa). SlpA has a demonstrated role in adhesion to Caco-2 intestinal epithelial cells in vitro, and has been shown to modulate dendritic cell (DC) and T-cell functionalities with murine DCs. In this study, a modification of a standard lithium chloride S-layer extraction revealed 37 proteins were solubilized from the S-layer wash fraction. Of these, 30 have predicted cleavage sites for secretion, 24 are predicted to be extracellular, six are lipid-anchored, three have N-terminal hydrophobic membrane spanning regions and four are intracellular, potentially moonlighting proteins. Some of these proteins, designated S-layer associated proteins (SLAPs), may be loosely associated with or embedded within the bacterial S-layer complex. Lba-1029, a putative SLAP gene, was deleted from the chromosome of L. acidophilus. Phenotypic characterization of the deletion mutant demonstrated that the SLAP LBA1029 contributes to a pro-inflammatory TNF-α response from murine DCs. This study identified extracellular proteins and putative SLAPs of L. acidophilus NCFM using LC-MS/MS. SLAPs appear to impart important surface display features and immunological properties to microbes that are coated by S-layers. PMID:24002751

  13. DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.

    PubMed

    Fontaine, Sarah N; Zheng, Dali; Sabbagh, Jonathan J; Martin, Mackenzie D; Chaput, Dale; Darling, April; Trotter, Justin H; Stothert, Andrew R; Nordhues, Bryce A; Lussier, April; Baker, Jeremy; Shelton, Lindsey; Kahn, Mahnoor; Blair, Laura J; Stevens, Stanley M; Dickey, Chad A

    2016-07-15

    It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease-associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, α-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 co-chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins. PMID:27261198

  14. Fibroblastic synoviocytes secrete plasma proteins via α2 -macroglobulins serving as intracellular and extracellular chaperones.

    PubMed

    Zhao, Ke-Wei; Murray, Elsa J Brochmann; Murray, Samuel S

    2015-11-01

    Changes in plasma protein levels in synovial fluid (SF) have been implicated in osteoarthritis and rheumatoid arthritis. It was previously thought that the presence of plasma proteins in SF reflected ultrafiltration or extravasation from the vasculature, possibly due to retraction of inflamed endothelial cells. Recent proteomic analyses have confirmed the abundant presence of plasma proteins in SF from control and arthritic patients. Systematic depletion of high-abundance plasma proteins from SF and conditioned media from synoviocytes cultured in serum, and protein analysis under denaturing/reducing conditions have limited our understanding of sources and the native structures of "plasma protein" complexes in SF. Using Western blotting, qPCR, and mass spectrometry, we found that Hig-82 lapine fibroblastic synovicytes cultured under serum-free conditions expressed and secreted plasma proteins, including the cytokine-binding protein secreted phosphoprotein 24 kDa (Spp24) and many of the proteases and protease inhibitors found in SF. Treating synoviocytes with TGF-β1 or BMP-2 for 24 h upregulated the expression of plasma proteins, including Spp24, α2 -HS-glycoprotein, α1 -antitrypsin, IGF-1, and C-reactive protein. Furthermore, many of the plasma proteins of mass <151 kDa were secreted as disulfide-bound complexes with members of the α2 -macroglobulin (A2M) family, which serve as intracellular and extracellular chaperones, not protease inhibitors. Using brefeldin A to block vesicular traffic and protease inhibitors to inhibit endogenous activation of naïve A2M, we demonstrated that the complexes were formed in the endoplasmic reticulum lumen and that Ca(2+) cysteine protease-dependent processes are involved. PMID:25900303

  15. A Relay Network of Extracellular Heme-Binding Proteins Drives C. albicans Iron Acquisition from Hemoglobin

    PubMed Central

    Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J.; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

    2014-01-01

    Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7−/− mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope. PMID:25275454

  16. Regulation of Extracellular Matrix Remodeling Proteins by Osteoblasts in Titanium Nanoparticle-Induced Aseptic Loosening Model.

    PubMed

    Xie, Jing; Hou, Yanhua; Fu, Na; Cai, Xiaoxiao; Li, Guo; Peng, Qiang; Lin, Yunfeng

    2015-10-01

    Titanium (Ti)-wear particles, formed at the bone-implant interface, are responsible for aseptic loosening, which is a main cause of total joint replacement failure. There have been many studies on Ti particle-induced function changes in mono-cultured osteoblasts and synovial cells. However, little is known on extracellular matrix remodeling displayed by osteoblasts when in coexistence with Synovial cells. To further mimic the bone-implant interface environment, we firstly established a nanoscaled-Ti particle-induced aseptic loosening system by co-culturing osteoblasts and Synovial cells. We then explored the impact of the Synovial cells on Ti particle-engulfed osteoblasts in the mimicked flamed niche. The matrix metalloproteinases and lysyl oxidases expression levels, two protein families which are critical in osseointegration, were examined under induction by tumor necrosis factor-alpha. It was found that the co-culture between the osteoblasts and Synovial cells markedly increased the migration and proliferation of the osteoblasts, even in the Ti-particle engulfed osteoblasts. Importantly, the Ti-particle engulfed osteoblasts, induced by TNF-alpha after the co-culture, enhanced the release of the matrix metalloproteinases and reduced the expressions of lysyl oxidases. The regulation of extracellular matrix remodeling at the protein level was further assessed by investigations on gene expression of the matrix metalloproteinases and lysyl oxidases, which also suggested that the regulation started at the genetic level. Our research work has therefore revealed the critical role of multi cell-type interactions in the extracellular matrix remodeling within the peri-prosthetic tissues, which provides new insights on aseptic loosening and brings new clues about incomplete osseointegration between the implantation materials and their surrounding bones. PMID:26502645

  17. Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis.

    PubMed

    Naba, Alexandra; Clauser, Karl R; Hynes, Richard O

    2015-01-01

    The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry. PMID:26273955

  18. Extracellular Toxoplasma gondii tachyzoites metabolize and incorporate unnatural sugars into cellular proteins.

    PubMed

    Nazarova, Lidia A; Ochoa, Roxanna J; Jones, Krysten A; Morrissette, Naomi S; Prescher, Jennifer A

    2016-03-01

    Toxoplasma gondii is an obligate intracellular parasite that infects all nucleated cell types in diverse warm-blooded organisms. Many of the surface antigens and effector molecules secreted by the parasite during invasion and intracellular growth are modified by glycans. Glycosylated proteins in the nucleus and cytoplasm have also been reported. Despite their prevalence, the complete inventory and biological significance of glycosylated proteins in Toxoplasma remain unknown. In this study, we aimed to globally profile parasite glycoproteins using a bioorthogonal chemical reporter strategy. This strategy involves the metabolic incorporation of unnatural functional groups (i.e., "chemical reporters") into Toxoplasma glycans, followed by covalent labeling with visual probes or affinity tags. The two-step approach enables the visualization and identification of newly biosynthesized glycoconjugates in the parasite. Using a buffer that mimics intracellular conditions, extracellular Toxoplasma tachyzoites were found to metabolize and incorporate unnatural sugars (equipped with bioorthogonal functional groups) into diverse proteins. Covalent chemistries were used to visualize and retrieve these labeled structures. Subsequent mass spectrometry analysis revealed 89 unique proteins. This survey identified novel proteins as well as previously characterized proteins from lectin affinity analyses. PMID:26687036

  19. Biglycan is an extracellular MuSK binding protein important for synapse stability

    PubMed Central

    Amenta, A.R.; Creely, H.E.; Mercado, M.L.; Hagiwara, H.; McKechnie, B. A.; Lechner, B.E.; Rossi, S. G.; Wang, Q.; Owens, R. T.; Marrero, E.; Mei, L.; Hoch, W.; Young, M. F.; McQuillan, D. J.; Rotundo, R. L.; Fallon, J.R.

    2012-01-01

    The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for pre-patterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn-/o) muscle is indistinguishable from wild type controls. However, by 5 wks after birth nerve-muscle synapses in bgn-/o mice are abnormal as judged by the presence of perijunctional folds, increased segmentation and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied pre- and post- synaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn-/o synapses. In bgn-/o myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability. PMID:22396407

  20. Structure Prediction of the Second Extracellular Loop in G-Protein-Coupled Receptors

    PubMed Central

    Kmiecik, Sebastian; Jamroz, Michal; Kolinski, Michal

    2014-01-01

    G-protein-coupled receptors (GPCRs) play key roles in living organisms. Therefore, it is important to determine their functional structures. The second extracellular loop (ECL2) is a functionally important region of GPCRs, which poses significant challenge for computational structure prediction methods. In this work, we evaluated CABS, a well-established protein modeling tool for predicting ECL2 structure in 13 GPCRs. The ECL2s (with between 13 and 34 residues) are predicted in an environment of other extracellular loops being fully flexible and the transmembrane domain fixed in its x-ray conformation. The modeling procedure used theoretical predictions of ECL2 secondary structure and experimental constraints on disulfide bridges. Our approach yielded ensembles of low-energy conformers and the most populated conformers that contained models close to the available x-ray structures. The level of similarity between the predicted models and x-ray structures is comparable to that of other state-of-the-art computational methods. Our results extend other studies by including newly crystallized GPCRs. PMID:24896119

  1. The widely expressed extracellular matrix protein SMOC-2 promotes keratinocyte attachment and migration

    SciTech Connect

    Maier, Silke; Paulsson, Mats; Hartmann, Ursula

    2008-08-01

    SMOC-2 is a recently discovered member of the BM-40/SPARC/osteonectin family of extracellular multidomain proteins of so far unknown function. While we have shown earlier that the homologous protein SMOC-1 is associated with basement membranes, in this study we demonstrate that, in the mouse, SMOC-2 could be detected in a large number of non-basement membrane localizations, often showing a diffuse tissue distribution. A more distinct expression pattern was seen in skin where SMOC-2 is mainly present in the basal layers of the epidermis. Functionally, recombinant SMOC-2 stimulated attachment of primary epidermal cells as well as several epidermal-derived cell lines but had no effect on the attachment of non-epidermal cells. Inhibition experiments using blocking antibodies against individual integrin subunits allowed the identification of {alpha}v{beta}6 and {alpha}v{beta}1 integrins as important cellular receptors for SMOC-2. Cell attachment as well as the formation of focal adhesions could be attributed to the extracellular calcium-binding domain. The calcium-binding domain also stimulated migration, but not proliferation of keratinocyte-like HaCaT cells. We conclude that SMOC-2, like other members of the BM40/SPARC family, acts as a regulator of cell-matrix interactions.

  2. The extracellular matrix protein Del1 induces apoptosis via its epidermal growth factor motif.

    PubMed

    Kitano, Hisataka; Kokubun, Shinichiro; Hidai, Chiaki

    2010-03-19

    Mouse Del1 is an extracellular matrix protein mainly expressed in the developing embryo. Del1 has three EGF motifs and two discoidin domains. The second EGF motif reportedly contains an RGD sequence that binds to integrin receptors. Here, we provide evidence that Del1 protein induces cell death in vitro. Chromatin condensation and DNA laddering were observed, suggestive of apoptosis. The results of analysis using the TUNEL method and annexin V staining were also consistent with apoptosis. The apoptosis-inducing activity of Del1 could be mapped to the third EGF motif, which fitted the consensus sequence CX(D/N)XXXX(F/Y)XCXC, wherein the aspartic acid residue (D) could be beta-hydroxylated. As little as twenty-five picomolar of recombinant E3 could induce apoptosis. PMID:20171188

  3. Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

    PubMed Central

    Yamashita, Takahiro; Morioka, Riki; Ohmori, Hideyuki

    2014-01-01

    Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (Ka) values of 5.2 × 106 to 44 × 106 M−1 among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures. PMID:25157080

  4. The Role of the Extracellular Matrix Protein Mindin in Airway Response to Environmental Airways Injury

    PubMed Central

    Frush, Sarah; Li, Zhuowei; Potts, Erin N.; Du, Wanglei; Eu, Jerry P.; Garantziotis, Stavros; He, You-Wen; Foster, W. Michael

    2011-01-01

    Background: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored. Objectives: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone. Methods: We exposed C57BL/6J and mindin-deficient (–/–) mice to aerosolized LPS (0.9 μg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline 
exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies. Results: After inhalation of LPS, mindin–/– mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin–/– mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin–/– mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin–/– mice demonstrated reduced constriction in response to carbachol. Conclusions: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of

  5. Insertion of Proteolipid Protein into Oligodendrocyte Mitochondria Regulates Extracellular pH and ATP

    PubMed Central

    Appikatla, Sunita; Bessert, Denise; Lee, Icksoo; Hüttemann, Maik; Mullins, Chadwick; Somayajulu-Nitu, Mallika; Yao, Fayi; Skoff, Robert P.

    2015-01-01

    Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 proteolipid protein gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher Disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases ATP in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and pro-inflammatory molecules are up-regulated in Plp1 transgenic mice (Tatar et al., 2010). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients. PMID:24382809

  6. Crystal structure of the extracellular domain of human myelin protein zero

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Brunzelle, Joseph S.; Kovari, Iulia A.; Sohi, Jasloveleen; Kamholz, John; Kovari, Ladislau C.

    2012-03-27

    Charcot-Marie-Tooth disease (CMT), a hereditary motor and sensory neuropathy, is the most common genetic neuropathy with an incidence of 1 in 2600. Several forms of CMT have been identified arising from different genomic abnormalities such as CMT1 including CMT1A, CMT1B, and CMTX. CMT1 with associated peripheral nervous system (PNS) demyelination, the most frequent diagnosis, demonstrates slowed nerve conduction velocities and segmental demyelination upon nerve biopsy. One of its subtypes, CMT1A, presents a 1.5-Mb duplication in the p11-p12 region of the human chromosome 17 which encodes peripheral myelin protein 22 (PMP22). CMT1B, a less common form, arises from the mutations in the myelin protein zero (MPZ) gene on chromosome 1, region q22-q23, which encodes the major structural component of the peripheral myelin. A rare type of CMT1 has been found recently and is caused by point mutations in early growth response gene 2 (EGR2), encoding a zinc finger transcription factor in Schwann cells. In addition, CMTX, an X-linked form of CMT, arises from a mutation in the connexin-32 gene. Myelin protein zero, associated with CMT1B, is a transmembrane protein of 219 amino acid residues. Human MPZ consists of three domains: 125 residues constitute the glycosylated immunoglobulin-like extracellular domain; 27 residues span the membrane; and 67 residues comprise the highly basic intracellular domain. MPZ makes up approximately 50% of the protein content of myelin, and is expressed predominantly in Schwann cells, the myelinating cell of the PNS. Myelin protein zero, a homophilic adhesion molecule, is a member of the immunoglobulin super-family and is essential for normal myelin structure and function. In addition, MPZ knockout mice displayed abnormal myelin that severely affects the myelination pathway, and overexpression of MPZ causes congenital hypomyelination of peripheral nerves. Myelin protein zero mutations account for {approx}5% of patients with CMT. To date, over 125

  7. Expression and purification of HER2 extracellular domain proteins in Schneider2 insect cells.

    PubMed

    Kanthala, Shanthi; Mill, Christopher P; Riese, David J; Jaiswal, Mihir; Jois, Seetharama

    2016-09-01

    Overexpression of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu) results in ligand independent activation of kinase signaling and is found in about 30% of human breast cancers, and is correlated with a more aggressive tumor phenotype. The HER2 extracellular domain (ECD) consists of four domains - I, II, III and IV. Although the role of each domain in the dimerization and activation of the receptor has been extensively studied, the role of domain IV (DIV) is not clearly understood yet. In our previous studies, we reported peptidomimetic molecules inhibit HER2:HER3 heterodimerization. In order to study the binding interactions of peptidomimetics with HER2 DIV in detail, properly folded recombinant HER2 protein in pure form is important. We have expressed and purified HER2 ECD and DIV proteins in the Drosophila melanogaster Schneider2 (S2) cell line. Using the commercial Drosophila expression system (DES), we transfected S2 cells with plasmids designed to direct the expression of secreted recombinant HER2 ECD and DIV proteins. The secreted proteins were purified from the conditioned medium by filtration, ultrafiltration, dialysis and nickel affinity chromatography techniques. The purified HER2 proteins were then analyzed using Western blot, mass spectrometry and circular dichroism (CD) spectroscopy. PMID:26363121

  8. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus

    PubMed Central

    Shah, Firoz; Rineau, Francois; Canbäck, Björn; Johansson, Tomas; Tunlid, Anders

    2013-01-01

    Proteins contribute to a major part of the organic nitrogen (N) in forest soils. This N is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. The mechanisms by which these fungi depolymerize proteins and assimilate the released N are poorly characterized. Biochemical analysis and transcriptome profiling were performed to examine the proteolytic machinery and the uptake system of the ectomycorrhizal basidiomycete Paxillus involutus during the assimilation of organic N from various protein sources and extracts of organic matter. All substrates induced secretion of peptidase activity with an acidic pH optimum, mostly contributed by aspartic peptidases. The peptidase activity was transiently repressed by ammonium. Transcriptional analysis revealed a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. For the first time the molecular components of the protein degradation pathways of an ectomycorrhizal fungus are described. The data suggest that the transcripts encoding these components are regulated in response to the chemical properties and the availability of the protein substrates. PMID:23902518

  9. Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps.

    PubMed

    Csomós, Krisztián; Kristóf, Endre; Jakob, Bernadett; Csomós, István; Kovács, György; Rotem, Omri; Hodrea, Judit; Bagoly, Zsuzsa; Muszbek, Laszlo; Balajthy, Zoltán; Csősz, Éva; Fésüs, László

    2016-01-01

    Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system. PMID:27512953

  10. OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

    PubMed Central

    Fernandes, Luis G. V.; Vieira, Monica L.; Kirchgatter, Karin; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Romero, Eliete C.

    2012-01-01

    Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical β-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 ± 871.03 nM and 1,239.23 ± 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 ± 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection. PMID:22802342

  11. Protein Modification by Deamidation Indicates Variations in Joint Extracellular Matrix Turnover*

    PubMed Central

    Catterall, Jonathan B.; Hsueh, Ming F.; Stabler, Thomas V.; McCudden, Christopher R.; Bolognesi, Michael; Zura, Robert; Jordan, Joanne M.; Renner, Jordan B.; Feng, Sheng; Kraus, Virginia B.

    2012-01-01

    As extracellular proteins age, they undergo and accumulate nonenzymatic post-translational modifications that cannot be repaired. We hypothesized that these could be used to systemically monitor loss of extracellular matrix due to chronic arthritic diseases such as osteoarthritis (OA). To test this, we predicted sites of deamidation in cartilage oligomeric matrix protein (COMP) and confirmed, by mass spectroscopy, the presence of deamidated (Asp64) and native (Asn64) COMP epitopes (mean 0.95% deamidated COMP (D-COMP) relative to native COMP) in cartilage. An Asp64, D-COMP-specific ELISA was developed using a newly created monoclonal antibody 6-1A12. In a joint replacement study, serum D-COMP (p = 0.017), but not total COMP (p = 0.5), declined significantly after replacement demonstrating a joint tissue source for D-COMP. In analyses of 450 participants from the Johnston County Osteoarthritis Project controlled for age, gender, and race, D-COMP was associated with radiographic hip (p < 0.0001) but not knee (p = 0.95) OA severity. In contrast, total COMP was associated with radiographic knee (p < 0.0001) but not hip (p = 0.47) OA severity. D-COMP was higher in soluble proteins extracted from hip cartilage proximal to OA lesions compared with remote from lesions (p = 0.007) or lesional and remote OA knee (p < 0.01) cartilage. Total COMP in cartilage did not vary by joint site or proximity to the lesion. This study demonstrates the presence of D-COMP in articular cartilage and the systemic circulation, and to our knowledge, it is the first biomarker to show specificity for a particular joint site. We believe that enrichment of deamidated epitope in hip OA cartilage indicates a lesser repair response of hip OA compared with knee OA cartilage. PMID:22179616

  12. Calcite formation in soft coral sclerites is determined by a single reactive extracellular protein.

    PubMed

    Rahman, M Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-09-01

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called "calcite and aragonite seas." Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that -OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

  13. Insertion of proteolipid protein into oligodendrocyte mitochondria regulates extracellular pH and adenosine triphosphate.

    PubMed

    Appikatla, Sunita; Bessert, Denise; Lee, Icksoo; Hüttemann, Maik; Mullins, Chadwick; Somayajulu-Nitu, Mallika; Yao, Fayi; Skoff, Robert P

    2014-03-01

    Proteolipid protein (PLP) and DM20, the most abundant myelin proteins, are coded by the human PLP1 and non-human Plp1 PLP gene. Mutations in the PLP1 gene cause Pelizaeus-Merzbacher disease (PMD) with duplications of the native PLP1 gene accounting for 70% of PLP1 mutations. Humans with PLP1 duplications and mice with extra Plp1 copies have extensive neuronal degeneration. The mechanism that causes neuronal degeneration is unknown. We show that native PLP traffics to mitochondria when the gene is duplicated in mice and in humans. This report is the first demonstration of a specific cellular defect in brains of PMD patients; it validates rodent models as ideal models to study PMD. Insertion of nuclear-encoded mitochondrial proteins requires specific import pathways; we show that specific cysteine motifs, part of the Mia40/Erv1 mitochondrial import pathway, are present in PLP and are required for its insertion into mitochondria. Insertion of native PLP into mitochondria of transfected cells acidifies media, partially due to increased lactate; it also increases adenosine triphosphate (ATP) in the media. The same abnormalities are found in the extracellular space of mouse brains with extra copies of Plp1. These physiological abnormalities are preventable by mutations in PLP cysteine motifs, a hallmark of the Mia40/Erv1 pathway. Increased extracellular ATP and acidosis lead to neuronal degeneration. Our findings may be the mechanism by which microglia are activated and proinflammatory molecules are upregulated in Plp1 transgenic mice (Tatar et al. (2010) ASN Neuro 2:art:e00043). Manipulation of this metabolic pathway may restore normal metabolism and provide therapy for PMD patients. PMID:24382809

  14. Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

    PubMed Central

    Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-01-01

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

  15. Extracellular Vesicle Proteins Associated with Systemic Vascular Events Correlate with Heart Failure: An Observational Study in a Dyspnoea Cohort

    PubMed Central

    Zhang, Ya-Nan; Vernooij, Flora; Ibrahim, Irwani; Ooi, Shirley; Gijsberts, Crystel M.; Schoneveld, Arjan H.; Sen, Kuan Win; den Ruijter, Hester M.; Timmers, Leo; Richards, Arthur Mark; Jong, Chun Tzen; Mazlan, Ibrahim; Wang, Jiong-Wei; Lam, Carolyn S. P.; de Kleijn, Dominique P. V.

    2016-01-01

    Background SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma extracellular vesicle (EV) -levels of these proteins have been reported to be associated with systemic vascular events. Evidence is accumulating that inflammatory processes may play a pivotal role both in systemic vascular events and in heart failure. Therefore, we studied the association between plasma extracellular vesicle SerpinF2-, SerpinG1-, CystatinC and CD14-levels and the occurrence of acute heart failure in patients. Methods and Result Extracellular vesicle protein levels of SerpinG1, SerpinF2, CystatinC and CD14 were measured in an observational study of 404 subjects presenting with dysponea at the emergency department (4B-cohort). Plasma extracellular vesicles were precipitated in a total extracellular vesicles (TEX)-fraction and in separate LDL- and HDL-subfractions. Extracellular vesicle protein levels were measured with a quantitative immune assay in all 3 precipitates. Out of 404 subjects, 141 (35%) were diagnosed with acutely decompensated heart failure. After correction for confounders (including comorbidities and medications), levels of CD14 in the HDL-fraction (OR 1.53, p = 0.01), SerpinF2 in the TEX-and LDL-fraction (ORs respectively 0.71 and 0.65, p<0.05) and SerpinG1 in the TEX-fraction (OR 1.55, p = 0.004) were statistically significantly related to heart failure. Furthermore, extracellular vesicle CD14- and SerpinF2-levels were significantly higher in heart failure patients with preserved ejection fraction than in those with reduced ejection fraction. Conclusion Extracellular vesicle levels of CD14, SerpinG1 and SerpinF2 are associated with the occurrence of heart failure in subjects suspected for acute heart failure, suggesting common underlying pathophysiological mechanisms for heart failure and vascular events. PMID:26820481

  16. An Ensemble Method with Hybrid Features to Identify Extracellular Matrix Proteins

    PubMed Central

    Yang, Runtao; Zhang, Chengjin; Gao, Rui; Zhang, Lina

    2015-01-01

    The extracellular matrix (ECM) is a dynamic composite of secreted proteins that play important roles in numerous biological processes such as tissue morphogenesis, differentiation and homeostasis. Furthermore, various diseases are caused by the dysfunction of ECM proteins. Therefore, identifying these important ECM proteins may assist in understanding related biological processes and drug development. In view of the serious imbalance in the training dataset, a Random Forest-based ensemble method with hybrid features is developed in this paper to identify ECM proteins. Hybrid features are employed by incorporating sequence composition, physicochemical properties, evolutionary and structural information. The Information Gain Ratio and Incremental Feature Selection (IGR-IFS) methods are adopted to select the optimal features. Finally, the resulting predictor termed IECMP (Identify ECM Proteins) achieves an balanced accuracy of 86.4% using the 10-fold cross-validation on the training dataset, which is much higher than results obtained by other methods (ECMPRED: 71.0%, ECMPP: 77.8%). Moreover, when tested on a common independent dataset, our method also achieves significantly improved performance over ECMPP and ECMPRED. These results indicate that IECMP is an effective method for ECM protein prediction, which has a more balanced prediction capability for positive and negative samples. It is anticipated that the proposed method will provide significant information to fully decipher the molecular mechanisms of ECM-related biological processes and discover candidate drug targets. For public access, we develop a user-friendly web server for ECM protein identification that is freely accessible at http://iecmp.weka.cc. PMID:25680094

  17. Effects of extracellular matrix proteins on the growth of haematopoietic progenitor cells.

    PubMed

    Celebi, Betül; Mantovani, Diego; Pineault, Nicolas

    2011-10-01

    Umbilical cord blood (UCB) transplantation and haematological recovery are currently limited by the amount of haematopoietic progenitor cells (HPCs) present in each unit. HPCs and haematopoietic stem cells (HSCs) normally interact with cells and extracellular matrix (ECM) proteins present within the endosteal and vascular niches. Hence, we investigated whether coating of culture surfaces with ECM proteins normally present in the marrow microenvironment could benefit the ex vivo expansion of HPCs. Towards this, collagen types I and IV (COL I and IV), laminin (LN) and fibronectin (FN) were tested individually or as component of two ECM-mix complexes. Individually, ECM proteins had both common and unique properties on the growth and differentiation of UCB CD34+ cells; some ECM proteins favoured the differentiation of some lineages over that of others (e.g. FN for erythroids), some the expansion of HPCs (e.g. LN and megakaryocyte (MK) progenitor) while others had less effects. Next, two ECM-mix complexes were tested; the first one contained all four ECM proteins (4ECMp), while the second 'basement membrane-like structure' was without COL I (3ECMp). Removal of COL I led to strong reductions in cell growth and HPCs expansion. Interestingly, the 4ECMp-mix complex reproducibly increased CD34+ (1.3-fold) and CD41+ (1.2-fold) cell expansions at day 6 (P < 0.05) versus control, and induced greater myeloid progenitor expansion (P < 0.05) than 3ECMp. In conclusion, these results suggest that optimization of BM ECM protein complexes could provide a better environment for the ex vivo expansion of haematopoietic progenitors than individual ECM protein. PMID:21931196

  18. Fundamental Investigations of the Extracellular Proteins Fibrin and Collagen in Microchannel Devices

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Koester, Sarah; Pfohl, Thomas

    2007-03-01

    Microfluidic structures are particularly amenable to controlled investigations of protein bundle and network formation. Hydrodynamic focusing is utilized to create a diffusion-controlled gradient of reactants, enabling non-equilibrium investigations. We present studies of the blood clotting protein fibrin, a three-dimensional network formed from the enzymatic cleavage of fibrinogen monomers by the protein thrombin. Fibrin is a vital component of blood clots, and has been implicated in a variety of diseases. Real-time fluorescence microscopy and x-ray micro-diffraction are used to quantify supramolecular assembly and provide snapshots of the evolution of fibrin network formation. We also show that collagen, a ubiquitous extracellular protein, can be bundled in situ through the use of a pH gradient. An outlook toward artificial blood vessels arises from the insight that both fibrin and collagen can easily be used to coat microchannel structures. The resulting mesh forms an ideal environment for red blood cells and other cell types.

  19. Cell response to RGD density in cross-linked artificial extracellular matrix protein films.

    PubMed

    Liu, Julie C; Tirrell, David A

    2008-11-01

    This study examines the adhesion, spreading, and migration of human umbilical vein endothelial cells on cross-linked films of artificial extracellular matrix (aECM) proteins. The aECM proteins described here were designed for application in small-diameter grafts and are composed of elastin-like structural repeats and fibronectin cell-binding domains. aECM-RGD contains the RGD sequence derived from fibronectin; the negative control protein aECM-RDG contains a scrambled cell-binding domain. The covalent attachment of poly(ethylene glycol) (PEG) to aECM substrates reduced nonspecific cell adhesion to aECM-RDG-PEG but did not preclude sequence-specific adhesion of endothelial cells to aECM-RGD-PEG. Variation in ligand density was accomplished by the mixing of aECM-RGD-PEG and aECM-RDG-PEG prior to cross-linking. Increasing the density of RGD domains in cross-linked films resulted in more robust cell adhesion and spreading but did not affect cell migration speed. Control of cell-binding domain density in aECM proteins can thus be used to modulate cell adhesion and spreading and will serve as an important design tool as these materials are further developed for use in surgery, tissue engineering, and regenerative medicine. PMID:18826275

  20. Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease

    PubMed Central

    Liang, Yan; Sheikh, Farah

    2016-01-01

    The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways may exert their regulatory functions in a more compartmentalized manner in cardiac muscle. Emerging data has uncovered specific MAPK scaffolding proteins that tether MAPK/ERK signaling specifically at the sarcomere and plasma membrane in cardiac muscle and show that deficiencies in these scaffolding proteins alter ERK activity and phosphorylation, which are then critical in altering the cardiac myocyte response to stress-induced hypertrophy and disease progression. In this review, we provide insights on ERK-associated scaffolding proteins regulating cardiac myofilament function and their impact on cardiac hypertrophy and disease. PMID:26973524

  1. The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles.

    PubMed

    Hajj, Glaucia N M; Arantes, Camila P; Dias, Marcos Vinicios Salles; Roffé, Martín; Costa-Silva, Bruno; Lopes, Marilene H; Porto-Carreiro, Isabel; Rabachini, Tatiana; Lima, Flávia R; Beraldo, Flávio H; Prado, Marco A M; Prado, Marco M A; Linden, Rafael; Martins, Vilma R

    2013-09-01

    The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. PMID:23543276

  2. Amyloid β-Protein as a Substrate Interacts with Extracellular Matrix to Promote Neurite Outgrowth

    NASA Astrophysics Data System (ADS)

    Koo, Edward H.; Park, Lisa; Selkoe, Dennis J.

    1993-05-01

    Progressive deposition of amyloid β-protein (Aβ) in brain parenchyma and blood vessels is a characteristic feature of Alzheimer disease. Recent evidence suggests that addition of solubilized synthetic Aβ to medium may produce toxic or trophic effects on cultured hippocampal neurons. Because soluble Aβ may not accumulate in significant quantities in brain, we asked whether immobilized Aβ peptide as a substrate alters neurite outgrowth from cultured rat peripheral sensory neurons. This paradigm may closely mimic the conditions in Alzheimer disease brain tissue, in which neurites contact insoluble, extracellular aggregates of β-amyloid. We detected no detrimental effects of Aβ substrate on neurite outgrowth. Rather, Aβ in combination with low doses of laminin or fibronectin enhanced neurite out-growth from these neuronal explants. Our results suggest that insoluble Aβ in the cerebral neuropil may serve as a neurite-promoting matrix, perhaps explaining the apparent regenerative response of neurites observed around amyloid plaques in Alzheimer disease. Moreover, in concert with the recent discovery of Aβ production by cultured neurons, our data suggest that Aβ plays a normal physiological role in brain by complexing with the extracellular matrix.

  3. Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3.

    PubMed

    Zhou, Yuehan; Skelton, Lara A; Xu, Lumei; Chandler, Margaret P; Berthiaume, Jessica M; Boron, Walter F

    2016-09-01

    Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis. PMID:26839367

  4. Extracellular protein disulfide isomerase regulates coagulation on endothelial cells through modulation of phosphatidylserine exposure

    PubMed Central

    Popescu, Narcis I.; Lupu, Cristina

    2010-01-01

    Tissue factor (TF) is the cellular receptor for plasma protease factor VIIa (FVIIa), and the TF-FVIIa complex initiates coagulation in both hemostasis and thrombosis. Cell surface-exposed TF is mainly cryptic and requires activation to fully exhibit the procoagulant potential. Recently, the protein disulfide isomerase (PDI) has been hypothesized to regulate TF decryption through the redox switch of an exposed disulfide in TF extracellular domain. In this study, we analyzed PDI contribution to coagulation using an in vitro endothelial cell model. In this model, extracellular PDI is detected by imaging and flow cytometry. Inhibition of cell surface PDI induces a marked increase in TF procoagulant function, whereas exogenous addition of PDI inhibits TF decryption. The coagulant effects of PDI inhibition were sensitive to annexin V treatment, suggesting exposure of phosphatidylserine (PS), which was confirmed by prothrombinase assays and direct labeling. In contrast, exogenous PDI addition enhanced PS internalization. Analysis of fluorescent PS revealed that PDI affects both the apparent flippase and floppase activities on endothelial cells. In conclusion, we identified a new mechanism for PDI contribution to coagulation on endothelial cells, namely, the regulation of PS exposure, where PDI acts as a negative regulator of coagulation. PMID:20448108

  5. Growth of an extracellular proteinase-deficient strain of Pseudomonas fluorescens on milk and milk proteins.

    PubMed

    Torrie, J P; Cholette, H; Froehlich, D A; McKellar, R C

    1983-08-01

    An extracellular proteinase-and lipase-deficient mutant of a psychrotroph, Pseudomonas fluorescens strain 32A, has been isolated and the absence of the proteinase enzyme confirmed by growth on differential media, enzyme assay and polyacrylamide gel electrophoresis. Competition between the parent and the mutant was observed when equal numbers of the 2 strains were inoculated together into raw skim-milk at 6 degrees C. Bitterness was detected at 6 degrees C in pasteurized skim-milk inoculated with the parent cells concurrent with the detection of proteolytic activity. In the case of the mutant, slight bitterness which did not increase with increasing cell numbers was detected in the absence of proteolysis. Mutant cells failed to grow on Na caseinate as the sole source of carbon. It was concluded that the extracellular proteinase, while not essential for growth in milk, does provide a selective advantage to the producer organism. This enzyme is, however, essential for growth on milk proteins and contributes to the development of bitterness in pasteurized milk. PMID:6413562

  6. Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain

    PubMed Central

    Cooley, Christina B.; Ryno, Lisa M.; Plate, Lars; Morgan, Gareth J.; Hulleman, John D.; Kelly, Jeffery W.; Wiseman, R. Luke

    2014-01-01

    Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation. PMID:25157167

  7. Transglutaminase 2 interactions with extracellular matrix proteins as probed with celiac disease autoantibodies.

    PubMed

    Cardoso, Inês; Stamnaes, Jorunn; Andersen, Jan Terje; Melino, Gerry; Iversen, Rasmus; Sollid, Ludvig M

    2015-06-01

    Transglutaminases have been implicated in various human diseases. A prominent example is the involvement of transglutaminase 2 (TG2) in the gluten-sensitive enteropathy celiac disease, where the enzyme is both the target of autoantibodies and responsible for the generation of immunogenic gluten epitopes. Here, we aimed to characterize the microenvironment of TG2 in the extracellular matrix (ECM) in order to gain insights into the antigenic structures that are recognized by autoantibodies in celiac disease. A panel of TG2-specific mAbs established from gut plasma cells of celiac disease patients was employed as probes to characterize the interactions between TG2 and ECM constituents. With immunofluorescence staining, microplate protein-binding and surface plasmon resonance assays, we found that the main epitope (epitope 1) recognized by TG2-specific gut plasma cells overlaps with the fibronectin (FN)-binding site of TG2. Furthermore, we found that the same TG2 amino acids that are involved in binding of epitope 1 mAbs are also important for efficient binding of FN. Notably, epitope 1 mAbs recognize TG2 in tissue sections, suggesting that some TG2 in the extracellular matrix has interaction partners in addition to FN. We demonstrate that collagen VI is a strong candidate, on the basis of its tissue expression pattern and ability to bind TG2. Collagen VI may thus serve as a matrix for deposition of TG2 in a context that can also be recognized by epitope 1-targeting autoantibodies. PMID:25808416

  8. The interaction of Tamm-Horsfall protein with the extracellular matrix.

    PubMed Central

    Lambert, C; Brealey, R; Steele, J; Rook, G A

    1993-01-01

    Tamm-Horsfall protein (THP) is the major glycoprotein component of urine, yet its biological role remains obscure. Recent reports have suggested that a concanavalin A (Con A)-binding fraction of THP from pregnancy urine can bind the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). In order to investigate this claim in relation to THP from normal adult urine we raised monoclonal antibodies to THP and sought THP/TNF-alpha interactions in three separate assay systems. We found no evidence that THP binds to TNF-alpha under physiological conditions, but we observed that it exerts a weak, probably not physiologically relevant, but reproducible inhibitory effect on the toxicity of TNF-alpha for monolayers of L929 cells, even when the cells are pretreated with the THP, and washed before addition of the cytokine. Since our preparations of THP do not interact directly with TNF-alpha we postulated an interaction with the cells themselves, or with their extracellular matrix. The THP was found by ELISA, immunoblotting and immunohistology, to bind to as yet unidentified components of the extracellular matrix in a manner dependent on cations, pH and carbohydrates. These data, considered in the light of the published amino acid sequence and biochemical properties, suggest that THP is a member of a structural glycoprotein family known to modulate cell adhesion. Images Figure 4 Figure 5 PMID:8344699

  9. Symposium: Role of the extracellular matrix in mammary development. Regulation of milk protein and basement membrane gene expression: The influence of the extracellular matrix

    SciTech Connect

    Aggeler, J.; Park, C.S.; Bissell, M.J.

    1988-10-01

    Synthesis and secretion of milk proteins ({alpha}-casein, {beta}-casein, {gamma}-casein, and transferrin) by cultured primary mouse mammary epithelial cells is modulated by the extracellular matrix. In cells grown on released or floating type I collagen gels, mRNA for {beta}-casein and transferrin is increased as much as 30-fold over cells grown on plastic. Induction of {beta}-casein expression depends strongly on the presence of lactogenic hormones, especially prolactin, in the culture. When cells are plated onto partially purified reconstituted basement membrane, dramatic changes in morphology and milk protein gene expression are observed. Cells cultured on the matrix for 6 to 8 d in the presence of prolactin, insulin, and hydrocortisone form hollow spheres and duct-like structures that are completely surrounded by matrix. The cells lining these spheres appear actively secretory and are oriented with their apices facing the lumen. Hybridization experiments indicate that mRNA for {beta}-casein can be increased as much as 70-fold in these cultures. Because > 90% of the cultured cells synthesize immunoreactive {beta}-casein, as compared with only 40% of cells in the late pregnant gland, the matrix appears to be able to induce protein expression in previously silent cells. Synthesis of laminin and assembly of a mammary-specific basal lamina by cells cultured on different extracellular matrices also appears to depend on the presence of lactogenic hormones. These studies provide support for the concept of dynamic reciprocity in which complex interactions between extracellular matrix and the cellular cytoskeleton contribute to the induction and maintenance of tissue-specific gene expression in the mammary gland.

  10. Cutting edge: extracellular high mobility group box-1 protein is a proangiogenic cytokine.

    PubMed

    Mitola, Stefania; Belleri, Mirella; Urbinati, Chiara; Coltrini, Daniela; Sparatore, Bianca; Pedrazzi, Marco; Melloni, Edon; Presta, Marco

    2006-01-01

    The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus. PMID:16365390

  11. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

    PubMed

    Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-07-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

  12. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix

    PubMed Central

    Williams, B. Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-01-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

  13. The Bfp60 surface adhesin is an extracellular matrix and plasminogen protein interacting in Bacteroides fragilis

    PubMed Central

    de Oliveira Ferreira, Eliane; Teixeira, Felipe; Cordeiro, Fabiana; Lobo, Leandro Araujo; Rocha, Edson R.; Smith, Jeffrey C.; Domingues, Regina M C P

    2014-01-01

    Plasminogen (Plg) is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by several pathogenic species of bacteria to manipulate the host plasminogen system and facilitate invasion of tissues during infection by modifying the activation of this process through the binding of Plg at their surface. Bacteroides fragilis is the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses and anaerobic bacteraemia. The ability of B. fragilis to convert plasminogen (Plg) into plasmin has been associated with an outer membrane protein named Bfp60. In this study, we characterized the function of Bfp60 protein in B. fragilis 638R by constructing the bfp60 defective strain and comparing its with that of the wild type regarding binding to laminin-1 (LMN-1) and activation of Plg into plasmin. Although the results showed in this study indicate that Bfp60 surface protein of B. fragilis is important for the recognition of LMN-1 and Plg activation, a significant slow activation of Plg into plasmin was observed in the mutant strain. For that reason, the possibility of another unidentified mechanism activating Plg is also present in B. fragilis can not be discarded. The results demonstrate that Bfp60 protein is responsible for the recognition of laminin and Plg-plasmin activation. Although the importance of this protein is still unclear in the pathogenicity of the species, it is accepted that since other pathogenic bacteria use this mechanism to disseminate through the extracellular matrix during the infection, it should also contribute to the virulence of B. fragilis. PMID:23850366

  14. Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injury

    PubMed Central

    Wang, Guan; Zhang, Jian-ning; Guo, Jia-kui; Cai, Ying; Sun, Hong-sheng; Dong, Kun; Wu, Cheng-gang

    2016-01-01

    Cold-inducible RNA-binding protein (CIRP), a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5°C on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP mRNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury. PMID:27335561

  15. Intense pulsed light induces synthesis of dermal extracellular proteins in vitro.

    PubMed

    Cuerda-Galindo, E; Díaz-Gil, G; Palomar-Gallego, M A; Linares-GarcíaValdecasas, R

    2015-09-01

    Intense pulsed light (IPL) devices have been shown to be highly effective for the skin rejuvenation. In our study, we try to elucidate effects of IPL in fibroblast proliferation, in gene expression, and in extracellular matrix protein production. 1BR3G human skin fibroblasts were used to test the effects of an IPL device (MiniSilk FT, Deka®). Fibroblasts were divided into three groups: group 1 was irradiated with filter 800-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm) twice; group 2 was irradiated with filter 550-1200 nm (double pulse 5 ms + 5 ms, delay 10 ms, fluence 13 J/cm2) twice; and group 3 was irradiated with filter 550-1200 nm (frequency 10 Hz, 15 s, fluence 60.1 J/cm2) twice. To determine changes in gene expression, messenger RNA (mRNA) levels for collagen types I and III and metalloproteinase 1 (MMP-1) were performed 48 h after irradiation. To determine changes in hyaluronic acid, versican, and decorin, mRNA and ELISA tests were performed after 48 h of treatment. In addition to this, a Picro-Sirius red staining for collagen was made. The study showed an increase of mRNA and hyaluronic acid, decorin, and versican production. With RT-PCR assays, an increase mRNA for collagen type I, type III, and MMP-1 was observed. Collagen and hyaluronic synthesis was increased in all groups with no differences among them, while decorin and versican synthesis was higher in those groups irradiated with 550-1200-nm filters with no dependence of type pulse or total energy dose. IPL applied in vitro cultured cells increases fibroblasts activity. Synthesis of extracellular proteins seems to be produced more specifically in determined wavelengths, which could demonstrate a biochemical mechanism light depending. PMID:26188855

  16. The neuronal extracellular matrix restricts distribution and internalization of aggregated Tau-protein.

    PubMed

    Suttkus, A; Holzer, M; Morawski, M; Arendt, T

    2016-01-28

    Alzheimer's disease (AD) is a chronic degenerative disorder characterized by fibrillary aggregates of Aß and Tau-protein. Formation and progression of these pathological hallmarks throughout the brain follow a specific spatio-temporal pattern which provides the basis for neuropathological staging. Previously, we could demonstrate that cortical and subcortical neurons are less frequently affected by neurofibrillary degeneration if they are enwrapped by a specialized form of the hyaluronan-based extracellular matrix (ECM), the so called 'perineuronal net' (PN). PNs are composed of large aggregating chondroitin sulfate proteoglycans connected to a hyaluronan backbone, stabilized by link proteins and cross-linked via tenascin-R. Recently, PN-associated neurons were shown to be better protected against iron-induced neurodegeneration compared to neurons without PN, indicating a neuroprotective function. Here, we investigated the role of PNs in distribution and internalization of exogenous Tau-protein by using organotypic slice cultures of wildtype mice as well as mice lacking the ECM-components aggrecan, HAPLN1 or tenascin-R. We could demonstrate that PNs restrict both distribution and internalization of Tau. Accordingly, PN-ensheathed neurons were less frequently affected by Tau-internalization, than neurons without PN. Finally, the PNs as well as their three investigated components were shown to modulate the processes of distribution as well as internalization of Tau. PMID:26621125

  17. Horizontal transfer of RNA and proteins between cells by extracellular microvesicles: 14 years later.

    PubMed

    Ratajczak, Mariusz Z; Ratajczak, Janina

    2016-12-01

    Extracellular microvesicles (ExMVs) are part of the cell secretome, and evidence has accumulated for their involvement in several biological processes. Fourteen years ago our team demonstrated for the first time that ExMVs carry functional RNA species and proteins from one cell to another, an observation that opened up the new research field of horizontal transfer of bioactive molecules in cell-to-cell communication. Moreover, the presence of mRNA, noncoding RNA, and miRNA in ExMVs in blood and other biological fluids opened up the possibility of employing ExMVs as new detection markers for pathological processes, and ExMVs became a target for "liquid biopsy" approaches. While ExMV-derived mRNAs may be translated in target cells into appropriate proteins, miRNAs regulate expression of corresponding mRNA species, and both RNA-depended ExMV-mediated mechanisms lead to functional changes in the target cells. Following from this observation, several excellent papers have been published that confirm the existence of the horizontal transfer of RNA. Moreover, in addition to RNA, proteins, bioactive lipids, infectious particles and intact organelles such as mitochondria may follow a similar mechanism. In this review we will summarize the impressive progress in this field-14 years after initial report. PMID:26943717

  18. Extracellular High-Mobility Group Box 1 Protein (HMGB1) as a Mediator of Persistent Pain

    PubMed Central

    Agalave, Nilesh M; Svensson, Camilla I

    2014-01-01

    Although originally described as a highly conserved nuclear protein, high-mobility group box 1 protein (HMGB1) has emerged as a danger-associated molecular pattern molecule protein (DAMP) and is a mediator of innate and specific immune responses. HMGB1 is passively or actively released in response to infection, injury and cellular stress, providing chemotactic and cytokine-like functions in the extracellular environment, where it interacts with receptors such as receptor for advanced glycation end products (RAGE) and several Toll-like receptors (TLRs). Although HMGB1 was first revealed as a key mediator of sepsis, it also contributes to a number of other conditions and disease processes. Chronic pain arises as a direct consequence of injury, inflammation or diseases affecting the somatosensory system and can be devastating for the affected patients. Emerging data indicate that HMGB1 is also involved in the pathology of persistent pain. Here, we give an overview of HMGB1 as a proinflammatory mediator, focusing particularly on the role of HMGB1 in the induction and maintenance of hypersensitivity in experimental models of pain and discuss the therapeutic potential of targeting HMGB1 in conditions of chronic pain. PMID:25222915

  19. Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein.

    PubMed

    Rockey, D D; Turaga, P S; Wiens, G D; Cook, B A; Kaattari, S L

    1991-10-01

    Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 degrees C but became readily apparent at 37 degrees C. Proteinase activity was detected at bacterial physiological temperatures (17 degrees C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 degrees C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol). PMID:1777853

  20. Ultrasound Technologies for the Spatial Patterning of Cells and Extracellular Matrix Proteins and the Vascularization of Engineered Tissue

    NASA Astrophysics Data System (ADS)

    Garvin, Kelley A.

    Technological advancements in the field of tissue engineering could save the lives of thousands of organ transplant patients who die each year while waiting for donor organs. Currently, two of the primary challenges preventing tissue engineers from developing functional replacement tissues and organs are the need to recreate complex cell and extracellular microenvironments and to vascularize the tissue to maintain cell viability and function. Ultrasound is a form of mechanical energy that can noninvasively and nondestructively interact with tissues at the cell and protein level. In this thesis, novel ultrasound-based technologies were developed for the spatial patterning of cells and extracellular matrix proteins and the vascularization of three-dimensional engineered tissue constructs. Acoustic radiation forces associated with ultrasound standing wave fields were utilized to noninvasively control the spatial organization of cells and cell-bound extracellular matrix proteins within collagen-based engineered tissue. Additionally, ultrasound induced thermal mechanisms were exploited to site-specifically pattern various extracellular matrix collagen microstructures within a single engineered tissue construct. Finally, ultrasound standing wave field technology was used to promote the rapid and extensive vascularization of three-dimensional tissue constructs. As such, the ultrasound technologies developed in these studies have the potential to provide the field of tissue engineering with novel strategies to spatially pattern cells and extracellular matrix components and to vascularize engineered tissue, and thus, could advance the fabrication of functional replacement tissues and organs in the field of tissue engineering.

  1. Expression of extracellular matrix proteins in cervical squamous cell carcinoma--a clinicopathological study.

    PubMed Central

    Goldberg, I; Davidson, B; Lerner-Geva, L; Gotlieb, W H; Ben-Baruch, G; Novikov, I; Kopolovic, J

    1998-01-01

    AIM: To evaluate the intracellular and peritumoral expression of matrix proteins in squamous cell carcinoma of the uterine cervix using immunohistochemistry. METHODS: 71 squamous cell carcinomas and 10 controls were stained for laminin, fibronectin, and collagen IV. Cytoplasmic staining in tumour cells and peritumoral deposition of matrix proteins were evaluated. The association between staining results and patient age, tumour stage, histological grade, and survival was studied. RESULTS: Positive cytoplasmic staining for laminin, fibronectin, and collagen IV was observed in 17 (23.9%), 27 (38%), and 10 (14.1%) cases, respectively. Staining for laminin was most pronounced in the invasive front of tumour islands, while for fibronectin and collagen IV it appeared to be diffuse. Peritumoral staining for laminin and collagen IV was detected in 12 cases (16.9%). Early stage (Ia1-Ia2) tumours were uniformly negative for all three proteins. Cytoplasmic staining for laminin correlated with positive staining for fibronectin and collagen IV, and with the presence of a peritumoral deposition of collagen IV and laminin. There was no correlation with any of the three markers between staining results and patient age, stage, grade, or survival. CONCLUSIONS: Expression of extracellular matrix proteins in some cervical squamous cell carcinomas might reflect the enhanced ability of these tumours to modify the peritumoral stroma. This ability seems to be absent in early stage tumours. The correlation between intracytoplasmic and peritumoral expression of matrix proteins supports the evidence of their synthesis by tumour cells. However, this property did not correlate with disease outcome in this study. Images PMID:10023343

  2. Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin.

    PubMed

    Beyeler, Michael; Schild, Christof; Lutz, Roman; Chiquet, Matthias; Trueb, Beat

    2010-04-15

    Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell

  3. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects

    SciTech Connect

    Desai, Jayashree; Shannon, Mark E.; Johnson, Mahlon D.; Ruff, David W.; Hughes, Lori A; Kerley, Marilyn K; Carpenter, D A; Johnson, Dabney K; Rinchik, Eugene M.; Culiat, Cymbeline T

    2006-01-01

    The mammalian Nell1 gene encodes a protein kinase C-b1 (PKC-b1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell16R, a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell16R has a T!A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell16R mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell16R mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

  4. Extracellular Regulation of Sperm Transmembrane Adenylyl Cyclase by a Forward Motility Stimulating Protein

    PubMed Central

    Dey, Souvik; Roy, Debarun; Majumder, Gopal C.; Bhattacharyya, Debdas

    2014-01-01

    Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation

  5. Induction of cell cycle changes and modulation of apoptogenic/anti-apoptotic and extracellular signaling regulatory protein expression by water extracts of I'm-Yunity™ (PSP)

    PubMed Central

    Hsieh, Tze-chen; Wu, Peili; Park, Spencer; Wu, Joseph M

    2006-01-01

    degree p50 forms of transcription factor NF-κB, which was accompanied by a reduction in the expression of cyclooxygenase 2 (COX2). I'm-Yunity™ (PSP) also elicited an increase in STAT1 (signal transducer and activator of transcription) and correspondingly, decrease in the expression of activated form of ERK (extracellular signal-regulated kinase). Conclusion Aqueous extracts of I'm-Yunity™ (PSP) induces cell cycle arrest and alterations in the expression of apoptogenic/anti-apoptotic and extracellular signaling regulatory proteins in human leukemia cells, the net result being suppression of proliferation and increase in apoptosis. These findings may contribute to the reported clinical and overall health effects of I'm-Yunity™ (PSP). PMID:16965632

  6. Putative role of protein kinase C in neurotoxic inflammation mediated by extracellular heat shock protein 70 after ischemia-reperfusion

    PubMed Central

    2014-01-01

    Background Sterile inflammation occurs in the absence of live pathogens and is an unavoidable consequence of ischemia-reperfusion (IR) injury in the central nervous system (CNS). It is known that toll-like receptor 4 (Tlr4) contributes to damage and sterile inflammation in the CNS mediated by IR. However, the mechanism of Tlr4 activation under sterile conditions in ischemic tissue is poorly understood. We performed this study to clarify the mechanism. To this end, we focused on the extracellular heat shock protein 70 (Hsp70), the prototypic Tlr4 ligand. Methods Tlr4-, Myd88- and Trif-knockout animals, as well as C57BL/6 mice, were used for the wild type control. For the in vivo study, we used a mouse model of retinal IR injury. To test the role of protein kinase C (PKC) in IR injury, IR retinas were treated with the PKC inhibitors (polymyxin B and Gö6976) and retinal damage was evaluated by directly counting neurons in the ganglion cell layer of flat-mounted retinas seven days after IR. Primary retinal neurons (retinal ganglion cells) and glial cells were used for in vitro experiments. Quantitative RT-PCR, ELISA and western blot analysis were used to study the production of pro-inflammatory factors in IR retinas and in primary cell cultures. Results We found significant accumulation of extracellular Hsp70 in a model of retinal IR injury. We noted that PKC was involved in Tlr4 signaling, and found that PKC inhibitors promoted neuroprotection by reducing pro-inflammatory activity in ischemic tissue. To put all of the pieces in the signaling cascade together, we performed an in vitro study. We found that PKC was critical to mediate the Hsp70-dependent pro-inflammatory response. At the same time, the contamination of Hsp70 preparations with low-dose endotoxin was not critical to mediate the production of pro-inflammatory factors. We found that extracellular Hsp70 can promote neuronal death at least, by mediating production of cytotoxic levels of tumor necrosis factor

  7. Sec24D-Dependent Transport of Extracellular Matrix Proteins Is Required for Zebrafish Skeletal Morphogenesis

    PubMed Central

    Sarmah, Swapnalee; Barrallo-Gimeno, Alejandro; Melville, David B.; Topczewski, Jacek; Solnica-Krezel, Lilianna; Knapik, Ela W.

    2010-01-01

    Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor β1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects. PMID:20442775

  8. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  9. Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR)

    PubMed Central

    Gallart-Palau, Xavier; Serra, Aida; Wong, Andrew See Weng; Sandin, Sara; Lai, Mitchell K. P.; Chen, Christopher P.; Kon, Oi Lian; Sze, Siu Kwan

    2015-01-01

    Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications. PMID:26419333

  10. Drosophila dec-1 eggshell proteins are differentially distributed via a multistep extracellular processing and localization pathway.

    PubMed

    Noguerón, M I; Mauzy-Melitz, D; Waring, G L

    2000-09-15

    In Drosophila the multilayered eggshell forms during late oogenesis between the oocyte and the overlaying follicle cells. Proper eggshell assembly requires wild-type dec-1 gene function. Alternatively spliced dec-1 transcripts encode three proproteins that are cleaved extracellularly in a stage-specific manner to at least five distinct derivatives. Using polyclonal antibodies raised against fusion proteins containing different regions of the dec-1 proteins, we have localized several dec-1 derivatives in the assembling and completed eggshell. Although all of the dec-1 derivatives are generated in the oocyte proximal vitelline membrane layer, they are differentially distributed in the mature egg. Some derivatives are gradually released from the vitelline membrane and become localized within distinct regions of the chorion, while others are taken up by the oocyte or become concentrated in the endochorionic spaces or cavities. The diverse distributions of the dec-1 derivatives suggest that each derivative plays a distinct role in eggshell assembly. These results also suggest that the vitelline membrane layer, by acting as a transient storage site, may control the availability of molecules active in eggshell assembly and by extension perhaps other follicle cell products important in early embryonic pattern formation. PMID:10985863

  11. Periostin is an extracellular matrix protein required for eruption of incisors in mice

    SciTech Connect

    Kii, Isao; Amizuka, Norio; Minqi, Li; Kitajima, Satoshi; Saga, Yumiko; Kudo, Akira . E-mail: akudo@bio.titech.ac.jp

    2006-04-14

    A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin {sup -/-} mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin {sup -/-} mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone.

  12. Mice lacking the extracellular matrix protein MAGP1 display delayed thrombotic occlusion following vessel injury

    PubMed Central

    Werneck, Claudio C.; Vicente, Cristina P.; Weinberg, Justin S.; Shifren, Adrian; Pierce, Richard A.; Broekelmann, Thomas J.; Tollefsen, Douglas M.

    2008-01-01

    Mice lacking the extracellular matrix protein microfibril-associated glycoprotein-1 (MAGP1) display delayed thrombotic occlusion of the carotid artery following injury as well as prolonged bleeding from a tail vein incision. Normal occlusion times were restored when recombinant MAGP1 was infused into deficient animals prior to vessel wounding. Blood coagulation was normal in these animals as assessed by activated partial thromboplastin time and prothrombin time. Platelet number was lower in MAGP1-deficient mice, but the platelets showed normal aggregation properties in response to various agonists. MAGP1 was not found in normal platelets or in the plasma of wild-type mice. In ligand blot assays, MAGP1 bound to fibronectin, fibrinogen, and von Willebrand factor, but von Willebrand factor was the only protein of the 3 that bound to MAGP1 in surface plasmon resonance studies. These findings show that MAGP1, a component of microfibrils and vascular elastic fibers, plays a role in hemostasis and thrombosis. PMID:18281502

  13. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-dependent transcriptional program controls activation of the early growth response 1 (EGR1) gene during amino acid limitation.

    PubMed

    Shan, Jixiu; Balasubramanian, Mukundh N; Donelan, William; Fu, Lingchen; Hayner, Jaclyn; Lopez, Maria-Cecilia; Baker, Henry V; Kilberg, Michael S

    2014-08-29

    Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism. PMID:25028509

  14. A Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK)-dependent Transcriptional Program Controls Activation of the Early Growth Response 1 (EGR1) Gene during Amino Acid Limitation*

    PubMed Central

    Shan, Jixiu; Balasubramanian, Mukundh N.; Donelan, William; Fu, Lingchen; Hayner, Jaclyn; Lopez, Maria-Cecilia; Baker, Henry V.; Kilberg, Michael S.

    2014-01-01

    Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism. PMID:25028509

  15. Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells

    SciTech Connect

    Tada, Hiroyuki; Nemoto, Eiji; Kanaya, Sousuke; Hamaji, Nozomu; Sato, Hisae; Shimauchi, Hidetoshi

    2010-04-16

    Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca{sup 2+}Ca{sub o}{sup 2+} has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca{sub o}{sup 2+} signaling in odontogenesis remains unclear. We found that elevated Ca{sub o}{sup 2+} increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca{sup 2+} increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca{sup 2+} channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca{sup 2+}, suggesting that the Ca{sup 2+} influx from Ca{sup 2+} channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca{sup 2+}-sensing receptors (CaSR) and only respond slightly to other cations such as Sr{sup 2+} and spermine, suggesting that dental pulp cells respond to Ca{sub o}{sup 2+} to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca{sub o}{sup 2+} among cations.

  16. Extracellular matrix protein in calcified endoskeleton: a potential additive for crystal growth and design

    NASA Astrophysics Data System (ADS)

    Azizur Rahman, M.; Fujimura, Hiroyuki; Shinjo, Ryuichi; Oomori, Tamotsu

    2011-06-01

    In this study, we demonstrate a key function of extracellular matrix proteins (ECMPs) on seed crystals, which are isolated from calcified endoskeletons of soft coral and contain only CaCO 3 without any living cells. This is the first report that an ECMP protein extracted from a marine organism could potentially influence in modifying the surface of a substrate for designing materials via crystallization. We previously studied with the ECMPs from a different type of soft coral ( Sinularia polydactyla) without introducing any seed crystals in the process , which showed different results. Thus, crystallization on the seed in the presence of ECMPs of present species is an important first step toward linking function to individual proteins from soft coral. For understanding this interesting phenomenon, in vitro crystallization was initiated in a supersaturated solution on seed particles of calcite (1 0 4) with and without ECMPs. No change in the crystal growth shape occurred without ECMPs present during the crystallization process. However, with ECMPs, the morphology and phase of the crystals in the crystallization process changed dramatically. Upon completion of crystallization with ECMPs, an attractive crystal morphology was found. Scanning electron microscopy (SEM) was utilized to observe the crystal morphologies on the seeds surface. The mineral phases of crystals nucleated by ECMPs on the seeds surface were examined by Raman spectroscopy. Although 50 mM Mg 2+ is influential in making aragonite in the crystallization process, the ECMPs significantly made calcite crystals even when 50 mM Mg 2+ was present in the process. Crystallization with the ECMP additive seems to be a technically attractive strategy to generate assembled micro crystals that could be used in crystals growth and design in the Pharmaceutical and biotechnology industries.

  17. Extracellular Superoxide Dismutase Regulates the Expression of Small GTPase Regulatory Proteins GEFs, GAPs, and GDI

    PubMed Central

    Laukkanen, Mikko O.; Cammarota, Francesca; Esposito, Tiziana; Salvatore, Marco; Castellone, Maria D.

    2015-01-01

    Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3–induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3–driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme. PMID:25751262

  18. Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

    PubMed

    Laukkanen, Mikko O; Cammarota, Francesca; Esposito, Tiziana; Salvatore, Marco; Castellone, Maria D

    2015-01-01

    Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3-induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3-driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme. PMID:25751262

  19. Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    PubMed

    Park, Chul; Helm, Richard F; Novak, John T

    2008-12-01

    The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study. PMID:19146099

  20. Accumulation of extracellular proteins bearing unique proline-rich motifs in intercellular spaces of the legume nodule parenchyma.

    PubMed

    Sherrier, D J; Taylor, G S; Silverstein, K A T; Gonzales, M B; VandenBosch, K A

    2005-04-01

    Nodulins encoding repetitive proline-rich cell wall proteins (PRPs) are induced during early interactions with rhizobia, suggesting a massive restructuring of the plant extracellular matrix during infection and nodulation. However, the proteins corresponding to these gene products have not been isolated or characterized, nor have cell wall localizations been confirmed. Posttranslational modifications, conformation, and interactions with other wall polymers are difficult to predict on the basis of only the deduced amino acid sequence of PRPs. PsENOD2 is expressed in nodule parenchyma tissue during nodule organogenesis and encodes a protein with distinctive PRP motifs that are rich in glutamate and basic amino acids. A database search for the ENOD2 signature motifs indicates that similar proteins may have a limited phylogenetic distribution, as they are presently only known from legumes. To determine the ultrastructural location of the proteins, antibodies were raised against unique motifs from the predicted ENOD2 sequence. The antibodies recognized nodule-specific proteins in pea (Pisum sativum), with a major band detected at 110 kDa, representing a subset of PRPs from nodules. The protein was detected specifically in organelles of the secretory pathway and intercellular spaces in the nodule parenchyma, but it was not abundant in primary walls. Similar proteins with an analogous distribution were detected in soybean (Glycine max). The use of polyclonal antibodies raised against signature motifs of extracellular matrix proteins thus appears to be an effective strategy to identify and isolate specific structural proteins for functional analysis. PMID:15868212

  1. Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation

    PubMed Central

    Moore, Nadia H.; Costa, Lucio G.; Shaffer, Scott A; Goodlett, David R.; Guizzetti, Marina

    2009-01-01

    Astrocytes play an important role in neuronal development through the release of soluble factors that affect neuronal maturation. Shotgun proteomics followed by Gene Ontology analysis was used in this study to identify proteins present in the conditioned medium of primary rat astrocytes. 133 secreted proteins were identified, the majority of which were never before reported to be produced by astrocytes. Extracellular proteins were classified based on their biological and molecular functions; most of the identified proteins were involved in neuronal development. Semi-quantitative proteomic analysis was carried out to identify changes in the levels of proteins released by astrocytes after stimulation with the cholinergic agonist carbachol, as we have previously reported that carbachol-treated astrocytes elicit neuritogenesis in hippocampal neurons through the release of soluble factors. Carbachol up-regulated the secretion of 15 proteins and down-regulated the release of 17 proteins. Changes in the levels of four proteins involved in neuronal differentiation (thrombospondin-1, fibronectin, plasminogen activator inhibitor-1, and plasminogen activator urokinase) were verified by Western blot or ELISA. In conclusion, this study identified a large number of proteins involved in neuronal development in the astrocyte secretome and implicated extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation. PMID:19077055

  2. Mass Spectrometry Analysis of the Extracellular Peptidome of Lactococcus lactis: Lines of Evidence for the Coexistence of Extracellular Protein Hydrolysis and Intracellular Peptide Excretion.

    PubMed

    Guillot, Alain; Boulay, Mylène; Chambellon, Émilie; Gitton, Christophe; Monnet, Véronique; Juillard, Vincent

    2016-09-01

    We report here the use of a peptidomic approach to revisit the extracellular proteolysis of Lactococcus lactis. More than 1800 distinct peptides accumulate externally during growth of the plasmid-free protease-negative strain L. lactis IL1403 in a protein- and peptide-free medium. These peptides mainly originate from cell-surface- and cytoplasmic-located proteins, despite the fact that no cell lysis could be evidenced. Positioning each identified peptide on its parental protein sequence demonstrated the involvement of exo- and endopeptidase activities. The endopeptidases responsible for the release of surface and cytoplasmic peptides had distinct specificities. The membrane-anchored protease HtrA was responsible for the release of only a part of the surface peptides, and its preference for branched-chain amino acids in the N-terminal side of the cleaved bond was established in situ. Other yet uncharacterized surface proteases were also involved. Several lines of evidence suggest that surface and cytoplasmic peptides were produced by different routes, at least part of the latter being most likely excreted as peptides from the cells. The mechanism by which these cytoplasmic peptides are excreted remains an open question, as it is still the case for excreted cytoplasmic proteins. PMID:27439475

  3. FGF signalling regulates bone growth through autophagy.

    PubMed

    Cinque, Laura; Forrester, Alison; Bartolomeo, Rosa; Svelto, Maria; Venditti, Rossella; Montefusco, Sandro; Polishchuk, Elena; Nusco, Edoardo; Rossi, Antonio; Medina, Diego L; Polishchuk, Roman; De Matteis, Maria Antonietta; Settembre, Carmine

    2015-12-10

    Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes. PMID:26595272

  4. Staphylococcus aureus Manganese Transport Protein C (MntC) Is an Extracellular Matrix- and Plasminogen-Binding Protein

    PubMed Central

    Salazar, Natália; Castiblanco-Valencia, Mónica Marcela; da Silva, Ludmila Bezerra; de Castro, Íris Arantes; Monaris, Denize; Masuda, Hana Paula; Barbosa, Angela Silva; Arêas, Ana Paula Mattos

    2014-01-01

    Infections caused by Staphylococcus aureus – particularly nosocomial infections - represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM). Manganese transport protein C (MntC), a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA). The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation. PMID:25409527

  5. Molecular Actions of Ovarian Cancer G Protein-Coupled Receptor 1 Caused by Extracellular Acidification in Bone

    PubMed Central

    Yuan, Feng-Lai; Zhao, Ming-Dong; Jiang, Li-Bo; Wang, Hui-Ren; Cao, Lu; Zhou, Xiao-Gang; Li, Xi-Lei; Dong, Jian

    2014-01-01

    Extracellular acidification occurs under physiologic and pathologic conditions, such as exercise, ischemia, and inflammation. It has been shown that acidosis has various adverse effects on bone. In recent years there has been increasing evidence which indicates that ovarian cancer G protein-coupled receptor 1 (OGR1) is a pH-sensing receptor and mediates a variety of extracellular acidification-induced actions on bone cells and other cell types. Recent studies have shown that OGR1 is involved in the regulation of osteoclast differentiation, survival, and function, as well as osteoblast differentiation and bone formation. Moreover, OGR1 also regulates acid-induced apoptosis of endplate chondrocytes in intervertebral discs. These observations demonstrate the importance of OGR1 in skeletal development and metabolism. Here, we provide an overview of OGR1 regulation ofosteoclasts, osteoblasts, and chondrocytes, and the molecular actions of OGR1 induced by extracellular acidification in the maintenance of bone health. PMID:25479080

  6. Protective Immunity to Vaccinia Virus Induced by Vaccination with Multiple Recombinant Outer Membrane Proteins of Intracellular and Extracellular Virions

    PubMed Central

    Fogg, Christiana; Lustig, Shlomo; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2004-01-01

    Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-μg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine. PMID:15367588

  7. Nanoscale viscoelasticity of extracellular matrix proteins in soft tissues: A multiscale approach.

    PubMed

    Miri, Amir K; Heris, Hossein K; Mongeau, Luc; Javid, Farhad

    2014-02-01

    It is hypothesized that the bulk viscoelasticity of soft tissues is determined by two length-scale-dependent mechanisms: the time-dependent response of the extracellular matrix (ECM) proteins at the nanometer scale and the biophysical interactions between the ECM solid structure and interstitial fluid at the micrometer scale. The latter is governed by poroelasticity theory assuming free motion of the interstitial fluid within the porous ECM structure. In a recent study (Heris, H.K., Miri, A.K., Tripathy, U., Barthelat, F., Mongeau, L., 2013. J. Mech. Behav. Biomed. Mater.), atomic force microscopy was used to measure the response of porcine vocal folds to a creep loading and a 50-nm sinusoidal oscillation. A constitutive model was calibrated and verified using a finite element model to accurately predict the nanoscale viscoelastic moduli of ECM. A generally good correlation was obtained between the predicted variation of the viscoelastic moduli with depth and that of hyaluronic acids in vocal fold tissue. We conclude that hyaluronic acids may regulate vocal fold viscoelasticity. The proposed methodology offers a characterization tool for biomaterials used in vocal fold augmentations. PMID:24317493

  8. Nanoscale Viscoelasticity of Extracellular Matrix Proteins in Soft Tissues: a Multiscale Approach

    PubMed Central

    Miri, Amir K.; Heris, Hossein K.; Mongeau, Luc; Javid, Farhad

    2013-01-01

    We propose that the bulk viscoelasticity of soft tissues results from two length-scale-dependent mechanisms: the time-dependent response of extracellular matrix proteins (ECM) at the nanometer scale and the biophysical interactions between the ECM solid structure and interstitial fluid at the micrometer scale. The latter was modeled using the poroelasticity theory with an assumption of free motion of the interstitial fluid within the porous ECM structure. Following a recent study (Heris, H.K., Miri, A.K., Tripathy, U., Barthelat, F., Mongeau, L., 2013. Journal of the Mechanical Behavior of Biomedical Materials), atomic force microscopy was used to perform creep loading and 50-nm sinusoidal oscillations on porcine vocal folds. The proposed model was calibrated by a finite element model to accurately predict the nanoscale viscoelastic moduli of ECM. A linear correlation was observed between the in-depth distribution of the viscoelastic moduli and that of hyaluronic acids in the vocal fold tissue. We conclude that hyaluronic acids may regulate the vocal fold viscoelasticity at nanoscale. The proposed methodology offers a characterization tool for biomaterials used in vocal fold augmentations. PMID:24317493

  9. Adolescent binge ethanol treatment alters adult brain regional volumes, cortical extracellular matrix protein and behavioral flexibility

    PubMed Central

    Coleman, Leon Garland; Liu, Wen; Oguz, Ipek; Styner, Martin; Crews, Fulton T.

    2014-01-01

    Adolescents binge drink more than any other age group, increasing risk of disrupting the development of the frontal cortex. We hypothesized that adolescent binge drinking would lead to persistent alterations in adulthood. In this study, we modeled adolescent weekend underage binge-drinking, using adolescent mice (post-natal days [P] 28–37). The adolescent intermittent binge ethanol (AIE) treatment includes 6 binge intragastric doses of ethanol in an intermittent pattern across adolescence. Assessments were conducted in adulthood following extended abstinence to determine if there were persistent changes in adults. Reversal learning, open field and other behavioral assessments as well as brain structure using magnetic imaging and immunohistochemistry were determined. We found AIE did not impact adult Barnes Maze learning. However, AIE did cause reversal learning deficits in adults. AIE also caused structural changes in the adult brain. AIE was associated with adulthood volume enlargements in specific brain regions without changes in total brain volume. Enlarged regions included the orbitofrontal cortex (OFC, 4%), cerebellum (4.5%), thalamus (2%), internal capsule (10%) and genu of the corpus callosum (7%). The enlarged OFC volume in adults after AIE is consistent with previous imaging studies in human adolescents. AIE treatment was associated with significant increases in the expression of several extracellular matrix (ECM) proteins in the adult OFC including WFA (55%), Brevican (32%), Neurocan (105%), Tenacin-C (25%), and HABP (5%). These findings are consistent with AIE causing persistent changes in brain structure that could contribute to a lack of behavioral flexibility. PMID:24275185

  10. Functions of the extracellular histidine residues of receptor activity-modifying proteins vary within adrenomedullin receptors

    SciTech Connect

    Kuwasako, Kenji Kitamura, Kazuo; Nagata, Sayaka; Kato, Johji

    2008-12-05

    Receptor activity-modifying protein (RAMP)-2 and -3 chaperone calcitonin receptor-like receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM receptor trafficking and receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [{sup 125}I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM receptor function.

  11. Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo

    PubMed Central

    1990-01-01

    The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis. PMID:2277082

  12. The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation

    SciTech Connect

    Coutinho Carneiro, Vitor; Moraes Maciel, Renata de; Caetano de Abreu da Silva, Isabel; Furtado Madeira da Costa, Rodrigo; Neto Paiva, Claudia; Torres Bozza, Marcelo; Rosado Fantappie, Marcelo

    2009-12-25

    Schistosoma mansoni HMGB1 (SmHMGB1) was revealed to be a substrate for the parasite histone acetyltransferases SmGCN5 and SmCBP1. We found that full-length SmHMGB1, as well as its HMG-box B (but not HMG-box A) were acetylated in vitro by SmGCN5 and SmCBP1. However, SmCBP1 was able to acetylate both substrates more efficiently than SmGCN5. Interestingly, the removal of the C-terminal acidic tail of SmHMGB1 (SmHMGB1{Delta}C) resulted in increased acetylation of the protein. We showed by mammalian cell transfection assays that SmHMGB1 and SmHMGB1{Delta}C were transported from the nucleus to the cytoplasm after sodium butyrate (NaB) treatment. Importantly, after NaB treatment, SmHMGB1 was also present outside the cell. Together, our data suggest that acetylation of SmHMGB1 plays a role in cellular trafficking, culminating with its secretion to the extracellular milieu. The possible role of SmHMGB1 acetylation in the pathogenesis of schistosomiasis is discussed.

  13. Neural cell alignment by patterning gradients of the extracellular matrix protein laminin

    PubMed Central

    Chelli, Beatrice; Barbalinardo, Marianna; Valle, Francesco; Greco, Pierpaolo; Bystrenova, Eva; Bianchi, Michele; Biscarini, Fabio

    2014-01-01

    Anisotropic orientation and accurate positioning of neural cells is achieved by patterning stripes of the extracellular matrix protein laminin on the surface of polystyrene tissue culture dishes by micromoulding in capillaries (MIMICs). Laminin concentration decreases from the entrance of the channels in contact with the reservoir towards the end. Immunofluorescence analysis of laminin shows a decreasing gradient of concentration along the longitudinal direction of the stripes. The explanation is the superposition of diffusion and convection of the solute, the former dominating at length scales near the entrance (characteristic length around 50 μm), the latter further away (length scale in excess of 900 μm). These length scales are independent of the channel width explored from about 15 to 45 μm. Neural cells are randomly seeded and selectively adhere to the pattern, leaving the unpatterned areas depleted even upon 6 days of incubation. Cell alignment was assessed by the orientation of the long axis of the 4′,6-diamidino-2-phenylindole-stained nuclei. Samples on patterned the laminin area exhibit a large orientational order parameter. As control, cells on the unpatterned laminin film exhibit no preferential orientation. This implies that the anisotropy of laminin stripes is an effective chemical stimulus for cell recruiting and alignment. PMID:24501672

  14. Novel extracellular matrix for cell sheet recovery using genetically engineered elastin-like protein.

    PubMed

    Mie, Masayasu; Mizushima, Yasunori; Kobatake, Eiry

    2008-07-01

    Elastin-like peptides (ELPs) sequences are repeats of the pentapeptide GVGVP, and they have the ability to coaggregate reversibly, depending on the temperature. By exploiting this characteristic, a novel extracellular matrix protein (ECM) containing ELP was developed genetically to harvest a cell sheet from a culture dish. One of the ELP constructs, G288, consisted of 288 repeats of the sequence GVGVGP (G); it was attached to a hydrophobic dish surface. Next, cells with the sequence His-G36RG36, which has a His tag and an RGD sequence (R) that promotes attachment of the cell between the G36 sequences, consisted of 36 repeats of the sequence GVGVP, were added to the dish. After these cells became confluent, the temperature was changed to 20 degrees C in order to reverse the coaggregation. At this temperature, cells could be detached from the dish as a cell sheet. This genetically engineering method for construction of thermoresponsive ECM would be suitable to modify ECM with further functional domains. PMID:18161837

  15. Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1*

    PubMed Central

    Wohl, Alexander P.; Troilo, Helen; Collins, Richard F.; Baldock, Clair

    2016-01-01

    Since the discovery of bone morphogenetic proteins (BMPs) as pluripotent cytokines extractable from bone matrix, it has been speculated how targeting of BMPs to the extracellular matrix (ECM) modulates their bioavailability. Understanding these processes is crucial for elucidating pathomechanisms of connective tissue disorders characterized by ECM deficiency and growth factor dysregulation. Here, we provide evidence for a new BMP targeting and sequestration mechanism that is controlled by the ECM molecule fibrillin-1. We present the nanoscale structure of the BMP-7 prodomain-growth factor complex using electron microscopy, small angle x-ray scattering, and circular dichroism spectroscopy, showing that it assumes an open V-like structure when it is bioactive. However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements. BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding. Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor. PMID:27059954

  16. The Extracellular Matrix Protein Brevican Limits Time-Dependent Enhancement of Cocaine Conditioned Place Preference.

    PubMed

    Lubbers, Bart R; Matos, Mariana R; Horn, Annemarie; Visser, Esther; Van der Loo, Rolinka C; Gouwenberg, Yvonne; Meerhoff, Gideon F; Frischknecht, Renato; Seidenbecher, Constanze I; Smit, August B; Spijker, Sabine; van den Oever, Michel C

    2016-06-01

    Cocaine-associated environmental cues sustain relapse vulnerability by reactivating long-lasting memories of cocaine reward. During periods of abstinence, responding to cocaine cues can time-dependently intensify a phenomenon referred to as 'incubation of cocaine craving'. Here, we investigated the role of the extracellular matrix protein brevican in recent (1 day after training) and remote (3 weeks after training) expression of cocaine conditioned place preference (CPP). Wild-type and Brevican heterozygous knock-out mice, which express brevican at ~50% of wild-type levels, received three cocaine-context pairings using a relatively low dose of cocaine (5 mg/kg). In a drug-free CPP test, heterozygous mice showed enhanced preference for the cocaine-associated context at the remote time point compared with the recent time point. This progressive increase was not observed in wild-type mice and it did not generalize to contextual-fear memory. Virally mediated overexpression of brevican levels in the hippocampus, but not medial prefrontal cortex, of heterozygous mice prevented the progressive increase in cocaine CPP, but only when overexpression was induced before conditioning. Post-conditioning overexpression of brevican did not affect remote cocaine CPP, suggesting that brevican limited the increase in remote CPP by altering neuro-adaptive mechanisms during cocaine conditioning. We provide causal evidence that hippocampal brevican levels control time-dependent enhancement of cocaine CPP during abstinence, pointing to a novel substrate that regulates incubation of responding to cocaine-associated cues. PMID:26711251

  17. Haemophilus influenzae P4 Interacts With Extracellular Matrix Proteins Promoting Adhesion and Serum Resistance.

    PubMed

    Su, Yu-Ching; Mukherjee, Oindrilla; Singh, Birendra; Hallgren, Oskar; Westergren-Thorsson, Gunilla; Hood, Derek; Riesbeck, Kristian

    2016-01-15

    Interaction with the extracellular matrix (ECM) is one of the successful colonization strategies employed by nontypeable Haemophilus influenzae (NTHi). Here we identified Haemophilus lipoprotein e (P4) as a receptor for ECM proteins. Purified recombinant P4 displayed a high binding affinity for laminin (Kd = 9.26 nM) and fibronectin (Kd = 10.19 nM), but slightly less to vitronectin (Kd = 16.51 nM). A P4-deficient NTHi mutant showed a significantly decreased binding to these ECM components. Vitronectin acquisition conferred serum resistance to both P4-expressing NTHi and Escherichia coli transformants. P4-mediated bacterial adherence to pharynx, type II alveolar, and bronchial epithelial cells was mainly attributed to fibronectin. Importantly, a significantly reduced bacterial infection was observed in the middle ear of the Junbo mouse model when NTHi was devoid of P4. In conclusion, our data provide new insight into the role of P4 as an important factor for Haemophilus colonization and subsequent respiratory tract infection. PMID:26153407

  18. Extracellular Regulation of Bone Morphogenetic Protein Activity by the Microfibril Component Fibrillin-1.

    PubMed

    Wohl, Alexander P; Troilo, Helen; Collins, Richard F; Baldock, Clair; Sengle, Gerhard

    2016-06-10

    Since the discovery of bone morphogenetic proteins (BMPs) as pluripotent cytokines extractable from bone matrix, it has been speculated how targeting of BMPs to the extracellular matrix (ECM) modulates their bioavailability. Understanding these processes is crucial for elucidating pathomechanisms of connective tissue disorders characterized by ECM deficiency and growth factor dysregulation. Here, we provide evidence for a new BMP targeting and sequestration mechanism that is controlled by the ECM molecule fibrillin-1. We present the nanoscale structure of the BMP-7 prodomain-growth factor complex using electron microscopy, small angle x-ray scattering, and circular dichroism spectroscopy, showing that it assumes an open V-like structure when it is bioactive. However, upon binding to fibrillin-1, the BMP-7 complex is rendered into a closed ring shape, which also confers latency to the growth factor, as demonstrated by bioactivity measurements. BMP-7 prodomain variants were used to map the critical epitopes for prodomain-growth factor and prodomain-prodomain binding. Together, these data show that upon prodomain binding to fibrillin-1, the BMP-7 complex undergoes a conformational change, which denies access of BMP receptors to the growth factor. PMID:27059954

  19. Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins

    PubMed Central

    Cooke, M. J.; Phillips, S. R.; Shah, D. S.H.; Athey, D.; Lakey, J. H.

    2008-01-01

    Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory. PMID:19002844

  20. Neural differentiation regulated by biomimetic surfaces presenting motifs of extracellular matrix proteins.

    PubMed

    Cooke, M J; Zahir, T; Phillips, S R; Shah, D S H; Athey, D; Lakey, J H; Shoichet, M S; Przyborski, S A

    2010-06-01

    The interaction between cells and the extracellular matrix (ECM) is essential during development. To elucidate the function of ECM proteins on cell differentiation, we developed biomimetic surfaces that display specific ECM peptide motifs in a controlled manner. Presentation of ECM domains for collagen, fibronectin, and laminin influenced the formation of neurites by differentiating PC12 cells. The effect of these peptide sequences was also tested on the development of adult neural stem/progenitor cells. In this system, collagen I and fibronectin induced the formation of beta-III-tubulin positive cells, whereas collagen IV reduced such differentiation. Biomimetic surfaces composed of multiple peptide types enabled the combinatorial effects of various ECM motifs to be studied. Surfaces displaying combined motifs were often predictable as a result of the synergistic effects of ECM peptides studied in isolation. For example, the additive effects of fibronectin and laminin resulted in greater expression of beta-III-tubulin positive cells, whereas the negative effect of the collagen IV domain was canceled out by coexpression of collagen I. However, simultaneous expression of certain ECM domains was less predictable. These data highlight the complexity of the cellular response to combined ECM signals and the need to study the function of ECM domains individually and in combination. PMID:19653304

  1. Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins.

    PubMed

    Cooke, M J; Phillips, S R; Shah, D S H; Athey, D; Lakey, J H; Przyborski, S A

    2008-02-01

    Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study we evaluate the application of a novel technology for the immobilisation of functional domains of known ECM proteins in a controlled manner on a surface. By examining the adherence of cultured PC12 cells to alternative growth surfaces, we show that surfaces coated with motifs from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with the corresponding whole molecules. Furthermore, we show that the adherence of cells can be controlled by modifying the hydropathic properties of the surface to either enhance or inhibit cell attachment. Collectively, these data demonstrate the application of a new technology to enable optimisation of cell growth in the tissue culture laboratory. PMID:19002844

  2. Hypoxia and Extracellular Matrix Proteins Influence Angiogenesis and Lymphangiogenesis in Mouse Embryoid Bodies

    PubMed Central

    Foskett, Andrea M.; Ezekiel, Uthayashanker R.; Trzeciakowski, Jerome P.; Zawieja, David C.; Muthuchamy, Mariappan

    2011-01-01

    Regulatory mechanisms for angiogenesis are relatively well established compared to lymphangiogenesis. Few studies have shown that a combination of vascular endothelial growth factor VEGF-A/C with hypoxia or collagen matrix promotes lymphatic structures along with blood vessel development in mouse embryoid bodies (EB). In this study we tested the hypothesis that while hypoxia combined with prolonged VEGF-A/C treatment would induce early lymphangiogenesis in addition to angiogenesis in mouse EBs, under similar conditions specific extracellular matrix (ECM) proteins would promote lymphatic vessel-like structures over angiogenesis. EBs were subjected to four conditions and were maintained under normoxia and hypoxia (21% and 2.6% O2, respectively) with or without VEGF-A/C. Microarray analyses of normoxic and hypoxic EBs, and immunofluorescence data showed very low expression of early lymphatic endothelial cell (LEC) markers, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), and prospero-related homeobox 1 (Prox1) at early time points. Double immunofluorescence using MECA-32 and Prox1/LYVE1 demonstrated that combined hypoxia and VEGF-A/C treatment promoted formation of blood vessel-like structures, whereas only Prox1+/LYVE1+ LECs were detected in EBs at E22.5. Furthermore, EBs were grown on laminin or collagen-I coated plates and were subjected to the four treatments as described above. Results revealed that LECs in EBs at E36.5 attached better to collagen-I, resulting in an organized network of lymphatic vessel-like structures as compared to EBs grown on laminin. However, blood vessel-like structures were less favored under these same conditions. Collectively, our data demonstrate that hypoxia combined with growth factors promotes angiogenesis, whereas combination of these conditions with specific ECM proteins favors lymphangiogenesis processes in mouse EBs. PMID:22194726

  3. Biomechanics of fibrous proteins of the extracellular matrix studied by Brillouin scattering

    PubMed Central

    Palombo, Francesca; Winlove, C. Peter; Edginton, Ryan S.; Green, Ellen; Stone, Nick; Caponi, Silvia; Madami, Marco; Fioretto, Daniele

    2014-01-01

    Brillouin light scattering (BLS) spectroscopy is a technique that is able to detect thermally excited phonons within a material. The speed of propagation of these phonons can be determined from the magnitude of the Brillouin frequency shift between incident and scattered light, thereby providing a measure of the mechanical properties of the material in the gigahertz range. The mechanical properties of the extracellular matrices of biological tissues and their constituent biopolymers are important for normal tissue function and disturbances in these properties are widely implicated in disease. BLS offers the prospect of measuring mechanical properties on a microscopic scale in living tissues, thereby providing insights into structure–function relationships under normal and pathological conditions. In this study, we investigated BLS in collagen and elastin—the fibrous proteins of the extracellular matrix (ECM). Measurements were made on type I collagen in rat tail tendon, type II collagen in articular cartilage and nuchal ligament elastin. The dependence of the BLS spectrum on fibre orientation was investigated in a backscattering geometry using a reflective substrate. Two peaks, a bulk mode arising from phonon propagation along a quasi-radial direction to the fibre axis and a mode parallel to the surface, depending on sample orientation relative to the fibre axis, could be distinguished. The latter peak was fitted to a model of wave propagation through a hexagonally symmetric elastic solid, and the five components of the elasticity tensor were combined to give axial and transverse Young's, shear and bulk moduli of the fibres. These were 10.2, 8.3, 3.2 and 10.9 GPa, and 6.1, 5.3, 1.9 and 8 GPa for dehydrated type I collagen and elastin, respectively. The former values are close to those previously reported. A microfocused BLS approach was also applied providing selection of single fibres. The moduli of collagen and elastin are much higher than those measured at

  4. Biomechanics of fibrous proteins of the extracellular matrix studied by Brillouin scattering.

    PubMed

    Palombo, Francesca; Winlove, C Peter; Edginton, Ryan S; Green, Ellen; Stone, Nick; Caponi, Silvia; Madami, Marco; Fioretto, Daniele

    2014-12-01

    Brillouin light scattering (BLS) spectroscopy is a technique that is able to detect thermally excited phonons within a material. The speed of propagation of these phonons can be determined from the magnitude of the Brillouin frequency shift between incident and scattered light, thereby providing a measure of the mechanical properties of the material in the gigahertz range. The mechanical properties of the extracellular matrices of biological tissues and their constituent biopolymers are important for normal tissue function and disturbances in these properties are widely implicated in disease. BLS offers the prospect of measuring mechanical properties on a microscopic scale in living tissues, thereby providing insights into structure-function relationships under normal and pathological conditions. In this study, we investigated BLS in collagen and elastin-the fibrous proteins of the extracellular matrix (ECM). Measurements were made on type I collagen in rat tail tendon, type II collagen in articular cartilage and nuchal ligament elastin. The dependence of the BLS spectrum on fibre orientation was investigated in a backscattering geometry using a reflective substrate. Two peaks, a bulk mode arising from phonon propagation along a quasi-radial direction to the fibre axis and a mode parallel to the surface, depending on sample orientation relative to the fibre axis, could be distinguished. The latter peak was fitted to a model of wave propagation through a hexagonally symmetric elastic solid, and the five components of the elasticity tensor were combined to give axial and transverse Young's, shear and bulk moduli of the fibres. These were 10.2, 8.3, 3.2 and 10.9 GPa, and 6.1, 5.3, 1.9 and 8 GPa for dehydrated type I collagen and elastin, respectively. The former values are close to those previously reported. A microfocused BLS approach was also applied providing selection of single fibres. The moduli of collagen and elastin are much higher than those measured at lower

  5. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    PubMed

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-01

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera. PMID:27386892

  6. Extracellular heat shock protein 90 binding to TGFβ receptor I participates in TGFβ-mediated collagen production in myocardial fibroblasts.

    PubMed

    García, Raquel; Merino, David; Gómez, Jenny M; Nistal, J Francisco; Hurlé, María A; Cortajarena, Aitziber L; Villar, Ana V

    2016-10-01

    The pathological remodeling heart shows an increase in left ventricular mass and an excess of extracellular matrix deposition that can over time cause heart failure. Transforming growth factor β (TGFβ) is the main cytokine controlling this process. The molecular chaperone heat shock protein 90 (Hsp90) has been shown to play a critical role in TGFβ signaling by stabilizing the TGFβ signaling cascade. We detected extracellular Hsp90 in complex with TGFβ receptor I (TGFβRI) in fibroblasts and determined a close proximity between both proteins suggesting a potential physical interaction between the two at the plasma membrane. This was supported by in silico studies predicting Hsp90 dimers and TGFβRI extracellular domain interaction. Both, Hsp90aa1 and Hsp90ab1 isoforms participate in TGFβRI complex. Extracellular Hsp90 inhibition lessened the yield of collagen production as well as the canonical TGFβ signaling cascade, and collagen protein synthesis was drastically reduced in Hsp90aa1 KO mice. These observations together with the significant increase in activity of Hsp90 at the plasma membrane pointed to a functional cooperative partnership between Hsp90 and TGFβRI in the fibrotic process. We propose that a surface population of Hsp90 extracellularly binds TGFβRI and this complex behaves as an active participant in collagen production in TGFβ-activated fibroblasts. We also offer an in vivo insight into the role of Hsp90 and its isoforms during cardiac remodeling in murine aortic banding model suffering from pathological cardiac remodeling and detect circulating Hsp90 overexpressed in remodeling mice. PMID:27418101

  7. Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis

    PubMed Central

    Iwata, Akiko; de Claro, R. Angelo; Morgan-Stevenson, Vicki L.; Tupper, Joan C.; Schwartz, Barbara R.; Liu, Li; Zhu, Xiaodong; Jordan, Katherine C.; Winn, Robert K.; Harlan, John M.

    2011-01-01

    Background Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival. Methodology/Principal Findings We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo. Conclusions/Significance Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins. PMID:21390214

  8. Expression of SPARC like protein 1 (SPARCL1), extracellular matrix-associated protein is down regulated in gastric adenocarcinoma

    PubMed Central

    Jakharia, Aniruddha; Borkakoty, Biswajyoti

    2016-01-01

    Background SPARC-like protein 1 (SPARCL1/Hevin), a member of the SPARC family is defined by the presence of a highly acidic domain-I, a follistatin-like domain, and an extracellular calcium (EC) binding domain. SPARCL1 has been shown to be down-regulated in many types of cancer and may serve as a negative regulator of cell growth and proliferation. Methods Both tumor and adjacent normal tissue were collected from patients with gastric adenocarcinoma. Monoclonal antibody developed against recombinant SPARCL1 was used to analyze the expression of SPARCL1 by immunohisto chemical and western blotting (WB) analysis. Results The expression of SPARCL1 was found to be significantly lower or negligible in gastric adenocarcinoma tissues in nearly all of the cases in comparison with adjacent normal tissue. This comparison was found to be independent of the patient’s age, sex, and stage of cancer. Conclusions We postulate that down regulation of SPARCL1 may be related to inactivation of its tumor suppressor functions and might play an important role in the development of gastric adenocarcinoma. PMID:27034797

  9. Extracellular functions of glycolytic enzymes of parasites: unpredicted use of ancient proteins.

    PubMed

    Gómez-Arreaza, Amaranta; Acosta, Hector; Quiñones, Wilfredo; Concepción, Juan Luis; Michels, Paul A M; Avilán, Luisana

    2014-02-01

    In addition of their usual intracellular localization where they are involved in catalyzing reactions of carbohydrate and energy metabolism by glycolysis, multiple studies have shown that glycolytic enzymes of many organisms, but notably pathogens, can also be present extracellularly. In the case of parasitic protists and helminths, they can be found either secreted or attached to the surface of the parasites. At these extracellular localizations, these enzymes have been shown to perform additional, very different so-called "moonlighting" functions, such as acting as ligands for a variety of components of the host. Due to this recognition, different extracellular glycolytic enzymes participate in various important parasite-host interactions such as adherence and invasion of parasites, modulation of the host's immune and haemostatic systems, promotion of angiogenesis, and acquisition of specific nutrients by the parasites. Accordingly, extracellular glycolytic enzymes are important for the invasion of the parasites and their establishment in the host, and in determining their virulence. PMID:24602601

  10. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    PubMed

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  11. Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs.

    PubMed

    Wang, Wei; Gao, Xuejiao; Guo, Mengyao; Zhang, Wenlong; Song, Xiaojing; Wang, Tiancheng; Zhang, Zecai; Jiang, Haichao; Cao, Yongguo; Zhang, Naisheng

    2014-10-01

    Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans. PMID:25153777

  12. Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

    PubMed Central

    Harwood, V J; Gordon, A S

    1994-01-01

    Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes. Images PMID:8031076

  13. DAMP and DIC: The role of extracellular DNA and DNA-binding proteins in the pathogenesis of DIC.

    PubMed

    Liaw, Patricia C; Ito, Takashi; Iba, Toshiaki; Thachil, Jecko; Zeerleder, Sacha

    2016-07-01

    Disseminated intravascular coagulation (DIC) is a heterogeneous group of disorders, which manifest as a spectrum of haemorrhage and thrombosis complicating many primary conditions including sepsis, trauma and malignancies. The pathophysiology of this condition is complex. In the recent years there is growing evidence that damage associated molecular patterns (DAMPs) play a crucial role in the pathogenesis of DIC. Upon cell-death and/or cell activation of hematopoietic and parenchymal cells extracellular cell-free DNA as well as DNA binding proteins (e.g. histones and high mobility group box 1 protein [HMGB1]) are released into circulation. This release is a highly regulated process mediated among others by serine proteases, such as factor VII-activating protease (FSAP) and DNase1. Circulating cell-free DNA has been demonstrated to influence primary and secondary hemostasis by inducing platelet aggregation, promoting coagulation activation, inhibition of fibrinolysis and directly interfering with clot stability. In this respect cell-free DNA in tissue as well as released into the circulation after neutrophil activation in the form of neutrophil extracellular traps (NETs) has been shown to be cytotoxic and highly procoagulant. DNA-binding proteins such as histones and HMGB1 are also strongly procoagulant and are involved in the pathogenesis of DIC. The present review gives an overview on how extracellular DNA is released into circulation and the structure of circulating DNA. In addition it summarizes the effect of extracellular DNA and DNA-binding proteins on platelet activation, plasmatic coagulation as well as fibrinolysis. PMID:26776504

  14. Association of levels of antibodies from patients with inflammatory bowel disease with extracellular proteins of food and probiotic bacteria.

    PubMed

    Hevia, Arancha; López, Patricia; Suárez, Ana; Jacquot, Claudine; Urdaci, María C; Margolles, Abelardo; Sánchez, Borja

    2014-01-01

    Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n = 50), Crohn's disease patients (CD, n = 37), and ulcerative colitis patients (UC, n = 15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P < 0.002; n = 52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. PMID:24991549

  15. A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix

    PubMed Central

    Shechter, Assaf; Glazer, Lilah; Cheled, Shira; Mor, Eyal; Weil, Simy; Berman, Amir; Bentov, Shmuel; Aflalo, Eliahu D.; Khalaila, Isam; Sagi, Amir

    2008-01-01

    Despite the proclamation of Lowenstam and Weiner that crustaceans are the “champions of mineral mobilization and deposition of the animal kingdom,” relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization. PMID:18480260

  16. Association of Levels of Antibodies from Patients with Inflammatory Bowel Disease with Extracellular Proteins of Food and Probiotic Bacteria

    PubMed Central

    Hevia, Arancha; López, Patricia; Suárez, Ana; Jacquot, Claudine; Urdaci, María C.; Margolles, Abelardo; Sánchez, Borja

    2014-01-01

    Inflammatory bowel disease (IBD) is an autoimmune disease characterized by a chronic inflammation of the gastrointestinal tract mucosa and is related to an abnormal immune response to commensal bacteria. Our aim of the present work has been to explore the levels of antibodies (IgG and IgA) raised against extracellular proteins produced by LAB and its association with IBD. We analyzed, by Western-blot and ELISA, the presence of serum antibodies (IgA and IgG) developed against extracellular protein fractions produced by different food bacteria from the genera Bifidobacterium and Lactobacillus. We used a sera collection consisting of healthy individuals (HC, n = 50), Crohn's disease patients (CD, n = 37), and ulcerative colitis patients (UC, n = 15). Levels of IgA antibodies developed against a cell-wall hydrolase from Lactobacillus casei subsp. rhamnosus GG (CWH) were significantly higher in the IBD group (P < 0.002; n = 52). The specificity of our measurements was confirmed by measuring IgA antibodies developed against the CWH peptide 365-VNTSNQTAAVSAS-377. IBD patients appeared to have different immune response to food bacteria. This paper sets the basis for developing systems for early detection of IBD, based on the association of high levels of antibodies developed against extracellular proteins from food and probiotic bacteria. PMID:24991549

  17. Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated

    SciTech Connect

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J.; Vander Wall, Todd A.; Groom, Joseph; Himmel, Michael E.; Westpheling, Janet

    2015-03-23

    Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes described with ability to digest lignocellulosic biomass without conventional pretreatment. The cellulolytic ability of different species varies dramatically and correlates with the presence of the multimodular cellulase CelA, which contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. This architecture exploits the cellulose surface ablation driven by its general cellulase processivity as well as excavates cavities into the surface of the substrate, revealing a novel paradigm for cellulase activity. We recently reported that a deletion of celA in C. bescii had a significant effect on its ability to utilize complex biomass. To analyze the structure and function of CelA and its role in biomass deconstruction, we constructed a new expression vector for C. bescii and were able, for the first time, to express significant quantities of full-length protein in vivo in the native host. The protein, which contains a Histidine tag, was active and excreted from the cell. Expression of CelA protein with and without its signal sequence allowed comparison of protein retained intracellularly to protein transported extracellularly. Analysis of protein in culture supernatants revealed that the extracellular CelA protein is glycosylated whereas the intracellular CelA is not, suggesting that either protein transport is required for this post-translational modification or that glycosylation is required for protein export. The mechanism and role of protein glycosylation in bacteria is poorly understood and the ability to express CelA in vivo in C. bescii will allow the study of the mechanism of protein glycosylation in this thermophile. Finally, it will also allow the study of glycosylation of CelA itself and its role in the

  18. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    PubMed Central

    2012-01-01

    Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins. PMID:22234238

  19. Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

    NASA Technical Reports Server (NTRS)

    Mooney, D. J.; Hansen, L. K.; Langer, R.; Vacanti, J. P.; Ingber, D. E.

    1994-01-01

    Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and

  20. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

    PubMed Central

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

    Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

  1. Controlling the spatial organization of cells and extracellular matrix proteins in engineered tissues using ultrasound standing wave fields

    PubMed Central

    Garvin, Kelley A.; Hocking, Denise C.; Dalecki, Diane

    2010-01-01

    Tissue engineering holds great potential for saving the lives of thousands of organ transplant patients who die each year while waiting for donor organs. However, to successfully fabricate tissues and organs in vitro, methodologies that recreate appropriate extracellular microenvironments to promote tissue regeneration are needed. In this study, we have developed an application of ultrasound standing wave field (USWF) technology to the field of tissue engineering. Acoustic radiation forces associated with USWF were used to non-invasively control the spatial distribution of mammalian cells and cell-bound extracellular matrix proteins within three-dimensional collagen-based engineered tissues. Cells were suspended in unpolymerized collagen solutions and were exposed to a continuous wave USWF, generated using a 1 MHz source, for 15 min at room temperature. Collagen polymerization occurred during USWF exposure resulting in the formation of three-dimensional collagen gels with distinct bands of aggregated cells. The density of cell bands was dependent on both the initial cell concentration and the pressure amplitude of the USWF. Importantly, USWF exposure did not decrease cell viability, but rather enhanced cell function. Alignment of cells into loosely clustered, planar cell bands significantly increased levels of cell-mediated collagen gel contraction and collagen fiber reorganization as compared to sham-exposed samples with a homogeneous cell distribution. Additionally, the extracellular matrix protein, fibronectin, was localized to cell banded areas by binding the protein to the cell surface prior to USWF exposure. By controlling cell and extracellular organization, this application of USWF technology is a promising approach for engineering tissues in vitro. PMID:20870341

  2. Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

    PubMed Central

    2010-01-01

    Background CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions. In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events. Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω) >1. Results Of the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid

  3. Identification and Characterization of a Novel 38.5-Kilodalton Cell Surface Protein of Staphylococcus aureus with Extended-Spectrum Binding Activity for Extracellular Matrix and Plasma Proteins

    PubMed Central

    Hussain, Muzaffar; Becker, Karsten; von Eiff, Christof; Schrenzel, Jacques; Peters, Georg; Herrmann, Mathias

    2001-01-01

    The ability to attach to host ligands is a well-established pathogenic factor in invasive Staphylococcus aureus disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (srtA) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene emp (1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR, emp was demonstrated in all 240 S. aureus isolates of a defined clinical strain collection as well as in 6 S. aureus laboratory strains, whereas it is lacking in all 10 S. epidermidis strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50, a significantly decreased adhesion of mEmp50 to immobilized fibronectin and fibrinogen, and restoration of these characteristics upon complementation of mEmp50. Emp expression was also demonstrable upon heterologous complementation of S. carnosus. rEmp expressed in Escherichia coli interacted with fibronectin, fibrinogen, and vitronectin in surface plasmon resonance experiments at a Kd of 21 nM, 91 nM, and 122 pM, respectively. In conclusion, the biologic characterization of Emp suggests that it is a member of the group of secreted S. aureus molecules that interact with an extended spectrum of host ligands and thereby contribute to S. aureus pathogenicity. PMID:11698365

  4. Ameloblastin, an Extracellular Matrix Protein, Affects Long Bone Growth and Mineralization.

    PubMed

    Lu, Xuanyu; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla A; Diekwisch, Thomas Gh; Luan, Xianghong

    2016-06-01

    Matrix molecules such as the enamel-related calcium-binding phosphoprotein ameloblastin (AMBN) are expressed in multiple tissues, including teeth, bones, and cartilage. Here we have asked whether AMBN is of functional importance for timely long bone development and, if so, how it exerts its function related to osteogenesis. Adolescent AMBN-deficient mice (AMBN(Δ5-6) ) suffered from a 33% to 38% reduction in femur length and an 8.4% shorter trunk spinal column when compared with WT controls, whereas there was no difference between adult animals. On a cellular level, AMBN truncation resulted in a shortened growth plate and a 41% to 49% reduction in the number of proliferating tibia chondrocytes and osteoblasts. Bone marrow stromal cells (BMSCs) isolated from AMBN mutant mice displayed defects in proliferation and differentiation potential as well as cytoskeleton organization. Osteogenesis-related growth factors, such as insulin-like growth factor 1 (IGF1) and BMP7, were also significantly (46% to 73%) reduced in AMBN-deficient BMSCs. Addition of exogenous AMBN restored cytoskeleton structures in AMBN mutant BMSCs and resulted in a dramatic 400% to 600% increase in BMP2, BMP7, and Col1A expression. Block of RhoA diminished the effect of AMBN on osteogenic growth factor and matrix protein gene expression. Addition of exogenous BMP7 and IGF1 rescued the proliferation and differentiation potential of AMBN-deficient BMSCs. Confirming the effects of AMBN on long bone growth, back-crossing of mutant mice with full-length AMBN overexpressors resulted in a complete rescue of AMBN(Δ5-6) bone defects. Together, these data indicate that AMBN affects extracellular matrix production and cell adhesion properties in the long bone growth plate, resulting in altered cytoskeletal dynamics, increased osteogenesis-related gene expression, as well as osteoblast and chondrocyte proliferation. We propose that AMBN facilitates rapid long bone growth and an important growth spurt during the

  5. Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering.

    PubMed

    Low, Pearlie S J; Tjin, Monica S; Fong, Eileen

    2015-01-01

    Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin. PMID:26132812

  6. Expression of the extracellular domain of OB-cadherin as an Fc fusion protein using bicistronic retroviral expression vector

    PubMed Central

    Lira, Cristina B. B.; Chu, Khoi; Lee, Yu-Chen; Hu, Mickey C-T.; Lin, Sue-Hwa

    2008-01-01

    Osteoblast cadherin (OB-cadherin, also known as cadherin-11) is a Ca2+-dependent homophilic cell adhesion molecule that is expressed mainly in osteoblasts. OB-cadherin is expressed in prostate cancer and may be involved in the homing of metastatic prostate cancer cells to bone. The extracellular domain of OB-cadherin may be used to inhibit the adhesion between prostate cancer cells and osteoblasts. In this report, we describe the expression of the extracellular domain of OB-cadherin as an Fc fusion protein (OB-CAD-Fc) in human embryonic kidney 293FT cells using a bicistronic retroviral vector. Coexpression of GFP and OB-CAD-Fc through the bicistronic vector permitted enrichment of OB-CAD-Fc–expressing cells by fluorescence-activated cell sorting. Recombinant OB-CAD-Fc proteins were secreted into cell medium, and about 0.85 mg of purified OB-CAD-Fc protein was purified from 1 liter of the conditioned medium using immobilized protein A-affinity chromatography. The purified OB-CAD-Fc was biologically active because it supported the adhesion of PC3 cells and L cells transduced with OB-cadherin. The availability of OB-CAD-Fc offers opportunities to test whether OB-CAD-Fc can be used to inhibit OB-cadherin–mediated prostate cancer bone metastasis in vivo or to generate antibodies for inhibiting the adhesion between prostate cancer cells and osteoblasts. PMID:18620062

  7. Proteins derived from neutrophil extracellular traps may serve as self-antigens and mediate organ damage in autoimmune diseases

    PubMed Central

    Knight, Jason S.; Carmona-Rivera, Carmelo; Kaplan, Mariana J.

    2012-01-01

    Neutrophils are the most abundant leukocytes in circulation and represent one of the first lines of defense against invading pathogens. Neutrophils possess a vast arsenal of antimicrobial proteins, which can be released from the cell by a death program termed NETosis. Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin decorated with granular and cytosolic proteins. Both exuberant NETosis and impaired clearance of NETs have been implicated in the organ damage of autoimmune diseases, such as systemic lupus erythematosus (SLE), small vessel vasculitis (SVV), and psoriasis. NETs may also represent an important source of modified autoantigens in SLE and SVV. Here, we review the autoimmune diseases linked to NETosis, with a focus on how modified proteins externalized on NETs may trigger loss of immune tolerance and promote organ damage. PMID:23248629

  8. Stimulation by extracellular ATP and UTP of the stress-activated protein kinase cascade in rat renal mesangial cells

    PubMed Central

    Huwiler, Andrea; van Rossum, Gerda; Wartmann, Markus; Pfeilschifter, Josef

    1997-01-01

    Extracellular adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP) have been shown to activate a nucleotide receptor (P2U receptor) in rat mesangial cells that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. This is followed by an increased activity of the mitogen-activated protein kinase cascade and cell proliferation. Here we show that ATP and UTP potently stimulate the stress-activated protein kinase pathway and phosphorylation of the transcription factor c-Jun.Both nucleotides stimulated a rapid (within 5 min) and concentration-dependent activation of stress-activated protein kinases as measured by the phosphorylation of c-Jun in a solid phase kinase assay.When added at 100 μM the rank order of potency of a series of nucleotide analogues for stimulation of c-Jun phosphorylation was UTP>ATP=UDP=ATPγS>2-methylthio-ATP>βγ-imido-ATP= ADP>AMP=UMP=adenosine=uridine. Activation of stress-activated protein kinase activity by ATP and UTP was dose-dependently attenuated by suramin.Down-regulation of protein kinase C-α, -δ and -ε isoenzymes by 24 h treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate did not inhibit ATP- and UTP-induced activation of c-Jun phosphorylation. Furthermore, the specific protein kinase C inhibitors, CGP 41251 and Ro 31-8220, did not inhibit nucleotide-stimulated c-Jun phosphorylation, suggesting that protein kinase C is not involved in ATP- and UTP-triggered stress-activated protein kinase activation.Pretreatment of the cells with pertussis toxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP- and UTP-induced c-Jun phosphorylation. Furthermore, N-acetyl-cysteine completely blocked the activation of stress-activated protein kinase in response to extracellular nucleotide stimulation.In summary, these results suggest that ATP and UTP trigger the activation of the stress-activated protein kinase module in mesangial cells by a

  9. Extracellular Matrix Proteins, Alkaline Phosphatase and Pyrophosphate as Molecular Determinants of Bone, Tooth, Kidney and Vascular Calcification

    NASA Astrophysics Data System (ADS)

    McKee, Marc D.

    2008-09-01

    Progress in biomineralization research in recent years has identified, characterized and described functions for key noncollagenous extracellular matrix proteins regulating crystal growth in the skeleton and dentition. Some of these same proteins expressed in soft tissues undergoing pathologic calcification also inhibit ectopic crystal growth. In addition to extracellular matrix proteins regulating matrix mineralization, the enzyme tissue-nonspecific alkaline phosphatase—which is highly expressed by cells in mineralized tissues—cleaves pyrophosphate, an anionic small-molecule inhibitor of mineralization. Together with the required mineral ion availability necessary for crystal growth, these molecular determinants appear to function in limiting the spread of pathologic calcification seen in soft tissues such as blood vessels and kidneys. Osteopontin, in particular, is a potent calcification inhibitor that accumulates in mineralized tissues and in calcified deposits during vascular calcification and nephrolithiasis/urolithiasis. Additional research is required to establish the exact temporal sequence in which the molecular determinants of pathologic calcification appear relative to mineral crystal growth in different tissues, and to establish their relationship (if any) to the activation of osteogenic differentiation programs.

  10. Human VE-Cadherin Fusion Protein as an Artificial Extracellular Matrix Enhancing the Proliferation and Differentiation Functions of Endothelial Cell.

    PubMed

    Xu, Ke; Shuai, Qizhi; Li, Xiaoning; Zhang, Yan; Gao, Chao; Cao, Lei; Hu, Feifei; Akaike, Toshihiro; Wang, Jian-xi; Gu, Zhongwei; Yang, Jun

    2016-03-14

    In an attempt to enhance endothelial cell capture and promote the vascularization of engineered tissue, we biosynthesized and characterized the recombinant fusion protein consisting of human vascular endothelial-cadherin extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a bioartificial extracellular matrix. The hVE-cad-Fc protein naturally formed homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on polystyrene plates. Atomic force microscop assay showed uniform hVE-cad-Fc distribution with nanorod topography. The hVE-cad-Fc matrix markedly promoted human umbilical vein endothelial cells (HUVECs) adhesion and proliferation with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix improved HUVECs migration, vWF expression, and NO release, which are closely related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous VE-cadherin/β-catenin proteins and effectively triggered the intracellular signals such as F-actin stress fiber, p-FAK, AKT, and Bcl-2. Taken together, hVE-cad-Fc could be a promising bioartificial matrix to promote vascularization in tissue engineering. PMID:26859785

  11. Multidrug resistance-associated protein 1 decreases the concentrations of antiepileptic drugs in cortical extracellular fluid in amygdale kindling rats

    PubMed Central

    Chen, Ying-hui; Wang, Cui-cui; Xiao, Xia; Wei, Li; Xu, Guoxiong

    2013-01-01

    Aim: To investigate whether multidrug resistance-associated protein 1 (MRP1) was responsible for drug resistence in refractory epilepsy in amygdale kindling rats. Methods: Rat amygdale kindling was used as a model of refractory epilepsy. The expression of MRP1 mRNA and protein in the brains was examined using RT-PCR and Western blot. MRP1-positive cells in the cortex and hippocampus were studied with immunohistochemical staining. The rats were intraperitoneally injected with phenytoin (50 mg/kg) or carbamazepine (20 mg/kg), and their concentrations in the cortical extracellular fluid were measured using microdialysis and HPLC. Probenecid, a MRP1 inhibitor (40 mmol/L, 50 μL) was administered through an inflow tube into the cortex 30 min before injection of the antiepileptic drugs. Results: The expression of MRP1 mRNA and protein was significantly up-regulated in the cortex and hippocampus in amygdale kindling rats compared with the control group. Furthermore, the number of MRP1-positive cells in the cortex and hippocampus was also significantly increased in amygdale kindling rats. Microdialysis studies showed that the concentrations of phenytoin and carbamazepine in the cortical extracellular fluid were significantly decreased in amygdale kindling rats. Pre-administration of probenecid could restore the concentrations back to their control levels. Conclusion: Up-regulation of MRP1 is responsible for the resistance of brain cells to antiepileptic drugs in the amygdale kindling rats. PMID:23474709

  12. Distinctive expression of extracellular matrix molecules at mRNA and protein levels during formation of cellular and acellular cementum in the rat.

    PubMed

    Sasano, Y; Maruya, Y; Sato, H; Zhu, J X; Takahashi, I; Mizoguchi, I; Kagayama, M

    2001-02-01

    Little is known about differential expression of extracellular matrices secreted by cementoblasts between cellular and acellular cementum. We hypothesize that cementoblasts lining acellular cementum express extracellular matrix genes differently from those lining cellular cementum, thereby forming two distinct types of extracellular matrices. To test this hypothesis, we investigated spatial and temporal gene expression of selected extracellular matrix molecules, that is type I collagen, bone sialoprotein, osteocalcin and osteopontin, during formation of both cellular and acellular cementum using in situ hybridization. In addition, their extracellularly deposited and accumulated proteins were examined immunohistochemically. The mRNA transcripts of pro-alpha1 (I) collagen were primarily localized in cementoblasts of cellular cementum and cementocytes, while those of bone sialoprotein were predominantly seen in cementoblasts lining acellular cementum. In contrast, osteocalcin was expressed by both types of cementoblasts and cementocytes and so was osteopontin but only transiently. Our immunohistochemical examination revealed that translated proteins were localized extracellularly where the genes had been expressed intracellularly. The present study demonstrated the distinctive expression of genes and proteins of the extracellular matrix molecules between cellular and acellular cementum. PMID:11432645

  13. An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility.

    PubMed

    Suarez, Andres; Huber, Robert J; Myre, Michael A; O'Day, Danton H

    2011-07-01

    CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote. PMID:21402150

  14. Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be modulated by small molecule ligands and, hence, these protein-protein interactions play a key role in biochemica...

  15. High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism

    PubMed Central

    Tullius, Michael V.; Harth, Günter; Horwitz, Marcus A.

    2001-01-01

    Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of Mycobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (glnA1 and sodA) from the rapid-growing, nonpathogenic Mycobacterium smegmatis, generated glnA1 and sodA mutants of M. smegmatis by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from M. tuberculosis. When each of the genes was expressed from a multicopy plasmid, M. smegmatis exported comparable proportions of both the M. tuberculosis and M. smegmatis GSs (in the glnA1 strain) or SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant M. smegmatis and M. tuberculosis strains even exported nonmycobacterial SODs. To determine the extent to which export of these large, leaderless proteins is expression dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenous M. tuberculosis GS and SOD. Similarly, the recombinant M. smegmatis strain exported bacterioferritin, a large (∼500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless

  16. Crosstalk between intracellular and extracellular signals regulating interneuron production, migration and integration into the cortex

    PubMed Central

    Peyre, Elise; Silva, Carla G.; Nguyen, Laurent

    2015-01-01

    During embryogenesis, cortical interneurons are generated by ventral progenitors located in the ganglionic eminences of the telencephalon. They travel along multiple tangential paths to populate the cortical wall. As they reach this structure they undergo intracortical dispersion to settle in their final destination. At the cellular level, migrating interneurons are highly polarized cells that extend and retract processes using dynamic remodeling of microtubule and actin cytoskeleton. Different levels of molecular regulation contribute to interneuron migration. These include: (1) Extrinsic guidance cues distributed along migratory streams that are sensed and integrated by migrating interneurons; (2) Intrinsic genetic programs driven by specific transcription factors that grant specification and set the timing of migration for different subtypes of interneurons; (3) Adhesion molecules and cytoskeletal elements/regulators that transduce molecular signalings into coherent movement. These levels of molecular regulation must be properly integrated by interneurons to allow their migration in the cortex. The aim of this review is to summarize our current knowledge of the interplay between microenvironmental signals and cell autonomous programs that drive cortical interneuron porduction, tangential migration, and intergration in the developing cerebral cortex. PMID:25926769

  17. Factor H–Related Protein 5 Interacts with Pentraxin 3 and the Extracellular Matrix and Modulates Complement Activation

    PubMed Central

    Csincsi, Ádám I.; Kopp, Anne; Zöldi, Miklós; Bánlaki, Zsófia; Uzonyi, Barbara; Hebecker, Mario; Caesar, Joseph J. E.; Pickering, Matthew C.; Daigo, Kenji; Hamakubo, Takao; Lea, Susan M.; Goicoechea de Jorge, Elena

    2015-01-01

    The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C–reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease. PMID:25855355

  18. Extracellular ATP promotes stomatal opening of Arabidopsis thaliana through heterotrimeric G protein α subunit and reactive oxygen species.

    PubMed

    Hao, Li-Hua; Wang, Wei-Xia; Chen, Chen; Wang, Yu-Fang; Liu, Ting; Li, Xia; Shang, Zhong-Lin

    2012-07-01

    In recent years, adenosine tri-phosphate (ATP) has been reported to exist in apoplasts of plant cells as a signal molecule. Extracellular ATP (eATP) plays important roles in plant growth, development, and stress tolerance. Here, extracellular ATP was found to promote stomatal opening of Arabidopsis thaliana in light and darkness. ADP, GTP, and weakly hydrolyzable ATP analogs (ATPγS, Bz-ATP, and 2meATP) showed similar effects, whereas AMP and adenosine did not affect stomatal movement. Apyrase inhibited stomatal opening. ATP-promoted stomatal opening was blocked by an NADPH oxidase inhibitor (diphenylene iodonium) or deoxidizer (dithiothreitol), and was impaired in null mutant of NADPH oxidase (atrbohD/F). Added ATP triggered ROS generation in guard cells via NADPH oxidase. ATP also induced Ca(2+) influx and H(+) efflux in guard cells. In atrbohD/F, ATP-induced ion flux was strongly suppressed. In null mutants of the heterotrimeric G protein α subunit, ATP-promoted stomatal opening, cytoplasmic ROS generation, Ca(2+) influx, and H(+) efflux were all suppressed. These results indicated that eATP-promoted stomatal opening possibly involves the heterotrimeric G protein, ROS, cytosolic Ca(2+), and plasma membrane H(+)-ATPase. PMID:22138967

  19. Diversity of extracellular proteins during the transition from the 'proto-apicomplexan' alveolates to the apicomplexan obligate parasites.

    PubMed

    Templeton, Thomas J; Pain, Arnab

    2016-01-01

    The recent completion of high-coverage draft genome sequences for several alveolate protozoans - namely, the chromerids, Chromera velia and Vitrella brassicaformis; the perkinsid Perkinsus marinus; the apicomplexan, Gregarina niphandrodes, as well as high coverage transcriptome sequence information for several colpodellids, allows for new genome-scale comparisons across a rich landscape of apicomplexans and other alveolates. Genome annotations can now be used to help interpret fine ultrastructure and cell biology, and guide new studies to describe a variety of alveolate life strategies, such as symbiosis or free living, predation, and obligate intracellular parasitism, as well to provide foundations to dissect the evolutionary transitions between these niches. This review focuses on the attempt to identify extracellular proteins which might mediate the physical interface of cell-cell interactions within the above life strategies, aided by annotation of the repertoires of predicted surface and secreted proteins encoded within alveolate genomes. In particular, we discuss what descriptions of the predicted extracellular proteomes reveal regarding a hypothetical last common ancestor of a pre-apicomplexan alveolate - guided by ultrastructure, life strategies and phylogenetic relationships - in an attempt to understand the evolution of obligate parasitism in apicomplexans. PMID:26585326

  20. Circulating Extracellular RNA Markers of Liver Regeneration

    PubMed Central

    Yan, Irene K.; Wang, Xue; Asmann, Yan W.; Haga, Hiroaki; Patel, Tushar

    2016-01-01

    Background and Aims Although a key determinant of hepatic recovery after injury is active liver regeneration, the ability to detect ongoing regeneration is lacking. The restoration of liver mass after hepatectomy involves systemic changes with coordinated changes in gene expression guiding regenerative responses, activation of progenitor cells, and proliferation of quiescent hepatocytes. We postulated that these responses involve intercellular communication involving extracellular RNA and that these could represent biomarkers of active regenerative responses. Methods RNA sequencing was performed to identify temporal changes in serum extracellular non-coding RNA after partial hepatectomy in C57BL/6 male mice. Tissue expression of selected RNA was performed by microarray analysis and validated using qRT-PCR. Digital PCR was used to detect and quantify serum expression of selected RNA. Results A peak increase in extracellular RNA content occurred six hours after hepatectomy. RNA sequencing identified alterations in several small non-coding RNA including known and novel microRNAs, snoRNAs, tRNA, antisense and repeat elements after partial hepatectomy. Combinatorial effects and network analyses identified signal regulation, protein complex assembly, and signal transduction as the most common biological processes targeted by miRNA that altered. miR-1A and miR-181 were most significantly altered microRNA in both serum and in hepatic tissues, and their presence in serum was quantitated using digital PCR. Conclusions Extracellular RNA selectively enriched during acute regeneration can be detected within serum and represent biomarkers of ongoing liver regeneration in mice. The ability to detect ongoing active regeneration would improve the assessment of hepatic recovery from liver injury. PMID:27415797

  1. Step-by-step strategy for protein enrichment and proteome characterisation of extracellular polymeric substances in wastewater treatment systems.

    PubMed

    Silva, Ana F; Carvalho, Gilda; Soares, Renata; Coelho, Ana V; Barreto Crespo, M Teresa

    2012-08-01

    Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4-12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25-32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples. PMID:22622841

  2. Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.

    PubMed Central

    Edwards, Marcus J.; White, Gaye F.; Norman, Michael; Tome-Fernandez, Alice; Ainsworth, Emma; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; Clarke, Thomas A.

    2015-01-01

    Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen. PMID:26126857

  3. Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer.

    DOE PAGESBeta

    Edwards, Marcus J.; White, Gaye F.; Norman, Michael; Tome-Fernandez, Alice; Ainsworth, Emma; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; et al

    2015-07-01

    Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX₈C disulfide that, when substituted for AX₈A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation ofmore » a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.« less

  4. Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer.

    SciTech Connect

    Edwards, Marcus J.; White, Gaye F.; Norman, Michael; Tome-Fernandez, Alice; Ainsworth, Emma; Shi, Liang; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.; Clarke, Thomas A.

    2015-07-01

    Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX₈C disulfide that, when substituted for AX₈A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.

  5. Aortic Wall Extracellular Matrix Proteins Correlate with Syntax Score in Patients Undergoing Coronary Artery Bypass Surgery

    PubMed Central

    Chiong, Terri; Cheow, Esther S. H.; Woo, Chin C.; Lin, Xiao Y.; Khin, Lay W.; Lee, Chuen N.; Hartman, Mikael; Sze, Siu K.; Sorokin, Vitaly A.

    2016-01-01

    Aims: The SYNTAX score correlate with major cardiovascular events post-revascularization, although the histopathological basis is unclear. We aim to evaluate the association between syntax score and extracellular matrix histological characteristics of aortic punch tissue obtained during coronary artery bypass surgery (CABG). This analysis compares coronary artery bypass surgery patients with High and Low syntax score which were followed up for one year period. Methods and Results: Patients with High (score ≥ 33, (n=77)) and Low Syntax Scores (score ≤ 22, (n=71)) undergoing elective CABG were recruited prospectively. Baseline clinical characteristics and surgical risks were well matched. At 1 year, EMACCE (Sum of cardiovascular death, stroke, congestive cardiac failure, and limb, gut and myocardial ischemia) was significantly elevated in the High syntax group (P=0.022). Mass spectrometry (MS)-based quantitative iTRAQ proteomic results validated on independent cohort by immunohistochemistry (IHC) revealed that the High syntax group had significantly upraised Collagen I (P<0.0001) and Elastin (P<0.0001) content in ascending aortic wall. Conclusion: This study shows that aortic extracellular matrix (ECM) differ between High and Low syntax groups with up-regulation of Collagen I and Elastin level in High Syntax Score group. This identifies aortic punches collected during CABG as another biomarker source related with atherosclerosis severity and possible clinical outcome. PMID:27347220

  6. Proteins of novel lactic acid bacteria from Apis mellifera mellifera: an insight into the production of known extra-cellular proteins during microbial stress

    PubMed Central

    2013-01-01

    Background Lactic acid bacteria (LAB) has been considered a beneficial bacterial group, found as part of the microbiota of diverse hosts, including humans and various animals. However, the mechanisms of how hosts and LAB interact are still poorly understood. Previous work demonstrates that 13 species of Lactobacillus and Bifidobacterium from the honey crop in bees function symbiotically with the honeybee. They protect each other, their hosts, and the surrounding environment against severe bee pathogens, bacteria, and yeasts. Therefore, we hypothesized that these LAB under stress, i.e. in their natural niche in the honey crop, are likely to produce bioactive substances with antimicrobial activity. Results The genomic analysis of the LAB demonstrated varying genome sizes ranging from 1.5 to 2.2 mega-base pairs (Mbps) which points out a clear difference within the protein gene content, as well as specialized functions in the honeybee microbiota and their adaptation to their host. We demonstrate a clear variation between the secreted proteins of the symbiotic LAB when subjected to microbial stressors. We have identified that 10 of the 13 LAB produced extra-cellular proteins of known or unknown function in which some are arranged in interesting putative operons that may be involved in antimicrobial action, host interaction, or biofilm formation. The most common known extra-cellular proteins secreted were enzymes, DNA chaperones, S-layer proteins, bacteriocins, and lysozymes. A new bacteriocin may have been identified in one of the LAB symbionts while many proteins with unknown functions were produced which must be investigated further. Conclusions The 13 LAB symbionts likely play different roles in their natural environment defending their niche and their host and participating in the honeybee’s food production. These roles are partly played through producing extracellular proteins on exposure to microbial stressors widely found in natural occurring flowers. Many of

  7. Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

    PubMed

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2013-06-01

    Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application. PMID:23647126

  8. Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton

    PubMed Central

    von Nandelstadh, Pernilla; Gucciardo, Erika; Lohi, Jouko; Li, Rui; Sugiyama, Nami; Carpen, Olli; Lehti, Kaisa

    2014-01-01

    Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP–negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain–containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion. PMID:24989798

  9. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

    PubMed

    Wei, Shipeng; Roessler, Bryan C; Icyuz, Mert; Chauvet, Sylvain; Tao, Binli; Hartman, John L; Kirk, Kevin L

    2016-03-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.-Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman IV, J. L., Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels. PMID:26606940

  10. Variable protein profiles in extracellular products of the protistan parasite Perkinsus olseni among regions of the Spanish coast.

    PubMed

    Fernández-Boo, Sergio; Villalba, Antonio; Cao, Asunción

    2015-11-01

    The variability of the protein expression profiling in the extracellular products (ECPs) of in vitro cultured Perkinsus olseni deriving from 4 regions of the Spanish coast was evaluated. The regions involved were the rías of Arousa and Pontevedra (Galicia, NW Spain), Carreras River (Andalusia, SW Spain) and Delta de l'Ebre (Catalonia, NE Spain). P. olseni in vitro clonal cultures were produced from parasite isolates from four clams from each region. Proteins released by the in vitro cultured parasites were isolated and separated by two dimensional electrophoresis (2DE). Qualitative comparison of protein expression profiles in the P. olseni ECPs among clones from all the regions was performed with PD Quest software. Around 130 spots were counted in the gels from ECPs of P. olseni clones from each region, of which 23 spots were shared by clones from all the regions and various spots were representative from clones of one region (appear in every clonal culture from that region but did not in every one of the other regions). A total of 34 spots were excised from the gels and analysed for sequencing. The protein cathepsin B, involved in proteolysis, the signal recognition particle receptor subunit β, involved in protein transport through membranes, and a protein belonging to N-acetyl transferase superfamily, involved in biosynthesis, were identified in spots shared by P. olseni ECPs from all regions. Pepsin A precursor, involved in proteolysis; heat shock protein (HSP) 60; and phosphoserine aminotransferase, involved in biosynthesis, were representative of P. olseni ECPs from Ría de Arousa, while peroxiredoxin V, involved in oxidation-reduction, was representative of P. olseni ECPs from Ría de Pontevedra. Differences in released proteins suggest different virulence or resistance to host attack between parasites from different locations. PMID:26555510

  11. The extracellular matrix proteins laminin and fibronectin contain binding domains for human plasminogen and tissue plasminogen activator.

    PubMed

    Moser, T L; Enghild, J J; Pizzo, S V; Stack, M S

    1993-09-01

    This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa laminin fragment. Amino acid sequence analysis demonstrated that the 5-kDa fragment originates with the fibronectin amino terminus whereas the laminin fragment was derived from the carboxyl-terminal globular domain of the laminin A chain. Ligand blotting experiments using isolated plasminogen domains were also used to identify distinct regions of the plasminogen molecule involved in fibronectin and laminin binding. Solution phase fibronectin binding to immobilized plasminogen was mediated primarily via lysine binding site-dependent interactions with plasminogen kringles 1-4. Lysine binding site-dependent binding of soluble laminin to immobilized plasminogen kringles 1-5 as well as an additional lysine binding site-independent interaction between mini-plasminogen and the 38-kDa laminin A chain fragment were also observed. These studies demonstrate binding of plasminogen and tissue-type plasminogen activator to specific regions of the extracellular matrix glycoproteins laminin and fibronectin and provide further insight into the mechanism of regulation of plasminogen activation by components of the extracellular matrix. PMID:8360181

  12. Glucosepane is a major protein cross-link of the senescent human extracellular matrix. Relationship with diabetes.

    PubMed

    Sell, David R; Biemel, Klaus M; Reihl, Oliver; Lederer, Markus O; Strauch, Christopher M; Monnier, Vincent M

    2005-04-01

    The extracellular matrix in most tissues is characterized by progressive age-related stiffening and loss of proteolytic digestibility that are accelerated in diabetes and can be duplicated by the nonenzymatic reaction of reducing sugars and extracellular matrix proteins. However, most cross-links of the Maillard reaction described so far are present in quantities too low to account for these changes. Here we have determined in human skin and glomerular basement membrane (GBM) collagen the levels of the recently discovered lysine-arginine cross-links derived from glucose, methylglyoxal, glyoxal, and 3-deoxyglucosone, i.e. glucosepane, MODIC, GODIC, and DOGDIC, respectively. Insoluble preparations of skin collagen (n = 110) and glomerular basement membrane (GBM, n = 28) were enzymatically digested, and levels were measured by isotope dilution technique using liquid chromatography/mass spectrometry. In skin, all cross-links increased with age (p < 0.0001) except DOGDIC (p = 0.34). In nondiabetic controls, levels at 90 years were 2000, 30, and 15 pmol/mg for glucosepane, MODIC, and GODIC, respectively. Diabetes, but not renal failure, increased glucosepane to 5000 pmol/mg (p < 0.0001), and for all others, increased it to <60 pmol/mg (p < 0.01). In GBMs, glucosepane reached up to 500 pmol/mg of collagen and was increased in diabetes (p < 0.0001) but not old age. In conclusion, glucosepane is the single major cross-link of the senescent extracellular matrix discovered so far, accounting for up to >120 mole% of triple helical collagen modification in diabetes. Its presence in high quantities may contribute to a number of structural and cell matrix dysfunctions observed in aging and diabetes. PMID:15677467

  13. Phosphatidic Acid-Mediated Signaling Regulates Microneme Secretion in Toxoplasma.

    PubMed

    Bullen, Hayley E; Jia, Yonggen; Yamaryo-Botté, Yoshiki; Bisio, Hugo; Zhang, Ou; Jemelin, Natacha Klages; Marq, Jean-Baptiste; Carruthers, Vern; Botté, Cyrille Y; Soldati-Favre, Dominique

    2016-03-01

    The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii, extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide-phospholipase C (PLC) activation, which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product, phosphatidic acid (PA), is essential for controlling microneme release. Governing this balance is the apicomplexan-specific DAG-kinase-1, which interconverts PA and DAG, and whose depletion impairs egress and causes parasite death. Additionally, we identify an acylated pleckstrin-homology (PH) domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa, these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis. PMID:26962945

  14. The feelgood mutation in zebrafish dysregulates COPII-dependent secretion of select extracellular matrix proteins in skeletal morphogenesis

    PubMed Central

    Melville, David B.; Montero-Balaguer, Mercedes; Levic, Daniel S.; Bradley, Kevin; Smith, Jeffrey R.; Hatzopoulos, Antonis K.; Knapik, Ela W.

    2011-01-01

    SUMMARY Craniofacial and skeletal dysmorphologies account for the majority of birth defects. A number of the disease phenotypes have been attributed to abnormal synthesis, maintenance and composition of extracellular matrix (ECM), yet the molecular and cellular mechanisms causing these ECM defects remain poorly understood. The zebrafish feelgood mutant manifests a severely malformed head skeleton and shortened body length due to defects in the maturation stage of chondrocyte development. In vivo analyses reveal a backlog of type II and type IV collagens in rough endoplasmic reticulum (ER) similar to those found in coat protein II complex (COPII)-deficient cells. The feelgood mutation hinders collagen deposition in the ECM, but trafficking of small cargos and other large ECM proteins such as laminin to the extracellular space is unaffected. We demonstrate that the zebrafish feelgood mutation causes a single amino acid substitution within the DNA-binding domain of transcription factor Creb3l2. We show that Creb3l2 selectively regulates the expression of genes encoding distinct COPII proteins (sec23a, sec23b and sec24d) but find no evidence for its regulation of sec24c expression. Moreover, we did not detect activation of ER stress response genes despite intracellular accumulation of collagen and prominent skeletal defects. Promoter trans-activation assays show that the Creb3l2 feelgood variant is a hypomorphic allele that retains approximately 50% of its transcriptional activity. Transgenic rescue experiments of the feelgood phenotype restore craniofacial development, illustrating that a precise level of Creb3l2 transcriptional activity is essential for skeletogenesis. Our results indicate that Creb3l2 modulates the availability of COPII machinery in a tissue- and cargo-specific manner. These findings could lead to a better understanding of the etiology of human craniofacial and skeletal birth defects as well as adult-onset diseases that are linked to dysregulated

  15. Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens.

    PubMed

    Persson, B David; Schmitz, Nikolaus B; Santiago, César; Zocher, Georg; Larvie, Mykol; Scheu, Ulrike; Casasnovas, José M; Stehle, Thilo

    2010-01-01

    The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system. PMID:20941397

  16. Association of cell surface heparan sulfate proteoglycans of Schwann cells with extracellular matrix proteins.

    PubMed

    Carey, D J; Crumbling, D M; Stahl, R C; Evans, D M

    1990-11-25

    The terminal differentiation of Schwann cells is dependent on contact with basement membrane. The present study was undertaken to investigate the role of cell surface heparan sulfate proteoglycans (HSPGs) in mediating Schwann cell responses to extracellular matrix contact. Phosphatidylinositol-specific phospholipase C-releasable cell surface HSPGs purified from cultures of neonatal rat Schwann cells were subjected to affinity chromatography on immobilized laminin and fibronectin. Binding of the HSPG to both affinity matrices was observed. The strength of the association, however, was sensitive to the ionic strength of the buffer. In 0.1 M Tris-HCl, HSPG binding was essentially irreversible whereas in physiological ionic strength buffer (e.g. 0.142 M NaCl, 10 mM Tris), weaker binding was detected as a delay in elution of the HSPG from the affinity columns. Further studies of HSPG-laminin binding suggested that the binding was mediated by the glycosaminoglycan chains of the proteoglycans. Results of equilibrium gel filtration chromatography provided additional evidence for a reversible association of the HSPG and laminin with a Kd of approximately 1 x 10(-6) M. When Schwann cells were plated on plastic dishes coated with laminin, the cells attached and extended long slender processes. Inclusion of heparin, but not chondroitin sulfate, in the assay medium resulted in partial inhibition of process extension, but at concentrations of heparin which were higher than that needed to disrupt laminin-HSPG association in vitro. Addition of anti-integrin receptor antibodies resulted in more extensive inhibition of laminin-dependent process extension. Anti-integrin antibodies plus heparin essentially totally inhibited laminin-dependent process extension. These results demonstrate that cell surface HSPGs are capable of reversible association with extracellular matrix molecules and suggest that HSPG-laminin interactions play a role in laminin-dependent Schwann cell spreading. PMID

  17. Purification of the extracellular domain of the membrane protein GlialCAM expressed in HEK and CHO cells and comparison of the glycosylation.

    PubMed

    Gaudry, Jean-Philippe; Arod, Christian; Sauvage, Christophe; Busso, Stephane; Dupraz, Philippe; Pankiewicz, Renata; Antonsson, Bruno

    2008-03-01

    Adhesion molecules are essential for a wide range of biological and physiological functions, including cell-cell interactions, cell interactions with the extracellular matrix, cell migration, proliferation and survival. Defects in cell adhesion have been associated with pathological conditions such as neoplasia, and neurodegenerative diseases. We have identified a new adhesion molecule of the immunoglobulin family, GlialCAM. The same protein was recently published under the name hepaCAM and was suggested to be associated with hepatocellular carcinoma. Here we have expressed and purified the extracellular domain of this molecule in two mammalian expression systems, HEK and CHO cells. A three step purification protocol gave an over 95% pure protein. The extracellular domain of GlialCAM possesses several potential N- and O-glycosylation sites. Glycosylation is one of the most common post-translational modifications of secreted proteins and of the extracellular domains of membrane bound proteins. It can influence both the activity and the stability of the protein. The glycosylation pattern has been shown to depend on the cell type where the protein is expressed. We examined if differences in the glycosylation of this protein could be detected when it was expressed in the two commonly used mammalian expression systems, HEK and CHO. Differences in the glycosylation were detected. PMID:18082421

  18. Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples

    PubMed Central

    Belov, Larissa; Matic, Kieran J.; Hallal, Susannah; Best, O. Giles; Mulligan, Stephen P.; Christopherson, Richard I.

    2016-01-01

    Extracellular vesicles (EV) are membranous particles (30–1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19+ EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals. PMID:27086589

  19. Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples.

    PubMed

    Belov, Larissa; Matic, Kieran J; Hallal, Susannah; Best, O Giles; Mulligan, Stephen P; Christopherson, Richard I

    2016-01-01

    Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals. PMID:27086589

  20. Matrilin-2, an extracellular adaptor protein, is needed for the regeneration of muscle, nerve and other tissues

    PubMed Central

    Korpos, Éva; Deák, Ferenc; Kiss, Ibolya

    2015-01-01

    The extracellular matrix (ECM) performs essential functions in the differentiation, maintenance and remodeling of tissues during development and regeneration, and it undergoes dynamic changes during remodeling concomitant to alterations in the cell-ECM interactions. Here we discuss recent data addressing the critical role of the widely expressed ECM protein, matrilin-2 (Matn2) in the timely onset of differentiation and regeneration processes in myogenic, neural and other tissues and in tumorigenesis. As a multiadhesion adaptor protein, it interacts with other ECM proteins and integrins. Matn2 promotes neurite outgrowth, Schwann cell migration, neuromuscular junction formation, skeletal muscle and liver regeneration and skin wound healing. Matn2 deposition by myoblasts is crucial for the timely induction of the global switch toward terminal myogenic differentiation during muscle regeneration by affecting transforming growth factor beta/bone morphogenetic protein 7/Smad and other signal transduction pathways. Depending on the type of tissue and the pathomechanism, Matn2 can also promote or suppress tumor growth. PMID:26199591

  1. Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinson's Disease, Glioma, and Schwannoma.

    PubMed

    Hall, Justin; Prabhakar, Shilpa; Balaj, Leonora; Lai, Charles P; Cerione, Richard A; Breakefield, Xandra O

    2016-04-01

    Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases. PMID:27017608

  2. Retbindin Is an Extracellular Riboflavin-binding Protein Found at the Photoreceptor/Retinal Pigment Epithelium Interface*

    PubMed Central

    Kelley, Ryan A.; Al-Ubaidi, Muayyad R.; Naash, Muna I.

    2015-01-01

    Retbindin is a novel retina-specific protein of unknown function. In this study, we have used various approaches to evaluate protein expression, localization, biochemical properties, and function. We find that retbindin is secreted by the rod photoreceptors into the inter-photoreceptor matrix where it is maintained via electrostatic forces. Retbindin is predominantly localized at the interface between photoreceptors and retinal pigment epithelium microvilli, a region critical for retinal function and homeostasis. Interestingly, although it is associated with photoreceptor outer segments, retbindin's expression is not dependent on their presence. In vitro, retbindin is capable of binding riboflavin, thus implicating the protein as a metabolite carrier between the retina and the retinal pigment epithelium. Altogether, our data show that retbindin is a novel photoreceptor-specific protein with a unique localization and function. We hypothesize that retbindin is an excellent candidate for binding retinal flavins and possibly participating in their transport from the extracellular space to the photoreceptors. Further investigations are warranted to determine the exact function of retbindin in retinal homeostasis and disease. PMID:25542898

  3. Downregulation of the Yes-Associated Protein Is Associated with Extracellular Matrix Disorders in Ascending Aortic Aneurysms

    PubMed Central

    Li, Haiyang; Jiang, Wenjian; Ren, Weihong; Guo, Dong; Guo, Jialong; Wang, Xiaolong; Liu, Yuyong; Lan, Feng; Du, Jie; Zhang, Hongjia

    2016-01-01

    Previous studies indicate that extracellular matrix (ECM) disorders lead to the apoptosis of Vascular Smooth Muscle Cells (VSMCs), which impairs the aortic wall by reducing the generation of elastic fibers, and ultimately result in ascending aortic aneurysm. The critical role of the Yes-associated protein (YAP) has been elucidated in cardiac/SMC proliferation during cardiovascular development. However, the association of YAP expression and extracellular matrix disorders in ascending aortic aneurysms is not clear. Here, we present for the first time that the downregulation of YAP in VSMCs is associated with ECM disorders of the media in ascending aortic aneurysms. We found that aortic ECM deteriorated with increased apoptotic VSMCs. Moreover, expression of YAP was dramatically reduced in the aortic walls of patients with ascending aortic aneurysms, while the normal aortic samples exhibited abundant YAP in the VSMCs. These results suggest that downregulation of YAP leads to apoptosis of VSMCs, which are essential for the homeostasis of the aortic wall. The resultant ECM disorders affect aortic structure and function and contribute to the development of ascending aortic aneurysms. In summary, through assessment of clinical samples, we revealed the association between downregulation of YAP in VSMCs and the development of ascending aortic aneurysms, providing new insight into the pathogenesis of this disease. PMID:26904131

  4. Extracellular vesicle-mediated transfer of CLIC1 protein is a novel mechanism for the regulation of glioblastoma growth

    PubMed Central

    Richichi, Cristina; Ortensi, Barbara; Del Bene, Massimiliano; Fornasari, Lorenzo; Beznoussenko, Galina; Mironov, Alexandre; Rappa, Germana; Cuomo, Alessandro; Faretta, Mario; Bonaldi, Tiziana; Lorico, Aurelio; Pelicci, Giuliana

    2015-01-01

    Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth. PMID:26429879

  5. Extracellular vesicles from bone marrow mesenchymal stem/stromal cells transport tumor regulatory microRNA, proteins, and metabolites.

    PubMed

    Vallabhaneni, Krishna C; Penfornis, Patrice; Dhule, Santosh; Guillonneau, Francois; Adams, Kristen V; Mo, Yin Yuan; Xu, Rui; Liu, Yiming; Watabe, Kounosuke; Vemuri, Mohan C; Pochampally, Radhika

    2015-03-10

    Human mesenchymal stem/stromal cells (hMSCs) have been shown to support breast cancer cell proliferation and metastasis, partly through their secretome. hMSCs have a remarkable ability to survive for long periods under stress, and their secretome is tumor supportive. In this study, we have characterized the cargo of extracellular vesicular (EV) fraction (that is in the size range of 40-150nm) of serum deprived hMSCs (SD-MSCs). Next Generation Sequencing assays were used to identify small RNA secreted in the EVs, which indicated presence of tumor supportive miRNA. Further assays demonstrated the role of miRNA-21 and 34a as tumor supportive miRNAs. Next, proteomic assays revealed the presence of ≈150 different proteins, most of which are known tumor supportive factors such as PDGFR-β, TIMP-1, and TIMP-2. Lipidomic assays verified presence of bioactive lipids such as sphingomyelin. Furthermore, metabolite assays identified the presence of lactic acid and glutamic acid in EVs. The co-injection xenograft assays using MCF-7 breast cancer cells demonstrated the tumor supportive function of these EVs. To our knowledge this is the first comprehensive -omics based study that characterized the complex cargo of extracellular vesicles secreted by hMSCs and their role in supporting breast cancers. PMID:25669974

  6. Design of an Osteoinductive Extracellular Fibronectin Matrix Protein for Bone Tissue Engineering

    PubMed Central

    Lee, Sujin; Lee, Dong-Sung; Choi, Ilsan; Pham, Le B. Hang; Jang, Jun-Hyeog

    2015-01-01

    Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05). PMID:25853265

  7. Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation.

    PubMed

    El Asmar, Zeina; Terrand, Jérome; Jenty, Marion; Host, Lionel; Mlih, Mohamed; Zerr, Aurélie; Justiniano, Hélène; Matz, Rachel L; Boudier, Christian; Scholler, Estelle; Garnier, Jean-Marie; Bertaccini, Diego; Thiersé, Danièle; Schaeffer, Christine; Van Dorsselaer, Alain; Herz, Joachim; Bruban, Véronique; Boucher, Philippe

    2016-03-01

    The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFβ-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (β) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 β-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases. PMID:26792864

  8. In vitro effects of the extracellular protein of Renibacterium salmoninarum on phagocyte function in brook trout (Salvelinus fontinalis).

    PubMed

    Densmore, C L; Smith, S A; Holladay, S D

    1998-04-30

    Renibacterium salmoninarum is a facultative intracellular pathogen often found in host phagocytes where it appears to successfully avoid the host fish's immunological defenses. The objective of this investigation was to determine if soluble extracellular protein (ECP) produced by R. salmoninarum may contribute to the immunomodulation in bacterial kidney disease (BKD) via inhibition of phagocyte respiratory burst and/or phagocytosis mechanisms. Splenic cells from adult brook trout (Salvelinus fontinalis) were incubated with two different concentrations of ECP (0.1 mg/ml and 1.0 mg/ml) and viable R. salmoninarum. Splenic cell cultures were evaluated for respiratory burst activity via flow cytometry with the dichlorofluorescin diacetate (DCF-DA) assay and for phagocytosis via light microscopic assessment of microsphere engulfment. Respiratory burst activity was significantly inhibited in all treatment groups as compared to untreated fish, while no differences were noted in phagocytic activity. PMID:9646439

  9. The Functions of Grainy Head-Like Proteins in Animals and Fungi and the Evolution of Apical Extracellular Barriers

    PubMed Central

    Paré, Adam; Kim, Myungjin; Juarez, Michelle T.; Brody, Stuart; McGinnis, William

    2012-01-01

    The Grainy head (GRH) family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi – organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl). We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2) mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins. PMID:22590528

  10. Are extracellular osmolality and sodium concentration determined by Donnan effects of intracellular protein charges and of pumped sodium?

    PubMed

    Kurbel, Sven

    2008-06-21

    Although we are used to attribute almost identical extracellular fluid (ECF) sodium concentrations in birds, amphibians, reptiles, and mammals to the composition of the primordial oceans in which, presumably, all life originated, this interpretation is not supported by geological data suggesting that the ocean salinity was never much lower than the present-day values, still four times higher than our plasma sodium. Here presented interpretation is that the similar ECF salt concentrations are dictated by the opposed Donnan effects on the cell membrane. The only way for the cell to reach the osmotic equilibrium is to alter cell volume, until concentration of nondiffusible intracellular ions (mainly charges on intracellular proteins) is equal to the ECF restricted ions (mainly Na+ ions, restricted by pumping out of cells). The achievement of electroneutrality requires that the sum of all anions equals concentration of positive ions in the cell (mainly K+). Negative charges on cytoplasmic proteins are the most stable component among ionized particles and other ions have to adapt to their concentration. Positive and negative soluble intracellular ions are all osmotically active and to achieve balance of osmotic forces on the cell membrane, the sum of their intracellular concentrations must equal the concentration of osmotically active extracellular particles. Since almost half the osmotically active ECF particles are sodium ions, the ECF sodium concentration seems related to concentration of charges on cytoplasmic proteins and concentration of intracellular phosphates. Our ancestors could not leave the salty ocean and move to brackish, or even fresh waters, without adequate regulation of their ECF sodium concentration and osmolality. Concentration of charges on cytoplasmic proteins or of intracellular phosphate buffers could not be altered, since this would compromise cell functioning. The remaining solution was to maintain the lowest ECF Na+ concentration effective in

  11. Cooperative regulation of substrate stiffness and extracellular matrix proteins in skin wound healing of axolotls.

    PubMed

    Huang, Ting-Yu; Wu, Cheng-Han; Wang, Mu-Hui; Chen, Bo-Sung; Chiou, Ling-Ling; Lee, Hsuan-Shu

    2015-01-01

    Urodele amphibians (Ambystoma mexicanum), unique among vertebrates, can regenerate appendages and other body parts entirely and functionally through a scar-free healing process. The wound epithelium covering the amputated or damaged site forms early and is essential for initiating the subsequent regenerative steps. However, the molecular mechanism through which the wound reepithelializes during regeneration remains unclear. In this study, we developed an in vitro culture system that mimics an in vivo wound healing process; the biomechanical properties in the system were precisely defined and manipulated. Skin explants that were cultured on 2 to 50 kPa collagen-coated substrates rapidly reepithelialized within 10 to 15 h; however, in harder (1 GPa) and other extracellular matrices (tenascin-, fibronectin-, and laminin-coated environments), the wound epithelium moved slowly. Furthermore, the reepithelialization rate of skin explants from metamorphic axolotls cultured on a polystyrene plate (1 GPa) increased substantially. These findings afford new insights and can facilitate investigating wound epithelium formation during early regeneration using biochemical and mechanical techniques. PMID:25839038

  12. Extracellular matrix proteins expression profiling in chemoresistant variants of the A2780 ovarian cancer cell line.

    PubMed

    Januchowski, Radosław; Zawierucha, Piotr; Ruciński, Marcin; Nowicki, Michał; Zabel, Maciej

    2014-01-01

    Ovarian cancer is the leading cause of death among gynaecological malignancies. Extracellular matrix (ECM) can affect drug resistance by preventing the penetration of the drug into cancer cells and increased resistance to apoptosis. This study demonstrates alterations in the expression levels of ECM components and related genes in cisplatin-, doxorubicin-, topotecan-, and paclitaxel-resistant variants of the A2780 ovarian cancer cell line. Affymetrix Gene Chip Human Genome Array Strips were used for hybridisations. The genes that had altered expression levels in drug-resistant sublines were selected and filtered by scatter plots. The genes that were up- or downregulated more than fivefold were selected and listed. Among the investigated genes, 28 genes were upregulated, 10 genes were downregulated, and two genes were down- or upregulated depending on the cell line. Between upregulated genes 12 were upregulated very significantly--over 20-fold. These genes included COL1A2, COL12A1, COL21A1, LOX, TGFBI, LAMB1, EFEMP1, GPC3, SDC2, MGP, MMP3, and TIMP3. Four genes were very significantly downregulated: COL11A1, LAMA2, GPC6, and LUM. The expression profiles of investigated genes provide a preliminary insight into the relationship between drug resistance and the expression of ECM components. Identifying correlations between investigated genes and drug resistance will require further analysis. PMID:24804215