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Sample records for extracellular signal-regulated protein

  1. Phosphorylation of the TAL1 oncoprotein by the extracellular-signal-regulated protein kinase ERK1.

    PubMed Central

    Cheng, J T; Cobb, M H; Baer, R

    1993-01-01

    Alteration of the TAL1 gene is the most common genetic lesion found in T-cell acute lymphoblastic leukemia. TAL1 encodes phosphoproteins, pp42TAL1 and pp22TAL1, that represent phosphorylated versions of the full-length (residues 1 to 331) and truncated (residues 176 to 331) TAL1 gene products, respectively. Both proteins contain the basic helix-loop-helix motif, a DNA-binding and protein dimerization motif common to several known transcriptional regulatory factors. We now report that serine residue 122 (S122) is a major phosphorylation site of pp42TAL1 in leukemic cell lines and transfected COS1 cells. In vivo phosphorylation of S122 is induced by epidermal growth factor with a rapid time course that parallels activation of the ERK/MAP2 protein kinases. Moreover, S122 is readily phosphorylated in vitro by the extracellular signal-regulated protein kinase ERK1. These data suggest that TAL1 residue S122 serves as an in vivo substrate for ERK/MAP2 kinases such as ERK1. Therefore, S122 phosphorylation may provide a mechanism whereby the properties of TAL1 polypeptides can be modulated by extracellular stimuli. Images PMID:8423803

  2. Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

    PubMed Central

    Jiang, Youwei; Cypess, Aaron M.; Muse, Evan D.; Wu, Cui-Rong; Unson, Cecilia G.; Merrifield, R. B.; Sakmar, Thomas P.

    2001-01-01

    We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation. PMID:11517300

  3. Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder.

    PubMed

    Wang, Hongyan; Zhang, Yingquan; Qiao, Mingqi

    2013-03-25

    The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

  4. Expression and activation of platelet-derived growth factor β receptor, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in canine mammary tumours.

    PubMed

    Altamura, Gennaro; Uberti, Barbara Degli; Galiero, Giorgio; Martano, Manuela; Pirro, Antonella; Russo, Marco; Borzacchiello, Giuseppe

    2017-02-01

    Canine mammary tumours are frequent neoplasms mostly affecting intact female dogs, for which no 100% efficient therapy is available. Platelet derived growth factor β receptor (PDGFβR) is a tyrosine kinase receptor (TKR) with a potential role in human breast cancer and a series of canine tumours. In this study we demonstrated, for the first time, expression of PDGFβR and its downstream transduction molecules, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), as well as their activated forms in canine mammary tumours by both biochemical analysis and immunohistochemistry. PDGFβR was expressed and hyperphosphorylated in the majority of tumour samples and tumour derived cell lines. Additionally, both MEK and ERK were expressed and activated in cell lines as well as biopsies. TKR inhibitors (TKRi) are currently under investigation as possible therapy in human breast and several canine tumours, thus our in vivo and in vitro findings pave the way for future studies aimed at establishing a potential therapeutic employment of TKRi for the treatment of canine mammary cancer.

  5. 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.

  6. 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.

  7. Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3

    SciTech Connect

    Zeliadt, Nicholette A.; Mauro, Laura J.; Wattenberg, Elizabeth V.

    2008-11-01

    Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

  8. Modulation of Brahma expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation.

    PubMed

    Mehrotra, Aanchal; Saladi, Srinivas Vinod; Trivedi, Archit R; Aras, Shweta; Qi, Huiling; Jayanthy, Ashika; Setaluri, Vijayasaradhi; de la Serna, Ivana L

    2014-12-01

    Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.

  9. Protein Kinase A-independent Ras Protein Activation Cooperates with Rap1 Protein to Mediate Activation of the Extracellular Signal-regulated Kinases (ERK) by cAMP.

    PubMed

    Li, Yanping; Dillon, Tara J; Takahashi, Maho; Earley, Keith T; Stork, Philip J S

    2016-10-07

    Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1, have been proposed to mediate this activation, with either Ras or Rap1 acting in distinct cell types. Using Hek293 cells, we show that both Ras and Rap1 are required for cAMP signaling to ERKs. The roles of Ras and Rap1 were distinguished by their mechanism of activation, dependence on the cAMP-dependent protein kinase (PKA), and the magnitude and kinetics of their effects on ERKs. Ras was required for the early portion of ERK activation by cAMP and was activated independently of PKA. Ras activation required the Ras/Rap guanine nucleotide exchange factor (GEF) PDZ-GEF1. Importantly, this action of PDZ-GEF1 was disrupted by mutation within its putative cyclic nucleotide-binding domain within PDZ-GEF1. Compared with Ras, Rap1 activation of ERKs was of longer duration. Rap1 activation was dependent on PKA and required Src family kinases and the Rap1 exchanger C3G. This is the first report of a mechanism for the cooperative actions of Ras and Rap1 in cAMP activation of ERKs. One physiological role for the sustained activation of ERKs is the transcription and stabilization of a range of transcription factors, including c-FOS. We show that the induction of c-FOS by cAMP required both the early and sustained phases of ERK activation, requiring Ras and Rap1, as well as for each of the Raf isoforms, B-Raf and C-Raf.

  10. Neuronal nitric oxide contributes to neuroplasticity-associated protein expression through cGMP, protein kinase G, and extracellular signal-regulated kinase.

    PubMed

    Gallo, Eduardo F; Iadecola, Costantino

    2011-05-11

    Nitric oxide (NO) synthesized by neuronal NO synthase (nNOS) has long been implicated in brain plasticity. However, it is unclear how this short-lived mediator contributes to the long-term molecular changes underlying neuroplasticity, which typically require activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway and gene expression. To address this issue, we used a neuroplasticity model based on treatment of neuronal cultures with bicuculline and a model of experience-dependent plasticity in the barrel cortex. In neuronal cultures, NOS inhibition attenuated the bicuculline-induced activation of ERK and the expression of c-Fos, Egr-1, Arc, and brain-derived neurotrophic factor (BDNF), proteins essential for neuroplasticity. Furthermore, inhibition of the NO target soluble guanylyl cyclase or of the cGMP effector kinase protein kinase G (PKG) reduced both ERK activation and plasticity-related protein expression. NOS inhibition did not affect phosphorylation of cAMP response element-binding protein (CREB), a well-established ERK nuclear target, but it attenuated the nuclear accumulation of the CREB coactivator TORC1 and suppressed the activation of Elk-1, another transcription factor target of ERK. Consistent with these in vitro observations, induction of c-Fos, Egr-1, and BDNF was attenuated in the D1 cortical barrel of nNOS(-/-) mice subjected to single whisker experience. These results establish nNOS-derived NO as a key factor in the expression of proteins involved in neuroplasticity, an effect mediated through cGMP, PKG, and ERK signaling. These actions of NO do not depend on CREB phosphorylation but may involve TORC1 and Elk-1. Our data unveil a previously unrecognized link between neuronal NO and the molecular machinery responsible for the sustained synaptic changes underlying neuroplasticity.

  11. 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

  12. Inhibition of mitogen-activated protein kinase-extracellular signal-regulated kinase disrupts latent inhibition of cued fear conditioning in C57BL/6 mice.

    PubMed

    Lewis, Michael C; Davis, Jennifer A; Gould, Thomas J

    2004-12-01

    The mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) cascade has been implicated in a variety of associative conditioning tasks. However, the role of the MAPK-ERK cascades in modulating conditioning is less clear. The authors examined the effect of the potent and selective MAPK-ERK inhibitor SL327 on latent inhibition of cued fear conditioning. The results demonstrate that 50 mg/kg and 100 mg/kg SL327 disrupt latent inhibition of cued fear conditioning. These data provide evidence for an essential role of the MAPK-ERK cascade in tasks that modulate the strength of associative conditioning. The results are discussed in relation to the molecular mechanisms that support latent inhibition of cued fear conditioning.

  13. β-Adrenergic stimulation activates protein kinase Cε and induces extracellular signal-regulated kinase phosphorylation and cardiomyocyte hypertrophy.

    PubMed

    Li, Lin; Cai, Hongyan; Liu, Hua; Guo, Tao

    2015-06-01

    The cardiac adrenergic signaling pathway is important in the induction of cardiac hypertrophy. The cardiac adrenergic pathway involves two main branches, phospholipase C (PLC)/protein kinase C (PKC) and the adenylate cyclase (cAMPase)/protein kinase A (PKA) signaling pathways. It is hypothesized that PLC/PKC and cAMPase/PKA are activated by the α‑adrenergic receptor (αAR) and the β‑adrenergic receptor (βAR), respectively. Previous studies have demonstrated that exchange protein directly activated by cAMP (Epac), a guanine exchange factor, activates phospholipase Cε. It is possible that there are βAR‑activated PKC pathways mediated by Epac and PLC. In the present study, the role of Epac and PLC in βAR activated PKC pathways in cardiomyocytes was investigated. It was found that PKCε activation and translocation were induced by the βAR agonist, isoproterenol (Iso). Epac agonist 8‑CPT‑2'OMe‑cAMP also enhanced PKCε activation. βAR stimulation activated PKCε in the cardiomyocytes and was regulated by Epac. Iso‑induced change in PKCε was not affected in the cardiomyocytes, which were infected with adenovirus coding rabbit muscle cAMP‑dependent protein kinase inhibitor. However, Iso‑induced PKCε activation was inhibited by the PLC inhibitor, U73122. The results suggested that Iso‑induced PKCε activation was independent of PKA, but was regulated by PLC. To further investigate the downstream signal target of PKCε activation, the expression of phosphorylated extracellular signal‑regulated kinase (pERK)1/2 and the levels of ERK phosphorylation was analyzed. The results revealed that Iso‑induced PKCε activation led to an increase in the expression of pERK1/2. ERK phosphorylation was inhibited by the PKCε inhibitor peptide. Taken together, these data demonstrated that the βAR is able to activate PKCε dependent on Epac and PLC, but independent of PKA.

  14. 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…

  15. PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma.

    PubMed

    Puustinen, Pietri; Junttila, Melissa R; Vanhatupa, Sari; Sablina, Anna A; Hector, Melissa E; Teittinen, Kaisa; Raheem, Olayinka; Ketola, Kirsi; Lin, Shujun; Kast, Juergen; Haapasalo, Hannu; Hahn, William C; Westermarck, Jukka

    2009-04-01

    Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway activity is regulated by the antagonist function of activating kinases and inactivating protein phosphatases. Sustained ERK pathway activity is commonly observed in human malignancies; however, the mechanisms by which the pathway is protected from phosphatase-mediated inactivation in the tumor tissue remain obscure. Here, we show that methylesterase PME-1-mediated inhibition of the protein phosphatase 2A promotes basal ERK pathway activity and is required for efficient growth factor response. Mechanistically, PME-1 is shown to support ERK pathway signaling upstream of Raf, but downstream of growth factor receptors and protein kinase C. In malignant gliomas, PME-1 expression levels correlate with both ERK activity and cell proliferation in vivo. Moreover, PME-1 expression significantly correlates with disease progression in human astrocytic gliomas (n=222). Together, these observations identify PME-1 expression as one mechanism by which ERK pathway activity is maintained in cancer cells and suggest an important functional role for PME-1 in the disease progression of human astrocytic gliomas.

  16. Research progress of the role and mechanism of extracellular signal-regulated protein kinase 5 (ERK5) pathway in pathological pain*

    PubMed Central

    Yu, Li-na; Sun, Li-hong; Wang, Min; Yan, Min

    2016-01-01

    Extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1 (MAPK1), is an important member of ERK family, which is a subfamily of the large MAPK family. ERK5 is expressed in many tissues, including the dorsal root ganglion (DRG) neurons and the spinal cord. In this review, we focus on elaborating ERK5-associated pathway in pathological pain, in which the ERK5/CREB (cyclic adenosine monophosphate (cAMP)-response element-binding protein) pathway plays a crucial role in the transduction of pain signal and contributes to pain hypersensitivity. ERK5 activation in the spinal dorsal horn occurs mainly in microglia. The activation of ERK5 can be mediated by N-methyl-D-aspartate (NMDA) receptors. We also elaborate the relationship between ERK5 activation and nerve growth factor-tyrosine kinase A (NGF-TrkA), and the connection between ERK5 activation and brain-derived neurotrophic factor (BDNF) in pathological pain in detail. PMID:27704743

  17. Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

    PubMed

    Chan, Chien-Pin; Tsai, Yao-Ting; Chen, Yao-Li; Hsu, Yu-Wen; Tseng, Joseph T; Chuang, Hung-Yi; Shiurba, Robert; Lee, Mei-Hsien; Wang, Jaw-Yuan; Chang, Wei-Chiao

    2015-02-01

    Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 μM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

  18. Blockade of ankyrin repeat-rich membrane spanning protein modulates extracellular signal-regulated kinase expression and inhibits allergic inflammation in ovalbumin-sensitized mice.

    PubMed

    Ni, Xiuqin; Li, Xing; Tao, Shuhua; Xu, Minghui; Ma, Hongmei; Wang, Xiuli

    2013-07-01

    Ankyrin repeat-rich membrane spanning protein (ARMS), also known as kinase D-interacting substrate of 220 kDa (Kidins220), is a transmembrane protein that has been reported to be involved in the pathogenesis of asthma through the nerve growth factor (NGF)/tyrosine kinase A (TrkA) receptor signaling pathway. To investigate whether NGF/TrkA-Kidins220/ARMS-extracellular signal-regulated kinase (ERK) signaling is activated in airway inflammation of asthma, BALB/c mice were sensitized and challenged with ovalbumin (OVA). The effects of Kidins220/ARMS on ERK, interleukin (IL)-1β, IL-4 and tumor necrosis factor (TNF)-α in lung tissues following the allergic airway challenge in mice were assessed by administering anti-ARMS antibody to the mice. Pathological changes in the bronchi and lung tissues were examined via hematoxylin and eosin staining. The phosphorylated ERK, IL-1β, IL-4 and TNF-α levels were determined using western blot analysis and ELISA and were found to be overexpressed in lung tissues following the allergen challenge. Moreover, after the mice were treated with anti-NGF, anti-TrkA or anti-ARMS, the levels of Kidins220/ARMS, phosphorylated ERK, IL-1β, IL-4, TNF-α and allergen-induced airway inflammation were downregulated. These results suggested that NGF/TrkA-Kidins220/ARMS-ERK signaling was activated in airway inflammation induced by the allergic airway challenge, possibly representing a new mechanism in asthma.

  19. Mitogen-activated protein kinase phosphatase-1 inhibition and sustained extracellular signal-regulated kinase 1/2 activation in camptothecin-induced human colon cancer cell death

    PubMed Central

    Lee, Minyoung; Young Kim, Sun; Kim, JongGuk; Kim, Hak-Su; Kim, Sang-Man; Kim, Eun Ju

    2013-01-01

    Camptothecins are commonly used chemotherapeutics; in some models, they enhance signaling via the mitogen-activated protein kinase (MAPK) pathway through effects on upstream kinases. To evaluate the impact of camptothecin (CPT) on MAPKs in human colon cancer, we studied HCT116 and CaCo2 colon cancer cells. We found that HCT116 cells highly express mitogen-activated protein kinase phosphatase-1 (MKP1), which selectively inactivates extracellular signal-regulated kinase (ERK), whereas MKP1 levels were undetectable in CaCo2 cells. CPT did not affect ERK activity in CaCo2 cells, but did induce a striking increase in ERK activity in HCT116 cells in association with a corresponding decrease in MKP1. The reduction in MKP1 expression occurred at a posttranscriptional level and was blocked by the proteasome inhibitor MG132, whereas that CPT-induced downregulation of MKP1 was not due to proteasome-mediated degradation. Treatment of HCT116 cells with CPT induced a sustained activation of nuclear ERK, which was required for CPT-induced apoptosis. P38 and JNK activity were unaffected by CPT, suggesting that the effects of CPT are mediated specifically by ERK. These results suggest that targeting dual-specificity MAPK phosphatases in colon cancer cells may be a viable strategy for optimizing camptothecin-based therapeutic protocols. PMID:24005240

  20. 17 beta-estradiol induces spermatogonial proliferation through mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activity in the lizard (Podarcis s. sicula).

    PubMed

    Chieffi, Paolo; Colucci D'Amato, Luca; Guarino, Fabio; Salvatore, Gaetano; Angelini, Francesco

    2002-02-01

    There are always more evidences indicating that 17beta-estradiol (E(2)) is necessary for normal male fertility. We have used a nonmammalian vertebrate model (the lizard Podarcis s. sicula) to investigate the regulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity in the testis during the annual sexual cycle and to study whether E(2) exerts a role in the spermatogenesis through ERK1/2 activity. Immunocytochemistry analysis shows that ERK1/2 proteins are present in the nucleus of the spermatogonia (SPG), and in primary (I) spermatocytes (SPC). The annual E(2) profile shows a progressive increase during the active spermatogenesis (from April to June) and a peak in the month of August (spermatogonial mitosis). In parallel, ERK1/2 (molecular weight 44 and 42 kDa, respectively) are highly phosphorylated during the period of active spermatogenesis and in post-refractory period (August) compared with the winter stasis (from November to March). Present results demonstrate that E(2) treatment induces spermatogonial proliferation, possibly via the activation of ERK1/2, and this effect is counteracted by the antiestrogen ICI 182-780.

  1. 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.

  2. ICAM-1-induced expression of proinflammatory cytokines in astrocytes: involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways.

    PubMed

    Lee, S J; Drabik, K; Van Wagoner, N J; Lee, S; Choi, C; Dong, Y; Benveniste, E N

    2000-10-15

    ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer's disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1beta and IL-1alpha protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1alpha and IL-1beta expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.

  3. Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum.

    PubMed Central

    Moughal, N; Stevens, P A; Kong, D; Pyne, S; Pyne, N J

    1995-01-01

    Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with

  4. Blockade of ankyrin repeat-rich membrane spanning protein modulates extracellular signal-regulated kinase expression and inhibits allergic inflammation in ovalbumin-sensitized mice

    PubMed Central

    NI, XIUQIN; LI, XING; TAO, SHUHUA; XU, MINGHUI; MA, HONGMEI; WANG, XIULI

    2013-01-01

    Ankyrin repeat-rich membrane spanning protein (ARMS), also known as kinase D-interacting substrate of 220 kDa (Kidins220), is a transmembrane protein that has been reported to be involved in the pathogenesis of asthma through the nerve growth factor (NGF)/tyrosine kinase A (TrkA) receptor signaling pathway. To investigate whether NGF/TrkA-Kidins220/ARMS-extracellular signal-regulated kinase (ERK) signaling is activated in airway inflammation of asthma, BALB/c mice were sensitized and challenged with ovalbumin (OVA). The effects of Kidins220/ARMS on ERK, interleukin (IL)-1β, IL-4 and tumor necrosis factor (TNF)-α in lung tissues following the allergic airway challenge in mice were assessed by administering anti-ARMS antibody to the mice. Pathological changes in the bronchi and lung tissues were examined via hematoxylin and eosin staining. The phosphorylated ERK, IL-1β, IL-4 and TNF-α levels were determined using western blot analysis and ELISA and were found to be overexpressed in lung tissues following the allergen challenge. Moreover, after the mice were treated with anti-NGF, anti-TrkA or anti-ARMS, the levels of Kidins220/ARMS, phosphorylated ERK, IL-1β, IL-4, TNF-α and allergen-induced airway inflammation were downregulated. These results suggested that NGF/TrkA-Kidins220/ARMS-ERK signaling was activated in airway inflammation induced by the allergic airway challenge, possibly representing a new mechanism in asthma. PMID:24649008

  5. Salvinorin A Pretreatment Preserves Cerebrovascular Autoregulation After Brain Hypoxic/Ischemic Injury via Extracellular Signal-Regulated Kinase / Mitogen-Activated Protein Kinase in Piglets

    PubMed Central

    Su, Diansan; Riley, John; Armstead, William M.; Liu, Renyu

    2012-01-01

    Background Cerebral hypoxia/ischemia during infant congenital heart surgery is not uncommon, and may induce devastating neurologic disabilities persistent over the lifespan. Hypoxia/ischemia-induced cerebrovascular dysfunction is thought to be an important contributor to neurological damage. No pharmacological agents have been found to prevent this. Mitogen activated protein kinase (MAPK), including extracellular signal regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38, is thought to contribute to ischemic preconditioning. We investigated whether pretreatment with salvinorin A, the only natural non-opioid kappa receptor agonist, could preserve autoregulation of the pial artery via MAPK. Methods The response of the pial artery to hypotension and hypercapnia was monitored in piglets equipped with a closed cranial window before and after hypoxia and ischemia in the presence or absence of U0126, an inhibitor for the protein kinase upstream of ERK, sp600125, an inhibitor of c-JNK or sb203580, an inhibitor of p38. Salvinorin A (10 μg/kg IV) was administered 30 minutes before hypoxia/ischemia in salvinorin-treated animals. Cerebrospinal fluid samples were collected before and 30 minutes after salvinorin A administration for the measurement of MAPK. Data (n=5) were analyzed by repeated-measures analysis of variance. Results Pial artery dilation to hypercapnia and hypotension was blunted after hypoxia/ischemia, but preserved well by pretreatment with salvinorin A. U0126, but not sp600125 or sb203580, abolished the preservative effects of salvinorin A on cerebral vascular autoregulation to hypotension and hypercapnia. The ratio of pERK/ERK in cerebrospinal fluid increased significantly in salvinorin-treated animals, which was inhibited by U0126. Conclusions Salvinorin A pretreatment preserves autoregulation of the pial artery to hypotension and hypercapnia after hypoxia/ischemia via ERK in a piglet model. PMID:22075021

  6. Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa.

    PubMed

    Cocchia, N; Pasolini, M P; Mancini, R; Petrazzuolo, O; Cristofaro, I; Rosapane, I; Sica, A; Tortora, G; Lorizio, R; Paraggio, G; Mancini, A

    2011-04-15

    New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50 IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24 h, 48 h and 7 2h. Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24 h, 48 h and 72 h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.

  7. Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein*

    PubMed Central

    Takahashi-Niki, Kazuko; Kato-Ose, Izumi; Murata, Hiroaki; Maita, Hiroshi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2015-01-01

    DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf. PMID:26048984

  8. Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein.

    PubMed

    Takahashi-Niki, Kazuko; Kato-Ose, Izumi; Murata, Hiroaki; Maita, Hiroshi; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2015-07-17

    DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.

  9. Effects of resistance exercise intensity on extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation in men.

    PubMed

    Taylor, Lem W; Wilborn, Colin D; Kreider, Richard B; Willoughby, Darryn S

    2012-03-01

    Extracellular signal-regulated kinase (ERK) 1/2 signaling has been shown to be increased after heavy resistance exercise and suggested to play a role in the hypertrophic adaptations that are known to occur with training. However, the role that ERK1/2 may play in response to lower intensities of resistance exercise is unknown. Therefore, the purpose of this study was to determine the effects of resistance exercise intensity on ERK1/2 activity in human skeletal muscle. Twelve recreationally active men completed separate bouts of single-legged resistance exercise with 8-10 repetitions (reps) at 80-85% 1 repetition maximum (1RM) (85%) and 18-20 reps at 60-65% 1RM (65%) in a randomized crossover fashion. For both resistance exercise sessions, vastus lateralis biopsies and blood draws were taken immediately before exercise (PRE) and at 30 minutes (30MPST), 2 hours (2HRPST), and 6 hours (6HRPST) post exercise, with an additional blood draw occurring immediately after exercise (POST). The phosphorylated levels of pIGF-1R, pMEK1, pERK1/2, and activated Elk-1 were assessed by phosphoELISA, and serum insulin-like growth factor 1 (IGF-1) was assessed via enzyme-linked immunosorbent assay. Statistical analyses used a 2 × 4 (muscle responses) and 2 × 5 (serum responses) multivariate analysis of variance on delta values from baseline (p < 0.05). Both exercise intensities significantly increased the activity of insulin-like growth factor 1 receptor (IGF-1R), mitogen-activated protein kinase 1, ERK1/2, and Elk-1, with peak activity occurring at 2HRPST (p < 0.001). However, 65% resulted in a preferential increase in IGF-1R and Elk-1 activation when compared with 85% (p < 0.05). No differences were observed for serum IGF-1 levels regardless of intensity and time. These findings demonstrate that resistance exercise upregulates ERK1/2 signaling in a manner that does not appear to be preferentially dependent on exercise intensity.

  10. Extracellular signal-regulated protein kinase activation in spinal cord contributes to pain hypersensitivity in a mouse model of type 2 diabetes.

    PubMed

    Xu, Xiang; Chen, Hui; Ling, Bing-Yu; Xu, Lan; Cao, Hong; Zhang, Yu-Qiu

    2014-02-01

    Painful peripheral neuropathy is a common complication of diabetes mellitus. The symptom of pain can become a major factor that decreases the quality of life of patients with diabetes, while effective treatment is lacking. In the present study, we aimed to investigate the changes of pain threshold in the early stage of diabetes in db/db mice, an animal model of type 2 diabetes mellitus, and the underlying molecular mechanisms. We found that (1) db/db mice (with a leptin receptor-null mutation and characterized by obesity and hyperglycemia) showed hypersensitivity to mechanical and thermal stimuli at the early stage of diabetes; (2) phosphorylated extracellular signal-regulated kinase (pERK), but not total ERK in the spinal cord and dorsal root ganglia in db/db mice significantly increased compared with wild-type mice. The increased pERK immunoreactivity occurred in both NeuN-expressing neurons and GFAP-expressing astrocytes, but not in Iba-1-expressing microglia; (3) both single and consecutive (for 5 days) intrathecal injections of U0126 (2 nmol per day), a selective MEK (an ERK kinase) inhibitor beginning at 8 weeks of age, attenuated the bilateral mechanical allodynia in the von-Frey test and heat hyperalgesia in Hargreave's test; and (4) db/db mice also displayed increased nocifensive behavior during the formalin test, and this was blocked by intrathecal injection of U0126. Also, the expression of pERK1 and pERK2 was upregulated following the formalin injection. Our results suggested that the activation of ERK in spinal neurons and astrocytes is correlated with pain hypersensitivity of the type 2 diabetes animal model. Inhibiting the ERK pathway may provide a new therapy for pain control in type 2 diabetes.

  11. 5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19.

    PubMed

    Wang, Qin; Terauchi, Akiko; Yee, Christopher H; Umemori, Hisashi; Traynor, John R

    2014-09-01

    The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor-Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.

  12. Extracellular Signal-Regulated Protein Kinase, c-Jun N-terminal Protein Kinase, and Calcineurin Regulate Transient Receptor Potential M3 (TRPM3) Induced Activation of AP-1.

    PubMed

    Lesch, Andrea; Rössler, Oliver G; Thiel, Gerald

    2017-01-23

    Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca(2+) /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. This article is protected by copyright. All rights reserved.

  13. Ras-induced and extracellular signal-regulated kinase 1 and 2 phosphorylation-dependent isomerization of protein tyrosine phosphatase (PTP)-PEST by PIN1 promotes FAK dephosphorylation by PTP-PEST.

    PubMed

    Zheng, Yanhua; Yang, Weiwei; Xia, Yan; Hawke, David; Liu, David X; Lu, Zhimin

    2011-11-01

    Protein tyrosine phosphatase (PTP)-PEST is a critical regulator of cell adhesion and migration. However, the mechanism by which PTP-PEST is regulated in response to oncogenic signaling to dephosphorylate its substrates remains unclear. Here, we demonstrate that activated Ras induces extracellular signal-regulated kinase 1 and 2-dependent phosphorylation of PTP-PEST at S571, which recruits PIN1 to bind to PTP-PEST. Isomerization of the phosphorylated PTP-PEST by PIN1 increases the interaction between PTP-PEST and FAK, which leads to the dephosphorylation of FAK Y397 and the promotion of migration, invasion, and metastasis of v-H-Ras-transformed cells. These findings uncover an important mechanism for the regulation of PTP-PEST in activated Ras-induced tumor progression.

  14. Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells.

    PubMed

    Marganski, William A; Gangopadhyay, Samudra S; Je, Hyun-Dong; Gallant, Cynthia; Morgan, Kathleen G

    2005-09-16

    Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.

  15. Extracellular-signal regulated kinase (Erk1/2), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and tristetraprolin (TTP) comprehensively regulate injury-induced immediate early gene (IEG) response in in vitro liver organ culture.

    PubMed

    Tran, Doan Duy Hai; Koch, Alexandra; Saran, Shashank; Armbrecht, Marcel; Ewald, Florian; Koch, Martina; Wahlicht, Tom; Wirth, Dagmar; Braun, Armin; Nashan, Björn; Gaestel, Matthias; Tamura, Teruko

    2016-05-01

    Differentiated hepatocytes are long-lived and normally do not undergo cell division, however they have the unique capacity to autonomously decide their replication fate after liver injury. In this context, the key players of liver regeneration immediately after injury have not been adequately studied. Using an in vitro liver culture system, we show that after liver injury, p38 mitogen-activated protein kinase (p38MAPK), mitogen-activated protein kinase-activated protein kinase 2 (MK2) and extracellular-signal regulated kinase (Erk)1/2 were activated within 15 min and continued to be phosphorylated for more than 2h. Both p38MAPK and Erk1/2 were activated at the edge of the cut as well as on the liver surface where the mesothelial cell sheet expresses several cytokines. Notably, in human liver Erk1/2 was also activated under the mesothelial cell sheet shortly after liver resections. Furthermore, in in vitro liver slice culture immediate early genes (IEGs) were upregulated within 1-2 h and the S phase marker proliferation-cell-nuclear-antigen (PCNA) appeared 24 h after injury. Although Erk1/2 was activated after injury, in MK2 depleted liver a set of IEGs, such as Dusp1, Cox2, or c-Myc and proliferation marker gene Ki67 were not induced. In addition, in immortalized hepatocyte cells, THLE-2, the same subset of genes was upregulated upon stimulation with lipopolysaccharide (LPS), but not in the presence of MK2 inhibitor. The protein level of tristetraprolin (TTP), a substrate for MK2 that plays a role in mRNA degradation, was increased in the presence of MK2 inhibitor. In this context, the depletion of TTP gene rescued Dusp1, Cox2, or c-Myc upregulation in the presence of MK2 inhibitor. These data imply that MK2 pathway is positively involved in Erk1/2 induced IEG response after liver injury. These data also suggest that in vitro liver culture may be a useful tool for measuring the proliferation potential of hepatocytes in individual liver.

  16. 8-Amino-adenosine induces loss of phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Akt kinase: role in induction of apoptosis in multiple myeloma.

    PubMed

    Ghias, Kulsoom; Ma, Chunguang; Gandhi, Varsha; Platanias, Leonidas C; Krett, Nancy L; Rosen, Steven T

    2005-04-01

    Multiple myeloma is a slowly proliferating B-cell malignancy that accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics that target rapidly replicating cells. Multiple myeloma remains an incurable disease in need of new therapeutic approaches. The purine nucleoside analogue, 8-amino-adenosine (8-NH2-Ado), exhibits potent activity in preclinical studies, inducing apoptosis in several multiple myeloma cell lines. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its triphosphate form, 8-amino-ATP, and results in a concomitant loss of endogenous ATP levels. Here, we show the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. Multiple myeloma cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and Akt kinase. Cells depleted of ATP independent of 8-NH2-Ado do not exhibit the same decrease in phosphorylation of vital cellular proteins. Therefore, the significant shifts in endogenous ATP pools caused by 8-NH2-Ado treatment cannot account for the changes in phosphorylation levels. Instead, 8-NH2-Ado may influence the activity of select regulatory protein kinases and/or phosphatases, with preliminary data suggesting that protein phophatase 2A activity is affected by 8-NH2-Ado. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analogue drug and is unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical prosurvival and apoptosis-regulating proteins suggests that the modulation of these proteins by dephosphorylation at early time points may be an important mechanistic step in 8-NH2-Ado-induced apoptosis.

  17. Rapid Turnover of Extracellular Signal-Regulated Kinase 3 by the Ubiquitin-Proteasome Pathway Defines a Novel Paradigm of Mitogen-Activated Protein Kinase Regulation during Cellular Differentiation

    PubMed Central

    Coulombe, Philippe; Rodier, Geneviève; Pelletier, Stéphane; Pellerin, Johanne; Meloche, Sylvain

    2003-01-01

    Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G1 arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability. PMID:12808096

  18. Transferrin receptor 2 and HFE regulate furin expression via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) signaling. Implications for transferrin-dependent hepcidin regulation

    PubMed Central

    Poli, Maura; Luscieti, Sara; Gandini, Valentina; Maccarinelli, Federica; Finazzi, Dario; Silvestri, Laura; Roetto, Antonella; Arosio, Paolo

    2010-01-01

    Background Impaired regulation of hepcidin in response to iron is the cause of genetic hemochromatosis associated with defects of HFE and transferrin receptor 2. However, the role of these proteins in the regulation of hepcidin expression is unclear. Design and Methods Hepcidin expression, SMAD and extracellular signal-regulated kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2 cells in which HFE and transferrin receptor 2 were down-regulated or expressed, or furin activity specifically inhibited. Furin expression was also analyzed in the liver of transferrin receptor 2 null mice. Results We showed that the silencing of HFE and transferrin receptor 2 reduced both Erk phosphorylation and furin expression, that the exogenous expression of the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin, but that this did not occur when the pathogenic HFE mutant C282Y was expressed. Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of furin activity caused a strong suppression of hepcidin mRNA, probably due to the inhibition of bone morphogenic protein maturation. Conclusions The data indicate that transferrin receptor 2 and HFE are involved in holotransferrin-dependent signaling for the regulation of furin which involved Erk phosphorylation. Furin in turn may control hepcidin expression. PMID:20634490

  19. 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

  20. Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice.

    PubMed

    Moriguchi, Shigeki; Yamamoto, Yui; Ikuno, Tatsuya; Fukunaga, Kohji

    2011-06-01

    Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK.

  1. 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

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

    2014-03-01

    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 signal-regulated 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 h but not 2 h post-training, delineating time points for STM (2 h) and LTM (4 h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24 h but not 2 h 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.

  2. Novel human neutrophil agonistic properties of arsenic trioxide: involvement of p38 mitogen-activated protein kinase and/or c-jun NH2-terminal MAPK but not extracellular signal-regulated kinases-1/2.

    PubMed

    Binet, François; Girard, Denis

    2008-12-01

    Arsenic trioxide (ATO) is known for treating acute promyelocytic leukemia and for inducing apoptosis and mitogen-activated protein kinases (MAPKs) in promyelocytes and cancer cells. We recently reported that ATO induces neutrophil apoptosis. The aim of this study was to establish whether or not ATO recruits MAPKs in neutrophils, as well as to further investigate its agonistic properties. We found that ATO activates p38 and that, unlike H2O2, this response was not inhibited by exogenous catalase. Also, we demonstrated that ATO-induced p38 activation occurs before H2O2 generation and without a calcium burst. We next established that ATO recruits c-jun NH2-terminal (JNK) but not extracellular signal-regulated kinase 1 and 2 (Erk-1/2). Using pharmacological inhibitors, we found that the proapoptotic activity of ATO occurs by a MAPK-independent mechanism. In contrast, the ability of ATO to enhance adhesion, migration, phagocytosis, release, and activity of gelatinase and degranulation of secretory, specific, and gelatinase, but not azurophilic granules, is dependent upon activation of p38 and/or JNK. This is the first study establishing that ATO possesses important agonistic properties in human neutrophils. Given the central role of neutrophils in various inflammatory disorders, we propose that ATO might have broader therapeutic implications in clinics, especially for regulating inflammation.

  3. 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

  4. Aurintricarboxylic acid protects against cell death caused by lipopolysaccharide in macrophages by decreasing inducible nitric-oxide synthase induction via IkappaB kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase inhibition.

    PubMed

    Tsi, Chin-Ju; Chao, Yee; Chen, Ching-Wen; Lin, Wan Wan

    2002-07-01

    To elucidate the mechanisms involved in cell protection by aurintricarboxylic acid (ATA), an endonuclease inhibitor, high nitric oxide (NO)-induced macrophage apoptosis was studied. In RAW 264.7 macrophages, a high level of NO production accompanied by cell apoptosis was apparent with lipopolysaccharide (LPS) treatment. Direct NO donor sodium nitroprusside (SNP) also dramatically induced cell death, with an EC(50) of 1 mM. Coincubation of ATA (1-500 microM) in LPS-stimulated RAW 264.7 cells resulted in a striking reduction of NO production and cell apoptosis, whereas only a partial cell protection was achieved in response to SNP. This suggests that abrogation of inducible nitric-oxide synthase (iNOS)-dependent NO production might contribute to ATA protection of LPS-treated cells. Immunoblotting and reverse transcription-polymerase chain reaction analysis revealed that ATA down-regulated iNOS protein through transcriptional inhibition of iNOS gene expression but was unrelated to iNOS protein stability. ATA not only inhibited nuclear factor-kappaB (NF-kappaB) activation through impairment of the targeting and degradation of IkappaBs but also reduced LPS-induced activator protein-1 (AP-1) activation. These actions of ATA were not caused by the influence on LPS binding to macrophage membrane. Kinase assays indicated that ATA inhibited IkappaB kinase (IKK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) activity both in vivo and in vitro, suggesting a direct interaction between ATA and these signaling molecules. Taken together, these results provide novel action targets of ATA and indicate that ATA protection of macrophages from LPS-mediated cell death is primarily the result of its inhibition of NO production, which closely relates to the inactivation of NF-kappaB and AP-1 and inhibition of IKK, ERK and p38 MAPK.

  5. 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

  6. 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.

  7. Oxidative stress induces extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase in cystic fibrosis lung epithelial cells: Potential mechanism for excessive IL-8 expression.

    PubMed

    Boncoeur, Emilie; Criq, Vinciane Saint; Bonvin, Elise; Roque, Telma; Henrion-Caude, Alexandra; Gruenert, Dieter C; Clement, Annick; Jacquot, Jacky; Tabary, Olivier

    2008-01-01

    Cystic fibrosis (CF) is a lethal disease caused by defective function of the cftr gene product, the CF transmembrane conductance regulator (CFTR) that leads to oxidative damage and excessive inflammatory response in lungs of CF patients. We here report the effects of oxidative stress (hyperoxia, 95% O(2)) on the expression of pro-inflammatory interleukin (IL)-8 and CXCR1/2 receptors in two human CF lung epithelial cell lines (IB3-1, with the heterozygous F508del/W1282X mutation and CFBE41o- with the homozygous F508del/F508del mutation) and two control non-CF lung epithelial cell lines (S9 cell line derived from IB3-1 after correction with wtCFTR and the normal bronchial cell line 16HBE14o-). Under oxidative stress, the expression of IL-8 and CXCR1/2 receptors was increased in CF, corrected and normal lung cell lines. The effects of oxidative stress were also investigated by measuring the transcription nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activities. Under oxidative stress, no increase of NF-kappaB activation was observed in CF lung cells in contrast to that observed in normal and corrected CF lung cells. The signalling of mitogen-activated protein (MAP) kinases was further studied. We demonstrated that extracellular signal-regulated kinase (ERK1/2) and AP-1 activity was markedly enhanced in CF but not non-CF lung cells under oxidative stress. Consistently, inhibition of ERK1/2 in oxidative stress-exposed CF lung cells strongly decreased both the IL-8 production and CXCR1/2 expression. Therefore, targeting of ERK1/2 MAP kinase may be critical to reduce oxidative stress-mediated inflammation in lungs of CF patients.

  8. Up-regulation of brain-derived neurotrophic factor is regulated by extracellular signal-regulated protein kinase 5 and by nerve growth factor retrograde signaling in colonic afferent neurons in colitis

    PubMed Central

    Yu, Sharon J; Grider, John R; Gulick, Melisa A; Xia, Chun-mei; Shen, Shanwei; Qiao, Li-Ya

    2012-01-01

    Brain-derived neurotrophic factor (BDNF) plays an essential role in sensory neuronal activation in response to visceral inflammation. Here we report that BDNF up-regulation in the primary afferent neurons in the dorsal root ganglia (DRG) in a rat model of colitis is mediated by the activation of endogenous extracellular signal-regulated protein kinases (ERK) 5 and by nerve growth factor (NGF) retrograde signaling. At 7 days of colitis, the expression level of BDNF is increased in conventional neuronal tracing dye Fast Blue labeled primary afferent neurons project to the distal colon. In these neurons, the phosphorylation (activation) level of ERK5 is also increased. In contrast, the level of phospho-ERK1/2 is not changed in the DRG during colitis. Prevention of the ERK5 activation in vivo with an intrathecal application of the MEK inhibitor PD98059 significantly attenuates the colitis-induced increases in BDNF expression in the DRG. Further studies show that BDNF up-regulation in the DRG is triggered by NGF retrograde signaling which also involves activation of the MEK/ERK pathways. Application of exogenous NGF exclusively to the compartment containing DRG nerve terminals in an ex vivo ganglia-nerve preparation has markedly increased the BDNF expression level in the DRG neuronal cell body that is placed in a different compartment; this BDNF elevation is attenuated by U0126, PD98059 and a specific ERK5 inhibitor BIX02188. These results demonstrate the mechanisms and pathways by which BDNF expression is elevated in primary sensory neurons following visceral inflammation that is mediated by increased activity of ERK5 and is likely to be triggered by the elevated NGF level in the inflamed viscera. PMID:22921460

  9. Prevention of tumour cell apoptosis associated with sustained protein kinase B phosphorylation is more sensitive to regulation by insulin signalling than stimulation of proliferation and extracellular signal-regulated kinase.

    PubMed

    Schmid, Christoph; Ghirlanda, Claudia; Niessen, Markus

    2017-03-18

    Insulin controls blood glucose while insulin-like growth factor (IGF) 1 is an important growth factor. Interestingly, both hormones have overlapping bioactivities and can activate the same intracellular signal transduction cascades. Growth control (mainly by IGF1) and metabolic function (predominantly by insulin) are believed to depend on activation of extracellular signal-regulated kinases (ERKs) 1/2 and protein kinase B (Akt/PKB), respectively. Therefore, insulin analogues that are used to normalize blood glucose are tested for their ability to preferentially activate Akt/PKB but not ERK1/2 and mitogenesis. Growth hormone, IGF1, and hyperinsulinemia are associated with increased risk of growth progression of some cancer types. To test if continuous exposure to insulin can favour tumour growth, we studied insulin/IGF1-dependent activation of ERK1/2 and Akt/PKB by Western blotting, inhibition of apoptosis by ELISA, and induction of proliferation by [(3)H]-thymidine incorporation in Saos-2/B10 osteosarcoma cells. IGF1 and insulin both induced proliferation and prevented apoptosis effectively. Regulation of apoptosis was far more sensitive than regulation of proliferation. IGF1 and insulin activated PKB (Akt/PKB) rapidly and consistently maintained its phosphorylation. Activation of ERK1/2 was only observed in response to IGF1. Loss of p-Akt/PKB (but not of p-ERK1/2) was associated with increased apoptosis, and protection from apoptosis was lost when activation of Akt/PKB was inhibited. These findings in Saos-2/B10 cells were also replicated in the A549 cell line, originally derived from a human lung carcinoma. Therefore, IGF1 and insulin more likely (at lower concentrations) enhance tumour cell survival than proliferation, via activation and maintenance of phosphatidylinositol 3-kinase activity and p-Akt/PKB.

  10. Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2.

    PubMed

    Perkovic, Sanja; Basic-Kinda, Sandra; Gasparovic, Vladimir; Krznaric, Zeljko; Babel, Jaksa; Ilic, Ivana; Aurer, Igor; Batinic, Drago

    2012-07-11

    Aggressive natural killer-cell leukaemia (ANKL) is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV) and P-glycoprotein (P-gp) expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56(+) NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

  11. Lipopolysaccharide Activation of the TPL-2/MEK/Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Cascade Is Regulated by IκB Kinase-Induced Proteolysis of NF-κB1 p105†

    PubMed Central

    Beinke, S.; Robinson, M. J.; Hugunin, M.; Ley, S. C.

    2004-01-01

    The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-κB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IκB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-κB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade. PMID:15485931

  12. Activation of extracellular signal-regulated kinase (ERK) and induction of mitogen-activated protein kinase phosphatase 1 (MKP-1) by perifused thyrotropin-releasing hormone (TRH) stimulation in rat pituitary GH3 cells.

    PubMed

    Oride, Aki; Kanasaki, Haruhiko; Mutiara, Sandra; Purwana, Indri Nuryani; Miyazaki, Kohji

    2008-12-16

    We investigated the pattern of extracellular signal-regulated kinase (ERK) phosphorylation and the induction of mitogen-activated protein kinase phosphatase 1 (MKP-1) by thyrotropin-releasing hormone (TRH) under various stimulation conditions in pituitary GH3 cells. In static culture, ERK activation by continuous TRH was maximal at 10 min and persisted for up to 60 min, with a return to the basal level by 2h. Stimulation with continuous TRH in perifused cells resulted in a similar level of ERK phosphorylation. MKP-1 was expressed 60 min following either static or perifused, continuous TRH stimulation. When cells were stimulated with pulsatile TRH every 30 min, ERK activation was maximal at 10 min and returned to its baseline level by 30 min. ERK was phosphorylated again with each subsequent pulse. Pulsatile TRH did not induce MKP-1. Prolactin promoter activity following continuous, static TRH stimulation was higher than that following perifused TRH stimulation. TRH at a frequency of one pulse every 30 min increased prolactin promoter activity similar to that of perifused, continuous TRH stimulation. Additionally, changes in pulse frequency resulted in alterations in the level of prolactin promoter. Following static stimulation, a 10 min exposure to TRH was sufficient to obtain full activation of the prolactin promoter. Additionally, a 5-10 min exposure of TRH was sufficient to maintain ERK activation. A single 5-min pulse of TRH stimulation resulted in low activation of the prolactin promoter. ERK activation was necessary for prolactin gene transcription; however, prolactin gene transcription is not entirely determined by the strength or duration of TRH-induced ERK activation.

  13. Capsaicin- and mustard oil-induced extracellular signal-regulated protein kinase phosphorylation in sensory neurons in vivo: effects of neurokinins 1 and 2 receptor antagonists and of a nitric oxide synthase inhibitor.

    PubMed

    Donnerer, Josef; Liebmann, Ingrid; Schuligoi, Rufina

    2009-01-01

    Stimulation of primary sensory neurons with capsaicin or mustard oil leads to phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) via activation of transient receptor potential V1 (TRPV1) or TRPA1, respectively. p-ERK1/2 was determined by Western immunoblotting in the dorsal root ganglia and in the sciatic nerve of rats following either systemic or perineural capsaicin treatment, or mustard oil application to the hind paw skin. To investigate the possible involvement of neurokinin 1 (NK(1)) and NK(2) receptors as well as of nitric oxide, the selective antagonists, SR140333 for NK(1) and SR48968 for NK(2), and the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), were employed. The increase of p-ERK1/2 after systemic capsaicin treatment was markedly attenuated by SR140333, while only the increase in the dorsal root ganglia was impaired by SR48968; in contrast, inhibition of nitric oxide synthase had no effect. Perineural capsaicin induced an increase in p-ERK1/2 in the ipsilateral sciatic nerve and in the dorsal root ganglia. This effect was not influenced by SR140333 or L-NAME. We found for the first time that mustard oil application to the hind paw skin caused an increase in p-ERK1/2 in the sciatic nerve and in the dorsal root ganglia and only the phosphorylation in the latter was attenuated by SR140333 while L-NAME showed no effect. From the present results, it may be assumed that capsaicin- or mustard oil-induced p-ERK1/2 in sensory neurons is not solely directly linked to TRPV1 or TRPA1 channels, but under certain conditions NK(1)- and NK(2)-mediated mechanisms are involved.

  14. Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells.

    PubMed

    Herraiz, Cecilia; Journé, Fabrice; Ghanem, Ghanem; Jiménez-Cervantes, Celia; García-Borrón, José C

    2012-12-01

    Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.

  15. Operant ethanol self-administration increases extracellular-signal regulated protein kinase (ERK) phosphorylation in reward-related brain regions: selective regulation of positive reinforcement in the prefrontal cortex of C57BL/6J mice

    PubMed Central

    Faccidomo, Sara; Salling, Michael C; Galunas, Christina; Hodge, Clyde W

    2015-01-01

    Rationale Extracellular-signal regulated protein kinase (ERK1/2) is activated by ethanol in reward-related brain regions. Accordingly, systemic inhibition of ERK1/2 potentiates ethanol reinforcement. However, the brain region(s) that mediate this effect are unknown. Objective To pharmacologically inhibit ERK1/2 in the medial prefrontal cortex (PFC), nucleus accumbens (NAC) and amygdala (AMY) prior to ethanol or sucrose self-administration, and evaluate effects of operant ethanol self-administration on ERK1/2 phosphorylation (pERK1/2). Methods Male C57BL/6J mice were trained to lever press on a fixed-ratio-4 schedule of 9% ethanol+2% sucrose (ethanol) or 2% sucrose (sucrose) reinforcement. Mice were sacrificed immediately after the 30th self-administration session and pERK1/2 immunoreactivity was quantified in targeted brain regions. Additional groups of mice were injected with SL 327 (0–1.7 μg/side) in PFC, NAC or AMY prior to self-administration. Results pERK1/2 immunoreactivity was significantly increased by operant ethanol (g/kg=1.21 g/kg; BAC=54.9 mg/dl) in the PFC, NAC (core and shell), and AMY (central nucleus) as compared to sucrose. Microinjection of SL 327 (1.7 μg) into the PFC selectively increased ethanol self-administration. Intra-NAC injection of SL 327 had no effect on ethanol- but suppressed sucrose-reinforced responding. Intra-AMY microinjection of SL 327 had no effect on either ethanol- or sucrose-reinforced responding. Locomotor activity was unaffected under all conditions. Conclusions Operant ethanol self-administration increases pERK1/2 activation in the PFC, NAC and AMY. However, ERK1/2 activity only in the PFC mechanistically regulates ethanol self-administration. These data suggest that ethanol-induced activation of ERK1/2 in the PFC is a critical pharmacological effect that mediates the reinforcing properties of the drug. PMID:26123321

  16. 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

  17. 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.

  18. Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

    PubMed Central

    Cerezo-Guisado, Maria Isabel; GarcíA-Román, Natalia; García-MaríN, Luis Jesús; Álvarez-Barrientos, Alberto; Bragado, Maria Julia; Lorenzo, Maria Jesús

    2006-01-01

    We have shown previously that lovastatin, a 3-hydroxy-3-methyl- glutaryl coenzyme A reductase inhibitor, induces apoptosis in spontaneously immortalized rat brain neuroblasts. In the present study, we analysed the intracellular signal transduction pathways by which lovastatin induces neuroblast apoptosis. We showed that lovastatin efficiently inhibited Ras activation, which was associ-ated with a significant decrease in ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Lovastatin also decreased CREB phosphorylation and CREB-mediated gene expression. The effects of lovastatin on the Ras/ERK1/2/CREB pathway were time- and concentration-dependent and fully prevented by meva-lonate. In addition, we showed that two MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitors, PD98059 and PD184352, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apop-tosis induced by lovastatin treatment. Furthermore, we showed that pharmacological inhibition of both MEK and phosphoinos-itide 3-kinase activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. Our results suggest that lovast-atin triggers neuroblast apoptosis by regulating several signalling pathways, including the Ras/ERK1/2 pathway. These findings might also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy. PMID:16952276

  19. 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.

  20. 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.

  1. Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a*

    PubMed Central

    Kakiya, Naomasa; Saito, Takashi; Nilsson, Per; Matsuba, Yukio; Tsubuki, Satoshi; Takei, Nobuyuki; Nawa, Hiroyuki; Saido, Takaomi C.

    2012-01-01

    Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain. PMID:22767595

  2. 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.

  3. Sleep deprivation impairs spatial memory and decreases extracellular signal-regulated kinase phosphorylation in the hippocampus.

    PubMed

    Guan, Zhiwei; Peng, Xuwen; Fang, Jidong

    2004-08-20

    Loss of sleep may result in memory impairment. However, little is known about the biochemical basis for memory deficits induced by sleep deprivation. Extracellular signal-regulated kinase (ERK) is involved in memory consolidation in different tasks. Phosphorylation of ERK is necessary for its activation and is an important step in mediating neuronal responses to synaptic activities. The aim of the present study was to determine the effects of total sleep deprivation (TSD) on memory and ERK phosphorylation in the brain. Rats were trained in Morris water maze to find a hidden platform (a spatial task) or a visible platform (a nonspatial task) after 6 h TSD or spontaneous sleep. TSD had no effect on spatial learning, but significantly impaired spatial memory tested 24 h after training. Nonspatial learning and memory were not impaired by TSD. Phospho-ERK levels in the hippocampus were significantly reduced after 6 h TSD compared to the controls and returned to the control levels after 2 h recovery sleep. Total ERK1 and ERK2 were slightly increased after 6 h TSD and returned to the control levels after 2 h recovery sleep. These alterations were not observed in the cortex after TSD. Protein phosphotase-1 and mitogen-activated protein kinase phosphatase-2, which dephosphorylates phospho-ERK, were also measured, but they were not altered by TSD. The impairments of both spatial memory and ERK phosphorylation indicate that the hippocampus is vulnerable to sleep loss. These results are consistent with the idea that decreased ERK activation in the hippocampus is involved in sleep deprivation-induced spatial memory impairment.

  4. Dynamic expression of extracellular signal-regulated kinase in rat liver tissue during hepatic fibrogenesis

    PubMed Central

    Zhang, Xiao-Lan; Liu, Jin-Ming; Yang, Chang-Chun; Zheng, Yi-Lin; Liu, Li; Wang, Zhan-Kui; Jiang, Hui-Qing

    2006-01-01

    AIM: To investigate whether extracellular signal-regulated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and Masson’s trichrome method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining. RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and perisinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vascular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK2 protein was up-regulated during the model course, and its level was the highest 4 wk after operation, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days after BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-SMA expression (r = 0.958,P < 0.05). CONCLUSION: The expression of ERK1 protein and mRNA is greatly increased in fibrotic rat liver tissues, which may play a

  5. 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.

  6. Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway*

    PubMed Central

    Quintanar-Audelo, Martina; Yusoff, Permeen; Sinniah, Saravanan; Chandramouli, Sumana; Guy, Graeme R.

    2011-01-01

    SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway. PMID:21531714

  7. Neuromedin U type 1 receptor stimulation of A-type K+ current requires the βγ subunits of Go protein, protein kinase A, and extracellular signal-regulated kinase 1/2 (ERK1/2) in sensory neurons.

    PubMed

    Zhang, Yiming; Jiang, Dongsheng; Zhang, Yuan; Jiang, Xinghong; Wang, Fen; Tao, Jin

    2012-05-25

    Although neuromedin U (NMU) has been implicated in analgesia, the detailed mechanisms still remain unclear. In this study, we identify a novel functional role of NMU type 1 receptor (NMUR1) in regulating the transient outward K(+) currents (I(A)) in small dorsal root ganglion (DRG) neurons. We found that NMU reversibly increased I(A) in a dose-dependent manner, instead the sustained delayed rectifier K(+) current (I(DR)) was not affected. This NMU-induced I(A) increase was pertussis toxin-sensitive and was totally reversed by NMUR1 knockdown. Intracellular application of GDPβS (guanosine 5'-O-(2-thiodiphosphate)), QEHA peptide, or a selective antibody raised against the Gα(o) or Gβ blocked the stimulatory effects of NMU. Pretreatment of the cells with the protein kinase A (PKA) inhibitor or ERK inhibitor abolished the NMU-induced I(A) response, whereas inhibition of phosphatidylinositol 3-kinase or PKC had no such effects. Exposure of DRG neurons to NMU markedly induced the phosphorylation of ERK (p-ERK), whereas p-JNK or p-p38 was not affected. Moreover, the NMU-induced p-ERK increase was attenuated by PKA inhibition and activation of PKA by foskolin would mimic the NMU-induced I(A) increase. Functionally, we observed a significant decrease of the firing rate of neuronal action potential induced by NMU and pretreatment of DRG neurons with 4-AP could abolish this effect. In summary, these results suggested that NMU increases I(A) via activation of NMUR1 that couples sequentially to the downstream activities of Gβγ of the G(o) protein, PKA, and ERK, which could contribute to its physiological functions including neuronal hypoexcitability in DRG neurons.

  8. Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia.

    PubMed

    Abraham, E; Arcaroli, J; Shenkar, R

    2001-01-01

    Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.

  9. 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.

  10. Extracellular signal-regulated kinase involved in NGF/VEGF-induced neuroprotective effect.

    PubMed

    Yang, Ji-Ping; Liu, Xin-Feng; Liu, Huai-Jun; Xu, Ge-Lin; Ma, Yu-Ping

    2008-03-28

    Compelling evidence has shown that extracellular signal-regulated kinase (ERK) is widely expressed in many tissues, including the brain. In the present work, we investigated the temporospatial alterations of ERK1 immunoreactivity in hippocampus and perifocal cortex, and the expression involved in NGF/VEGF-induced neuroprotective effect. We demonstrated that ERK1 expression was first increased in hippocampal CA3/DG 1 h after reperfusion, then it was also increased 6 h after reperfusion in other brain regions, with a peak at day 1-3, and then gradually decreased to basal level at day 14. The expression of caspase-3 was strongly increased 1 h after reperfusion, with peak demonstrated at 3d. NGF/VEGF significantly inhibited the expression of ERK1 and caspase-3. These results suggest that ERK1 signaling pathway may be involved in neuronal cell death and NGF/VEGF-induced neuroprotective effect and there appeared an association between ERK and caspase-3. Inhibition of the ERK signaling pathway might therefore provide an efficient way to prevent neuronal cell death after ischemic cerebral injuries.

  11. NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

    PubMed Central

    Kuek, Vincent; Yang, Zhifan; Chim, Shek Man; Zhu, Sipin; Xu, Huazi; Chow, Siu To; Tickner, Jennifer; Rosen, Vicki; Erber, Wendy; Li, Xiucheng; An, Qin; Qian, Yu; Xu, Jiake

    2016-01-01

    Angiogenesis plays an important role in bone development and remodeling and is mediated by a plethora of potential angiogenic factors. However, data regarding specific angiogenic factors that are secreted within the bone microenvironment to regulate osteoporosis is lacking. Here, we report that Nephronectin (NPNT), a member of the epidermal growth factor (EGF) repeat superfamily proteins and a homologue of EGFL6, is expressed in osteoblasts. Intriguingly, the gene expression of NPNT is reduced in the bone of C57BL/6J ovariectomised mice and in osteoporosis patients. In addition, the protein levels of NPNT and CD31 are also found to be reduced in the tibias of OVX mice. Exogenous addition of mouse recombinant NPNT on endothelial cells stimulates migration and tube-like structure formation in vitro. Furthermore, NPNT promotes angiogenesis in an ex vivo fetal mouse metatarsal angiogenesis assay. We show that NPNT stimulates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated kinase (MAPK) in endothelial cells. Inhibition of ERK1/2 impaired NPNT-induced endothelial cell migration, tube-like structure formation and angiogenesis. Taken together, these results demonstrate that NPNT is a paracrine angiogenic factor and may play a role in pathological osteoporosis. This may lead to new targets for treatment of bone diseases and injuries. PMID:27782206

  12. Sex differences in contextual fear conditioning are associated with differential ventral hippocampal extracellular signal-regulated kinase activation.

    PubMed

    Gresack, J E; Schafe, G E; Orr, P T; Frick, K M

    2009-03-17

    Although sex differences have been reported in hippocampal-dependent learning and memory, including contextual fear memories, the underlying molecular mechanisms contributing to such differences are not well understood. The present study examined the extent to which sex differences in contextual fear conditioning are related to differential activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), a protein kinase critically involved in memory formation. We first show that male rats exhibit more long-term retention of contextual fear conditioning than female rats. During a tone test, females spent more time freezing than males, although both sexes exhibited robust retention of auditory fear learning. Using Western blot analysis, we then show that phosphorylated ERK levels in ventral, but not dorsal, hippocampus are higher in males than females, relative to same-sex controls, 60 minutes after fear conditioning. Post-conditioning increases in ERK activation were observed in the amygdala in both males and females, suggesting a selective effect of sex on hippocampal ERK activation. Together, these findings suggest that differential activation of the ERK signal transduction pathway in male and female rats, particularly in the ventral hippocampus, is associated with sex differences in contextual fear.

  13. Induction of interleukin-8 by Naegleria fowleri lysates requires activation of extracellular signal-regulated kinase in human astroglial cells.

    PubMed

    Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Sang-Hee; Kwon, Daeho; Shin, Ho-Joon

    2012-08-01

    Naegleria fowleri is a pathogenic free-living amoeba which causes primary amoebic meningoencephalitis in humans and experimental animals. To investigate the mechanisms of such inflammatory diseases, potential chemokine gene activation in human astroglial cells was investigated following treatment with N. fowleri lysates. We demonstrated that N. fowleri are potent inducers for the expression of interleukin-8 (IL-8) genes in human astroglial cells which was preceded by activation of extracellular signal-regulated kinase (ERK). In addition, N. fowleri lysates induces the DNA binding activity of activator protein-1 (AP-1), an important transcription factor for IL-8 induction. The specific mitogen-activated protein kinase kinase/ERK inhibitor, U0126, blocks N. fowleri-mediated AP-1 activation and subsequent IL-8 induction. N. fowleri-induced IL-8 expression requires activation of ERK in human astroglial cells. These findings indicate that treatment of N. fowleri on human astroglial cells leads to the activation of AP-1 and subsequent expression of IL-8 which are dependent on ERK activation. These results may help understand the N. fowleri-mediated upregulation of chemokine and cytokine expression in the astroglial cells.

  14. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  15. 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.

  16. 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.

  17. Using Caenorhabditis elegans as a model organism for evaluating extracellular signal-regulated kinase docking domain inhibitors

    PubMed Central

    Chen, Fengming; MacKerell, Alexander D.; Luo, Yuan

    2008-01-01

    We have recently identified several novel ATP-independent inhibitors that target the extracellular signal-regulated kinase-2 (ERK2) protein and inhibit substrate phosphorylation. To further characterize these compounds, we describe the use of C. elegans as a model organism. C. elegans is recognized as a versatile and cost effective model for use in drug development. These studies take advantage of the well characterized process of vulva development and egg laying, which requires MPK-1, the homolog to human ERK2. It is shown that treatment of C. elegans eggs or larvae prior to vulva formation with a previously identified lead compound (76) caused up to 50% reduction in the number of eggs produced from the adult worm. In contrast, compound 76 had no effect on egg laying in young adult or adult worms with fully formed vulva. The reduction in egg laying by the test compound was not due to effects on C. elegans life span, general toxicity, or non-specific stress. However, compound 76 did show selective inhibition of phosphorylation of LIN-1, a MPK-1 substrate essential for vulva precursor cell formation. Moreover, compound 76 inhibited cell fusion necessary for vulva maturation and reduced the multivulva phenotype in LET-60 (Ras) mutant worms that have constitutive activation of MPK-1. These findings support the use of C. elegans as a model organism to evaluate the selectivity and specificity of novel ERK targeted compounds. PMID:19105050

  18. N-Terminal Ubiquitination of Extracellular Signal-Regulated Kinase 3 and p21 Directs Their Degradation by the Proteasome

    PubMed Central

    Coulombe, Philippe; Rodier, Geneviève; Bonneil, Eric; Thibault, Pierre; Meloche, Sylvain

    2004-01-01

    Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH2 terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized. PMID:15226418

  19. 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.

  20. 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.

  1. α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

    PubMed Central

    Liu, Fei; He, Kangmin; Yang, Xinxing; Xu, Ning; Liang, Zhangyi; Xu, Ming; Zhao, Xinsheng; Han, Qide; Zhang, Youyi

    2011-01-01

    G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling. PMID:21738688

  2. Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins.

    PubMed

    Chowdhury, Indrajit; Thompson, Winston E; Welch, Crystal; Thomas, Kelwyn; Matthews, Roland

    2013-12-01

    Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1

  3. p53 Protein-mediated Up-regulation of MAP Kinase Phosphatase 3 (MKP-3) Contributes to the Establishment of the Cellular Senescent Phenotype through Dephosphorylation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2)*

    PubMed Central

    Zhang, Hui; Chi, Yuan; Gao, Kun; Zhang, Xiling; Yao, Jian

    2015-01-01

    Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced β-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogen-activated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-α. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence. PMID:25414256

  4. The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth*

    PubMed Central

    Rosati, Rayna; Patki, Mugdha; Chari, Venkatesh; Dakshnamurthy, Selvakumar; McFall, Thomas; Saxton, Janice; Kidder, Benjamin L.; Shaw, Peter E.; Ratnam, Manohar

    2016-01-01

    The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1. The ELK1 polypeptide motifs required for co-activation by AR versus those required for activation of ELK1 by ERK were systematically mapped using a mammalian two-hybrid system and confirmed using a co-immunoprecipitation assay. The mapping precisely identified the two ERK-docking sites in ELK1, the D-box and the DEF (docking site for ERK, FXFP) motif, as the essential motifs for its cooperation with AR(A/B) or WTAR. In contrast, the transactivation domain in ELK1 was only required for activation by ERK. ELK1-mediated transcriptional activity of AR(A/B) was optimal in the absence of ELK1 binding partners, ERK1/2 and serum-response factor. Purified ELK1 and AR bound with a dissociation constant of 1.9 × 10−8 m. A purified mutant ELK1 in which the D-box and DEF motifs were disrupted did not bind AR. An ELK1 mutant with deletion of the D-box region had a dominant-negative effect on androgen-dependent growth of PCa cells that were insensitive to MEK inhibition. This novel mechanism in which a nuclear receptor impinges on a signaling pathway by co-opting protein kinase docking sites to constitutively activate growth genes could enable rational design of a new class of targeted drug interventions. PMID:27793987

  5. Phosphorylation of TPL-2 on Serine 400 Is Essential for Lipopolysaccharide Activation of Extracellular Signal-Regulated Kinase in Macrophages▿

    PubMed Central

    Robinson , M. J.; Beinke , S.; Kouroumalis, A.; Tsichlis, P. N.; Ley, S. C.

    2007-01-01

    Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-κB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1−/− macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1−/− macrophages. TPL-2S400A expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8−/− macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-κB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step. PMID:17709378

  6. Measurement of phosphorylated extracellular signal-regulated kinase 1 and 2 in an undergraduate teaching laboratory with ALPHAscreen technology.

    PubMed

    Hay, Debbie L

    2009-03-17

    Teaching the practical aspects of signal transduction to large undergraduate classes can be challenging when there is only a finite time frame in which to engage in laboratory activities. This teaching resource describes the use of bead-based ALPHAscreen technology for a class of 300 second-year biochemistry students, exposing the next generation of researchers to cutting-edge technology. Although in this case phosphorylated extracellular signal-regulated kinase 1 and 2 were measured, this technology is applicable to the measurement of many different signaling components. This resource provides a practical guide for instructors and exemplifies how such traditionally high-throughput research technologies can be used as teaching tools.

  7. Both mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 and phosphatidylinositide-3-OH kinase (PI3K)/Akt pathways regulate activation of E-twenty-six (ETS)-like transcription factor 1 (Elk-1) in U138 glioblastoma cells.

    PubMed

    Mut, Melike; Lule, Sevda; Demir, Ozlem; Kurnaz, Isil Aksan; Vural, Imran

    2012-02-01

    Epidermal growth factor (EGF) and its receptor (EGFR) have been shown to play a significant role in the pathogenesis of glioblastoma. In our study, the EGFR was stimulated with EGF in human U138 glioblastoma cells. We show that the activated mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) 1/2 pathway phosphorylated the E twenty-six (ETS)-like transcription factor 1 (Elk-1) mainly at serine 383 residue. Mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, UO126 and ERK inhibitor II, FR180204 blocked the Elk-1 phosphorylation and activation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt pathway was also involved in the Elk-1 activation. Activation of the Elk-1 led to an increased survival and a proliferative response with the EGF stimulation in the U138 glioblastoma cells. Knocking-down the Elk-1 using an RNA interference technique caused a decrease in survival of the unstimulated U138 glioblastoma cells and also decreased the proliferative response to the EGF stimulation. The Elk-1 transcription factor was important for the survival and proliferation of U138 glioblastoma cells upon the stimulation of EGFR with EGF. The MAPK/ERK1/2 and PI3K/Akt pathways regulated this response via activation of the Elk-1 transcription factor. The Elk-1 may be one of the convergence points for pathways located downstream of EGFR in glioblastoma cells. Utilization of the Elk-1 as a therapeutic target may lead to a novel strategy in treatment of glioblastoma.

  8. Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways: role of c-Jun N-terminal kinase in paraquat-induced cell death.

    PubMed

    Niso-Santano, Mireia; Morán, José M; García-Rubio, Lourdes; Gómez-Martín, Ana; González-Polo, Rosa A; Soler, Germán; Fuentes, José M

    2006-08-01

    Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP(+)), a well-known neurotoxin which causes a clinical syndrome similar to Parkinson's disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB) activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059 (2'-amino-3'-methoxyflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 had no effect on paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented clearly suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and support the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment.

  9. Neuroprotection of brain-derived neurotrophic factor against hypoxic injury in vitro requires activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

    PubMed

    Sun, Xiaomei; Zhou, Hui; Luo, Xiaoli; Li, Shengfu; Yu, Dan; Hua, Jiping; Mu, Dezhi; Mao, Meng

    2008-01-01

    Intrauterine asphyxia is one of the major contributors for perinatal death, mental and physical disorders of surviving children. Brain-derived neurotrophic factor (BDNF) provides a promising solution to hypoxic injury due to its survival-promoting effects. In an attempt to identify possible molecular mechanisms underlying the neuroprotective role of BDNF, we studied extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI-3-K) and p38 mitogen-activated protein kinase (MAPK) pathways. We demonstrated that BDNF protected cortical neurons against hypoxic injury in vitro via activation of both the ERK and PI-3-K pathways but not the p38 MAPK pathway. We also showed that both hypoxic stimuli and exogenous BDNF treatment phosphorylated the cyclic AMP response element-binding protein (CREB) and that CREB phosphorylation induced by BDNF was mediated via the ERK pathway in cultured cortical neurons.

  10. Food restriction increases NMDA receptor-mediated calcium-calmodulin kinase II and NMDA receptor/extracellular signal-regulated kinase 1/2-mediated cyclic amp response element-binding protein phosphorylation in nucleus accumbens upon D-1 dopamine receptor stimulation in rats.

    PubMed

    Haberny, S L; Carr, K D

    2005-01-01

    Biological drive states exert homeostatic control in part by increasing the reinforcing effects of environmental incentive stimuli. An apparent by-product of this adaptive response is the enhanced acquisition of drug self-administration behavior in food-restricted (FR) animals. While previous research has demonstrated increased central sensitivity to rewarding effects of abused drugs and direct dopamine (DA) receptor agonists in FR subjects, the underlying neurobiology is not well understood. Recently, it was demonstrated that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958 produces a stronger activation of striatal extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element-binding protein (CREB) in FR relative to ad libitum (AL) fed rats. The main purpose of the present study was to characterize the involvement and mechanisms of interaction between NMDA receptor function and the augmented cellular responses to D-1 DA receptor stimulation in nucleus accumbens (NAc) of FR rats. In experiment 1, Western immunoblotting was used to demonstrate that i.c.v. injection of SKF-82958 (20 microg) produces greater phosphorylation of the NMDA NR1 subunit and calcium-calmodulin kinase II (CaMK II) in NAc of FR as compared with AL rats. In experiment 2, pretreatment of subjects with the NMDA antagonist, MK-801 (1.0 mg/kg, i.p.) decreased SKF-82958-induced activation of CaMK II, ERK1/2 and CREB, and reversed the augmenting effect of FR on activation of all three proteins. In experiment 3, pretreatment with the mitogen-activated protein kinase/ERK kinase inhibitor SL-327 (60 mg/kg, i.p.) suppressed SKF-82958- induced activation of ERK1/2 and reversed the augmenting effect of FR on CREB activation. These results point to specific neuroadaptations in the NAc of FR rats whereby D-1 DA receptor stimulation leads to increased NMDA NR1 subunit phosphorylation and consequent increases in NMDA receptor-dependent CaMK II and ERK1

  11. 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.

  12. Erythropoietin inhibits osmotic swelling of retinal glial cells by Janus kinase- and extracellular signal-regulated kinases1/2-mediated release of vascular endothelial growth factor.

    PubMed

    Krügel, K; Wurm, A; Linnertz, R; Pannicke, T; Wiedemann, P; Reichenbach, A; Bringmann, A

    2010-02-17

    The volume homeostasis of retinal glial cells is mediated by an autocrine purinergic mechanism of ion channel opening which is activated in response to a decrease in the extracellular osmolarity. Here, we show that erythropoietin (EPO) prevents the osmotic swelling of glial somata in retinal slices and of isolated glial cells from control and diabetic rats, with a half-maximal effect at approximately 0.01 nM. The downstream signaling evoked by EPO includes a release of vascular endothelial growth factor from the cells which was blocked by Janus kinase and extracellular signal-regulated kinases (ERK)1/2 inhibitors. Transactivation of kinase insert domain-containing receptor/fms-like tyrosine kinase 1 (KDR/flk-1) evokes a calcium-dependent, exocytotic release of glutamate, followed by activation of group I/II metabotropic glutamate receptors which results in calcium-independent release of ATP and adenosine from the cells. The final step in this cascade is the activation of adenosine A(1) receptors which results in protein kinase A- and phosphoinositide 3-kinase-mediated opening of potassium and chloride channels. EPO receptor protein was immunohistochemically localized to the inner retina and photoreceptor inner segments. In isolated glial cells, EPO receptor protein is selectively localized to fibers which traverse the inner nuclear layer in situ. Inhibition of glial swelling might contribute to the neuroprotective action of EPO in the retina under pathological conditions.

  13. Role of the extracellular signal-regulated kinase (Erk) signal transduction cascade in alpha(2) adrenoceptor-mediated vasoconstriction in porcine palmar lateral vein.

    PubMed

    Roberts, R E

    2001-07-01

    The mechanism of alpha(2) adrenoceptor-mediated vasoconstriction is unknown, but may involve activation of voltage-sensitive calcium channels, and/or a protein tyrosine kinase. Recently the extracellular signal-regulated kinase (Erk) cascade, often an event downstream of tyrosine kinase activation, has been shown to mediate vasoconstriction to a variety of agents. The aim of this present study was to determine the involvement of the Erk signal transduction cascade in alpha(2) adrenoceptor-mediated vasoconstriction, and to confirm the involvement of activation of voltage-sensitive calcium channels, and protein tyrosine kinase. Contractions to the alpha(2) adrenoceptor agonist UK14304 in the porcine palmar lateral vein in vitro were reduced 70 - 80% by the MEK inhibitors PD98059 (10 - 50 microM) and U0126 (10 - 50 microM), indicating the involvement of the Erk signal transduction cascade. Immunoblots also demonstrated an increase in the phosphorylated (activated) form of Erk in palmar lateral vein segments after contraction with UK14304, which was inhibited by PD98059 and U0126. The calcium channel blockers nifedipine and verapamil, or removal of extracellular calcium inhibited UK14304-induced contractions and phosphorylation of Erk, demonstrating the importance of an influx of extracellular calcium. UK14304-induced contractions were inhibited by PP2 (1 - 10 microM), a selective inhibitor of Src tyrosine kinases, but not by PP3, an inactive analogue. PP2 also prevented the phosphorylation of Erk by UK14304. These data demonstrate that alpha(2) adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the Erk signal transduction cascade, which is downstream of an influx of extracellular calcium, and activation of Src tyrosine kinases.

  14. 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.

  15. Extracellular signal-regulated kinase activation in spinal astrocytes and microglia contributes to cancer-induced bone pain in rats.

    PubMed

    Wang, X-W; Li, T-T; Zhao, J; Mao-Ying, Q-L; Zhang, H; Hu, S; Li, Q; Mi, W-L; Wu, G-C; Zhang, Y-Q; Wang, Y-Q

    2012-08-16

    Cancer pain, especially cancer-induced bone pain, affects the quality of life of cancer patients, and current treatments for this pain are limited. The present study demonstrates that spinal extracellular signal-regulated kinase (ERK) activation in glial cells plays a crucial role in cancer-induced bone pain. From day 4 to day 21 after the intra-tibia inoculation with Walker 256 mammary gland carcinoma cells, significant mechanical allodynia was observed as indicated by the decrease of mechanical withdrawal thresholds in the von Frey hair test. Intra-tibia inoculation with carcinoma cells induced a vast and persistent (>21 D) activation of ERK in the bilateral L2-L3 and L4-L5 spinal dorsal horn. The increased pERK1/2-immunoreactivity was observed in both Iba-1-expressing microglia and GFAP-expressing astrocytes but not in NeuN-expressing neurons. A single intrathecal injection of the selective MEK (ERK kinase) inhibitors PD98059 (10 μg) on day 12 and U0126 (1.25 and 3 μg) on day 14, attenuated the bilateral mechanical allodynia in the von Frey hair test. Altogether, our results suggest that ERK activation in spinal microglia and astrocytes is correlated with the onset of allodynia and is important for allodynia maintenance in the cancer pain model. This study indicated that inhibition of the ERK pathway may provide a new therapy for cancer-induced bone pain.

  16. 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.

  17. Genistein modulates prostate epithelial cell proliferation via estrogen- and extracellular signal-regulated kinase-dependent pathways.

    PubMed

    Wang, Xingya; Clubbs, Elizabeth A; Bomser, Joshua A

    2006-03-01

    Epidemiological evidence suggests that consumption of soy is associated with a decreased risk for prostate cancer. Genistein, the most abundant isoflavone present in soy, is thought to be responsible, in part, for these anticancer effects. The present study examined the effects of genistein on cellular proliferation, extracellular signal-regulated kinase (ERK1/2) activity and apoptosis in a nontumorigenic human prostate epithelial cell line (RWPE-1). Low concentrations of genistein (0-12.5 micromol/L) significantly increased cell proliferation and ERK1/2 activity (P<.01) in RWPE-1 cells, while higher concentrations (50 and 100 micromol/L) of genistein significantly inhibited cell proliferation and ERK1/2 activity (P<.001). A similar biphasic effect of genistein on MEK1 activity, an ERK1/2 kinase, was also observed. Pretreatment of cells with a MEK1 inhibitor (PD 098059) significantly blocked genistein-induced proliferation and ERK1/2 activity (P<.01). In addition, treatment of cells with ICI 182,780, a pure antiestrogen, inhibited genistein-induced RWPE-1 proliferation and ERK1/2 signaling. Taken together, these results suggest that genistein modulates RWPE-1 cell proliferation and signal transduction via an estrogen-dependent pathway involving ERK1/2 activation.

  18. Traditional Chinese medicine suppresses left ventricular hypertrophy by targeting extracellular signal-regulated kinases signaling pathway in spontaneously hypertensive rats

    PubMed Central

    Xiong, Xingjiang; Yang, Xiaochen; Duan, Lian; Liu, Wei; Zhang, Yun; Liu, Yongmei; Wang, Pengqian; Li, Shengjie; Li, Xiaoke

    2017-01-01

    Chinese herbal medicine Bu-Shen-Jiang-Ya decoction (BSJYD) is reported to be beneficial for hypertension. Over expression of extracellular signal regulated kinases (ERK) pathway plays an important role in left ventricular hypertrophy (LVH). This study aimed to observe effects of BSJYD on LVH in spontaneously hypertensive rats (SHRs) and explore its possible mechanism on regulation of ERK pathway. Sixty 12-week-old SHRs were randomly allocated into 5 groups: BSJYD high dose group, middle dose group, low dose group, captopril group, and control group. Besides, a control group of Wistar-Kyoto rats was established. All rats were treated for 8 weeks. Systolic blood pressure (SBP), heart rate (HR), pathology, and left ventricular mass index (LVMI) were measured. Western blotting and Real-time PCR were used to assess the expressions of BDNF, Ras, ERK1/2, and c-fox levels. SBP and HR were significantly decreased compared with the control group and LVMI was markedly improved by BSJYD treatment in a dose-dependent manner. BSJYD inhibited the expression of BDNF, Ras, ERK1/2, and c-fox mRNA in LVH. In conclusion, BSJYD suppressed hypertension-induced cardiac hypertrophy by inhibiting the expression of ERK pathway. These changes in gene expression may be a possible mechanism by which BSJYD provides myocardial protection from hypertension. PMID:28225023

  19. Pleiotrophin promotes microglia proliferation and secretion of neurotrophic factors by activating extracellular signal-regulated kinase 1/2 pathway.

    PubMed

    Miao, Jiayin; Ding, Minghui; Zhang, Aiwu; Xiao, Zijian; Qi, Weiwei; Luo, Ning; Di, Wei; Tao, Yuqian; Fang, Yannan

    2012-12-01

    Pleiotrophin (PTN) is an effective neuroprotective factor and its expression is strikingly increased in microglia after ischemia/reperfusion injury. However, whether PTN could provide neurotrophic support to neurons by regulating microglia function is not clear. In this study, we demonstrated that the expression of PTN was induced in microglia after oxygen-glucose deprivation/reperfusion. PTN promoted the proliferation of microglia by enhancing the G1 to S phase transition. PTN also stimulated the secretion of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) in microglia, but did not upregulate the expression of proinflammatory factors such as TNF-α, IL-1β and iNOS. Mechanistically, we found that PTN increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in microglia in both concentration-dependent and time-dependent manners. In addition, ERK1/2 inhibitor U0126 abolished the proliferation and G1 to S phase transition of microglia stimulated by PTN, and inhibited the production of BDNF, CNTF and NGF induced by PTN. In conclusion, our results demonstrated that PTN-ERK1/2 pathway plays important role in regulating microglia growth and secretion of neurotrophic factors. These findings provide new insight into the neuroprotective role of PTN and suggest that PTN is a new target for therapeutic intervention of stroke.

  20. Traditional Chinese medicine suppresses left ventricular hypertrophy by targeting extracellular signal-regulated kinases signaling pathway in spontaneously hypertensive rats.

    PubMed

    Xiong, Xingjiang; Yang, Xiaochen; Duan, Lian; Liu, Wei; Zhang, Yun; Liu, Yongmei; Wang, Pengqian; Li, Shengjie; Li, Xiaoke

    2017-02-22

    Chinese herbal medicine Bu-Shen-Jiang-Ya decoction (BSJYD) is reported to be beneficial for hypertension. Over expression of extracellular signal regulated kinases (ERK) pathway plays an important role in left ventricular hypertrophy (LVH). This study aimed to observe effects of BSJYD on LVH in spontaneously hypertensive rats (SHRs) and explore its possible mechanism on regulation of ERK pathway. Sixty 12-week-old SHRs were randomly allocated into 5 groups: BSJYD high dose group, middle dose group, low dose group, captopril group, and control group. Besides, a control group of Wistar-Kyoto rats was established. All rats were treated for 8 weeks. Systolic blood pressure (SBP), heart rate (HR), pathology, and left ventricular mass index (LVMI) were measured. Western blotting and Real-time PCR were used to assess the expressions of BDNF, Ras, ERK1/2, and c-fox levels. SBP and HR were significantly decreased compared with the control group and LVMI was markedly improved by BSJYD treatment in a dose-dependent manner. BSJYD inhibited the expression of BDNF, Ras, ERK1/2, and c-fox mRNA in LVH. In conclusion, BSJYD suppressed hypertension-induced cardiac hypertrophy by inhibiting the expression of ERK pathway. These changes in gene expression may be a possible mechanism by which BSJYD provides myocardial protection from hypertension.

  1. 5-HT7 receptor-mediated fear conditioning and possible involvement of extracellular signal-regulated kinase.

    PubMed

    Takeda, Kotaro; Tsuji, Minoru; Miyagawa, Kazuya; Takeda, Hiroshi

    2017-01-18

    Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The present study examined the involvement of extracellular signal-regulated kinase 1/2 (ERK) signaling on the serotonin (5-HT)7 receptor-mediated fear conditioning. Conditioning was performed in a trial in which a tone was followed by an electrical foot-shock. Context- and tone-dependent fear were examined in tests conducted 24 and 48h after conditioning, respectively. The selective 5-HT7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,-tetrahydrobenzo(c,d)indol-2-(1H)-one (DR4004) (5mg/kg), when administered intraperitoneally (i.p.) immediately after conditioning, caused a significant decrease in both context- and tone-dependent fear responses (freezing behavior). A significant increase in ERK activity was observed in the amygdala of mice that displayed context- or tone-dependent fear responses, and these changes were also inhibited by the administration of DR4004 (5mg/kg, i.p.) immediately after conditioning. In contrast, the increase in hippocampal ERK activity in mice that displayed context-dependent fear responses was further enhanced by the administration of DR4004 (5mg/kg, i.p.). These results suggest that 5-HT7 receptor-mediated ERK signaling may play a significant role in the processes of emotional learning and memory.

  2. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter.

    PubMed

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-07-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPK(ERK)) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters.

  3. Retinoic Acid Receptors Inhibit AP1 Activation by Regulating Extracellular Signal-Regulated Kinase and CBP Recruitment to an AP1-Responsive Promoter

    PubMed Central

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-01-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPKERK) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters. PMID:12052862

  4. Early postnatal stress alters extracellular signal-regulated kinase signaling in the corticolimbic system modulating emotional circuitry in adult rats.

    PubMed

    Ishikawa, Shuhei; Saito, Yasuhiro; Yanagawa, Yoshiki; Otani, Satoru; Hiraide, Sachiko; Shimamura, Kei-ichi; Matsumoto, Machiko; Togashi, Hiroko

    2012-01-01

    The present study elucidated whether early life stress alters the extracellular signal-regulated kinase (ERK) pathway that underlies fear retrieval and fear extinction based on a contextual fear conditioning paradigm, using a juvenile stress model. Levels of phospho-ERK (pERK), the active form of ERK, increased after fear retrieval in the hippocampal CA1 region but not in the medial prefrontal cortex (mPFC). ERK activation in the CA1 following fear retrieval was not observed in adult rats who received aversive footshock (FS) stimuli during the second postnatal period (2wFS), which exhibited low levels of freezing. In fear extinction, pERK levels in the CA1 were increased by repeated extinction trials, but they were not altered after extinction retrieval. In contrast, pERK levels in the mPFC did not change during extinction training, but were enhanced after extinction retrieval. These findings were compatible in part with electrophysiological data showing that synaptic transmission in the CA1 field and mPFC was enhanced during extinction training and extinction retrieval, respectively. ERK activation in the CA1 and mPFC associated with extinction processes did not occur in rats that received FS stimuli during the third postnatal period (3wFS), which exhibited sustained freezing behavior. The repressed ERK signaling and extinction deficit observed in the 3wFS group were ameliorated by treatment with the partial N-methyl-D-aspartate receptor agonist D-cycloserine. These findings suggest that early postnatal stress induced the downregulation of ERK signaling in distinct brain regions through region-specific regulation, which may lead to increased behavioral abnormalities or emotional vulnerabilities in adulthood.

  5. Involvement of activation of NADPH oxidase and extracellular signal-regulated kinase (ERK) in renal cell injury induced by zinc.

    PubMed

    Matsunaga, Yoshiko; Kawai, Yoshiko; Kohda, Yuka; Gemba, Munekazu

    2005-05-01

    Zinc is employed as a supplement; however, zinc-related nephropathy is not generally known. In this study, we investigated zinc-induced renal cell injury using a pig kidney-derived cultured renal epithelial cell line, LLC-PK(1), with proximal kidney tubule-like features, and examined the involvement of free radicals and extracellular signal-regulated kinase (ERK) in the cell injury. The LLC-PK(1) cells showed early uptake of zinc (30 microM), and the release of lactate dehydrogenase (LDH), an index of cell injury, was observed 24 hr after uptake. Three hours after zinc exposure, generation of reactive oxygen species (ROS) was increased. An antioxidant, N, N'-diphenyl-p-phenylenediamine (DPPD), inhibited a zinc-related increase in ROS generation and zinc-induced renal cell injury. An NADPH oxidase inhibitor, diphenyleneiodonium (DPI), inhibited a zinc-related increase in ROS generation and cell injury. We investigated translocation from the cytosol fraction of the p67(phox) subunit, which is involved in the activation of NADPH oxidase, to the membrane fraction, and translocation was induced 3 hr after zinc exposure. We examined the involvement of ERK1/2 in the deterioration of zinc-induced renal cell injury, and the association between ERK1/2 and an increase in ROS generation. Six hours after zinc exposure, the activation (phosphorylation) of ERK1/2 was observed. An antioxidant, DPPD, inhibited the zinc-related activation of ERK1/2. An MAPK/ERK kinase (MEK1/2) inhibitor, U0126, almost completely inhibited zinc-related cell injury (the release of LDH), but did not influence ROS generation. These results suggest that early intracellular uptake of zinc by LLC-PK(1) cells causes the activation of NADPH oxidase, and that ROS generation by the activation of the enzyme leads to the deterioration of renal cell injury via the activation of ERK1/2.

  6. 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

  7. Pattern-dependent role of NMDA receptors in action potential generation: consequences on extracellular signal-regulated kinase activation.

    PubMed

    Zhao, Meilan; Adams, J Paige; Dudek, Serena M

    2005-07-27

    Synaptic long-term potentiation is maintained through gene transcription, but how the nucleus is recruited remains controversial. Activation of extracellular signal-regulated kinases (ERKs) 1 and 2 with synaptic stimulation has been shown to require NMDA receptors (NMDARs), yet stimulation intensities sufficient to recruit action potentials (APs) also appear to be required. This has led us to ask the question of whether NMDARs are necessary for AP generation as they relate to ERK activation. To test this, we examined the effects of NMDAR blockade on APs induced with synaptic stimulation using whole-cell current-clamp recordings from CA1 pyramidal cells in hippocampal slices. NMDAR antagonists were found to potently inhibit APs generated with 5 and 100 Hz synaptic stimulation. Blockade of APs and ERK activation could be overcome with the addition of the GABAA antagonist bicuculline, indicating that APs are sufficient to activate signals such as ERK in the nucleus and throughout the neuron in the continued presence of NMDAR antagonists. Interestingly, no effects of the NMDAR antagonists were observed when theta-burst stimulation (TBS) was used. This resistance to the antagonists is conferred by temporal summation during the bursts. These results clarify findings from a previous study showing that ERK activation induced with TBS is resistant to 2-amino-5-phosphonovalerate, in contrast to that induced with 5 or 100 Hz stimulation, which is sensitive. By showing that NMDAR blockade inhibits AP generation, we demonstrate that a major role that NMDARs play in cell-wide and nuclear ERK activation is through their contribution to action potential generation.

  8. The noble gas argon modifies extracellular signal-regulated kinase 1/2 signaling in neurons and glial cells.

    PubMed

    Fahlenkamp, Astrid V; Rossaint, Rolf; Haase, Hajo; Al Kassam, Hussam; Ryang, Yu-Mi; Beyer, Cordian; Coburn, Mark

    2012-01-15

    Recently, the noble gas argon has been identified as a potent neuroprotective agent, but little is known about its cellular effects. In this in vitro study, we investigated argon's influence on the extracellular signal-regulated kinase (ERK) 1/2, a ubiquitous enzyme with numerous functions in cell proliferation and survival. Primary neuronal and astroglial cell cultures and the microglial cell line BV-2 were exposed to 50 vol.% argon. Further possible effects were studied following stimulation of microglia with 50 ng/ml LPS. ERK 1/2 activation was assessed by phosphorylation state-specific western blotting, cytokine levels by real-time PCR and western blotting. Total phosphotyrosine phosphatase activity was examined with p-nitrophenylphosphate. After 30 min exposure, argon significantly activated ERK 1/2 signaling in microglia. Enhanced phosphorylation of ERK 1/2 was also found in astrocytes and neurons following argon exposure, but it lacked statistical significance. In microglia, argon did not substantially interfere with LPS-induced ERK1/2 activation and inflammatory cytokine induction. Addition of the MEK-Inhibitor U0126 abolished the induced ERK 1/2 phosphorylation. Cellular phosphatase activity and the inactivation of phosphorylated ERK 1/2 were not altered by argon. In conclusion, argon enhanced ERK 1/2 activity in microglia via the upstream kinase MEK, probably through a direct mode of activation. ERK 1/2 signaling in astrocytes and neurons in vitro was also influenced, although not with statistical significance. Whether ERK 1/2 activation by argon affects cellular functions like differentiation and survival in the brain in vivo will have to be determined in future experiments.

  9. PB1 Domain-Dependent Signaling Complex Is Required for Extracellular Signal-Regulated Kinase 5 Activation

    PubMed Central

    Nakamura, Kazuhiro; Uhlik, Mark T.; Johnson, Nancy L.; Hahn, Klaus M.; Johnson, Gary L.

    2006-01-01

    MEKK2, MEK5, and extracellular signal-regulated kinase 5 (ERK5) are members of a three-kinase cascade for the activation of ERK5. MEK5 is the only MAP2K to express a PB1 domain, and we have shown that it heterodimerizes with the PB1 domain of MEKK2. Here we demonstrate the MEK5 PB1 domain is a scaffold that also binds ERK5, functionally forming a MEKK2-MEK5-ERK5 complex. Reconstitution assays and CFP/YFP imaging (fluorescence resonance energy transfer [FRET]) measuring YFP-MEKK2/CFP-MEK5 and CFP-MEK5/YFP-ERK5 interactions define distinct MEK5 PB1 domain binding sites for MEKK2 and ERK5, with a C-terminal extension of the PB1 domain contributing to ERK5 binding. Stimulus-dependent CFP/YFP FRET in combination with mutational analysis was used to define MEK5 PB1 domain residues critical for the interaction of MEKK2/MEK5 and MEK5/ERK5 required for activation of the ERK5 pathway in living cells. Fusion of the MEK5 PB1 domain to the N terminus of MEK1 confers ERK5 regulation by a MAP2K normally regulating only ERK1/2. The MEK5 PB1 domain confers stringent MAP3K regulation of ERK5 relative to more promiscuous MAP3K control of ERK1/2, JNK, and p38. PMID:16507987

  10. Extracellular signal-regulated kinase signaling in the ventral tegmental area mediates cocaine-induced synaptic plasticity and rewarding effects.

    PubMed

    Pan, Bin; Zhong, Peng; Sun, Dalong; Liu, Qing-song

    2011-08-03

    Drugs of abuse such as cocaine induce long-term synaptic plasticity in the reward circuitry, which underlies the formation of drug-associated memories and addictive behavior. We reported previously that repeated cocaine exposure in vivo facilitates long-term potentiation (LTP) in dopamine neurons of the ventral tegmental area (VTA) by reducing the strength of GABAergic inhibition and that endocannabinoid-dependent long-term depression at inhibitory synapses (I-LTD) constitutes a mechanism for cocaine-induced reduction of GABAergic inhibition. The present study investigated the downstream signaling mechanisms and functional consequences of I-LTD in the VTA in the rat. Extracellular signal-regulated kinase (ERK) signaling has been implicated in long-term synaptic plasticity, associative learning, and drug addiction. We tested the hypothesis that VTA ERK activity is required for I-LTD and cocaine-induced long-term synaptic plasticity and behavioral effects. We show that the activation of receptors required for I-LTD increased ERK1/2 phosphorylation and inhibitors of ERK activation blocked I-LTD. We further demonstrate that ERK mediates cocaine-induced reduction of GABAergic inhibition and facilitation of LTP induction. Finally, we show that cocaine conditioned place preference (CPP) training (15 mg/kg; four pairings) increased ERK1/2 phosphorylation in the VTA, while bilateral intra-VTA injections of a CB(1) antagonist or an inhibitor of ERK activation attenuated ERK1/2 phosphorylation and the acquisition, but not the expression, of CPP to cocaine. Our study has identified the CB(1) and ERK signaling cascade as a key mediator of several forms of cocaine-induced synaptic plasticity and provided evidence linking long-term synaptic plasticity in the VTA to rewarding effects of cocaine.

  11. A novel role for extracellular signal-regulated kinase in maintaining long-term memory-relevant excitability changes.

    PubMed

    Cohen-Matsliah, Sivan Ida; Brosh, Inbar; Rosenblum, Kobi; Barkai, Edi

    2007-11-14

    Pyramidal neurons in the piriform cortex from olfactory-discrimination-trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the postburst afterhyperpolarization (AHP), which is generated by repetitive spike firing. AHP reduction is attributable to decreased conductance of a calcium-dependent potassium current, the sI(AHP). We have previously shown that such learning-induced AHP reduction is maintained by PKC activation. However, the molecular machinery underlying such long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the extracellular signal-regulated kinase I/II (ERKI/II) pathway, which is known to be crucial in learning, memory, and synaptic plasticity processes, is instrumental for the long-term maintenance of learning-induced AHP reduction. PD98059 or UO126, which selectively block MEK, the upstream kinase of ERK, increased the AHP in neurons from trained rats but not in neurons from naive and pseudo-trained rats. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls were abolished. This effect was not mediated by modulation of basic membrane properties. In accordance with its effect on neuronal excitability, the level of activated ERK in the membranal fraction was significantly higher in piriform cortex samples taken from trained rats. In addition, the PKC activator OAG (1-oleoyl-20acety-sn-glycerol), which was shown to reduce the AHP in neurons from control rats, had no effect on these neurons in the presence of PD98059. Our data show that ERK has a key role in maintaining long-lasting learning-induced enhancement of neuronal excitability.

  12. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  13. 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

  14. Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation.

    PubMed

    Kim, Wun-Jae; Lee, Se-Jung; Choi, Young Deuk; Moon, Sung-Kwon

    2010-04-01

    Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

  15. Oxytocin in the regulation of social behaviours in medial amygdala-lesioned mice via the inhibition of the extracellular signal-regulated kinase signalling pathway.

    PubMed

    Wang, Yu; Zhao, Shanshan; Wu, Zhe; Feng, Yu; Zhao, Chuansheng; Zhang, Chaodong

    2015-05-01

    The neuropeptide oxytocin (OXT) has been implicated in the pathophysiology of behavioural deficits among patients with autism spectrum disorder (ASD). However, the molecular mechanisms underlying its role in ASD remain unclear. In the present study, a murine model with ASD-like phenotypes was induced by intra-medial amygdala injection of N-methyl-d-aspartate, and it was used to investigate the role of OXT in behaviour regulation. Behavioural tests were performed to verify the ASD-like phenotypes of N-methyl-d-aspartate-treated mice, and the results showed that mice with bilateral medial amygdala lesions presented significant behavioural deficits, including impaired learning and memory and increased anxiety and depression. We also observed a notably decreased level of OXT in both the plasma and the hypothalamus of medial amygdala-lesioned mice, and the extracellular signal-regulated kinase (ERK) was activated. Further studies demonstrated that the administration of OXT alleviated ASD-like symptoms and significantly inhibited phosphorylation of ERK; the inhibitory effect was similar to that of U0126, an ERK signalling inhibitor. In addition, OXT administration modulated the expression of downstream proteins of the ERK signalling pathway, such as cyclic adenosine monophosphate response element binding and c-fos. Taken together, our data indicate that OXT plays an important role in ameliorating behavioural deficits in an ASD-like mouse model, which was mediated by inhibiting the ERK signalling pathway and its downstream proteins.

  16. Role of TCR-induced extracellular signal-regulated kinase activation in the regulation of early IL-4 expression in naive CD4+ T cells.

    PubMed

    Jorritsma, Patricia J; Brogdon, Jennifer L; Bottomly, Kim

    2003-03-01

    Although extracellular signal-regulated kinase (Erk) activation influences IL-4 production in various experimental systems, its role during Th differentiation is unclear. In this study, we show that Erk plays a critical role in IL-4 expression during TCR-induced Th differentiation of naive CD4(+) T cells. Stimulation of CD4(+) T cells with a high affinity peptide resulted in sustained Erk activation and Th1 differentiation. However, reduction of Erk activity led to a dramatic increase in IL-4 production and Th2 generation. Analysis of RNA and nuclear proteins of CD4(+) T cells 48 h after stimulation revealed that this was due to early IL-4 expression. Interestingly, transient Erk activation resulted in altered AP-1 DNA binding activity and the induction of an AP-1 complex that was devoid of Fos protein and consisted of Jun-Jun dimers. These data show that in the presence of a strong TCR signal, IL-4 expression can be induced in naive CD4(+) T cells by altering the strength of Erk activation. In addition, these data suggest that TCR-induced Erk activation is involved in the regulation of IL-4 expression by altering the composition of the AP-1 complex and its subsequent DNA binding activity.

  17. IκB Kinase 2 Regulates TPL-2 Activation of Extracellular Signal-Regulated Kinases 1 and 2 by Direct Phosphorylation of TPL-2 Serine 400

    PubMed Central

    Roget, Karine; Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Gantke, Thorsten; Yang, Huei-Ting; Janzen, Julia; Morrice, Nick; Abbott, Derek

    2012-01-01

    Tumor progression locus 2 (TPL-2) functions as a MEK-1/2 kinase, which is essential for Toll-like receptor 4 (TLR4) activation of extracellular signal-regulated kinase 1 and 2 (ERK-1/2) mitogen-activated protein (MAP) kinases in lipopolysaccharide (LPS)-stimulated macrophages and for inducing the production of the proinflammatory cytokines tumor necrosis factor and interleukin-1β. In unstimulated cells, association of TPL-2 with NF-κB1 p105 prevents TPL-2 phosphorylation of MEK-1/2. LPS stimulation of TPL-2 MEK-1/2 kinase activity requires TPL-2 release from p105. This is triggered by IκB kinase 2 (IKK-2) phosphorylation of the p105 PEST region, which promotes p105 ubiquitination and degradation by the proteasome. LPS activation of ERK-1/2 additionally requires transphosphorylation of TPL-2 on serine 400 in its C terminus, which controls TPL-2 signaling to ERK-1/2 independently of p105. However, the identity of the protein kinase responsible for TPL-2 serine 400 phosphorylation remained unknown. In the present study, we show that TPL-2 serine 400 phosphorylation is mediated by IKK2. The IKK complex therefore regulates two of the key regulatory steps required for TPL-2 activation of ERK-1/2, underlining the close linkage of ERK-1/2 MAP kinase activation to upregulation of NF-κB-dependent transcription. PMID:22988300

  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. 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

  20. 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.

  1. Wnt5a-Dopamine D2 Receptor Interactions Regulate Dopamine Neuron Development via Extracellular Signal-regulated Kinase (ERK) Activation*

    PubMed Central

    Yoon, Sehyoun; Choi, Mi-hyun; Chang, Min Seok; Baik, Ja-Hyun

    2011-01-01

    The dopamine D2 receptor (D2R) plays an important role in mesencephalic dopaminergic neuronal development, particularly coupled with extracellular signal-regulated kinase (ERK) activation. Wnt5a protein is known to regulate the development of dopaminergic neurons. We analyzed the effect of Wnt5a on dopaminergic neuron development in mesencephalic primary cultures from wild-type (WT) and D2R knock-out (D2R−/−) mice. Treatment with Wnt5a increased the number and neuritic length of dopamine neurons in primary mesencephalic neuronal cultures from WT mice, but not from D2R−/− mice. The effect of Wnt5a was completely blocked by treatment with D2R antagonist or inhibitors of MAPK or EGFR. Wnt5a-mediated ERK activation in mesencephalic neuronal cultures was inhibited by treatment of D2R antagonist and EGFR inhibitors in WT mice. However, these regulations were not observed for D2R−/− mice. Co-immunoprecipitation and displacement of [3H]spiperone from D2R by Wnt5a demonstrated that Wnt5a could bind with D2R. This interaction was confirmed by GST pulldown assays demonstrating that the domain including transmembrane domain 4, second extracellular loop, and transmembrane domain 5 of D2R binds to Wnt5a. These results suggest that the interaction between D2R and Wnt5a has an important role in dopamine neuron development in association with EGFR and the ERK pathway. PMID:21454669

  2. Integrin-mediated Ras–Extracellular Regulated Kinase (ERK) Signaling Regulates Interferon γ Production in Human Natural Killer Cells

    PubMed Central

    Mainiero, Fabrizio; Gismondi, Angela; Soriani, Alessandra; Cippitelli, Marco; Palmieri, Gabriella; Jacobelli, Jordan; Piccoli, Mario; Frati, Luigi; Santoni, Angela

    1998-01-01

    Recent evidence indicates that integrin engagement results in the activation of biochemical signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that β1 integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc–growth factor receptor–bound protein 2 (Grb2) and Shc–proline-rich tyrosine kinase 2–Grb2 complexes are the receptor-proximal events accompanying the β1 integrin–mediated Ras activation. In addition, we demonstrate that ligation of β1 integrins results in the stimulation of interferon γ (IFN-γ) production, which is under the control of extracellular signal–regulated kinase 2 activation. Overall, our data indicate that β1 integrins, by delivering signals capable of triggering IFN-γ production, may function as NK-activating receptors. PMID:9763606

  3. 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

  4. 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

  5. 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 7.5 mg/ml was comparable to that of ambroxol at 20 mg/ml i.v. and dexamethasone at 0.5 mg/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.

  6. Chrysophanic acid reduces testosterone-induced benign prostatic hyperplasia in rats by suppressing 5α-reductase and extracellular signal-regulated kinase.

    PubMed

    Youn, Dong-Hyun; Park, Jinbong; Kim, Hye-Lin; Jung, Yunu; Kang, JongWook; Jeong, Mi-Young; Sethi, Gautam; Seok Ahn, Kwang; Um, Jae-Young

    2017-02-07

    Benign prostatic hyperplasia (BPH) is one of the most common chronic diseases in male population, of which incidence increases gradually with age. In this study, we investigated the effect of chrysophanic acid (CA) on BPH. BPH was induced by a 4-week injection of testosterone propionate (TP). Four weeks of further injection with vehicle, TP, TP + CA, TP + finasteride was carried on. In the CA treatment group, the prostate weight was reduced and the TP-induced histological changes were restored as the normal control group. CA treatment suppressed the TP-elevated prostate specific antigen (PSA) expression. In addition, 5α-reductase, a crucial factor in BPH development, was suppressed to the normal level close to the control group by CA treatment. The elevated expressions of androgen receptor (AR), estrogen receptor α and steroid receptor coactivator 1 by TP administration were also inhibited in the CA group when compared to the TP-induced BPH group. Then we evaluated the changes in three major factors of the mitogen-activated protein kinase chain during prostatic hyperplasia; extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38). While ERK was elevated in the process of BPH, JNK and p38 was not changed. This up-regulated ERK was also reduced as normal by CA treatment. Further in vitro studies with RWPE-1 cells confirmed TP-induced proliferation and elevated AR, PSA and p-ERK were all reduced by CA treatment. Overall, these results suggest a potential pharmaceutical feature of CA in the treatment of BPH.

  7. Desnitro-imidacloprid activates the extracellular signal-regulated kinase cascade via the nicotinic receptor and intracellular calcium mobilization in N1E-115 cells.

    PubMed

    Tomizawa, Motohiro; Casida, John E

    2002-11-01

    Imidacloprid (IMI) is the principal neonicotinoid (the only major new class of synthetic insecticides of the past three decades). The excellent safety profile of IMI is not shared with a metabolite, desnitro-IMI (DNIMI), which displays high toxicity to mammals associated with agonist action at the alpha4beta2 nicotinic acetylcholine receptor (nAChR) in brain. This study examines the hypothesis that IMI, DNIMI, and (-)-nicotine activate the extracellular signal-regulated kinase (ERK) cascade via primary interaction with the alpha4beta2 nAChR in mouse neuroblastoma N1E-115 cells. These three nicotinic agonists induce phosphorylation of ERK (p44/p42) in a concentration-dependent manner with an optimal incubation period of 30 min. DNIMI (1 microM)-induced ERK activation is blocked by nicotinic antagonist mecamylamine but not by alpha-bungarotoxin and muscarinic antagonist atropine. This activation is prevented by intracellular Ca(2+) chelator BAPTA-AM but not by removal of external Ca(2+) using EGTA and Ca(2+)-free medium. 2-Aminoethoxy-diphenylborate, a blocker for inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from intracellular stores, inhibits DNIMI-induced ERK activation but a high level of ryanodine (to block ryanodine receptor-mediated Ca(2+) release) does not. The inhibitor U-73122 for phospholipase C (to suppress IP(3) production) prevents ERK activation evoked by DNIMI. Inhibitors for protein kinase C (PKC) (GF109203X) and ERK kinase (PD98059) block this activation whereas an inhibitor (H-89) for cyclic AMP-dependent protein kinase does not. Thus, neonicotinoids activate the ERK cascade triggered by primary action at the alpha4beta2 nAChR with an involvement of intracellular Ca(2+) mobilization possibly mediated by IP(3). It is further suggested that intracellular Ca(2+) activates a sequential pathway from PKC to ERK.

  8. Extracellular signal-regulated kinases 1/2 and Akt contribute to triclosan-stimulated proliferation of JB6 Cl 41-5a cells.

    PubMed

    Wu, Yuanfeng; Beland, Frederick A; Chen, Si; Fang, Jia-Long

    2015-08-01

    Triclosan is a broad spectrum anti-bacterial agent widely used in many personal care products, household items, medical devices, and clinical settings. Human exposure to triclosan is mainly through oral and dermal routes. In previous studies, we found that sub-chronic dermal exposure of B6C3F1 mice to triclosan induced epidermal hyperplasia and focal necrosis; however, the mechanisms for these responses remain elusive. In this study, using mouse epidermis-derived JB6 Cl 41-5a cells, we found that triclosan stimulated cell growth in a concentration- and time-dependent manner. Enhanced cell proliferation was demonstrated by a substantial increase in the percentage of BrdU-positive cells, an elevation in the protein levels of cyclin D1 and cyclin A, and a reduction in the protein level of p27(Kip1). Western blotting analysis revealed that triclosan induced the activation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), p38, and Akt. Pre-treatment of the cells with PD184352, an inhibitor of the upstream kinase MEK1/2, or with wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked triclosan-mediated phosphorylation of ERK1/2 and Akt, respectively, and substantially suppressed triclosan-stimulated cell proliferation, whereas the JNK inhibitor SP600125 or the p38 inhibitor SB203580 had little to no effect on triclosan-stimulated cell proliferation. The phosphorylation activation of ERK1/2 and Akt was further confirmed on the skin of mice dermally administered triclosan. These data suggest that the activation of ERK1/2 and Akt is involved in triclosan-stimulated proliferation of JB6 Cl 41-5a cells.

  9. 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.

  10. 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

  11. PACAP stimulation of maturational gonadotropin secretion in goldfish involves extracellular signal-regulated kinase, but not nitric oxide or guanylate cyclase, signaling.

    PubMed

    Chang, John P; Sawisky, Grant R; Mitchell, Gabriel; Uretsky, Aubrey D; Kwong, Patrick; Grey, Caleb L; Meints, Amanda N; Booth, Morgan

    2010-01-01

    In goldfish, nitric oxide synthase (NOS) immunoreactivity is present in gonadotropes and extracellular signal-regulated protein kinase (ERK) mediates GnRH stimulation of gonadotropin release and synthesis. In this study, we tested the possible involvement of nitric oxide (NO) and ERK in mediating PACAP-stimulated maturational gonadotropin (GTH-II) release from primary cultures of dispersed goldfish pituitary cells. In static incubation experiments, PACAP-induced GTH-II release was unaffected by two inhibitors of NOS synthase, AGH and 1400W; whereas addition of a NO donor, SNAP, elevated GTH-II secretion. In perifusion experiments, neither NOS inhibitors (AGH, 1400W and 7-Ni) nor NO scavengers (PTIO and rutin hydrate) attenuated the GTH-II response to pulse applications of PACAP. In addition, the GTH-II responses to PACAP and the NO donor SNP were additive while PTIO blocked SNP action. Although dibutyryl cGMP increased GTH-II secretion in static incubation, inhibition of guanylate cyclase (GC), a known down-stream target for NO signaling, did not reduce the GTH-II response to pulse application of PACAP. On the other hand, GTH-II responses to PACAP in perifusion were attenuated in the presence of two inhibitors of ERK kinase (MEK), U 0126 and PD 98059. These results suggest that although increased availability of NO and cGMP can lead to increased GTH-II secretion, MEK/ERK signaling, rather than NOS/NO/GC activation, mediates PACAP action on GTH-II release in goldfish.

  12. Extracellular Signal-Regulated Kinase Is an Endogenous Signal Retaining the Nuclear Constitutive Active/Androstane Receptor (CAR) in the Cytoplasm of Mouse Primary Hepatocytes

    PubMed Central

    Koike, Chika; Moore, Rick; Negishi, Masahiko

    2007-01-01

    The nuclear receptor constitutive active/androstane receptor (CAR) is sequestered in the cytoplasm of liver cells before its activation by therapeutic drugs and xenobiotics such as phenobarbital (PB) and 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in mouse liver, the regulatory mechanism of which remains poorly understood. Given the finding that epidermal growth factor repressed PB activation of CAR-mediated transcription (Mol Pharmacol 65:172–180, 2004), here we investigated the regulatory role of hepatocyte growth factor (HGF)-mediated signal in sequestering CAR in the cytoplasm of mouse primary hepatocytes. HGF treatment effectively repressed the induction of endogenous CYP2b10 gene by PB and TCPOBOP in mouse primary hepatocytes. On the other hand, inhibition by 1,4-diamino-2,3-dicyano-1,4-bis(methyl-thio)butadiene (U0126) of an HGF downstream kinase mitogen-activated protein kinase kinase (MEK) induced the Cyp2b10 gene and up-regulated the CAR-regulated promoter activity in the absence of TCPOBOP. HGF treatment increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in the cytosol, thus decreasing the TCPOBOP-induced nuclear accumulation of CAR. In contrast, U0126 dephosphorylated ERK1/2 and increased nuclear CAR accumulation in the absence of TCPOBOP. These results are consistent with the conclusion that the HGF-dependent phosphorylation of ERK1/2 is the endogenous signal that sequesters CAR in the cytoplasm of mouse primary hepatocytes. PMID:17314319

  13. Phosphorylation of Nuclear Phospholipase C β1 by Extracellular Signal-Regulated Kinase Mediates the Mitogenic Action of Insulin-Like Growth Factor I

    PubMed Central

    Xu, Aimin; Suh, Pann-Ghill; Marmy-Conus, Nelly; Pearson, Richard B.; Seok, Oh Yong; Cocco, Lucio; Gilmour, R. Stewart

    2001-01-01

    It is well established that a phosphoinositide (PI) cycle which is operationally distinct from the classical plasma membrane PI cycle exists within the nucleus, where it is involved in both cell proliferation and differentiation. However, little is known about the regulation of the nuclear PI cycle. Here, we report that nucleus-localized phospholipase C (PLC) β1, the key enzyme for the initiation of this cycle, is a physiological target of extracellular signal-regulated kinase (ERK). Stimulation of Swiss 3T3 cells with insulin-like growth factor I (IGF-I) caused rapid nuclear translocation of activated ERK and concurrently induced phosphorylation of nuclear PLC β1, which was completely blocked by the MEK inhibitor PD 98059. Coimmunoprecipitation detected a specific association between the activated ERK and PLC β1 within the nucleus. In vitro studies revealed that recombinant PLC β1 could be efficiently phosphorylated by activated mitogen-activated protein kinase but not by PKA. The ERK phosphorylation site was mapped to serine 982, which lies within a PSSP motif located in the characteristic carboxy-terminal tail of PLC β1. In cells overexpressing a PLC β1 mutant in which serine 982 is replaced by glycine (S982G), IGF-I failed to activate the nuclear PI cycle, and its mitogenic effect was also markedly attenuated. Expression of S982G was found to inhibit ERK-mediated phosphorylation of endogenous PLC β1. This result suggests that ERK-evoked phosphorylation of PLC β1 at serine 982 plays a critical role in the activation of the nuclear PI cycle and is also crucial to the mitogenic action of IGF-I. PMID:11287604

  14. Extracellular-signal regulated kinase 8 of Trypanosoma brucei uniquely phosphorylates its proliferating cell nuclear antigen homolog and reveals exploitable properties

    PubMed Central

    Valenciano, Ana L.; Knudsen, Giselle M.; Mackey, Zachary B.

    2016-01-01

    ABSTRACT The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC50 for TbERK8 that is several hundred times higher than its reported IC50 for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite. PMID:27589575

  15. Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

    PubMed Central

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara

    2013-01-01

    Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the 106PNTP109 motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, 183KKRKRKCLLL192 (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1. PMID:24043306

  16. Extracellular Signal-regulated Kinase Mediates Phosphorylation of Tropomyosin-1 to Promote Cytoskeleton Remodeling in Response to Oxidative Stress: Impact on Membrane Blebbing

    PubMed Central

    Houle, François; Rousseau, Simon; Morrice, Nick; Luc, Mario; Mongrain, Sébastien; Turner, Christopher E.; Tanaka, Sakae; Moreau, Pierre; Huot, Jacques

    2003-01-01

    Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 min of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the mitogen-activated protein kinase kinase (MEK) inhibitor PD098059 (2′-amino-3′-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H2O2 resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H2O2 induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PD098059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEKCA), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated cells, tropomyosin-1 was found diffuse in the cells, whereas it quickly colocalized with actin and stress fibers upon stimulation of ERK by H2O2 or by expression of MEKCA. We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7 (1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated

  17. 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.

  18. Extracellular secretion of recombinant proteins

    DOEpatents

    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.

  19. 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

  20. Expression of Extracellular Signal-regulated Kinase 5 and Ankyrin Repeat Domain 1 in Composite Pheochromocytoma and Ganglioneuroblastoma Detected Incidentally in the Adult Adrenal Gland.

    PubMed

    Suenaga, Shinta; Ichiyanagi, Osamu; Ito, Hiromi; Naito, Sei; Kato, Tomoyuki; Nagaoka, Akira; Kato, Tomoya; Yamakawa, Mitsunori; Obara, Yutaro; Tsuchiya, Norihiko

    Composite pheochromocytoma (cPC) is extremely rare, arising in the adrenal medulla as a mixture of PC and other tumors of neural origin. We herein report on a case of adrenal incidentaloma post-operatively diagnosed as cPC with ganglioneuroblastoma (GNBL). The PC component had 7 points on the PASS, a Ki-67 index of 5.1%, a focal absence of sustentacular cells, and no genetic aberrations in succinate dehydrogenase subunit B. The GNBL component exhibited no N-myc amplification. Tumor cells of both components were stained positively for extracellular signal-regulated kinase 5 and ankyrin repeat domain 1. The aberrant activation of growth signaling may play a role in the marginal malignancy of cPC.

  1. Expression of Extracellular Signal-regulated Kinase 5 and Ankyrin Repeat Domain 1 in Composite Pheochromocytoma and Ganglioneuroblastoma Detected Incidentally in the Adult Adrenal Gland

    PubMed Central

    Suenaga, Shinta; Ichiyanagi, Osamu; Ito, Hiromi; Naito, Sei; Kato, Tomoyuki; Nagaoka, Akira; Kato, Tomoya; Yamakawa, Mitsunori; Obara, Yutaro; Tsuchiya, Norihiko

    2016-01-01

    Composite pheochromocytoma (cPC) is extremely rare, arising in the adrenal medulla as a mixture of PC and other tumors of neural origin. We herein report on a case of adrenal incidentaloma post-operatively diagnosed as cPC with ganglioneuroblastoma (GNBL). The PC component had 7 points on the PASS, a Ki-67 index of 5.1%, a focal absence of sustentacular cells, and no genetic aberrations in succinate dehydrogenase subunit B. The GNBL component exhibited no N-myc amplification. Tumor cells of both components were stained positively for extracellular signal-regulated kinase 5 and ankyrin repeat domain 1. The aberrant activation of growth signaling may play a role in the marginal malignancy of cPC. PMID:27980262

  2. Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation

    PubMed Central

    Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

    2016-01-01

    Background: Rice (Oryza sativa) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. Materials and Methods: In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Results: We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. Conclusion: This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. SUMMARY Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin

  3. 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.

  4. Dehydroepiandrosterone Stimulates Endothelial Proliferation and Angiogenesis through Extracellular Signal-Regulated Kinase 1/2-Mediated Mechanisms

    PubMed Central

    Liu, Dongmin; Iruthayanathan, Mary; Homan, Laurie L.; Wang, Yiqiang; Yang, Lingling; Wang, Yao; Dillon, Joseph S.

    2008-01-01

    Dehydroepiandrosterone (DHEA) activates a plasma membrane receptor on vascular endothelial cells and phosphorylates ERK 1/2. We hypothesize that ERK1/2-dependent vascular endothelial proliferation underlies part of the beneficial vascular effect of DHEA. DHEA (0.1–10 nm) activated ERK1/2 in bovine aortic endothelial cells (BAECs) by 15 min, causing nuclear translocation of phosphorylated ERK1/2 and phosphorylation of nuclear p90 ribosomal S6 kinase. ERK1/2 phosphorylation was dependent on plasma membrane-initiated activation of Gi/o proteins and the upstream MAPK kinase because the effect was seen with albumin-conjugated DHEA and was blocked by pertussis toxin or PD098059. A 15-min incubation of BAECs with 1 nm DHEA (or albumin-conjugated DHEA) increased endothelial proliferation by 30% at 24 h. This effect was not altered by inhibition of estrogen or androgen receptors or nitric oxide production. There was a similar effect of DHEA to increase endothelial migration. DHEA also increased the formation of primitive capillary tubes of BAECs in vitro in solubilized basement membrane. These rapid DHEA-induced effects were reversed by the inhibition of either Gi/o-proteins or ERK1/2. Additionally, DHEA enhanced angiogenesis in vivo in a chick embryo chorioallantoic membrane assay. These findings indicate that exposure to DHEA, at concentrations found in human blood, causes vascular endothelial proliferation by a plasma membrane-initiated activity that is Gi/o and ERK1/2 dependent. These data, along with previous findings, define an important vascular endothelial cell signaling pathway that is activated by DHEA and suggest that this steroid may play a role in vascular function. PMID:18079198

  5. Xanthohumol inhibits the extracellular signal regulated kinase (ERK) signalling pathway and suppresses cell growth of lung adenocarcinoma cells.

    PubMed

    Sławińska-Brych, Adrianna; Zdzisińska, Barbara; Dmoszyńska-Graniczka, Magdalena; Jeleniewicz, Witold; Kurzepa, Jacek; Gagoś, Mariusz; Stepulak, Andrzej

    2016-05-16

    Aberrant activation of the Ras/MEK/ERK signaling pathway has been frequently observed in non-small-cell lung carcinoma (NSCLC) and its important role in cancer progression and malignant transformation has been documented. Hence, the ERK1/2 kinase cascade becomes a potential molecular target in cancer treatment. Xanthohumol (XN, a prenylated chalcone derived from hope cones) is known to possess a broad spectrum of chemopreventive and anticancer activities. In our studies, the MTT and BrdU assays revealed that XN demonstrated greater antiproliferative activity against A549 lung adenocarcinoma cells than against the lung adenocarcinoma H1563 cell line. We observed that XN was able to suppress the activities of ERK1/2 and p90RSK kinases, followed by inhibition of phosphorylation and activation of the CREB protein. Additionally, the XN treatment of the cancer cells caused upregulation of key cell cycle regulators p53 and p21 as well as downregulation of cyclin D1. As a result, the cytotoxic effect of XN was attributed to the cell cycle arrest at G1 phase and induction of apoptosis indicated by increased caspase-3 activity. Thus, XN might be a promising anticancer drug candidate against lung carcinomas.

  6. Distribution of extracellular signal-regulated kinase 1- and 2-activated neurons in the rat periaqueductal gray matter after noxious stimulation.

    PubMed

    Gioia, Magda; Moscheni, Claudia; Gagliano, Nicoletta

    2005-05-01

    The periaqueductal gray matter (PAG), the midbrain region made up of neuronal columns encircling the cerebral aqueduct, plays a key role in nociception. As the extracellular signal-regulated kinases (ERKs) 1 and 2 are activated after noxious stimulation, we analyzed the distribution of ERK-activated neurons in the PAG after visceral noxious stimulation. Ether- and urethane-anesthetized rats received an intraperitoneal injection of acetic acid or were left untreated and were perfused after 2 hr. Serial sections immunoreacted with an antibody selective for the activated ERKs. Significant ERK activation occurred only in the ether-anesthetized noxious stimulated rats. In these rats, we evaluated the number of ERK-activated neurons and their density as the ratio of the number of immunolabeled neurons to the extension of the region where they were located. ERK-activated neurons were more numerous in the lateral (LPAG) and ventrolateral (VLPAG) columns, but without significant differences. No ERK activation was seen in neurons of the most rostral PAG. The ERK-activated neurons were significantly denser at the intermediate level of the PAG. At the caudal level, they were denser in the LPAG and VLPAG columns, and in the DPAG column at the intermediate and rostral level. These findings suggest that noxious stimulation activates ERKs in neurons involved in the different functional activities related to nociception, overlapping in the PAG columns, and strengthens the role of PAG in integration.

  7. Repositioning of amprenavir as a novel extracellular signal-regulated kinase-2 inhibitor and apoptosis inducer in MCF-7 human breast cancer.

    PubMed

    Jiang, Wenchun; Li, Xin; Li, Tongyu; Wang, Hailian; Shi, Wei; Qi, Ping; Li, Chunyang; Chen, Jie; Bao, Jinku; Huang, Guodong; Wang, Yi

    2017-03-01

    Computational drug repositioning by virtually screening existing drugs for additional therapeutic usage could efficiently accelerate anticancer drug discovery. Herein, a library of 1447 Food and Drug Administration (FDA)-approved small molecule drugs was screened in silico for inhibitors of extracellular signal-regulated kinase 2 (ERK2). Then, in vitro kinase assay demonstrated amprenavir, a HIV-1 protease inhibitor, as a potential kinase inhibitor of ERK2. The in vivo kinase assay indicated that amprenavir could inhibit ERK2-mediated phosphorylation of BimEL at Ser69. Amprenavir could suppress this phosphorylation in MCF-7 cells, which may further facilitate the association of BimEL with several pro-survival molecules. Additionally, inhibition of ERK2-BimEL signaling pathway by amprenavir could contribute to its anti-proliferative and apoptosis-inducing activity in MCF-7 cells. Finally, in vivo tumor growth and immunohistochemical studies confirmed that amprenavir remarkably suppressed tumor proliferation and induce apoptosis in MCF-7 xenografts. Taken together, amprenavir can effectively inhibit the kinase activity of ERK2, and thus induces apoptosis and inhibits tumor growth in human MCF-7 cancer cells both in vitro and in vivo, making amprenavir a promising candidate for future anticancer therapeutics.

  8. Prenatal ethanol exposure persistently impairs N-methyl-D-aspartate receptor-dependent activation of extracellular signal-regulated kinase in the mouse dentate gyrus

    PubMed Central

    Samudio-Ruiz, Sabrina L.; Allan, Andrea M.; Valenzuela, C. Fernando; Perrone-Bizzozero, Nora I.; Caldwell, Kevin K.

    2009-01-01

    The dentate gyrus (DG) is the central input region to the hippocampus and is known to play an important role in learning and memory. Previous studies have shown that prenatal alcohol is associated with hippocampal-dependent learning deficits and a decreased ability to elicit long term potentiation (LTP) in the DG in adult animals. Given that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade by N-methyl-D-aspartate (NMDA) receptors is required for various forms of learning and memory, as well as LTP, in hippocampal regions, including the DG, we hypothesized that fetal alcohol-exposed (FAE) adult animals would have deficits in hippocampal NMDA receptor-dependent ERK1/2 activation. We used immunoblotting and immunohistochemistry techniques to detect NMDA-stimulated ERK1/2 activation in acute hippocampal slices prepared from adult FAE mice. We present the first evidence linking prenatal alcohol exposure to deficits in NMDA receptor-dependent ERK1/2 activation specifically in the DG of adult offspring. This deficit may account for the LTP deficits previously observed in the DG, as well as the life-long cognitive deficits, associated with prenatal alcohol exposure. PMID:19317851

  9. Carbonic anhydrase activation enhances object recognition memory in mice through phosphorylation of the extracellular signal-regulated kinase in the cortex and the hippocampus.

    PubMed

    Canto de Souza, Lucas; Provensi, Gustavo; Vullo, Daniela; Carta, Fabrizio; Scozzafava, Andrea; Costa, Alessia; Schmidt, Scheila Daiane; Passani, Maria Beatrice; Supuran, Claudiu T; Blandina, Patrizio

    2017-03-09

    Rats injected with by d-phenylalanine, a carbonic anhydrase (CA) activator, enhanced spatial learning, whereas rats given acetazolamide, a CA inhibitor, exhibited impairments of fear memory consolidation. However, the related mechanisms are unclear. We investigated if CAs are involved in a non-spatial recognition memory task assessed using the object recognition test (ORT). Systemic administration of acetazolamide to male CD1 mice caused amnesia in the ORT and reduced CA activity in brain homogenates, while treatment with d-phenylalanine enhanced memory and increased CA activity. We provided also the first evidence that d-phenylalanine administration rapidly activated extracellular signal-regulated kinase (ERK) pathways, a critical step for memory formation, in the cortex and the hippocampus, two brain areas involved in memory processing. Effects elicited by d-phenylalanine were completely blunted by co-administration of acetazolamide, but not of 1-N-(4-sulfamoylphenyl-ethyl)-2,4,6-trimethylpyridinium perchlorate ((C18),) a CA inhibitor that, differently from acetazolamide, does not cross the blood brain barrier. Our results strongly suggest that brain but not peripheral CAs activation potentiates memory as a result of ERK pathway enhanced activation.

  10. 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.

  11. Hydrogen sulfide potentiates interleukin-1{beta}-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells

    SciTech Connect

    Jeong, Sun-Oh; Pae, Hyun-Ock; Oh, Gi-Su; Jeong, Gil-Saeng; Lee, Bok-Soo; Lee, Seoul; Kim, Du Yong; Rhew, Hyun Yul; Lee, Kang-Min; Chung, Hun-Taeg . E-mail: htchung@wonkwang.ac.kr

    2006-07-07

    Hydrogen sulfide (H{sub 2}S) and nitric oxide (NO) are endogenously synthesized from L-cysteine and L-arginine, respectively. They might constitute a cooperative network to regulate their effects. In this study, we investigated whether H{sub 2}S could affect NO production in rat vascular smooth muscle cells (VSMCs) stimulated with interleukin-1{beta} (IL-1{beta}). Although H{sub 2}S by itself showed no effect on NO production, it augmented IL-{beta}-induced NO production and this effect was associated with increased expression of inducible NO synthase (iNOS) and activation of nuclear factor (NF)-{kappa}B. IL-1{beta} activated the extracellular signal-regulated kinase 1/2 (ERK1/2), and this activation was also enhanced by H{sub 2}S. Inhibition of ERK1/2 activation by the selective inhibitor U0126 inhibited IL-1{beta}-induced NF-{kappa}B activation, iNOS expression, and NO production either in the absence or presence of H{sub 2}S. Our findings suggest that H{sub 2}S enhances NO production and iNOS expression by potentiating IL-1{beta}-induced NF-{kappa}B activation through a mechanism involving ERK1/2 signaling cascade in rat VSMCs.

  12. 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.

  13. Aqueous extract of Allium sativum L bulbs offer nephroprotection by attenuating vascular endothelial growth factor and extracellular signal-regulated kinase-1 expression in diabetic rats.

    PubMed

    Shiju, T M; Rajkumar, R; Rajesh, N G; Viswanathan, Pragasam

    2013-02-01

    To investigate the nephroprotective effect of garlic and elucidate the mechanism by which it prevents the progression of diabetic nephropathy in diabetic rats, diabetes was induced by a single ip injection of streptozotocin (45 mg/kg body weight). Garlic extract (500 mg/kg body weight) and aminoguanidine (1 g/L) were supplemented in the treatment groups. Histopathological examination using H&E, PAS staining and the immunohistochemical analysis of vascular endothelial growth factor (VEGF) and extracellular signal-regulated kinase-1 (ERK-1) expression were performed on kidney sections at the end of 12 weeks. Significant change in both, the urine and serum biochemistry confirmed kidney damage in diabetic animals which was further confirmed by the histological changes such as mesangial expansion, glomerular basement membrane thickening, glycosuria and proteinuria. However, the diabetic animals treated with garlic extract showed a significant change in urine and serum biochemical parameters such as albumin, urea nitrogen and creatinine compared to that of diabetic rats. Further, the garlic supplemented diabetic rats showed a significant decrease in the expression of VEGF and ERK-1 compared to diabetic rats, attenuating mesangial expansion and glomerulosclerosis. Thus, garlic extract rendered nephroprotection in diabetic rats.

  14. Neurotrophins elevate cAMP to reach a threshold required to overcome inhibition by MAG through extracellular signal-regulated kinase-dependent inhibition of phosphodiesterase.

    PubMed

    Gao, Ying; Nikulina, Elena; Mellado, Wilfredo; Filbin, Marie T

    2003-12-17

    Inhibitors of regeneration in myelin, such as myelin-associated glycoprotein (MAG), play an important role in preventing regeneration after CNS injury. Elevation of cAMP, either with dibutyryl-cAMP (db-cAMP) or by priming with a variety of neurotrophins, overcomes inhibition by MAG and myelin. However, activation of cAMP is not generally regarded as a signaling pathway for neurotrophins. Here we show that the NGF-like neurotrophins overcome inhibition by MAG by activating tyrosine kinase receptors. We also show that activation of extracellular signal-regulated kinase (Erk) by BDNF is required to overcome inhibition by MAG, and that activated Erk transiently inhibits phosphodiesterase 4 (PDE4), the enzyme that hydrolyzes cAMP. Inhibition of PDE4 then allows cAMP to increase and so initiates the pathway to overcome inhibition. Furthermore, we also show that basal levels of Erk activation and basal cAMP levels contribute to the effects of db-cAMP by pushing the combined levels of cAMP above a threshold required to overcome inhibition. Together, these results not only show how NGF-like neurotrophins can elevate cAMP and overcome inhibition but also point to a novel mechanism of cross talk in neurons from the Erk to the cAMP signaling pathways.

  15. Extracellular matrix proteins of dentine.

    PubMed

    Butler, W T; Ritchie, H H; Bronckers, A L

    1997-01-01

    Bone and dentine extracellular matrix proteins are similar, consisting primarily of type I collagen, acidic proteins and proteoglycans. Although collagen forms the lattice for deposition of calcium and phosphate for formation of carbonate apatite, the non-collagenous proteins are believed to control initiation and growth of the crystals. Despite this similarity, dentine contains three unique proteins apparently absent from bone and other tissue: dentine phosphophoryn (DPP), dentine matrix protein 1 (DMP1) and dentine sialoprotein (DSP). DPP and DMP1 are acidic phosphoproteins probably involved in the control of mineralization processes. DPP may localize in gap regions of collagen and initiate apatite crystal formation by binding large quantities of calcium in a conformation that promotes this process. Extensive studies have been conducted in our laboratory on the nature, biosynthesis, localization and gene structure of DSP. Immunolocalization studies showed that rat DSP, a 53 kDa sialic acid-rich glycoprotein, was synthesized by young and mature odontoblasts, and by dental pulp cells and pre-ameloblasts, but not by ameloblasts, osteoblasts, chondrocytes or other cell types. The cDNA sequence indicated that DSP was a 366-residue protein with several potential N-glycosylation sites, as well as phosphorylation sites, but that the amino acid sequence was dissimilar to that of other known proteins. Northern blot analysis detected several mRNA species near 4.6 and 1.5 kb, indicative of alternative splicing events. Evidence for two DSP genes was obtained, further complicating this picture. Recent in situ hybridization studies utilizing rat and mouse molars and incisors indicated that DSP mRNA was expressed by young odontoblasts and odontoblasts in animals of all ages. Transcripts were also observed in pre-ameloblasts. The expression of DSP mRNA ceased when these cells matured to become secretory ameloblasts. DSP transcripts were not detected in osteoblasts or other cell

  16. 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.

  17. Leukaemia inhibitory factor mediated proliferation of HTR-8/SVneo trophoblast cells is dependent on activation of extracellular signal-regulated kinase 1/2.

    PubMed

    Prakash, Golla Jaya; Suman, Pankaj; Morales Prieto, Diana M; Markert, Udo R; Gupta, Satish K

    2011-01-01

    Leukaemia inhibitory factor (LIF) is one of the cytokines that is indispensable for embryo implantation. The aim of the present study was to investigate the role of activation of extracellular signal-regulated kinase (ERK) 1/2 in LIF-mediated proliferation of HTR-8/SVneo cells. Stimulation of HTR-8/SVneo cells with LIF (50 ng mL(-1)) resulted in an increase in cell proliferation (P < 0.05) via increased transition of cells to the G(2)/M phase of cell cycle. Stimulation with LIF resulted in the activation of both signal transducer and activator of transcription (STAT) 3 Tyr(705) and ERK1/2, but inhibition of ERK1/2 signalling by pretreatment of cells with U0126 (10 µM) for 2h resulted in abrogation of LIF-mediated increases in G(2)/M transition, with a significant decrease (P < 0.05) in absolute cell numbers compared with control. Although STAT3 silencing had no effect on LIF-dependent proliferation of HTR-8/SVneo cells, it did result in an increase in cell apoptosis, which increased further upon inhibition of ERK1/2 activation irrespective of LIF stimulation. Stimulation of cells with LIF increased the Bcl-2/Bax ratio, whereas ERK1/2 inhibition decreased the Bcl-2/Bax ratio, even after LIF stimulation. Hence, it can be inferred that ERK1/2 activation is essential for LIF-mediated increases in proliferation and that both STAT3 and ERK1/2 activation are important for the survival of HTR-8/SVneo cells.

  18. Phospholipase A(2) activation by poultry particulate matter is mediated through extracellular signal-regulated kinase in lung epithelial cells: regulation of interleukin-8 release.

    PubMed

    Kotha, Sainath R; Piper, Melissa G; Patel, Rishi B; Sliman, Sean; Malireddy, Smitha; Zhao, Lingying; Baran, Christopher P; Nana-Sinkam, Patrick S; Wewers, Mark D; Romberger, Debra; Marsh, Clay B; Parinandi, Narasimham L

    2013-11-01

    The mechanisms of poultry particulate matter (PM)-induced agricultural respiratory disorders are not thoroughly understood. Hence, it is hypothesized in this article that poultry PM induces the release of interleukin-8 (IL-8) by lung epithelial cells that is regulated upstream by the concerted action of cytosolic phospholipase A2 (cPLA2) and extracellular signal-regulated kinase (ERK). To test this hypothesis, the widely used cultured human lung epithelial cells (A549) were chosen as the model system. Poultry PM caused a significant activation of PLA2 in A549 cells, which was attenuated by AACOCF3 (cPLA2 inhibitor) and PD98059 (ERK-1/2 upstream inhibitor). Poultry PM induced upstream ERK-1/2 phosphorylation and downstream cPLA2 serine phosphorylation, in a concerted fashion, in cells with enhanced association of ERK-1/2 and cPLA2. The poultry PM-induced cPLA2 serine phosphorylation and IL-8 release were attenuated by AACOCF3, PD98059, and by transfection with dominant-negative ERK-1/2 DNA in cells. The poultry PM-induced IL-8 release by the bone marrow-derived macrophages of cPLA2 knockout mice was significantly lower. For the first time, this study demonstrated that the poultry PM-induced IL-8 secretion by human lung epithelial cells was regulated by cPLA2 activation through ERK-mediated serine phosphorylation, suggesting a mechanism of airway inflammation among poultry farm workers.

  19. 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

    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 help

  20. The effect of active and passive intravenous cocaine administration on the extracellular signal-regulated kinase (ERK) activity in the rat brain.

    PubMed

    Miszkiel, Joanna; Detka, Jan; Cholewa, Joanna; Frankowska, Małgorzata; Nowak, Ewa; Budziszewska, Bogusława; Przegaliński, Edmund; Filip, Małgorzata

    2014-08-01

    According to a current hypothesis of learning processes, recent papers pointed out to an important role of the extracellular signal-regulated kinase (ERK), in drug addiction. We employed the Western blotting techniques to examine the ERK activity immediately after cocaine iv self-administration and in different drug-free withdrawal periods in rats. To distinguish motivational vs. pharmacological effects of the psychostimulant intake, a "yoked" procedure was used. Animals were decapitated after 14 daily cocaine self-administration sessions or on the 1st, 3rd or 10th extinction days. At each time point the activity of the ERK was assessed in several brain structures, including the prefrontal cortex, hippocampus, dorsal striatum and nucleus accumbens. Passive, repeated iv cocaine administration resulted in a 45% increase in ERK phosphorylation in the hippocampus while cocaine self-administration did not change brain ERK activity. On the 1st day of extinction, the activity of the ERK in the prefrontal cortex was decreased in rats with a history of cocaine chronic intake: by 66% for "active" cocaine group and by 35% for "yoked" cocaine group. On the 3rd day the reduction in the ERK activity (25-34%) was observed in the hippocampus for both cocaine-treated groups, and also in the nucleus accumbens for "yoked" cocaine group (40%). On the 10th day of extinction there was no significant alteration in ERK activity in any group of rats. Our findings suggest that cortical ERK is involved in cocaine seeking behavior in rats. They also indicate the time and regional adaptations in this enzyme activity after cocaine withdrawal.

  1. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway.

    PubMed

    Hara, Tomoya; Kakudo, Natsuko; Morimoto, Naoki; Ogawa, Takeshi; Lai, Fangyuan; Kusumoto, Kenji

    2016-12-01

    Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P < 0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P < 0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

  2. 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

  3. 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 Central

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

    2016-01-01

    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

  4. Extracellular signal-regulated kinase (ERK) dictates osteogenic and/or chondrogenic lineage commitment of mesenchymal stem cells under dynamic compression.

    PubMed

    Pelaez, Daniel; Arita, Nestor; Cheung, Herman S

    2012-01-27

    Elucidating the intracellular signaling cascades which lead to differentiation programs can be a daunting but necessary task. Even more so when the nature of the differentiating stimuli can elicit different biochemical responses yet achieve the same functional outcome. In the field of cartilage and bone regeneration the importance of the extracellular signal-regulated kinase (ERK) pathway has been a controversial issue as of late. Whether differentiation results from a soluble chemical induction or a microenvironmental cue on the cells seems to have a determining effect on the role that this pathway plays in ultimate cell fate. Here we explore the role of the ERK1/2 pathway on the mechanical induction of chondrogenesis of bone marrow mesenchymal stem cells (MSC). The cells were encapsulated in fibrin gel scaffolds and subjected to a dynamic mechanical compression stimulus previously demonstrated to induce chondrogenic differentiation of the cells with and without the addition of PD98059, a selective inhibitor for the ERK1/2 pathway. Samples were then analyzed by RT-PCR and histochemical staining for markers of both chondrogenic and osteogenic differentiation. Our results show that dynamic compression induces the chondrogenic differentiation of the cells and that inhibition of the ERK1/2 pathway completely abolishes this chondrogenic response. On the other hand, inhibition of ERK1/2 under dynamic compression augments the osteogenic response of the cells and significantly increases their expression of alkaline phosphatase (ALP), collagen type I (COLI) and osteocalcin (OCN) (P<0.05). These results were confirmed by the histochemical staining where dynamically compressed samples show staining for sulfated glycosaminoglycans (sGAG) while the inhibited and compressed samples show no sGAG but present positive staining for microcalcifications. These results would suggest that the activation of ERK1/2 can determine the ultimate cell fate between the chondrogenic and

  5. Mitogenic properties of major extracellular proteins.

    PubMed

    Lévesque, J P; Hatzfeld, A; Hatzfeld, J

    1991-08-01

    The major plasma and extracellular matrix proteins are multifunctional molecules. Some, such as fibrinogen or C3, have one domain that binds adhesion receptors and another that specifically binds and activates a separate, mitogenic receptor. In this review, Jean-Pierre Lévesque, Antoinette Hatzfeld and Jacques Hatzfeld describe adhesion and mitogenic receptors that bind to distinct domains of the same extracellular matrix protein and discuss the possibility of common ancestral genes for cell adhesion molecules, extracellular matrix proteins, integrins, immunoglobulins, growth factors and their receptors.

  6. cAMP inhibits transforming growth factor-beta-stimulated collagen synthesis via inhibition of extracellular signal-regulated kinase 1/2 and Smad signaling in cardiac fibroblasts.

    PubMed

    Liu, Xiaoqiu; Sun, Shu Qiang; Hassid, Aviv; Ostrom, Rennolds S

    2006-12-01

    Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Cytokines such as transforming growth factor-beta (TGF-beta) play a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and other profibrotic responses, but less is known about pathways that might inhibit fibrosis. Increased cAMP formation inhibits myofibroblast differentiation and collagen production by cardiac fibroblasts, but the mechanism of this inhibition is not known. We sought to characterize the signaling pathways by which cAMP-elevating agents alter collagen expression and myofibroblast differentiation. Treatment with 10 microM forskolin or isoproterenol increased cAMP production and cAMP response element binding protein (CREB) phosphorylation in cardiac fibroblasts and inhibited serum- or TGF-beta-stimulated collagen synthesis by 37% or more. These same cAMP-elevating agents blunted TGF-beta-stimulated expression of collagen I, collagen III, and alpha-smooth muscle actin. Forskolin or isoproterenol treatment blocked the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by TGF-beta despite the fact that these cAMP-elevating agents stimulated ERK1/2 activation on their own. cAMP-elevating agents also attenuated the activation of c-Jun NH(2)-terminal kinase and reduced binding of the transcriptional coactivator CREB-binding protein 1 to transcriptional complexes containing Smad2, Smad3, and Smad4. Pharmacological inhibition of ERK completely blocked TGF-beta-stimulated collagen gene expression, but expression of an active mutant of MEK was additive with TGF-beta treatment. Thus, cAMP-elevating agents inhibit the profibrotic effects of TGF-beta in cardiac fibroblasts largely through inhibiting ERK1/2 phosphorylation but also by reducing Smad-mediated recruitment of transcriptional coactivators.

  7. A-raf and B-raf are dispensable for normal endochondral bone development, and parathyroid hormone-related peptide suppresses extracellular signal-regulated kinase activation in hypertrophic chondrocytes.

    PubMed

    Provot, Sylvain; Nachtrab, Gregory; Paruch, Jennifer; Chen, Adele Pin; Silva, Alcino; Kronenberg, Henry M

    2008-01-01

    Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.

  8. The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways.

    PubMed

    Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T; Portell, Craig A; Lannutti, Brian J; Almasan, Alexandru; Hsi, Eric D

    2013-10-01

    Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

  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. Thrombin-induced regulation of CD95(Fas) expression in the N9 microglial cell line: evidence for involvement of proteinase-activated receptor(1) and extracellular signal-regulated kinase 1/2.

    PubMed

    Weinstein, Jonathan R; Zhang, Matthew; Kutlubaev, Mansur; Lee, Richard; Bishop, Caroline; Andersen, Henrik; Hanisch, Uwe-Karsten; Möller, Thomas

    2009-03-01

    Microglia are the immune cells of the CNS. Brain injury triggers phenotypic changes in microglia including regulation of surface antigens. The serine proteinase alpha-thrombin can induce profound changes in neural cell physiology via cleavage of proteinase-activated receptors (PARs). We recently demonstrated that pharmaceutical-grade recombinant human alpha-thrombin (rh-thr) induces a restricted set of proteolysis-dependent changes in microglia. CD95(Fas) is a cell-death receptor that is up-regulated in microglia by inflammatory stimuli. Here we characterized the effect of rh-thr on CD95(Fas) expression in the N9 microglial cell line. Dose-response and time course studies demonstrated maximal effects at 100 U/ml and 24 h, respectively. Regulation of expression was seen at both the surface protein and steady-state mRNA levels. The rh-thr-induced effects were mimicked by PAR(1) agonist peptides and blocked by pharmacologic inhibitors selective for extracellular signal-regulated kinase 1/2 (ERK 1/2). Rh-thr also induced a rapid and sustained phosphorylation of ERK 1/2. Thrombin-induced regulation of CD95(Fas) could modulate the neuroinflammatory response in a variety of neurological disorders.

  11. Irciniastatin A induces potent and sustained activation of extracellular signal-regulated kinase and thereby promotes ectodomain shedding of tumor necrosis factor receptor 1 in human lung carcinoma A549 cells.

    PubMed

    Quach, Hue Tu; Hirano, Seiya; Fukuhara, Sayuri; Watanabe, Tsubasa; Kanoh, Naoki; Iwabuchi, Yoshiharu; Usui, Takeo; Kataoka, Takao

    2015-01-01

    Irciniastatin A is a pederin-type marine product that potently inhibits translation. We have recently shown that irciniastatin A induces ectodomain shedding of tumor necrosis factor (TNF) receptor 1 with slower kinetics than other translation inhibitors. In human lung carcinoma A549 cells, irciniastatin A induced a marked and sustained activation of extracellular signal-regulated kinase (ERK) and induced little activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). Moreover, the TNF receptor 1 shedding induced by irciniastatin A was blocked by the MAP kinase/ERK kinase inhibitor U0126, but not by the p38 MAP kinase inhibitor SB203580 or the JNK inhibitor SP600125. Thus unlike other translation inhibitors that trigger ribotoxic stress response, our results show that irciniastatin A is a unique translation inhibitor that induces a potent and sustained activation of the ERK pathway, and thereby promotes the ectodomain shedding of TNF receptor 1 in A549 cells.

  12. Sulfur Dioxide Inhibits Extracellular Signal-regulated Kinase Signaling to Attenuate Vascular Smooth Muscle Cell Proliferation in Angiotensin II-induced Hypertensive Mice

    PubMed Central

    Wu, Hui-Juan; Huang, Ya-Qian; Chen, Qing-Hua; Tian, Xiao-Yu; Liu, Jia; Tang, Chao-Shu; Jin, Hong-Fang; Du, Jun-Bao

    2016-01-01

    Background: Clarifying the mechanisms underlying vascular smooth muscle cell (VSMC) proliferation is important for the prevention and treatment of vascular remodeling and the reverse of hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the mechanism for the inhibition of the angiotensin II (AngII)-induced VSMC proliferation by SO2 has not been fully elucidated. This study was designed to investigate if SO2 inhibited VSMC proliferation in mice with hypertension induced by AngII. Methods: Thirty-six male C57 mice were randomly divided into control, AngII, and AngII + SO2 groups. Mice in AngII group and AngII + SO2 group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng·kg−1·min−1. In addition, mice in AngII + SO2 received intraperitoneal injections of SO2 donor. Arterial blood pressure of tail artery was determined. The thickness of the aorta was measured by elastic fiber staining, and proliferating cell nuclear antigen (PCNA) and phosphorylated-extracellular signal-regulated kinase (P-ERK) were detected in aortic tissues. The concentration of SO2 in serum and aortic tissue homogenate supernatant was measured using high-performance liquid chromatography with fluorescence determination. In the in vitro study, VSMC of A7R5 cell lines was divided into six groups: control, AngII, AngII + SO2, PD98059 (an inhibitor of ERK phosphorylation), AngII + PD98059, and AngII + SO2 + PD98059. Expression of PCNA, ERK, and P-ERK was determined by Western blotting. Results: In animal experiment, compared with the control group, AngII markedly increased blood pressure (P < 0.01) and thickened the aortic wall in mice (P < 0.05) with an increase in the expression of PCNA (P < 0.05). SO2, however, reduced the systemic hypertension and the wall thickness induced by AngII (P < 0.05). It inhibited the increased expression of PCNA and P

  13. 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

  14. 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

    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-beta1) 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-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, 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-beta1-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-beta1. TGF-beta1 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-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 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-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 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

  15. Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein.

    PubMed

    Lezcano, N; Mrzljak, L; Eubanks, S; Levenson, R; Goldman-Rakic, P; Bergson, C

    2000-03-03

    The synergistic response of cells to the stimulation of multiple receptors has been ascribed to receptor cross talk; however, the specific molecules that mediate the resultant signal amplification have not been defined. Here a 24-kilodalton single transmembrane protein, designated calcyon, we functionally characterize that interacts with the D1 dopamine receptor. Calcyon localizes to dendritic spines of D1 receptor-expressing pyramidal cells in prefrontal cortex. These studies delineate a mechanism of Gq- and Gs-coupled heterotrimeric GTP-binding protein-coupled receptor cross talk by which D1 receptors can shift effector coupling to stimulate robust intracellular calcium (Ca2+i) release as a result of interaction with calcyon. The role of calcyon in potentiating Ca2+-dependent signaling should provide insight into the D1 receptor-modulated cognitive functions of prefrontal cortex.

  16. Bis-Retinoid A2E Induces an Increase of Basic Fibroblast Growth Factor via Inhibition of Extracellular Signal-Regulated Kinases 1/2 Pathway in Retinal Pigment Epithelium Cells and Facilitates Phagocytosis

    PubMed Central

    Balmer, Delphine; Bapst-Wicht, Linda; Pyakurel, Aswin; Emery, Martine; Nanchen, Natacha; Bochet, Christian G.; Roduit, Raphael

    2017-01-01

    Age-related macular degeneration (ARMD) is the leading cause of vision loss in developed countries. Hallmarks of the disease are well known; indeed, this pathology is characterized by lipofuscin accumulation, is principally composed of lipid-containing residues of lysosomal digestion. The N-retinyl-N-retinylidene ethanolamine (A2E) retinoid which is thought to be a cytotoxic component for RPE is the best-characterized component of lipofuscin so far. Even if no direct correlation between A2E spatial distribution and lipofuscin fluorescence has been established in aged human RPE, modified forms or metabolites of A2E could be involved in ARMD pathology. Mitogen-activated protein kinase (MAPK) pathways have been involved in many pathologies, but not in ARMD. Therefore, we wanted to analyze the effects of A2E on MAPKs in polarized ARPE19 and isolated mouse RPE cells. We showed that long-term exposure of polarized ARPE19 cells to low A2E dose induces a strong decrease of the extracellular signal-regulated kinases' (ERK1/2) activity. In addition, we showed that A2E, via ERK1/2 decrease, induces a significant decrease of the retinal pigment epithelium-specific protein 65 kDa (RPE65) expression in ARPE19 cells and isolated mouse RPE. In the meantime, we showed that the decrease of ERK1/2 activity mediates an increase of basic fibroblast growth factor (bFGF) mRNA expression and secretion that induces an increase in phagocytosis via a paracrine effect. We suggest that the accumulation of deposits coming from outer segments (OS) could be explained by both an increase of bFGF-induced phagocytosis and by the decrease of clearance by A2E. The bFGF angiogenic protein may therefore be an attractive target to treat ARMD. PMID:28298893

  17. Extracellular recombinant protein production from Escherichia coli.

    PubMed

    Ni, Ye; Chen, Rachel

    2009-11-01

    Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.

  18. 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.

  19. Bone Morphogenetic Protein Signaling Regulates Development and Activation of CD4(+) T Cells.

    PubMed

    Kuczma, Michal; Kraj, Piotr

    2015-01-01

    Bone morphogenetic proteins (BMPs) are growth factors belonging to the TGF-β (transforming growth factor β) superfamily. BMPs were found to regulate multiple cell processes such as proliferation, survival, differentiation, and apoptosis. They were originally described to play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites but were found to play a significant role in embryogenesis and development of multiple tissues and organs. Activities of BMPs are regulated by a number of secreted proteins, which modulate their availability to bind cellular receptors. The functions of individual BMPs are highly redundant due to binding the same receptors and inducing overlapping signal transduction pathways. Recently, BMPs were found to regulate cells of the innate and adaptive immune system. BMPs are involved in thymic development of T cells at the early, double negative, as well as later, double positive, stages of thymopoesis. They specifically modulate thymic development of regulatory T cells (T(reg)). In the periphery, BMPs affect T cell activation, promoting generation of T(reg) cells. We found that mice deficient for one of the receptors activated by BMPs demonstrated slower growth of transplantable melanoma tumors.

  20. Morphine activates the E twenty six-like transcription factor-1/serum response factor pathway via extracellular signal-regulated kinases 1/2 in F11 cells derived from dorsal root ganglia neurons.

    PubMed

    Rothe, Kathrin; Solinski, Hans Jürgen; Boekhoff, Ingrid; Gudermann, Thomas; Breit, Andreas

    2012-07-01

    Morphine-induced signaling via opioid receptors (ORs) in dorsal root ganglia (DRG) neurons, the spinal cord, and various brain regions has been shown to modulate gene activity. Hitherto, little attention has been paid to extracellular signal-regulated kinases-1/2 (ERK-1/2)-mediated activation of the serum response factor (SRF) and ternary complex factors (TCFs) such as the E twenty six-like transcription factor-1 (ELK-1) in this context. Using TCF/SRF-dependent reporter gene constructs, a specific ERK-1/2 inhibitor and a dominant-negative ELK-1 mutant, we show herein that morphine activates ELK-1 via ERK-1/2 in DRG-derived F11 cells endogenously expressing μ and δ ORs. Previous studies with glioma cell lines such as NG108-15 cells attributed morphine-induced gene expression to the activation of the cAMP-responsive element binding protein (CREB). Thus, we also analyzed morphine-dependent activation of CREB in F11 and NG108-15 cells. In contrast to the CREB stimulation found in NG108-15 cells, we observed an inhibitory effect of morphine in F11 cells, indicating cell type-specific regulation of CREB by morphine. To obtain data about putative target genes of morphine-induced ELK-1/SRF activation, we analyzed mRNA levels of 15 ELK-1/SRF-dependent genes in cultured rat DRG neurons and F11 cells. We identified the early growth response protein-4 (EGR-4) as the strongest up-regulated gene in both cell types and observed ELK-1 activity-dependent activation of an EGR-4-driven reporter in F11 cells. Overall, we reveal an important role of ELK-1 for morphine-dependent gene induction in DRG-derived cells and propose that ELK-1 and EGR-4 contribute to the effects of morphine on neuronal plasticity.

  1. The D1 dopamine receptor agonist, SKF83959, attenuates hydrogen peroxide-induced injury in RGC-5 cells involving the extracellular signal-regulated kinase/p38 pathways

    PubMed Central

    Li, Guang-Yu; Li, Ting; Fan, Bin; Zheng, Yong-Chen

    2012-01-01

    Purpose Oxidative stress is widely implicated in the death of retinal ganglion cells associated with various optic neuropathies. Agonists of the dopamine D1 receptor have recently been found to be potentially neuroprotective against oxidative stress–induced injury. The goal of this study was to investigate whether SKF83959, a next-generation high-affinity D1 receptor agonist, could protect retinal ganglion cell 5 (RGC-5) cells from H2O2-induced damage and the molecular mechanism involved. Methods We examined expression of the D1 receptor in RGC-5 cells with reverse-transcription–PCR and immunoblotting and assessed neuroprotection using propidium iodide staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, we monitored the activation and involvement of members of mitogen-activated protein kinase family, extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase, with western blot and specific inhibitors. Results We found that the D1 receptor was expressed in RGC-5 cells, but the sequence analysis suggested this cell line is from mouse and not rat origin. SKF83959 exhibited a remarkable neuroprotective effect on H2O2-damaged RGC-5 cells, which was blocked by the specific D1 receptor antagonist, SCH23390. ERK and p38 were activated by SKF83959, and pretreatment with their inhibitors U0126 and SB203580, respectively, significantly blunted the SKF83959-induced cytoprotection. However, the specific c-Jun NH2-terminal kinase inhibitor, SP600125, had no effect on the SKF83959-induced protection. Conclusions We conclude that SKF83959 attenuates hydrogen peroxide–induced injury in RGC-5 cells via a mechanism involving activation of the ERK and p38 pathways and the D1 receptor is a potential molecular target for developing neuroprotective drugs. PMID:23233790

  2. 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

  3. 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.

  4. 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.

  5. Heat-shock protein 70 modulates apoptosis signal-regulating kinase 1 in stressed hepatocytes of Mugil cephalus.

    PubMed

    Padmini, Ekambaram; Tharani, Jayachandran

    2014-10-01

    Oxidative stress causes damage at the cellular level and activates a number of signaling pathways. Heat-shock proteins (HSPs) play an important role in repair and protective mechanisms under cell response to stress conditions. HSP70 has been shown to act as an inhibitor of apoptosis. Apoptosis signal-regulating kinase-1 (ASK1) activity is regulated at multiple levels, one of which is through inhibition by cytosolic chaperons HSP70. The current study was aimed to investigate the alteration in signaling molecules that allow the fish to survive under stressed natural field conditions. The study also investigates the variation in biomolecular composition of hepatocytes by using Fourier transform infrared spectroscopy. The impact of stress on hepatocytes was assessed by measuring the level of lipid peroxides (LPO), catalase activity (CAT) and assessing the changes in hepatocytes of Mugil cephalus inhabiting Kovalam and Ennore estuaries. The expression of HSP70 and ASK1 were analyzed by immunoblot analysis and ELISA, respectively. The spectral analysis showed variations in biomolecular composition of hepatocytes at a wave number region of 4,000-400 cm(-1). There was significant decrease of CAT activity (p < 0.01) (25 %) with significant increase of LPO (p < 0.001) (35 %) and HSP70 (p < 0.001) and insignificant increase of ASK1 (p < 0.05) (16 %) in fish hepatocytes inhabiting Ennore estuary than Kovalam estuary. In conclusion, the present study suggests that the survival of fish in the Ennore estuary under stressed condition may be due to the upregulation of HSP70 that mediates the altered signal pathway which promotes cellular resistance against apoptosis.

  6. Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

    PubMed

    Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

    1995-03-01

    Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context

  7. 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

  8. 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

  9. Kinetic and biochemical correlation between sustained p44ERK1 (44 kDa extracellular signal-regulated kinase 1) activation and lysophosphatidic acid-stimulated DNA synthesis in Rat-1 cells.

    PubMed Central

    Cook, S J; McCormick, F

    1996-01-01

    Rat-1 fibroblasts were used to study the role of the sustained activation of extracellular signal-regulated kinase 1 (ERK1) in lysophosphatidic acid (LPA)-stimulated mitogenic signalling. Mitogenic doses of LPA, like serum, stimulated biphasic, sustained, ERK activation that persisted towards the G1/S boundary. The EC50 for LPA-stimulated ERK activation after 10 min, the time of peak response, was 2 orders of magnitude to the left of that for the sustained response after 3 h or that for DNA synthesis after 22 h, with the result that non-mitogenic doses stimulated a maximal peak response but no second phase. To complement these studies, we examined the role of different signal pathways in regulating the sustained and acute phases of ERK activation using defined biochemical inhibitors and mimetics. Activation of protein kinase C and Ca2+ fluxes played a minor and transient role in regulation of ERK1 activity by LPA in Rat-1 cells. Sustained ERK1 activation stimulated by LPA was completely inhibited by pertussis toxin, whereas the early peak response was only partly affected; this is correlated with the specific inhibition of LPA-stimulated DNA synthesis by pertussis toxin. The selective tyrosine kinase inhibitor herbimycin A completely inhibited sustained ERK1 activation by LPA but, again, the early phase of the response was only partially inhibited. In addition, low doses of staurosporine inhibited ERK1 activation by LPA. The effects of herbimycin A and staurosporine were selective for the response to LPA but did not affect that to epidermal growth factor. The results suggest a strong correlation between sustained ERK1 activation and DNA synthesis in LPA-stimulated Rat-1 cells. Furthermore, the two discrete phases of ERK activation by LPA are regulated by a combination of at least two different signalling pathways; the sustained activation of ERK1 in Rat-1 cells proceeds via a G1- or Gzero-mediated pathway which may also involve a tyrosine kinase. PMID:8947493

  10. Protein Dynamics in the Plant Extracellular Space

    PubMed Central

    Guerra-Guimarães, Leonor; Pinheiro, Carla; Chaves, Inês; Barros, Danielle R.; Ricardo, Cândido P.

    2016-01-01

    The extracellular space (ECS or apoplast) is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF). The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions. PMID:28248232

  11. Protein Dynamics in the Plant Extracellular Space.

    PubMed

    Guerra-Guimarães, Leonor; Pinheiro, Carla; Chaves, Inês; Barros, Danielle R; Ricardo, Cândido P

    2016-07-13

    The extracellular space (ECS or apoplast) is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF). The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in "cell wall organization and biogenesis", "response to stimulus" and "protein metabolism". It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions.

  12. Insights into Basal Signaling Regulation, Oligomerization, and Structural Organization of the Human G-Protein Coupled Receptor 83

    PubMed Central

    Scheerer, Patrick; Biebermann, Heike; Kleinau, Gunnar

    2016-01-01

    The murine G-protein coupled receptor 83 (mGPR83) is expressed in the hypothalamus and was previously suggested to be involved in the regulation of metabolism. The neuropeptide PEN has been recently identified as a potent GPR83 ligand. Moreover, GPR83 constitutes functionally relevant hetero-oligomers with other G-protein coupled receptors (GPCR) such as the ghrelin receptor (GHSR) or GPR171. Previous deletion studies also revealed that the long N-terminal extracellular receptor domain (eNDo) of mGPR83 may act as an intra-molecular ligand, which participates in the regulation of basal signaling activity, which is a key feature of GPCR function. Here, we investigated particular amino acids at the eNDo of human GPR83 (hGPR83) by side-directed mutagenesis to identify determinants of the internal ligand. These studies were accompanied by structure homology modeling to combine functional insights with structural information. The capacity for hetero-oligomer formation of hGPR83 with diverse family A GPCRs such as the melanocortin-4 receptor (MC4R) was also investigated, with a specific emphasis on the impact of the eNDo on oligomerization and basal signaling properties. Finally, we demonstrate that hGPR83 exhibits an unusual basal signaling for different effectors, which also supports signaling promiscuity. hGPR83 interacts with a variety of hypothalamic GPCRs such as the MC4R or GHSR. These interactions are not dependent on the ectodomain and most likely occur at interfaces constituted in the transmembrane regions. Moreover, several amino acids at the transition between the eNDo and transmembrane helix 1 were identified, where mutations lead also to biased basal signaling modulation. PMID:27936173

  13. 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.

  14. Erythropoietin Rescues Primary Rat Cortical Neurons by Altering the Nrf2:Bach1 Ratio: Roles of Extracellular Signal-Regulated Kinase 1/2.

    PubMed

    Zhang, Li-Min; Zhang, Dong-Xue; Zhao, Xiao-Chun; Sun, Wenbo

    2017-01-12

    While inhalation anesthetics are indispensable, and generally considered safe and effective, there is growing concern about the selective neurotoxicity of these agents, especially sevoflurane. Erythropoetin (EPO)-induced protection against sevoflurane-induced neuronal death is an effective intervention, but the underlying mechanism is poorly understood. Extracellular signal-related kinases (Erk) 1/2 plays a pivotal role in cell growth and proliferation. Alteration of the nuclear factor erythroid 2-related factor (Nrf2)/BTB-to-CNC homology 1 (Bach1) ratio by Erk1/2 ameliorates the oxidative stress which occurs in human macrophages. Primary cortical neuron cultures exposed to sevoflurane were assessed for Nrf2, Bach1, total Erk1/2, and phosphorylated Erk1/2 with the following: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; propidium iodide uptake; lactate dehydrogenase; malondialdehyde (MDA); superoxide dismutase (SOD); and Western blot. Sevoflurane exposure increased cell death, injury, and MDA (n = 9, P < 0.05), but decreased cell viability and the Nrf2:Bach1 ratio (n = 9, P < 0.05) and down-regulated SOD (n = 9, P < 0.05), while EPO partially rescued the neurotoxicity induced by sevoflurane (n = 9, P < 0.05). Inhibition of Erk1/2 phosphorylation via PD98059 reversed the protective effect of EPO (n = 9, P < 0.05). Thus, protection of EPO markedly attenuated death of neurons exposed to sevoflurane by altering the Nrf2:Bach1 ratio mediated by phosphorylation and activation of Erk1/2.

  15. Potential involvement of extracellular signal-regulated kinase 1 and 2 in encystation of a primitive eukaryote, Giardia lamblia. Stage-specific activation and intracellular localization.

    PubMed

    Ellis, John G; Davila, Monica; Chakrabarti, Ratna

    2003-01-17

    Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44- and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is approximately 5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial approximately 2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.

  16. Rosiglitazone attenuates the metalloprotease/anti-metalloprotease imbalance in emphysema induced by cigarette smoke: involvement of extracellular signal-regulated kinase and NFκB signaling

    PubMed Central

    Hou, Gang; Yin, Yan; Han, Dan; Wang, Qiu-yue; Kang, Jian

    2015-01-01

    Objective We investigated how rosiglitazone attenuated cigarette smoke (CS)-induced emphysema in a rat model. In particular, we focused on its possible effects on the imbalance between metalloprotease (MMP) and anti-MMP activity, mitogen-activated protein kinase (MAPK) phosphorylation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling pathway over-activation. Methods A total of 36 Wistar rats were divided into three groups (n=12 each): animals were exposed to CS for 12 weeks in the absence (the CS group) or presence of 30 mg/kg rosiglitazone (the rosiglitazone-CS [RCS] group); a control group was treated with the rosiglitazone vehicle only, without any CS exposure. Histopathology of lung tissue in all groups was evaluated to grade severity of the disease. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ), MMP2, and MMP9 in lung tissue were determined and compared using Western blotting and immunohistochemistry. Activation of MAPKs, NFκB, and the nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα) phosphorylation in lung tissue was examined by Western blotting. Results Emphysema-related pathology, based on inter-alveolar wall distance and alveolar density, was less severe in the RCS group than in the CS group. Compared with the CS group, levels of PPARγ were higher in the RCS group, and levels of MMP2 and MMP9 proteins were lower in the RCS rats. Levels of activated MAPKs and NFκB were also lower, while the IκBαphosphorylation was increased in the lung tissue of RCS rats. Conclusion Our findings suggest that oral administration of rosiglitazone attenuates the metalloprotease activity induced by CS, and the underlying mechanism might involve the activation of signaling pathways dependent on MAPKs or NFκB. Our results further suggest that PPARγ contributes to the pathogenesis of emphysema as well as airway inflammation induced by CS. PMID:25897215

  17. 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.

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

    PubMed

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

    2017-04-15

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

  19. 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

  20. Autophagy-related gene 7 (ATG7) and reactive oxygen species/extracellular signal-regulated kinase regulate tetrandrine-induced autophagy in human hepatocellular carcinoma.

    PubMed

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

    2012-10-12

    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.

  1. Glycodelin-A stimulates interleukin-6 secretion by human monocytes and macrophages through L-selectin and the extracellular signal-regulated kinase pathway.

    PubMed

    Lee, Cheuk-Lun; Lam, Eve Y F; Lam, Kevin K W; Koistinen, Hannu; Seppälä, Markku; Ng, Ernest H Y; Yeung, William S B; Chiu, Philip C N

    2012-10-26

    Macrophages represent the second major type of decidual leukocytes at the fetomaternal interface. Changes in macrophage number and activity are associated with fetal loss and pregnancy complications. Glycodelin-A (GdA) is an abundant glycoprotein in the first-trimester decidua. It is involved in fetomaternal defense and early placental development through its regulatory activities in various immune cells. The N-glycosylation of GdA mediates the binding and therefore the activities of the molecule. In this study, we studied the biological activities of GdA in the functions of human monocytes/macrophages. GdA was purified from amniotic fluid by affinity chromatography. GdA treatment did not affect the viability, cell death, or phagocytic activity of the monocytes/macrophages. GdA, but not recombinant glycodelin without glycosylation, induced IL-6 production as demonstrated by cytokine array, intracellular staining, and ELISA. GdA also induced phosphorylation of ERK in monocytes/macrophages. The involvement of ERKs in IL-6 induction was confirmed using pharmacological inhibitors. Co-immunoprecipitation showed that L-selectin on the monocytes/macrophages was the binding protein of GdA. Treatment with anti-L-selectin antibody reduced GdA binding and GdA-induced IL-6 production. GdA-treated macrophages suppressed IFN-γ expression by co-cultured T-helper cells in an IL-6-dependent manner. These results show that GdA interacts with L-selectin to induce IL-6 production in monocytes/macrophages by activating the ERK signaling pathway. In turn, the increased IL-6 production suppresses IFN-γ expression in T-helper cells, which may play an important role in inducing a Th-2-polarized cytokine environment that flavors the immunotolerance of the fetoplacental unit.

  2. Elemene Induces Apoptosis of Human Gastric Cancer Cell Line BGC-823 via Extracellular Signal-Regulated Kinase (ERK) 1/2 Signaling Pathway

    PubMed Central

    Li, Pihong; Zhou, Xiang; Sun, Weijian; Sheng, Weiwei; Tu, Yangyang; Yu, Yaojun; Dong, Jianda; Ye, Bing; Zheng, Zhiqiang; Lu, Mingdong

    2017-01-01

    Background Elemene is extracted from a traditional herbal medicine and is commonly used in the treatment of cancer in China. However, its effect on gastric cancer cells remains unknown. The goal of this study was to investigate its effect on human gastric cancer cells. Material/Methods Human gastric cancer BGC-823 cells and a tumor-bearing mouse model were employed to be divided into 4 groups: control group, elemene group, PD98059 group (an ERK 1/2 signaling pathway inhibitor), and the combined group (elemene plus PD98059). The tumor size, cell proliferation, expression of ERK 1/2 and phosphorylated ERK 1/2 (p-ERK 1/2), Bcl-2 mRNA, and Bax mRNA were measured. Moreover, cell apoptosis was detected and the apoptosis index was calculated. Results Elemene and PD98059 each significantly inhibited the proliferation of gastric cancer cells BGC-823, and their combination showed higher synergistic inhibitory effect (P<0.05). We also found increased expression levels of p-ERK l/2 protein and Bax mRNA, but reduced level of Bcl-2 mRNA expression (P<0.05). Elemene presented higher apoptosis rate in a dose-dependent manner (P<0.05). Furthermore, the injection of elemene decreased the weight of transplanted tumors. Conclusions Elemene can inhibit the proliferation and induce the apoptosis of gastric cancer cells associated with the ERK 1/2 signaling pathway and expression levels of Bax mRNA and Bcl-2 mRNA. PMID:28196062

  3. Economical evolution: microbes reduce the synthetic cost of extracellular proteins.

    PubMed

    Smith, Daniel R; Chapman, Matthew R

    2010-08-24

    Protein evolution is not simply a race toward improved function. Because organisms compete for limited resources, fitness is also affected by the relative economy of an organism's proteome. Indeed, many abundant proteins contain relatively high percentages of amino acids that are metabolically less taxing for the cell to make, thus reducing cellular cost. However, not all abundant proteins are economical, and many economical proteins are not particularly abundant. Here we examined protein composition and found that the relative synthetic cost of amino acids constrains the composition of microbial extracellular proteins. In Escherichia coli, extracellular proteins contain, on average, fewer energetically expensive amino acids independent of their abundance, length, function, or structure. Economic pressures have strategically shaped the amino acid composition of multicomponent surface appendages, such as flagella, curli, and type I pili, and extracellular enzymes, including type III effector proteins and secreted serine proteases. Furthermore, in silico analysis of Pseudomonas syringae, Mycobacterium tuberculosis, Saccharomyces cerevisiae, and over 25 other microbes spanning a wide range of GC content revealed a broad bias toward more economical amino acids in extracellular proteins. The synthesis of any protein, especially those rich in expensive aromatic amino acids, represents a significant investment. Because extracellular proteins are lost to the environment and not recycled like other cellular proteins, they present a greater burden on the cell, as their amino acids cannot be reutilized during translation. We hypothesize that evolution has optimized extracellular proteins to reduce their synthetic burden on the cell.

  4. Icariin inhibits oxidized low-density lipoprotein-induced proliferation of vascular smooth muscle cells by suppressing activation of extracellular signal-regulated kinase 1/2 and expression of proliferating cell nuclear antigen.

    PubMed

    Hu, Yanwu; Liu, Kai; Yan, Mengtong; Zhang, Yang; Wang, Yadi; Ren, Liqun

    2016-03-01

    Icariin, a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to possess anti-inflammatory, anti‑oxidant and anti-atherosclerotic activities in vivo and in vitro. The aim of the present study was to investigate the effects of icariin on oxidized low‑density lipoprotein (ox-LDL)-induced proliferation of vascular smooth muscle cells (VSMCs) and the possible underlying mechanism. VSMCs were cultured and pre‑treated with various concentrations of icariin (0, 10, 20 or 40 µm) prior to stimulation by ox‑LDL (50 µg/ml). Cell proliferation was evaluated by an MTT assay. Flow cytometry was used to study the influence of icariin on the cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 were detected by western blot analysis. The results indicated that icariin significantly inhibited ox‑LDL‑induced proliferation of VSMCs and phosphorylation of ERK1/2. Furthermore, icariin also blocked the ox‑LDL‑induced cell‑cycle progression at G1/S‑interphase and downregulated the expression of PCNA in VSMCs. In conclusion, the present study indicated for the first time that icariin reduced the amount of ox‑LDL‑induced proliferation of VSMCs through suppression of PCNA expression and inactivation of ERK1/2.

  5. Clonorchis sinensis excretory-secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression.

    PubMed

    Pak, Jhang Ho; Shin, Jimin; Song, In-Sung; Shim, Sungbo; Jang, Sung-Wuk

    2017-01-01

    Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory-secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory-secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory-secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory-secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory-secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory-secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma.

  6. Effects of DA-9701, a Novel Prokinetic Agent, on Phosphorylated Extracellular Signal-Regulated Kinase Expression in the Dorsal Root Ganglion and Spinal Cord Induced by Colorectal Distension in Rats

    PubMed Central

    Lee, Sang Pyo; Lee, Kang Nyeong; Lee, Hang Lak; Jun, Dae Won; Yoon, Byung Chul; Choi, Ho Soon; Hwang, Se Jin; Lee, Seo Eun

    2014-01-01

    Background/Aims DA-9701, a standardized extract of Pharbitis Semen and Corydalis Tuber, is a new prokinetic agent that exhibits an analgesic effect on the abdomen. We investigated whether DA-9701 affects visceral pain induced by colorectal distension (CRD) in rats. Methods A total of 21 rats were divided into three groups: group A (no CRD+no drug), group B (CRD+no drug), and group C (CRD+DA-9701). Expression of pain-related factors, substance P (SP), c-fos, and phosphorylated extracellular signal-regulated kinase (p-ERK) in the dorsal root ganglion (DRG) and spinal cord was determined by immunohistochemical staining and Western blotting. Results The proportions of neurons in the DRG and spinal cord expressing SP, c-fos, and p-ERK were higher in group B than in group A. In the group C, the proportion of neurons in the DRG and spinal cord expressing p-ERK was lower than that in group B. Western blot results for p-ERK in the spinal cord indicated a higher level of expression in group B than in group A and a lower level of expression in group C than in group B. Conclusions DA-9701 may decrease visceral pain via the downregulation of p-ERK in the DRG and spinal cord. PMID:24672654

  7. Polycystin-1 Regulates Extracellular Signal-Regulated Kinase-Dependent Phosphorylation of Tuberin To Control Cell Size through mTOR and Its Downstream Effectors S6K and 4EBP1 ▿

    PubMed Central

    Distefano, Gianfranco; Boca, Manila; Rowe, Isaline; Wodarczyk, Claas; Ma, Li; Piontek, Klaus B.; Germino, Gregory G.; Pandolfi, Pier Paolo; Boletta, Alessandra

    2009-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease characterized by bilateral renal cyst formation. Both hyperproliferation and hypertrophy have been previously observed in ADPKD kidneys. Polycystin-1 (PC-1), a large orphan receptor encoded by the PKD1 gene and mutated in 85% of all cases, is able to inhibit proliferation and apoptosis. Here we show that overexpression of PC-1 in renal epithelial cells inhibits cell growth (size) in a cell cycle-independent manner due to the downregulation of mTOR, S6K1, and 4EBP1. Upregulation of the same pathway leads to increased cell size, as found in mouse embryonic fibroblasts derived from Pkd1−/− mice. We show that PC-1 controls the mTOR pathway in a Tsc2-dependent manner, by inhibiting the extracellular signal-regulated kinase (ERK)-mediated phosphorylation of tuberin in Ser664. We provide a detailed molecular mechanism by which PC-1 can inhibit the mTOR pathway and regulate cell size. PMID:19255143

  8. Extracellular-signal regulated kinase 1-dependent metabotropic glutamate receptor 5-induced long-term depression in the bed nucleus of the stria terminalis is disrupted by cocaine administration.

    PubMed

    Grueter, Brad A; Gosnell, Heather B; Olsen, Christopher M; Schramm-Sapyta, Nicole L; Nekrasova, Tanya; Landreth, Gary E; Winder, Danny G

    2006-03-22

    The bed nucleus of the stria terminalis (BNST) is a key component of the CNS stress and reward circuit. Synaptic plasticity in this region could in part underlie the persistent behavioral alterations in generalized anxiety and addiction. Group I metabotropic glutamate receptors (mGluRs) have been implicated in stress, addiction, and synaptic plasticity, but their roles in the BNST are unknown. We find that activation of group I mGluRs in the dorsal BNST induces depression of excitatory synaptic transmission through two distinct mechanisms. First, a combined activation of group I mGluRs (mGluR1 and mGluR5) induces a transient depression that is cannabinoid 1 receptor dependent. Second, as with endocannabinoid-independent group I mGluR long-term depression (LTD) in the adult hippocampus, we find that activation of mGluR5 induces an extracellular signal-regulated kinase (ERK)-dependent LTD. Surprisingly, our data demonstrate that this LTD requires the ERK1 rather than ERK2 isoform, establishing a key role for this isoform in the CNS. Finally, we find that this LTD is dramatically reduced after multiple exposures but not a single exposure to cocaine, suggesting a role for this form of plasticity in the actions of psychostimulants on anxiety and reward circuitries and their emergent control of animal behavior.

  9. 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...

  10. Extracellular Signal-regulated Kinases (ERKs) Phosphorylate Lin28a Protein to Modulate P19 Cell Proliferation and Differentiation.

    PubMed

    Liu, Xiangyuan; Chen, Min; Li, Long; Gong, Liyan; Zhou, Hu; Gao, Daming

    2017-03-10

    Lin28a, originally discovered in the nematode Caenorhabditis elegans and highly conserved across species, is a well characterized regulator of let-7 microRNA (miRNA) and is implicated in cell proliferation and pluripotency control. However, little is known about how Lin28a function is modulated at the post-translational level and thereby responds to major signaling pathways. Here we show that Lin28a is directly phosphorylated by ERK1/2 kinases at Ser-200. By editing lin28a gene with the CRISPR/Cas9-based method, we generated P19 mouse embryonic carcinoma stem cells expressing Lin28a-S200A (phospho-deficient) and Lin28a-S200D (phospho-mimetic) mutants, respectively, to study the functional impact of Ser-200 phosphorylation. Lin28a-S200D-expressing cells, but not Lin28a-S200A-expressing or control P19 embryonic carcinoma cells, displayed impaired inhibition of let-7 miRNA and resulted in decreased cyclin D1, whereas Lin28a-S200A knock-in cells expressed less let-7 miRNA, proliferated faster, and exhibited differentiation defect upon retinoic acid induction. Therefore our results support that ERK kinase-mediated Lin28a phosphorylation may be an important mechanism for pluripotent cells to facilitate the escape from the self-renewal cycle and start the differentiation process.

  11. 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.

  12. Selective inhibition of extracellular signal-regulated kinases 1/2 blocks nerve growth factor to brain-derived neurotrophic factor signaling and suppresses the development of and reverses already established pain behavior in rats.

    PubMed

    Matsuoka, Y; Yang, J

    2012-03-29

    Brain-derived neurotrophic factor (BDNF) plays a key role in the development of pathological pain. Although it is known that nerve growth factor (NGF) induces BDNF mRNA through extracellular signal-regulated kinases (ERK), whether ERK1/2 or ERK5, two closely related members of the ERK family, mediate this signal is still unclear because classical MEK inhibitors block both pathways. We studied the involvement of ERK-signaling in NGF induction of BDNF in PC12 cells, cultured dorsal root ganglia neurons, and in rats subjected to neuropathic pain models using ERK1/2- and ERK5-specific tools. Selective activation of ERK1/2 upregulated BDNF mRNA in PC12 cells, whereas selective ERK5 activation did not. AZD6244, a potent selective inhibitor of ERK1/2 activation, blocked NGF induction of BDNF mRNA in vitro suggesting that NGF induction of BDNF is mediated by ERK1/2. siRNA experiments indicated that both ERK1 or ERK2 can signal suggesting that both pathways must be blocked to prevent NGF-induced increase in BDNF mRNA. I.p. injection of AZD6244 prevented the development of pain in rats subjected to the chronic constriction injury and reversed already established pain in the spared nerve injury model. Immunohistochemical studies showed decreased phospho-ERK1/2-immunoreactivity in dorsal root ganglia and BDNF immunoreactivity in ipsilateral spinal dorsal horn in the drug-treated rats. Our results suggest the possible use of AZD6244, already in human clinical trials as an anticancer agent, for the treatment of pathological pain.

  13. Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2.

    PubMed

    Sanchez, A; Tripathy, D; Yin, X; Luo, J; Martinez, J; Grammas, P

    2012-01-01

    Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H₂O₂ as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H₂O₂ treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H₂O₂ exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H₂O₂-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.

  14. Extra-cellular signal-regulated kinase 1/2 (ERK1/2) activated in the hippocampal CA1 neurons is critical for retrieval of auditory trace fear memory.

    PubMed

    Huang, Ching-Hsun; Chiang, Yu-Wei; Liang, Keng-Chen; Thompson, Richard F; Liu, Ingrid Y

    2010-04-22

    The brain regions involved with trace fear conditioning (TFC) and delayed fear conditioning (DFC) are well-characterized, but little is known about the cellular representation subsuming these types of classical conditioning. Previous evidence has shown that activation of the amygdala is required for both TFC and DFC, while TFC also involves the hippocampus for forming conditioned response to tone. Lesions of the hippocampus did not affect tone learning in DFC, but it impaired learning in TFC. Synaptic plasticity in the hippocampus, underlying a cellular representation subsuming learning and memory, is in part modulated by extra-cellular signal-regulated kinase (ERK) signaling pathway. ERK1/2 activation is required for both TFC and DFC during memory formation, but whether this pathway is involved in memory retrieval of TFC is still unknown. In the present study, we investigated changes in ERK1/2 phosphorylation after memory retrieval in groups of mice that received TFC, DFC, tone-shock un-paired conditioning, and naïve control. Our results showed that ERK1/2 phosphorylation was elevated in the hippocampal CA1 region after retrieval of all conditioned fear responses. In particular, in the TFC group, immunohistochemistry indicated higher level of ERK1/2 phosphorylation in the hippocampal pyramidal neurons 30min after tone testing. Inhibition of the ERK1/2 signaling pathway diminished fear memory elicited by a tone in TFC. Together these results suggest that the memory retrieval process in TFC is more dependent on ERK1/2 signaling pathway than that in DFC. ERK1/2 signaling is critical for retrieval associative memory of temporally noncontiguous stimuli.

  15. α-Mangostin suppresses lipopolysaccharide-induced invasion by inhibiting matrix metalloproteinase-2/9 and increasing E-cadherin expression through extracellular signal-regulated kinase signaling in pancreatic cancer cells

    PubMed Central

    YUAN, JIANGTAO; WU, YAOLU; LU, GUIFANG

    2013-01-01

    Invasion and metastasis are major factors in the poor prognosis of pancreatic cancer, which remains one of the most aggressive and lethal diseases worldwide. α-mangostin, a major xanthone compound identified in the pericarp of mangosteen (Garcinia mangostana, Linn; GML), possesses unique biological activities, including antioxidant, antitumor and anti-inflammatory effects. Whether α-mangostin is able to inhibit the invasive ability of pancreatic cancer cells has not been elucidated. In the present study, α-mangostin was shown to inhibit the invasive ability of the pancreatic cancer cell lines MIAPaCa-2 and BxPC-3. The results showed that α-mangostin inhibited the growth of the pancreatic cancer cells in a dose- and time-dependent manner. At concentrations of <5 μM, α-mangostin had no significant effects on cytotoxicity, but significantly inhibited the invasion and migration of pancreatic cancer cells and the expression of matrix metalloproteinase (MMP)-2 and MMP-9, while increasing the expression of E-cadherin. The present data also showed that α-mangostin exerted an inhibitory effect on the phosphorylation of extracellular-signal-regulated kinase (ERK). Furthermore, the reduction of ERK phosphorylation by small interfering RNA (siRNA) potentiated the effect of α-mangostin. Taken together, the data suggest that α-mangostin inhibited the invasion and metastasis of pancreatic cancer cells by reducing MMP-2 and MMP-9 expression, increasing E-cadherin expression and suppressing the ERK signaling pathway. The present study suggests that α-mangostin may be a promising agent against pancreatic cancer. PMID:23833675

  16. c-Met Overexpression Contributes to the Acquired Apoptotic Resistance of Nonadherent Ovarian Cancer Cells through a Cross Talk Mediated by Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/212

    PubMed Central

    Tang, Maggie K S; Zhou, Hong Y; Yam, Judy W P; Wong, Alice S T

    2010-01-01

    Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications. PMID:20126471

  17. In vitro Determination of Extracellular Proteins from Xylella fastidiosa

    PubMed Central

    Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

  18. 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.

  19. Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A

    PubMed Central

    Zhang, Xiangmin; Ma, Danjun; Caruso, Michael; Lewis, Monique; Qi, Yue; Yi, Zhengping

    2014-01-01

    Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes. PMID:24972320

  20. G protein-coupled receptors: bridging the gap from the extracellular signals to the Hippo pathway.

    PubMed

    Zhou, Xin; Wang, Zhen; Huang, Wei; Lei, Qun-Ying

    2015-01-01

    The Hippo pathway is crucial in organ size control, whereas its dysregulation contributes to organ degeneration or tumorigenesis. The kinase cascade of MST1/2 and LATS1/2 and the coupling transcription co-activators YAP/TAZ represent the core components of the Hippo pathway. Extensive studies have identified a number of upstream regulators of the Hippo pathway, including contact inhibition, mechanic stress, extracellular matrix stiffness, cytoskeletal rearrangement, and some molecules of cell polarity and cell junction. However, how the diffuse extracellular signals regulate the Hippo pathway puzzles the researchers for a long time. Unexpectedly, recent elegant studies demonstrated that stimulation of some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid receptor, sphingosine-1-phosphate receptor, and the protease activated receptor PAR1, causes potent YAP/TAZ dephosphorylation and activation by promoting actin cytoskeleton assemble. In this review, we briefly describe the components of the Hippo pathway and focus on the recent progress with respect to the regulation of the Hippo pathway by GPCRs and G proteins in cancer cells. In addition, we also discuss the potential therapeutic roles targeting the Hippo pathway in human cancers.

  1. 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.

  2. Differential involvement of medial prefrontal cortex and basolateral amygdala extracellular signal-regulated kinase in extinction of conditioned taste aversion is dependent on different intervals of extinction following conditioning.

    PubMed

    Lin, P-Y; Wang, S-P; Tai, M-Y; Tsai, Y-F

    2010-11-24

    Extinction reflects a decrease in the conditioned response (CR) following non-reinforcement of a conditioned stimulus. Behavioral evidence indicates that extinction involves an inhibitory learning mechanism in which the extinguished CR reappears with presentation of an unconditioned stimulus. However, recent studies on fear conditioning suggest that extinction erases the original conditioning if the time interval between fear acquisition and extinction is short. The present study examined the effects of different intervals between acquisition and extinction of the original memory in conditioned taste aversion (CTA). Male Long-Evans rats acquired CTA by associating a 0.2% sucrose solution with malaise induced by i.p. injection of 4 ml/kg 0.15 M LiCl. Two different time intervals, 5 and 24 h, between CTA acquisition and extinction were used. Five or 24 h after CTA acquisition, extinction trials were performed, in which a bottle containing 20 ml of a 0.2% sucrose solution was provided for 10 min without subsequent LiCl injection. If sucrose consumption during the extinction trials was greater than the average water consumption, then rats were considered to have reached CTA extinction. Rats subjected to extinction trials lasting 24 h, but not 5 h, after acquisition re-exhibited the extinguished CR following injection of 0.15 M LiCl alone 7 days after acquisition. Extracellular signal-regulated kinase (ERK) in the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) was examined by Western blot after the first extinction trial. ERK activation in the mPFC was induced after the extinction trial beginning 5 h after acquisition, whereas the extinction trial performed 24 h after acquisition induced ERK activation in the BLA. These data suggest that the original conditioning can be inhibited or retained by CTA extinction depending on the time interval between acquisition and extinction and that the ERK transduction pathway in the mPFC and BLA is

  3. 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.

  4. Inhibition of Protein Kinase Akt1 by Apoptosis Signal-regulating Kinase-1 (ASK1) Is Involved in Apoptotic Inhibition of Regulatory Volume Increase*

    PubMed Central

    Subramanyam, Muthangi; Takahashi, Nobuyuki; Hasegawa, Yuichi; Mohri, Tatsuma; Okada, Yasunobu

    2010-01-01

    Most animal cell types regulate their cell volume after an osmotic volume change. The regulatory volume increase (RVI) occurs through uptake of NaCl and osmotically obliged water after osmotic shrinkage. However, apoptotic cells undergo persistent cell shrinkage without showing signs of RVI. Persistence of the apoptotic volume decrease is a prerequisite to apoptosis induction. We previously demonstrated that volume regulation is inhibited in human epithelial HeLa cells stimulated with the apoptosis inducer. Here, we studied signaling mechanisms underlying the apoptotic inhibition of RVI in HeLa cells. Hypertonic stimulation was found to induce phosphorylation of a Ser/Thr protein kinase Akt (protein kinase B). Shrinkage-induced Akt activation was essential for RVI induction because RVI was suppressed by an Akt inhibitor, expression of a dominant negative form of Akt, or small interfering RNA-mediated knockdown of Akt1 (but not Akt2). Staurosporine, tumor necrosis factor-α, or a Fas ligand inhibited both RVI and hypertonicity-induced Akt activation in a manner sensitive to a scavenger for reactive oxygen species (ROS). Any of apoptosis inducers also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in a ROS-dependent manner. Suppression of (ASK1) expression blocked the effects of apoptosis, in hypertonic conditions, on both RVI induction and Akt activation. Thus, it is concluded that in human epithelial cells, shrinkage-induced activation of Akt1 is involved in the RVI process and that apoptotic inhibition of RVI is caused by inhibition of Akt activation, which results from ROS-mediated activation of ASK1. PMID:20048146

  5. Extracellular proteins of Cryptococcus neoformans and host antibody response.

    PubMed Central

    Chen, L C; Pirofski, L A; Casadevall, A

    1997-01-01

    Proteins secreted by the fungal pathogen Cryptococcus neoformans may be involved in invasion and could be useful in vaccine design. Despite the medical importance of this fungus, little is known about its extracellular proteins or the immune response to these antigens. To study C. neoformans extracellular proteins, 12 strains were metabolically radiolabeled and protein supernatants were analyzed. Both strain- and growth condition-dependent differences were observed. Enzymatic assays of filtered culture supernatants revealed butyrate esterase and caprylate esterase lipase activity for 11 of 12 strains, as well as acid phosphatase, naphthol-AS-BI-phosphohydrolase, and beta-glucosidase activities in some strains. Serum from infected rodents immunoprecipitated several secreted proteins, consistent with in vivo expression and development of an antibody response. For strain 24067, two immunodominant species, of approximately 75 and 30 kDa, were recognized. The relative intensity of the autoradiographic bands depended on the route of infection for both rats and mice. In summary, our results indicate that (i) there are multiple proteins in C. neoformans culture supernatants, (ii) there are strain differences in supernatant protein profiles, (iii) there are differences in supernatant protein profile depending on the growth conditions, (iv) there are several new extracellular and/or cell-associated enzymatic activities, and (v) antibodies to several supernatant proteins are made in the course of infection. PMID:9199426

  6. 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

  7. Role of extracellular signal-regulated kinase in synaptic transmission and plasticity of a nociceptive input on capsular central amygdaloid neurons in normal and acid-induced muscle pain mice.

    PubMed

    Cheng, Sin-Jhong; Chen, Chien-Chang; Yang, Hsiu-Wen; Chang, Ya-Ting; Bai, Shin-Wen; Chen, Chih-Cheng; Yen, Chen-Tung; Min, Ming-Yuan

    2011-02-09

    Application of phorbol 12,13-diacetate (PDA) caused marked enhancement of synaptic transmission of nociceptive parabrachio-amygdaloid (PBA) input onto neurons of the capsular central amygdaloid (CeAC) nucleus. The potentiation of PBA-CeAC EPSCs by PDA involved a presynaptic protein kinase C (PKC)-dependent component and a postsynaptic PKC-extracellular-regulated kinase (ERK)-dependent component. NMDA glutamatergic receptor (NMDAR)-dependent long-term potentiation (LTP) of PBA-CeAC EPSCs, which was also dependent on the PKC-ERK signaling pathway, was induced by tetanus stimulation at 100 Hz. In slices from mice subjected to acid-induced muscle pain (AIMP), phosphorylated ERK levels in the CeAC increased, and PBA-CeAC synaptic transmission was postsynaptically enhanced. The enhanced PBA-CeAC synaptic transmission in AIMP mice shared common mechanisms with the postsynaptic potentiation effect of PDA and induction of NMDAR-dependent LTP by high-frequency stimulation in normal slices, both of which required ERK activation. Since the CeAC plays an important role in the emotionality of pain, enhanced synaptic function of nociceptive (PBA) inputs onto CeAC neurons might partially account for the supraspinal mechanisms underlying central sensitization.

  8. 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.

  9. 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. PMID:27375598

  10. Extracellular Antibody Drug Conjugates Exploiting the Proximity of Two Proteins

    PubMed Central

    Marshall, David J; Harried, Scott S; Murphy, John L; Hall, Chad A; Shekhani, Mohammed S; Pain, Christophe; Lyons, Conner A; Chillemi, Antonella; Malavasi, Fabio; Pearce, Homer L; Thorson, Jon S; Prudent, James R

    2016-01-01

    The human Na+/K+-ATPase (NKA) is a plasma membrane ion pump that uses ATP to help maintain the resting potential of all human cells. Inhibition of the NKA leads to cell swelling and death. The results of this investigation show that on cancer cells, the NKA either comes in close proximity to, associate with or complexes to important cancer-related proteins, and thus can be targeted with a new type of precision therapy called the extracellular drug conjugate or EDC. The EDCs reported here exhibit EC50 values in the low to mid-picomolar range, and signal to noise ratios > 1,000:1, both of which are dependent on the cell surface expression of the NKA and corresponding cancer-related target. We demonstrate that a potent small molecule inhibitor of the NKA can be covalently attached to antibodies targeting CD20, CD38, CD56, CD147, or dysadherin, to create a series of selective and powerful EDCs that kill cancer cells extracellularly by a mechanism resembling necrosis. This is therefore a framework for the development of a new type of precision therapy wherein exquisite selectivity is achieved for targeting extracellular disease-related proteins. PMID:27434591

  11. Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

    PubMed

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

    2014-12-01

    Nontypeable 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- and 96-h NTHi biofilms contained polysaccharides and proteinaceous components as detected by nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (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-h biofilms, two were found only in 96-h biofilms, and fifteen were present in the ECM of both 24- and 96-h NTHi biofilms. All proteins identified were either associated with bacterial membranes or 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.

  12. Secretion and extracellular space travel of Wnt proteins.

    PubMed

    Gross, Julia Christina; Boutros, Michael

    2013-08-01

    Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion.

  13. 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

  14. [Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

    PubMed

    Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

    2013-01-01

    After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

  15. Designing an extracellular matrix protein with enhanced mechanical stability

    PubMed Central

    Ng, Sean P.; Billings, Kate S.; Ohashi, Tomoo; Allen, Mark D.; Best, Robert B.; Randles, Lucy G.; Erickson, Harold P.; Clarke, Jane

    2007-01-01

    The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by ≈20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions. PMID:17535921

  16. An innovative protocol for schwann cells extracellular matrix proteins extraction.

    PubMed

    Parisi, L; Zomer Volpato, F; Cagol, N; Siciliano, M; Migliaresi, C; Motta, A; Sala, R

    2016-12-01

    The evidence that extracellular matrix (ECM) components could represent new targets for drugs designed to approach degenerative disease, requires their analysis. Before the analysis, proteins should be extracted from ECM and solubilized. Currently, few protocols for ECM proteins extraction and solubilization are available in literature, and most of them are based mainly on the use of proteolytic enzymes, such as trypsin, which often lead to proteins damage. Moreover, no methods have been so far proposed to solubilize Schwann Cell ECM, which may represent an important target for the therapy of neurodegenerative disorders. In our study, we propose to solubilize SC ECM through the use of surfactants and urea. We compared our method of solubilization, with one of that proposed in literature for a general ECM, mainly based on the use of enzymes. We want to highlight the benefit of solubilizing SC ECM, avoiding the use of proteolytic enzymes. To compare the amount of proteins extracted with both methods, MicroBCA assay was used, while the quality of the proteins extracted was observed through the SDS-PAGE. The results obtained confirm a better solubilization of SC ECM proteins with the proposed protocol, both quantitatively and qualitatively, showing a higher concentration of proteins extracted and a better enrichment of protein fractions, if compared to the enzyme-based protocol. Our results show that SC ECM could be efficiently solubilized through the use of surfactant and urea, avoiding the use of enzyme-base methods. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3175-3180, 2016.

  17. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy.

    PubMed

    Edginton, Ryan S; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C Peter; Palombo, Francesca

    2016-09-15

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

  18. Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

    PubMed Central

    Edginton, Ryan S.; Mattana, Sara; Caponi, Silvia; Fioretto, Daniele; Green, Ellen; Winlove, C. Peter; Palombo, Francesca

    2016-01-01

    Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis. PMID:27684584

  19. Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer

    PubMed Central

    van der Mijn, Johannes C.; Sol, Nik; Mellema, Wouter; Jimenez, Connie R.; Piersma, Sander R.; Dekker, Henk; Schutte, Lisette M.; Smit, Egbert F.; Broxterman, Henk J.; Skog, Johan; Tannous, Bakhos A.; Wurdinger, Thomas; Verheul, Henk M. W.

    2014-01-01

    Background Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. Methods EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. Results In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005). Conclusion Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer. PMID:25491250

  20. 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

  1. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis.

    PubMed

    Watson, Steve P

    2009-01-01

    The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms possessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of

  2. Transforming growth factor-β1 induces type II collagen and aggrecan expression via activation of extracellular signal-regulated kinase 1/2 and Smad2/3 signaling pathways.

    PubMed

    Zhu, Yanhui; Tao, Hairong; Jin, Chen; Liu, Yonzhang; Lu, Xiongwei; Hu, Xiaopeng; Wang, Xiang

    2015-10-01

    Transforming growth factor (TGF)‑β regulates the anabolic metabolism of articular cartilage and prevents cartilage degradation. TGF‑β1 influences cellular proliferation, differentiation and the extracellular matrix through activation of the extracellular signal‑regulated kinase (ERK)1/2 and Smad2/3 signaling pathways. However, it has remained to be fully elucidated precisely how the ERK1/2 and Smad2/3 signaling pathways mediate anabolic processes of articular cartilage. The present study investigated how ERK1/2 and Smad2/3 signaling mediate TGF‑β1‑stimulated type II collagen and aggrecan expression in rat chondrocytes. The results confirmed that TGF‑β1 stimulates type II collagen and aggrecan expression in rat chondrocytes, and furthermore, that the ERK1/2 and Smad2/3 signaling pathways were activated by TGF‑β1. Conversely, the TGF‑β receptor I (ALK5) kinase inhibitor SB525334 significantly impaired TGF‑β1‑induced type II collagen and aggrecan expression, coinciding with a reduction of ERK1/2 and Smad3 phosphorylation. In addition, TGF‑β1‑induced type II collagen and aggrecan expression were significantly suppressed by ERK1/2 inhibitor PD98059. Similarly, TGF‑β1‑stimulated type II collagen and aggrecan expression were decreased in the presence of a Smad3 phosphorylation inhibitor SIS3. Therefore, the present study demonstrated that the ERK1/2 and Smad2/3 signaling pathways regulate type II collagen and aggrecan expression in rat chondrocytes.

  3. 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.

  4. 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

  5. Sevoflurane Post-conditioning Protects Primary Rat Cortical Neurons Against Oxygen-Glucose Deprivation/Resuscitation: Roles of Extracellular Signal-Regulated Kinase 1/2 and Bid, Bim, Puma.

    PubMed

    Zhang, Limin; Zhao, Xiaochun; Jiang, Xiaojing

    2015-08-01

    Temporal post-conditioning to induce neuroprotection against brain ischemia-reperfusion injury insult is considered to be an effective intervention, but the exact mechanisms of sevoflurane post-conditioning are poorly understood. Extracellular signal-related kinases 1/2 (Erk1/2) play a pivotal role in the cell growth and proliferation. The essential axis of activator Bid, Bim, Puma (BH3s) and BAX, BAK in activating the mitochondrial death program might offer common ground for cell death signal. We hypothesized that, sevoflurane post-conditioning might inhibit the expression of Bid, Bim and Puma and is activated by phosphor-Erk1/2 to reduce neuronal death. To test this hypothesis, we exposed primary cultured cortical neurons to oxygen-glucose deprivation for 1 h and resuscitation for 24 h (OGD/R). The assays of MTT, propidium iodide uptake, JC-1 fluorescence and western blot demonstrated that OGD/R exposure reduced cell viability, increased cell death, decreased mitochondrial membrane potential and the expressions of Bid, Bim, and Puma. Inhibition of Erk1/2 phosphorylation could partially attenuate 2 % of sevoflurane post-conditioning mediated increase in neuronal viability and mitochondrial membrane potential, and also a decrease in cell death and expression of Bid, Bim and Puma after OGD/R treatment. The results demonstrated that, the protection of sevoflurane post-conditioning markedly reducing death of cortical neurons exposed to OGD/R could be correlated with down-regulation of Bid, Bim and Puma expression mediated by phosphorylation/activation of Erk1/2.

  6. 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

  7. Extracellular proteins from Lactobacillus plantarum BMCM12 prevent adhesion of enteropathogens to mucin.

    PubMed

    Sánchez, Borja; Urdaci, María C

    2012-06-01

    The aim of this study was to study the interference of the extracellular proteins produced by Lactobacillus plantarum BMCM12 with the adhesion of some well-known gut pathogens. The extracellular proteins secreted by L. plantarum BMCM12 in MRS broth were precipitated, resolved by SDS-PAGE, and identified by tandem mass spectrometry. Discordances between the observed and the theoretical molecular masses of several proteins suggested the presence of protein glycosylation, corroborated with specific glycoprotein staining after protein de-glycosylation using trifluoromethanesulfonic acid. Experiments of exclusion, competition, or prevention of the pathogen adhesion to mucin were performed using BMCM12 extracellular proteins, using Escherichia coli LMG2092 and Salmonella enterica subsp. enterica LMG15860. Extracellular proteins from BMCM12 reduced significantly the adhesion of the pathogens when they were added prior to adhesion assays. These proteins play thus important roles in preventing pathogen adhesion to the mucin layer.

  8. High glucose stimulates adipogenic and inhibits osteogenic differentiation in MG-63 cells through cAMP/protein kinase A/extracellular signal-regulated kinase pathway.

    PubMed

    Wang, Weiwei; Zhang, Xiaolin; Zheng, Jiaqiang; Yang, Jianhong

    2010-05-01

    Patients with diabetes tend to have an increased incidence of osteoporosis that may be related to hyperglycemia. In this study, we investigated the effects of high glucose on differentiation of human osteoblastic MG-63 cells and involved intracellular signal transduction pathways. Here, we showed that high glucose suppressed the cell growth, mineralization, and expression of osteogenic markers including Runx2, collagen I, osteocalcin, osteonectin, but inversely promoted expression of adipogenic markers including PPARgamma, aP2, resistin, and adipsin. Moreover, high glucose significantly increased the intracellular cAMP level in a time-dependent manner and induced ERK1/2 activation. Meanwhile, supplementation of H89, a specific inhibitor of PKA, and PD98059, a specific inhibitor of MAPK/ERK kinase, reversed the cell growth inhibition, the down-regulation of osteogenic markers and the up-regulation of adipogenic markers as well as the activation of ERK under high glucose. These results indicate that high glucose can increase adipogenic and inhibit osteogenic differentiation by activating cAMP/PKA/ERK pathway in MG-63 cells, thereby providing further insight into the molecular mechanism of diabetic osteoporosis.

  9. Acrylonitrile-induced extracellular signal-regulated kinase (ERK) activation via protein kinase C (PKC) in SK-N-SH neuroblastoma cells.

    PubMed

    Chantara, Wantika; Watcharasit, Piyajit; Thiantanawat, Apinya; Satayavivad, Jutamaad

    2006-01-01

    Acrylonitrile (ACN) is classified by IARC as a probable carcinogen. Chronic exposure to ACN increases the incidence of tumors in various organs of test animals, including the brain and lung. ERK1/2 activation plays crucial roles in cell proliferation and is involved in many steps of tumor progression. Therefore, this study examined whether ACN altered the activation state of ERK1/2 in human neuroblastoma SK-N-SH cells. Treatment of these cells with ACN greatly increased phosphorylation of ERK1/2 in dose- and time-dependent manners. This effect was inhibited by PD 98059 and U 0126, specific inhibitors of MEK, indicating that MEK, an upstream activator of ERK1/2, was directly involved in ACN-induced ERK1/2 activation. Furthermore, the activation of ERK1/2 by ACN was attenuated by inhibition of PKC with GF 109203X, rottlerin and prolonged incubation with PMA (phorbol 12-myristate 13-acetate). This demonstrated the participation of PKC in the ACN-stimulated activation of ERK1/2. Taken together, our results indicate that ACN-induced ERK1/2 activation involves PKC through a MEK-dependent pathway.

  10. Extracellular proteins secreted by probiotic bacteria as mediators of effects that promote mucosa-bacteria interactions.

    PubMed

    Sánchez, Borja; Urdaci, María C; Margolles, Abelardo

    2010-11-01

    During the last few years, a substantial body of scientific evidence has accumulated suggesting that certain surface-associated and extracellular components produced by probiotic bacteria could be responsible for some of their mechanisms of action. These bacterial components would be able to directly interact with the host mucosal cells; they include exopolysaccharides, bacteriocins, lipoteichoic acids and surface-associated and extracellular proteins. Extracellular proteins include proteins that are actively transported to the bacterial surroundings through the cytoplasmic membrane, as well as those that are simply shed from the bacterial surface. Compared to the other bacterial components, the interactive ability of extracellular proteins/peptides has been less extensively studied. In this review, current findings supporting an interaction between extracellular proteins/peptides produced by probiotic bacteria (strains of the genera Bifidobacterium, Lactobacillus and Escherichia) and host mucosal cells are discussed. Research needs and future trends are also considered.

  11. The potential for targeting extracellular LOX proteins in human malignancy.

    PubMed

    Mayorca-Guiliani, Alejandro; Erler, Janine T

    2013-11-25

    The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network.

  12. The potential for targeting extracellular LOX proteins in human malignancy

    PubMed Central

    Mayorca-Guiliani, Alejandro; Erler, Janine T

    2013-01-01

    The extracellular matrix (ECM) is the physical scaffold where cells are organized into tissues and organs. The ECM may be modified during cancer to allow and promote proliferation, invasion, and metastasis. The family of lysyl oxidase (LOX) enzymes cross-links collagens and elastin and, therefore, is a central player in ECM deposition and maturation. Extensive research has revealed how the LOX proteins participate in every stage of cancer progression, and two family members, LOX and LOX-like 2, have been linked to metastasis, the final stage of cancer responsible for over 90% of cancer patient deaths. However, LOX biosynthesis results in by-product with antiproliferative properties in certain cancers, and LOX enzymes may have different effects depending on the molecular network in which they are active. Therefore, the design of therapies targeting the LOX family needs to be guided by the molecular makeup of the individual disease and will probably require other agents to act on both the LOX enzymes and their associated network. PMID:24348049

  13. Extracellular matrix protein CCN1 limits oncolytic efficacy in glioma.

    PubMed

    Haseley, Amy; Boone, Sean; Wojton, Jeffrey; Yu, Lianbo; Yoo, Ji Young; Yu, Jianhua; Kurozumi, Kazuhiko; Glorioso, Joseph C; Caligiuri, Michael A; Kaur, Balveen

    2012-03-15

    Oncolytic viral therapy has been explored widely as an option for glioma treatment but its effectiveness has remained limited. Cysteine rich 61 (CCN1) is an extracellular matrix (ECM) protein elevated in cancer cells that modulates their adhesion and migration by binding cell surface receptors. In this study, we examined a hypothesized role for CCN1 in limiting the efficacy of oncolytic viral therapy for glioma, based on evidence of CCN1 induction that occurs in this setting. Strikingly, we found that exogenous CCN1 in glioma ECM orchestrated a cellular antiviral response that reduced viral replication and limited cytolytic efficacy. Gene expression profiling and real-time PCR analysis revealed a significant induction of type-I interferon responsive genes in response to CCN1 exposure. This induction was accompanied by activation of the Jak/Stat signaling pathway, consistent with induction of an innate antiviral cellular response. Both effects were mediated by the binding of CCN1 to the cell surface integrin α6β1, activating its signaling and leading to rapid secretion of interferon-α, which was essential for the innate antiviral effect. Together, our findings reveal how an integrin signaling pathway mediates activation of a type-I antiviral interferon response that can limit the efficacy of oncolytic viral therapy. Furthermore, they suggest therapeutic interventions to inhibit CCN1-integrin α6 interactions to sensitize gliomas to viral oncolysis.

  14. Comprehensive analysis of the extracellular proteins from Xanthomonas campestris pv. campestris B100.

    PubMed

    Watt, Steven Alexander; Wilke, Andreas; Patschkowski, Thomas; Niehaus, Karsten

    2005-01-01

    The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.

  15. Structural Basis for the Interaction of a Human Small Heat Shock Protein with the 14-3-3 Universal Signaling Regulator.

    PubMed

    Sluchanko, Nikolai N; Beelen, Steven; Kulikova, Alexandra A; Weeks, Stephen D; Antson, Alfred A; Gusev, Nikolai B; Strelkov, Sergei V

    2017-02-07

    By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein, HSPB6, within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering, and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants.

  16. Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator

    PubMed Central

    Sluchanko, Nikolai N.; Beelen, Steven; Kulikova, Alexandra A.; Weeks, Stephen D.; Antson, Alfred A.; Gusev, Nikolai B.; Strelkov, Sergei V.

    2017-01-01

    Summary By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein HSPB6 within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first-ever atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants. PMID:28089448

  17. The progesterone-induced enhancement of object recognition memory consolidation involves activation of the extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) pathways in the dorsal hippocampus.

    PubMed

    Orr, Patrick T; Rubin, Amanda J; Fan, Lu; Kent, Brianne A; Frick, Karyn M

    2012-04-01

    Although much recent work has elucidated the biochemical mechanisms underlying the modulation of memory by 17β-estradiol, little is known about the signaling events through which progesterone (P) regulates memory. We recently demonstrated that immediate post-training infusion of P into the dorsal hippocampus enhances object recognition memory consolidation in young ovariectomized female mice (Orr et al., 2009). The goal of the present study was to identify the biochemical alterations that might underlie this mnemonic enhancement. We hypothesized that the P-induced enhancement of object recognition would be dependent on activation of the ERK and mTOR pathways. In young ovariectomized mice, we found that bilateral dorsal hippocampal infusion of P significantly increased levels of phospho-p42 ERK and the mTOR substrate S6K in the dorsal hippocampus 5 min after infusion. Phospho-p42 ERK levels were downregulated 15 min after infusion and returned to baseline 30 min after infusion, suggesting a biphasic effect of P on ERK activation. Dorsal hippocampal ERK and mTOR activation were necessary for P to facilitate memory consolidation, as suggested by the fact that inhibitors of both pathways infused into the dorsal hippocampus immediately after training blocked the P-induced enhancement of object recognition. Collectively, these data provide the first demonstration that the ability of P to enhance memory consolidation depends on the rapid activation of cell signaling and protein synthesis pathways in the dorsal hippocampus.

  18. Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis.

    PubMed

    Trigo, Gabriela; Ferreira, Paula; Ribeiro, Niza; Dinis, Márcia; Andrade, Elva Bonifácio; Melo-Cristino, José; Ramirez, Mário; Tavares, Delfina

    2008-11-01

    Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.

  19. 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

  20. AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

    PubMed

    Cho, Kwang-Jin; Casteel, Darren E; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A; Kim, Choel; Capon, Robert J; Lacey, Ernest; Cunha, Shane R; Gorfe, Alemayehu A; Hancock, John F

    2016-12-15

    K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

  1. DNA methyltransferase 3a and mitogen-activated protein kinase signaling regulate the expression of fibroblast growth factor-inducible 14 (Fn14) during denervation-induced skeletal muscle atrophy.

    PubMed

    Tajrishi, Marjan M; Shin, Jonghyun; Hetman, Michal; Kumar, Ashok

    2014-07-18

    The TWEAK-fibroblast growth factor-inducible 14 (Fn14) system is a critical regulator of denervation-induced skeletal muscle atrophy. Although the expression of Fn14 is a rate-limiting step in muscle atrophy on denervation, mechanisms regulating gene expression of Fn14 remain unknown. Methylation of CpG sites within promoter region is an important epigenetic mechanism for gene silencing. Our study demonstrates that Fn14 promoter contains a CpG island close to transcription start site. Fn14 promoter also contains multiple consensus DNA sequence for transcription factors activator protein 1 (AP1) and specificity protein 1 (SP1). Denervation diminishes overall genomic DNA methylation and causes hypomethylation at specific CpG sites in Fn14 promoter leading to the increased gene expression of Fn14 in skeletal muscle. Abundance of DNA methyltransferase 3a (Dnmt3a) and its interaction with Fn14 promoter are repressed in denervated skeletal muscle of mice. Overexpression of Dnmt3a inhibits the gene expression of Fn14 and attenuates skeletal muscle atrophy upon denervation. Denervation also causes the activation of ERK1/2, JNK1/2, and ERK5 MAPKs and AP1 and SP1, which stimulate the expression of Fn14 in skeletal muscle. Collectively, our study provides novel evidence that Dnmt3a and MAPK signaling regulate the levels of Fn14 in skeletal muscle on denervation.

  2. Altered protein levels in the isolated extracellular matrix of failing human hearts with dilated cardiomyopathy.

    PubMed

    DeAguero, Joshua L; McKown, Elizabeth N; Zhang, Liwen; Keirsey, Jeremy; Fischer, Edgar G; Samedi, Von G; Canan, Benjamin D; Kilic, Ahmet; Janssen, Paul M L; Delfín, Dawn A

    Dilated cardiomyopathy (DCM) is associated with extensive pathological cardiac remodeling and involves numerous changes in the protein expression profile of the extracellular matrix of the heart. We obtained seven human, end-stage, failing hearts with DCM (DCM-failing) and nine human, nonfailing donor hearts and compared their extracellular matrix protein profiles. We first showed that the DCM-failing hearts had indeed undergone extensive remodeling of the left ventricle myocardium relative to nonfailing hearts. We then isolated the extracellular matrix from a subset of these hearts and performed a proteomic analysis on the isolated matrices. We found that the levels of 26 structural proteins were altered in the DCM-failing isolated cardiac extracellular matrix compared to nonfailing isolated cardiac extracellular matrix. Overall, most of the extracellular matrix proteins showed reduced levels in the DCM-failing hearts, while all of the contractile proteins showed increased levels. There was a mixture of increased and decreased levels of cytoskeletal and nuclear transport proteins. Using immunoprobing, we verified that collagen IV (α2 and α6 isoforms), zyxin, and myomesin protein levels were reduced in the DCM-failing hearts. We expect that these data will add to the understanding of the pathology associated with heart failure with DCM.

  3. A generalised module for the selective extracellular accumulation of recombinant proteins

    PubMed Central

    2012-01-01

    Background It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. Results Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. Conclusions The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications. PMID:22640772

  4. Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation

    PubMed Central

    Cvjetkovic, Aleksander; Jang, Su Chul; Konečná, Barbora; Höög, Johanna L.; Sihlbom, Carina; Lässer, Cecilia; Lötvall, Jan

    2016-01-01

    Extracellular vesicles (EVs) are important mediators of intercellular communication that change the recipient cell by shuttling lipids, RNA, or protein cargo between cells. Here, we investigate the topology of the protein cargo found in EVs, as this topology can fundamentally influence the biological effects of EVs. A multiple proteomics approach, combining proteinase treatment and biotin tagging, shows that many proteins of cytosolic origin are localized on the surface of EVs. A detailed analysis of the EV proteome at the peptide level revealed that a number of EV membrane proteins are present in a topologically reversed orientation compared to what is annotated. Two examples of such proteins, SCAMP3 and STX4, were confirmed to have a reversed topology. This reversed typology was determined using flow cytometry and fluorescent microscopy with antibodies directed toward their cytoplasmic epitopes. These results describe a novel workflow to define the EV proteome and the orientation of each protein, including membrane protein topology. These data are fundamentally important to understanding the EV proteome and required to fully explain EV biogenesis as well as biological function in recipient cells. PMID:27821849

  5. Targeting the extracellular matrix: matricellular proteins regulate cell-extracellular matrix communication within distinct niches of the intervertebral disc.

    PubMed

    Bedore, Jake; Leask, Andrew; Séguin, Cheryle A

    2014-07-01

    The so-called "matricellular" proteins have recently emerged as important regulators of cell-extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell-ECM interactions, ECM synthesis and disc tissue homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration.

  6. Activation of the adenosine A3 receptor in RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-alpha release by reducing calcium-dependent activation of nuclear factor-kappaB and extracellular signal-regulated kinase 1/2.

    PubMed

    Martin, Lynn; Pingle, Sandeep C; Hallam, Daniel M; Rybak, Leonard P; Ramkumar, Vickram

    2006-01-01

    Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-alpha and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-alpha release in human UB939 macrophages by activating an adenosine A(3) receptor (A(3)AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A(3)AR-dependent inhibition of TNF-alpha release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 mug/ml) increased TNF-alpha release, which was reduced in a dose-dependent manner by adenosine analogs N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A(3)AR blockade. The increase in TNF-alpha release was preceded by an increase in intracellular Ca(2+) levels. Inhibition of intracellular Ca(2+) release by IB-MECA, a selective agonist of the A(3)AR, or with BAPTA-AM, an intracellular Ca(2+) chelator, reduced LPS-stimulated TNF-alpha release. Activation of the A(3)AR or inhibition of intracellular Ca(2+) release also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A(3)AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-alpha and NO via the A(3)AR, involves suppression of intracellular Ca(2+)signaling, leading to suppression of NF-kappaB and ERK1/2 pathways.

  7. The forced swimming-induced behavioural immobility response involves histone H3 phospho-acetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/mitogen- and stress-activated kinase signalling pathway.

    PubMed

    Chandramohan, Yalini; Droste, Susanne K; Arthur, J Simon C; Reul, Johannes M H M

    2008-05-01

    The hippocampus is involved in learning and memory. Previously, we have shown that the acquisition of the behavioural immobility response after a forced swim experience is associated with chromatin modifications and transcriptional induction in dentate gyrus granule neurons. Given that both N-methyl-D-aspartate (NMDA) receptors and the extracellular signal-regulated kinases (ERK) 1/2 signalling pathway are involved in neuroplasticity processes underlying learning and memory, we investigated in rats and mice whether these signalling pathways regulate chromatin modifications and transcriptional events participating in the acquisition of the immobility response. We found that: (i) forced swimming evoked a transient increase in the number of phospho-acetylated histone H3-positive [P(Ser10)-Ac(Lys14)-H3(+)] neurons specifically in the middle and superficial aspects of the dentate gyrus granule cell layer; (ii) antagonism of NMDA receptors and inhibition of ERK1/2 signalling blocked forced swimming-induced histone H3 phospho-acetylation and the acquisition of the behavioural immobility response; (iii) double knockout (DKO) of the histone H3 kinase mitogen- and stress-activated kinases (MSK) 1/2 in mice completely abolished the forced swimming-induced increases in histone H3 phospho-acetylation and c-Fos induction in dentate granule neurons and the behavioural immobility response; (iv) blocking mineralocorticoid receptors, known not to be involved in behavioural immobility in the forced swim test, did not affect forced swimming-evoked histone H3 phospho-acetylation in dentate neurons; and (v) the pharmacological manipulations and gene deletions did not affect behaviour in the initial forced swim test. We conclude that the forced swimming-induced behavioural immobility response requires histone H3 phospho-acetylation and c-Fos induction in distinct dentate granule neurons through recruitment of the NMDA/ERK/MSK 1/2 pathway.

  8. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    PubMed

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  9. Exercise-induced extracellular 72 kDa heat shock protein (Hsp72) stimulates neutrophil phagocytic and fungicidal capacities via TLR-2.

    PubMed

    Giraldo, Esther; Martin-Cordero, Leticia; Garcia, Juan Jose; Gehrmann, Mathias; Gerhmann, Mathias; Multhoff, Gabriele; Ortega, Eduardo

    2010-01-01

    This study evaluated the role of toll like receptor 2 (TLR-2) in the interaction of 72 kDa extracellular heat shock protein (Hsp72, a stress-inducible protein) with neutrophils and the participation on TLR-2 in the stimulation of neutrophil phagocytic and fungicidal capacities by post-exercise physiological concentrations of Hsp72. Human peripheral blood neutrophils were incubated with fluorescein isothiocyanate-conjugated Hsp72, and were analyzed by immunofluorescence microscopy and flow cytometry. Both methods revealed an interaction of Hsp72 with neutrophils. In addition, when neutrophils were pre-incubated with an anti-TLR-2 antibody this interaction was clearly decreased. Post-exercise circulating concentration of Hsp72 (8.6 ng/ml) stimulated the phagocytic and fungicidal capacities of neutrophils and this effect could be also blocked using an antibody against TLR-2. Phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated kinase (ERK) and the nuclear transcription factor kappa beta (NF-kappabeta) were found to be involved in the signaling process, confirming the participation of TLR-2 in the stimulation of neutrophil function by Hsp72. In conclusion, TLR-2 is involved at least in part, in the stimulation of neutrophil phagocytic and fungicidal capacities induced by post-exercise physiological concentrations of Hsp72.

  10. Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis.

    PubMed

    Gilbert, Charlie; Howarth, Mark; Harwood, Colin R; Ellis, Tom

    2017-03-07

    The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics, and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis. Using this approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling, and we show that cellular consortia can be engineered to self-assemble functional protein-protein conjugates with tunable composition. This novel genetically encoded modular system provides a flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.

  11. 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.

  12. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  13. A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins

    PubMed Central

    Tejeda-Dominguez, Farid; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia

    2017-01-01

    ABSTRACT The type 3 secretion system (T3SS) is essential for bacterial virulence through delivering effector proteins directly into the host cytosol. Here, we identified an alternative delivery mechanism of virulence factors mediated by the T3SS, which consists of the association of extracellularly secreted proteins from bacteria with the T3SS to gain access to the host cytosol. Both EspC, a protein secreted as an enteropathogenic Escherichia coli (EPEC) autotransporter, and YopH, a protein detected on the surface of Yersinia, require a functional T3SS for host cell internalization; here we provide biophysical and molecular evidence to support the concept of the EspC translocation mechanism, which requires (i) an interaction between EspA and an EspC middle segment, (ii) an EspC translocation motif (21 residues that are shared with the YopH translocation motif), (iii) increases in the association and dissociation rates of EspC mediated by EspA interacting with EspD, and (iv) an interaction of EspC with the EspD/EspB translocon pore. Interestingly, this novel mechanism does not exclude the injection model (i.e., EspF) operating through the T3SS conduit; therefore, T3SS can be functioning as an internal conduit or as an external railway, which can be used to reach the translocator pore, and this mechanism appears to be conserved among different T3SS-dependent pathogens. PMID:28351918

  14. Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein.

    PubMed

    Sommer, Benjamin; Friehs, Karl; Flaschel, Erwin; Reck, Michael; Stahl, Frank; Scheper, Thomas

    2009-03-25

    Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to alpha-1,4-glucans.

  15. Importance of extracellular proteins in maintaining structural integrity of aerobic granules.

    PubMed

    Xiong, Yanghui; Liu, Yu

    2013-12-01

    Aerobic granules developed through self-immobilization of microorganisms are compact and structured microbial consortia embedded in a matrix of extracellular polymeric substances (EPS). This study investigated the contribution of extracellular proteins (PN) to maintaining the structural integrity of aerobic granule. It was found that hydrolysis of PN induced by Proteinase K led to significant disintegration of aerobic granules, whereas a substantial reduction of extracellular polysaccharides (PS) was also observed. It was proposed that hydrolysis of extracellular proteins present in the EPS matrix of aerobic granules led to collapse of the EPS matrix, and subsequent disintegration of aerobic granule. These suggested that extracellular proteins would be essential for maintaining structural stability of EPS matrix of aerobic granules. In addition, it was revealed that production of signaling molecules, such as autoinducer-2 (AI-2) and N-acyl homoserine lactones (AHLs) was also inhibited probably due to hydrolysis of quorum sensing receptor proteins by Proteinase K. This in turn provided an additional explanation for the observed Proteinase K-triggered dispersal of aerobic granules.

  16. Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

    PubMed Central

    Langó, Tamás; Róna, Gergely; Hunyadi-Gulyás, Éva; Turiák, Lilla; Varga, Julia; Dobson, László; Várady, György; Drahos, László; Vértessy, Beáta G.; Medzihradszky, Katalin F.; Szakács, Gergely; Tusnády, Gábor E.

    2017-01-01

    Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins. PMID:28211907

  17. Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins.

    PubMed

    Langó, Tamás; Róna, Gergely; Hunyadi-Gulyás, Éva; Turiák, Lilla; Varga, Julia; Dobson, László; Várady, György; Drahos, László; Vértessy, Beáta G; Medzihradszky, Katalin F; Szakács, Gergely; Tusnády, Gábor E

    2017-02-13

    Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins.

  18. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    PubMed

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  19. Extracellular heat shock protein 70 has novel functional effects on sea urchin eggs and coelomocytes.

    PubMed

    Browne, Carole L; Swan, Justin B; Rankin, Ellen E; Calvert, Hayes; Griffiths, Shylise; Tytell, Michael

    2007-04-01

    Numerous reports document that the 70 kDa heat shock proteins are not only intracellular proteins but are also present in blood and other extracellular compartments. How they affect cell function from the extracellular space remains unclear. Using two well-characterized cell types from the sea urchin, we show that extracellular mixtures of the constitutive and inducible forms of the 70 kDa heat shock proteins (Hsc70 and Hsp70, respectively) have dramatic effects on initiation of cell division in fertilized eggs and on the clotting reaction of hypotonically stressed coelomocytes. In suspensions of fertilized eggs to which Hsc70 or a 2:3 mixture of Hsc and Hsp70 was added, progression to the first mitotic division was accelerated. Evidence is provided that the extracellular Hsc70 passes into the egg cells in an unconventional manner, being distributed through the cytoplasm, and that it may alter the intracellular signaling cascade initiated by sperm penetration. In coelomocytes that were stimulated by hypotonic shock to mimic injury, the spreading reaction of the clotting response was significantly inhibited when either Hsp70 or Hsc70 was in the medium. These results suggest that the presence of Hsc and/or Hsp70 in the extracellular fluid may promote mitosis of dividing cells and suppress the reactivity of immune system cells.

  20. The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

    PubMed

    Eisenbeis, Janina; Peisker, Henrik; Backes, Christian S; Bur, Stephanie; Hölters, Sebastian; Thewes, Nicolas; Greiner, Markus; Junker, Christian; Schwarz, Eva C; Hoth, Markus; Junker, Kerstin; Preissner, Klaus T; Jacobs, Karin; Herrmann, Mathias; Bischoff, Markus

    2017-02-01

    Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the

  1. Role of oligomerization domains in thrombospondins and other extracellular matrix proteins.

    PubMed

    Engel, Jürgen

    2004-06-01

    Coiled coils, collagen triple helices and globular oligomerization domains mediate the subunit assembly of many proteins in vertebrates and invertebrates. Oligomerization offers functional advantages including multivalency, increased binding strength and the combined function of different domains. These features are seen in natural proteins and may be introduced by protein engineering. The special focus of this review is on oligomerization domain of extracellular matrix proteins. For thrombospondins, initial interesting results on the functional role of oligomerization have been published. Other features remain to be explored. For example, it is not clear why thrombospondin-1 and thrombospondin-2 are trimers whereas thrombospondins-3 to -5 are pentamers. To stimulate this type of research, this review makes a survey of oligomerization domains and their functional role in extracellular matrix proteins.

  2. Insider trading: Extracellular matrix proteins and their non-canonical intracellular roles.

    PubMed

    Hellewell, Andrew L; Adams, Josephine C

    2016-01-01

    In metazoans, the extracellular matrix (ECM) provides a dynamic, heterogeneous microenvironment that has important supportive and instructive roles. Although the primary site of action of ECM proteins is extracellular, evidence is emerging for non-canonical intracellular roles. Examples include osteopontin, thrombospondins, IGF-binding protein 3 and biglycan, and relate to roles in transcription, cell-stress responses, autophagy and cancer. These findings pose conceptual problems on how proteins signalled for secretion can be routed to the cytosol or nucleus, or can function in environments with diverse redox, pH and ionic conditions. We review evidence for intracellular locations and functions of ECM proteins, and current knowledge of the mechanisms by which they may enter intracellular compartments. We evaluate the experimental methods that are appropriate to obtain rigorous evidence for intracellular localisation and function. Better insight into this under-researched topic is needed to decipher the complete spectrum of physiological and pathological roles of ECM proteins.

  3. 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.

  4. The mouse secretome: functional classification of the proteins secreted into the extracellular environment.

    PubMed

    Grimmond, Sean M; Miranda, Kevin C; Yuan, Zheng; Davis, Melissa J; Hume, David A; Yagi, Ken; Tominaga, Naoko; Bono, Hidemasa; Hayashizaki, Yoshihide; Okazaki, Yasushi; Teasdale, Rohan D

    2003-06-01

    We have developed a computational strategy to identify the set of soluble proteins secreted into the extracellular environment of a cell. Within the protein sequences predominantly derived from the RIKEN representative transcript and protein set, we identified 2033 unique soluble proteins that are potentially secreted from the cell. These proteins contain a signal peptide required for entry into the secretory pathway and lack any transmembrane domains or intracellular localization signals. This class of proteins, which we have termed the mouse secretome, included >500 novel proteins and 92 proteins <100 amino acids in length. Functional analysis of the secretome included identification of human orthologs, functional units based on InterPro and SCOP Superfamily predictions, and expression of the proteins within the RIKEN READ microarray database. To highlight the utility of this information, we discuss the CUB domain-containing protein family.

  5. The Mouse Secretome: Functional Classification of the Proteins Secreted Into the Extracellular Environment

    PubMed Central

    Grimmond, Sean M.; Miranda, Kevin C.; Yuan, Zheng; Davis, Melissa J.; Hume, David A.; Yagi, Ken; Tominaga, Naoko; Bono, Hidemasa; Hayashizaki, Yoshihide; Okazaki, Yasushi; Teasdale, Rohan D.

    2003-01-01

    We have developed a computational strategy to identify the set of soluble proteins secreted into the extracellular environment of a cell. Within the protein sequences predominantly derived from the RIKEN representative transcript and protein set, we identified 2033 unique soluble proteins that are potentially secreted from the cell. These proteins contain a signal peptide required for entry into the secretory pathway and lack any transmembrane domains or intracellular localization signals. This class of proteins, which we have termed the mouse secretome, included >500 novel proteins and 92 proteins <100 amino acids in length. Functional analysis of the secretome included identification of human orthologs, functional units based on InterPro and SCOP Superfamily predictions, and expression of the proteins within the RIKEN READ microarray database. To highlight the utility of this information, we discuss the CUB domain-containing protein family. PMID:12819133

  6. 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...

  7. 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.

  8. Responses in the expression of extracellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone.

    PubMed

    Visutthi, Monton; Srimanote, Potjanee; Voravuthikunchai, Supayang Piyawan

    2011-12-01

    Rhodomyrtone from a medicinal plant species, Rhodomyrtus tomentosa, is a challenged effective agent against Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). The present study was undertaken to provide insight into MRSA extracellular protein expression following rhodomyrtone treatment. Secreteomic approach was performed on a representative clinical MRSA isolate exposing to subinhibitory concentration rhodomyrtone (0.174 μg/ml). The identified extracellular proteins of a response of MRSA to rhodomyrtone treated condition were both suppressed and overexpressed. Staphylococcal antigenic proteins, immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA) involved in cell wall hydrolysis were downregulated after the treatment. The results suggested that rhodomyrtone may interfere with WalK/WalR (YycG/YycF) system. Other enzymes such as lipase precursor and another lipase, glycerophosphoryl diester phosphodiesterase, were absent. In contrast, cytoplasmic proteins such as SpoVG and glycerol phosphate lipoteichoic acid synthase, and ribosomal proteins were found in the treated sample. Appearance of several cytoplasmic proteins in the treated culture supernatant revealed that the bacterial cell wall biosynthesis was disturbed. This finding provides a proteomic mapping of extracellular proteins after rhodomytone treatment. Extensive investigation is required for this natural compound as it has a great potency as an alternative anti-MRSA drug.

  9. An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks.

    PubMed

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

    2013-07-03

    Extracellular domains of cell surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein "interactome" data sets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on a set of 202 proteins composed of the Drosophila melanogaster immunoglobulin superfamily (IgSF), fibronectin type III (FnIII), and leucine-rich repeat (LRR) families, which are known to be important in neuronal and developmental functions. Out of 20,503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We "deorphanized" the 20 member subfamily of defective-in-proboscis-response 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 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.

  10. 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

  11. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  12. A protein disulfide isomerase gene fusion expression system that increases the extracellular productivity of Bacillus brevis.

    PubMed

    Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H

    2000-02-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

  13. EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora.

    PubMed

    Torto, Trudy A; Li, Shuang; Styer, Allison; Huitema, Edgar; Testa, Antonino; Gow, Neil A R; van West, Pieter; Kamoun, Sophien

    2003-07-01

    Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including

  14. Extracellular regulation of type IIa receptor protein tyrosine phosphatases: mechanistic insights from structural analyses

    PubMed Central

    Coles, Charlotte H.; Jones, E. Yvonne; Aricescu, A. Radu

    2016-01-01

    The receptor protein tyrosine phosphatases (RPTPs) exhibit a wide repertoire of cellular signalling functions. In particular, type IIa RPTP family members have recently been highlighted as hubs for extracellular interactions in neurons, regulating neuronal extension and guidance, as well as synaptic organisation. In this review, we will discuss the recent progress of structural biology investigations into the architecture of type IIa RPTP ectodomains and their interactions with extracellular ligands. Structural insights, in combination with biophysical and cellular studies, allow us to begin to piece together molecular mechanisms for the transduction and integration of type IIa RPTP signals and to propose hypotheses for future experimental validation. PMID:25234613

  15. Human cecum content modulates production of extracellular proteins by food and probiotic bacteria.

    PubMed

    Sánchez, Borja; Ruiz, Lorena; Suárez, Adolfo; de Los Reyes-Gavilán, Clara G; Margolles, Abelardo

    2011-11-01

    Lactic acid bacteria (LAB) are responsible for different types of food fermentations that provide humans with many different classes of fermented products. During the 20th century, some LAB strains as well as several members of the genus Bifidobacterium started to be extensively used in human nutrition as probiotics because of their health-promoting effects. Nowadays, the subset of extracellular proteins is being investigated as potential mediators of the process known as bacteria-host molecular crosstalk. Inclusion of human cecum extracts in laboratory culture medium modified the production of extracellular proteins by food and probiotic microorganisms. By proteomic and genetic means, the specific overproduction of two proteins was revealed to occur at transcriptional level. This work sheds light on the potential molecular effectors that food bacteria could use for interacting with the human gut and revealed that they may be produced under very specific environmental conditions.

  16. Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins.

    PubMed

    Bayer-Santos, Ethel; Aguilar-Bonavides, Clemente; Rodrigues, Silas Pessini; Cordero, Esteban Maurício; Marques, Alexandre Ferreira; Varela-Ramirez, Armando; Choi, Hyungwon; Yoshida, Nobuko; da Silveira, José Franco; Almeida, Igor C

    2013-02-01

    Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells.

  17. Role of Extracellular Matrix Proteins and Their Receptors in the Development of the Vertebrate Neuromuscular Junction

    PubMed Central

    Singhal, Neha; Martin, Paul T.

    2012-01-01

    The vertebrate neuromuscular junction 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 neuromuscular junction, 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 has 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 extracellular matrix proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability and transmission. PMID:21766463

  18. 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

  19. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.

    PubMed

    Kamphorst, Jurre J; Nofal, Michel; Commisso, Cosimo; Hackett, Sean R; Lu, Wenyun; Grabocka, Elda; Vander Heiden, Matthew G; Miller, George; Drebin, Jeffrey A; Bar-Sagi, Dafna; Thompson, Craig B; Rabinowitz, Joshua D

    2015-02-01

    Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate, and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiologic albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC.

  20. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein

    PubMed Central

    Kamphorst, Jurre J.; Nofal, Michel; Commisso, Cosimo; Hackett, Sean R.; Lu, Wenyun; Grabocka, Elda; Vander Heiden, Matthew G.; Miller, George; Drebin, Jeffrey A.; Bar-Sagi, Dafna; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-01-01

    Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiological albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids, and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC. PMID:25644265

  1. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.

    PubMed

    Urban, Constantin F; Ermert, David; Schmid, Monika; Abu-Abed, Ulrike; Goosmann, Christian; Nacken, Wolfgang; Brinkmann, Volker; Jungblut, Peter R; Zychlinsky, Arturo

    2009-10-01

    Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

  2. Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus replication and reactivates virus from latency.

    PubMed Central

    Levy, D N; Refaeli, Y; Weiner, D B

    1995-01-01

    The vpr gene product of human immunodeficiency virus (HIV) and simian immunodeficiency virus is a virion-associated regulatory protein that has been shown using vpr mutant viruses to increase virus replication, particularly in monocytes/macrophages. We have previously shown that vpr can directly inhibit cell proliferation and induce cell differentiation, events linked to the control of HIV replication, and also that the replication of a vpr mutant but not that of wild-type HIV type 1 (HIV-1) was compatible with cellular proliferation (D. N. Levy, L. S. Fernandes, W. V. Williams, and D. B. Weiner, Cell 72:541-550, 1993). Here we show that purified recombinant Vpr protein, in concentrations of < 100 pg/ml to 100 ng/ml, increases wild-type HIV-1 replication in newly infected transformed cell lines via a long-lasting increase in cellular permissiveness to HIV replication. The activity of extracellular Vpr protein could be completely inhibited by anti-Vpr antibodies. Extracellular Vpr also induced efficient HIV-1 replication in newly infected resting peripheral blood mononuclear cells. Extracellular Vpr transcomplemented a vpr mutant virus which was deficient in replication in promonocytic cells, restoring full replication competence. In addition, extracellular Vpr reactivated HIV-1 expression in five latently infected cell lines of T-cell, B-cell, and promonocytic origin which normally express very low levels of HIV RNA and protein, indicating an activation of translational or pretranslational events in the virus life cycle. Together, these results describe a novel pathway governing HIV replication and a potential target for the development of anti-HIV therapeutics. PMID:7815499

  3. Moderate cyclic tensile strain alters the assembly of cartilage extracellular matrix proteins in vitro.

    PubMed

    Bleuel, Judith; Zaucke, Frank; Brüggemann, Gert-Peter; Heilig, Juliane; Wolter, Marie-Louise; Hamann, Nina; Firner, Sara; Niehoff, Anja

    2015-06-01

    Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

  4. Extracellular Proteins in Pea Root Tip and Border Cell Exudates1[OA

    PubMed Central

    Wen, Fushi; VanEtten, Hans D.; Tsaprailis, George; Hawes, Martha C.

    2007-01-01

    Newly generated plant tissue is inherently sensitive to infection. Yet, when pea (Pisum sativum) roots are inoculated with the pea pathogen, Nectria haematococca, most newly generated root tips remain uninfected even though most roots develop lesions just behind the tip in the region of elongation. The resistance mechanism is unknown but is correlated spatially with the presence of border cells on the cap periphery. Previously, an array of >100 extracellular proteins was found to be released while border cell separation proceeds. Here we report that protein secretion from pea root caps is induced in correlation with border cell separation. When this root cap secretome was proteolytically degraded during inoculation of pea roots with N. haematococca, the percentage of infected root tips increased from 4% ± 3% to 100%. In control experiments, protease treatment of conidia or roots had no effect on growth and development of the fungus or the plant. A complex of >100 extracellular proteins was confirmed, by multidimensional protein identification technology, to comprise the root cap secretome. In addition to defense-related and signaling enzymes known to be present in the plant apoplast were ribosomal proteins, 14-3-3 proteins, and others typically associated with intracellular localization but recently shown to be extracellular components of microbial biofilms. We conclude that the root cap, long known to release a high molecular weight polysaccharide mucilage and thousands of living cells into the incipient rhizosphere, also secretes a complex mixture of proteins that appear to function in protection of the root tip from infection. PMID:17142479

  5. 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

  6. 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.

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

    DOE PAGES

    Chung, Daehwan; Young, Jenna; Bomble, Yannick J.; ...

    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

  8. 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

  9. Expression and purification of the extracellular domain of human myelin protein zero.

    PubMed

    Bond, J P; Saavedra, R A; Kirschner, D A

    2001-12-01

    Myelin protein zero (P0), an adhesion protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. Protein zero is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and the intracellular domains. Mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies of varying severity. Comparisons between the human and rat isoforms, whose three-dimensional structure has been determined by X-ray crystallography, suggest that these disease-associated genetic alterations lead to structural changes in the protein that alter P0-P0 interactions and hence affect myelin functionality. Knowing the crystal structures of native and altered human P0 isoforms could help to elucidate the structural changes in myelin membrane packing that underlie the altered functionality. Alterations of P0 extracellular domain (P0-ED) are of additional interest as previous X-ray diffraction studies on myelin membrane packing suggest that P0-ED molecules can assume distinct adhesive arrangements. Here, we describe an improved method to express and purify human P0-ED (hP0-ED) suitable for crystallographic analysis. A fusion protein consisting of maltose binding protein fused to hP0-ED was secreted to the periplasm of Escherichia coli to allow an appropriate folding pathway. The fusion protein was extracted via osmotic shock and purified by affinity chromatography. Factor Xa was used to cleave the fusion protein, and a combination of affinity and ion-exchange chromatography was used to further purify hP0-ED. We document several significant improvements to previous protocols, including bacterial growth to approximately 15 OD using orbital shakers and the use of diafiltration, which result in yields of approximately 150 mg highly pure protein per liter of medium.

  10. Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins.

    PubMed

    Chernov, Vladislav M; Mouzykantov, Alexey A; Baranova, Natalia B; Medvedeva, Elena S; Grygorieva, Tatiana Yu; Trushin, Maxim V; Vishnyakov, Innokentii E; Sabantsev, Anton V; Borchsenius, Sergei N; Chernova, Olga A

    2014-10-14

    Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen-host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects.

  11. Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells

    PubMed Central

    Gautier, Violette; Cayrol, Corinne; Farache, Dorian; Roga, Stéphane; Monsarrat, Bernard; Burlet-Schiltz, Odile; Gonzalez de Peredo, Anne; Girard, Jean-Philippe

    2016-01-01

    IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells. PMID:27694941

  12. Yeast Two-Hybrid Screening for Proteins that Interact with the Extracellular Domain of Amyloid Precursor Protein.

    PubMed

    Yu, You; Li, Yinan; Zhang, Yan

    2016-04-01

    Alzheimer's disease (AD) is a neurodegenerative disorder in which amyloid β plaques are a pathological characteristic. Little is known about the physiological functions of amyloid β precursor protein (APP). Based on its structure as a type I transmembrane protein, it has been proposed that APP might be a receptor, but so far, no ligand has been reported. In the present study, 9 proteins binding to the extracellular domain of APP were identified using a yeast two-hybrid system. After confirming the interactions in the mammalian system, mutated PLP1, members of the FLRT protein family, and KCTD16 were shown to interact with APP. These proteins have been reported to be involved in Pelizaeus-Merzbacher disease (PMD) and axon guidance. Therefore, our results shed light on the mechanisms of physiological function of APP in AD, PMD, and axon guidance.

  13. 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

  14. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    SciTech Connect

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K.

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  15. Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

    PubMed

    M, Saritha; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

    2016-11-01

    The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-β-glucanase, β-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers.

  16. 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

  17. Abnormal recruitment of extracellular matrix proteins by excess Notch3 ECD: a new pathomechanism in CADASIL.

    PubMed

    Monet-Leprêtre, Marie; Haddad, Iman; Baron-Menguy, Céline; Fouillot-Panchal, Maï; Riani, Meriem; Domenga-Denier, Valérie; Dussaule, Claire; Cognat, Emmanuel; Vinh, Joelle; Joutel, Anne

    2013-06-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross

  18. Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

    PubMed

    Castaneda, Julie T; Harui, Airi; Kiertscher, Sylvia M; Roth, Jeffrey D; Roth, Michael D

    2013-03-01

    mRNA encoding for the CB(2) cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB(2) and GFP, we developed a flow cytometry assay for characterizing CB(2) protein expression. A monoclonal antibody directed against human CB(2) selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB(2) corresponded closely with the level of GFP expression. When exposed to Δ(9)-tetrahydrocannabinol, CB(2)-expressing cells internalized cell surface CB(2) receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB(2) protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB(2) expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB(2) mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB(2) protein but that its distribution is predominantly intracellular with only B cells expressing CB(2) protein at the extracellular membrane. The differential role of intracellular and extracellular CB(2) receptors in mediating ligand signaling and immune function remains to be determined.

  19. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

    PubMed Central

    Torii, K U; Mitsukawa, N; Oosumi, T; Matsuura, Y; Yokoyama, R; Whittier, R F; Komeda, Y

    1996-01-01

    Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID:8624444

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

    PubMed

    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.

  1. 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.

  2. Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

    PubMed

    Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

    2004-10-01

    Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

  3. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    NASA Astrophysics Data System (ADS)

    Nakanishi, Jun; Nakayama, Hidekazu; Yamaguchi, Kazuo; Garcia, Andres J.; Horiike, Yasuhiro

    2011-08-01

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG7). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7-10) to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  4. Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

    PubMed

    Healy, Laura D; Puy, Cristina; Fernández, José A; Mitrugno, Annachiara; Keshari, Ravi S; Taku, Nyiawung A; Chu, Tiffany T; Xu, Xiao; Gruber, András; Lupu, Florea; Griffin, John H; McCarty, Owen J T

    2017-04-13

    Activated protein C (APC) is a multi-functional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a non-human primate model of E. coli-induced sepsis, pre-treatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

  5. 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.

  6. Extracellular proteins of Clostridium chauvoei are protective in a mouse model.

    PubMed

    Mattar, María A; Cortiñas, Teresa I; Stefanini, Ana M

    2007-06-01

    The anaerobic bacillus Clostridium chauvoei is the causative agent of blackleg, a lethal disease that has an important impact on the sheep and cattle industry worldwide. Immunity to C. chauvoei is considered to be mainly anticellular, and for this reason there is scarce information about the immunogenicity of extracellular proteins. In this work variations in protein profiles, immune response by ELISA and protective capacity of culture supernatants of three C. chauvoei strains, collected at different growth phases, are reported. Sera raised against extracellular antigens also recognised cellular antigens of the same molecular masses. Partially purified cell-free supernatants and those concentrated 10 times by ultrafiltration (C-CFS), obtained at the early stationary phase of growth, induced a strong immunoprotective response, even at low doses, that was more marked for C. chauvoei strain ATCC 10092 (p < or = 0.05). With C-CFS formulations, a clear relationship was observed between IgG titres, protective capacity and concentration of the antigen doses, indicating a specific immune response.

  7. 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.

  8. The extracellular matrix protein WARP is a novel component of a distinct subset of basement membranes.

    PubMed

    Allen, Justin M; Brachvogel, Bent; Farlie, Peter G; Fitzgerald, Jamie; Bateman, John F

    2008-05-01

    WARP is a recently described member of the von Willebrand factor A domain superfamily of extracellular matrix proteins, and is encoded by the Vwa1 gene. We have previously shown that WARP is a multimeric component of the chondrocyte pericellular matrix in articular cartilage and intervertebral disc, where it interacts with the basement membrane heparan sulfate proteoglycan perlecan. However, the tissue-specific expression of WARP in non-cartilaginous tissues and its localization in the extracellular matrix of other perlecan-containing tissues have not been analyzed in detail. To visualize WARP-expressing cells, we generated a reporter gene knock-in mouse by targeted replacement of the Vwa1 gene with beta-galactosidase. Analysis of reporter gene expression and WARP protein localization by immunostaining demonstrates that WARP is a component of a limited number of distinct basement membranes. WARP is expressed in the vasculature of neural tissues and in basement membrane structures of the peripheral nervous system. Furthermore, WARP is also expressed in the apical ectodermal ridge of developing limb buds, and in skeletal and cardiac muscle. These findings are the first evidence for WARP expression in non-cartilaginous tissues, and the identification of WARP as a component of a limited range of specialized basement membranes provides further evidence for the heterogeneous composition of basement membranes between different tissues.

  9. Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins.

    PubMed

    Rutter, Brian D; Innes, Roger W

    2017-01-01

    Exosomes are extracellular vesicles (EVs) that play a central role in intercellular signaling in mammals by transporting proteins and small RNAs. Plants are also known to produce EVs, particularly in response to pathogen infection. The contents of plant EVs have not been analyzed, however, and their function is unknown. Here, we describe a method for purifying EVs from the apoplastic fluids of Arabidopsis (Arabidopsis thaliana) leaves. Proteomic analyses of these EVs revealed that they are highly enriched in proteins involved in biotic and abiotic stress responses. Consistent with this finding, EV secretion was enhanced in plants infected with Pseudomonas syringae and in response to treatment with salicylic acid. These findings suggest that EVs may represent an important component of plant immune responses.

  10. Extracellular Vesicles Isolated from the Leaf Apoplast Carry Stress-Response Proteins1[OPEN

    PubMed Central

    2017-01-01

    Exosomes are extracellular vesicles (EVs) that play a central role in intercellular signaling in mammals by transporting proteins and small RNAs. Plants are also known to produce EVs, particularly in response to pathogen infection. The contents of plant EVs have not been analyzed, however, and their function is unknown. Here, we describe a method for purifying EVs from the apoplastic fluids of Arabidopsis (Arabidopsis thaliana) leaves. Proteomic analyses of these EVs revealed that they are highly enriched in proteins involved in biotic and abiotic stress responses. Consistent with this finding, EV secretion was enhanced in plants infected with Pseudomonas syringae and in response to treatment with salicylic acid. These findings suggest that EVs may represent an important component of plant immune responses. PMID:27837092

  11. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    PubMed

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process.

  12. 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

  13. Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps

    PubMed Central

    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

  14. A bioinformatics analysis of alternative exon usage in human genes coding for extracellular matrix proteins.

    PubMed

    Sakabe, Noboru Jo; Vibranovski, Maria Dulcetti; de Souza, Sandro José

    2004-12-30

    Alternative splicing increases protein diversity through the generation of different mRNA molecules from the same gene. Although alternative splicing seems to be a widespread phenomenon in the human transcriptome, it is possible that different subgroups of genes present different patterns, related to their biological roles. Analysis of a subgroup may enhance common features of its members that would otherwise disappear amidst a heterogeneous population. Extracellular matrix (ECM) proteins are a good set for such analyses since they are structurally and functionally related. This family of proteins is involved in a large variety of functions, probably achieved by the combinatorial use of protein domains through exon shuffling events. To determine if ECM genes have a different pattern of alternative splicing, we compared clusters of expressed sequences of ECM to all other genes regarding features related to the most frequent type of alternative splicing, alternative exon usage (AEU), such as: the number of alternative exon-intron structures per cluster, the number of AEU events per exon-intron structure, the number of exons per event, among others. Although we did not find many differences between the two sets, we observed a higher frequency of AEU events involving entire protein domains in the ECM set, a feature that could be associated with their multi-domain nature. As other subgroups or even the ECM set in different tissues could present distinct patterns of AEU, it may be premature to conclude that alternative splicing is homogeneous among groups of related genes.

  15. Extracellular Stiffness Modulates the Expression of Functional Proteins and Growth Factors in Endothelial Cells.

    PubMed

    Santos, Lívia; Fuhrmann, Gregor; Juenet, Maya; Amdursky, Nadav; Horejs, Christine-Maria; Campagnolo, Paola; Stevens, Molly M

    2015-08-13

    Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.

  16. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus.

    PubMed

    Shah, Firoz; Rineau, Francois; Canbäck, Björn; Johansson, Tomas; Tunlid, Anders

    2013-11-01

    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.

  17. Lactobacillus plantarum extracellular chitin-binding protein and its role in the interaction between chitin, Caco-2 cells, and mucin.

    PubMed

    Sánchez, Borja; González-Tejedo, Carmen; Ruas-Madiedo, Patricia; Urdaci, María C; Margolles, Abelardo

    2011-02-01

    In the present work, we describe the adhesion capabilities of a recombinant Lactococcus lactis strain producing an extracellular protein from Lactobacillus plantarum. Our results show that this protein may offer the bacterium a mechanism to bind to N-acetylglucosamine-containing polymers, such as human mucins, present in different environments.

  18. 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

  19. Quantitative proteomics analysis integrated with microarray data reveals that extracellular matrix proteins, catenins, and p53 binding protein 1 are important for chemotherapy response in ovarian cancers.

    PubMed

    Pan, Sheng; Cheng, Lihua; White, James T; Lu, Wei; Utleg, Angelita G; Yan, Xiaowei; Urban, Nicole D; Drescher, Charles W; Hood, Leroy; Lin, Biaoyang

    2009-08-01

    Chemotherapy with carboplatin and paclitaxel is the standard treatment for ovarian cancer patients. Although most patients initially respond to this treatment, few are cured. Resistance to chemotherapy is the major cause of treatment failure. We applied a quantitative proteomic approach based on ICAT/MS/MS technology to analyze tissues harvested at primary debulking surgery before the initiation of combination chemotherapy in order to identify potential naive or intrinsic chemotherapy response proteins in ovarian cancers. We identified 44 proteins that are overexpressed, and 34 proteins that are underexpressed in the chemosensitive tissue compared to the chemoresistant tissue. The overexpressed proteins identified in the chemoresistant tissue include 10 proteins (25.6%) belonging to the extracellular matrix (ECM), including decorin, versican, basigin (CD147), fibulin-1, extracellular matrix protein 1, biglycan, fibronectin 1, dermatopontin, alpha-cardiac actin (smooth muscle actin), and an EGF-containing fibulin-like extracellular matrix protein 1. Interesting proteins identified as overexpressed in the chemosensitive tissue include gamma-catenin (junction plakoglobin) and delta-catenin, tumor suppressor p53-binding protein 1 (53BP1), insulin-like growth factor-binding protein 2 (IGFBP2), proliferating cell nuclear antigen (PCNA), annexin A11, and 53 kDa selenium binding protein 1. Integrative analysis with expression profiling data of eight chemoresistant tissues and 13 chemosensitive tissues revealed that 16 proteins showed consistent changes at both the protein and the RNA levels. These include P53 binding protein 1, catenin delta 1 and plakoglobin, EGF-containing fibulin-like extracellular matrix protein 1 and voltage-dependent anion-selective channel protein 1. Our results suggest that chemotherapy response may be determined by multiple and complex system properties involving extracellular-matrix, cell adhesion and junction proteins.

  20. The covalent linkage of protein to carbohydrate in the extracellular protein-polysaccharide from the red alga Porphyridium cruentum.

    PubMed Central

    Heaney-Kieras, J; Rodén, L; Chapman, D J

    1977-01-01

    The extracellular anionic polysaccharide isolated from cultures of a unicellular red alga, Porphyridium cruentum, contains a small amount of protein after extensive purification. The polysaccharide and protein are recovered in the same fraction after isopycnic CsCl-density-gradient centrifugation in 4M-guanidinium chloride, under conditions designed to separate proteins from polysaccharide. The peptide portion of the protein-polysaccharide is released from the polysaccharide by alkali under conditions for beta-elimination. The released peptide is non-diffusible, but in can be separated from the polysaccharide by precipitation of the polysaccharide as the cetylpyridinium complex. Under conditions for beta-elimination of certain O-glycosidic carbohydrate-protein linkages, selective destruction of serine and threonine occurs. The addition of a reducing agent to the alkali mixture produces a selective increase in alanine and alpha-aminobutyric acid. Addition of a tritiated reducing agent to the alkali mixture produces radioactive alanine and alpha-aminobutyric acid, and xylitol as the only sugar alcohol. Similar results are obtained from glycopeptides isolated from partial acid hydrolysates. A macromolecular structure of the protein-polysaccharide is suggested by a comparison of the intrinsic viscosity of material before and after treatment with alkali and proteolytic enzymes. PMID:889565

  1. Calcite formation in soft coral sclerites is determined by a single reactive extracellular protein.

    PubMed

    Rahman, M Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-09-09

    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.

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. Proteomic Analysis of Extracellular Proteins from Aspergillus oryzae Grown under Submerged and Solid-State Culture Conditions

    PubMed Central

    Oda, Ken; Kakizono, Dararat; Yamada, Osamu; Iefuji, Haruyuki; Akita, Osamu; Iwashita, Kazuhiro

    2006-01-01

    Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as α-amylase (TAA) and β-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture. PMID:16672490

  11. Extracellular matrix proteins as temporary coating for thin-film neural implants

    NASA Astrophysics Data System (ADS)

    Ceyssens, Frederik; Deprez, Marjolijn; Turner, Neill; Kil, Dries; van Kuyck, Kris; Welkenhuysen, Marleen; Nuttin, Bart; Badylak, Stephen; Puers, Robert

    2017-02-01

    Objective. This study investigates the suitability of a thin sheet of extracellular matrix (ECM) proteins as a resorbable coating for temporarily reinforcing fragile or ultra-low stiffness thin-film neural implants to be placed on the brain, i.e. microelectrocorticographic (µECOG) implants. Approach. Thin-film polyimide-based electrode arrays were fabricated using lithographic methods. ECM was harvested from porcine tissue by a decellularization method and coated around the arrays. Mechanical tests and an in vivo experiment on rats were conducted, followed by a histological tissue study combined with a statistical equivalence test (confidence interval approach, 0.05 significance level) to compare the test group with an uncoated control group. Main results. After 3 months, no significant damage was found based on GFAP and NeuN staining of the relevant brain areas. Significance. The study shows that ECM sheets are a suitable temporary coating for thin µECOG neural implants.

  12. Interaction with the adaptor protein Shc prevents aberrant Erk activation in the absence of extracellular stimulus

    PubMed Central

    Suen, Kin Man; Lin, Chi-Chuan; George, Roger; Melo, Fernando A.; Biggs, Eleanor R.; Ahmed, Zamal; Drake, Melanie N.; Arur, Swathi; Arold, Stefan T.; Ladbury, John E.

    2014-01-01

    Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a novel mechanism by which the adaptor protein Shc binds directly to the MAP-kinase Erk, preventing its activation in the absence of extracellular stimulus. The Shc–Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex is formed through unique binding sites on both the Shc PTB domain and N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc—induced through interaction with the phosphorylated receptor—releases Erk allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP-kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered as a tumor suppressor in human cells. PMID:23584453

  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. Endometrial inflammation and abnormal expression of extracellular matrix proteins induced by Mycoplasma bovis in dairy cows.

    PubMed

    Guo, Mengyao; Wang, Guoqing; Lv, Tingting; Song, Xiaojing; Wang, Tiancheng; Xie, Guanghong; Cao, Yongguo; Zhang, Naisheng; Cao, Rongfeng

    2014-03-15

    Mycoplasma bovis infection can cause endometrial inflammation leading to infertility and involuntary culling in dairy cows. Because extracellular matrix (ECM) proteins affect the adherence of mycoplasma to eukaryotic cell surface, they may play a role in the pathogenesis of the bacteria. The objective of the present study was to evaluate the endometrial inflammatory response and ECM protein expression induced by M bovis. Endometrial concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and mRNA and protein expression of collagen IV (CL-IV), fibronectin (FN), and laminin (LN) were evaluated 10, 20, and 30 days after M bovis intrauterine infusion in breed cows 18 days postpartum. The presence of the bacteria in the uterus was detected by nested polymerase chain reaction and denaturing gradient gel electrophoresis. Endometrial TNF-α, IL-1β, and IL-6 concentrations in the treatment group were greater (P < 0.05) than in the positive and negative control groups 20 and 30 days after infusion. Endometrial CL-IV, FN, and LN mRNA and protein expression increased (P < 0.01) 20 days after infusion in all groups. However, the increase was more pronounced in the treatment group and reactive expressions were greater (P < 0.05) than in the positive and negative control groups 10, 20, and 30 days after infusion. In conclusion, M bovis triggered endometrial inflammatory response and increased CL-IV, FN, and LN mRNA and protein expression. The abnormal expression of ECM these proteins may promote the pathogenic effects of M bovis that lead to endometrial tissue damage and infertility.

  15. Characterization and identification of a novel candidate vaccine protein through systematic analysis of extracellular proteins of Erysipelothrix rhusiopathiae.

    PubMed

    Shi, Fang; Ogawa, Yohsuke; Sano, Akiyuki; Harada, Tomoyuki; Hirota, Jiro; Eguchi, Masahiro; Oishi, Eiji; Shimoji, Yoshihiro

    2013-12-01

    Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

  16. Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

    PubMed Central

    Shi, Fang; Ogawa, Yohsuke; Sano, Akiyuki; Harada, Tomoyuki; Hirota, Jiro; Eguchi, Masahiro; Oishi, Eiji

    2013-01-01

    Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen. PMID:24019408

  17. Interactions of promonocytic U937 cells with proteins of the extracellular matrix.

    PubMed Central

    Pucillo, C E; Colombatti, A; Vitale, M; Salzano, S; Rossi, G; Formisano, S

    1993-01-01

    Monocyte interaction with proteins of the extracellular matrix (ECM) is regulated by expression of specific cell-surface receptors. 12-O-tetradecanoyl phorbol-13-acetate (TPA) has been shown to induce the promonocytic cell line U937 to a more differentiated monocyte-like state. In this study we have analysed the attachment of U937 cells to ECM proteins and the effects of treatment with TPA on this process. Non-induced U937 cells attach to fibronectin- and Matrigel-coated surfaces without TPA stimulation, but TPA further increases adherence to these substrates as measured by an enhanced binding and by the lower concentration of proteins needed in the substrate to achieve 50% of maximal cell adhesion. Attachment to type I collagen was seen only with activated U937 cells, whereas no measurable attachment to bovine serum albumin, vitronectin, and type IV collagen was detected. TPA-activated U937 cells showed a two-fold increase in the expression of the RGD-dependent integrin receptors alpha 3 and alpha 5, and a reduction in the expression of alpha 4, another fibronectin-specific receptor, whereas the common beta 1 chain was unchanged. Attachment of U937 cells to fibronectin was primarily mediated by the alpha 3 and alpha 5 integrins, as revealed by the ability of GRGDS peptides to inhibit attachment, whereas the CS-1 peptide, containing the alpha 4 binding site, was largely ineffective in blocking attachment. PMID:8262552

  18. 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.

  19. Mechanical Stimulation of Piezo1 Receptors Depends on Extracellular Matrix Proteins and Directionality of Force.

    PubMed

    Gaub, Benjamin M; Müller, Daniel J

    2017-02-08

    Piezo receptors convert mechanical forces into electrical signals. In mammals, they play important roles in basic physiological functions including proprioception, sensation of touch, and vascular development. However, basic receptor properties like the gating mechanism, the interaction with extracellular matrix (ECM) proteins, and the response to mechanical stimulation, remain poorly understood. Here, we establish an atomic force microscopy (AFM)-based assay to mechanically stimulate Piezo1 receptors in living animal cells, while monitoring receptor activation in real-time using functional calcium imaging. Our experiments show that in the absence of ECM proteins Piezo1 receptors are relatively insensitive to mechanical forces pushing the cellular membrane, whereas they can hardly be activated by mechanically pulling the membrane. Yet, if conjugated with Matrigel, a mix of ECM proteins, the receptors become sensitized. Thereby, forces pulling the cellular membrane activate the receptor much more efficiently compared to pushing forces. Finally, we found that collagen IV, a component of the basal lamina, which forms a cohesive network and mechanical connection between cells, sensitizes Piezo1 receptors to mechanical pulling.

  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. 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

  2. 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.

  3. Binding of Galectin-3, a β-Galactoside-binding Lectin, to MUC1 Protein Enhances Phosphorylation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) and Akt, Promoting Tumor Cell Malignancy.

    PubMed

    Mori, Yugo; Akita, Kaoru; Yashiro, Masakazu; Sawada, Tetsuji; Hirakawa, Kosei; Murata, Takeomi; Nakada, Hiroshi

    2015-10-23

    Both mucin 1 (MUC1) and galectin-3 are known to be overexpressed in various malignant tumors and associated with a poor prognosis. It has been extensively reported that MUC1 is involved in potentiation of growth factor-dependent signal transduction. Because some carbohydrate moieties carried on MUC1 change to preferable ones for binding of galectin-3 in cancer cells, we speculated that MUC1-mediated signaling may occur through direct binding of galectin-3. Immunochemical studies showed that the distribution of galectin-3 coincided with that of MUC1 in various human tumor tissues but not in human nonmalignant tissues, and the level of galectin-3 retained on the surface of various cancer cells paralleled that of MUC1. Treatment of MUC1-expressing cells with galectin-3 induced phosphorylation of ERK1/2 and Akt following enhanced phosphorylation of MUC1 C-terminal domain, consistently promoting tumor cell malignancy. It is also noted that this enhanced phosphorylation occurred independently of EGF receptor-mediated signaling in both EGF receptor- and MUC1-expressing cells, and multivalency of galectin-3 was important for initiation of MUC1-mediated signaling. Expectedly, both silencing of endogenous galectin-3 and treatment with galectin-3 antagonists down-regulated cell proliferation of MUC1-expressing cells. These results suggest that the binding of galectin-3 to MUC1 plays a key role in MUC1-mediated signaling. Thus, constitutive activation of MUC1-mediated signaling in an autocrine/paracrine manner caused by ligation of galectin-3 promotes uncontrolled tumor cell malignancy. This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated signaling pathway.

  4. 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

  5. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins.

    PubMed Central

    Wretlind, B; Pavlovskis, O R

    1984-01-01

    We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme. PMID:6427194

  6. 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.

  7. Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events

    PubMed Central

    1990-01-01

    Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine- aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important

  8. ALK1 heterozygosity increases extracellular matrix protein expression, proliferation and migration in fibroblasts.

    PubMed

    Muñoz-Félix, José M; Perretta-Tejedor, Nuria; Eleno, Nélida; López-Novoa, José M; Martínez-Salgado, Carlos

    2014-06-01

    Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.

  9. Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands

    PubMed Central

    Thompson, Christopher; Hielscher, Abigail

    2017-01-01

    The extracellular matrix (ECM) is abnormal in breast tumors and has been reported to contribute to breast tumor progression. One factor, which may drive ongoing matrix synthesis in breast tumors, is the loss of stromal caveolin-1 (cav-1), a scaffolding protein of caveolae, which has been linked to breast tumor aggressiveness. To determine whether loss of cav-1 results in the abnormal expression of matrix proteins, mammary glands from cav- 1-/- and cav- 1 +/+ mice were investigated for differences in expression of several ECM proteins. In addition, the presence of myofibroblasts, changes in the vessel density, and differences in duct number and size were assessed in the mammary glands of both animal models. Using immunohistochemistry, expression of fibronectin, tenascin-C, collagens and αSMA were significantly increased in the mammary glands of cav-1-/- mice. Second harmonic generation revealed more organized collagen fibers in cav-1 -/- glands and supported immunohistochemical analyses of increased collagen abundance in the glands of cav-1 -/- mice. Analysis of the ductal structure demonstrated a significant increase in the number of proliferating ducts in addition to significant increases in the duct circumference and area in cav-1 -/- glands compared to cav- 1 +/+ glands. Differences in microvessel density weren’t apparent between the animal models. In summary, we found that the loss of cav-1 resulted in increased ECM and α-SMA protein expression in murine mammary glands. Furthermore, we found that an abnormal ductal architecture accompanied the loss of cav-1. These data support a role for cav-1 in maintaining mammary gland structure. PMID:28187162

  10. Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands.

    PubMed

    Thompson, Christopher; Rahim, Sahar; Arnold, Jeremiah; Hielscher, Abigail

    2017-01-01

    The extracellular matrix (ECM) is abnormal in breast tumors and has been reported to contribute to breast tumor progression. One factor, which may drive ongoing matrix synthesis in breast tumors, is the loss of stromal caveolin-1 (cav-1), a scaffolding protein of caveolae, which has been linked to breast tumor aggressiveness. To determine whether loss of cav-1 results in the abnormal expression of matrix proteins, mammary glands from cav- 1-/- and cav- 1 +/+ mice were investigated for differences in expression of several ECM proteins. In addition, the presence of myofibroblasts, changes in the vessel density, and differences in duct number and size were assessed in the mammary glands of both animal models. Using immunohistochemistry, expression of fibronectin, tenascin-C, collagens and αSMA were significantly increased in the mammary glands of cav-1-/- mice. Second harmonic generation revealed more organized collagen fibers in cav-1 -/- glands and supported immunohistochemical analyses of increased collagen abundance in the glands of cav-1 -/- mice. Analysis of the ductal structure demonstrated a significant increase in the number of proliferating ducts in addition to significant increases in the duct circumference and area in cav-1 -/- glands compared to cav- 1 +/+ glands. Differences in microvessel density weren't apparent between the animal models. In summary, we found that the loss of cav-1 resulted in increased ECM and α-SMA protein expression in murine mammary glands. Furthermore, we found that an abnormal ductal architecture accompanied the loss of cav-1. These data support a role for cav-1 in maintaining mammary gland structure.

  11. The extracellular loop of IRT1 ZIP protein--the chosen one for zinc?

    PubMed

    Potocki, Slawomir; Valensin, Daniela; Camponeschi, Francesca; Kozlowski, Henryk

    2013-10-01

    Zinc complexes with the extracellular loop of IRT1 (iron-regulated transporter 1), a ZIP (ZRT/IRT - Related Protein) family protein from Arabidopsis thaliana, have been studied. This unstructured fragment is responsible for metal selectivity and is located between the II and III transmembrane domains of IRT1. Zinc complexes with the Ac-(95)MHVLPDSFEMLSSICLEENPWHK(117)-NH2 peptide (IRT1), revealed surprisingly high thermodynamic stability. Additionally, an N-terminal fragment of human/mouse ZIP 13 zinc transporter (MPGCPCPGCGMACPR-NH2, later called ZIP13+C), has been chosen for the thermodynamic stability comparison studies. The relative ZIP13+C stability has been shown using several Zn(2+) complexes with artificially arranged multi-cysteine sequences. An interesting coordination mode has been proposed for the IRT1-Zn(2+) complex, in which imidazoles from two histidines (His-96 and His-116), a cysteine thiolate (Cys-109) and one of a glutamic acid carboxyl group are involved. All data were collected using potentiometric, NMR and mass spectrometric methods.

  12. Influence of Extracellular Matrix Proteins and Substratum Topography on Corneal Epithelial Cell Alignment and Migration

    PubMed Central

    Raghunathan, VijayKrishna; McKee, Clayton; Cheung, Wai; Naik, Rachel; Nealey, Paul F.; Russell, Paul

    2013-01-01

    The basement membrane (BM) of the corneal epithelium presents biophysical cues in the form of topography and compliance that can impact the phenotype and behaviors of cells and their nuclei through modulation of cytoskeletal dynamics. In addition, it is also well known that the intrinsic biochemical attributes of BMs can modulate cell behaviors. In this study, the influence of the combination of exogenous coating of extracellular matrix proteins (ECM) (fibronectin-collagen [FNC]) with substratum topography was investigated on cytoskeletal architecture as well as alignment and migration of immortalized corneal epithelial cells. In the absence of FNC coating, a significantly greater percentage of cells aligned parallel with the long axis of the underlying anisotropically ordered topographic features; however, their ability to migrate was impaired. Additionally, changes in the surface area, elongation, and orientation of cytoskeletal elements were differentially influenced by the presence or absence of FNC. These results suggest that the effects of topographic cues on cells are modulated by the presence of surface-associated ECM proteins. These findings have relevance to experiments using cell cultureware with biomimetic biophysical attributes as well as the integration of biophysical cues in tissue-engineering strategies and the development of improved prosthetics. PMID:23488816

  13. Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR).

    PubMed

    Gallart-Palau, Xavier; Serra, Aida; Wong, Andrew See Weng; Sandin, Sara; Lai, Mitchell K P; Chen, Christopher P; Kon, Oi Lian; Sze, Siu Kwan

    2015-09-30

    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.

  14. Influence of extracellular matrix proteins and substratum topography on corneal epithelial cell alignment and migration.

    PubMed

    Raghunathan, Vijaykrishna; McKee, Clayton; Cheung, Wai; Naik, Rachel; Nealey, Paul F; Russell, Paul; Murphy, Christopher J

    2013-08-01

    The basement membrane (BM) of the corneal epithelium presents biophysical cues in the form of topography and compliance that can impact the phenotype and behaviors of cells and their nuclei through modulation of cytoskeletal dynamics. In addition, it is also well known that the intrinsic biochemical attributes of BMs can modulate cell behaviors. In this study, the influence of the combination of exogenous coating of extracellular matrix proteins (ECM) (fibronectin-collagen [FNC]) with substratum topography was investigated on cytoskeletal architecture as well as alignment and migration of immortalized corneal epithelial cells. In the absence of FNC coating, a significantly greater percentage of cells aligned parallel with the long axis of the underlying anisotropically ordered topographic features; however, their ability to migrate was impaired. Additionally, changes in the surface area, elongation, and orientation of cytoskeletal elements were differentially influenced by the presence or absence of FNC. These results suggest that the effects of topographic cues on cells are modulated by the presence of surface-associated ECM proteins. These findings have relevance to experiments using cell cultureware with biomimetic biophysical attributes as well as the integration of biophysical cues in tissue-engineering strategies and the development of improved prosthetics.

  15. Alterations in junctional proteins, inflammatory mediators and extracellular matrix molecules in eosinophilic esophagitis.

    PubMed

    Abdulnour-Nakhoul, Solange M; Al-Tawil, Youhanna; Gyftopoulos, Alex A; Brown, Karen L; Hansen, Molly; Butcher, Kathy F; Eidelwein, Alexandra P; Noel, Robert A; Rabon, Edd; Posta, Allison; Nakhoul, Nazih L

    2013-08-01

    Eosinophilic esophagitis (EoE), an inflammatory atopic disease of the esophagus, causes massive eosinophil infiltration, basal cell hyperplasia, and sub-epithelial fibrosis. To elucidate cellular and molecular factors involved in esophageal tissue damage and remodeling, we examined pinch biopsies from EoE and normal pediatric patients. An inflammation gene array confirmed that eotaxin-3, its receptor CCR3 and interleukins IL-13 and IL-5 were upregulated. An extracellular matrix (ECM) gene array revealed upregulation of CD44 & CD54, and of ECM proteases (ADAMTS1 & MMP14). A cytokine antibody array showed a marked decrease in IL-1α and IL-1 receptor antagonist and an increase in eotaxin-2 and epidermal growth factor. Western analysis indicated reduced expression of intercellular junction proteins, E-cadherin and claudin-1 and increased expression of occludin and vimentin. We have identified a number of novel genes and proteins whose expression is altered in EoE. These findings provide new insights into the molecular mechanisms of the disease.

  16. 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.

  17. Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid).

    PubMed

    Cai, Ning; Gong, Yingxue; Chian, Kerm Sin; Chan, Vincent; Liao, Kin

    2008-03-01

    Effective attachment of esophageal cells on biomaterials is one important requirement in designing engineered esophagus substitute for esophageal cancer treatment. In this study, poly(lactic acid) (PLA) was subjected to surface modification by coupling extracellular matrix (ECM) proteins on its surface to promote cell adhesion. Two typical ECM proteins, collagen type I (COL) and fibronectin (FN), were immobilized on the PLA surface with the aid of glutaraldehyde as a cross linker between aminolyzed PLA and ECM proteins. By using confocal reflectance interference contrast microscopy (C-RICM) integrating with phase contrast microscopy, the long-term adhesion dynamics of porcine esophageal fibroblasts (PEFs) on four types of surfaces (unmodified PLA, PLA-COOH, PLA-COL and PLA-FN) was investigated during 24 h of culture. It is demonstrated by C-RICM results that PEFs form strong adhesion contact on all four types of surfaces at different stages of cell seeding. Among the four surfaces, PEFs on the PLA-FN surface reach the maximum adhesion energy (9.5 x 10(-7) J m(-2)) in the shortest time (20 min) during the initial stage of cell seeding. After adhesion energy reaches the maximum value, PEFs maintain their highly deformed geometries till they reached a steady state after 20 h of culture. F-actin immunostaining results show that the evolvement of spatial organization of F-actin is tightly correlated with the formation of adhesion contact and cell spreading. Furthermore, the cell attachment ratio of PEFs on PLA in 2 h is only 26% compared with 88% on PLA-FN, 73% on PLA-COL and 36% on PLA-COOH. All the results demonstrate the effect of surface functionalization on the biophysical responses of PEFs in cell adhesion. Fibronectin-immobilized PLA demonstrates promising potential for application as an engineered esophagus substitute.

  18. 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.

  19. Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

    PubMed

    Kunadt, Marcel; Eckermann, Katrin; Stuendl, Anne; Gong, Jing; Russo, Belisa; Strauss, Katrin; Rai, Surya; Kügler, Sebastian; Falomir Lockhart, Lisandro; Schwalbe, Martin; Krumova, Petranka; Oliveira, Luis M A; Bähr, Mathias; Möbius, Wiebke; Levin, Johannes; Giese, Armin; Kruse, Niels; Mollenhauer, Brit; Geiss-Friedlander, Ruth; Ludolph, Albert C; Freischmidt, Axel; Feiler, Marisa S; Danzer, Karin M; Zweckstetter, Markus; Jovin, Thomas M; Simons, Mikael; Weishaupt, Jochen H; Schneider, Anja

    2015-05-01

    Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.

  20. A Standard Operating Procedure (SOP) for the preparation of intra- and extracellular proteins of Clostridium acetobutylicum for proteome analysis.

    PubMed

    Schwarz, Katrin; Fiedler, Tomas; Fischer, Ralf-Jörg; Bahl, Hubert

    2007-02-01

    We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.

  1. 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

  2. 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

  3. Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

    PubMed

    Moore, Nadia H; Costa, Lucio G; Shaffer, Scott A; Goodlett, David R; Guizzetti, Marina

    2009-02-01

    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. One hundred and thirty three 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 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.

  4. Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering*

    PubMed Central

    Hill, Ryan C.; Calle, Elizabeth A.; Dzieciatkowska, Monika; Niklason, Laura E.; Hansen, Kirk C.

    2015-01-01

    The use of extracellular matrix (ECM)1 scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs. PMID:25660013

  5. Trafficking protein particle complex 6A delta (TRAPPC6AΔ) is an extracellular plaque-forming protein in the brain

    PubMed Central

    Chang, Jean-Yun; Lee, Ming-Hui; Lin, Sing-Ru; Yang, Li-Yi; Sun, H. Sunny; Sze, Chun-I; Hong, Qunying; Lin, Yee-Shin; Chou, Ying-Tsen; Hsu, Li-Jin; Jan, Ming-Shiou; Gong, Cheng-Xin; Chang, Nan-Shan

    2015-01-01

    Tumor suppressor WWOX is involved in the progression of cancer and neurodegeneration. Here, we examined whether protein aggregation occurs in the brain of nondemented, middle-aged humans and whether this is associated with WWOX downregulation. We isolated an N-terminal internal deletion isoform, TPC6AΔ, derived from alternative splicing of the TRAPPC6A (TPC6A) gene transcript. TPC6AΔ proteins are present as aggregates or plaques in the extracellular matrix of the brain such as in the cortex. Filter retardation assays revealed that aggregate formation of TPC6AΔ occurs preceding Aβ generation in the hippocampi of middle-aged postmortem normal humans. In a Wwox gene knockout mouse model, we showed the plaques of pT181-Tau and TPC6AΔ in the cortex and hippocampus in 3-week-old mice, suggesting a role of WWOX in limiting TPC6AΔ aggregation. To support this hypothesis, in vitro analysis revealed that TGF-β1 induces dissociation of the ectopic complex of TPC6AΔ and WWOX in cells, and then TPC6AΔ undergoes Ser35 phosphorylation-dependent polymerization and induces caspase 3 activation and Aβ production. Similarly, knockdown of WWOX by siRNA resulted in dramatic aggregation of TPC6AΔ. Together, when WWOX is downregulated, TPC6AΔ is phosphorylated at Ser35 and becomes aggregated for causing caspase activation that leads to Tau aggregation and Aβ formation. PMID:25650666

  6. Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins.

    PubMed

    Benito, María J; Rodríguez, Mar; Núñez, Félix; Asensio, Miguel A; Bermúdez, María E; Córdoba, Juan J

    2002-07-01

    An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.

  7. Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

    PubMed Central

    Benito, María J.; Rodríguez, Mar; Núñez, Félix; Asensio, Miguel A.; Bermúdez, María E.; Córdoba, Juan J.

    2002-01-01

    An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products. PMID:12089038

  8. Mechanical and failure properties of extracellular matrix sheets as a function of structural protein composition.

    PubMed

    Black, Lauren D; Allen, Philip G; Morris, Shirley M; Stone, Phillip J; Suki, Béla

    2008-03-01

    The goal of this study was to determine how alterations in protein composition of the extracellular matrix (ECM) affect its functional properties. To achieve this, we investigated the changes in the mechanical and failure properties of ECM sheets generated by neonatal rat aortic smooth muscle cells engineered to contain varying amounts of collagen and elastin. Samples underwent static and dynamic mechanical measurements before, during, and after 30 min of elastase digestion followed by a failure test. Microscopic imaging was used to measure thickness at two strain levels to estimate the true stress and moduli in the ECM sheets. We found that adding collagen to the ECM increased the stiffness. However, further increasing collagen content altered matrix organization with a subsequent decrease in the failure strain. We also introduced collagen-related percolation in a nonlinear elastic network model to interpret these results. Additionally, linear elastic moduli correlated with failure stress which may allow the in vivo estimation of the stress tolerance of ECM. We conclude that, in engineered replacement tissues, there is a tradeoff between improved mechanical properties and decreased extensibility, which can impact their effectiveness and how well they match the mechanical properties of native tissue.

  9. 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.

  10. 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.

  11. Elastin, a novel extracellular matrix protein adhering to mycobacterial antigen 85 complex.

    PubMed

    Kuo, Chih-Jung; Ptak, Christopher P; Hsieh, Ching-Lin; Akey, Bruce L; Chang, Yung-Fu

    2013-02-08

    The antigen 85 complex (Ag85) consists of three predominantly secreted proteins (Ag85A, Ag85B, and Ag85C), which play a key role in the mycobacterial pathogenesis and also possess enzymatic mycolyltransferase activity involved in cell wall synthesis. Ag85 is not only considered to be a virulence factor because its expression is essential for intracellular survival within macrophages, but also because it contributes to adherence, invasion, and dissemination of mycobacteria in host cells. In this study, we report that the extracellular matrix components, elastin and its precursor (tropoelastin) derived from human aorta, lung, and skin, serve as binding partners of Ag85 from Mycobacterium tuberculosis. The binding affinity of M. tuberculosis Ag85 to human tropoelastin was characterized (K(D) = 0.13 ± 0.006 μm), and a novel Ag85-binding motif, AAAKAA(K/Q)(Y/F), on multiple tropoelastin modules was identified. In addition, the negatively charged Glu-258 of Ag85 was demonstrated to participate in an electrostatic interaction with human tropoelastin. Moreover, binding of Ag85 on elastin siRNA-transfected Caco-2 cells was significantly reduced (34.3%), implying that elastin acts as an important ligand contributing to mycobacterial invasion.

  12. Extracellular matrix protein Reelin promotes myeloma progression by facilitating tumor cell proliferation and glycolysis

    PubMed Central

    Qin, Xiaodan; Lin, Liang; Cao, Li; Zhang, Xinwei; Song, Xiao; Hao, Jie; Zhang, Yan; Wei, Risheng; Huang, Xiaojun; Lu, Jin; Ge, Qing

    2017-01-01

    Reelin is an extracellular matrix protein that is crucial for neuron migration, adhesion, and positioning. We examined the expression of Reelin in a large cohort of multiple myeloma patients recorded in Gene Expression Omnibus (GEO) database and used over-expression and siRNA knockdown of Reelin to investigate the role of Reelin in myeloma cell growth. We find that Reelin expression is negatively associated with myeloma prognosis. Reelin promotes myeloma cell proliferation in vitro as well as in vivo. The Warburg effect, evidenced by increased glucose uptake and lactate production, is also enhanced in Reelin-expressing cells. The activation of FAK/Syk/Akt/mTOR and STAT3 pathways contributes to Reelin-induced cancer cell growth and metabolic reprogramming. Our findings further reveal that activated Akt and STAT3 pathways induce the upregulation of HIF1α and its downstream targets (LDHA and PDK1), leading to increased glycolysis in myeloma cells. Together, our results demonstrate the critical contributions of Reelin to myeloma growth and metabolism. It presents an opportunity for myeloma therapeutic intervention by inhibiting Reelin and its signaling pathways. PMID:28345605

  13. Adolescent binge ethanol treatment alters adult brain regional volumes, cortical extracellular matrix protein and behavioral flexibility.

    PubMed

    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 that 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.

  14. 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

  15. Extracellular self-assembly of virus-like particles from secreted recombinant polyoma virus major coat protein.

    PubMed

    Ng, J; Koechlin, O; Ramalho, M; Raman, D; Krauzewicz, N

    2007-12-01

    Mouse polyoma virus major coat protein (VP1) expressed from a recombinant baculovirus is efficiently transported to infected cell nuclei and assembles into protein nanospheres morphologically similar to natural capsids. The nanospheres readily combine with plasmid DNA to form a hybrid gene therapy agent known as virus-like particles (VLPs). To facilitate large-scale production of VLPs free from cellular contaminants, the use of stable Drosophila cell lines expressing either wild-type protein, or VP1 tagged with a secretion signal for targeting to the extracellular medium, was investigated. Both wild-type and tagged VP1 expressed at 2-4 mg VP1/litre of culture. As expected, the wild-type protein self-assembled into VLPs. The tagged VP1 was efficiently secreted to the extracellular medium but was also glycosylated, unlike wild-type VP1. Despite this fact, a small fraction of the recombinant secreted protein assembled into VLP-like structures that had altered disulphide bonding, but were still biologically active. These results demonstrate the considerable tolerance in the nanosphere assembly to structural (i.e. aberrant glycosylation) and environmental (i.e. extracellular medium vs. nuclear milieu) changes. Thus, with modifications to improve nanosphere assembly, the secretion method could be adapted to large-scale preparation of VLPs, providing significant advantages over current methods of production of the vector.

  16. Changes of defense proteins in the extracellular proteome of grapevine (Vitis vinifera cv. Gamay) cell cultures in response to elicitors.

    PubMed

    Martinez-Esteso, M J; Sellés-Marchart, S; Vera-Urbina, J C; Pedreño, M A; Bru-Martinez, R

    2009-12-01

    In plant cells, elicitors induce defense responses that resemble those triggered by pathogen attack, such as the synthesis of phytoalexins and pathogen-related proteins which accumulate in the extracellular space. In the search for the particular proteins involved in defense responses, we investigated the changes in the extracellular proteome of a grapevine (Vitis vinifera cv. Gamay) cell suspension in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA). Twenty-five of the 39 spots differentially expressed in 2-D gels were identified and found to be encoded by 10 different genes: three secretory peroxidases, chitinase-III, beta-1,3-glucanase, thaumatin-like, SGNH plant lipase-like, NtPR27-like, xyloglucan endotransglycosylase and subtilisin-like protease. Most of them belong to the pathogenesis-related type proteins. A new class III secretory basic peroxidase and chitinase III were strongly induced in cultures treated with MBCD alone or combined with MeJA, while cultures treated with MeJA alone displayed a general repression of most of the extracellular proteins. Some of the proteins induced in grapevine cell cultures by MBCD are induced in other species by activators of systemic acquired resistance (SAR), a form of plant immunity. Collectively, the results suggest that treatment with MBCD resembles the effect of SAR induction agents in cell cultures.

  17. Expression of TGF-β Signaling Regulator RBPMS (RNA-Binding Protein With Multiple Splicing) Is Regulated by IL-1β and TGF-β Superfamily Members, and Decreased in Aged and Osteoarthritic Cartilage

    PubMed Central

    Shanmugaapriya, S.; van Caam, A.; de Kroon, L.; Vitters, Elly L.; Walgreen, B; van Beuningen, H.; Davidson, E. Blaney; van der Kraan, Peter M.

    2016-01-01

    Objective RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and enhance transforming growth factor-β (TGF-β)–mediated Smad2/3 transcriptional activity in mammalian cells. Objective of this study was to examine whether expression of RBPMS is regulated by interleukin-1β (IL)-1β and TGF-β superfamily growth factors and whether expression of RBPMS is altered during aging and experimental osteoarthritis. Methods Expression of RBPMS protein was investigated in chondrocyte cell lines of murine (H4) and human (G6) origin using Western blot analysis. Regulation of RBPMS expression in H4 chondrocytes at mRNA level was done by reverse transcriptase–quantitative polymerase chain reaction. Furthermore, characterization of Smad signaling pathways regulating RBPMS expression was performed by blocking studies using small molecule inhibitors or by transfection studies with adenoviral vector constructs (constitutive-active ALK1 and constitutive-active ALK5). Expression of RBPMS in cartilage of different age groups of C57BL/6N mice (6 months and 20 months) and in a surgically induced osteoarthritis (OA) mouse model was analyzed using immunohistochemistry. Results RBPMS was shown to be expressed in chondrocytes and cartilage of murine, human, and bovine origin. TGF-β inhibited RBPMS expression while BMP2 and IL-1β increased its expression. TGF-β-induced inhibition was blocked by ALK5 inhibitor. Overexpression of ca-ALK1 stimulated RBPMS expression. Moreover, RBPMS expression was found to be reduced with ageing and in OA pathogenesis. Conclusions Expression of RBPMS in chondrocytes is regulated by TGF-β superfamily members and IL-1β, indicating a counter-regulatory mechanism. Expression of RBPMS, in cartilage and its reduction during ageing and OA might suggest its potential role in the maintenance of normal articular cartilage. PMID:27688842

  18. Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

    PubMed

    Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

    2017-02-01

    Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell.

  19. 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

  20. 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

    2014-12-06

    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

  1. Fibronectin Fragment Activation of Proline-rich Tyrosine Kinase PYK2 Mediates Integrin Signals Regulating Collagenase-3 Expression by Human Chondrocytes through a Protein Kinase C-dependent Pathway*

    PubMed Central

    Loeser, Richard F.; Forsyth, Christopher B.; Samarel, Allen M.; Im, Hee-Jeong

    2010-01-01

    Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the α5β1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the α5β1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCδ inhibitor rottlerin. Furthermore, PKCδ translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after co-transfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves

  2. 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.

  3. EP2 Receptor Signaling Regulates Microglia Death

    PubMed Central

    Yang, Myung-Soon; Jiang, Jianxiong; Ganesh, Thota; Joe, Eunhye; Dingledine, Raymond

    2015-01-01

    The timely resolution of inflammation prevents continued tissue damage after an initial insult. In the brain, the death of activated microglia by apoptosis has been proposed as one mechanism to resolve brain inflammation. How microglial death is regulated after activation is still unclear. We reported that exposure to lipopolysaccharide (LPS) and interleukin (IL)-13 together initially activates and then kills rat microglia in culture by a mechanism dependent on cyclooxygenase-2 (COX-2). We show here that activation of the E prostanoid receptor 2 (EP2, PTGER2) for prostaglandin E2 mediates microglial death induced by LPS/IL-13, and that EP2 activation by agonist alone kills microglia. Both EP2 antagonists and reactive oxygen scavengers block microglial death induced by either LPS/IL-13 or EP2 activation. By contrast, the homeostatic induction of heme oxygenase 1 (Hmox1) by LPS/IL-13 or EP2 activation protects microglia. Both the Hmox1 inducer cobalt protoporphyrin and a compound that releases the Hmox1 product carbon monoxide (CO) attenuated microglial death produced by LPS/IL-13. Whereas CO reduced COX-2 protein expression, EP2 activation increased Hmox1 and COX-2 expression at both the mRNA and protein level. Interestingly, caspase-1 inhibition prevented microglial death induced by either LPS/IL-13 or low (but not high) concentrations of butaprost, suggestive of a predominantly pyroptotic mode of death. Butaprost also caused the expression of activated caspase-3 in microglia, pointing to apoptosis. These results indicate that EP2 activation, which initially promotes microglial activation, later causes delayed death of activated microglia, potentially contributing to the resolution phase of neuroinflammation. PMID:25715797

  4. 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

    2016-08-05

    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.

  5. Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics.

    PubMed

    Rocco, C J; Davey, M E; Bakaletz, L O; Goodman, S D

    2017-04-01

    Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co-aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity.

  6. The Staphylococcus aureus extracellular adherence protein promotes bacterial internalization by keratinocytes independent of fibronectin-binding proteins.

    PubMed

    Bur, Stephanie; Preissner, Klaus T; Herrmann, Mathias; Bischoff, Markus

    2013-08-01

    Staphylococcus aureus, the leading causal pathogen of skin infections, is strongly associated with skin atopy, and a number of bacterial adhesins allow the microbe to adhere to and invade eukaryotic cells. One of these adhesive molecules is the multifunctional extracellular adherence protein (Eap), which is overexpressed in situ in authentic human wounds and was shown to delay wound healing in experimental models. Yet, its role during invasion of keratinocytes is not clearly defined. By using a gentamicin/lysostaphin protection assay we demonstrate here that preincubation of HaCaT cells or primary keratinocytes with Eap results in a concentration-dependent significant increase in staphylococcal adhesion, followed by an even more pronounced internalization of bacteria by eukaryotic cells. Flow cytometric analysis revealed that Eap increased both the number of infected eukaryotic cells and the bacterial load per infected cell. Moreover, treatment of keratinocytes with Eap strongly enhanced the internalization of coagulase-negative staphylococci, as well as of E. coli, and markedly promoted staphylococcal invasion into extended-culture keratinocytes, displaying expression of keratin 10 and involucrin as differentiation markers. Thus, wound-related staphylococcal Eap may provide a major cellular invasin function, thereby enhancing the pathogen's ability to hide from the host immune system during acute and chronic skin infection.

  7. 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.

  8. Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils

    PubMed Central

    Funchal, Giselle A.; Jaeger, Natália; Czepielewski, Rafael S.; Machado, Mileni S.; Muraro, Stéfanie P.; Stein, Renato T.; Bonorino, Cristina B. C.; Porto, Bárbara N.

    2015-01-01

    Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis. PMID:25856628

  9. 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

  10. 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.

  11. 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

  12. Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo.

    PubMed Central

    Juhasz, I.; Murphy, G. F.; Yan, H. C.; Herlyn, M.; Albelda, S. M.

    1993-01-01

    Although changes in extracellular matrix proteins during wound healing have been well documented, little is known about the regulation of corresponding extracellular matrix adhesion receptors (integrins). To study this process in a human in vivo model, full thickness human skin grafts were transplanted onto severe combined immunodeficient mice and deep excisional wounds involving both the epidermal and dermal layers were then made. The changes in the expression of cell matrix proteins and epithelial integrins over time were analyzed with specific antibodies using immunohistochemistry. Wounding was associated with alterations in extracellular matrix proteins, namely, loss of laminin and type IV collagen in the region of the wound and expression of tenascin and fibronectin. Changes were also noted in the integrins on the migrating keratinocytes. There was marked up-regulation of the alpha v subunit and de novo expression of the fibronectin receptor (alpha 5 beta 1) during the stage of active migration (days 1 to 3 after wounding). In the later stages of wound healing, after epithelial integrity had been established, redistribution of the alpha 2, alpha 3, alpha 6, and beta 4 collagen/laminin-binding integrin subunits to suprabasal epidermal layers was noted. Thus, during cutaneous wound healing, keratinocytes up-regulate fibronectin/fibrinogen-binding integrins and redistribute collagen/laminin-binding integrins. This study demonstrates that the human skin/severe combined immunodeficient chimera provides a useful model to study events during human wound repair. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7694470

  13. 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

  14. 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

  15. Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

    PubMed Central

    Savas, Jeffrey N.; De Wit, Joris; Comoletti, Davide; Zemla, Roland; Ghosh, Anirvan

    2015-01-01

    Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligand – receptor interactions. PMID:25101821

  16. Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: interaction, regulation and role in chondrogenesis.

    PubMed

    Acharya, Chitrangada; Yik, Jasper H N; Kishore, Ashleen; Van Dinh, Victoria; Di Cesare, Paul E; Haudenschild, Dominik R

    2014-07-01

    Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a 'lattice' to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions.

  17. A two-plasmid system for stable, selective-pressure-independent expression of multiple extracellular proteins in mycobacteria.

    PubMed

    Harth, Günter; Maslesa-Galić, Sasa; Horwitz, Marcus A

    2004-07-01

    Recombinant mycobacteria expressing Mycobacterium tuberculosis extracellular proteins are leading candidates for new vaccines against tuberculosis and other mycobacterial diseases, and important tools both in antimycobacterial drug development and basic research in mycobacterial pathogenesis. Recombinant mycobacteria that stably overexpress and secrete major extracellular proteins of M. tuberculosis in native form on plasmids pSMT3 and pNBV1 were previously constructed by the authors. To enhance the versatility of this plasmid-based approach for mycobacterial protein expression, the Escherichia coli/mycobacteria shuttle plasmid pGB9 was modified to accommodate mycobacterial genes expressed from their endogenous promoters. Previous studies showed that the modified plasmid, designated pGB9.2, derived from the cryptic Mycobacterium fortuitum plasmid pMF1, was present at a low copy number in both E. coli and mycobacteria, and expression of recombinant M. tuberculosis proteins was found to be at levels paralleling its copy number, that is, approximating their endogenous levels. Plasmid pGB9.2 was compatible with the shuttle vectors pSMT3 and pNBV1 and in combination with them it simultaneously expressed the M. tuberculosis 30 kDa extracellular protein FbpB. Plasmid pGB9.2 was stably maintained in the absence of selective pressure in three mycobacterial species: Mycobacterium bovis BCG, M. tuberculosis and M. smegmatis. Plasmid pGB9.2 was found to be self-transmissible between both fast- and slow-growing mycobacteria, but not from mycobacteria to E. coli or between E. coli