Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo^−/− myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.     

Notch signaling suppresses p38 MAPK activity via induction of MKP-1 in myogenesis

Cross-talks among intracellular signaling pathways are important for the regulation of cell fate decisions and cellular responses to extracellular signals. Both the Notch pathway and the MAPK pathways play important roles in many biological processes, and the Notch pathway has been shown to interact with the ERK-type MAPK pathway. However, its interaction with the other MAPK pathways is unknown. Here we show that Notch signaling activation in C2C12 cells suppresses the activity of p38 MAPK to inhibit myogenesis. Our results show that Notch specifically induces expression of MKP-1, a member of the dual-specificity MAPK phosphatase, which directly inactivates p38 to negatively regulate C2C12 myogenesis. The Notch-induced expression of MKP-1 is shown to depend on RBP-J. Moreover, inhibition of MKP-1 expression by short interfering RNA suppresses p38 inactivation and partially rescues the negative regulation of myogenesis. These results reveal a novel cross-talk between the Notch pathway and the p38 MAPK pathway that is mediated by Notch induction of MKP-1.     

Thrombin Enhances NGF-Mediated Neurite Extension via Increased and Sustained Activation of p44/42 MAPK and p38 MAPK

Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling.     

Activation of p38 MAPK induces cell cycle arrest via inhibition of Raf/ERK pathway during muscle differentiation

Cell cycle arrest is essential for initiation of muscle differentiation in myoblasts. Given the previously described essential role for p38 MAPK in myogenesis, we undertook the present study to investigate the role of p38 MAPK in the cell cycle arrest that initiates muscle differentiation. p38 MAPK activity increased during, and was required for, muscle differentiation. Inhibition of p38 MAPK stimulated Raf and ERK activities, and induced cell proliferation in differentiation medium. The concomitant inhibition of p38 MAPK and ERK, however, failed to induce differentiation or proliferation. In conclusion, inhibition of the Raf/ERK pathway and the consequent cell cycle arrest is one of the major functions of p38 MAPK during muscle differentiation.     

Rotenone Directly Induces BV2 Cell Activation via the p38 MAPK Pathway

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Although its pathogenesis is still unclear, increasing evidence suggests that mitochondrial dysfunction induced by environmental toxins, such as mitochondrial complex I inhibitors, plays a significant role in the disease process. The microglia in PD brains are highly activated, and inflammation is also an essential element in PD pathogenesis. However, the means by which these toxins activate microglia is still unclear. In the present study, we found that rotenone, a mitochondrial complex I inhibitor, could directly activate microglia via the nuclear factor kappa B (NF-κB) signaling pathway, thereby inducing significantly increased expression of inflammatory cytokines. We further observed that rotenone induced caspase-1 activation and mature IL-1β release, both of which are strictly dependent on p38 mitogen-activated protein kinase (MAPK). The activation of p38 is associated with the presence of reactive oxygen species (ROS) produced by rotenone. Removal of these ROS abrogated the activation of the microglia. Therefore, our data suggest that the environmental toxin rotenone can directly activate microglia through the p38 MAPK pathway.     

Activation of p38MAPK contributes to expanded polyglutamine-induced cytotoxicity

Background

The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood.

Methodologies/Principal Findings

Herein we demonstrate that expanded polyglutamine protein cytotoxicity is mediated primarily through activation of p38MAPK and that the atypical PKC iota (PKCι) enzyme antagonizes polyglutamine-induced cell death through induction of the ERK signaling pathway. We show that pharmacological blockade of p38MAPK rescues cells from polyglutamine-induced cell death whereas inhibition of ERK recapitulates the sensitivity observed in cells depleted of PKCι by RNA interference. We provide evidence that two unrelated proteins with expanded polyglutamine repeats induce p38MAPK in cultured cells, and demonstrate induction of p38MAPK in an in vivo model of neurodegeneration (spinocerebellar ataxia 1, or SCA-1).

Conclusions/Significance

Taken together, our data implicate activated p38MAPK in disease progression and suggest that its inhibition may represent a rational strategy for therapeutic intervention in the polyglutamine disorders.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Microtubular stability affects pVHL-mediated regulation of HIF-1alpha via the p38/MAPK pathway in hypoxic cardiomyocytes

Background

Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α.

Methodology/Principal Findings

In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule–depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells.

Conclusions/Significance

This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.     

A Cdo-Bnip-2-Cdc42 signaling pathway regulates p38alpha/beta MAPK activity and myogenic differentiation

The p38alpha/beta mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38alpha/beta pathway scaffold protein JLP and, via JLP, p38alpha/beta itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38alpha/beta activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38alpha/beta activation during cell differentiation.     

IGF2BP1: a novel binding protein of p38 MAPK

Maneb (MB) and paraquat (PQ) provoke oxidative stress-mediated cell damage. Role of xanthine oxidase (XO) in oxidative stress and its association with nitric oxide (NO)/NO synthase (NOS) have been widely reported. While inducible NOS (iNOS) is implicated in MB+PQ-induced toxicity in rat polymorphonuclear leukocytes (PMNs), role of XO and its alliance with iNOS have not yet been established. The study investigated the role of XO in MB+PQ-induced oxidative stress in rat PMNs and its regulation by iNOS and inflammatory cytokines. MB+PQ-augmented reactive oxygen species (ROS), superoxide, nitro-tyrosine, lipid peroxidation (LPO), and nitrite levels along with the catalytic activity of iNOS, superoxide dismutase (SOD), and XO. XO inhibitor, allopurinol (AP), alleviated MB+PQ-induced changes except nitrite content and iNOS activity. Conversely, an iNOS inhibitor, aminoguanidine, mitigated MB+PQ-induced LPO, nitrite, iNOS, and nitro-tyrosine levels; however, no change was observed in ROS, SOD, and XO. Nuclear factor-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), tumor necrosis factor-alpha (TNF-α) inhibitor, pentoxyfylline, and an anti-inflammatory agent, dexamethasone, attenuated MB+PQ-induced increase in XO, superoxide, and ROS with parallel reduction in the expression of interferon-gamma (IFN-γ), TNF-α, and interleukin-1β (IL-1β) in rat PMNs. Exogenous IFN-γ, TNF-α, and IL-1β enhanced superoxide, ROS, and XO in the PMNs of control and MB+PQ-treated rats; however, IFN- γ was found to be the most potent inducer. Moreover, AP ameliorated cytokine-induced free radical generation and restored XO activity towards normalcy. The results thus demonstrate that XO mediates oxidative stress in MB+PQ-treated rat PMNs via iNOS-independent but cytokine (predominantly IFN-γ)-dependent mechanism.     

Inhibition of p38 MAPK alpha/beta reduces ischemic injury and does not block protective effects of preconditioning

We examined the effect of inhibition of p38 mitogen-activated protein kinase (MAPK) alpha/beta during ischemia and preconditioning by using the inhibitor SB-202190. Isolated rat hearts were perfused with Krebs-Henseleit buffer, while left ventricular developed pressure (LVDP) and (31)P nuclear magnetic resonance spectra were acquired continuously. After 20 min of ischemia and 25 min of reperfusion, recovery of LVDP in untreated hearts was 32 +/- 4%, whereas hearts treated with SB-202190 5 min before ischemia recovered 59 +/- 7% of their pretreatment LVDP. Preconditioning improved functional recovery to 65 +/- 5%, which was unaffected by SB-202190 treatment, added either throughout the preconditioning protocol (56 +/- 5% recovery) or during the final reperfusion period of preconditioning (71 +/- 11% recovery). Necrosis was assessed after 40 min of ischemia and 2 h of reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC) staining and creatine kinase release. The untreated group had 54 +/- 8% necrotic myocardium, whereas the SB-202190-treated group had 32 +/- 7% and the preconditioned group had 21 +/- 4% necrotic tissue by TTC staining.     

Triptolide promotes autophagy to inhibit mesangial cell proliferation in IgA nephropathy via the CARD9/p38 MAPK pathway

BackgroundMesangial cell proliferation is the most basic pathological feature of immunoglobulin A nephropathy (IgAN); however, the specific underlying mechanism and an appropriate therapeutic strategy are yet to be unearthed. This study aimed to investigate the therapeutic effect of triptolide (TP) on IgAN and the mechanism by which TP regulates autophagy and proliferation of mesangial cells through the CARD9/p38 MAPK pathway.MethodsWe established a TP‐treated IgAN mouse model and produced IgA1‐induced human mesangial cells (HMC) and divided them into control, TP, IgAN, and IgAN+TP groups. The levels of mesangial cell proliferation (PCNA, cyclin D1, cell viability, and cell cycle) and autophagy (P62, LC3 II, and autophagy flux rate) were measured, with the autophagy inhibitor 3‐Methyladenine used to explore the relationship between autophagy and proliferation. We observed CARD9 expression in renal biopsies from patients and analyzed its clinical significance. CARD9 siRNA and overexpression plasmids were constructed to investigate the changes in mesangial cell proliferation and autophagy as well as the expression of CARD9 and p‐p38 MAPK/p38 MAPK following TP treatment.ResultsAdministering TP was safe and effectively alleviated mesangial cell proliferation in IgAN mice. Moreover, TP inhibited IgA1‐induced HMC proliferation by promoting autophagy. The high expression of CARD9 in IgAN patients was positively correlated with the severity of HMC proliferation. CARD9/p38 MAPK was involved in the regulation of HMC autophagy and proliferation, and TP promoted autophagy to inhibit HMC proliferation by downregulating the CARD9/p38 MAPK pathway in IgAN.ConclusionTP promotes autophagy to inhibit mesangial cell proliferation in IgAN via the CARD9/p38 MAPK pathway.

The proliferation of glomerular mesangial cells is the most basic pathological feature for IgA nephropathy (IgAN). Galactose‐deficient IgA1 (Gd‐IgA1) immune complex deposition activates mesangial cells to mediate specific intracellular signal transduction, promotes mesangial cell proliferation, and initiates kidney damage. Autophagy is involved in mesangial cell proliferation. CARD9 is a risk gene for IgAN. The triptolide (TP), purified from Tripterygium wilfordii hook F., promotes autophagy to inhibit mesangial cell proliferation in IgAN via the CARD9/p38 MAPK pathway.     

Activation of p38 MAPK by oxidative stress underlying epirubicin-induced vascular endothelial cell injury

Epirubicin, an anthracycline antitumor drug, often causes vascular injury such as vascular pain, phlebitis, and necrotizing vasculitis. However, an effective prevention for the epirubicin-induced vascular injury has not been established. The purpose of this study is to identify the mechanisms of cell injury induced by epirubicin in porcine aorta endothelial cells (PAECs). PAECs were exposed to epirubicin for 10 min followed by further incubation without epirubicin. The exposure to epirubicin (3-30 μM) decreased the cell viability concentration and time dependently. Epirubicin increased the activity of caspase-3/7, apoptotic cells, and intracellular lipid peroxide levels, and also induced depolarization of mitochondrial membranes. These intracellular events were reversed by glutathione (GSH) and N-acetylcysteine (NAC), while epirubicin rather increased intracellular GSH slightly and L-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, had no effect on the epirubicin-induced cell injury. The epirubicin-induced cell injury and increase of caspase-3/7 activity were also attenuated by p38 mitogen-activated protein kinase (MAPK) inhibitors, SB203580 and PD169316. Moreover, epirubicin significantly enhanced the phosphorylation of p38 MAPK, and these effects were attenuated by GSH and NAC. In contrast, a c-Jun N-terminal kinase inhibitor SP600125, an extracellular signal-regulated kinase inhibitor PD98059, and a p53 inhibitor pifithrin α did not affect the epirubicin-induced cell injury and increase of caspase-3/7 activity. These results indicate that an activation of p38 MAPK by oxidative stress is involved in the epirubicin-induced endothelial cell injury.     

Activation by 2-arachidonoylglycerol of platelet p38MAPK/cPLA2 pathway

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 μM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.     

A Novel MAPK phosphatase MKP-7 acts preferentially on JNK/SAPK and p38 alpha and beta MAPKs

Mitogen-activated protein kinases (MAPKs) are inactivated via dephosphorylation of either the threonine or tyrosine residue or both in the P-loop catalyzed by protein phosphatases which include serine/threonine phosphatases, tyrosine phosphatases, and dual specificity phosphatases. Nine members of the dual specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has its own substrate specificity, tissue distribution, and subcellular localization. In this study, we have cloned and characterized a novel MKP, designated MKP-7. MKP-7 is most similar to hVH5, a member of previously known MKPs, in the primary structure. MKP-7 is predominantly localized in the cytoplasm when expressed in cultured cells, whereas hVH5 is both in the nucleus and the cytoplasm. MKP-7 binds to and inactivates p38 MAPK and JNK/SAPK, but not ERK. Furthermore, we have found that MKPs have the substrate specificity toward the isoforms of the p38 family (alpha, beta, gamma, and delta). MKP-7 binds to and inactivates p38 alpha and -beta, but not gamma or delta. MKP-5 and CL100/MKP-1 also bind to p38 alpha and -beta, but not gamma or delta. Finally, we propose a tentative classification of MKPs based on the sequence characteristics of their MAPK-docking site.     

Arf6 promotes cell proliferation via the PLD-mTORC1 and p38MAPK pathways

The small G-protein ADP-ribosylation factor 6 (Arf6) belongs to the Ras GTPases superfamily and is mostly known for its actin remodeling functions and involvement in the processes of plasma membrane reorganization and vesicular transport. The majority of data indicates that Arf6 contributes to cancer progression through activation of cell motility and invasion. Alternatively, we found that the expression of a wild-type or a constitutively active Arf6 does not influence tumor cell motility and invasion but instead significantly stimulates cell proliferation and activates phospholipase D (PLD). Conversely the expression of a mutant Arf6 (Arf6N48I), that is, unable to interact with PLD has no effect on proliferation but promotes motility, invasion, and matrix degradation by uPA extracellular proteinase. Studying the mechanisms of Arf6-dependent stimulation of cell proliferation, we found some signaling pathways contributing to Arf6 promitogenic activity. Namely, we showed that Arf6 in a PLD-mTORC1-dependent manner activates S6K1 kinase, a well-known regulator of mitogen-stimulated translation initiation. Furthermore, we demonstrated an Arf6-dependent phosphorylation of mTORC1 downstream targets, 4E-BP1 and ribosomal S6 protein, confirming an existence of Arf6-PLD-mTORC1-S6K1/4E-BP1 signaling pathway and also demonstrated its impact on proliferation stimulation. Next, we found that Arf6 activation potentiates Erk1/2 and p38MAP kinases phosphorylation. Surprisingly, p38 opposite to Erk1/2 significantly contributes to Arf6-dependent proliferation increase promoting S6 ribosomal protein phosphorylation at Ser235/236 residues. Therefore, we demonstrated Arf6 proliferation stimulating activity and revealed PLD-mTORC1 and p38MAP kinase as Arf6 partners mediating promitogenic activity. These results highlight a new aspect of Arf6 functioning in cancer cell biology.