Phosphorylation Of MSK1 Research Articles

Epigallocatechin-3-gallate inhibits interleukin-1β-induced MUC5AC gene expression and MUC5AC secretion in normal human nasal epithelial cells

It has been reported that the proinflammatory cytokine interleukin-1β (IL-1β) induces mucus hypersecretion in normal human nasal epithelial (NHNE) cells and that the MAP kinase pathway may be an important signal pathway in IL-1β-induced MUC5AC gene expression. Green tea ( Camellia sinensis) polyphenols are potent anti-inflammatory agents and have been shown to inhibit inflammation in tumor cell lines and cultured respiratory epithelial cells. In this study, we examined the effect of (−)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, on IL-1β-induced MUC5AC gene expression and secretion in NHNE cells. After cells had been treated with IL-1β (10 ng/ml) and pretreated with EGCG (10, 50 and 100 μM), mRNA expression of MUC5AC was determined by real-time polymerase chain reaction. The suppression of each signal pathway protein was determined by Western blot analysis after treatment with IL-1β and EGCG, respectively. IL-1β increased MUC5AC gene expression and MUC5AC secretion. EGCG markedly suppressed IL-1β-induced MUC5AC gene expression and MUC5AC secretion via suppression of the phosphorylation of ERK MAP kinase, MSK1, and transcription factor, cAMP response element-binding protein. IL-1β increased the number of cells staining positive with MUC5AC antibodies, and EGCG treatment decreased this number. Our data suggest that EGCG may be an effective inhibitor of IL-1β-induced mucus hypersecretion.

TNFR1 and TNFR2 Signaling Interplay in Cardiac Myocytes

Tumor necrosis factor alpha (TNFalpha) plays a major role in chronic heart failure, signaling through two different receptor subtypes, TNFR1 and TNFR2. Our aim was to further delineate the functional role and signaling pathways related to TNFR1 and TNFR2 in cardiac myocytes. In cardiac myocytes isolated from control rats, TNFalpha induced ROS production, exerted a dual positive and negative action on [Ca(2+)] transient and cell fractional shortening, and altered cell survival. Neutralizing anti-TNFR2 antibodies exacerbated TNFalpha responses on ROS production and cell death, arguing for a major protective role of the TNFR2 pathway. Treatment with either neutralizing anti-TNFR1 antibodies or the glutathione precursor, N-acetylcysteine (NAC), favored the emergence of TNFR2 signaling that mediated a positive effect of TNFalpha on [Ca(2+)] transient and cell fractional shortening. The positive effect of TNFalpha relied on TNFR2-dependent activation of the cPLA(2) activity, independently of serine 505 phosphorylation of the enzyme. Together with cPLA(2) redistribution and AA release, TNFalpha induced a time-dependent phosphorylation of ERK, MSK1, PKCzeta, CaMKII, and phospholamban on the threonine 17 residue. Taken together, our results characterized a TNFR2-dependent signaling and illustrated the close interplay between TNFR1 and TNFR2 pathways in cardiac myocytes. Although apparently predominant, TNFR1-dependent responses were under the yoke of TNFR2, acting as a critical limiting factor. In vivo NAC treatment proved to be a unique tool to selectively neutralize TNFR1-mediated effects of TNFalpha while releasing TNFR2 pathways.

Diffusion and Not Active Transport Underlies and Limits ERK1/2 Synapse-to-Nucleus Signaling in Hippocampal Neurons

The propagation of signals from synapses and dendrites to the nucleus is crucial for long lasting adaptive changes in the nervous system. The ERK-MAPK pathway can link neuronal activity and cell surface receptor activation to the regulation of gene transcription, and it is often considered the principal mediator of synapse-to-nucleus communication in late-phase plasticity and learning. However, the mechanisms underlying ERK1/2 trafficking in dendrites and nuclear translocation in neurons remain to be determined leaving it unclear whether ERK1/2 activated at the synapse can contribute to nuclear signaling and transcriptional regulation. Using the photobleachable and photoactivable fluorescent tag Dronpa on ERK1 and ERK2, we show here that ERK1/2 translocation to the nucleus of hippocampal neurons is induced by the stimulation of N-methyl-D-aspartate receptors or TrkB stimulation and is apparently mediated by facilitated diffusion. In contrast, ERK1/2 trafficking within dendrites is not signal-regulated and is mediated by passive diffusion. Within dendrites, the reach of a locally activated pool of ERK1/2 is very limited and follows an exponential decay with distance. These results indicate that successful signal propagation to the nucleus by the ERK-MAPK pathway depends on the distance of the nucleus from the site of ERK1/2 activation. ERK1/2 activated within or near the soma may rapidly reach the nucleus to induce gene expression, whereas ERK1/2 activated at distal synapses may only contribute to local signaling.

P38 MAPK and MSK1 mediate caspase-8 activation in manganese-induced mitochondria-dependent cell death

Heavy metals are important regulators of cell apoptosis. Manganese (Mn(2+)) is a potent inducer of apoptosis in different cell types, but the precise mechanisms that mediate such effects are not well defined. We previously reported that Mn(2+) was a potent apoptotic agent in human B cells, including lymphoma B cell lines. We show here that Mn(2+)-induced cell death in human B cells is associated with caspase-8-dependent mitochondrial activation leading to caspase-3 activity and apoptosis. We used specific caspase-8 interfering shRNAs to reduce caspase-8 expression, and this also reduced Mn(2+)-induced caspase-3 activation and apoptosis. Mn(2+)-triggered caspase-8 activation is associated with a specific pathway, which is independent of Fas-associated death domain protein, and dependent on the sequential activation of p38-mitogen-activated protein kinase (p38 MAPK) and mitogen- and stress-response kinase 1 (MSK1). Inhibition of p38 activity using either pharmacological inhibitors or dominant-negative mutant forms of p38 blocked Mn(2+)-mediated phosphorylation of MSK1 and blocked subsequent caspase-8 activation. However, specific inhibitors and the expression of a dominant-interfering mutant of MSK1 only inhibited caspase-8 activation, but not p38 activity. These findings suggest a novel model for the regulation of caspase-8 during Mn(2+)-induced apoptosis based on the sequential activation of p38 MAPK, MSK1, caspase-8 and mitochondria, respectively.

MK2 controls the level of negative feedback in the NF-κB pathway and is essential for vascular permeability and airway inflammation

We demonstrate that mitogen-activated protein kinase–activated kinase-2 (MK2) is essential for localized Th2-type inflammation and development of experimental asthma. MK2 deficiency does not affect systemic Th2 immunity, but reduces endothelial permeability, as well as adhesion molecule and chemokine expression. NF-κB regulates transcription of adhesion molecules and chemokines. We show that MK2 and its substrate HSP27 are essential for sustained NF-κB activation. MK2 and HSP27 prevent nuclear retention of p38 by sequestering it in the cytosol. As a result, MK2 precludes excessive phosphorylation of MSK1. By reducing MSK1 activity, MK2 prevents p65 NF-κB hyperphosphorylation and excessive IκBα transcription. IκBα mediates nuclear export of p65. By reducing IκBα level, MK2 prevents premature export of NF-κB from the nucleus. Thus, the MK2–HSP27 pathway regulates the NF-κB transcriptional output by switching the activation pattern from high level, but short lasting, to moderate-level, but long lasting. This pattern of activation is essential for many NF-κB–regulated genes and development of inflammation. Thus, the MK2–HSP27 pathway is an excellent target for therapeutic control of localized inflammatory diseases.

Neuron‐specific phosphorylation of mitogen‐ and stress‐activated protein kinase‐1 involved in cerebral hypoxic preconditioning of mice

Studies have demonstrated the involvement of mitogen-activated protein kinase (MAPK) cascade pathways in the development of cerebral ischemic/hypoxic preconditioning (I/HPC). However, the role of mitogen- and stress-activated protein kinase 1 (MSK1), an important downstream kinase of MAPK signaling pathways, in cerebral I/HPC is unclear. By using Western blot and immunostaining methods, we applied our unique "autohypoxia"-induced I/HPC mouse model to investigate the effects of repetitive hypoxic exposure (H0-H6, n=6 for each group) on phosphorylation and protein expression levels of MSK1 in the brain of mice. We found that the levels of phosphorylation on threonine 645 (Thr645) and serine 375 (Ser375) of MSK1, but not the protein expression, increased significantly both in hippocampus and in cortex of mice from H1-H6 groups (P<0.05) over that of the normoxic group (H0, n=6). Similarly, enhanced phosphorylations on Thr645 and Ser375 of MSK1 were also observed by immunostaining in both the cortex and the hippocampus of mice following three series of hypoxic exposures (H3). In addition, we found by using double-immunofluorescence labeling that phosphorylated Thr645-MSK1 colocalized with a neuron-specific protein, neurogranin, in both cortex and hippocampus of I/HPC mice (H3). These results suggest that the increased neuron-specific phosphorylation of MSK1 on Thr645 and Ser375, not protein expression, might be involved in the development of cerebral I/HPC in mice.

Converging on the Promoter

Steroid hormones modulate gene transcription through interactions of their receptors with hormone-responsive elements on target genes. Noting that steroid receptors also activate the extracellular signal-regulated kinase (ERK) signaling pathway, Vicent et al . used the T47D-MTVL breast cancer cell line--which carries a chromosomally integrated copy of the luciferase gene under the control of the MMTV-LTR promoter--to investigate the role of ERK signaling in progestin action. Treatment with the progesterone receptor (PR) agonist R5020 led to rapid phosphorylation of ERK1/2, the progesterone receptor-B isoform (PR B ), and the ERK target MSK1 (mitogen- and stress-activated protein kinase 1) as well as to transcriptional activation of the MMTV reporter. Hormone-dependent phosphorylation of PR and MSK1 required ERK activation, as did the hormone-dependent induction of MMTV (which also required MSK1 activation) and other progesterone target genes. R5020 stimulated recruitment to the MMTV promoter of PR and a complex containing phosphorylated PR B , phosphorylated ERK, and phosphorylated MSK1. Furthermore, R5020 elicited a MSK1-dependent increase in phosphorylation on serine 10 of histone H3 of the MMTV promoter, associated with the MSK1-dependent loss of HP1γ (heterochromatin protein 1γ) and recruitment of chromatin-remodeling factors and RNA polymerase II. Thus, activation of the ERK signaling pathway appears to play a critical role in progestin-dependent gene transcription that involves MSK1-dependent phosphorylation of histone H3, enabling the loss of repressive complexes from the promoters of target genes and the recruitment of chromatin-remodeling factors. G. P. Vicent, C. Ballaré, A. S. Nacht, J. Clausell, A. Subtil-Rodríguez, I. Quiles, A. Jordan, M. Beato, Induction of progesterone target genes requires activation of Erk and Msk kinases and phosphorylation of histone H3. Mol. Cell. 24 , 367-381 (2006). [PubMed]

Mitogen- and Stress-Activated Protein Kinase 1 Is Activated in Lesional Psoriatic Epidermis and Regulates the Expression of Pro-Inflammatory Cytokines

Mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of both the p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPKs). MSK1 stimulates transcription of different pro-inflammatory genes through activation of transcription factors. The purpose of this study was to investigate the expression and activation of MSK1 in lesional psoriatic skin and its role in cytokine production in cultured normal human keratinocytes. Western blotting revealed a consistent and significant increase in phosphorylated (activated) MSK1(Ser376) in lesional psoriatic skin. Immunofluorescence staining revealed the phosphorylated MSK1(Thr581) to be localized in the basal layers of the epidermis in lesional psoriatic skin. No staining was found in non-lesional psoriatic skin. Cultured human keratinocytes incubated with anisomycin or IL-1beta resulted in the phosphorylation of the p38 MAPK and MSK1(Ser376). MSK1(Ser376) phosphorylation was inhibited by pre-incubation with the p38 inhibitor SB 202190. Transfection of the keratinocytes with specific MSK1 small interfering RNA resulted in 80% reduction of MSK1 expression and 51, 40, and 31% decrease in IL-6, IL-8, and tumor necrosis factor-alpha protein production, respectively. This study demonstrates for the first time the expression of MSK1 in epidermal keratinocytes and increased activation focally in psoriatic epidermis. As MSK1 regulates the production of pro-inflammatory cytokines, it may play a role in the pathogenesis of psoriasis.

Involvement of JNKs and p38-MAPK/MSK1 pathways in H 2O 2-induced upregulation of heme oxygenase-1 mRNA in H9c2 cells

One of the most important challenges that cardiomyocytes experience is an increase in the levels of reactive oxygen species (ROS), i.e., during ischemia, reperfusion as well as in the failing myocardium. HOX-1 has been found to protect cells and tissues against oxidative damage; therefore, we decided to study the signalling cascades involved in its transcriptional regulation. HOX-1 mRNA levels were found to be maximally induced after 6 h of treatment with 200 μM H 2O 2 and remained elevated for at least 24 h. Inhibition of JNKs, p38-MAPK and MSK1 pathways, by pharmacological inhibitors, reduced HOX-1 mRNA levels in H 2O 2-treated H9c2 cells. In parallel, we observed that all three subfamilies of the mitogen-activated protein kinases (MAPKs) attained their maximal phosphorylation levels at 5–15 min of H 2O 2 treatment, with mitogen- and stress-activated-protein kinase 1 (MSK1) also being maximally phosphorylated at 15 min. H 2O 2-induced MSK1 phosphorylation was completely abrogated in the presence of the selective p38-MAPK inhibitor SB203580. In an effort to define possible substrates of MSK1, we found that ATF2 as well as cJun phosphorylation were equally induced after 30 min and 60 min, respectively, a response inhibited by SP600125 (JNKs inhibitor) and H89 (MSK1 inhibitor), indicating the involvement of these kinases in the observed response. This finding was further substantiated with the detection of a potential signalling complex composed of either p-MSK1 and p-cJun or p-MSK1 and p-ATF2 (co-immunoprecipitation). ATF2 and cJun are known AP1 components. Given the presence of an AP-1 site in HOX-1 promoter region, the activity of AP1 transcription factor was examined. Electrophoretic mobility shift assays performed showed a maximal upregulation of AP1 binding activity after 60 min of H 2O 2 treatment, which was significantly inhibited by SP600125 and H89. Our results show for the first time the potential role of JNKs, p38-MAPK and MSK1 in the mechanism of transducing the oxidative stress-signal to HOX-1, possibly promoting cell survival and preserving homeostasis.

Platelet‐derived growth factor‐BB‐induced hypertrophy of peritubular smooth muscle cells is mediated by activation of p38 MAP‐kinase and of Rho‐kinase

Peritubular smooth muscle cells (PSMC) from rat testis in primary serum-free cultures unexpectedly undergo contraction and subsequent cell hypertrophy in response to the growth factor PDGF-BB, remaining stationary. The present study investigates the transduction pathways involved in the observed paradoxical upregulation of the differentiated phenotype and induction of hypertrophy in PSMC. PI3K, ERK, JNK, and p38 kinases, known to mediate PDGF-BB signaling in the canonic dedifferentiative and proliferative response of smooth muscle cells (SMC) were rapidly activated by PDGF-BB but only p38 remained activated after 2-day stimulation. Immunofluorescence and immunoblotting experiments showed that in 4-day treatment: (i) continuous inhibition of PI3K, of ERK, of JNK, failed to inhibit either cell enlargement and formation of prominent alpha-SM actin containing stress fibers or the typical increase in alpha-SM actin; (ii) when stimulated in the presence of the p38 inhibitor SB203580 both responses were significantly inhibited and cytofluorimetric analysis of cell size showed a remarkable reduction of the hypertrophic response. PDGF-BB was also found to activate the small GTPase RhoA and inhibition of Rho-dependent kinase ROCK by Y27632 counteracted the effects of PDGF-BB similarly to SB203580. Both the transcription factor ATF2 and the nucleosomal kinase MSK1, downstream targets of p38, were activated by PDGF-BB, but p38 inhibitor SB203580 inhibited only the phosphorylation of MSK1 which appeared unaffected by ROCK inhibitor Y27632. In concluding, p38 and the Rho-ROCK system were found to play prominent, probably independent roles in the upregulation of PSMC differentiated phenotype and induction of hypertrophy by PDGF-BB.

Light Stimulates MSK1 Activation in the Suprachiasmatic Nucleus via a PACAP-ERK/MAP Kinase-Dependent Mechanism

Signaling via the p42/44 mitogen-activated protein kinase (MAPK) pathway has been shown to be a key intracellular signaling event that couples light to entrainment of the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Because many of the physiological effects of the MAPK pathway are mediated by extracellular signal-regulated kinase (ERK)-regulated kinases, it was of interest to identify kinase targets of ERK in the SCN. In this study, we examined whether mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of ERK in the SCN and whether it couples to clock gene expression. Here we show that photic stimulation during the subjective night stimulates MSK1 phosphorylation at serine 360, an event required for robust kinase activation. Activated ERK and MSK1 were colocalized in SCN cell nuclei after photic stimulation. The in vivo administration of the MAP kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene] attenuated MSK1 phosphorylation. MSK1 phosphorylation was more responsive to late-night than early-night photic stimulation, indicating that MSK1 may differentially contribute to light-induced phase advancing and phase delaying of the clock. The potential connection between pituitary adenylate cyclase-activating polypeptide (PACAP) (a regulator of clock entrainment) and MSK1 phosphorylation was examined. PACAP infusion stimulated MSK1 phosphorylation, whereas PACAP receptor antagonist infusion attenuated light-induced MSK1 phosphorylation in the SCN. In reporter gene assays, MSK1 was shown to couple to mPeriod1 via a cAMP response element-binding protein-dependent mechanism. Together, these data identify MSK1 as both a downstream target of the MAPK cascade within the SCN and a regulator of clock gene expression.

Clostridium difficile Toxin A Regulates Inducible Cyclooxygenase-2 and Prostaglandin E2 Synthesis in Colonocytes via Reactive Oxygen Species and Activation of p38 MAPK

Clostridium difficile toxin A induces acute colitis with neutrophil infiltration and up-regulation of numerous pro-inflammatory mediators, but the contribution of cyclooxygenase-2 (COX-2) induction in this infection is unknown. We report here that toxin A induces expression of COX-2 and secretion of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cultured NCM460 human colonocytes and in human intestinal xenografts. This induction was blocked by SB203580, a p38 MAPK inhibitor, which also decreased the phosphorylation of MSK-1, CREB/ATF-1, and COX-2 promoter activity following toxin A stimulation. Gel shift assays indicated that CREB/ATF-1 was the major proteins binding to the COX-2-CRE. Moreover, colonocytes exposed to toxin A produced reactive oxygen species (ROS), which activated p38 MAPK, MSK-1, and CREB/ATF-1, leading to subsequent COX-2 induction and PGE2 secretion. In intact mice, blockage of p38 MAPK inhibited toxin A-mediated induction of COX-2 in enterocytes as well as lamina propria cells, and significantly blocked the toxin A-induced ileal secretion of fluid and PGE2. Furthermore, a selective COX-2 inhibitor also diminished toxin A-associated ileal fluid and PGE2 secretion. The main signaling pathway for toxin A induction of human COX-2 involves ROS-mediated activation of p38 MAPK, MSK-1, CREB, and ATF-1. Toxin A triggers ileal inflammation and secretion of fluid via COX-2 induction and release of PGE2.

N-Methyl-D-aspartate and Brain-Derived Neurotrophic Factor Induce Distinct Profiles of Extracellular Signal-Regulated Kinase, Mitogen- and Stress-Activated Kinase, and Ribosomal S6 Kinase Phosphorylation in Cortical Neurons

Stimulation of N-methyl-D-aspartate (NMDA) receptors is believed to underlie long-term memory formation, and excessive NMDA receptor activation has been linked to several neuropathological conditions. Phosphorylation and activation of p42/44 mitogen-activated protein kinase (ERK) is believed to mediate many of these effects, but the downstream targets of ERK in response to NMDA activation have not been determined. In primary cultures of rat cortical neurons, we found that NMDA was able to elevate phosphorylation of mitogen- and stress-activated kinase 1 (MSK1) as well as ERK. Likewise, brain-derived neurotrophic factor (BDNF) treatment increased phosphorylation of MSK1 and ERKs. The NMDA-induced MSK1 phosphorylation was sensitive to the MEK inhibitor 2'-amino-3'-methoxyflavone (PD98059) and the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580). MSK1 activation by NMDA was transient, although ERK remained phosphorylated within the neuronal cytoplasm for several hours. Although BDNF increased ribosomal S6 kinase (RSK) phosphorylation, NMDA had no discernable effect on the phosphorylation of RSKs. Thus, phosphorylation and activation of MSK1 but not RSK could be an important step in the pathway linking NMDA-induced ERK phosphorylation to the activation of transcription factors required for the formation of long-term memory.

Interleukin-1β and Tumor Necrosis Factor-α Induce MUC5AC Overexpression through a Mechanism Involving ERK/p38 Mitogen-activated Protein Kinases-MSK1-CREB Activation in Human Airway Epithelial Cells

Mucin hypersecretion is commonly observed in many inflammatory diseases of the respiratory tract. MUC5AC is generally recognized to be a major airway mucin because MUC5AC is highly expressed in the goblet cells of human airway epithelium. Moreover, it is regulated by various inflammatory cytokines. However, the mechanisms by which the interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha induce MUC5AC gene expression in normal nasal epithelial cells, and the signal molecules involved, especially in the downstream signaling of mitogen-activated protein (MAP) kinases, remain unclear. Here we show that pharmacologic or genetic inhibition of either ERK or p38 MAP kinase pathway abolished IL-1beta- and TNF-alpha-induced MUC5AC gene expression in normal human nasal epithelial cells. Our results also indicate that the activation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-response element-binding protein and cAMP-response element signaling cascades via ERK and p38 MAP kinases are crucial aspects of the intracellular mechanisms that mediate MUC5AC gene expression. Taken together, these studies give additional insights into the molecular mechanism of IL-1beta- and TNF-alpha-induced MUC5AC gene expression and enhance our understanding on mucin hypersecretion during inflammation.

Regulation of the ER81 transcription factor and its coactivators by mitogen- and stress-activated protein kinase 1 (MSK1).

The transcription factor ER81 has been shown to be involved in ontogenesis and breast tumor formation. ER81 is activated by many signals through phosphorylation directly mediated by mitogen-activated protein kinases (MAPKs), but also by an unknown protein kinase(s). Here, mitogen- and stress-activated protein kinase 1 (MSK1), which itself is directly activated by distinct classes of MAPKs, is identified to regulate ER81 function. MSK1 expression enhances ER81-dependent transcription upon stimulation of especially the p38-MAPK pathway. Two serine residues in ER81 are phosphorylated by MSK1, and mutating these serine residues to alanines dramatically diminishes the ability of MSK1 to stimulate ER81. However, mutation of the MSK1 phosphorylation sites in ER81 does not completely abrogate the ability of MSK1 to activate ER81 function, suggesting that MSK1 may also target cofactors of ER81. Consistently, MSK1 interacts with two homologous coactivators of ER81, CBP and p300, and stimulates the transactivation domains of CBP. Thus, MSK1 may regulate ER81-dependent transcription via direct phosphorylation of ER81 as well as via stimulation of CBP/p300, which might be important for ER81's normal function and during mammary tumor formation.

Phosphorylation and activation of mitogen- and stress-activated protein kinase-1 in adult rat cardiac myocytes by G-protein-coupled receptor agonists requires both extracellular-signal-regulated kinase and p38 mitogen-activated protein kinase.

G-protein-coupled receptor agonists are powerful stimulators of mitogen-activated protein kinase (MAPK) cascades in cardiac myocytes. However, little is known regarding the physiological activation of enzymes downstream of MAPKs. We examined the activation of mitogen- and stress-activated protein kinase-1 (MSK1), a downstream target of MAPKs, in adult rat cardiac myocytes by phenylephrine and endothelin-1. Both agonists induced the phosphorylation of MSK1 at Thr-581 and Ser-376 but not at Ser-360. Maximal phosphorylation was observed at 10-15 min after stimulation and it correlated with increased activity. Maximal activation of MSK1 in adult cardiomyocytes temporally coincided with maximal p38 MAPK activation while activation of the extracellular-signal-regulated kinase (ERK) cascade was more rapid. Phosphorylation and activation of MSK1 was completely inhibited by either PD98059 (ERK1/2 pathway inhibitor) or SB203580 (p38 MAPK inhibitor) alone. These data demonstrate that MSK1 activation in adult rat cardiac myocytes by G-protein-coupled receptor agonists requires the simultaneous activation of both the ERK and p38 MAPK pathways. However, the lack of phosphorylation at Ser-360, an identified phosphorylation site targeted by MAPKs, may indicate that MSK1 is not a direct substrate of ERK1/2 and p38 MAPK in adult rat cardiomyocytes.

Role of p38 mitogen-activated protein kinase (p38 MAPK) in cytokine-induced rat islet cell apoptosis

The signaling pathways mediating nitric oxide production and apoptosis in pancreatic β-cells are not fully understood. We investigated cytokine-induced protein phosphorylation events in insulin-producing cells and evaluated their role in inducible nitric oxide synthase (iNOS) induction and cell death. Interleukin-1β (IL-1β), but not interferon-γ (IFN-γ), induced phosphorylation of p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and mitogen- and stress-activated protein kinase 1 (MSK1) in rat insulin-producing RINm5F cells. This was paralleled by an increased phosphorylation of the transcription factors activating transcription factor-2 (ATF-2) and cAMP-responsive element-binding protein (CREB). The p38 inhibitor SB203580 prevented cytokine-induced phosphorylation of CREB and MSK1, but not of ATF-2. IFN-γ induced the phosphorylation of signal transducer and activator of transcription 1. The combination of IL-1β and IFN-γ increased both apoptosis and necrosis in rat islet cells. SB203580, but not the extracellular signal-regulated kinase inhibitor PD98059, partially prevented cytokine-induced apoptosis, an effect that was not associated with reduced nitrite production or lowered iNOS expression. In conclusion, cytokine-induced p38 activation participates in β-cell apoptosis, possibly by a nitric oxide-independent mechanism or by enhancing the sensitivity to nitric oxide.