Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.     

Map kinase phosphatases (MKP's) are early responsive genes during induction of cerulein hyperstimulation pancreatitis

Mitogen-activated protein kinase (MAPK) family members such as c-jun N-terminal kinase (JNK) may act as signal transducers early during pancreatitis development and evidence indicates that MAPK phosphatases (MKP) downregulate MAPK. We therefore investigated expression and regulation of pancreatic MKP in vivo. Pancreatic MKP mRNA levels were near or below the detection threshold in unstimulated animals. Cerulein hyperstimulation strongly induced MKP-1, MKP-3, and MKP-5 expression, peaking 30 to 60 min after treatment. Thus, MKP's clearly are early responsive genes during pancreatitis induction. Interestingly, inhibition of MKP-1 expression by Ro-31-8220 maximally induced activation of JNK but not of p38 and ERK in acutely isolated acini. These effects indicate that JNK may indeed be a preferred MKP-1 substrate in vivo.     

Insulin stimulation of a MEK-dependent but ERK-independent SOS protein kinase.

The Ras guanylnucleotide exchange protein SOS undergoes feedback phosphorylation and dissociation from Grb2 following insulin receptor kinase activation of Ras. To determine the serine/threonine kinase(s) responsible for SOS phosphorylation in vivo, we assessed the role of mitogen-activated, extracellular-signal-regulated protein kinase kinase (MEK), extracellular-signal-regulated protein kinase (ERK), and the c-JUN protein kinase (JNK) in this phosphorylation event. Expression of a dominant-interfering MEK mutant, in which lysine 97 was replaced with arginine (MEK/K97R), resulted in an inhibition of insulin-stimulated SOS and ERK phosphorylation, whereas expression of a constitutively active MEK mutant, in which serines 218 and 222 were replaced with glutamic acid (MEK/EE), induced basal phosphorylation of both SOS and ERK. Although expression of the mitogen-activated protein kinase-specific phosphatase (MKP-1) completely inhibited the insulin stimulation of ERK activity both in vitro and in vivo, SOS phosphorylation and the dissociation of the Grb2-SOS complex were unaffected. In addition, insulin did not activate the related protein kinase JNK, demonstrating the specificity of insulin for the ERK pathway. The insulin-stimulated and MKP-1-insensitive SOS-phosphorylating activity was reconstituted in whole-cell extracts and did not bind to a MonoQ anion-exchange column. In contrast, ERK1/2 protein was retained by the MonoQ column, eluted with approximately 200 mM NaCl, and was MKP-1 sensitive. Although MEK also does not bind to MonoQ, immunodepletion analysis demonstrated that MEK is not the insulin-stimulated SOS-phosphorylating activity. Together, these data demonstrate that at least one of the kinases responsible for SOS phosphorylation and functional dissociation of the Grb2-SOS complex is an ERK-independent but MEK-dependent insulin-stimulated protein kinase.     

Preheating accelerates mitogen-activated protein (MAP) kinase inactivation post-heat shock via a heat shock protein 70-mediated increase in phosphorylated MAP kinase phosphatase-1

Heat shock (HS) activates mitogen-activated protein (MAP) kinases. Although prior exposure to nonlethal HS makes cells refractory to the lethal effect of a subsequent HS, it is unclear whether this also occurs in MAP kinase activation. This study was undertaken to evaluate the effect of a heat pretreatment on MAP kinase activation by a subsequent HS and to elucidate its possible mechanism. Preheating did not make BEAS-2B cells refractory to extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) activation by a second HS but accelerated their inactivation after HS. The rapid inactivation of ERK and JNK was dependent on de novo protein synthesis and associated with the up-regulation of heat shock protein 70 (HSP70). Moreover, the inhibition of phosphatase activity reversed this rapid inactivation. MAP kinase phosphatase-1 (MKP-1) expression was increased by HS, and the presence of its phosphorylated form (p-MKP-1) correlated with the observed rapid ERK and JNK inactivation. Blocking induction of p-MKP-1 with antisense MKP-1 oligonucleotides suppressed the rapid inactivation of ERK and JNK in preheated cells. HSP70 overexpression caused the early phosphorylation of MKP-1. Moreover, MKP-1 phosphorylation and the rapid inactivation of ERK were inhibited by blocking HSP70 induction in preheated cells. In addition, MKP-1 was insolubilized by HS, and HSP70 associated physically with MKP-1, suggesting that a chaperone effect of HSP70 might have caused the early phosphorylation of MKP-1. These results indicate that preheating accelerated MAP kinase inactivation after a second HS and that this is related to a HSP70-mediated increase in p-MKP-1.     

Discordance between the binding affinity of mitogen-activated protein kinase subfamily members for MAP kinase phosphatase-2 and their ability to activate the phosphatase catalytically

MKP-2 is a member of the mitogen-activated protein (MAP) kinase phosphatase family which has been suggested to play an important role in the feedback control of MAP kinase-mediated gene expression. Although MKP-2 preferentially inactivates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) MAP kinase subfamilies, the mechanisms underlying its own regulation remain unclear. In this report, we have examined the MKP-2 interaction with and catalytic activation by distinct MAP kinase subfamilies. We found that the catalytic activity of MKP-2 was enhanced dramatically by ERK and JNK but was affected only minimally by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, whereas JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the NH(2)-terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised the catalytic activation of MKP-2 by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 whereas MKP-2 mutants incapable of binding to ERK/p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for the substrate preference of MKP-2 and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function.     

Substitution of two insulin receptor carboxy-terminal tyrosines with phenylalanine impairs the expression of MAP kinase phosphatase-1 (MKP-1) mRNA

Cells expressing mutant insulin receptors (Y/F2), in which tyrosines 1316 and 1322 have been replaced with phenylalanine, exhibit enhanced insulin-induced MAP kinase activity and DNA synthesis in comparison with cells expressing wild type insulin receptors (Hirc B). To elucidate the mechanism of enhanced responsiveness, the expression of MAP kinase phosphatase-1 (MKP-1), a negative regulator of MAP kinase activity, was measured in Hirc B and Y/F2 cells incubated in the absence and presence of insulin for various periods of time, and over increasing concentrations of the ligand. Treatment of both cell lines with insulin induced a time and concentration-dependent relative increase in MKP-1 mRNA expression. However, in Y/F2 cells both basal and insulin-stimulated MKP-1 mRNA levels were more than 60% lower than that observed in cells transfected with the wild-type receptors. Cyclic AMP analog (8-Br-cAMP)/inducer (Forskoline) increased MKP-1 mRNA levels in both cell lines, and to a lesser extent in Y/F2 cells. In contrast to insulin the relative increase in MKP-1 mRNA expression induced by 8-Br-cAMP or forskoline was similar in Y/F2 and Hirc B cells. The overexpression of MKP-1 in Y/F2 cells inhibited insulin stimulated DNA synthesis. Transfection of wild type insulin receptors into Y/F2 cells increased basal levels of MKP-1. These results suggest that insulin receptor tyrosine residues 1316 and 1322 play an important role in the regulation of MKP-1 expression both under basal and insulin stimulated conditions, and are not necessary for the induction of MKP-1 mRNA by cAMP. Furthermore, the enhanced insulin induced mitogenic signaling seen in Y/F2 cells is, at least in part, due to impaired MKP-1 expression.     

The kinase domain of MEKK1 induces apoptosis by dysregulation of MAP kinase pathways

MAP kinase pathways comprise a group of parallel protein phosphorylation cascades, which are involved in signaling triggered by a variety of stimuli. Previous findings suggested that the ERK and the JNK pathways have opposing roles in regulating proliferation and survival or apoptosis and that apoptosis can be promoted by inhibiting the ERK pathway or by activation of the JNK pathway. In order to test this hypothesis and explore whether it can be exploited as a strategy for killing human cancer cells, we used gene transfer experiments with a range of cancer cell lines. We expressed the catalytic fragment of human MEKK1 to activate JNK and the Ras-binding domain (RBD) of Raf-1 to inhibit the Ras-ERK pathway. In addition, we designed several RBD-MEKK1 fusion proteins aiming to simultaneously activate the JNK and block the ERK pathway. We found that the MEKK1 proteins as well as the RBD alone could reduce colony formation in all cell lines. The survival time of MEKK1-expressing cells depended on the cell line. In HeLa cells, survival could be prolonged by inhibition of caspases but not by coexpression of the anti-apoptotic protein Bcl-2. Due to a lower kinase activity the RBD-MEKK1 fusion proteins were less effective in apoptosis induction than the MEKK1 kinase domain alone. Using mutant forms of Ras and Raf-1 we could show that the reduced kinase activity of RBD-MEKK1 fusion proteins was caused by binding to the Ras protein. The expression of lethal doses of MEKK1 resulted in a strong activation of all three major MAP kinase families JNK, ERK, and p38. Blocking these pathways either by coexpressing a dominant negative form of MKK4 or with inhibitors of MEK or p38 failed to inhibit apoptosis. This suggests that MEKK1 induces apoptosis by causing a general deregulation of MAP kinase signaling rather than by the activation of a single pathway.     

Restraint of proinflammatory cytokine biosynthesis by mitogen-activated protein kinase phosphatase-1 in lipopolysaccharide-stimulated macrophages

Exposure of macrophages to LPS elicits the production of proinflammatory cytokines, such as TNF-alpha, through complex signaling mechanisms. Mitogen-activated protein (MAP) kinases play a critical role in this process. In the present study, we have addressed the role of MAP kinase phosphatase-1 (MKP-1) in regulating proinflammatory cytokine production using RAW264.7 macrophages. Analysis of MAP kinase activity revealed a transient activation of c-Jun N-terminal kinase (JNK) and p38 after LPS stimulation. Interestingly, MKP-1 was induced concurrently with the inactivation of JNK and p38, whereas blocking MKP-1 induction by triptolide prevented this inactivation. Ectopic expression of MKP-1 accelerated JNK and p38 inactivation and substantially inhibited the production of TNF-alpha and IL-6. Induction of MKP-1 by LPS was found to be extracellular signal-regulated kinase dependent and involved enhanced gene expression and increased protein stability. Finally, MKP-1 expression was also induced by glucocorticoids as well as cholera toxin B subunit, an agent capable of preventing autoimmune diseases in animal models. These findings highlight MKP-1 as a critical negative regulator of the macrophage inflammatory response, underscoring its premise as a potential target for developing novel anti-inflammatory drugs.     

The P2Y(2) nucleotide receptor mediates tissue factor expression in human coronary artery endothelial cells

The discovery of the role of P2Y(12) receptor in platelet aggregation leads to a new anti-thrombotic drug Plavix; however, little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) mediates up-regulation of tissue factor (TF), the initiator of coagulation cascade. Stimulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased the mRNA level of TF but not of its counterpart-tissue factor pathway inhibitor, which was accompanied by up-regulation of TF protein and cell surface activity. RT-PCR revealed a selective expression of P2Y(2) and P2Y(11) receptors in HCAEC. Consistent with this, TF up-regulation was inhibited by suramin or by siRNA silencing of P2Y(2) receptor, but not by NF-157, a P2Y(11)-selective antagonist, suggesting a role for the P2Y(2) receptor. In addition, P2Y(2) receptor activated ERK1/2, JNK, and p38 MAPK pathways without affecting the positive NF-κB and negative AKT regulatory pathways of TF expression. Furthermore, TF up-regulation was abolished or partially suppressed by inhibition of p38 or JNK but not ERK1/2. Interestingly, blockade of the PLC/Ca(2+) pathway did not affect P2Y(2) receptor activation of p38, JNK, and TF induction. However, blockade of Src kinase reduced phosphorylation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced phosphorylation of JNK but not p38, decreasing TF expression. These findings demonstrate that P2Y(2) receptor mediates TF expression in HCAEC through new mechanisms involving Src/p38 and Rho/JNK pathways, possibly contributing to a pro-thrombotic status after vascular injury.     

The function of mitogen-activated protein kinase phosphatase-1 in peptidoglycan-stimulated macrophages

Mitogen-activated protein (MAP) kinases play a pivotal role in the macrophages in the production of proinflammatory cytokines triggered by lipopolysaccharides. However, their function in the responses of macrophages to Gram-positive bacteria is poorly understood. Even less is known about the attenuation of MAP kinase signaling in macrophages exposed to Gram-positive bacteria. In the present study, we have investigated the regulation of MAP kinases and the role of MAP kinase phosphatase (MKP)-1 in the production of pro-inflammatory cytokines using murine RAW264.7 and primary peritoneal macrophages after peptidoglycan stimulation. Treatment of macrophages with peptidoglycan resulted in a transient activation of JNK, p38, and extracellular signal-regulated kinase. Most interestingly, MKP-1 expression was potently induced by peptidoglycan, and this induction was concurrent with MAP kinase dephosphorylation. Triptolide, a diterpenoid triepoxide, potently blocked the induction of MKP-1 by peptidoglycan and prolonged the activation of JNK and p38. Overexpression of MKP-1 substantially attenuated the production of tumor necrosis factor (TNF)-alpha induced by peptidoglycan, whereas knockdown of MKP-1 by small interfering RNA substantially increased the production of both TNF-alpha and interleukin-1 beta. Finally, we found that in primary murine peritoneal macrophages, MKP-1 induction following peptidoglycan stimulation also coincided with inactivation of JNK and p38. Blockade of MKP-1 induction resulted in a sustained activation of both JNK and p38 in primary macrophages. Our results reveal that MKP-1 critically regulates the expression of TNF-alpha and interleukin-1 beta in RAW264.7 cells and further suggest a central role for this phosphatase in controlling the inflammatory responses of primary macrophages to Gram-positive bacterial infection.     

p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.     

Conditional expression of MAP kinase phosphatase-2 protects against genotoxic stress-induced apoptosis by binding and selective dephosphorylation of nuclear activated c-jun N-terminal kinase

MAP Kinase Phosphatase-2 (MKP-2) is a dual specific nuclear phosphatase which is selective for both ERK and JNK, MAP kinases implicated in the regulation of apoptosis in response to genotoxic stress. Here we report the conditional expression of MKP-2 in human embryonic kidney cells 293. We demonstrate that Flag-WT-MKP-2 is able to rescue cells from apoptotic commitment when subjected to UV-C or cisplatin treatment. We establish that upon stimulation all three major MAP kinase families (ERK, JNK and p38 MAP kinases) are activated. However, MKP-2 is surprisingly only able to deactivate JNK in vivo. Furthermore, whilst pre-treatment of cells with either the JNK inhibitor SP600125, or the MEK-1 inhibitor PD98059, also reverses UV-C and cisplatin-induced apoptosis, the anti-apoptotic effect of MKP-2 overexpression is not additive with SP600125 but is with PD098059, suggesting that MKP-2 is involved in specifically terminating JNK activity and not ERK. The inability of MKP-2 to dephosphorylate ERK in vivo is also not due to the inability of Flag-MKP-2 to bind both ERK and JNK; phosphorylated forms of each kinase are co-precipitated with both WT and CI-MKP-2. Immunofluorescence studies however demonstrate that ERK is exclusively cytosolic in origin and not translocated to the nucleus following UV-C and cisplatin treatment whilst JNK is principally nuclear. These studies demonstrate the in vivo specificity of MKP-2 for JNK and not ERK and show that nuclear-targeted JNK is involved in genotoxic stress-induced apoptosis.     

Mitogen-activated protein kinases and mitogen-activated protein kinase phosphatases mediate the inhibitory effects of all-trans retinoic acid on the hypertrophic growth of cardiomyocytes

All-trans retinoic acid (RA) has been implicated in mediation of cardiac growth inhibition in neonatal cardiomyocytes. However, the associated signaling mechanisms remain unclear. Utilizing neonatal cardiomyocytes, we demonstrated that RA suppressed the hypertrophic features induced by cyclic stretch or angiotensin II (Ang II). Cyclic stretch- or Ang II-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAP kinase) was dose- and time-dependently inhibited by RA. Significant inhibition was observed by 5 microm RA, from 8 to 24 h of pretreatment. This inhibitory effect was not mediated at the level of mitogen-activated protein kinase kinases (MKKs), because RA had no effect on stretch- or Ang II-induced phosphorylation of MEK1/2, MKK4, and MKK3/6. However, the phosphatase inhibitor vanadate reversed the inhibitory effect of RA on MAP kinases and protein synthesis. RA up-regulated the expression level of MAP kinase phosphatase-1 (MKP-1) and MKP-2, and the time course was correlated with the inhibitory effect of RA on activation of MAP kinases. Overexpression of wild-type MKP-1 inhibited the phosphorylation of JNK and p38 in cardiomyocytes. These data indicated that MKPs were involved in the inhibitory effect of RA on MAP kinases. Using specific RAR and RXR antagonists, we demonstrated that both RARs and RXRs were involved in regulating stretch- or Ang II-induced activation of MAP kinases. Our findings provide the first evidence that the anti-hypertrophic effect of RA is mediated by up-regulation of MKPs and inhibition of MAP kinase signaling pathways.