Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activation of both the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK) signal transduction pathways. The MAPK phosphatase, MKP-1, is capable of inactivating both SAPK and p38 MAPK in vivo. To determine whether MKP-1-mediated inhibition of SAPK and/or p38 MAPK activity provided cytoprotection against UV-induced apoptosis, a U937 cell line conditionally expressing MKP-1 from the human metallothionein IIa promoter was established. Conditional expression of MKP-1 was found to abolish UV-induced SAPK and p38 MAPK activity, and inhibit UV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 was also found to inhibit other biochemical events associated with apoptosis, including activation of caspase-3 and the proteolytic cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase. These findings demonstrate that MKP-1 acts at a site upstream of caspase activation within the apoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its ability to inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 had no effect on UV-induced apoptosis in U937 cells. Furthermore, by titrating the level of MKP-1 expression it was found that MKP-1 inhibited UV-induced SAPK activity, DNA fragmentation, and caspase-3 activation in a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibit UV-induced activation of SAPK, did not provide a similar cytoprotection against UV-induced apoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection against UV-induced apoptosis by inhibiting UV-induced SAPK activity.     

Regulation of mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle cells

Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dual-specificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs.     

Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase

MAP kinase phosphatase-3 (MKP-3) dephosphorylates phosphotyrosine and phosphothreonine and inactivates selectively ERK family mitogen-activated protein (MAP) kinases. MKP-3 was activated by direct binding to purified ERK2. Activation was independent of protein kinase activity and required binding of ERK2 to the noncatalytic amino-terminus of MKP-3. Neither the gain-of-function Sevenmaker ERK2 mutant D319N nor c-Jun amino-terminal kinase-stress-activated protein kinase (JNK/SAPK) or p38 MAP kinases bound MKP-3 or caused its catalytic activation. These kinases were also resistant to enzymatic inactivation by MKP-3. Another homologous but nonselective phosphatase, MKP-4, bound and was activated by ERK2, JNK/SAPK, and p38 MAP kinases. Catalytic activation of MAP kinase phosphatases through substrate binding may regulate MAP kinase activation by a large number of receptor systems.     

The dual specificity phosphatases M3/6 and MKP-3 are highly selective for inactivation of distinct mitogen-activated protein kinases

The mitogen-activated protein (MAP) kinase family includes extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38/RK/CSBP (p38) as structurally and functionally distinct enzyme classes. Here we describe two new dual specificity phosphatases of the CL100/MKP-1 family that are selective for inactivating ERK or JNK/SAPK and p38 MAP kinases when expressed in COS-7 cells. M3/6 is the first phosphatase of this family to display highly specific inactivation of JNK/SAPK and p38 MAP kinases. Although stress-induced activation of p54 SAPKbeta, p46 SAPKgamma (JNK1) or p38 MAP kinases is abolished upon co-transfection with increasing amounts of M3/6 plasmid, epidermal growth factor-stimulated ERK1 is remarkably insensitive even to the highest levels of M3/6 expression obtained. In contrast to M3/6, the dual specificity phosphatase MKP-3 is selective for inactivation of ERK family MAP kinases. Low level expression of MKP-3 blocks totally epidermal growth factor-stimulated ERK1, whereas stress-induced activation of p54 SAPKbeta and p38 MAP kinases is inhibited only partially under identical conditions. Selective regulation by M3/6 and MKP-3 was also observed upon chronic MAP kinase activation by constitutive p21(ras) GTPases. Hence, although M3/6 expression effectively blocked p54 SAPKbeta activation by p21(rac) (G12V), ERK1 activated by p21(ras) (G12V) was insensitive to this phosphatase. ERK1 activation by oncogenic p21(ras) was, however, blocked totally by co-expression of MKP-3. This is the first report demonstrating reciprocally selective inhibition of different MAP kinases by two distinct dual specificity phosphatases.     

The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity

We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S. (1996) J. Biol. Chem. 271, 27205-27208). We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected. Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases. A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta. Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity. In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations. Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta. Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.     

Interleukin-1beta-induced cyclooxygenase-2 expression requires activation of both c-Jun NH2-terminal kinase and p38 MAPK signal pathways in rat renal mesangial cells

The inflammatory cytokine interleukin-1beta (IL-1beta) induces cyclooxygenase-2 (Cox-2) expression with a concomitant release of prostaglandins from glomerular mesangial cells. We reported previously that IL-1beta rapidly activates the c-Jun NH2-terminal/stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein kinase (MAPK) and also induces Cox-2 expression and prostaglandin E2 (PGE2) production. The current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 JNK2/SAPKbeta reduces Cox-2 expression and PGE2 production stimulated by IL-1beta. Similarly, overexpression of the kinase-dead form of p38 MAPK also inhibits IL-1beta-induced Cox-2 expression and PGE2 production. These results suggest that activation of both JNK/SAPK and p38 MAPK is required for Cox-2 expression after IL-1beta activation. Furthermore, our experiments confirm that IL-1beta activates MAP kinase kinase-4 (MKK4)/SEK1, MKK3, and MKK6 in renal mesangial cells. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta- induced Cox-2 expression with inhibition of both JNK/SAPK and p38 MAPK phosphorylation. Overexpression of the kinase-dead form of MKK3 or MKK6 demonstrated that either of these two mutant kinases inhibited IL-1beta-induced p38 MAPK phosphorylation and Cox-2 expression but not JNK/SAPK phosphorylation and activation. This study suggests that the activation of both JNK/SAPK and p38 MAPK signaling cascades is required for IL-1beta-induced Cox-2 expression and PGE2 synthesis.     

Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.     

The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation

Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.     

Adrenocorticotropin induction of stress-activated protein kinase in the adrenal cortex in vivo

A broad array of stressors induce ACTH release from the anterior pituitary, with consequent stimulation of the adrenal cortex and release of glucocorticoids critical for survival of the animal. ACTH stimulates adrenocortical gene expression in vivo and inhibits adrenocortical cell proliferation. Binding of ACTH to its G-protein-coupled receptor stimulates the production of cAMP and activation of the protein kinase A pathway. The stress-activated protein kinases (SAPKs) (or c-Jun N-terminal kinases) and the extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase family of serine/threonine kinases, which have recently been implicated in G-protein-coupled receptor intracellular signaling. The SAPKs are preferentially induced by osmotic stress and UV light, whereas the ERKs are preferentially induced by growth factors and proliferative signals in cultured cells. In these studies, ACTH stimulated SAPK activity 3-4-fold both in the adrenal cortex in vivo and in the Y1 adrenocortical cell line. 12-O-Tetradecanoylphorbol-13-acetate but not cAMP induced SAPK activity in Y1 cells. The isoquinolinesulfonamide inhibitors H-8 and H-89 blocked ACTH induction of SAPK activity at protein kinase C inhibitory doses but not at protein kinase A inhibitory doses. The calcium chelating agent EGTA inhibited ACTH-induced SAPK activity and the calcium ionophore A23187 induced SAPK activity 3-fold. In contrast with the induction of SAPK by ACTH, ERK activity was inhibited in the adrenal cortex in vivo and in Y1 adrenal cells. Together these findings suggest that ACTH induces SAPK activity through a PKC and Ca+2-dependent pathway. The induction of SAPK and inhibition of ERK by ACTH in vivo may preferentially regulate target genes involved in the adrenocortical stress responses in the whole animal.     

Murine hepatitis virus strain 3 induces the macrophage prothrombinase fgl-2 through p38 mitogen-activated protein kinase activation

The clinical syndrome of acute liver failure produced by fulminant viral hepatitis can be reproduced in mice by infection with murine hepatitis virus strain 3 (MHV-3). Although it is clear that MHV-3-induced hepatitis depends upon macrophage activation and the expression of a specific prothrombinase, fgl-2, the signaling pathways involved in virally stimulated cell activation are unclear. Since we had previously found that MHV-3 induces the tyrosine phosphorylation of cellular proteins, we investigated the roles of the mitogen-activated protein kinase (MAPK) proteins. In a series of Western blots, immunoprecipitation and in vitro kinase assay studies, we found that both the extracellular signal-related kinase (ERK) and p38 MAPK proteins are tyrosine-phosphorylated and activated following exposure of murine peritoneal exudative macrophages (PEM) to MHV-3. Although p38 phosphorylation and activity are induced soon after MHV-3 exposure, peaking by 1-5 min, ERK phosphorylation and activity increase more gradually, peaking at 20-30 min and gradually fading thereafter. Interestingly, whereas selective p38 inhibition with SB203580 (1-20 microM) abolished the virally stimulated induction of fgl-2 mRNA, protein, and functional activity, selective ERK inhibition with PD98059 (1-50 microM) limited fgl-2 functional activity but had little to no effect on fgl-2 mRNA or protein levels. Moreover, whereas inhibition of ERK had no effect on p38 activity, p38 inhibition consistently increased MHV-3-induced ERK activity. To ensure that these pathways were relevant in vivo, MHV-3 was injected intraperitoneally, and peritoneal exudative macrophages were collected. Again, MHV-3 exposure led to increased p38 and ERK tyrosine phosphorylation. These data argue that MHV-3 induces tightly interconnected ERK and p38 MAPK cascades in the macrophage both in vitro and in vivo. Although the ERK and p38 MAPK proteins have discordant effects at the level of fgl-2 expression, both converge at the level of its activity, suggesting that targeted MAPK inhibition may ultimately be useful in the modulation of viral hepatitis.     

Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes

Mammalian cells contain at least three signaling systems which are structurally related to the mitogen-activated protein kinase (MAPK) pathway. Growth factors acting through Ras primarily stimulate the Raf/MEK/MAPK cascade of protein kinases. In contrast, many stress-related signals such as heat shock, inflammatory cytokines, and hyperosmolarity induce the MEKK/SEK(MKK4)/SAPK(JNK) and/or the MKK3 or MKK6/p38(hog) pathways. Physiological agonists of these pathway types are either qualitatively or quantitatively distinct, suggesting few common proximal signaling elements, although past studies performed in vitro, or in cells using transient over-expression, reveal interaction between the components of all three pathways. These studies suggest a high degree of cross-talk apparently not seen in vivo. We have examined the possible molecular basis of the differing agonist profiles of these three MAPK pathways. We report preferential association between MAP kinases and their activators in eukaryotic cells. Furthermore, using the yeast 2-hybrid system, we show that association between these components can occur independent of additional eukaryotic proteins. We show that SAPK(JNK) or p38(hog) activation is specifically impaired by co-expression of cognate dominant negative MAP kinase kinase mutants, demonstrating functional specificity at this level. Further divergence and insulation of the stress pathways occurs proximal to the MAPK kinases since activation of the MAPK kinase kinase MEKK results in SAPK(JNK) activation but does not cause p38(hog) phosphorylation. Therefore, in intact cells, the three MAPK pathways may be independently regulated and their components show specificity in their interaction with cognate cascade members. The degree of intermolecular specificity suggests that mammalian MAPK signaling pathways may remain distinct without the need for specific scaffolding proteins to sequester components of individual pathways.     

Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase

Collagenase-3 (matrix metalloproteinase-13, MMP-13) is a recently identified human MMP with an exceptionally wide substrate specificity and restricted tissue-specific expression. Here we show that MMP-13 expression is induced in normal human skin fibroblasts cultured within three-dimensional collagen gel resulting in production and proteolytic activation of MMP-13. Induction of MMP-13 mRNAs by collagen gel was potently inhibited by blocking antibodies against alpha1 and alpha2 integrin subunits and augmented by activating antibody against beta1 integrin subunit, indicating that both alpha1 beta1 and alpha2 beta1 integrins mediate the MMP-13-inducing cellular signal generated by three-dimensional collagen. Collagen-related induction of MMP-13 expression was dependent on tyrosine kinase activity, as it was abolished by treatment of fibroblasts with tyrosine kinase inhibitors genistein and herbimycin A. Contact of fibroblasts to three-dimensional collagen resulted in simultaneous activation of mitogen-activated protein kinases (MAPKs) in three distinct subgroups: extracellular signal-regulated kinase (ERK)1 and ERK2, Jun N-terminal kinase/stress-activated protein kinase, and p38. Induction of MMP-13 expression was inhibited by treatment of fibroblasts with a specific p38 inhibitor, SB 203580, whereas blocking the ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by PD 98059, a selective inhibitor of MEK1,2 activation potently augmented MMP-13 expression. Furthermore, specific activation of ERK1,2 pathway by 12-O-tetradecanoylphorbol-13-acetate markedly suppressed MMP-13 expression in dermal fibroblasts in collagen gel. These results show that collagen-dependent induction of MMP-13 in dermal fibroblasts requires p38 activity, and is inhibited by activation of ERK1,2. Therefore, the balance between the activity of ERK1,2 and p38 MAPK pathways appears to be crucial in regulation of MMP-13 expression in dermal fibroblasts, suggesting that p38 MAPK may serve as a target for selective inhibition of collagen degradation, e.g. in chronic dermal ulcers.     

Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB

We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.     

Differential activation of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases confers cadmium-induced HSP70 expression in 9L rat brain tumor cells

We have reported that treatment with CdCl2 at 40-100 microM induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 microM) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38(MAPK)), while that in cells treated with a low concentration (60 microM) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 microM cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38(MAPK). By contrast, in 60 microM cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38(MAPK) and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38(MAPK) and ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.     

Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stress-activated protein kinases, in in vitro propagated endothelial cells

We investigated the involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways in human endothelial cells in response to shear stress and alterations of these kinases in in vitro-propagated endothelial cells (ECs). Potent activation (10-fold) of extracellular signal-regulated kinase (ERK2), a member of the MAPK family, occurred within 10 min of shear stress (5 dynes/cm2), whereupon rapid inactivation ensued. Shear stress also induced activation of stress-activated protein kinase (SAPK) or c-Jun NH2-terminal protein kinase (JNK) in ECs. Suramin pretreatment completely inhibited shear stress stimulation of ERK2, but not SAPK/JNK, highlighting a role for growth factor receptors in ERK activation. Translocation of ERK2 from the cytoplasm to the nucleus was observed in shear-stressed endothelial cells. In addition, we compared activities of MAPKs in shear-stressed cells derived from passages 4 and 10 (older). The magnitude of ERK2 activation was significantly lower in aged ECs compared to those of passage 4, while SAPK/JNK was not altered in the in vitro aged ECs. A similar level of ERK2 activation was found in both young and older cells stimulated with phorbol-12-myristate-13-acetate (PMA), indicating an age-related alteration of the plasma membrane. Taken together, these findings suggest that MAP kinase activation may be crucial for the expression of many genes in ECs stimulated by shear stress, and that an alteration in MAPK activities could contribute to the age-related decline in proliferative capacity.     

Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest

The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK), and p38. Although MAPK is essential for meiotic processes in Xenopus oocytes and the spindle assembly checkpoint in Xenopus egg extracts, the role of members of the MAPK superfamily in M phase or the spindle assembly checkpoint during somatic cell cycles has not been elucidated. The kinase p38, but not MAPK or JNK, was activated in mammalian cultured cells when the cells were arrested in M phase by disruption of the spindle with nocodazole. Addition of activated recombinant p38 to Xenopus cell-free extracts caused arrest of the extracts in M phase, and injection of activated p38 into cleaving embryos induced mitotic arrest. Treatment of NIH 3T3 cells with a specific inhibitor of p38 suppressed activation of the checkpoint by nocodazole. Thus, p38 functions as a component of the spindle assembly checkpoint in somatic cell cycles.     

Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis

Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.