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.     

Atorvastatin suppresses oxidized LDL-induced dendritic cell-like differentiation of RAW264.7 cells regulated by the p38 MAPK pathway

The molecular signaling events leading to protection from oxidative stress-induced apoptosis upon contact inhibition have not been fully investigated. Previous research has indicated a role for mitogen-activated protein kinases (MAPKs) in the regulation of contact inhibition, and these proteins have also been associated with cell cycle regulation and stress-induced apoptosis. The potential role of the MAPK JNK-1 in the stress-response of actively proliferating and contact-inhibited cells was investigated. Actively proliferating normal fibroblasts (BJ) and fibrosarcoma cells (HT-1080) were stressed with H2O2, and levels of activated JNK-1 and cleaved PARP were ascertained. Similarly, these results were compared with levels of activated JNK-1 and cleaved PARP detected in H2O2-stressed confluent fibrosarcoma or contact-inhibited fibroblast cells. Contact-inhibited fibroblasts were protected from apoptosis in comparison to subconfluent fibroblasts, concurrent with decreased JNK-1 activation. Increased culture density of fibrosarcoma cells was not protective against apoptosis, and these cells did not demonstrate density-dependent alterations in the JNK-1 stress response. This decreased activation of JNK-1 in stressed, contact-inhibited cells did not appear to be dependent upon increased expression of MKP-1; however, over-expression of MKP-1 was sufficient to result in a slight decrease in H2O2-stimulated PARP cleavage. Increasing the antioxidant capacity of fibroblasts through NAC-treatment not only lessened H2O2-stimulated JNK-1 activation, but also did not influence the expression of MKP-1. Taken together, these results suggest that regulation of negative regulation of JNK-1 upon contact inhibition is protective against apoptosis, and that this regulation is independent of MKP-1.     

AtMEK1 mediates stress-induced gene expression of CAT1 catalase by triggering H2O2 production in Arabidopsis

Catalase and hydrogen peroxide (H(2)O(2)) have been extensively studied for their roles in various stress responses. However, little is known about the triggering mechanisms for stress-induced catalase gene expression or about H(2)O(2) production as a stress signal. It is reported here that ABA-, drought-, and salt stress-induced gene expression of CAT1 catalase is mediated by AtMEK1, an Arabidopsis MAPK kinase, by triggering H(2)O(2) signal production. Both CAT1 expression and AtMEK1 activity were activated by ABA, drought, and salt stresses. The mek1 mutant totally blocked stress-induced CAT1 expression and, interestingly, stress-induced H(2)O(2) production was also blocked. Over-expression of AtMEK1 significantly promoted stress-induced CAT1 expression, and also promoted H(2)O(2) production. These results conclusively indicate that stress-induced CAT1 expression is mediated by AtMEK1 and, furthermore, that the triggering of H(2)O(2) production might be involved in this process, as further proved by the observation that CAT1 expression was induced by applied H(2)O(2.) Surprisingly, the signalling mechanisms for stress-induced gene expression of CAT2 and CAT3 were very different from that of CAT1. Except for drought stress, expression of CAT2 or CAT3 was also activated by salt stress or ABA treatment, and AtMEK1 was not proved to be involved in the drought-induced expression of CAT2 or CAT3. Further studies showed that stomatal movement was much less sensitive to ABA in AtMEK1 mutant (mek1), and over-expression of AtMEK1 in Arabidopsis increased plant resistance to drought or salt stress, which further demonstrated that AtMEK1 is a crucial mediator in plant stress signal transduction.     

Deletion of the dual specific phosphatase-4 (DUSP-4) gene reveals an essential non-redundant role for MAP kinase phosphatase-2 (MKP-2) in proliferation and cell survival

Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP) implicated in a number of cancers. We examined the role of MKP-2 in the regulation of MAP kinase phosphorylation, cell proliferation, and survival responses in mouse embryonic fibroblasts (MEFs) derived from a novel MKP-2 (DUSP-4) deletion mouse. We show that serum and PDGF induced ERK-dependent MKP-2 expression in wild type MEFs but not in MKP-2(-/-) MEFs. PDGF stimulation of sustained ERK phosphorylation was enhanced in MKP-2(-/-) MEFs, whereas anisomycin-induced JNK was only marginally increased. However, marked effects upon cell growth parameters were observed. Cellular proliferation rates were significantly reduced in MKP-2(-/-) MEFs and associated with a significant increase in cell doubling time. Infection with adenoviral MKP-2 reversed the decrease in proliferation. Cell cycle analysis revealed a block in G(2)/M phase transition associated with cyclin B accumulation and enhanced cdc2 phosphorylation. MEFs from MKP-2(-/-) mice also showed enhanced apoptosis when stimulated with anisomycin correlated with increased caspase-3 cleavage and γH2AX phosphorylation. Increased apoptosis was reversed by adenoviral MKP-2 infection and correlated with selective inhibition of JNK signaling. Collectively, these data demonstrate for the first time a critical non-redundant role for MKP-2 in regulating cell cycle progression and apoptosis.     

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.     

AP-1-independent sensitization to oxidative stress-induced apoptosis by proteasome inhibitors

Hydrogen peroxide (H(2)O(2)) induces apoptosis of mesangial cells via c-Jun N-terminal kinase (JNK)-activator protein-1 (AP-1) and extracellular signal-regulated kinase (ERK)-AP-1 pathways. We recently found that subtoxic doses of proteasome inhibitors, MG132 and lactacystin, dramatically enhanced H(2)O(2)-induced apoptosis in mesangial cells. In this report, we examined molecular mechanisms involved in this phenomenon, especially focusing on AP-1 pathways. Reporter assays showed that MG132 induced activation of AP-1. However, pharmacological inhibitors of AP-1, retinoic acid, and curcumin, did not suppress the proapoptotic effect of MG132. Suppression of JNK-AP-1 by transfection with either a dominant-negative mutant of JNK or a dominant-negative mutant of c-Jun did not attenuate the apoptosis enhancement by MG132. Similarly, suppression of ERK-AP-1 by PD98059 or dominant-negative mutants of ERK did not affect the apoptosis-promoting effect of MG132. Interestingly, pretreatment with MG132 did not enhance activation of AP-1 by H(2)O(2). These data suggested a novel, AP-1-independent promotion of apoptosis by proteasome inhibitors.     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

Volume-sensitive outwardly rectifying chloride channels are involved in oxidative stress-induced apoptosis of mesangial cells

Volume-sensitive outwardly rectifying (VSOR) Cl- channels have been electrophysiologically identified in human and mouse mesangial cells, but the functional role of VSOR Cl- channels in mesangial cell apoptosis is not clear. The aim of the present study was to demonstrate the role of VSOR Cl- channels in oxidative stress-induced mesangial cell apoptosis. H2O2-induced Cl- currents showed phenotypic properties of VSOR Cl- channels, including outward rectification, voltage-dependent inactivation at more positive potentials, sensitivity to hyperosmolarity, and inhibition by VSOR Cl- channel blockers. Moreover, blockage of VSOR Cl- channels by DIDS (100 microM), NPPB (10 microM) or niflumic acid (10 microM) rescued mesangial cell apoptosis induced by H2O2. Treatment with 150 microM H2O2 for 2h resulted in significant reduction of cell volume, in contrast, nuclear condensation and/or fragmentation were not observed and the caspase-3 activity was also not increased. The early-phase alterations in cell volume were markedly abolished by pretreatment with VSOR Cl- channel blockers. We conclude that VSOR Cl- channels are involved in H2O2-induced apoptosis in cultured mesangial cells and its mechanism is associated with apoptotic volume decrease processes.     

Mitofusin-2 is a major determinant of oxidative stress-mediated heart muscle cell apoptosis

An inexorable loss of terminally differentiated heart muscle cells is a crucial causal factor for heart failure. Here, we have provided several lines of evidence to demonstrate that mitofusin-2 (Mfn-2; also called hyperplasia suppressor gene), a member of the mitofusin family, is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. First, oxidative stress with H(2)O(2) led to concurrent increases in Mfn-2 expression and apoptosis in cultured neonatal rat cardiomyocytes. Second, overexpression of Mfn-2 to a level similar to that induced by H(2)O(2) was sufficient to trigger myocyte apoptosis, which is associated with profound inhibition of Akt activation without altering ERK1/2 signaling. Third, Mfn-2 silencing inhibited oxidative stress-induced apoptosis in H9C2 cells, a cardiac muscle cell line. Furthermore, Mfn-2-induced myocyte apoptosis was abrogated by inhibition of caspase-9 (but not caspase-8) and by overexpression of Bcl-x(L) or enhanced activation of phosphatidylinositol 3-kinase-Akt, suggesting that inhibition of Akt signaling and activation of the mitochondrial death pathway are essentially involved in Mfn-2-induced heart muscle cell apoptosis. These results indicate that increased cardiac Mfn-2 expression is both necessary and sufficient for oxidative stress-induced heart muscle cell apoptosis, suggesting that Mfn-2 deregulation may be a crucial pathogenic element and a potential therapeutic target for heart failure.     

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.     

Alterations in subcellular localization of p38 MAPK potentiates endothelin-stimulated COX-2 expression in glomerular mesangial cells

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with mitogenic actions linked to activation of tyrosine kinase signaling pathways. ET-1 induces cyclooxygenase-2 (COX-2), an enzyme that converts arachidonic acid to pro-inflammatory eicosanoids. Activation of each of the three major mitogen-activated protein kinase (MAPK) pathways, ERK1/2, JNK/SAPK, and p38 MAPK (p38), have been shown to enhance the expression of COX-2. Negative regulation of MAPK may occur via a family of dual specificity phosphatases referred to as mitogen-activated protein kinase phosphatases (MKP). The goal of this work was to test the hypothesis that wild type MKP-1 regulates the expression of ET-1-induced COX-2 expression by inhibiting the activation of p38 in cultured glomerular mesangial cells (GMC). An adenovirus expressing both wild type and a catalytically inactive mutant of MKP-1 (MKP-1/CS) were constructed to study ET-1-regulated MAPK signaling and COX-2 expression in cultured GMC. ET-1 stimulated the phosphorylation of ERK and p38 alpha MAPK and induced the expression of COX-2. Expression of COX-2 was partially blocked by U0126, a MEK inhibitor, and SB 203580, a p38 MAPK inhibitor. Adenoviral expression of MKP-1/CS augmented basal and ET-1-induced phosphorylation of p38 alpha MAPK with less pronounced effects on ERK1/2 phosphorylation. Ectopic expression of wild type MKP-1 blocked the phosphorylation of p38 alpha MAPK by ET-1 but increased the phosphorylation of p38 gamma MAPK. Co-precipitation studies demonstrated association of MKP-1 with p38 alpha MAPK and ERK1/2. Immunofluorescent image analysis demonstrated trapping of phospho-p38 MAPK in the cytoplasm by MKP-1/CS/green fluorescent protein. ET-1-stimulated expression of COX-2 was increased in MKP-1/CS versus LacZ or green fluorescent protein-infected control cells. These results indicate that MKP-1 demonstrates a relative selectivity for p38 alpha MAPK versus p38 gamma MAPK in GMC and is likely to indirectly regulate the expression of COX-2.     

Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H(2)O(2)). A yeast two-hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1. PP5 directly dephosphorylated an essential phospho-threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo. The interaction between PP5 and ASK1 was induced by H(2)O(2) treatment and was followed by the decrease in ASK1 activity. PP5 inhibited not only H(2)O(2)-induced sustained activation of ASK1 but also ASK1-dependent apoptosis. Thus, PP5 appears to act as a physiological inhibitor of ASK1-JNK/p38 pathways by negative feedback.     

Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation

The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca²⁺ signalling regulates the ASK1–p38 MAP kinase cascade. Ca²⁺ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca²⁺ signalling between CaMKII and p38 MAP kinase.     

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.