Rat parathyroid hormone 1-34 signals through the MEK/ERK pathway to induce cardiac hypertrophy

This study aimed to characterize the role of the mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in cardiac hypertrophy induced by parathyroid hormone (PTH). Various concentrations of rat PTH1-34 were used to induce hypertrophy in neonatal rat ventricular cardiomyocytes, and the effects were compared with control cells and those treated with PD98059, a selective inhibitor of MEK1. Hypertrophy was assessed in terms of cell diameter, atrial natriuretic peptide (ANP) mRNA expression and protein synthesis; the MEK/ERK pathway was assessed by measuring levels of phosphorylated ERK1/2. Treatment with PTH1-34 at 100 nM for 24 h effectively induced cardiac hypertrophy (increased cell diameter, protein synthesis and ANP mRNA expression) and also increased levels of phosphorylated ERK1/2 compared with normal control cells. Treatment with PTH1-34 plus PD98059 significantly attenuated these changes. These results demonstrate that inhibition of the MEK/ERK pathway blocks PTH1-34-induced cardiac hypertrophy, suggesting that PTH1-34 might signal through the MAPK pathway to induce hypertrophy in cardiomyocytes.     

Mechanical allodynia but not thermal hyperalgesia is impaired in mice deficient for ERK2 in the central nervous system

Extracellular signal-regulated kinase (ERK) plays critical roles in pain plasticity. However, the specific contribution of ERK2 isoforms to pain plasticity is not necessarily elucidated. Here we investigate the function of ERK2 in mouse pain models. We used the Cre-loxP system to cause a conditional, region-specific, genetic deletion of Erk2. To induce recombination in the central nervous system, Erk2-floxed mice were crossed with nestin promoter-driven cre transgenic mice. In the spinal cord of resultant Erk2 conditional knockout (CKO) mice, ERK2 expression was abrogated in neurons and astrocytes, but indistinguishable in microglia compared to controls. Although Erk2 CKO mice showed a normal baseline paw withdrawal threshold to mechanical stimuli, these mice had a reduced nociceptive response following a formalin injection to the hind paw. In a partial sciatic nerve ligation model, Erk2 CKO mice showed partially restored mechanical allodynia compared to control mice. Interestingly, thermal hyperalgesia was indistinguishable between Erk2 CKO and control mice in this model. In contrast to Erk2 CKO mice, mice with a targeted deletion of ERK1 did not exhibit prominent anomalies in these pain models. In Erk2 CKO mice, compensatory hyperphosphorylation of ERK1 was detected in the spinal cord. However, ERK1 did not appear to influence nociceptive processing because the additional inhibition of ERK1 phosphorylation using MEK (MAPK/ERK kinase) inhibitor SL327 did not produce additional changes in formalin-induced spontaneous behaviors in Erk2 CKO mice. Together, these results indicate that ERK2 plays a predominant and/or specific role in pain plasticity, while the contribution of ERK1 is limited.     

FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants.     

Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes.

Stimulation of T cells with antibodies directed towards the T cell receptor complex results in the activation of mitogen-associated protein kinase (MAPK). Two pathways have been described in other cell types that can lead to MAPK activation. One of these pathways involves the activation of Ras, leading to the activation of Raf-1, and the subsequent activation of MEK (MAPK or ERK kinase). The contribution of this pathway in T cells for anti-CD3 or phorbol myristate acetate (PMA)-mediated MAPK activation was examined. We detected the kinase activities of Raf-1 and MEK towards their substrates (MEK for Raf-1 and MAPK for MEK) in this pathway leading to the activation of MAPK. Stimulation of the T cells with either anti-CD3 antibody or PMA resulted in a rapid activation of both Ras and Raf-1. MEK activity towards kinase-active or -inactive recombinant MAPK also increased upon stimulation. In addition, both MAPK and p90rsk were activated in these cells. We suggest that activation of MAPK and the subsequent activation of ribosomal S6 kinase (p90rsk) occurs by the Ras/Raf-1/MEK cascade in T lymphocytes stimulated by ligation of the T cell receptor complex.     

Raf-1 antagonizes erythroid differentiation by restraining caspase activation

The Raf kinases are key signal transducers activated by mitogens or oncogenes. The best studied Raf isoform, Raf-1, was identified as an inhibitor of apoptosis by conventional and conditional gene ablation in mice. c-raf-1(-)(/)(-) embryos are growth retarded and anemic, and die at midgestation with anomalies in the placenta and fetal liver. Here, we show that Raf-1-deficient primary erythroblasts cannot be expanded in culture due to their accelerated differentiation into mature erythrocytes. In addition, Raf-1 expression is down-regulated in differentiating wild-type cells, whereas overexpression of activated Raf-1 delays differentiation. As recently described for human erythroid precursors, we find that caspase activation is necessary for the differentiation of murine fetal liver erythroblasts. Differentiation-associated caspase activation is accelerated in erythroid progenitors lacking Raf-1 and delayed by overexpression of the activated kinase. These results reveal an essential function of Raf-1 in erythropoiesis and demonstrate that the ability of Raf-1 to restrict caspase activation is biologically relevant in a context distinct from apoptosis.     

Protective role of Raf-1 in Salmonella-induced macrophage apoptosis

Invasive Salmonella induces macrophage apoptosis via the activation of caspase-1 by the bacterial protein SipB. Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation. Raf-1 degradation is SipB- and caspase-1-dependent, and is prevented by proteasome inhibitors. To study the functional significance of Raf-1 in this process, the c-raf-1 gene was inactivated by Cre-loxP-mediated recombination in vivo. Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis. Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappaB pathways is normal in Raf-1-deficient macrophages, and mitochondrial fragility is not increased. Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.     

Rapamycin and UCN-01 synergistically induce apoptosis in human leukemia cells through a process that is regulated by the Raf-1/MEK/ERK, Akt, and JNK signal transduction pathways

Interactions between the protein kinase C and Chk1 inhibitor UCN-01 and rapamycin in human leukemia cells have been investigated in relation to apoptosis induction. Treatment of U937 monocytic leukemia cells with rapamycin (10 nmol/L) in conjunction with a minimally toxic concentration of UCN-01 (100 nmol/L) for 36 hours resulted in marked potentiation of mitochondrial injury (i.e., loss of mitochondrial membrane potential and cytosolic release of cytochrome c, AIF, and Smac/DIABLO), caspase activation, and apoptosis. The release of cytochrome c, AIF, and Smac/DIABLO were inhibited by BOC-D-fmk, indicating that their release was caspase dependent. These events were associated with marked down-regulation of Raf-1, MEK, and ERK phosphorylation, diminished Akt activation, and enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK). Coadministration of UCN-01 and rapamycin reduced the expression levels of the antiapoptotic members of the Bcl-2 family Mcl-1 and Bcl-xL and diminished the expression of cyclin D1 and p34(cdc2). Furthermore, enforced expression of a constitutively active MEK1 or, to a lesser extent, myristoylated Akt construct partially but significantly attenuated UCN-01/rapamycin-mediated lethality in both U937 and Jurkat cell systems. Finally, inhibition of the stress-related JNK by SP600125 or by the expression of a dominant-negative mutant of c-Jun significantly attenuated apoptosis induced by rapamycin/UCN-01. Together, these findings indicate that the mammalian target of rapamycin inhibitor potentiates UCN-01 cytotoxicity in a variety of human leukemia cell types and suggest that inhibition of both Raf-1/MEK/ERK and Akt cytoprotective signaling pathways as well as JNK activation contribute to this phenomenon.     

The RAS/Raf1/MEK/ERK signaling pathway facilitates VSV-mediated oncolysis: implication for the defective interferon response in cancer cells.

Vesicular stomatitis virus (VSV) can replicate in malignant cells more efficiently than in normal cells. Although the selective replication appears to be caused by defects in the interferon (IFN) system in malignant cells, the mechanisms which render these cells less responsive to IFN remain poorly understood. Here we present evidence that an activated RAS/Raf1/MEK/ERK pathway plays a critical role in the defects. NIH 3T3 or human primary cells stably expressing active RAS or Raf1 were rapidly killed by VSV. Although IFNalpha treatment no longer protected the RAS- or Raf1-overexpressing cells from VSV infection, responsiveness to IFNalpha was restored following treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Similarly, human cancer-derived cell lines became more responsive to IFNalpha in conjunction with U0126 treatment. Intriguingly, dual treatment with both IFNalpha and U0126 severely reduced the levels of viral RNAs in the infected cells. Moreover, cancer cells showed defects in inducing an IFNalpha-responsive factor, MxA, which is known to block VSV RNA synthesis, and U0126 restored the MxA expression. Our observations suggest that activation of the extracellular signal-regulated protein kinase (ERK) signaling leads to the defect in IFNalpha-mediated upregulation of MxA protein, which facilitates VSV oncolysis. In view of the fact that 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway, VSV would be an ideal oncolytic virus for targeting such cancers.     

Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy

Activation of mammalian sterile 20-like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity-dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy.     

Proapoptotic Rassf1A/Mst1 signaling in cardiac fibroblasts is protective against pressure overload in mice

Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Drosophila Hippo, the master regulator of cell death, proliferation, and organ size in flies. It is the chief component of the mammalian Hippo pathway and promotes apoptosis and inhibits compensatory cardiac hypertrophy, playing a critical role in mediating heart failure. How Mst1 is regulated, however, remains unclear. Using genetically altered mice in which expression of the tumor suppressor Ras-association domain family 1 isoform A (Rassf1A) was modulated in a cell type-specific manner, we demonstrate here that Rassf1A is an endogenous activator of Mst1 in the heart. Although the Rassf1A/Mst1 pathway promoted apoptosis in cardiomyocytes, thereby playing a detrimental role, the same pathway surprisingly inhibited fibroblast proliferation and cardiac hypertrophy through both cell-autonomous and autocrine/paracrine mechanisms, playing a protective role during pressure overload. In cardiac fibroblasts, the Rassf1A/Mst1 pathway negatively regulated TNF-α, a key mediator of hypertrophy, fibrosis, and resulting cardiac dysfunction. These results suggest that the functional consequence of activating the proapoptotic Rassf1A/Mst1 pathway during pressure overload is cell type dependent in the heart and that suppressing this mechanism in cardiac fibroblasts could be detrimental.     

Significant neuroprotection against ischemic brain injury by inhibition of the MEK1 protein kinase in mice: exploration of potential mechanism associated with apoptosis

MEK1/2 is a serine/threonine protein kinase that phosphorylates and activates extracellular signal-responsive kinase (ERK)1/2. In the present study we explored the role of MEK1/2 in ischemic brain injury using a selective MEK1/2 inhibitor, SL327, in mice. C57BL/6 mice were subjected to a 30-min occlusion of the middle cerebral artery (MCAO) followed by reperfusion. Western blot analysis demonstrated the immediate activation of MEK/ERK after reperfusion (within the first 10 min) in the ischemic brain; this activation was dose dependently blocked by SL327 (10-100 mg/kg, i.p.). A single dose of SL327 (100 mg/kg) administered 15 min before or 25 min after the onset of ischemia resulted in 63.6% (n = 18, p < 0.001) and 50.7% (n = 18, p < 0.01) reduction in infarct size, respectively, compared with vehicle-treated mice. Similarly, SL327 significantly reduced neurological deficits 1 to 3 days after reperfusion (n = 12, p < 0.01). The salutary effect of SL327-induced neuroprotection was independent of mitochondrial cytochrome c release or caspase-8-mediated apoptosis; however, SL327 markedly suppressed the levels of active caspase-3 and DNA fragmentation (as a measure of apoptosis) after ischemia/reperfusion. Our data suggest that the inhibition of MEK1/2 results in neuroprotection from reperfusion injury and that this protection may be associated with the reduction in apoptosis.     

The role of Raf-1 in the regulation of extracellular signal-regulated kinase 2 by the T cell antigen receptor

Triggering of the T cell antigen receptor (TCR) complex activates the serine/threonine kinase Raf-1 whose function is necessary for TCR induction of the interleukin 2 gene. Raf-1 has been identified as a candidate mitogen-activated protein (MAP) kinase kinase kinase (MKKK) and thus has the potential to couple the TCR to the activation of the MAP kinases such as ERK2. In the present study, the role of Raf-1 in ERK2 regulation of ERK2 in T cells has been explored. A constitutively active Raf-1 kinase, v-raf, or a dominant inhibitory Raf-1 mutant were expressed transiently from the pEF BOS vector in Jurkat cells and the effects of these Raf-1 mutants on a coexpressed ERK2 reporter was assessed. The action of the constitutively active Raf-1 was to stimulate the ERK2 kinase, whereas the dominant negative version of Raf- 1 inhibited the ERK2 activation induced by triggering of the TCR. These data indicate a role for Raf-1 in the regulation of ERK2 in T cells.     

The MEKK1-JNK pathway plays a protective role in pressure overload but does not mediate cardiac hypertrophy

Mitogen-activated protein kinase kinase kinase (MEKK1) mediates activation of c-Jun NH(2)-terminal kinase (JNK). Although previous studies using cultured cardiac myocytes have suggested that the MEKK1-JNK pathway plays a key role in hypertrophy and apoptosis, its effects in cardiac hypertrophy and apoptosis are not fully understood in adult animals in vivo. We examined the role of the MEKK1-JNK pathway in pressure-overloaded hearts by using mice deficient in MEKK1. We found that transverse aortic banding significantly increased JNK activity in Mekk1(+/+) but not Mekk1(-/-) mice, indicating that MEKK1 mediates JNK activation by pressure overload. Nevertheless, pressure overload caused significant levels of cardiac hypertrophy and expression of atrial natriuretic factor in Mekk1(-/-) animals, which showed higher mortality and lung/body weight ratio than were seen in controls. Fourteen days after banding, Mekk1(-/-) hearts were dilated, and their left ventricular ejection fraction was low. Pressure overload caused elevated levels of apoptosis and inflammatory lesions in these mice and produced a smaller increase in TGF-beta and TNF-alpha expression than occurred in wild-type controls. Thus, MEKK1 appears to be required for pressure overload-induced JNK activation and cytokine upregulation but to be dispensable for pressure overload-induced cardiac hypertrophy. MEKK1 also prevents apoptosis and inflammation, thereby protecting against heart failure and sudden death following cardiac pressure overload.     

Signal transduction in heart failure

Heart failure is the final culmination of protracted disease status precipitated by underlying ischemic disease, valvular insufficiency and viral myocarditis. The factors that lead to the development of heart failure are still not fully understood. In mammalian cells, four parallel kinase cascades have been described that finally lead to the activation of members of the mitogen-activated protein kinase(MAPK) family, such as ERKs (p42 and p44), JNK and p38 protein kinase. Apoptosis signal-regulating kinase 1 (ASK1), an upstream activator of JNK and p38, was shown to promote heart dysfunction and dilation as well as cardiac fibrosis. Meanwhile, not only myocyte apoptosis but also myocardial interstitial changes such as extracellular matrix deposition, activation of fibroblasts, and narrowing of vessel lumens play important roles for the progression of heart failure.     

Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats.

A growing body of evidence has suggested that oxidative stress causes cardiac injuries during ischemia/reperfusion. Extracellular signal-regulated kinases (ERKs) have been reported to play pivotal roles in many aspects of cell functions and to be activated by oxidative stress in some types of cells. In this study, we examined oxidative stress-evoked signal transduction pathways leading to activation of ERKs in cultured cardiomyocytes of neonatal rats, and determined their role in oxidative stress-induced cardiomyocyte injuries. ERKs were transiently and concentration-dependently activated by hydrogen peroxide (H2O2) in cardiac myocytes. A specific tyrosine kinase inhibitor, genistein, suppressed H2O2-induced ERK activation, while inhibitors of protein kinase A and C or Ca2+ chelators had no effects on the activation. When CSK, a negative regulator of Src family tyrosine kinases, or dominant-negative mutant of Ras or of Raf-1 kinase was overexpressed, activation of transfected ERK2 by H2O2 was abolished. The treatment with H2O2 increased the number of cells stained positive by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and induced formation of DNA ladder and activation of CPP32, suggesting that H2O2 induced apoptosis of cardiac myocytes. When H2O2-induced activation of ERKs was selectively inhibited by PD98059, the number of cardiac myocytes which showed apoptotic death was increased. These results suggest that Src family tyrosine kinases, Ras and Raf-1 are critical for ERK activation by hydroxyl radicals and that activation of ERKs may play an important role in protecting cardiac myocytes from apoptotic death following oxidative stress.     

Therapies to slow polycystic kidney disease

Advances in the understanding of cystogenesis and availability of animal models orthologous to human autosomal dominant polycystic kidney disease (ADPKD) and recessive polycystic kidney disease (ARPKD) will likely facilitate the development of treatments for these diseases. Proteins mutated in ADPKD and ARPKD, as well as in several animal models, are localized to renal primary cilia. These are thought to have a sensory function and contribute to the regulation of the intracellular calcium ([Ca2+]i). It seems likely that the maintenance of a differentiated renal epithelial phenotype, characterized by controlled fluid secretion and cell proliferation, requires precise functional coordination of cAMP and Ras/Raf/MEK/ERK signaling by [Ca2+]i. [Ca2+]i alterations, linked to genetic defects causing polycystic kidney disease, may hinder negative feedback mechanisms that control cAMP and Ras/Raf/MEK/ERK signaling, and result in increased fluid secretion and cell proliferation. cAMP levels, Raf kinase activities and ERK phosphorylation are increased in polycystic kidneys. There is also evidence of abnormal cross-talk between cAMP and MAPK pathways, that can be reproduced in wild-type cells by altering [Ca2+]i. While cAMP inhibits Ras-Raf-1-stimulated phosphorylation of ERK in normal kidney cells, it markedly increases B-Raf kinase activity and ERK phosphorylation in polycystic kidney cells. Treatment strategies should probably be aimed at increasing [Ca2+]i, inhibiting Ras/Raf/MEK/ERK signaling or lowering cAMP in the distal nephron and collecting duct. Vasopressin is the major adenylyl cyclase agonist in the collecting duct principal cells via a V2 receptor. OPC31260, a V2 receptor antagonist, lowers renal cAMP and markedly inhibits cystogenesis in four animal models of polycystic kidney disease, three of which are orthologous to human diseases (PCK rat, ARPKD; pcy mouse, adolescent nephronophthisis; Pkd2WS25/- mouse, ADPKD). The renal selectivity and safety profile of this class of drugs make it an excellent candidate for clinical trials.     

Kinase suppressor of Ras-1 protects intestinal epithelium from cytokine-mediated apoptosis during inflammation

TNF plays a pathogenic role in inflammatory bowel diseases (IBDs), which are characterized by altered cytokine production and increased intestinal epithelial cell apoptosis. In vitro studies suggest that kinase suppressor of Ras-1 (KSR1) is an essential regulatory kinase for TNF-stimulated survival pathways in intestinal epithelial cell lines. Here we use a KSR1-deficient mouse model to study the role of KSR1 in regulating intestinal cell fate during cytokine-mediated inflammation. We show that KSR1 and its target signaling pathways are activated in inflamed colon mucosa. Loss of KSR1 increases susceptibility to chronic colitis and TNF-induced apoptosis in the intestinal epithelial cell. Furthermore, disruption of KSR1 expression enhances TNF-induced apoptosis in mouse colon epithelial cells and is associated with a failure to activate antiapoptotic signals including Raf-1/MEK/ERK, NF-kappaB, and Akt/protein kinase B. These effects are reversed by WT, but not kinase-inactive, KSR1. We conclude that KSR1 has an essential protective role in the intestinal epithelial cell during inflammation through activation of cell survival pathways.