Apoptosis signal-regulating kinase 1 is an intracellular inducer of p38 MAPK-mediated myogenic signalling in cardiac myoblasts

Myogenic differentiation is an essential process for the myogenesis in response to various extracellular stimuli. p38 MAPK is a core signalling molecule in myogenic differentiation. The activation of p38 MAPK is required for myogenic differentiation; however, the mechanism for this activation remains undefined. ASK1 is a member of the MAP3K family that activates both JNK and p38 MAPK pathways in response to an array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx. Here, we reported that TNFα was significantly released from H9c2 cardiac myoblast in differentiation medium. Furthermore, the oxidant H₂O₂ acted as a messenger in the TNFα signalling pathway to disrupt the complex of ASK1-Trx, which was followed by the activation of ASK1 in cardiac myogenic differentiation. Subsequently, the activated ASK1 stimulated MKK3/6-p38MAPK signalling cascade to induce specific myogenic differentiation. In addition, exogenous TNFα added to the medium at physiological levels enhanced the ASK1-p38 MAPK signalling pathway through the increased generation of H₂O₂. Interestingly, inhibition of p38 MAPK abrogated the production of H₂O₂, suggesting that there might be a positive feedback loop in the myogenic-redox signalling pathway. These results indicate that ASK1 is a new intracellular regulator of activation of the p38 MAPK in cardiac myogenic differentiation.     

p38MAPK介导的胶质细胞iNOS的转录激活机制

丝裂原激活蛋白激酶(MAPK)酶级联反应系统参与胶质细胞中iNOS的合成.通过瞬时转染p38MAPK途径中上游激酶,MAPK激酶3(MKK3)和MAPK激酶6 (MKK6 )表达质粒,进一步了解p38MAPK级联传导信号系统调节iNOS基因在胶质细胞中的转录激活机制.MKK3或MKK6表达质粒与接有荧光素酶(luciferase ,Luc)的大鼠iNOS启动基因质粒(iNOS Luc)联合转染C6星形胶质细胞株引起iNOS Luc的激活,并且使细胞因子诱导的iNOSmRNA的表达增强.这两种效应都能够被p38MAPK抑制剂SB2 0 35 80所抑制.MKK3 6也可以诱导核因子κB(NFκB Luc)依赖的转录活性.这些分子水平的研究结果为p38MAPK信号级联传导途径在调节大鼠胶质细胞中iNOS基因转录激活中的重要作用,包括转录因子NFκB的作用提供了证据.通过阻断iNOS表达或NO的生成,抑制细胞炎症发生,为防治神经细胞炎症反应性疾病提供实验依据.     

Oxidative stress induces an ATM-independent senescence pathway through p38 MAPK-mediated lamin B1 accumulation

We report crosstalk between three senescence-inducing conditions, DNA damage response (DDR) defects, oxidative stress (OS) and nuclear shape alterations. The recessive autosomal genetic disorder Ataxia telangiectasia (A-T) is associated with DDR defects, endogenous OS and premature ageing. Here, we find frequent nuclear shape alterations in A-T cells, as well as accumulation of the key nuclear architecture component lamin B1. Lamin B1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild-type cells, and normalizing lamin B1 levels in A-T cells reciprocally reduces both nuclear shape alterations and senescence. We further show that OS increases lamin B1 levels through p38 Mitogen Activated Protein kinase activation. Lamin B1 accumulation and nuclear shape alterations also occur during stress-induced senescence and oncogene-induced senescence (OIS), two canonical senescence situations. These data reveal lamin B1 as a general molecular mediator that controls OS-induced senescence, independent of established Ataxia Telangiectasia Mutated (ATM) roles in OIS.     

Ras-GRF1 signaling is required for normal beta-cell development and glucose homeostasis

Development of diabetes generally reflects an inadequate mass of insulin-producing beta-cells. beta-cell proliferation and differentiation are regulated by a variety of growth factors and hormones, including insulin-like growth factor I (IGF-I). GRF1 is a Ras-guanine nucleotide exchange factor known previously for its restricted expression in brain and its role in learning and memory. Here we demonstrate that GRF1 is also expressed in pancreatic islets. Interestingly, our GRF1-deficient mice exhibit reduced body weight, hypoinsulinemia and glucose intolerance owing to a reduction of beta-cells. Whereas insulin resistance is not detected in peripheral tissues, GRF1 knockout mice are leaner due to increased lipid catabolism. The reduction in circulating insulin does not reflect defective glucose sensing or insulin production but results from impaired beta-cell proliferation and reduced neogenesis. IGF-I treatment of isolated islets from GRF1 knockouts fails to activate critical downstream signals such as Akt and Erk. The observed phenotype is similar to manifestations of preclinical type 2 diabetes. Thus, our observations demonstrate a novel and specific role for Ras-GRF1 pathways in the development and maintenance of normal beta-cell number and function.     

Cell size homeostasis is maintained by CDK4-dependent activation of p38 MAPK

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Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function

The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus (see [1] [2] [3] [4] for reviews). The Mad and Bub proteins were identified as key components of the spindle checkpoint through budding yeast genetics [5] [6] and are highly conserved [3]. Most of the spindle checkpoint proteins have been localised to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p [7], and has been shown to recruit Mad2p to kinetochores [8]. Similarly, Bub3p binds to Bub1p [9] and may target it to kinetochores [10]. Here, we show that budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and that this complex is found at significantly higher levels once the spindle checkpoint is activated. We find that formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, we identify a conserved motif within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation. Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism.     

Regulation of Th2 cytokine genes by p38 MAPK-mediated phosphorylation of GATA-3

GATA-3 plays a critical role in allergic diseases by regulating the release of cytokines from Th2 lymphocytes. However, the molecular mechanisms involved in the regulation of GATA-3 in human T lymphocytes are not yet understood. Using small interfering RNA to knock down GATA-3, we have demonstrated its critical role in regulating IL-4, IL-5, and IL-13 release from a human T cell line. Specific stimulation of T lymphocytes by costimulation of CD3 and CD28 to mimic activation by APCs induces translocation of GATA-3 from the cytoplasm to the nucleus, with binding to the promoter region of Th2 cytokine genes, as determined by chromatin immunoprecipitation. GATA-3 nuclear translocation is dependent on its phosphorylation on serine residues by p38 MAPK, which facilitates interaction with the nuclear transporter protein importin-alpha. This provides a means whereby allergen exposure leads to the expression of Th2 cytokines, and this novel mechanism may provide new approaches to treating allergic diseases.     

BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway

The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells.     

Src family kinases regulate p38 MAPK-mediated IL-6 production in Kupffer cells following hypoxia

Tissue hypoxia is a common sequel of trauma-hemorrhage but can occur even without blood loss under hypoxic conditions. Although hypoxia is known to upregulate Kupffer cells (KC) to release cytokines, the precise mechanism of release remains unknown. We hypothesized that Src family kinases play a role in mediating KC mitogen-activated protein kinase (MAPK) signaling and their cytokine production after hypoxia. Male C3H/HeN mice received either Src inhibitor PP1 (1.5 mg/kg body wt) or vehicle 1 h before hypoxia. KCs were isolated 1 h after hypoxia, lysed, and immunoblotted with antibodies to Src, p38, ERK1/2, or JNK proteins. In addition, KCs were cultured to measure interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production. Hypoxia produced a significant increase in KC Src and MAPK (p38, ERK, JNK) activity compared with normoxic controls. This was associated with an increase in IL-6 and MCP-1 production. Treatment with PP1 abolished the increase in KC Src activation as well as p38 activity. However, PP1 did not prevent the increase in KC ERK1/2 or JNK phosphorylation. Furthermore, administration of PP1 prevented the hypoxia-induced increase in IL-6 but not MCP-1 release by KC. Additional in vitro results suggest that p38 but not ERK1/2 or JNK are critical for KC IL-6 production. In contrast, the production of MCP-1 by KC was found to be independent of MAPK. Thus hypoxia increases KC IL-6 production by p38 MAPK activation via Src-dependent pathway. Src kinases may therefore be a novel therapeutic target for preventing immune dysfunction following low-flow conditions in trauma patients. innate immunity; macrophages; cell signaling     

Calcium-dependent regulation of glucose homeostasis in the liver

A major role of the liver is to integrate multiple signals to maintain normal blood glucose levels. The balance between glucose storage and mobilization is primarily regulated by the counteracting effects of insulin and glucagon. However, numerous signals converge in the liver to ensure energy demand matches the physiological status of the organism. Many circulating hormones regulate glycogenolysis, gluconeogenesis and mitochondrial metabolism by calcium-dependent signaling mechanisms that manifest as cytosolic Ca²⁺ oscillations. Stimulus-strength is encoded in the Ca²⁺ oscillation frequency, and also by the range of intercellular Ca²⁺ wave propagation in the intact liver. In this article, we describe how Ca²⁺ oscillations and waves can regulate glucose output and oxidative metabolism in the intact liver; how multiple stimuli are decoded though Ca²⁺ signaling at the organ level, and the implications of Ca²⁺ signal dysregulation in diseases such as metabolic syndrome and non-alcoholic fatty liver disease.     

The regulation of Th1 responses by the p38 MAPK

IL-12 is a dimeric cytokine that is produced primarily by APCs. In this study we examined the role that the p38 MAPKs (MAPK/p38) play in regulating IL-12 production. We show that inhibition of p38 dramatically increased IL-12 production upon stimulation, while decreasing TNF-α. This reciprocal effect on these two cytokines following MAPK/p38 inhibition occurred in many different APCs, following a variety of different stimuli. IL-12 production was also increased in macrophages treated with small interfering RNA to limit p38α expression, and in macrophages deficient in MKK3, a kinase upstream of p38. The increase in IL-12 production following MAPK/p38 inhibition appears to be due to enhanced IL-12 (p40) mRNA stability. We show that MAPK/p38 inhibition can promote Th1 immune responses and thereby enhance vaccine efficacy against leishmaniasis. In a mouse model of Leishmania major infection, vaccination with heat-killed L. major plus CpG and SB203580 elicited complete protection against infection compared with heat-killed L. major plus CpG without SB203580. Thus, this work suggests that MAPK/p38 inhibitors may be applied as adjuvants to bias immune responses and improve vaccinations against intracellular pathogens.     

6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.     

NMDA receptor-driven calcium influx promotes ischemic human cardiomyocyte apoptosis through a p38 MAPK-mediated mechanism

N-methyl-D-aspartate receptor (NMDAR) activity plays a key role in cerebral ischemia. Although NMDAR is also expressed in cardiomyocytes, little research has been performed on NMDAR activity in myocardial ischemia. Here, using an in vitro oxygen-glucose deprivation (OGD) cardiomyocyte model, we evaluated the effects of NMDAR activity upon calcium influx, viability, apoptosis, and investigated the roles of several key mitogen-activated protein kinases (MAPKs). Primary human neonatal cardiomyocytes were cultured under OGD conditions to mimic in vivo ischemic conditions. Enhancing NMDAR activity via NMDA significantly promoted calcium influx, decreased cell viability, increased apoptosis, and enhanced p38 MAPK phosphorylation in OGD cardiomyocytes (all P < 0.05). These effects were rescued by several calcium-channel blockers (ie, MK-801, La³⁺, Gap26 peptide, 18β-glycyrrhetinic acid) but most potently rescued via the NMDAR-specific antagonist MK-801 or removal of extracellular free calcium (all P < 0.05). Knocking-down p38 MAPK activity by small-molecule inhibition or genetic methods significantly increased cell viability and reduced apoptosis (all P < 0.05). Enhancing p38 MAPK activity abolished MK-801′s apoptosis-reducing effects in a p38 MAPK-dependent manner. In conclusion, NMDAR-driven calcium influx promotes apoptosis in ischemic human cardiomyocytes, an effect which can be attributed to enhanced p38 MAPK activity.     

Specific regulation of cytokine-dependent p38 MAP kinase activation by p62/SQSTM1

We have previously shown that p62/SQSTM1 binds to p38. In this study, we identified two association domains of p62 to p38 by conducting co-immunoprecipitation experiments. One domain comprises the amino acids 173-182, named N-terminal p38 interaction (NPI) domain, and the other domain comprises the amino acids 335-344, named C-terminal p38 interaction (CPI) domain. An aspartic acid tripeptide located at 335-337 was required for their association. However, the direct interaction was only observed between the recombinant p38 and the peptide of the NPI domain, but not that of the CPI domain in the surface plasmon resonance analyses. These results suggest that the CPI domain may serve to form a certain conformation suitable for the association with p38. Furthermore, we showed that knockdown of p62 expression by siRNA led to impaired p38 phosphorylation only when HeLa cells were stimulated by cytokine. The critical role of p62 in cytokine-dependent p38 signalling pathway was further confirmed by measuring IL-8 mRNA. Cytokine mRNA is often stabilized via p38 pathway. In the absence of p62, IL-8 mRNA induced by IL-1beta became more fragile. These data show that p62 specifically regulates cytokine-dependent p38 signalling pathway.     

Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP

Autophagy, a lysosomal degradation pathway, is essential for homeostasis, development, neurological diseases, and cancer. Regulation of autophagy in human disease is not well understood. Atg9 is a transmembrane protein required for autophagy, and it has been proposed that trafficking of Atg9 may regulate autophagy. Mammalian Atg9 traffics between the TGN and endosomes in basal conditions, and newly formed autophagosomes in response to signals inducing autophagy. We identified p38IP as a new mAtg9 interactor and showed that this interaction is regulated by p38α MAPK. p38IP is required for starvation‐induced mAtg9 trafficking and autophagosome formation. Manipulation of p38IP and p38α alters mAtg9 localization, suggesting p38α regulates, through p38IP, the starvation‐induced mAtg9 trafficking to forming autophagosomes. Furthermore, we show that p38α is a negative regulator of both basal autophagy and starvation‐induced autophagy, and suggest that this regulation may be through a direct competition with mAtg9 for binding to p38IP. Our results provide evidence for a link between the MAPK pathway and the control of autophagy through mAtg9 and p38IP.