Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation. This receptor-induced inhibition of proliferation was blocked by a kinase-deficient MKK4 and by the p38 MAPK inhibitor SB203580. Additionally, transfection of constitutively activated Galphaq into cells also led to inhibition of proliferation in a JNK- and p38 MAPK-dependent manner. These results demonstrate that the alpha1B-adrenergic receptor/Galphaq signaling inhibits cell proliferation through pathways involving JNK and p38 MAPK.     

The p38 mitogen-activated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation

Changes in the cytoskeleton of endothelial cells (ECs) play important roles in mediating neutrophil migration during inflammation. Previous studies demonstrated that neutrophil adherence to TNF-alpha-treated pulmonary microvascular ECs induced cytoskeletal remodeling in ECs that required ICAM-1 ligation and oxidant production and was mimicked by cross-linking ICAM-1. In this study, we examined the role of ICAM-1-induced signaling pathways in mediating actin cytoskeletal remodeling. Cross-linking ICAM-1 induced alterations in ICAM-1 distribution, as well as the filamentous actin rearrangements and stiffening of ECs shown previously. ICAM-1 cross-linking induced phosphorylation of the p38 mitogen-activated protein kinase (MAPK) that was inhibited by allopurinol and also induced an increase in the activity of the p38 MAPK that was inhibited by SB203580. However, SB203580 had no effect on oxidant production in ECs or ICAM-1 clustering. ICAM-1 cross-linking also induced phosphorylation of heat shock protein 27, an actin-binding protein that may be involved in filamentous actin polymerization. The time course of heat shock protein 27 phosphorylation paralleled that of p38 MAPK phosphorylation and was completely inhibited by SB203580. In addition, SB203580 blocked the EC stiffening response induced by either neutrophil adherence or ICAM-1 cross-linking. Moreover, pretreatment of ECs with SB203580 reduced neutrophil migration toward EC junctions. Taken together, these data demonstrate that activation of p38 MAPK, mediated by xanthine oxidase-generated oxidant production, is required for cytoskeletal remodeling in ECs induced by ICAM-1 cross-linking or neutrophil adherence. These cytoskeletal changes in ECs may in turn modulate neutrophil migration toward EC junctions.     

Resveratrol downregulates acute pulmonary thromboembolism-induced pulmonary artery hypertension via p38 mitogen-activated protein kinase and monocyte chemoattractant protein-1 signaling in rats

AimsIn the present study, we explored the hypothesis that initiation of PH involves the upregulation of monocyte chemoattractant protein-1 (MCP-1) in acute PTE. We evaluated the effects of resveratrol and the role of p38 mitogen-activated protein kinase (MAPK) in this process.Main methodsA rat model of acute PTE was established by infusion of an autologous blood clot into the pulmonary artery through a polyethylene catheter. Rats were randomly divided into 1, 4, and 8 hour time groups. Resveratrol, C1142 (a rodent chimeric mAb that neutralizes rat MCP-1) or SB203580 (a p38MAPK specific inhibitor) was administered to the animals beginning 1 h prior to the start of the acute PTE protocol. At each time point, the mean pulmonary artery pressure (mPAP), mRNA and protein expressions of MCP-1 were measured. The phosphorylation of p38 MAPK (p-pMAPK) was also detected.Key findingsAcute PTE elicited significant increases in mean pulmonary artery pressure (mPAP), and up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1) and phosphorylation of p38 mitogen-activated protein kinase (p-p38 MAPK). Administration of C1142 markedly reduced mPAP. Furthermore, pre-treatment of rats with resveratrol significantly reduced mPAP and down-regulated the expression of MCP-1, which was associated with robustly suppressed acute PTE-induced p-p38MAPK expression.SignificanceThese findings suggested that MCP-1 was involved in the formation of acute PTE-induced PH, and resveratrol down-regulated the expression of MCP-1 by inhibiting acute PTE-induced p-p38MAPK activation, which contributed to the decrease in PH.     

Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 μM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 μM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 μM) or Rho kinase (ROCK) (Y-27632, 10 μM; H1152, 0.5 μM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 μM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 μg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.     

p38 mitogen-activated protein kinase regulates human T cell IL-5 synthesis.

Involvement of p38 mitogen-activated protein (MAP) kinase in human T cell cytokine synthesis was investigated. p38 MAP kinase was clearly induced in human Th cells activated through the TCR. SB203580, a highly selective inhibitor of p38 MAP kinase, inhibited the induction of p38 MAP kinase in human Th cells. Major T cell cytokines, IL-2, IL-4, IL-5, and IFN-gamma, were produced by Der f 2-specific Th clones upon stimulation through the TCR. IL-5 synthesis alone was significantly inhibited by SB203580 in a dose-dependent manner, whereas the production of IL-2, IL-4, and IFN-gamma was not affected. The proliferation of activated T cells was not affected. IL-5 synthesis of human Th clones induced upon stimulation with rIL-2, phorbol ester plus anti-CD28 mAb, and immobilized anti-CD3 mAb plus soluble anti-CD28 mAb was also suppressed by SB203580 in the same concentration response relationship. The results clearly indicated that IL-5 synthesis by human Th cells is dependent on p38 MAP kinase activity, and is regulated distinctly from IL-2, IL-4, and IFN-gamma synthesis. Selective control of IL-5 synthesis will provide a novel treatment devoid of generalized immune suppression for bronchial asthma and atopic dermatitis that are characterized by eosinophilic inflammation.     

The many faces of p38 mitogen-activated protein kinase in progenitor/stem cell differentiation

SIRT3 is a member of the sirtuin family of protein deacetylases that is preferentially localized to mitochondria. Prominent among the proteins targeted by SIRT3 are enzymes involved in energy metabolism processes, including the respiratory chain, tricarboxylic acid cycle, fatty acid β-oxidation and ketogenesis. Through these actions, SIRT3 controls the flow of mitochondrial oxidative pathways and, consequently, the rate of production of reactive oxygen species. In addition, SIRT3-mediated deacetylation activates enzymes responsible for quenching reactive oxygen species, and thereby exerts a profound protective action against oxidative stress-dependent pathologies, such as cardiac hypertrophy and neural degeneration. SIRT3 also plays a role in multiple additional metabolic processes, from acetate metabolism to brown adipose tissue thermogenesis, often by controlling mitochondrial pathways through the deacetylation of target enzymes. In general, SIRT3 activity and subsequent control of enzymes involved in energy metabolism is consistent with an overall role of protecting against age-related diseases. In fact, experimental and genetic evidence has linked SIRT3 activity with increased lifespan. In the coming years, the identification of drugs and nutrients capable of increasing SIRT3 expression or modulating SIRT3 activity can be expected to provide promising strategies for ameliorating the metabolic syndrome and other oxidative stress-related diseases that appear preferentially with aging, such as cancer, cardiac dysfunction and neural degeneration.     

Antiproliferative effect of IL-1 is mediated by p38 mitogen-activated protein kinase in human melanoma cell A375.

The role of p38 mitogen-activated protein kinase (MAPK) in IL-1-induced growth inhibition was investigated using IL-1-sensitive human melanoma A375-C2-1 cells and IL-1-resistant A375-R8 cells. In both cells, p38 MAPK was activated by IL-1. A selective inhibitor for p38 MAPK, SB203580, almost completely recovered the IL-1-induced growth inhibition in A375-C2-1 cells. IL-1-induced IL-6 production was also suppressed by SB203580. However, the reversal effect of SB203580 was not due to the suppression of IL-6 production because the SB203580 effect was still observed in the presence of exogenous IL-6. Down-regulation of ornithine decarboxylase (ODC) activity as well as its protein level has been shown to be essential for IL-1-induced growth inhibition. SB203580 also reversed the IL-1-induced down-regulation of ODC activity and intracellular polyamine levels without affecting ODC mRNA levels in A375-C2-1 cells. In IL-1-resistant R8 cells, however, IL-1 only slightly suppressed ODC activity. In A375-C2-1 cells, the mRNA expression level of antizyme (AZ), a regulatory factor of ODC activity, has been shown to be up-regulated by IL-1. IL-1-induced up-regulation of AZ mRNA level was not affected by SB203580. These findings demonstrate that p38 MAPK plays an important role in IL-1-induced growth inhibition in A375 cells through down-regulating ODC activity without affecting the level of ODC mRNA and AZ mRNA. In IL-1-resistant A375-R8 cells, IL-1 signaling pathway is deficient between p38 MAPK activation and down-regulation of ODC activity.     

Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation

Early inflammatory events include cytokine release, activation, and rapid accumulation of neutrophils, with subsequent recruitment of mononuclear cells. The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway plays a central role in regulating a wide range of inflammatory responses in many different cells. A murine model of mild LPS-induced lung inflammation was developed to investigate the role of the p38 MAPK pathway in the initiation of pulmonary inflammation. A novel p38 MAPK inhibitor, M39, was used to determine the functional consequences of p38 MAPK activation. In vitro exposure to M39 inhibited p38 MAPK activity in LPS-stimulated murine and human neutrophils and macrophages, blocked TNF-alpha and macrophage inflammatory protein-2 (MIP-2) release, and eliminated migration of murine neutrophils toward the chemokines MIP-2 and KC. In contrast, alveolar macrophages required a 1000-fold greater concentration of M39 to block release of TNF-alpha and MIP-2. Systemic inhibition of p38 MAPK resulted in significant decreases in the release of TNF-alpha and neutrophil accumulation in the airspaces following intratracheal administration of LPS. Recovery of MIP-2 and KC from the airspaces was not affected by inhibition of p38 MAPK, and accumulation of mononuclear cells was not significantly reduced. When KC was instilled as a proinflammatory stimulus, neutrophil accumulation was significantly decreased by p38 MAPK inhibition independent of TNF-alpha or LPS. Together, these results demonstrate a much greater dependence on the p38 MAPK cascade in the neutrophil when compared with other leukocytes, and suggest a means of selectively studying and potentially modulating early inflammation in the lung.     

p38 mitogen-activated protein kinase activation is required for fibroblast growth factor-2-stimulated cell proliferation but not differentiation.

Basic fibroblast growth factor (FGF-2) is a member of a family of polypeptides that have roles in a wide range of biological processes. To determine why different cell types show distinct responses to treatment with FGF-2, the array of FGF receptors present on the surface of a cell which differentiates in response to FGF-2 (PC12 cells) was compared with that present on the surface of a cell that proliferates in response to FGF-2 (Swiss 3T3 fibroblasts). Both cell types express exclusively FGFR1, suggesting that there are cell type-specific FGFR1 signaling pathways. Since mitogen-activated protein kinases function as mediators of cellular responses to a variety of stimuli, the roles of these proteins in FGF-mediated responses were examined. FGF-2 activates extracellular signal-regulated kinases with similar kinetics in both fibroblasts and PC12 cells, and a specific inhibitor of extracellular signal-regulated kinase activation blocks differentiation but has little effect on proliferation. In contrast, while p38 mitogen-activated protein kinase is activated weakly and transiently in PC12 cells treated with FGF-2, a much stronger and sustained activation of this kinase is seen in FGF-2-treated fibroblasts. Furthermore, specific inhibitors of this kinase block proliferation but have no effect on differentiation. This effect on proliferation is specific for FGF-2 since the same concentrations of inhibitors have little or no effect on proliferation induced by serum.     

A novel protein kinase A-independent, beta-arrestin-1-dependent signaling pathway for p38 mitogen-activated protein kinase activation by beta2-adrenergic receptors

A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by beta(2)-adrenergic receptors (beta(2)-ARs). However, the detailed mechanism of beta(2)-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by beta(2)-ARs in human embryonic kidney 293A cells. The beta(2)-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of beta-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of beta-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by beta-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47(phox) knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a beta-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for beta(2)-AR-mediated early activation of p38 MAPK.     

Endothelin-1 induces vascular endothelial growth factor synthesis in osteoblasts: involvement of p38 mitogen-activated protein kinase

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.     

A novel function of p38-regulated/activated kinase in endothelial cell migration and tumor angiogenesis

The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in both suppression and promotion of tumorigenesis. It remains unclear how these 2 opposite functions of p38 operate in vivo to impact cancer development. We previously reported that a p38 downstream kinase, p38-regulated/activated kinase (PRAK), suppresses tumor initiation and promotion by mediating oncogene-induced senescence in a murine skin carcinogenesis model. Here, using the same model, we show that once the tumors are formed, PRAK promotes the growth and progression of skin tumors. Further studies identify PRAK as a novel host factor essential for tumor angiogenesis. In response to tumor-secreted proangiogenic factors, PRAK is activated by p38 via a vascular endothelial growth factor receptor 2 (VEGFR2)-dependent mechanism in host endothelial cells, where it mediates cell migration toward tumors and incorporation of these cells into tumor vasculature, at least partly by regulating the phosphorylation and activation of focal adhesion kinase (FAK) and cytoskeletal reorganization. These findings have uncovered a novel signaling circuit essential for endothelial cell motility and tumor angiogenesis. Moreover, we demonstrate that the tumor-suppressing and tumor-promoting functions of the p38-PRAK pathway are temporally and spatially separated during cancer development in vivo, relying on the stimulus, and the tissue type and the stage of cancer development in which it is activated.     

p38 mitogen-activated protein kinase/Hog1p regulates translation of the AU-rich-element-bearing MFA2 transcript

AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3' untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3'-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3'-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3'-UTR in a regulated manner to control MFA2 3'-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3'-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3'-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3'-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.     

Stem cell factor-induced migration of mast cells requires p38 mitogen-activated protein kinase activity.

Stem cell factor (SCF) can be considered a cardinal cytokine in mast cell biology as it affects mast cell differentiation, survival, and migration. The objective of this study was to investigate the role of two mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) and p38, in SCF-induced cell migration. This was examined in mouse mast cells by using PD 098059 and SB203580, which are specific inhibitors of mitogen-induced extracellular kinase (MEK) and p38 MAP kinase, respectively. SCF induced a rapid and transient activation of ERK and p38 in a dose-dependent manner. Inhibition of p38 activity by SB203580 was paralleled with a marked reduction of migration toward SCF, whereas the effect of the MEK inhibitor was less pronounced. This is the first report of a physiological function of SCF-dependent activation of p38. Whether p38-mediated mast cell migration is a possible target for suppression of mast cell hyperplasia remains to be determined.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.