Upregulation Of P38 Mitogen-activated Protein Kinase Research Articles

Incretin accelerates platelet-derived growth factor-BB-induced osteoblast migration via protein kinase A: The upregulation of p38 MAP kinase

Incretins, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine cells after food ingestion, are currently recognized to regulate glucose metabolism through insulin secretion. We previously demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces the migration of osteoblast-like MC3T3-E1 cells through mitogen-activated protein (MAP) kinases, including p38 MAP kinase. In the present study, we investigated whether or not incretins affect the osteoblast migration. The PDGF-BB-induced cell migration was significantly reinforced by GLP-1, GIP or cAMP analogues in MC3T3-E1 cells and normal human osteoblasts. The upregulated migration by GLP-1 or cAMP analogues was suppressed by H89, an inhibitor of protein kinase A. The amplification by GLP-1 of migration induced by PDGF-BB was almost completely reduced by SB203580, a p38 MAP kinase inhibitor in MC3T3-E1 cells and normal human osteoblasts. In addition, GIP markedly strengthened the PDGF-BB-induced phosphorylation of p38 MAP kinase. Exendin-4, a GLP-1 analogue, induced Rho A expression and its translocation from cytoplasm to plasma membranes in osteoblasts at the epiphyseal lines of developing mouse femurs in vivo. These results strongly suggest that incretins accelerates the PDGF-BB-induced migration of osteoblasts via protein kinase A, and the up-regulation of p38 MAP kinase is involved in this acceleration. Our findings may highlight the novel potential of incretins to bone physiology and therapeutic strategy against bone repair.

Flumequine-Mediated Upregulation of p38 MAPK and JNK Results in Melanogenesis in B16F10 Cells and Zebrafish Larvae.

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo. According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

Hyperinsulinemia impairs functions of circulating endothelial progenitor cells.

Circulating endothelial progenitor cells (EPCs) play a key role in maintaining endothelial function. Dysfunction of EPCs is associated with the cardiovascular complication of diabetes. The purpose of this study is to investigate the direct effects of hyperinsulinemia on EPCs and the underlying mechanisms. EPCs isolated from healthy adults were cultured with various concentrations of insulin (control group, without insulin; physiological insulin group, 10nM insulin and hyperinsulinemia group, 100nM insulin) with or without phosphatidylinositol-3-kinase (PI3-K) inhibitor (LY294002, 5µM), endothelial nitric oxide synthase (eNOS) inhibitor (L-NG-nitro-arginine methyl ester (L-NAME), 100µM), sodium nitroprusside (SNP, 25µM), p38 mitogen-activated protein kinase(MAPK) inhibitor (SB203580, 5µM) or extracellular signal-regulated kinases (ERK) 1/2 inhibitor (PD98059, 10µM). Proliferation, tube formation, and apoptosis of EPCs were determined. Expressions of eNOS, PI3-K, protein kinase B (Akt), p38 MAPK, and ERK 1/2 were assessed. Hyperinsulinemia caused a significant decrease in proliferation and tube formation abilities than control group. Hyperinsulinemia increased apoptosis rate of EPCs than control group. Furthermore, hyperinsulinemia downregulated phosphorylation of eNOS, PI3-K and Akt, and upregulated phosphorylation of p38 MAPK and ERK. SNP could restore impaired tube formation induced by hyperinsulinemia. P38 MAPK inhibitor but not ERK inhibitor could decrease apoptosis induced by hyperinsulinemia. Hyperinsulinemia impaired EPCs' tube formation ability by downregulation of PI-3K/Akt/eNOS pathway. Hyperinsulinemia induced apoptosis of EPCs via upregulation of p38 MAPK.

Decreased microRNA-125a-3p contributes to upregulation of p38 MAPK in rat trigeminal ganglions with orofacial inflammatory pain.

Orofacial inflammatory pain is a difficult clinical problem, and the specific molecular mechanisms for this pain remain largely unexplained. The present study aimed to determine the differential expression of microRNAs (miRNAs) and disclose the underlying role of miR-125a-3p in orofacial inflammatory pain induced by complete Freund's adjuvant (CFA). Thirty-two differentially expressed miRNAs were first screened using a microarray chip in ipsilateral trigeminal ganglions (TGs) following CFA injection into the orofacial skin innervated by trigeminal nerve, and a portion of them, including miR-23a*, -24-2*, -26a, -92a, -125a-3p, -183 and -299 were subsequently selected and validated by qPCR. The target genes were predicted based on the miRWalk website and were further analyzed by gene ontology (GO). Further studies revealed miR-125a-3p expression was down-regulated, whereas both the expression of p38 MAPK (mitogen-activated protein kinase) alpha and CGRP (calcitonin gene-related peptide) were up-regulated in ipsilateral TGs at different time points after CFA injection compared with control. Furthermore, mechanistic study revealed that miR-125a-3p negatively regulates p38 alpha gene expression and is positively correlated with the head withdrawal threshold reflecting pain. Luciferase assay showed that binding of miR-125a-3p to the 3′UTR of p38 alpha gene suppressed the transcriptional activity, and overexpression of miR-125a-3p significantly inhibited the p38 alpha mRNA level in ND8/34 cells. Taken together, our results show that miR-125a-3p is negatively correlated with the development and maintenance of orofacial inflammatory pain via regulating p38 MAPK.

62: Hyperglycemia impairs cytotrophoblast function via stress signaling

Diabetes mellitus is a risk factor for preeclampsia (preE). The normal extent of cytotrophoblast (CTB) invasion of the decidua, which is facilitated by plasmin, may be inhibited in preE. Inactive plasminogen is converted to plasmin by urokinase plasminogen activator (uPA), and uPA is regulated by plasminogen activator inhibitor 1 (PAI-1). Peroxisome proliferator-activated receptor gamma (PPARγ) also is implicated in the dysfunction of CTBs in hyperglycemic conditions. This study assesses the signaling mechanisms of hyperglycemia-induced CTB dysfunction. Human CTBs (Sw. 71) were treated with 45, 135, 225, 495 or 945 mg/dL glucose for 48h. Some cells were pretreated with a p38 inhibitor (SB203580) or a PPARγ ligand (rosiglitazone). Thereafter, cell lysates were utilized to measure uPA, PAI-1 and PPARγ expression and p38 Mitogen Activated Protein Kinase (MAPK) phosphorylation by western blot. The mRNA expression of uPA and PAI-1 in CTB lysates was measured by qPCR. Levels of angiogenic (soluble fms-like tyrosine kinase-1 (sFLT-1), soluble endoglin (sENG)) and anti-angiogenic factors (VEGF, PlGF) and IL-6 were measured in the culture media by ELISA. Both uPA and PAI-1 protein and mRNA expression were downregulated (p<0.05) in CTBs treated with >135 mg/dL glucose compared to basal (45 mg/dL). Anti-angiogenic factors (sENG, sFLT-1) and IL-6 were up-regulated, while the angiogenic factors (VEGF, PlGF) were down-regulated in the presence of >135 mg/dL glucose. The p38 MAPK phosphorylation and PPARγ expression were up-regulated (p<0.05) in hyperglycemia-exposed CTBs. SB203580 and rosiglitazone pretreatment attenuated glucose-induced down-regulation of uPA and up-regulation of p38 MAPK. Exposure of CTBs to excess glucose inhibits the invasive profile of CTBs by decreasing the expression of uPA and PAI-1; by downregulating of VEGF and PlGF; and upregulating of sENG, sFLT-1, and IL-6. Attenuation of CTB dysfunction by SB203580 or rosiglitazone pretreatment suggests the involvement of stress signaling.

Fenofibrate suppresses melanogenesis in B16-F10 melanoma cells via activation of the p38 mitogen-activated protein kinase pathway

Fenofibrate and ciglitazone belong to the classes of fibrates and thiazolidinediones, respectively. Their pharmacological actions on peroxisome proliferator-activated receptors (PPARs) present a potential therapy for hyperlipidemia and hyperglycemia. However, the melanogenesis affected by PPAR ligands in melanocytes has not been well investigated. By determining the melanin content of cells treated with PPAR agonists, we showed that fenofibrate significantly reduced melanin synthesis, but its major active metabolite, fenofibric acid, did not. Notably, the suppression of melanogenesis by fenofibrate could not be prevented by the PPARα specific antagonist GW6471. In addition, T0901317, a liver X receptor (LXR) agonist, restored the antimelanogenic activity of fenofibrate. Accordingly, fenofibrate may suppress melanogenesis through a PPARα-independent pathway. Treatment of cells with fenofibrate led to the down-regulated gene expression of melanocortin 1 receptor (MC1R). Fenofibrate also attenuated the dihydroxyphenylalanine (DOPA)-staining activity and expression of tyrosinase as well as the expression of microphthalmia-associated transcription factor (MITF). The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was stimulated by fenofibrate. Furthermore, the p38 MAPK inhibitor SB203580 prevented the repressive effects of fenofibrate on the melanin production. Taken together, the results of the present study suggest that fenofibrate inhibits melanin synthesis via the down-regulation of MC1R, the up-regulation of p38 MAPK, and interference with LXR signaling pathways to decrease the expression of tyrosinase in B16-F10 melanoma cells.

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.

P38 MAP kinase is necessary for melanoma-mediated regulation of VE-cadherin disassembly

Vascular endothelial (VE)-cadherin is localized to the endothelial borders and the adherens junctions, which are regulated by changes in mitogen-activated protein (MAP) kinases, GTPases, and intracellular calcium. We previously showed that melanoma cells induce VE-cadherin disassembly through contact with human umbilical vein endothelial cells in coculture. However, the exact mechanism by which melanoma cells signal endothelial cells to induce VE-cadherin junction disassembly is not well understood. In this study, VE-cadherin junction disassembly was further examined under fluorescence microscopy. We found that melanoma-induced VE-cadherin junction disassembly and upregulation of p38 MAP kinase in endothelial cells is regulated by both soluble factors from melanomas, particularly interleukin (IL)-8, IL-6, and IL-1beta, and through vascular cell adhesion molecule-1. Neutralizing melanoma-secreted soluble factors reduced endothelial gap formation. Endothelial cells transfected with MAP kinase kinase 6, a direct activator of p38 MAP kinase, increased VE-cadherin-mediated gap formation, facilitating melanoma transendothelial migration. In contrast, endothelial cells transfected with small-interfering RNA against p38 MAP kinase expression largely prevented melanoma transendothelial migration in Boyden chamber experiments. These findings indicate that p38 MAP kinase proteins regulate VE-cadherin junction disassembly, facilitating melanoma migration across endothelial cells.

Eugenosedin-A amelioration of lipopolysaccharide-induced up-regulation of p38 MAPK, inducible nitric oxide synthase and cyclooxygenase-2

In this study, we investigate the protective effects of eugenosedin-A on p38 mitogen-activated protein kinase (MAPK), inflammatory nitric oxide (NO) and cyclooxygenase-2 (COX-2) pathways in a rat model of endotoxin shock. Rats were pretreated with eugenosedin-A, trazodone, yohimbine (1 mg kg(-1), i.v.), aminoguanidine or ascorbic acid (15 mg kg(-1), i.v.) 30 min before endotoxin challenge. Endotoxaemia was induced by a single i.v. injection of lipopolysaccharide (LPS, 10 mg kg(-1)). In rats not treated with eugenosedin-A, LPS increased plasma concentrations of NO and prostaglandin E(2) (PGE(2)), and levels of p38 MAPK, inducible NO synthase (iNOS) and COX-2 proteins in the liver, lung, aorta and lymphocytes. In the pre-treated rats, eugenosedin-A not only inhibited the LPS-induced NO and PGE(2) levels but also attenuated the LPS-induced increase in p38 MAPK and iNOS levels in the liver, aorta and lymphocytes. Eugenosedin-A also reduced LPS-induced COX-2 proteins in the aorta and lymphocytes. Likewise, aminoguanidine, ascorbic acid, yohimbine and trazodone were also found to decrease NO and PGE(2) concentrations after endotoxin challenge. While aminoguanidine and ascorbic acid also attenuated the LPS-induced increase in p38 MAPK, iNOS and COX-2 proteins in the aorta and lymphocytes, trazodone and yohimbine inhibited only the increase in p38 MAPK, iNOS and COX-2 proteins in lymphocytes. Finally, eugenosedin-A (10(-10)-10(-8) M) significantly inhibited the biphasic response induced by hydrogen peroxide (10(-6)-3 x 10(-5) M) in rat denudated aorta. Taken together, the results of this study indicate that eugenosedin-A, as well as ascorbic acid, can attenuate free-radical-mediated aortic contraction and relaxation. It may therefore be able to reduce the damage caused by septic shock by inhibiting formation of p38 MAPK, iNOS, COX-2 and free radicals.

Methamphetamine enhances HIV-1 infectivity in monocyte derived dendritic cells.

The US is currently experiencing an epidemic of methamphetamine (Meth) use as a recreational drug. Recent studies also show a high prevalence of HIV-1 infection among Meth users. We report that Meth enhances HIV-1 infectivity of dendritic cells as measured by multinuclear activation of a galactosidase indicator (MAGI) cell assay, p24 assay, and LTR-RU5 amplification. Meth induces increased HIV-1 infection in association with an increase in the HIV-1 coreceptors, CXCR4 and CCR5, and infection is mediated by downregulation of extracellular-regulated kinase (ERK2) and the upregulation of p38 mitogen-activated protein kinase (MAPK). A p38 inhibitor (SB203580) specifically reversed the Meth-induced upregulation of the CCR5 HIV-1 coreceptor. The dopamine D2 receptor antagonist RS +/- sulpiride significantly reversed the Meth-induced upregulation of CCR5, demonstrating that the Meth-induced effect is mediated via the D2 receptor. These studies report for the first time that Meth fosters HIV-1 infection, potentially via upregulating coreceptor gene expression. Further, Meth mediates its regulatory effects via dopamine receptors and via downregulating ERK2 with a reciprocal upregulation of p38 MAPK. Elucidation of the role of Meth in HIV-1 disease susceptibility and the mechanism through which Meth mediates its effects on HIV-1 infection may help to devise novel therapeutic strategies against HIV-1 infection in high-risk Meth-using HIV-1-infected subjects.

Role of p38 mitogen-activated protein kinase pathway in estrogen-mediated cardioprotection following trauma-hemorrhage

p38 mitogen-activated protein kinase (MAPK) activates a number of heat shock proteins (HSPs), including HSP27 and alpha(B)-crystallin, in response to stress. Activation of HSP27 or alpha(B)-crystallin is known to protect organs/cells by increasing the stability of actin microfilaments. Although our previous studies showed that 17beta-estradiol (E(2)) improves cardiovascular function after trauma-hemorrhage, whether the salutary effects of E(2) under those conditions are mediated via p38 MAPK remains unknown. Male rats (275-325 g body wt) were subjected to soft tissue trauma and hemorrhage (35-40 mmHg mean blood pressure for approximately 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were injected intravenously with vehicle, E(2) (1 mg/kg body wt), E(2) + the p38 MAPK inhibitor SB-203580 (2 mg/kg body wt), or SB-203580 alone, and various parameters were measured 2 h thereafter. Cardiac functions that were depressed after trauma-hemorrhage were returned to normal levels by E(2) administration, and phosphorylation of cardiac p38 MAPK, HSP27, and alpha(B)-crystallin was increased. The E(2)-mediated improvement of cardiac function and increase in p38 MAPK, HSP27, and alpha(B)-crystallin phosphorylation were abolished with coadministration of SB-203580. These results suggest that the salutary effect of E(2) on cardiac function after trauma-hemorrhage is in part mediated via upregulation of p38 MAPK and subsequent phosphorylation of HSP27 and alpha(B)-crystallin.

BIRB 796 enhances cytotoxicity triggered by bortezomib, heat shock protein (Hsp) 90 inhibitor, and dexamethasone via inhibition of p38 mitogen‐activated protein kinase/Hsp27 pathway in multiple myeloma cell lines and inhibits paracrine tumour growth

We have previously shown that heat shock protein (Hsp) 27 or its upstream activator p38 mitogen-activated protein kinase (MAPK) confers resistance to bortezomib and dexamethasone (Dex) in multiple myeloma (MM) cells. This study examined anti-MM activity of a novel p38 MAPK inhibitor, BIRB 796, alone and in combination with conventional and novel therapeutic agents. BIRB 796 blocked baseline and bortezomib-triggered upregulation of p38 MAPK and Hsp27 phosphorylation, thereby enhancing cytotoxicity and caspase activation. The Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) upregulated protein expression and phosphorylation of Hsp27; conversely, BIRB 796 inhibited this phosphorylation and enhanced 17-AAG-induced cytotoxicity. Importantly, BIRB 796 inhibited Hsp27 phosphorylation induced by 17-AAG plus bortezomib, thereby enhancing cytotoxicity. In bone marrow stromal cells (BMSC), BIRB 796 inhibited phosphorylation of p38 MAPK and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor triggered by either tumour necrosis factor-alpha or tumour growth factor-beta1. BIRB 796 also inhibited IL-6 secretion induced in BMSCs by adherence to MM cells, thereby inhibiting tumour cell proliferation. These studies therefore suggest that BIRB 796 overcomes drug-resistance in the BM microenvironment, providing the framework for clinical trials of a p38 MAPK inhibitor, alone and in combination with bortezomib, Hsp90 inhibitor, or Dex, to improve patient outcome in MM.