Anisomycin induces COX-2 mRNA expression through p38(MAPK) and CREB independent of small GTPases in intestinal epithelial cells

Cyclooxygenase (COX)-2 expression in intestinal epithelial cells is associated with colorectal carcinogenesis. COX-2 expression is induced by numerous growth factors and gastrointestinal hormones through multiple protein kinase cascades. Here, the role of mitogen activated protein kinases (MAPKs) and small GTPases in COX-2 expression was investigated. Anisomycin and sorbitol induced COX-2 expression in non-transformed, intestinal epithelial IEC-18 cells. Both anisomycin and sorbitol activated p38(MAPK) followed by phosphorylation of CREB. SB202190 and PD169316 but neither PD98059 nor U0126 blocked COX-2 expression and CREB phosphorylation by anisomycin or sorbitol. Clostridium difficile toxin B inhibition of small GTPases did not affect anisomycin-induced COX-2 mRNA expression or phosphorylation of p38MAPK and CREB but did inhibit sorbitol-dependent COX-2 expression and phosphorylation of p38MAPK and CREB. Angiotensin (Ang) II-dependent induction of COX-2 mRNA and induced phosphorylation of p38MAPK and CREB were inhibited by toxin B. Reduction of CREB protein in cells transfected with CREB siRNAs inhibited anisomycin-induced COX-2 expression. These results indicate that activation of p38MAPK signaling is sufficient for COX-2 expression in IEC-18 cells. Ang II and sorbitol require small GTPase activity for COX-2 expression via p38MAPK while anisomycin-induced COX-2 expression by p38MAPK does not require small GTPases. This places small GTPase activity down-stream of the AT1 receptor and hyperosmotic stress and up-stream of p38MAPK and CREB.     

Activation of p38 MAP kinase enhances sensitivity of muscle glucose transport to insulin

Muscle contractile activity is followed by an increase in the sensitivity of glucose transport to insulin. There is evidence suggesting that activation of p38 MAP kinase (p38) is involved in the stimulation of glucose transport by insulin and contractions. Exercise results in an increase in p38 phosphorylation that lasts for hours. In this context, we tested the hypothesis that activation of p38 results in an increase in insulin sensitivity. Muscles were exposed to anisomycin for 30 min to activate p38. Anisomycin increased p38 phosphorylation approximately 2.5-fold and glucose transport activity 2- to 3-fold. Three hours after anisomycin treatment, by which time the acute effect on glucose transport had partially worn off, sensitivity of muscle glucose transport to 60 microU/ml insulin was markedly increased. Both the activation of p38 and the increase in insulin sensitivity induced by anisomycin were completely prevented by pretreatment of muscles with the p38 inhibitor SB-202190. However, in contrast to the finding with anisomycin, inhibition of p38 activation did not prevent the contraction-induced increase in insulin sensitivity. Thus our results show that activation of p38 is followed by an increase in insulin sensitivity of muscle glucose transport. However, activation of p38 is not necessary for induction of an increase in muscle insulin sensitivity by contractions. This finding provides evidence that contractions have an additional effect that makes p38 activation unnecessary for enhancement of insulin sensitivity by contractile activity.     

Multiple members of the mitogen-activated protein kinase family are necessary for PED/PEA-15 anti-apoptotic function.

293 kidney embryonic cells feature very low levels of the anti-apoptotic protein PED. In these cells, expression of PED to levels comparable with those occurring in normal adult cells inhibits apoptosis induced by growth factor deprivation and by exposure to H(2)O(2) or anisomycin. In PED-expressing 293 cells (293(PED)), inhibition of apoptosis upon growth factor deprivation was paralleled by decreased phosphorylation of JNK1/2. In 293(PED) cells, decreased apoptosis induced by anisomycin and H(2)O(2) was also accompanied by block of JNK1/2 and p38 phosphorylations, respectively. Impaired activity of these stress kinases by PED correlated with inhibition of stress-induced Cdc-42, MKK4, and MKK6 activation. At variance with JNK1/2 and p38, PED expression increased basal and growth factor-stimulated Ras-Raf-1 co-precipitation and MAPK phosphorylation and activity. Treatment of 293(PED) cells with the MEK inhibitor PD98059 blocked ERK1/2 phosphorylations with no effect on inhibition of JNK1/2 and p38 activities. Complete rescue of JNK and p38 functions in 293(PED) cells by overexpressing JNK1 or p38, respectively, enabled only partial recovery of apoptotic response to growth factor deprivation and anisomycin. However, simultaneous rescue of JNK and p38 activities accompanied by block of ERK1/2 fully restored these responses. Thus, PED controls activity of the ERK, JNK, and p38 subfamilies of MAPKs. PED anti-apoptotic function in the 293 cells requires PED simultaneous activation of ERK1/2 and inhibition of the JNK/p38 signaling systems by PED.     

p42/p44-MAPK and PI3K are sufficient for IL-6 family cytokines/gp130 to signal to hypertrophy and survival in cardiomyocytes in the absence of JAK/STAT activation

The effect of differential signalling by IL-6 and leukaemia inhibitory factor (LIF) which signal by gp130 homodimerisation or LIFRβ/gp130 heterodimerisation on survival and hypertrophy was studied in neonatal rat cardiomyocytes. Both LIF and IL-6 [in the absence of soluble IL-6 receptor (sIL-6Rα)] activated Erk1/2, JNK1/2, p38-MAPK and PI3K signalling peaking at 20 min and induced cytoprotection against simulated ischemia-reperfusion injury which was blocked by the MEK1/2 inhibitor PD98059 but not the p38-MAPK inhibitor SB203580. In the absence of sIL-6R, IL-6 did not induce STAT1/3 phosphorylation, whereas IL-6/sIL-6R and LIF induced STAT1 and STAT3 phosphorylation. Furthermore, IL-6/sIL-6R induced phosphorylation of STAT1 Tyr⁷⁰¹ and STAT3 Tyr⁷⁰⁵ were enhanced by SB203580. IL-6 and pheneylephrine (PE), but not LIF, induced cardiomyocyte iNOS expression and nitric oxide (NO) production. IL-6, LIF and PE induced cardiomyocyte hypertrophy, but with phenotypic differences in ANF and SERCA2 expression and myofilament organisation with IL-6 more resembling PE than LIF. Transfection of cardiomyocytes with full length or truncated chimaeric gp130 cytoplasmic domain/Erythropoietin receptor (EpoR) extracellular domain fusion constructs showed that the membrane proximal Box 1 and Box 2 containing region of gp130 was necessary and sufficient for MAPK and PI3K activation; hypertrophy; SERCA2 expression and iNOS/NO induction in the absence of JAK/STAT activation. In conclusion, IL-6 can signal in cardiomyocytes independent of sIL-6R and STAT1/3 and furthermore, that Erk1/2 and PI3K activation by IL-6 are both necessary and sufficient for induced cardioprotection. In addition, p38-MAPK may act as a negative feedback regulator of JAK/STAT activation in cardiomyocytes.     

Multiple activation mechanisms of p38alpha mitogen-activated protein kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.     

Bigendothelin-1 via p38-MAPK-dependent mechanism regulates adult rat ventricular myocyte contractility in sepsis

We tested the hypothesis that exogenous administration of the ET-1 precursor, bigET-1, would regulate adult rat ventricular myocyte (ARVM) contractility in a p38-mitogen activated protein kinase (p38-MAPK)-dependent mechanism during sepsis. Ventricular myocytes from adult rat hearts (both sham and septic) were stimulated to contract at 0.5 Hz and mechanical properties were evaluated using an IonOptix Myocam system. Immunoblot analysis was used to determine the phosphorylation of p38-MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2). ARVMs were treated with vehicle, bigET-1 and inhibitors for 24 h and then subjected to functional and biochemical estimations. Septic ARVM displayed a distorted cell membrane and irregular network within the cells along with increased cell contractility as evidenced by elevated peak shortening (PS), maximal velocity of shortening (+dL/dt) and relengthening (-dL/dt) in comparison to sham ARVM. BigET-1 treatment caused ARVM enlargement in both sham and sepsis groups. BigET-1 (100 nM) produced an increase in ARVM contractility in sham group as compared to vehicle treatment. However, septic ARVM treated with bigET-1 exhibited unaltered ARVM contractility, and upregulated ET(B) receptors as compared to respective sham group. BigET-1 increased the concentration of ET-1 and upregulated phosphorylation of p38-MAPK but not of ERK1/2 in sham and septic ARVM. Furthermore, inhibition of p38-MAPK by SB203580 (10 microM) increased ARVM contractility in sham but not in sepsis group. BigET-1 reversed SB203580-induced increase in PS in sham group but accentuated it in sepsis group. BigET-1 also reversed SB203580-induced inhibition of p38-MAPK phosphorylation in sham but not in septic ARVM. SB203580 pretreatment followed by bigET-1 administration significantly decreased p38-MAPK phosphorylation and downregulated ET(B) receptor expression as compared to bigET-1 treatment per se in sepsis group but not in sham. We concluded that a bigET-1-induced non-responsive effect on septic ARVM contractile function could be due to upregulation of p38-MAPK phosphorylation and ET(B) receptor expression.     

模式识别受体激活状态对小胶质细胞p38-MAPK磷酸化水平的影响

为了探讨小胶质细胞模式识别受体（pattern recognition receptor,PRR）激活状态对Aβ42诱导下的细胞应激性p38丝裂原活化蛋白激酶（p38-mitogen activated protein kinase,p38-MAPK）磷酸化及细胞吞噬能力的影响,在不同模式识别受体（SRA,TRL2,SRB,CD36）的抗体存在的条件下用寡聚物Aβ42蛋白（oAβ42）孵育并活化小胶质细胞,测定细胞活化后的p38-MAPK激酶磷酸化水平和小胶质细胞模式识别受体TLR2的阻断对细胞Aβ42吞噬量的影响。结果发现,与正常小胶质细胞对比,模式识别受体SRA（scavenger receptor A）及TLR2受体（toll-like receptor 2）被相应抗体阻断的小胶质细胞在受到Aβ42蛋白激活时,p38-MAPK的磷酸化水平显著降低;TLR2受体被其抗体阻断后小胶质细胞对Aβ42的吞噬量降低,说明该模式识别受体激活状态影响胶质细胞吞噬能力,该过程与p38-MAPK的磷酸化水平相关。而受体内吞抑制剂对小胶质细胞的Aβ42蛋白的吞入量没有显著影响,说明上述细胞对寡聚物Aβ42的吞入过程并非经典的受体内吞过程。TLR家族受体有望成为阿尔茨海默病(Alzheimei Disease, AD)免疫治疗的潜在治疗靶标。     

Oxidized low density lipoprotein activates peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma through MAPK-dependent COX-2 expression in macrophages

It has been reported that oxidized low density lipoprotein (Ox-LDL) can activate both peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma. However, the detailed mechanisms of Ox-LDL-induced PPARalpha and PPARgamma activation are not fully understood. In the present study, we investigated the effect of Ox-LDL on PPARalpha and PPARgamma activation in macrophages. Ox-LDL, but not LDL, induced PPARalpha and PPARgamma activation in a dose-dependent manner. Ox-LDL transiently induced cyclooxygenase-2 (COX-2) mRNA and protein expression, and COX-2 specific inhibition by NS-398 or meloxicam or small interference RNA of COX-2 suppressed Ox-LDL-induced PPARalpha and PPARgamma activation. Ox-LDL induced phosphorylation of ERK1/2 and p38 MAPK, and ERK1/2 specific inhibition abrogated Ox-LDL-induced COX-2 expression and PPARalpha and PPARgamma activation, whereas p38 MAPK-specific inhibition had no effect. Ox-LDL decreased the amounts of intracellular long chain fatty acids, such as arachidonic, linoleic, oleic, and docosahexaenoic acids. On the other hand, Ox-LDL increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) level through ERK1/2-dependent overexpression of COX-2. Moreover, 15d-PGJ(2) induced both PPARalpha and PPARgamma activation. Furthermore, COX-2 and 15d-PGJ(2) expression and PPAR activity were increased in atherosclerotic lesions of apoE-deficient mice. Finally, we investigated the involvement of PPARalpha and PPARgamma on Ox-LDL-induced mRNA expression of ATP-binding cassette transporter A1 and monocyte chemoattractant protein-1. Interestingly, specific inhibition of PPARalpha and PPARgamma suppressed Ox-LDL-induced ATP-binding cassette transporter A1 mRNA expression and enhanced Ox-LDL-induced monocyte chemoattractant protein-1 mRNA expression. In conclusion, Ox-LDL-induced increase in 15d-PGJ(2) level through ERK1/2-dependent COX-2 expression is one of the mechanisms of PPARalpha and PPARgamma activation in macrophages. These effects of Ox-LDL may control excess atherosclerotic progression.     

Effect of endothelin-1(1-31) on p38 mitogen-activated protein kinase in cultured human mesangial cells

It was reported that human chymase cleaves big endothelins (ETs) at the Tyr31-Gly32 bond and produces 31-amino acid ETs(1-31). In this study, we investigated the effect of ET-1(1-31) on p38 mitogen-activated protein kinase (p38-MAPK) activity in human mesangial cells (HMCs). By measuring the kinase activity, we demonstrated that ET-1 (1-31) activated the p38-MAPK dose-dependently (10(-9) M to 10(-7) M), which was inhibited by SB203580. The p38-MAPK activation induced by ET-1(1-31) peaked at 10 minutes. BQ123 almost abolished ET-1(1-31)-induced p38-MAPK activation, whereas BQ788 failed to inhibit it. These findings suggest that the stimulatory effect of ET-1(1-31) on p38-MAPK activation is mediated through ET(A) or ET(A)-like receptor. In conclusion, ET-1(1-31) induced increase in p38-MAPK activation in cultured HMCs.     

p38alpha antagonizes p38gamma activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.     

Stress-induced inhibition of ERK1 and ERK2 by direct interaction with p38 MAP kinase

We have identified a direct physical interaction between the stress signaling p38alpha MAP kinase and the mitogen-activated protein kinases ERK1 and ERK2 by affinity chromatography and coimmunoprecipitation studies. Phosphorylation and activation of p38alpha enhanced its interaction with ERK1/2, and this correlated with inhibition of ERK1/2 phosphotransferase activity. The loss of epidermal growth factor-induced activation and phosphorylation of ERK1/2 but not of their direct activator MEK1 in HeLa cells transfected with the p38alpha activator MKK6(E) indicated that activated p38alpha may sequester ERK1/2 and sterically block their phosphorylation by MEK1.