15-Deoxy-Delta(12,14)-prostaglandin J(2) suppresses IL-6-induced STAT3 phosphorylation via electrophilic reactivity in endothelial cells

In this study, the effects of 15d-PGJ(2) were investigated in IL-6-activated endothelial cells (ECs). 15d-PGJ(2) was found to abrogate phosphorylation on tyr705 of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner, but did not inhibit serine phosphorylation of STAT3 and the upperstream JAK2 phosphorylation. Other PPAR activators, such as WY1643 or ciglitazone, had no effect upon IL-6-induced STAT3 phosphorylation. Additionally, neither orthovanadate nor l-NAME treatment reverses the inhibition of STAT3 phosphorylation by 15d-PGJ(2). Otherwise, the effect of 15d-PGJ(2) requires the alpha,beta-unsaturated carbonyl group in the cyclopentane ring. A 15d-PGJ(2) analog, 9,10-Dihydro-15d-PGJ(2), which lack alpha,beta-unsaturated carbonyl group showed no increase in ROS production and no effect in inhibition of IL-6-induced STAT3 phosphorylation. The electrophilic compound, acrolein, mimics the inhibition effect of 15d-PGJ(2). Among the antioxidants, only NAC and glutathione reversed the effects of 15d-PGJ(2). NAC, glutathione and DTT all reversed the inhibition of STAT3 phosphorylation when preincubated with 15d-PGJ(2). The inhibition of ICAM-1 gene expression by 15d-PGJ(2) was abrogated by NAC and glutathione in IL-6-treated ECs. Taken together, these results suggest that 15d-PGJ(2) inhibits IL-6-stimulated phosphorylation on tyr705 of STAT3 dependent on its own electrophilic reactivity in ECs.     

15-deoxy-Delta(12,14)-prostaglandin J2 induces vascular endothelial cell apoptosis through the sequential activation of MAPKS and p53

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a potent anti-angiogenic factor and induces endothelial cell apoptosis, although the mechanism remains unclear. In this study, 15d-PGJ(2) was found to increase p53 levels of the human umbilical vein endothelial cells by stabilizing p53. Both 15d-PGJ(2)-induced apoptosis and the induction of p21(Waf1) and Bax can be abolished by p53 small interfering RNA but not by peroxisome proliferator-activated receptor gamma inhibitors. Moreover, 15d-PGJ(2) activated JNK and p38 MAPK while inducing p53 phosphorylation at sites responsible for p53 activity. JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) pretreatment attenuated 15d-PGJ(2)-mediated apoptosis and suppressed the p21(Waf1) and Bax expressions without affecting p53 protein accumulation. Pretreatment with SP600125 partially prevented the phosphorylation of p53 at serines 33 and 392 induced by 15d-PGJ(2). 15d-PGJ(2) was also found to induce reactive oxygen species generation and partially blocked nuclear factor-kappaB activity. Pretreatment with antioxidant N-acetylcysteine prevented the p53 accumulation, the phosphorylations of JNK and p38 MAPK, the inhibition of NF-kappaB activity, as well as the apoptosis induced by 15d-PGJ(2). Using a mouse model of corneal neovascularization, it was demonstrated in vivo that 15d-PGJ(2) induced reactive oxygen species generation, activated JNK and p38 MAPK, induced p53 accumulation/phosphorylation, and induced vascular endothelial cell apoptosis, which could be abolished by N-acetylcysteine, SP600125, SB203580, or a virus-derived amphipathic peptides-based p53 small interfering RNA. This is the first study that 15d-PGJ(2) induces vascular endothelial cell apoptosis through the signaling of JNK and p38 MAPK-mediated p53 activation both in vitro and in vivo, further establishing the potential of 15d-PGJ(2) as an anti-angiogenesis agent.     

Identification of p38MAPK-dependent genes with changed transcript abundance in H2O2-induced premature senescence of IMR-90 hTERT human fibroblasts

Premature senescence of IMR-90 human diploid fibroblasts expressing telomerase (hTERT) establishes after exposure to an acute sublethal concentration of H2O2. We showed herein that p38(MAPK) was phosphorylated after exposure of IMR-90 hTERT cells to H2O2. Selective inhibition of p38(MAPK) activity attenuated the increase in the proportion of cells positive for senescence associated beta-galactosidase activity. We generated a low density DNA array to study gene expression profiles of 240 senescence-related genes. Using this array, p38(MAPK) inhibitor and p38(MAPK) small interferent RNA, we identified several p38(MAPK)-target genes differentially expressed in H2O2-stressed IMR-90 hTERT fibroblasts.     

Selenazoles (selenium compounds) facilitate survival of cultured rat pheochromocytoma PC12 cells after serum-deprivation and stimulate their neuronal differentiation via activation of Akt and mitogen-activated protein kinase, respectively

The activation of extracellular receptor kinase (ERK) is one of the checkpoints to assess the activation of the classical Ras/mitogen-activated protein kinase (MAPK) cascade. Therefore, we tested more than 100 selenium-containing compounds for their ability to activate the MAPK signal pathway. Among them, we found that three selenazoles, 5-chloroacetyl-2-piperidino-1,3-selenazole (CS1), 5-chloroacetyl-2-morpholino-1,3-selenazole (CS2), and 5-chloroacetyl-2-dimethylamino-1,3-selenazole (CS3), induced the phosphorylation of ERK. These compounds also enhanced the phosphorylation of Akt, a signal transducing protein kinase for cell survival; and this phosphorylation was followed by suppression of cell death, thus suggesting that they had anti-apoptotic effects. Moreover, CSs 1-3 induced neurite outgrowth and facilitated the expression of neurofilament-M of PC12 cells, demonstrating that they induced neuronal differentiation of these cells. On the other hand, the CS-induced phosphorylation of MAPK was enhanced by buthionine sulfoximine (BSO), an activator of protein tyrosine phosphatases (PTPs), but inhibited by N-acetyl-l-cysteine (NAC), an inhibitor of receptor tyrosine kinase. These results imply that activation of some receptor tyrosine kinase(s) is involved in the mechanism of action of CSs 1-3. The activation of MAPK by CSs 1-3 was suppressed by U0126, a MEK inhibitor, but not by K252a, an inhibitor of TrkA; AG1478, an antagonist of epidermal growth factor receptor (EGFR); or by pertussis toxin. These results demonstrate that the CS-induced phosphorylation of Akt and MAP kinase (receptor tyrosine kinase(s)-MEK1/2-ERK1/2) cascades was responsible for suppression of apoptosis and facilitation of neuronal differentiation of PC12 cells, respectively. Our results suggest that CSs 1-3 are promising candidates as neuroprotective and/or neurotrophic agents for the treatment of various neurodegenerative neurological disorders.     

15-Deoxy-delta(12,14)-prostaglandin J(2) down-regulates CXCR4 on carcinoma cells through PPARgamma- and NFkappaB-mediated pathways

The chemokine receptor CXCR4 plays a key role in the metastasis of colorectal cancer and its growth at metastatic sites. Here, we have investigated the mechanisms by which CXCR4 on cancer cells might be regulated by eicosanoids present within the colorectal tumor microenvironment. We show that prostaglandins PGE(2), PGA(2), PGD(2), PGJ(2) and 15dPGJ(2) each down-regulates CXCR4 receptor expression on human colorectal carcinoma cells to differing degrees. The most potent of these were PGD(2) and its metabolites PGJ(2) and 15dPGJ(2). Down-regulation was most rapid with the end-product 15dPGJ(2) and was accompanied by a marked reduction in CXCR4 mRNA. 15dPGJ(2) is known to be a ligand for the nuclear receptor PPARgamma. Down-regulation of CXCR4 was also observed with the PPARgamma agonist rosiglitazone, while 15dPGJ(2)-induced CXCR4 down-regulation was substantially diminished by the PPARgamma antagonists GW9662 and T0070907. These data support the involvement of PPARgamma. However, the 15dPGJ(2) analogue CAY10410, which can act on PPARgamma but which lacks the intrinsic cyclopentenone structure found in 15dPGJ(2), down-regulated CXCR4 substantially less potently than 15dPGJ(2). The cyclopentenone grouping is known to inhibit the activity of NFkappaB. Consistent with an additional role for NFkappaB, we found that the cyclopentenone prostaglandin PGA(2) and cyclopentenone itself could also down-regulate CXCR4. Immunolocalization studies showed that the cellular context was sufficient to trigger a focal nuclear pattern of NFkappaB p50 and that 15dPGJ(2) interfered with this p50 nuclear localization. These data suggest that 15dPGJ(2) can down-regulate CXCR4 on cancer cells through both PPARgamma and NFkappaB. 15dPGJ(2), present within the tumor microenvironment, may act to down-regulate CXCR4 and impact upon the overall process of tumor expansion.     

Suppression of chondrosarcoma cells by 15-deoxy-Delta 12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21

We previously reported that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the most potent agonist for peroxisome proliferator-activated receptor gamma (PPAR gamma), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ(2)-induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ(2)-induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ(2), but p16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ(2) was partly, but not completely, blocked by PPAR gamma antagonist (GW9662) suggesting the 15d-PGJ(2) exerted its effect by PPAR gamma-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ(2) in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma.     

15-deoxy-Delta12,14-prostaglandin J2-induced down-regulation of endothelial nitric oxide synthase in association with HSP70 induction

A natural ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), decreases endothelial nitric oxide synthase (eNOS) expression by an unknown mechanism. Here we found that 15d-PGJ(2)-induced eNOS reduction is inversely associated with heat shock protein 70 (HSP70) induction in endothelial cells. Treatment of cells with 15d-PGJ(2) decreased eNOS protein expression in a concentration- and time-dependent manner, but independently of PPARgamma with no effect on mRNA levels. Although 15d-PGJ(2) elicited endothelial apoptosis, inhibition of both pan-caspases and cathepsins failed to reverse reduction of eNOS protein. Interestingly, we observed that 15d-PGJ(2) induced HSP70 in a dose-dependent manner. Immunoprecipitation and heat shock treatment demonstrated that eNOS reduction was strongly related to HSP70 induction. Cellular fractionation revealed that treatment with 15d-PGJ(2) increased eNOS distribution 2.5-fold from soluble to insoluble fractions. These findings provide new insights into mechanisms whereby eNOS regulation by 15d-PGJ(2) is related to HSP70 induction.     

Distinct functions of Cdk5(Y15) phosphorylation and Cdk5 activity in stress fiber formation and organization

Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.     

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A₂ (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.