Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38

Early events in the response of cells to lipopolysaccharide (LPS) include activation of NF-kappaB and stress-activated MAP kinase p38. Recent studies have shown that the human Toll-like receptor 2 (TLR2) mediates activation of NF-kappaB in response to commercial preparations of LPS (comLPS), membrane lipoproteins, and Gram-positive bacterial products. Here, we show that expression of TLR2 in human embryonic kidney 293 cells enabled p38 phosphorylation in response to comLPS, a synthetic bacterial lipoprotein, and B. subtilis. Activation of p38 was confirmed by an in vitro kinase assay using ATF2 as substrate and by an assay measuring activation of the downstream effector of p38, MAP kinase-activated protein kinase in cells. Thus, TLR2 initiated the signaling pathway for p38 in response to bacterial products.     

ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK

Endoplasmic reticulum protein 29 (ERp29) is an ER luminal protein that has a role in protein unfolding and secretion, but its role in cancer is unclear. Recently, we reported that overexpression of ERp29 significantly inhibited cell proliferation and prevented tumorigenesis in highly proliferative MDA-MB-231 breast cancer cells. Here, we show that ERp29-induced cancer cell growth arrest is modulated by the interplay between the concomitant phosphorylation of p38 and upregulation of the inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, p58(IPK). In this cell model, ERp29 overexpression significantly downregulates modulators of cell proliferation, namely urokinase plasminogen activator receptor, β(1)-integrin and epidermal growth factor receptor. Furthermore, ERp29 significantly (P<0.001) increases phosphorylation of p38 (p-p38) and reduces matrix metalloproteinase-9 secretion. The role of ERp29 in upregulating cyclin-dependent kinase inhibitors (p15 and p21) and in downregulating cyclin D(2) is demonstrated in slowly proliferating ERp29-overexpressing MDA-MB-231 cells, whereas the opposite response was observed in ERp29-knockdown MCF-7 cells. Pharmacological inhibition of p-p38 downregulates p15 and p21 and inhibits eIF2α phosphorylation, indicating a role for p-p38 in this process. Furthermore, p58(IPK) expression was increased in ERp29-overexpressing MDA-MB-231 cells and highly decreased in ERp29-knockdown MCF-7 cells. This upregulation of p58(IPK) by ERp29 suppresses the activation of p-p38/p-PERK/p-eIF2α by repressing eIF2α phosphorylation. In fact, reduction of p58(IPK) expression by RNA interference stimulated eIF2α phosphorylation. The repression of eIF2α phosphorylation by p58(IPK) prevents ERp29-transfected cells from undergoing ER-dependent apoptosis driven by the activation of ATF4/CHOP/caspase-3. Hence, the interplay between p38 phosphorylation and p58(IPK) upregulation has key roles in modulating ERp29-induced cell-growth arrest and survival.     

Thiol depletion induces apoptosis in cultured lung fibroblasts.

Thiol antioxidants are implicated in the protection of cells from oxidative injury. We studied the role of thiols in the regulation of apoptosis in cultured lung fibroblasts. Thiol depletion by culturing fibroblasts in cystine-free medium or with thiol-depleting agents induced oxidant accumulation and cell death by apoptosis. The cell death was prevented by the antioxidants ascorbic acid (AA) and catalase. Thiol depletion also induced leukotriene (LT) C4, LTD4, and LTE4 production and selective phosphorylation of p38-mitogen-activated protein kinase (MAPK) and its nuclear substrate ATF2. LT production and p38-MAPK phosphorylation were required for induction of apoptosis because thiol depletion-induced apoptosis was completely blocked by the 5-lipoxygenase inhibitor AA861, the LT antagonists FPL55712 and ONO1078, and the p38-MAPK inhibitor SB203580. LT production was inhibited by AA and p38-MAPK phosphorylation was inhibited by AA, AA861, and FPL55712. In an in vitro scratch wound model, repopulating fibroblasts at the wound margin, but not quiescent cells at the intact site, selectively underwent thiol depletion- induced apoptosis that was completely blocked by AA861, FPL55712, and SB203580. Thus, thiol depletion induces apoptosis through an ordered pathway involving oxidant accumulation, LT production, and p38-MAPK activation. Apoptosis of wound fibroblasts may be responsible for impaired wound healing in various organs, including the lung.     

Cyclic adenosine monophosphate inhibits nitric oxide-induced apoptosis of cardiac muscle cells in a c-Jun N-terminal kinase-dependent manner

Cyclic adenosine monophosphate (cAMP) modulates various agent-induced apoptosis. In this study, we observed that cAMP had a significantly protective effect on nitric oxide (NO)-induced cytotoxicity in H9c2 cardiac muscle cells. Pretreatment with DBcAMP (cAMP analogue) or forskolin (adenylyl cyclase activator) also significantly prevented the SNP-induced apoptosis in H9c2 cells. In contrast, H-89 or KT5720 (PKA inhibitor) reversed the protective effects of DBcAMP. In this study, DBcAMP or forskolin reduced SNP-induced JNK/SAPK activation to the basal level, but KT5720 reversed the inhibitory effects of these two agents. In contrast to JNK/SAPK activation, DBcAMP and forskolin significantly enhanced SNP-activated p38 MAPK phosphorylation and did not affect SNP-mediated ERK activation. KT5720 reversed the effects of DBcAMP and forskolin on p38 MAPK phosphorylation. The inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK/SAPK markedly reduced the extent of SNP-induced cell death. Taken together, we suggest that JNK/SAPK is related to cAMP-protective effect in SNP-induced apoptosis. In addition, c-AMP relating agents protected SNP-induced cell death in neonatal rat ventricular cardiomyocytes. The cAMP-relating agent-induced protective effect is not resricted in H9c2 cardiac muscle cells.     

CXCL16 silencing alleviates hepatic ischemia reperfusion injury during liver transplantation by inhibiting p38 phosphorylation

CXC chemokine ligand 16 (CXCL16) has been reported to exacerbate acute kidney injury induced by ischemia-reperfusion (IR). This study aimed to investigate the probable role of CXCL16 in hepatic IR injury during liver transplantation. The expression patterns of CXCL16 and its receptor CXC chemokine receptor 6 (CXCR6) were detected in mouse models of IR injury during liver transplantation and cell models of oxygen-glucose deprivation and reoxygenation (OGD/R)-induced hepatocyte injury using RT-qPCR, Western blot analysis and ELISA. CXCL16 expression was silenced in AML12 cells exposed to OGD/R conditions to determine the role of CXCL16 in cell apoptosis and injury. After treatment with CXCL16 and a p38 phosphorylation inhibitor, SB203580, we examined whether CXCL16 regulated p38 phosphorylation to impact hepatocyte injury. To verify the effects of CXCL16 and p38 phosphorylation in vivo, CXCL16 was silenced and p38 phosphorylation was activated in IR-treated mice. CXCL16 and CXCR6 were highly expressed in mouse models of IR injury and cell models of OGD/R-induced hepatocyte injury. Silencing of CXCL16 in AML12 cells resulted in diminished CXCR6 expression and alleviated OGD/R-exposed cell injury. CXCL16 treatment in AML12 cells brought about increased protein expressions of CXCR6 and p38 phosphorylation and elevated apoptosis rate of hepatocytes. Inhibition of p38 phosphorylation neutralized CXCL16-induced apoptosis of AML12 cells. Furthermore, CXCL16 knockdown in vivo relieved hepatic injury, which was reversed by activation of p38 phosphorylation. Taken together, silencing of CXCL16 might protect against IR injury during liver transplantation by reducing p38 phosphorylation, highlighting the potential of CXCL16 as a promising target for treatment against hepatic IR injury.     

Inhibition of the p38 mitogen-activated protein kinase (MAPK) pathway attenuates cerebral vasospasm following experimental subarachnoid hemorrhage in rabbits

The p38 mitogen-activated protein kinase (MAPK) plays an important role in apoptosis and is also involved in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Here, we sought to examine whether inhibition of p38 MAPK could attenuate cerebral vasospasm and investigate the underlying mechanisms in a rabbit SAH model. SAH was established in rabbits (n=12/group) using the double-hemorrhage method. We observed apparent vasospasm in the basilar arteries of rabbits with SAH, which was significantly attenuated by SB203580, a selective p38MAPK inhibitor. Immunoblotting assays showed enhanced phosphorylation of p38 MAPK and ATF2 and increased caspase-3 cleavage following SAH, which were, however, markedly suppressed by SB203580. TUNEL staining further revealed significant apoptosis in the basilar arteries of rabbits with SAH, which was scantly present in rabbits treated with SB203580. Our results demonstrated that p38 MAPK was activated in cerebral vasospasm and associated with increased apoptosis in the basilar arteries and p38 MAPK inhibition suppressed apoptosis, suggesting that p38 MAPK could be a novel therapeutic target for cerebral vasospasm.     

Aberrant integrin activation induces p38 MAPK phosphorylation resulting in suppressed Fas-mediated apoptosis in T cells: Implications for rheumatoid arthritis

Delayed Fas-mediated apoptosis in T cells is associated with inflammatory diseases including rheumatoid arthritis (RA). CD3⁺ T cells in RA synovia expressed high amounts of phospho-p38 MAPK. Exposure to RA synovial fluid or soluble collagen, a degradation product of extracellular matrix abundant in RA synovium, induced the phosphorylation of p38 MAPK in Jurkat T cells accompanied by resistance against Fas-mediated apoptosis. Blocking β1 integrin by antibody diminished this effect. In addition, ectopic expression of auto-activated β1 integrin variant in T cells profoundly induced the phosphorylation of p38 MAPK. Suppression of p38 MAPK sensitized T cells to Fas-mediated apoptosis and increased caspase-8 and caspase-3 cleavage. A physical interaction of p38 MAPK and caspase-8 was demonstrated by using confocal microscopic imaging and co-immunoprecipitation assay. RA synovial fluid markedly increased the formation of phospho-p38 MAPK/caspase-8 complex in Jurkat T cells. In conclusion, abnormal activation of p38 MAPK to prevent Fas-mediated apoptosis may represent a common survival mechanism of RA synovial T cells contributing to the persistent inflammation of affected synovium.     

Cyclic Adenosine Monophosphate Inhibits Nitric Oxide‐Induced Apoptosis of Cardiac Muscle Cells in a c‐Jun N‐Terminal Kinase‐Dependent Manner

Cyclic adenosine monophosphate (cAMP) modulates various agent‐induced apoptosis. In this study, we observed that cAMP had a significantly protective effect on nitric oxide (NO)‐induced cytotoxicity in H9c2 cardiac muscle cells. Pretreatment with DBcAMP (cAMP analogue) or forskolin (adenylyl cyclase activator) also significantly prevented the SNP‐induced apoptosis in H9c2 cells. In contrast, H‐89 or KT5720 (PKA inhibitor) reversed the protective effects of DBcAMP. In this study, DBcAMP or forskolin reduced SNP‐induced JNK/SAPK activation to the basal level, but KT5720 reversed the inhibitory effects of these two agents. In contrast to JNK/SAPK activation, DBcAMP and forskolin significantly enhanced SNP‐activated p38 MAPK phosphorylation and did not affect SNP‐mediated ERK activation. KT5720 reversed the effects of DBcAMP and forskolin on p38 MAPK phosphorylation. The inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK/SAPK markedly reduced the extent of SNP‐induced cell death. Taken together, we suggest that JNK/SAPK is related to cAMP‐protective effect in SNP‐induced apoptosis. In addition, c‐AMP relating agents protected SNP‐induced cell death in neonatal rat ventricular cardiomyocytes. The cAMP‐relating agent‐induced protective effect is not resricted in H9c2 cardiac muscle cells.     

A mechanism in Epstein-Barr virus oncogenesis: inhibition of transforming growth factor-beta 1-mediated induction of MAPK/p21 by LMP1

Epstein-Barr virus (EBV)-infected, gastric epithelial cell line GT38 is resistant to TGF-beta 1-mediated growth inhibition and apoptosis, although TGF-beta 1 partially induces EBV reactivation in the cells. These findings indicate that abnormalities exist in these cells in the TGF-beta 1-mediated signaling pathway, influencing growth inhibition and apoptosis. In order to characterize the steps with abnormalities, we analyzed the TGF-beta 1/MAPK/p21 pathway in the cells. TGF-beta 1 activated MAPK (ERK 1/2) and p21 in the TGF-beta 1-susceptible cell line HSC-39 but not in GT38 cells. GT38 cells had higher constitutive levels of ERK 1/2 phosphorylation and p21 expression than did HSC-39 cells. U0126, a specific inhibitor of MEK, suppressed TGF-beta 1-mediated ERK 1/2 phosphorylation and p21 induction in HSC-39 cells and constitutive ERK 1/2 phosphorylation in GT38 cells. EBV latent membrane protein 1 (LMP1) induced constitutive ERK 1/2 phosphorylation and NF-kappa B activation in LMP1-transfected HSC-39 cells, which then became resistant to TGF-beta 1-mediated growth inhibition, TGF-beta 1-mediated ERK 1/2 phosphorylation, and p21 induction, and proliferated in low-serum medium. These results are consistent with the conclusion that the TGF-beta 1/MAPK/p21 pathway is required for TGF-beta 1-mediated growth inhibition, and that the resistance to TGF in GT38 cells is derived from constitutive MAPK phosphorylation induced by LMP1.     

Maintenance of Bad phosphorylation prevents apoptosis of rat hepatic sinusoidal endothelial cells in vitro and in vivo

To elucidate the mechanism of apoptosis of liver sinusoidal endothelial cells (SECs), we examined the phosphorylation status of Bad and its upstream signaling molecules during apoptosis in culture and after ischemia-reperfusion injury. Rat SECs were isolated by the immunomagnetic method, and 2 days after culture, most SECs underwent apoptosis, which was associated with decreased tyrosine phosphorylation of cellular proteins. Addition of orthovanadate (OV), a protein tyrosine phosphatase inhibitor, sustained cellular protein phosphorylation and strongly inhibited apoptosis. Bad was dephosphorylated at Ser-112 and Ser-136 during apoptosis, but the phosphorylation status of Bad was maintained in the presence of OV. OV activated the Akt, extracellular signal-regulated protein kinase, and p38 mitogen-activated protein kinase pathways, which are involved in Bad phosphorylation. In the absence of OV, depletion of Bad by RNA interference conferred resistance to apoptosis. Hepatic injury after ischemia-reperfusion was alleviated by OV treatment, with significant inhibition of SEC apoptosis. SEC apoptosis in vivo was associated with dephosphorylation of Bad, Akt, and extracellular signal-regulated protein kinase, which was blocked by OV treatment. Our data suggest that maintenance of Bad phosphorylation is important in the prevention of SEC apoptosis and that the anti-apoptotic property of OV might have therapeutic utility.     

Insertional mutation of a gene involved in growth regulation of the early mouse embryo.

A transgenic mouse strain derived from embryonic stem (ES) cells infected with multiple copies of a retroviral vector carries a recessive insertional mutation resulting in prenatal lethality. A detailed histological analysis of developing embryos has shown that the mutation results in hyperplasia of both embryonic and extraembryonic ectoderm and failure of mesoderm formation in the egg cylinder stage embryo. The number of cells in each lineage of normal and mutant embryos was estimated using stereological analysis of serial sections taken from implantation sites. We observed a 2-fold increase in the number of embryonic ectoderm cells in mutant embryos at 7.5 days postcoitum (dpc). In addition, we found that mutant embryonic ectoderm cells are only 0.6 times as large as normal cells. The number of extraembryonic ectoderm cells in mutant embryos at 7.5 dpc is also increased, by almost 4-fold. Mutant extraembryonic ectoderm cells are also smaller than normal, being only two-thirds the size of wild-type cells. The mutant phenotype suggests that the gene identified by this insertional mutation plays an important role in the growth control of early embryonic lineages.     

P38及上游激酶MKK6对热损伤诱导Raw264.7细胞凋亡的调控作用

目的探讨P38及其上游激酶MKK6对热损伤诱导单核细胞系Raw264.7细胞凋亡的调控作用。方法应用脂质体介导的基因转染将P38的显性负效突变体P38(AF)及具有持续活性的组成性活性突变体MKK6b(E)导入Raw264.7细胞,观察P38对细胞凋亡的调控作用。结果荧光染色及Westernblot检测显示,外源基因转染进细胞,并在细胞中得到表达;蛋白激酶活性检测显示,MKK6b(E)转染细胞系P38激酶活性明显升高,而P38(AF)转染细胞系P38激酶未被激活;流式细胞术检测显示,MKK6b(E)转染可明显诱导Raw264.7细胞凋亡,并且MKK6b(E)基因转染促进热损伤诱导的Raw264.7细胞凋亡,而P38(AF)基因转染抑制热损伤诱导的Raw264.7细胞凋亡。结论MKK6-P38信号通路参与介导热损伤诱导的Raw264.7细胞凋亡。     

肝纤维化大鼠肝脏中内质网应激相关分子CHOP和TRB3的表达变化

 目的： 观察内质网应激相关分子CCAAT/增强子结合蛋白同源蛋白(CHOP）和Tribbles同源蛋白3（TRB3）在四氯化碳（CCl4）致大鼠肝纤维化过程中的表达变化,探讨其在肝纤维化过程中的可能作用。方法： 体重180～200　g的雄性Wistar大鼠随机分为正常4周组、正常8周组、肝纤维化4周组和肝纤维化8周组,肝纤维化组大鼠皮下注射40%CCl4制备肝纤维化模型,分别在4周和8周处死大鼠,观察肝组织病理改变,Western blotting检测肝脏活化转录因子6（ATF6）蛋白,免疫组化、Western blotting和real-time PCR分别检测肝脏CHOP和TRB3蛋白和mRNA表达变化,TUNEL法检测肝脏细胞凋亡。结果： 肝纤维化组大鼠肝脏可见假小叶形成,p90ATF6蛋白表达量较正常组明显减少（P<0.01）,p50ATF6蛋白表达量较正常组明显增加（P<0.01）,肝细胞胞浆CHOP和TRB3蛋白及mRNA表达量较正常组显著增加（P<0.01）,细胞凋亡率较正常组显著增加（P<0.01）。结论： 内质网应激相关分子CHOP和TRB3在CCl4致大鼠肝纤维化过程中蛋白及mRNA表达水平明显增加,其变化趋势与大鼠肝细胞凋亡率一致,提示内质网应激可能通过CHOP和TRB3促进肝细胞凋亡,参与肝纤维化发生发展。     

Activation of c-Jun NH(2)-terminal kinase is required for porcine reproductive and respiratory syndrome virus-induced apoptosis but not for virus replication

Apoptosis of host cells plays a critical role in pathogenesis of virus infection. MAPK kinases especially stress-activated protein kinases c-Jun NH(2)-terminal kinase (SAPK/JNK) and p38 are often involved in virus-mediated apoptosis. It has been shown that porcine reproductive and respiratory syndrome virus (PRRSV) infection resulted in apoptosis of the host cells both in vitro and in vivo. The current investigation was initiated to determine whether stress-activated protein kinases JNK and p38 play a role in apoptosis induction by PRRSV infection. We examined phosphorylation of JNK and p38, and found that JNK but not p38 was activated in response to PRRSV infection. We then examined effects of this kinase on apoptosis induction and virus replication by using specific inhibitor. We found that JNK inhibition by its inhibitor SP600125 led to the abolishment of PRRSV-mediated apoptosis, but did not suppress virus replication. Further studies demonstrated that ROS generation was involved in JNK activation, and Bcl-2 family anti-apoptotic proteins Mcl-1 and Bcl-xl were downstream targets of JNK to mediate apoptosis. We conclude that activation of JNK signaling pathway is essential for PRRSV-mediated apoptosis but not for virus replication.