In vitro evidence for role of ERK, p38, and JNK in exocrine pancreatic cytokine production

Elucidation of mechanisms of acinar cell cytokine production is essential for a better understanding of acute pancreatitis pathogenesis. We hypothesize that the stress kinases ERK, p38, and JNK play an important role in acinar cell cytokine production. Rat pancreatic fragments were incubated with 100 nM concentration of the cholecystokinin analog caerulein or 100 nM caerulein and specific ERK inhibitor (100 μM PD98059), specific p38 inhibitor (10 μM SB203580), or specific JNK inhibitor (20 μM SP600125). After 3 hours of caerulein treatment, pancreatic fragments were homogenized and assayed for total and phosphorylated ERK, p38, and JNK, and for tumor necrosis factor-α or interleukin-1β concentrations (ELISA). Pancreatic fragments stimulated with caerulein showed activation of ERK, p38, and JNK and increased cytokine concentrations (ANOVA, P<0.05). Specific stress kinase inhibitors significantly attenuated caerulein-induced activation of the corresponding stress kinase and cytokine production; however, the effect of the JNK inhibitor was comparatively less convincing. Increased activation of ERK, p38, and JNK in pancreatic fragments was not associated with significant increases in total ERK, total p38, or total JNK concentrations. The stress kinases ERK and p38 play an important role in caerulein-stimulated exocrine pancreatic overproduction of cytokines. The role of JNK needs further evaluation in this experimental model. This work was presented at the Forty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, Los Angeles, CA, May 22, 2006. Dr. Samuel was supported for this research by an American College of Surgeons Faculty Research Fellowship (2003–2005) and a National Institutes of Health NIDDK Career Development Award (grant K08-DK062805).     

Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model

OBJECTIVE: To examine the expression of mitogen-activated protein kinases (MAPKs) in human chondrocytes, to investigate whether selective activation of MAPKs is involved in up-regulation of proteoglycan (PG) synthesis following cyclical mechanical stimulation (MS), and to examine whether MS is associated with integrin-dependent or independent activation of MAPKs. METHODS: The C-28/I2 and C-20/A4 human chondrocyte cell lines were mechanically stimulated in monolayer cell culture. PG synthesis was assessed by [(35)S]-sulphate incorporation in the presence and absence of the p38 inhibitor SB203580, and the extracellular-regulated kinase (ERK1/2) inhibitor PD98059. Kinase expression and activation were assessed by Western blotting using phosphorylation status-dependent and independent antibodies, and by kinase assays. The Jun N-terminal kinase (JNK) inhibitor SP600125 and the anti-beta(1) integrin (CD29) function-blocking antibody were used to assess JNK activation and integrin dependence, respectively. RESULTS: Increased PG synthesis following 3 h of cyclic MS was abolished by pretreatment with 10 microM SB203580, but was not affected by 50 microM PD98059. The kinases p38, ERK1/ERK2 and JNKs were expressed in both stimulated and unstimulated cells. Phosphorylated p38 was detected at various time points following 0.5, 1, 2 and 3 h MS in C-28/I2, but not detected in C-20/A4 cell lines. Phosphorylation of ERK1 and ERK2 was not significantly affected by MS. Phosphorylation of the 54 and 46 kDa JNKs increased following 0.5, 1, 2 and 3 h of MS, and following CO(2) deprivation. MS-induced JNK phosphorylation was inhibited by SB203580 at concentrations > or =5 microM and activation of JNK1 following MS was blocked by SP600125 and partially inhibited by anti-CD29. CONCLUSIONS: The data suggest JNK, rather than p38 or ERK dependent increases in PG synthesis, and selective, partially integrin-dependent, activation of JNK kinases in human chondrocyte cell lines following cyclical MS. JNK activation is also very sensitive to changes in CO(2)/pH in this chondrocyte culture model.     

Activation of TLR signalling regulates microwave radiation‐mediated impairment of spermatogenesis in rat testis

Microwave radiation could increase the expression of pro‐inflammatory cytokines in rat Sertoli cells, which may impair spermatogenesis. However, the mechanisms that microwave radiation induces the cytokine expression in Sertoli cells remain to be clarified. The activation of TLRs by their ligands can trigger a common signalling pathway to upregulate inflammatory cytokines such as IL‐1, IL‐6, IL‐12 and TNF‐α. Microwave radiation can increase the expression of TLRs in lymphocytes. The purpose of this study was to determine the effect of microwave radiation on the TLRs in rat testis. We focus on the effect of TLR2‐5 (which is expressed relatively highly) by microwave radiation. The results showed that the expression of TLR2‐5 and the pro‐inflammatory cytokines (IL‐1β, IL‐6 and TNF‐α) was increased both in mRNA and in protein. Furthermore, p‐p38, p‐ERK1/2, p‐JNK and p‐NF‐κB p65, the key factors of TLR signalling, were also elevated by microwave exposure. And the NF‐κB can be induced more dominantly. These results suggest that TLRs signalling can be activated by microwave radiation in testis, which may provide the molecular basis for the in‐depth study.     

Influence of Pituitary Adenylate Cyclase-Activating Polypeptide on the Activation of Mitogen Activated Protein Kinases Following Small Bowel Cold Preservation

Cold preservation prior to small bowel transplantation can moderate tissue oxidative injury. This stress triggers several intracellular pathways via mitogen activated protein (MAP) kinases. MAP kinases include the extracellular signal related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a central role in intestinal physiology. We sought to investigate the effect of PACAP on the activation of MAP kinases during cold preservation of the small bowel. Total orthotopic intestinal autotransplantation was performed on 40 Wistar rats. Perfused grafts were stored in University of Wisconsin (UW) solution for 1 (GI), 2 (GII), 3 (GIII), or 6 hours (GIV) without or with 30 PACAP, namely 1 (GV), 2 (GVI), 3 (GVII), or 6 hours (GVIII). After 3 hours of reperfusion in all groups, the activation of MAP kinases were measured using immunocytochemistry of small bowel tissue. Among the UW preserved grafts (GI-GIV), phosphorylated ERK1/2 level were decreased, while phosphorylated JNK1/2 and p38 MAP kinase activation were elevated compared with control levels. In GV-GVIII PACAP we observed enhanced phospho-ERK1/2 appearance with decreased JNK and p38 MAP kinase activity at the end of the reperfusion periods. We concluded that cold preservation decreased phosphorylated ERK1/2 levels and increased JNK1/2 and p38 MAP kinase activities, which meant that cold storage triggered apoptotic cell death. In contrast, PACAP treatment induced signalling pathways protective against oxidative injury by MAP kinases in bowel tissue.     

Endothelial cell response to different mechanical forces

PURPOSE: Endothelial cells (ECs) are subjected to the physical forces induced by blood flow. The aim of this study was to directly compare the EC signaling pathway in response to cyclic strain and shear stress in cultured bovine aortic ECs. MATERIALS AND METHODS: The ECs were seeded on flexible collagen I-coated silicone membranes to examine the effect of cyclic strain. The membranes were deformed with a 150-mm Hg vacuum at a rate of 60 cycle/min for up to 120 minutes. For a comparison of the effect of shear stress, ECs from the same batch as used in the strain experiments were seeded on collagen I-coated silicone sheets. The ECs were then subjected to 10 dyne/cm(2) shear with the use of a parallel flow chamber for up to 120 minutes. Activation of the mitogen- activated protein kinases was assessed by determining phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 with immunoblotting. RESULTS: ERK, JNK, and p38 were activated by both cyclic strain and shear stress. Both cyclic strain and shear stress activated JNK with a similar temporal pattern and magnitude and a peak at 30 minutes. However, shear stress induced a more robust and rapid activation of ERK and p38, compared with cyclic strain. CONCLUSIONS: Our results indicate that different mechanical forces induced differential activation of mitogen-activated protein kinases. This suggests that there may be different mechanoreceptors in ECs to detect the different forces or alternative coupling pathways from a single receptor.     

Disrupted expression of intermediate filaments in the testis of rhesus monkey after experimental cryptorchidism

Cytoskeletons in Sertoli cell play an important role in process of spermatogenesis. The expression and distribution of the intermediate filaments, vimentin, keratin and desmin, were studied in the Sertoli cells of the cryptorchid testis of rhesus monkey. Vimentin was localized in the perinuclear region of Sertoli cells of the normal testis. An intense increase in vimentin immunoreactivity was observed with appearance of disorganized staining in the Sertoli cells of the cryptorchid testes. Cytokeratin 18, a marker of immature Sertoli cells, re-expressed in the cells of the adult cryptorchid testes. Desmin was also observed in the Sertoli cells in addition to the peritubular myoid cells on 30 days after the cryptorchid operation. These data suggest that Sertoli cells in primate can be affected by the heat stress. The altered changes in intermediate filaments could be possible to induce the Sertoli cell functional changes that would partially contribute to the germ cell apoptosis leading to azoospermia or oligozoospermia.     

GnRH通过ERK途径来调控大鼠睾丸间质细胞类固醇激素的合成

GnRH是由下丘脑神经元分泌,且在脊椎动物生殖功能中非常的必要。GnRH还被发现存在于脑以外的组织中,在睾丸间质细胞的炎阎醇激素合成过程中发挥了重要的作用。然而,信号通路对其的调节作用仍然不清楚。本研究我们主要检测MAPK信号通路是否参与GnRH激动剂（GnRHa）诱导的雄激素的合成。我们建立了间质细胞的原代培养的方法。不同浓度GnRHa刺激间质细胞不同时间后3D—HSD和苇酮的量通过RT-PCR,Westernblot和RIA测定。在MAPK抑制剂PD-98059存在或不存在的情况下,我们也通过Westernblot的方法检测GnRHa对ERK1／2,JNK和p38的作用。结果显示GnRHa诱导睾酮的合成并且上调3β-HSDmRNA水平以及蛋A水平,同时也激活了ERK1／2水平,但是对JNK和p38的活化没有作用。虽然GnRHa最佳的浓度是100nmnol L-1,最佳时间是24h,但是GnRHa~]激细胞5min后ERK1／2活化作用达到最高水平,60min之内恢复到本地水平。而PD-98059则能完全阻滞ERKl／2活化,3β-HSD的上调以及睾酮合成。我们的数据显示GnRH通过ERK途径来调控大鼠睾丸间质细胞雄性激素的合成。GnRH对ERK1／2的活化诱导了3β—HSD基因和蛋白的表达,最终调节大鼠间质细胞类固醇激素的合成。     

The impact of altered annexin I protein levels on apoptosis and signal transduction pathways in prostate cancer cells

BACKGROUND: Although reduced expression levels of annexin I (ANX I) protein is a common finding in all stages of prostate cancer a causative relationship between ANX I dysregulation and prostate cancer development has yet to be established. METHODS: Annexin I expression was restored in LNCaP and MDA PCa 2b that normally express low or undetectable levels of ANX I protein. The impact of restoring ANX I expression on cell viability, colony formation in soft agar, apoptosis, and extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK) activation was examined. RESULTS: Restoring ANX I expression reduced cell viability, colony formation, in addition to inducing apoptosis. The proliferative response of epidermal growth factor was blocked by restoring ANX I expression. Furthermore, increasing basal and induced levels of phosphorylated p38 and JNK were observed in prostate cancer cells following restoration of ANX I expression. CONCLUSIONS: Annexin I may have tumor suppressor functions in prostate cancer. The pro-apoptotic effect of ANX I involves the activation of p38 and JNK, which appears to shift the balance of signal transduction away from proliferation and toward apoptosis.     

Anti-apoptotic effect of quercetin: intervention in the JNK- and ERK-mediated apoptotic pathways

BACKGROUND: Bioflavonoid quercetin inhibits hydrogen peroxide (H2O2)-induced apoptosis via intervention in the activator protein 1 (AP-1)-mediated apoptotic pathway. In this report, we investigated molecular events involved in the anti-apoptotic effect of quercetin, focusing especially on its effects on the family of mitogen-activated protein (MAP) kinases. METHODS: Cultured mesangial cells were exposed to H2O2, and activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and p38 MAP kinase was evaluated in the presence or absence of quercetin. Using pharmacological and genetic inhibitors, the roles for individual MAP kinases in H2O2-induced apoptosis were examined. Involvement of ERKs in the induction and activation of AP-1 was also investigated using Northern blot analysis and a reporter assay. RESULTS: Mesangial cells exposed to H2O2 exhibited rapid phosphorylation of JNK, ERKs, and p38 MAP kinase. Quercetin abrogated the activation of all three MAP kinases in response to H2O2. Pretreatment with MAP kinase kinase inhibitor PD098059 or JNK-c-Jun/AP-1 inhibitor curcumin attenuated the H2O2-induced apoptosis. In contrast, the p38 MAP kinase inhibitor SB203580 did not improve the cell survival. Consistently, transfection with dominant-negative mutants of ERK1 and ERK2 or a dominant-negative mutant of JNK inhibited H2O2-induced apoptosis. Transfection with a dominant-negative p38 MAP kinase did not attenuate the apoptotic process. Inhibition of ERKs by PD098059 suppressed induction of c-fos without affecting early induction of c-jun, leading to attenuated activation of AP-1 in response to H2O2. CONCLUSIONS: These results suggested that (1) activation of JNK and ERKs, but not p38 kinase, is required for the H2O2-induced apoptosis; and (2) suppression of the JNK-c-Jun/AP-1 pathway and the ERK-c-Fos/AP-1 pathway is involved in the anti-apoptotic effect of quercetin.     

Prostaglandin-dependent activation of ERK mediates cell proliferation induced by transforming growth factor beta in mouse osteoblastic cells

Transforming growth factor beta (TGF(beta)) is a major coupling factor for bone turnover and is known to stimulate osteoblastic proliferation. Recent information indicates that, in addition to the Smad pathway, TGF(beta) also activates MAP kinases in osteoblastic cells. The role of these signaling cascades in cell proliferation induced by TGF(beta) as well as the cellular and molecular mechanisms of their activation by TGF(beta) has been investigated in this study. In MC3T3-E1 cells, TGF(beta) enhanced cell proliferation by about 2-fold and induced activation of the three MAP kinases, extracellular regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Surprisingly, however, whereas activation of Smad2 was rapid and maximal after 15-min incubation, activation of MAP kinases was delayed with p38 stimulation detected after 1-h exposure and activation of ERK and JNK after 3 h, suggesting indirect activation of MAP kinases by TGF(beta). Among factors known to be released in response to TGF(beta) in osteoblastic cells and influence their growth, prostaglandins (PGs) were good candidates that were further investigated for mediating TGF(beta)-induced activation of MAP kinases and cell proliferation. Indomethacin, a selective inhibitor of PG synthesis, completely blunted cell proliferation induced by TGF(beta) and markedly reduced activation of MAP kinases without influencing Smad2 phosphorylation. EP4A, a specific PGE2 receptor antagonist, also blunted TGF(beta)-induced osteoblastic proliferation. In addition to these effects, PGE2 rapidly activated MAP kinases in MC3T3-E1 cells and increased cell proliferation by about 2-fold. The role of each MAP kinases in mediating TGF(beta)- and PGE2-induced cell proliferation was investigated using selective inhibitors. U0126, a specific inhibitor of the ERK pathway, completely blocked both TGF(beta)- and PGE2-induced cell proliferation whereas SB203580 and SP600125, which are selective inhibitors of, respectively, p38 and JNK pathways, had no effect. Finally, the effect of PGE2 on activation of ERK was mimicked by phorbol esters and not by forskolin, and was associated with activation of protein kinase C. This latter effect and the stimulation of ERK induced by PGE2 were completely blocked by a specific inhibitor of PKC. In conclusion, data presented in this study strongly suggest that the local release of PGE2 is involved in cell proliferation induced by TGF(beta) in osteoblastic cells. This effect is mediated by the ERK pathway activated by a PKC-dependent mechanism.     

白蛋白诱导的肾小管细胞凋亡与丝裂素激活蛋白激酶信号通路

目的 探讨白蛋白诱导肾小管上皮细胞凋亡以及诱导凋亡的信号传导机制&#65377; 方法 将培养的大鼠肾小管细胞NRK-52E分别与不同浓度(10&#65380; 20&#65380; 30 mg/ml)的去脂无内毒素牛血清白蛋白(BSA)共同孵育6&#65380; 12&#65380; 18和24 h&#65377;透射电镜&#65380;共聚焦激光显微镜和流式细胞仪检测细胞凋亡&#65377;BSA 20 mg/ml刺激NRK-52E细胞15&#65380; 30&#65380; 60和120 min后, Westen印迹测定p38&#65380;氨基末端激酶(JNK)和细胞外信号调节激酶(ERK)活性&#65377;将SB202190(20 μmol/L, p38抑制剂)&#65380;SP600125(10 μmol/L, JNK抑制剂)和PD98059(20 μmol/L, ERK抑制剂)分别与白蛋白和NRK-52E细胞共同孵育24 h后检测细胞凋亡&#65377;结果 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡&#65377;白蛋白与NRK-52E细胞共孵育后,p38和JNK活性明显升高,ERK活性显著降低&#65377;SB202190和SP600125可分别抑制白蛋白诱导NRK-52E细胞凋亡,而PD98059促进白蛋白诱导的NRK-52E细胞凋亡&#65377;结论 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡,而p38和JNK激活与ERK抑制介导了白蛋白诱导的肾小管细胞凋亡&#65377;     

Cholinergic receptor induction and JNK activation in acute pancreatitis

BACKGROUND: Cholecystokinin-A (CCK-A) and cholinergic receptor pathways, capable of activating stress kinases p38 mitogen-activated protein kinase (p38(MAPK)) and cJUN N-terminal kinase (JNK), are implicated in the pathogenesis of ligation-induced acute pancreatitis in rats. As ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38(MAPK) activation, and as receptor induction could amplify acinar hyperstimulation and exacerbate cell stress, we tested the hypothesis that the cholinergic M3 receptor is induced and JNK is activated in this model. METHODS: Cholinergic M3 receptor expression and JNK activation was compared in rats 1, 3, or 24 hours after sham operation or duct ligation. RESULTS: Immunoblot analysis of pancreatic homogenates showed a time-dependent increase in cholinergic M3 receptor protein, total JNK, and phospho-JNK after duct ligation. CONCLUSIONS: There is a rapid and progressive cholinergic M3 receptor induction and JNK activation in ligation-induced acute pancreatitis in rats. These findings may have significance in the mechanism of disease pathogenesis.     

Differential contribution of three mitogen-activated protein kinases to PDGF-BB-induced mesangial cell proliferation and gene expression

This study examined the role of mitogen-activated protein (MAP) kinase in PDGF-BB-induced proliferation and gene expression of human mesangial cells (MC). PDGF-BB stimulation of MC increased mRNA for transforming growth factor-beta1 (TGF-beta1), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) and increased the cell numbers. To inhibit activation of extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, MC were infected with recombinant adenovirus containing dominant-negative mutants of ERK, JNK, and p38 (Ad-DN-ERK, Ad-DN-JNK, Ad-DN-p38, respectively), respectively. Infection of MC with Ad-DN-ERK or Ad-DN-JNK inhibited PDGF-BB-induced increase in [(3)H]thymidine incorporation and cell numbers, whereas Ad-DN-p38 did not. Ad-DN-ERK inhibited MCP-1 and PAI-1 mRNA expression in MC, but not TGF-beta1. Ad-DN-JNK and Ad-DN-p38 inhibited TGF-beta1 and MCP-1 mRNA expression, but not PAI-1. The inhibition of activator protein-1 (AP-1) in MC, by adenovirus containing dominant-negative mutant of c-Jun (Ad-DN-c-Jun), inhibited PDGF-BB-induced cell proliferation and TGF-beta1, MCP-1, and PAI-1 expressions. Furthermore, Ad-DN-JNK or Ad-DN-p38, but not Ad-DN-ERK, attenuated PDGF-BB-induced AP-1 activation in MC, indicating the involvement of JNK and p38 in AP-1 activation. Our results indicated that ERK and JNK, but not p38, participated in PDGF-BB-induced MC proliferation. PDGF-BB-induced expression of TGF-beta1 was mediated by JNK and p38, MCP-1 expression was through ERK, JNK, and p38, whereas PAI-1 expression was due to only ERK. AP-1 activation, which was partially due to JNK and p38 activations, was involved in MC proliferation and these three gene expressions. Thus, three MAP kinases seem to contribute to progression of glomerular disease via different molecular mechanisms.     

戊乙奎醚预处理对脓毒症小鼠肺损伤时MAPK信号转导通路的影响

目的 探讨戊乙奎醚(PHC)预处理对脓毒症小鼠肺损伤时丝裂原活化蛋白激酶(MAPK)信号转导通路的影响.方法 健康雌性昆明小鼠105只,体重20～25 g,随机分为3组(n=35):假手术组(S组)、脓毒症(CLP)组和戊乙奎醚(PHC)组.采用盲肠结扎并穿孔法制备脓毒症模型.PHC组于造模前1 h腹腔注射戊乙奎醚0.45 mg/kg,s组和CLP组于造模前1 h注射等容量生理盐水.于造模后即刻测定肺微血管通透性;造模后12 h时进行动脉血气分析,观察肺组织病理结果,测定肺组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性和磷酸化的p38丝裂原活化蛋白激酶(p38MAPK)、细胞外信号调节激酶(ERK1/ERK2)和c-jun氨基末端蛋白激酶(JNK)表达.结果 与S组比较,CLP组PaO2、PaO2/FiO2和pH值降低,肺微血管通透性和肺组织MDA含量升高,SOD活性降低,磷酸化的p38MAPK、ERK1/ERK2和JNK表达上调(P<0.05或0.01);与CLP组比较,PHC组PaO2、PaO2/FiO2和pH值升高,肺微血管通透性和肺组织MDA含量降低,SOD活性升高,磷酸化的p38MAPK和ERK1/ERK2表达下调(P<0.05或0.01).结论 戊乙奎醚预处理可通过抑制MAPK信号转导通路(p38MAPK和ERK1/ERK2)的激活,从而减轻脓毒症小鼠肺损伤.     

Apoptosis induced by chemotherapeutic agents involves c-Jun N-terminal kinase activation in sarcoma cell lines.

Molecular mechanisms underlying chemotherapeutic agent-induced apoptosis in sarcoma cells are not well known. Induction of apoptosis is regulated by several components including mitogen-activated protein kinases (MAPKs) comprising ERK, p38MAPKs, and c-Jun N-terminal kinase (JNK). In the present study, we examined whether activation of JNK is induced by the chemotherapeutic agents cis-diaminedichloroplatinum (cisplatin, CDDP) or doxorubicin (DXR), and whether the ectopic expression of constitutively active (MKK7-JNK1) or dominant-negative form of JNK (dnJNK) influenced apoptosis in response to the CDDP or DXR in sarcoma cell lines MG-63 and SaOS-2. The CDDP or DXR induced JNK activation in the both cell lines, as assessed by Western blotting using phosphospecific antibodies. A transient expression of the activated form of JNK sensitized the MG-63 and SaOS-2 cells to the drug-induced apoptosis, while dnJNK1 reduced the proportion of apoptotic cell death. Apoptosis was determined by flow cytometry using annexin-V Cy5. Collectively, our results indicate that JNK activation is involved in apoptotic cell death in sarcoma cell lines following stimulation with CDDP or DXR.     

Inhibition of MKP-1 expression potentiates JNK related apoptosis in renal cancer cells

PURPOSE: Mitogen-activated protein kinases (MAPKs) comprise 3 subgroups, that is extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK) and p38 MAPK (p38). In this study we analyzed the role of JNK as well as the expression of MAPK phosphatase-1 (MKP-1) in renal cancers. MATERIALS AND METHODS: Four renal cell carcinoma (RCC) cell lines were used. The effects of anisomycin (JNK activator) and Ro-318220 (MKP-1 expression inhibitor) were analyzed by alamar blue assay. Apoptosis was determined by flow cytometric TUNEL analysis, nuclear morphological alternations and the detection of DNA fragmentation. Changes in MKP-1 expression as well as the activation of extracellular signal-regulated protein kinases and JNK were analyzed by Western blotting. RESULTS: All cell lines treated with anisomycin resulted in a transient activation of JNK without inducing apoptosis. Since we hypothesized that elevated MKP-1 expression could possibly prevent persistent JNK activation, Ro-318220 was used. When cells were treated with Ro-318220, MKP-1 expression decreased in Caki-1 and KU 20-01 cells but not in ACHN or 769P cells. Combined treatment of Caki-1 and KU 20-01 cells with anisomycin and Ro-318220 resulted in a decrease in MKP-1 expression concomitant with persistent JNK activation. Apoptosis was induced in each cell line. CONCLUSIONS: These results suggest that prevalent MKP-1 expression in RCC contributes to cancer cell survival by attenuating an apoptosis inducing signal cascade via JNK. Since Ro-318220 potentiated JNK related apoptosis, JNK activation by blocking MKP-1 expression may be an effective therapeutic approach to RCC.     

Preconditioning effects of steroids and hyperoxia on cardiac ischemia-reperfusion injury and vascular reactivity.

OBJECTIVE: Corticosteroids and hyperoxia protect the heart against ischemia-reperfusion injury and may attenuate vascular reactivity. We hypothesized that (1) combining these two pretreatments induces an additive cardioprotection, (2) protection depends on activation of survival kinases and/or heat shock proteins, and (3) these interventions would change vascular reactivity into a more relaxed state. METHODS: Male rats were randomized (n=10 in each group): 1. control, 2. dexamethasone (3mg/kg) injected 24 and 12 h before harvesting the hearts, 3. 60 min of hyperoxia (90-95% O(2)) immediately before harvest, 4. combination of dexamethasone and hyperoxia as in groups 2 and 3. The hearts were Langendorff-perfused and exposed to 30 min of global ischemia and reperfused for 120 min. Cardiac function was monitored and infarct size determined. Isometric tension to vasoconstrictive and vasodilatory agents was measured in femoral artery rings. Phosphorylation of survival kinases (protein kinase B/AKT, extracellular signal-regulated kinases (ERK1/2), the stress-activated/c-Jun NH2 terminal kinases (SAPK/JNK) and p38 MAPK), adenosine monophosphate dependent kinase (AMPK) and expression of heat shock protein 72 (HSP72) in hearts was evaluated by immunoblotting. RESULTS: Infarct size was attenuated in all pretreated groups versus controls: 29% reduction in the combined group (p<0.01), 23% in hyperoxia group (p<0.05) and 31% in dexamethasone group (p<0.01). There was no significant difference between the treated groups. Combined pretreatment improved postischemic left ventricular end diastolic pressure compared to all other groups (p<0.001 vs controls, p=0.002 vs dexamethasone, p=0.005 vs hyperoxia). Combined pretreatment improved left ventricular developed pressure and coronary flow compared to controls (p<0.001 for both) and hyperoxia (p=0.0047 and p=0.0024, respectively). Combined pretreatment enhanced endothelium-independent relaxation (p=0.0032) compared to controls. Excepting ERK1/2 phosphorylation in the combined group during early reperfusion, there was no increased phosphorylation of the survival kinases AKT, p38, JNK, or AMPK and no increase of HSP72 expression. CONCLUSION: Combined pretreatment by hyperoxia and dexamethasone improved postischemic heart function, but did not reduce infarct size compared to single pretreatment groups. Except of a possible role of ERK1/2, protection depended neither on survival kinases nor heat shock protein 72.