Hypoxia activates jun-N-terminal kinase, extracellular signal-regulated protein kinase, and p38 kinase in pulmonary arteries

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.     

Differential activation and regulation of mitogen-activated protein kinases through the antigen receptor and CD40 in human B cells.

In human B cells, antigen receptor ligation and CD40 ligation are known to activate the extracellular-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) pathways, which in turn regulate many important B cell functions. We previously reported that antigen receptor ligation activated the ERK pathway whereas CD40 ligation activated the JNK/stress-activated protein kinase (SAPK) pathway. Here, we demonstrate that another SAPK, p38/Hog1, is activated by both antigen receptor ligation or CD40 ligation in a human B-lymphoblastoid cell line and tonsillar B cells. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially inhibited ERK2 and p38 activation triggered through the B cell receptor whereas activation of JNK1 and p38 through CD40 was not affected. PD98059, a specific inhibitor of mitogen-activated extracellular response kinase kinase (MEK), significantly inhibited ERK2 activation and partially inhibited p38 activation triggered by anti-IgM antibody treatment, but did not affect CD40-dependent signaling events. In addition, anti-IgM antibody-induced signaling pathways were shown to be PKC-dependent in contrast to the CD40-induced signaling pathways. Thus, the B cell receptor and CD40 recruit the ERK, JNK and p38 pathways by using different upstream effectors.     

Close association of p38 and JNK with plasminogen-dependent upregulation of PAI-1 in rat astrocytes in vitro

As reported previously, stimulation of astrocytes with plasminogen (PLGn) remarkably enhances their production/release of plasminogen activator inhibitor-1 (PAI-1). In addition, both p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) are activated in these astrocytes. However, it remains to be determined whether the MAPK activation is associated with the PAI-1 induction in PLGn-stimulated astrocytes. In the present study, we investigated the relationship between MAPK activity and PAI-1 induction in PLGn-stimulated astrocytes. PLGn stimulation led to definitive phosphorylation of three MAPKs: external signal regulated kinase (ERK), JNK and p38. These results suggest that all of these MAPKs, either alone or in combination, are involved in PAI-1 induction. To verify this association, an inhibition experiment was carried out by using inhibitors specific for each MAPK. The results of the immunoblotting analysis indicated that 20 μM SB203580 (the p38 inhibitor) or SP600125 (the JNK inhibitor) suppressed approximately 85% or 40% of PLGn-inducible PAI-1, respectively. Only 20% inhibition was achieved by pretreatment of astrocytes with 20 μM PD98059 (the inhibitor of MEK1/2, an upstream kinase of ERK). In conclusion, p38 and JNK were shown to be the major MAPKs involved in the signaling cascade leading to PAI-1 induction in astrocytes stimulated with PLGn.     

Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)

Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (JNK). Following the exposure of PC12 cells to an NO donor, (+)-(E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 muM), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and JNK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or JNK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO.     

Involvement of MAPKs in ICAM-1 expression in glomerular endothelial cells in diabetic nephropathy

Inflammatory processes are involved in the pathogenesis of diabetic nephropathy. The aim of this study was to clarify the role of mitogen-activated protein kinase (MAPK) pathways for induction of intercellular adhesion molecule-1 (ICAM-1) expression in glomerular endothelial cells under diabetic conditions. We examined the expression of ICAM-1 in the kidneys of experimental diabetic rats. Human glomerular endothelial cells (GE cells) were exposed to normal glucose concentration, high glucose concentration (HG), or high mannitol concentration (HM), and then the expression of the ICAM-1 protein and the phosphorylation of the 3 subfamilies of mitogen-activated protein kinase (MAPK) were determined using Western blot analysis. Next, to evaluate the involvement of MAPKs in HG- or HM-induced ICAM-1 expression, we preincubated GE cells with the inhibitors for ERK, p38 or JNK 1h prior to the application of glucose or mannitol. Expression of ICAM-1 was increased in the glomeruli of diabetic rats. Both HG and HM induced ICAM-1 expression and phosphorylation of ERK1/2, p38 and JNK in GE cells. Expression of ICAM-1 was significantly attenuated by inhibitors of ERK, p38 and JNK. We conclude that activation of ERK1/2, p38 and JNK cascades may be involved in ICAM-1 expression in glomerular endothelial cells under diabetic conditions.     

三七皂苷Rg1抑制脂多糖诱导小胶质细胞激活的机制研究

目的:探讨中药有效成分三七皂苷Rg1(Ginsenoside Rg1,Rg1)对抑制脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞株BV-2细胞激活的机制。方法:用LPS刺激BV-2细胞构建激活模型,采用四甲基偶氮唑蓝比色法(MTT)检测Rg1对BV-2细胞的活力影响,蛋白质免疫印迹(Western Blot)方法检测不同浓度Rg1(10、20和40μmol/L)对磷酸化的核因子-κB抑制蛋白-α(inhibitorκB-α,IκB-α)和反应结合蛋白(cAMP-responseelement binding protein,CREB)以及促分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)家族的细胞外信号调节激酶(extracellular signal-regulated kinase 1/2,ERK1/2)、c-Jun氨基端激酶(c-Jun N-terminal kinase,JNK)和p38促分裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)等细胞信号通路蛋白的表达及其变化规律。结果:不同浓度的Rg1明显抑制了LPS诱导的磷酸化IκB-α和CREB蛋白表达以及MAPKs通路(ERK1/2,JNK,p38 MAPK)磷酸化蛋白表达,并且对p38 MAPK表达的影响呈剂量依赖性。结论:Rg1可能通过抑制MAPKs的磷酸化来调控LPS诱导的小胶质细胞株BV-2细胞激活,发挥其神经抗炎的作用。     

Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide

Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as increased expression of mitogen-activated protein kinase like extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The present study tested the hypothesis that administration of l-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, prior to hypoxia prevents the hypoxia-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) and in the cerebral cortex of the term guinea pig fetus. To test this hypothesis normoxic (Nx, n=6), hypoxic (Hx, n=7) and hypoxic pretreated with l-NAME (Hx+L-NAME, n=6) guinea pig fetuses at 60 days gestation were studied to determine the phosphorylated p38, ERK and JNK. Hypoxia was induced by exposing pregnant guinea pigs to FiO2 of 0.07 for 1h. l-NAME (30mg/kg i.p.) was administered to pregnant mothers 60min prior to hypoxia. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated, purified and proteins separated using 12% SDS-PAGE, and then probed with specific phosphorylated ERK, JNK and p38 antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and expressed as absorbance (ODxmm2). The relative level of p-p38 was 51.41+/-9.80 (Nx), 173.67+/-3.63 (Hx), 58.56+/-3.40 (Hx+L-NAME), p<0.05 vs. Hx. The relative level p-ERK was 44.91+/-4.20 (Nx), 135.12+/-17.02 (Hx), 58.37+/-9.5 (Hx+L-NAME), p<0.05 vs. Hx. The relative level of p-JNK was 34.86+/-6.77 (Nx), 97.36+/-19.24 (Hx), 46.65+/-12.81 (Hx+L-NAME), p<0.05 vs. Hx. The data show that administration of l-NAME prior to hypoxia decreased the relative level of phosphorylated p38, ERK and JNK at term gestation. Since a NOS inhibitor prevented the hypoxia-induced phosphorylation of p38, ERK and JNK, we conclude that the hypoxia-induced activation of p38, ERK and JNK in the cerebral cortical nuclei of guinea pig fetus at term is NO-mediated. We speculate that NO-mediated modification of cysteine residue leading to inhibition of MAP kinase phosphatases results in increased activation of p38, ERK and JNK in the guinea pig fetus at term.     

Role of apoptosis signal-regulating kinase 1 in complement-mediated glomerular epithelial cell injury

In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 activates protein kinases in glomerular epithelial cells (GEC), and induces sublethal GEC injury and proteinuria. Complement induces production of reactive oxygen species (ROS) via the NAPDH oxidase, and stimulates phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase in a ROS-dependent manner. In the present study, we demonstrate that apoptosis signal-regulating kinase 1 (ASK1) was activated in glomeruli of rats with PHN, and that incubation of GEC in culture with antibody and sublytic C5b-9 stimulated ASK1 activity. The latter was, in part, mediated via the NADPH oxidase and ROS. Sublytic complement induced JNK and p38 phosphorylation, which was amplified in GEC that stably overexpress ASK1, as compared with Neo (control) GEC. Complement-induced lysis was enhanced in GEC that overexpress ASK1, as compared with Neo, and was attenuated in GEC that overexpress a dominant negative ASK1 mutant. Inhibition of p38, but not JNK, attenuated complement lysis in GEC that overexpress ASK1, but not in Neo GEC. In Neo GEC, generation of ROS restricted complement-mediated GEC injury but the protective effect of ROS was lost when ASK1 was overexpressed. We propose that the level of ASK1 expression determines the functional effect of p38 activation, i.e. when ASK1 is overexpressed, p38 activation is amplified, and C5b-9 assembly leads to GEC injury via ASK1 and p38. The present study thus defines a novel role for ASK1 as a mediator of C5b-9-dependent cell injury.     

MST1-JNK promotes apoptosis via caspase-dependent and independent pathways

BACKGROUND: MST1 is an upstream kinase of the JNK and p38 MAPK pathways whose expression induces apoptotic morphological changes such as nuclear condensation. During apoptosis, caspase cleavage of MST1 removes a C-terminal regulatory domain, increasing the kinase activity of the MST1 N-terminal domain. Downstream pathways of MST1 in the induction of apoptosis remain to be clarified. RESULTS: In this study, we found that the expression of MST1 resulted in caspase-3 activation. Therefore, MST1 is not only a target of caspases but also an activator of caspases. This caspase activation and apoptotic changes occur through JNK, since the co-expression of a dominant-negative mutant of JNK inhibited MST1-induced morphological changes as well as caspase activation. In contrast, neither a dominant-negative p38 nor the p38 inhibitor SB203580 inhibited them. MST1 induced nucleosomal DNA fragmentation, which was suppressed by caspase inhibitors or ICAD (Inhibitor of Caspase-Activated DNase). Surprisingly, however, other changes such as membrane blebbing and chromatin condensation were not inhibited by caspase inhibitors. CONCLUSION: These results suggest that MST1 most likely promotes two events through JNK activation; first, MST1 induces the activation of caspases, resulting in CAD-mediated DNA fragmentation, and second, MST1 induces chromatin condensation and membrane blebbing without utilizing downstream caspases.     

Targeting apoptosis signal-regulating kinase 1 in acute and chronic kidney disease

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase (MAP3K) family which acts as an upstream regulator for the activation of p38 MAPK and c-Jun N-terminal kinase (JNK). Experimental studies have demonstrated a pathogenic role for p38 MAPK and JNK activation in a number of kidney disease models; however, clinical studies targeting these kinases directly have been problematic due to their role in homeostatic functions. In comparison, ASK1 is activated in pathological states and is not essential for homeostatic functions, suggesting that ASK1 may be a safe and effective therapeutic target to inhibit p38 MAPK and JNK signaling in disease. Animal model studies using Ask1 gene deficient mice or a selective ASK1 inhibitor have demonstrated that ASK1 blockade is effective in a variety of acute and chronic kidney diseases; preventing cell injury, inflammation, fibrosis, albuminuria, and renal function impairment. Positive outcomes from these experimental studies have led to the current evaluation of an ASK1 inhibitor in patients with moderate to advanced diabetic kidney disease. This review summarizes the preclinical studies of ASK1 blockade in models of acute and chronic kidney injury and a clinical study examining ASK1 inhibitor treatment in diabetic kidney disease.     

Spleen tyrosine kinase promotes acute neutrophil-mediated glomerular injury via activation of JNK and p38 MAPK in rat nephrotoxic serum nephritis

Glomerular antibody deposition induces acute neutrophil-mediated glomerular injury via activation of c-Jun amino terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). However, the link between antibody deposition and activation of JNK/p38 MAPK signalling is unclear. This study tested the postulate that spleen tyrosine kinase (Syk), which is activated via Fcγ-receptor ligation, is required for activation of JNK and p38 signalling and acute neutrophil-mediated glomerular injury. We used a Syk inhibitor (SYKi) in rat nephrotoxic serum nephritis (NTN) in which neutrophil-mediated glomerular injury is dependent upon JNK and p38 signalling. SYKi or vehicle treatment of Sprague-Dawley rats began 30 min before administration of anti-GBM serum with rats killed 3 or 24 h later. Immunostaining identified de novo glomerular Syk activation (p-Tyr 525/526) in untreated NTN, being most prominent in neutrophils. Vehicle and untreated NTN exhibited heavy proteinuria and glomerular thrombosis at 24 h with P-selectin and fibrin immunostaining within capillaries, glomerular macrophage and T cell infiltration, activation of JNK and p38 MAPK signalling, and upregulation of glomerular mRNA levels of pro-inflammatory molecules (TNF-α, NOS2, MMP-12 and CCL2). In contrast, SYKi treatment provided complete protection from proteinuria, with a profound reduction in glomerular thrombosis and immunostaining for P-selectin and fibrin, and a substantial reduction in glomerular mRNA levels of pro-inflammatory molecules. SYKi treatment also reduced the acute glomerular neutrophil influx and pro-inflammatory response at 3 h in NTN. These protective effects were associated with a significant reduction in glomerular JNK and p38 MAPK activation. In addition, activation of Syk, JNK and p38 was identified in human biopsy samples of acute crescentic glomerulonephritis. In conclusion, this study demonstrates that Syk signalling is required for JNK and p38 MAPK signalling and acute neutrophil-dependent glomerular injury in rat NTN. These findings identify Syk as a potential therapeutic target in antibody-dependent kidney disease.     

Differential modulation of MAP kinases by zinc deficiency in IMR-32 cells: role of H(2)O(2)

The influence of zinc deficiency on the modulation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK) was studied. Using human IMR-32 cells as a model of neuronal cells, the role of oxidants on MAPKs and activator protein-1 (AP-1) activation in zinc deficiency was investigated, characterizing the participation of these events in the triggering of apoptosis. Relative to controls, cells incubated in media with low zinc concentrations showed increased cell oxidants and hydrogen peroxide (H(2)O(2)) release, increased JNK and p38 activation, high nuclear AP-1-DNA binding activity, and AP-1-dependent gene expression. Catalase addition to the media prevented the increase of cellular oxidants and inhibited JNK, p38, and AP-1 activation. Low levels of ERK1/2 phosphorylation were observed in the zinc-deficient cells in association with a reduction in cell proliferation. Catalase treatment did not prevent the above events nor the increased rate of apoptosis in the zinc-deficient cells. It is first demonstrated that a decrease in cellular zinc triggers H(2)O(2)-independent, as well as H(2)O(2)-dependent effects on MAPKs. Zinc deficiency-induced increases in cellular H(2)O(2) can trigger the activation of JNK and p38, leading to AP-1 activation, events that are not involved in zinc deficiency-induced apoptosis.     

Activation and autophosphorylation of apoptosis signal-regulating kinase 1 (ASK1) following cerebral ischemia in rat hippocampus

We investigated the expression, activation and autophosphorylation of apoptosis signal-regulating kinase 1 (ASK1) in rat hippocampus after cerebral ischemia. The in vitro kinase assay showed that ASK1 activity gradually increased while the autophosphorylation of ASK1 gradually reduced during 5, 15 and 30 min of cerebral ischemia. At various time points of reperfusion, the activation and autophosphorylation of ASK1 reached a high point at 30 min and reduced to basal level at 6 h and then slightly increased at 3 d compared with sham operation. Both of the increases of ASK1 activation and autophosphorylation were suppressed by N-acetylcysteine, a well-known antioxidant, which was administered to the Sprague-Dawley rat 20 min before cerebral ischemia. Immunoprecipitation and Western blotting assay showed that there was no obvious change in the amount of ASK1 at each time point compared with sham control. Our results suggest that ASK1 protein which is known as an upstream mediator of JNK/p38 mitogen-actived protein kinase (MAPK) activation may play an important role in signal transduction in response to ischemic stress, given the fact that activation of JNK/p38 MAPK and subsequent phosphorylation of c-Jun are involved in the apoptotic pathway in cerebral ischemia.