缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.     

Induction of hypoxia-inducible-factor 1 by nitric oxide is mediated via the PI 3K pathway

Adaptation to hypoxic stress provokes activation of the hypoxia-inducible-factor-1 (HIF-1) which mediates gene expression of, e.g., erythropoietin or vascular endothelial growth factor. Detailed information on signaling pathways that stabilize HIF-1 is missing, but reactive oxygen species degrade the HIF-1 alpha subunit, whereas phosphorylation causes its stabilization. It was believed that hypoxia resembles the only HIF-1 inducer but recent evidence characterized other activators of HIF-1 such as nitric oxide (NO). Herein, we concentrated on NO-evoked HIF-1 induction as a heretofore unappreciated inflammatory response in association with massive NO formation. We demonstrated that S-nitrosoglutathione induces HIF-1 alpha accumulation and concomitant DNA binding. The response was attenuated by the kinase inhibitor genistein and blockers of phosphatidylinositol 3-kinase such as Ly 294002 or wortmannin. Whereas mitogen-activated protein kinases were not involved, we noticed phosphorylation/activation of Akt in correlation with HIF-1 alpha stabilization. NO appears to regulate HIF-1 alpha via the PI 3K/Akt pathway under normoxic conditions.     

Ginkgolides mimic the effects of hypoxic preconditioning to protect C6 cells against ischemic injury by up-regulation of hypoxia-inducible factor-1 alpha and erythropoietin

Hypoxic preconditioning can play a significant neuroprotective role. However, it has not been employed clinically because of safety concerns. To find a safer preconditioning stimulus that is both practical and effective, we investigated whether ginkgolides are capable of preconditioning as hypoxia to protect C6 cells against ischemic injury. We demonstrated that both ginkgolides (37.5microg/mL) and hypoxia (1% O(2) for 16h) can significantly increase cell viabilities and expression of phosphorylated glycogen synthase kinase (p-GSK), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) in ischemic cells. The inhibitors of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3'-kinase (PI3K) significantly but not completely reduced the enhanced expression of these proteins and cell viabilities induced by ginkgolides and hypoxic preconditioning. These indicated that ginkgolides could mimic hypoxic preconditioning by increasing expression of HIF-1alpha as well as its target protein EPO and that the ginkgolides and hypoxic preconditioning role might be partly mediated by the activation of the p42/p44-mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT/glycogen synthase kinase 3beta pathways. The similar tendency in the changes of protein expression, cell viabilities and responses to MAPK or PI3K inhibitors of the cells treated with ginkgolides and hypoxia suggests that ginkgolides and hypoxic preconditioning might operate by similar mechanisms. The findings also imply that ginkgolides might have the potential for clinical use to prevent injury in high-risk conditions.     

Positive regulation of ERK activation and MKP-1 expression by peroxovanadium complex bpV (phen)

Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 mol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.     

Possible involvement of mitogen-activated protein kinase phosphatase-1 (MKP-1) in thyrotropin-releasing hormone (TRH)-induced prolactin gene expression

The role of extracellular signal-regulated kinase (ERK) in mediating the ability of thyrotropin-releasing hormone (TRH) to stimulate the prolactin gene has been well elucidated. ERK is inactivated by a dual specificity phosphatase, mitogen-activated protein kinase phosphatase (MKP). In this study, we examined the induction of MKP-1 protein by thyrotropin-releasing hormone (TRH) in pituitary GH3 cells, and investigated the possible role for MKP-1 in TRH-induced prolactin gene expression. MKP-1 protein was induced significantly from 60 min after TRH stimulation, and remained elevated at 4 h. The effect of TRH on MKP-1 expression was completely prevented in the presence of the MEK inhibitor, U0126. In the experiments using triptolide, a potent blocker for MKP-1, MKP-1 induction by TRH was completely inhibited in a dose-dependent manner. TRH-induced ERK activation was significantly enhanced in this condition. Prolactin promoter activity, activated by TRH, was reduced to the control level in the presence of triptolide in a dose-dependent manner. In GH3 cells, which were transfected with MKP-1 specific siRNA, both the basal and TRH-stimulated activities of the prolactin promoter were significantly reduced compared to the cells transfected with negative control siRNA. Our present results support a critical role of MKP-1 in TRH-induced, ERK-dependent, prolactin gene expression.     

Protein kinase Czeta attenuates hypoxia-induced proliferation of fibroblasts by regulating MAP kinase phosphatase-1 expression

We have previously found that hypoxia stimulates proliferation of vascular fibroblasts through Galphai-mediated activation of ERK1/2. Here, we demonstrate that hypoxia also activates the atypical protein kinase Czeta (PKCzeta) isozyme and stimulates the expression of ERK1/2-specific phosphatase, MAP kinase phosphatase-1 (MKP-1), which attenuates ERK1/2-mediated proliferative signals. Replication repressor activity is unique to PKCzeta because the blockade of classical and novel PKC isozymes does not affect fibroblast proliferation. PKCzeta is phosphorylated upon prolonged (24 h) exposure to hypoxia, whereas ERK1/2, the downstream kinases, are maximally activated in fibroblasts exposed to acute (10 min) hypoxia. However, PKCzeta blockade results in persistent ERK1/2 phosphorylation and marked increase in hypoxia-induced replication. Similarly prolonged ERK1/2 phosphorylation and increase in hypoxia-stimulated proliferation are also observed upon blockade of MKP-1 activation. Because of the parallel suppressive actions of PKCzeta and MKP-1 on ERK1/2 phosphorylation and proliferation, the role of PKCzeta in the regulation of MKP-1 expression was evaluated. PKCzeta attenuation reduces MKP-1 expression, whereas PKCzeta overexpression increases MKP-1 levels. In conclusion, our results indicate for the first time that hypoxia activates PKCzeta, which acts as a terminator of ERK1/2 activation through the regulation of downstream target, MKP-1 expression and thus serves to limit hypoxia-induced proliferation of fibroblasts.     

ERK1/2 achieves sustained activation by stimulating MAPK phosphatase-1 degradation via the ubiquitin-proteasome pathway

Sustained extracellular signal-regulated kinase 1/2 (ERK1/2) activation does not always correlate with its upstream Ras-Raf-mitogen-activated protein kinase kinase 1/2 (MKK1/2) signal cascade in cancer cells, and the mechanism remains elusive. Here we report a novel mechanism by which sustained ERK1/2 activation is established. We demonstrate that Pb(II), a carcinogenic metal, persistently induces ERK1/2 activity in CL3 human lung cancer cells and that Ras-Raf-MKK1/2 signaling cannot fully account for such activation. It is intriguing that Pb(II) treatment reduces mitogen-activated protein kinase phosphatase 1 (MKP-1) protein levels in time- and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb(II)-treated cells, MKP-1 is polyubiquitinated, and proteasome inhibitors markedly alleviate the ubiquitination and degradation of MKP-1. Inhibiting the Pb(II)-induced ERK1/2 activation by PD98059 greatly suppresses MKP-1 ubiquitination and degradation. It is remarkable that constitutive activation of MKK1/2 triggers endogenous MKP-1 ubiquitination and degradation in various mammalian cell lines. Furthermore, expression of functional MKP-1 decreases ERK1/2 activation and the c-Fos protein level and enhances cytotoxicity under Pb(II) exposure. Taken together, these results demonstrate that activated ERK1/2 can trigger MKP-1 degradation via the ubiquitin-proteasome pathway, thus facilitating long-term activation of ERK1/2 against cytotoxicity.     

Crosstalk of hypoxia-mediated signaling pathways in upregulating plasminogen activator inhibitor-1 expression in keloid fibroblasts

Keloids are skin fibrotic conditions characterized by an excess accumulation of extracellular matrix (ECM) components secondary to trauma or surgical injuries. Previous studies have shown that plasminogen activator inhibitor-1 (PAI-1) can be upregulated by hypoxia and may contribute to keloid pathogenesis. In this study we investigate the signaling mechanisms involved in hypoxia-mediated PAI-1 expression in keloid fibroblasts. Using Northern and Western blot analysis, transient transfections, and pharmacological agents, we demonstrate that hypoxia-induced upregulation of PAI-1 expression is mainly controlled by hypoxia inducible factors-1alpha (HIF-1alpha) and that hypoxia leads to a rapid and transient activation of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2). Treatment of cells with PI-3K/Akt inhibitor (LY294002) and tyrosine protein kinase inhibitor (genistein) significantly attenuated hypoxia-induced PAI-1 mRNA and protein expression as well as promoter activation, apparently via an inhibition of the hypoxia-induced stabilization of HIF-1alpha protein, attenuation of the steady-state level of HIF-1alpha mRNA, and its DNA-binding activity. Even though disruption of ERK1/2 signaling pathway by PD98059 abolished hypoxia-induced PAI-1 promoter activation and mRNA/protein expression in keloid fibroblasts, it did not inhibit the hypoxia-mediated stabilization of HIF-1alpha protein and the steady-state level of HIF-1alpha mRNA nor its DNA binding activity. Our findings suggest that a combination of several signaling pathways, including ERK1/2, PI3-K/Akt, and protein tyrosine kinases (PTKs), may contribute to the hypoxia-mediated induction of PAI-1 expression via activation of HIF-1alpha in keloid fibroblasts.