Heart HIF-1alpha and MAP kinases during hypoxia: are they associated in vivo?

To study the in vivo dynamics of hypoxia-inducible factor 1alpha (HIF-1alpha), master regulator of O(2)-dependent gene expression, and mitogen-activated protein kinases (MAPKs) in the hypoxic myocardium, Sprague-Dawley rats (n = 4 to 6 per group) were exposed to 1-hr hypoxia (10% O(2)), 23-hr hypoxia, and 23-hr hypoxia, followed by reoxygenation. HIF-1alpha increased 15-fold after 1-hr hypoxia, remained constant for 23 hrs, and returned to baseline on reoxygenation. Extracellular signal-regulated kinases (ERK1/2) were unchanged throughout. Phosphorylated p38 increased 4-fold after 1-hr hypoxia and returned to baseline within 23-hr hypoxia. The activity of stress-activated protein kinases/c-Jun NH(2)-terminal kinases (JNKs), measured as phosphorylated c-Jun, increased 3-fold after 1-hr hypoxia and remained sustained afterward. Furthermore, HIF-1alpha was halved in rats that were administered with the p38 inhibitor SB202190 and made hypoxic for 1 hr. In conclusion, although very sensitive to the reoxygenation, HIF-1alpha is overexpressed in vivo in the hypoxic myocardium, and its acute induction by hypoxia is correlated with that of p38.     

RNA干扰沉默低氧诱导因子-1α对低氧下大鼠肺动脉平滑肌细胞增殖的影响

本研究应用基因克隆技术,将合成的发卡样特异性低氧诱导因子-1α（hypoxia inducible factor-1alpha,HIF-1α）干扰寡核苷酸（siRNA）序列插入真核表达载体中,构建出特异性HIF-1α基因RNA干扰（RNAi）真核表达载体。采用组织块种植法,原代培养大鼠肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMCs）,将构建出的特异性HIF-1αRNAi真核表达载体转染到PASMCs;分别在常氧和低氧下进行细胞培养,采用RT-PCR检测PASMCsHIF-1αmRNA表达水平,用MTT和流式细胞仪检测细胞增殖水平,探讨低氧条件下HIF-1αRNAi真核表达载体对PASMCs增殖的影响。结果表明,低氧培养48h后,正常PASMCs和转染了HIF-1αsiRNA阴性表达载体的细胞增殖显著,HIF-1αmRNA表达水平也显著升高;而转染了HIF-1αsiRNA阳性表达质粒的细胞增殖不显著,HIF-1αmRNA表达水平较低。结果提示：HIF-1αRNAi真核表达载体能显著干扰培养的PASMCsHIF-1αmRNA表达,同时抑制低氧环境下PASMCs的增殖。     

Regulation of ferrochelatase gene expression by hypoxia

Ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of iron into protoporphyrin to form heme. This pathway provides heme for hemoglobin and other essential hemoproteins. The regulatory role of oxygen in the pathway has not been clearly established. In this study, we examined whether FECH gene expression is upregulated during hypoxia by a mechanism which involves the hypoxia-inducible factor 1 (HIF-1). Two HIF-1 binding motifs were identified within the -150 bp FECH minimal promoter sequence. Exposure of HEL, K562, and Hep-G2 cells to hypoxia for 18 hours resulted in a significant increase in FECH mRNA expression (p < 0.05). Hypoxia also transactivated the minimal promoter for the FECH gene in the cells. Transient co-expression of wild-type HIF-1alpha or a dominant negative HIF-1alpha with the FECH minimal promoter luciferase construct stimulated or blocked FECH promoter activity, respectively. Expression of the von Hippel-Lindau (VHL) tumor suppressor factor blocked the expression of both FECH mRNA and HIF-1alpha protein during normoxic culture of renal carcinoma cell line (RCC4). The results suggest that the FECH gene is a target for HIF-1 during hypoxia.     

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。     

A prolyl-hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, induces haem oxygenase-1 expression in human cells through a mechanism independent of hypoxia-inducible factor-1alpha

Hypoxia-inducible factor (HIF)-1 is important for cellular homeostasis under hypoxia. Expression of haem oxygenase-1 (HO-1), an essential enzyme in haem catabolism, varies under hypoxia, depending on cell types. Here, we studied the role of HIF-1alpha, a component of HIF-1, in the regulation of HO-1 expression using three human cell lines: HeLa cervical cancer, and ARPE-19 and D407 retinal pigment epithelial cells. Under hypoxia (1% O(2)), the expression of HO-1 mRNA was decreased in HeLa cells, increased in D407 cells, and unchanged in ARPE-19 cells, while HIF-1alpha protein was accumulated in these cell lines. Thus, HIF-1alpha is unlikely to function as a key regulator for HO-1 expression under hypoxia. We then used ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl hydroxylases, to accumulate HIF-1alpha protein under normoxia. Treatment with EDHB (250-500 microM) increased HIF-1alpha protein levels in HeLa and D407 cells, but not in ARPE-19 cells, whereas EDHB at lower concentrations (50-100 microM) consistently induced HO-1 mRNA expression (about 20-fold) in these three cell lines. Moreover, EDHB increased the HO-1 gene promoter activity via the enhancer that lacks a HIF-1-binding site. In conclusion, the signals evoked by hypoxia and after EDHB treatment differentially regulate HO-1 mRNA expression through HIF-1alpha-independent mechanisms.     

HIF-1alpha, HIF-2alpha and VHL mRNA expression in different cell lines during hypoxia

Hypoxia inducible factor-1alpha (HIF-1alpha) mRNA expression is significantly decreased under hypoxia in different cell lines exposed directly to hypoxia or treated with dimethyloxalylglycine which mimics hypoxic effects under normoxic conditions. However, the decreased expression of HIF-1alpha mRNA is accompanied by an increase of HIF-1alpha protein (pHIF-1alpha) level as well as by overexpression of known HIF-dependent genes (VEGF, Glut1, PFKFB-3 and PFKFB-4) under hypoxic conditions or with the use of dimethyloxalylglycine. Expression of HIF-1alpha mRNA also depends on iron because desferrioxamine and cobalt chloride produce similar to hypoxia effects on the levels of this mRNA. It was shown that HIF-1alpha mRNA expression did not change significantly in some cell lines (SKBR3, MDA-MB468 and BT549) under hypoxia. However, in these cell lines hypoxia decreases expression of HIF-2alpha mRNA, another member of HIF-alpha gene family, as a result of cell specific regulation of HIF-alpha genes under hypoxia. Moreover, hypoxia slightly induces expression of PFKFB-4 mRNA in SKBR3, MDA-MB468 and BT549 as compared to other cell lines where this effect of hypoxia was much stronger and adaptation to hypoxia is controlled by HIF-1alpha. Hypoxia slightly reduces expression of tumor suppressor VHL which targets HIF-1alpha for ubiquitination. Thus, our results clearly demonstrated down regulation of HIF-1alpha or HIF-2alpha in different cell lines by hypoxia.     

Hypoxic induction of human visfatin gene is directly mediated by hypoxia-inducible factor-1

Visfatin has been originally identified as a growth factor for early stage B cells and recently known as an adipokine. Here, we report that hypoxia induces the visfatin mRNA and protein levels in MCF7 breast cancer cells. We also demonstrate that induction of visfatin gene is regulated by hypoxia-inducible factor-1alpha (HIF-1alpha). Moreover, 5'-flanking promoter region of human visfatin gene contains two functional HIF responsive elements (HREs), activating the expression of visfatin. Mutation of these HREs in the visfatin promoter abrogates activation of a luciferase reporter gene driven by visfatin promoter under hypoxia. Taken together, our results demonstrate that visfatin is a new hypoxia-inducible gene of which expression is stimulated through the interaction of HIF-1 with HRE sites in its promoter region.