Hypoxia Induced Autocrine Of Tumor Necrosis Factor α By Hypoxia-inducidble Factor-1α In Cardiomyocytes

PartⅠHypoxia induced autocrine of TNF-a by hypoxia-inducible factor-la in cardiomyocytesAims:Growing evidence indicates that excessive tumor necrosis factor-a (TNF-a) expression are detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-a is mainly released from macrophages, with persisent ischemia, TNF-a can originate from cardiomyocytes. We hypothesized the direct effects of hypoxia on TNF-a expression by hypoxia-inducible factor-la (HIF-la) in cardiomyocytes.Methods and results:Neonatal rat cardiomyocytes were exposed in a hypoxic incubator containing 1% oxygen for 5 minutes,15 minutes,30 minutes,1 hour,2 hours,4 hours,6 hours,12 hours and 24 hours. TNF-a mRNA increased slightly as early as 30 minutes (1.99±0.27 fold compared to control group,p<0.01), peaked at 12 hours (4.06±0.84 fold,p＜0.01) and 24 hours (4.09±0.81 fold,p＜0.01). TNF-a secretion became detectable as early as 6 hours and peaked at 12 hours (48.12±11.52 pg/ml) by ELISA. Hypoxia had no effect on HIF-la mRNA change (p>0.05) by real-time RT-PCR assay. Protein expression of HIF-1αwas detectable at 30 minutes and peaked at 1 hour by western blot. Immunofluorescent staining showed HIF-1αwas obviously observed as early as 15 minutes, meanwhile, it had the trends to translocate into cell nucleus. HIF-1αmRNA was downregulated effectively by siRNA through Nucleofection, meanwhile, TNF-αmRNA elevation after 12-hour hypoxia was obviously inhibited (3.75±0.45 fold vs 1.38±0.76 fold, p<0.01). Expression of HIF-la was inhibited effectively by 2-Methoxyestradiol (2ME2) after hypoxia, and 2ME2 also decreased TNF-αmRNA after 12-hour hypoxia (2.82±0.41 fold vs 1.31±0.37 fold,p＜0.01) and TNF-a secretion (48.12±11.52pg/ml vs 23.22±2.62, p＜0.05).Conclusions:Hypoxia induces autocrine of TNF-αby cardiomyocytes in vitro. HIF-1αplays an important role in hypoxia mediated TNF-αexpression and secretion in cardiomyocytes. PartⅡRegulation of TNF-a promoter by hypoxia inducible factor-la under hypoxia or CoCl2 mimicked hypoxiaAims:We investigated the direct effects of hypoxia on tumor necrosis factor-a (TNF-α) expression, and the role of hypoxia inducible factor-la (HIF-la) in TNF-a regulation.Methods and results:HEK293 and HepG2 cells were exposed in a hypoxia (1% O2) chamber or treated with CoCl2 to mimic hypoxia. HIF-la nuclear protein expression was detected by western blot, TNF-a mRNA levels were assayed by real time PCR. Significant up-regulation of HIF-la nuclear protein could be seen in both cell lines after 6-hour hypoxia and 3-hour CoCl2 mimic hypoxia stimulation. Obvious up-regulation of TNF-αmRNA levels was observed after 12-hour hypoxia and 12-hour CoCl2 mimic hypoxia. Inhibition of HIF-1αby 2-methoxyestradiol (2ME2) inhibited CoCl2 induced HIF-1αnuclear protein accumulation and TNF-a mRNA elevation. We found seven possible hypoxia response elements (HREs) at regions of 5'to the proximal 5000-bp fragment of the TNF-αpromoter relative to the transcription start site. Eight reporter gene plasmids were constructed after promoter truncation analysis, and then transiently transfected into HEK293 and HepG2 cells. Promoter activity was detected by dual-luciferase reporter assay after 24-hour hypoixa treatment. The results showed HIF consensus binding sites spanning bp-1470 relative to the transcription start site were functional for TNF-a promoter activity induction by hypoxia.Conclusions:We demonstrate that TNF-a is a hypoxia-inducible gene, whose transcription is stimulated through HIF-1αinteraction with HRE sites located at-1470 of the TNF-a promoter.