Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: initiated by hypoxia inducible factor 1α, presented by exosomes.

Excessive tumor necrosis factor-α (TNF-α) expression is increasingly thought to be detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-α is mainly released from macrophages, but with persistent ischemia, it can originate from cardiomyocytes and contribute to cardiac remodeling. The initiating factor and exact molecular mechanism of TNF-α release from cardiomyocytes is presently unclear. In this study, we investigated direct effects of hypoxia on TNF-α expression of cardiomyocytes, the role of hypoxia inducible factor-1α (HIF-1α) in TNF-α regulation and potential secretory pathway of TNF-α. Elevated TNF-α expression and HIF-1α activation in primary cultured cardiomyocytes under hypoxia were detected by real-time PCR, Western blotting and immunofluorescence. TNF-α mRNA elevation and protein secretion were obviously inhibited by nucleofection of HIF-1α small interfering RNA (siRNA) and treatment with 2-methoxyestradiol (inhibitor of HIF-1α protein). Similar results were observed in HEK293 and HepG2 cells. Putative hypoxia response elements were identified in the human TNF-α gene promoter. Deletion analysis and site-directed mutagenesis demonstrated that HIF consensus binding sites spanning bp-1295 to bp-1292 relative to the transcription start site were functional for activation of the TNF-α promoter which was confirmed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. Exosomes (vesicles mediating a non-classical route of protein secretion) in supernatants from hypoxic cardiomyocytes were identified by an anti-CD63 antibody in Western blot and observed by electron microscopy. The presence of TNF-α within exosomes precipitated from supernatants of hypoxic cardiomyocytes was verified by immunoelectron microscopy and immunoblotting. Results of this study indicate that under hypoxia, HIF-1α initiates expression of TNF-α, mediated by exosomes in cardiomyocytes.

Yu X ，Deng L ，Wang D ，Li N ，Chen X ，Cheng X ，Yuan J ，Gao X ，Liao M ，Wang M ，Liao Y ... -  《-》

Effect of hypoxia-inducible factor 1-alpha on hypoxia/reoxygenation-induced apoptosis in primary neonatal rat cardiomyocytes.

We studied the role of hypoxia-inducible factor 1-alpha (HIF-1α) in hypoxia/reoxygenation (H/R)-induced apoptosis in primary neonatal rat cardiomyocytes and its possible molecular mechanisms. Isolated neonatal and adult rat cardiac myocytes were cultured for 48h and were submitted to 5h of hypoxia followed by 2, 6, or 12h of reoxygenation. Small interfering RNA was used to target the HIF-1α gene. Cardiac myocyte apoptosis induced by H/R was assessed by Annexin V-FITC apoptosis assay. HIF-1α, Bnip3 and caspase-3 levels were determined by real-time reverse transcription polymerase chain reaction and western blot for mRNA and protein, respectively. H/R resulted in severe injury in cultured rat cardiomyocytes and it upregulated HIF-1α and proapoptotic Bnip3 mRNA and protein expression. HIF-1α activity inhibited by siRNA significantly decreased (P<0.01) the rate of apoptotic cardiomyocytes induced by 5h of hypoxia followed by 6h of reoxygenation compared with cardiomyocytes without siRNA treatment. Additionally, the expression of Bnip3 and caspase-3 was also markedly reduced. We conclude that HIF-1α is a key regulator of apoptosis of cardiomyocytes induced by H/R. H/R enhances primary neonatal rat cardiomyocyte apoptosis through the activation of HIF-1α and the mechanism might involve Bnip3 and caspase-3. HIF-1α may be a possible therapeutic target to limit myocardial injury after myocardial infarction.

Wang X ，Ma S ，Qi G 《-》

Molecular regulation of the PAI-1 gene by hypoxia: contributions of Egr-1, HIF-1alpha, and C/EBPalpha.

Liao H ，Hyman MC ，Lawrence DA ，Pinsky DJ ... -  《-》

[Role of hypoxia-inducible factor-1alpha in the prevention of cardiomyocyte injury induced by hypoxic preconditioning].

Xu FF ，Liu XH ，Cai LR 《-》

Opposite functions of HIF-α isoforms in VEGF induction by TGF-β1 under non-hypoxic conditions.

Transforming growth factor (TGF)-β1 has biphasic functions in prostate tumorigenesis, having a growth-inhibitory effect in the early stages, but in the late stages promoting tumor angiogenesis and metastasis. We demonstrate here that tumor-producing TGF-β1 induces vascular endothelial growth factor (VEGF) in prostate cancer cells, and hypoxia-inducible factor (HIF)-1α and HIF-2α has opposite functions in TGF-β1 regulation of VEGF expression under non-hypoxic conditions. The promoter response of VEGF to TGF-β1 was upregulated by the transfection of HIF-2α or siHIF-1α but downregulated by HIF-1α and siHIF-2α. Both HIF-1α and HIF-2α were induced by TGF-β1 at mRNA and protein levels, however, their nuclear translocation was differentially regulated by TGF-β1, suggesting its association with their opposite effects. VEGF induction by TGF-β1 occurred in a Smad3-dependent manner, and the Smad-binding element 2 (SBE2, -992 to -986) and hypoxia response element (-975 to -968) in the VEGF promoter were required for the promoter response to TGF-β1. Smad3 cooperated with HIF-2α in TGF-β1 activation of VEGF transcription and Smad3 binding to the SBE2 site was greatly impaired by knockdown of HIF-2α expression. Moreover, the VEGF promoter response to TGF-β1 was synergistically elevated by co-transfection of Smad3 and HIF-2α but attenuated by HIF-1α in a dose-dependent manner. Additionally, TGF-β1 was found to increase the stability of VEGF transcript by facilitating the cytoplasmic translocation of a RNA-stabilizing factor HuR. Collectively, our data show that tumor-producing TGF-β1 induces VEGF at the both transcription and post-transcriptional levels through multiple routes including Smad3, HIF-2α and HuR. This study thus suggests that autocrine TGF-β1 production may contribute to tumor angiogenesis via HIF-2α signaling under non-hypoxic conditions, providing a selective growth advantage for prostate tumor cells.

Chae KS ，Kang MJ ，Lee JH ，Ryu BK ，Lee MG ，Her NG ，Ha TK ，Han J ，Kim YK ，Chi SG ... -  《-》