title: Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: initiated by hypoxia inducible factor 1α, presented by exosomes.
abstract: 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.
authors.affiliation: Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Xian
authors.initials: X
authors.lastname: Yu
authors.forename: Lingyan
authors.initials: L
authors.lastname: Deng
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Xiao
authors.initials: X
authors.lastname: Chen
authors.forename: Xiang
authors.initials: X
authors.lastname: Cheng
authors.forename: Jin
authors.initials: J
authors.lastname: Yuan
authors.forename: Xingli
authors.initials: X
authors.lastname: Gao
authors.forename: Mengyang
authors.initials: M
authors.lastname: Liao
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Liao
mesh_terms: D000818:Animals; D019898:Autocrine Communication; D015687:Cell Hypoxia; D055354:Exosomes / Q000378:metabolism*; D005786:Gene Expression Regulation; D023061:Gene Order; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D015398:Signal Transduction; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014409:Tumor Necrosis Factor-alpha