P347Suppression of Hif-1a by a natural antisense transcript in human macrophages - Consequences for the hypoxic gene regulation

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During the atherosclerotic process hypoxic areas occur due to the increase in wall thickness. Mainly macrophages were found to be located within these areas. Essential for the hypoxic gene regulation are the active α-subunits of the hypoxia-inducible-factors (HIF-1α, HIF-2α, HIF-3α). These transcription factors are regulated by stabilization of the α-subunits, but also alternative regulations are known. One of these is the regulation of Hif-1α by a natural cis-antisense transcript. However, the role of ahif within the vascular system and its mode of regulation and function are barely investigated. Therefore, this study focusses on the HIF-1α-ahif interaction within vascular cells, especially macrophages. Compared to vascular smooth muscle cells and human umbilical vein endothelial cells, macrophages show the most distinct effect regarding the upregulation of ahif and the downregulation of Hif-1α after 24 h hypoxia (0.5% O2). Thus, macrophages were chosen to further investigate the interaction of HIF-1α and ahif. Hypoxia initially stabilizes HIF-1α and -2α protein, but sustained hypoxia (24 h) downregulates HIF-1α, whereas HIF-2α protein further increases. In contrast to HIF-1α, the mRNA of HIF-2α also remains unchanged. Using an adenoviral overexpression approach and siRNA-mediated silencing of Hif-1α and -2α, it could be shown that both Hif-α subunits are involved in ahif-regulation. The analysis of the putative promoter of ahif revealed that it contains several hypoxia-response-elements (HRE), the recognition sequence of the HIF-transcription-factor. The electromobility-shift-assay shows a binding of HIF-1α and -2α to a tandem HRE out of the ahif promotor. The modulation of HIF-1α expression by ahif was studied by adenoviral overexpression of ahif, which leads to reduced HIF-1α mRNA levels hypoxia, whereas HIF-2α mRNA is not affected. HIF-1α protein levels are reduced in ahif-overexpressing cells. The expression of Enolase2, an glycolytic enzyme and known target gene of the HIF-pathway, was analyzed. Hypoxia increases the expression of Enolase2 6fold. The overexpression of ahif leads to a 35% reduction of the hypoxia-driven mRNA-induction. In addition, siRNA-mediated silencing of ahif leads to elevated Hif-1α mRNA and protein expression. In conclusion, this study describes an alternative regulation-pathway of the HIF-system, which is responsible for a different regulation of HIF-1 and HIF-2 under prolonged hypoxia in human macrophages. This negative feedback regulation is mediated by ahif, which acts as a mediator between the both HIF isoforms.

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