reverse protocol, 12 w plate

Macrophages are often associated to pathophysiological processes and were found at hypoxic areas. However, cell adaption greatly depends on hypoxia-inducible factors (HIF). Activation of these transcription factors is induced by heterodimerization of an alpha-(HIF-1alpha, -2alpha, -3alpha) and HIF-1beta subunit. The main regulatory pathway is represented by alpha-subunit stability. Beside, little is known about the exact mechanisms of fine-tuning in Hif-regulation. The present study characterizes the hypoxia-induced regulation of HIF-1alpha and -2alpha in human macrophages. The hypoxic increase of both subunits is initially mediated by protein stabilization. Sustained hypoxia caused a distinct regulation of HIF-1alpha and -2alpha. The striking increase of HIF-2alpha protein expression was contrasted by a dramatic decrease of HIF-1alpha. The long-term downregulation of HIF-1alpha is due to downregulation of its mRNA. This decrease was accompanied by increased expression of ahif, a natural cis-antisense transcript of HIF-1alpha. The ahif-transcript was strongly inducible by hypoxia and rapidly degraded under reoxygenation. Using an adenoviral overexpression and siRNA silencing approach revealed that the targeted regulation of ahif is mediated by the HIF-system itself. Furthermore it could be shown that ahif indeed is able to modulate the hypoxic expression of HIF-1alpha and influences the expression of the HIF-target gene Enolase-2. Taken together, this study characterizes a new regulation process of the HIF-transcription factor-system in human macrophages under hypoxia. For the first time evidence is provided that ahif is regulated by the HIF-system and influences HIF-1alpha expression in primary human macrophages.