The hypoxia-inducible factor (HIF) system plays a key role in the adaptation to decreased oxygen tension. In its active form, HIF is a heterodimer consisting of a constitutively expressed beta- subunit (ARNT) and a regulated alpha-subunit. While the functions of human HIF-1alpha and HIF-2alpha have been well characterized, the functional role of human HIF-3alpha is less understood.

We found thatHIF3AmRNA is expressed in human microvascular endothelial cells (EC). Exposure of EC to severe hypoxia (0.1% oxygen) increasedHIF3AmRNA levels within 8 to 16 h. This response was diminished by depletion of HIF-1alpha by RNAi. In a luciferase assay, over-expressed HIF-3alpha decreased hypoxia-stimulated promoter activity of the HIF-target gene plasminogen activator-inhibitor-1 (PAI1). Oppositely, depletion ofHIF3Aincreased expression ofPAI1at later time points of hypoxia (>24h) while knockdown ofHIF2Abut notHIF1AabolishedPAI1expression in EC under hypoxia.

Co-immunoprecipitation assays revealed that HIF-3alpha interacted with HIF-1alpha, HIF-2alpha and ARNT. Gel shift assays demonstrated that HIF-3alpha/ARNT but not alpha/alpha dimers bind to HIF-response elements on DNA. Chromatin immunoprecipitation analysis revealed that HIF-2alpha binding to thePAI1promoter was slightly increased at 48h of hypoxia but was not detectable under normoxia. Knockdown ofHIF3Aincreased HIF-2alpha occupation of thePAI1promoter at 48h of hypoxia but not under normoxia. In contrast, ARNT occupation of thePAI1promoter was stable at hypoxia. Depletion of HIF-2alpha and HIF-3alpha abolished ARNT binding to thePAI1promoter indicating that a HIF-3alpha/ARNT complex blocks DNA binding of HIF-2alpha after prolonged hypoxia thereby decreasing target gene expression. This inhibitory effect was also shown in a functional angiogenesis assay: HIF-3alpha depletion increased tube formation whereas knockdown of HIF-2alpha decreased formation of tube like structures.

Collectively, we showed that HIF-3alpha inhibits HIF-2 activity by blocking thePAI1promoter under prolonged hypoxia, exerting a counterregulatory mechanism. Considering HIF-1alpha and HIF-2alpha being expressed in hypoxic regions of many solid tumors, understanding of its regulation by HIF-3alpha could be of potential therapeutical value.