Integrins are heterodimeric cell-surface adhesion receptors that play an important role in mediating numerous physiological processes, including inflammation, migration, adhesion, and proliferation. The beta subunits consist of a large extracellular domain, a single transmembrane segment, and a relatively short cytoplasmic tail. The cytoplasmic domains do not contain intrinsic tyrosine kinase activity, and therefore signaling occurs primarily via recruitment of intracellular signaling molecules. In particular, β3-integrins have gained interest due to their implications in regulating angiogenesis.

β3-endonexin has been identified as a β3-integrin binding protein which is expressed in endothelial cells and has been suggested to act as a shuttle protein between membrane and nucleus. However, its functional role in endothelial angiogenic processes is unclear. Since hypoxia is an important stimulus for angiogenesis we investigated the functional role of β3-endonexin in the hypoxic angiogenic response.

Using a matrigel tube formation assay we observed increased tube formation upon exposure to hypoxia. Surprisingly, overexpression of β3-endonexin prevented the hypoxia angiogenic response, while depletion of β3-endonexin by shRNA not only enhanced the basal angiogenic response but also promoted the response to hypoxia.

Similar results were obtained for endothelial proliferative activity as determined by BrdU incorporation.

Since the transcription factor HIF-1 is a major regulator of hypoxic angiogenesis we determined HIF activity in the presence or absence of β3-endonexin. Interestingly, hypoxic HIF activity was diminished when β3-endonexin was overexpressed but was enhanced in β3-endonexin depleted cells. Depletion of β3-endonexin resulted not only in enhanced HIF-1a protein levels but also in a marked increase in HIF-1a mRNA levels under hypoxic conditions. Reporter gene experiments using the human HIF-1a promoter confirmed increased reporter activity in the absence of β3-endonexin. However, when using a HIF1a promoter construct where the NFkB consensus sequence was mutated, this stimulatory effect was abolished. In line, depletion of β3-endonexin enhanced NFkB driven reporter gene activity under hypoxia, while overexpression of this protein prevented hypoxic reporter gene activation. Subsequently, expression of dominant-negative IkB prevented HIF-1a mRNA induction in the absence of β3-endonexin. Together with our findings that β3-endonexin shuttles to the nucleus in response to hypoxia these data indicate that β3-endonexin is a novel negative regulator of the HIF system by interfering with the NFkB system thus preserving HIF-1a mRNA levels under hypoxic conditions and finally promoting hypoxic angiogenic response.