Hypoxia-induced TIMAP upregulation in endothelial cells and TIMAP-dependent tumor angiogenesis

Abstract

TIMAP, the endothelial cell-predominant protein phosphatase 1β regulatory subunit also known as PPP1R16B, promotes in vitro endothelial cell proliferation and angiogenic sprouting. TIMAP was first identified as a target of TGF-β1 mediated repression, but the molecular pathways regulating its expression in endothelial cells are not well-defined. This study examined the role of BMP9, hypoxia and angiogenic growth factors in the regulation of TIMAP expression and determined whether TIMAP plays a role in tumor angiogenesis and growth in vivo. BMP9, which potently activated the SMAD1/5/8 pathway in endothelial cells, significantly reduced TIMAP mRNA and protein expression. Conversely, hypoxia and the prolyl hydroxylase inhibitor Roxadustat raised TIMAP mRNA and protein levels by inhibiting the BMP9 pathway. Angiogenic growth factors, most prominent among them VEGFA and IGF-I, raised endothelial TIMAP levels partly by attenuating BMP9 pathway activation, but also through BMP pathway-independent mechanisms. Cultured breast cancer E0771 cells released mediators that raised TIMAP expression in endothelial cells, effects that were inhibited by the VEGF inhibitor Sunitinib in conjunction with the IGF-1 inhibitor Picropodophyllin. In the mouse E0771 breast cancer model in vivo, tumor growth and tumor angiogenesis were markedly attenuated in TIMAP deficient, compared to wild-type littermates. These findings indicate that TIMAP plays a critical pro-angiogenic function during tumor angiogenesis in vivo, likely through hypoxia-driven inhibition of the BMP9 pathway and through elaboration of angiogenic growth factors by tumor cells.