Hypoxia‐Induced TIMAP Upregulation Promotes Tumor Angiogenesis by Attenuating ALK1‐Mediated TIMAP Repression

Abstract

Introduction TIMAP (TGF-β1 inhibited membrane associated protein) is an endothelial cell (EC)-predominant inhibitory protein phosphatase 1β (PP1β) regulatory subunit in the myosin phosphatase targeting (MYPT) family (J. Biol. Chem. 2294:13280, 2019). TIMAP promotes proliferation and survival of EC and enhances in vitro sprouting angiogenesis (Am. J. Physiol. - Renal Physiol. 307: F623, 2014). Hypothesis Hypoxia stimulates TIMAP expression in EC, enhancing in vivo tumor angiogenesis. Methods Mammary adenocarcinoma cells (E0771, syngeneic for C57BL/6 mice) were injected into the mammary glands of five pairs of female TIMAP+/+ / TIMAP-/- C57BL/6 mice at 6-8 weeks of age. For each pair, mice were euthanized on the day the tumor diameter exceeded 1.5 cm in one mouse. Tumor weight, mean tumor diameter, and tumor vascular density were then quantified. For quantification of vascular density, the EC markers were PECAM1 and Tie-2 for fluorescence microscopy (IF) and western blot (WB) analysis, respectively. TIMAP+/+ mice were also exposed to progressive, chronic hypoxia by reducing the ambient O2 concentration from 18 to 10 % over two weeks and then maintaining it at 10% for 3 weeks. Lung tissue was then evaluated for TIMAP abundance by WB analysis, and TIMAP localization was determined by IF. Finally, the mechanism regulating TIMAP expression was evaluated in cultured EC in the presence of 21% and 1% O2 with or without TGF-β pathway activation or inhibition. Results Tumor size was similar in TIMAP+/+ and TIMAP-/- mice through days 8-10 after injection of E0771 cells. In 4 of 5 TIMAP-/-, but not TIMAP+/+ mice, the tumors then eroded partially through the skin and regressed gradually. At the time of euthanasia tumor size (Fig. 1A), weight (Fig. 1B) and blood vessel density (Fig.1C, D&E) were significantly lower in TIMAP-/- compared to TIMAP+/+ mice. In the chronic hypoxia model, TIMAP protein abundance in lung lysates was significantly higher in hypoxic compared to control mice, and IF using anti-TIMAP and anti-PECAM1 antibodies revealed that the TIMAP protein was restricted to lung EC. In cultured EC, HIF-1α and TIMAP protein abundance were significantly higher in EC exposed to 1% than 21% O2, (Fig. 1F,G). The effect of 1% O2 on TIMAP was only observed in the presence of fresh 1% serum or BMP-9 (1-10 ng/ml), while the hypoxia-induced increase in HIF-1α abundance was independent of serum or BMP-9. In the absence of hypoxia 1% serum and BMP-9 strongly stimulated Smad 1/5/8 but not Smad 2/3 phosphorylation and significantly reduced TIMAP protein levels. The ALK1 inhibitor K02288 but not the ALK 5 inhibitor LY-364947 blocked 1% serum and BMP-9 stimulated Smad 1/5/8 phosphorylation and raised the EC TIMAP abundance. Notably, hypoxia mimicked the ALK1 inhibitor, significantly blunting 1% serum and BMP-9-stimulated Smad 1/5/8 phosphorylation. Conclusion Hence, hypoxia induces expression of the pro-angiogenic PP1β inhibitor TIMAP due, at least in part, to inhibition of BMP-9/ALK1-mediated repression of TIMAP. Furthermore, TIMAP is necessary for effective tumor angiogenesis in vivo. We therefore speculate that tumor hypoxia stimulates EC TIMAP expression by inhibiting the ALK1 pathway.