The Regulatory Activity of GIPC1 on RhoA‐Mediated bFGF Angiogenic Functions

Abstract

Introduction Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the best characterized pro-angiogenic factors involved in tumor angiogenesis. Acquired resistance upon anti-VEGF therapy remains a serious disadvantage of current approaches. Here we show that the small GTPase RhoA, which is known to regulate VEGF-induced angiogenesis, also regulates bFGF-induced angiogenesis and we further explore the role of the scaffolding protein GAIP Interacting Protein C-terminus (GIPC1) as a regulator of RhoA activation and bFGF angiogenic outcomes. Methods We performed in vitro angiogenesis assays (cell migration, tube formation, spheroid sprouting) to assess the impact of pharmacological inhibition or knockdown of endothelial RhoA on bFGF-induced angiogenesis using human umbilical vein endothelial cells (HUVECs). RhoA and GEF Pull-down experiments, along with other biochemical assays were performed to identify the molecular mechanism(s) of bFGF-induced RhoA activation and regulatory function of GIPC1. In vivo, endothelial-specific inducible RhoA-deficient mice with a fluorescent reporter were used in a modified matrigel plug angiogenesis assay and in the ex vivo aortic ring assay. Results In vitro, bFGF-induced angiogenesis was abrogated by pharmacological RhoA inhibition or RhoA knockdown. In vivo, tamoxifen-induced endothelial RhoA-deficiency blocked bFGF-induced angiogenesis. Mechanistically, bFGF activates RhoA, through the RhoA-specific GEFs GEFH1, P115, LARG and PDZ-RhoGEF. Among the bFGF receptors, FGFR1 is predominantly expressed in HUVECs and FGFR1 knockdown abrogated bFGF-induced RhoA activation and angiogenesis. The scaffolding protein GIPC1 interacts with FGFR1 and has an inhibitory effect, since its downregulation induced RhoA activation as well as basal and bFGF-induced angiogenesis. Ongoing experiments are aiming to further delineate the FGFR1-GIPC1-RhoA relationship. Conclusions Our data suggest that GIPC1 is an important element of bFGF-induced angiogenesis through its regulatory effect on endothelial RhoA.