Gαi1/3 mediate Netrin-1-CD146-activated signaling and angiogenesis.

Netrin-1 binds to the high-affinity receptor CD146 to activate downstream signaling and angiogenesis. Here, we examine the role and underlying mechanisms of G protein subunit alpha i1 (Gαi1) and Gαi3 in Netrin-1-induced signaling and pro-angiogenic activity. In mouse embryonic fibroblasts (MEFs) and endothelial cells, Netrin-1-induced Akt-mTOR (mammalian target of rapamycin) and Erk activation was largely inhibited by silencing or knockout of Gαi1/3, whereas signaling was augmented following Gαi1/3 overexpression. Netrin-1 induced Gαi1/3 association with CD146, required for CD146 internalization, Gab1 (Grb2 associated binding protein 1) recruitment and downstream Akt-mTOR and Erk activation. Netrin-1-induced signaling was inhibited by CD146 silencing, Gab1 knockout, or Gαi1/3 dominant negative mutants. Netrin-1-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation were inhibited by Gαi1/3 short hairpin RNA (shRNA), but were potentiated by ectopic Gαi1/3 overexpression. In vivo, intravitreous injection of Netrin-1 shRNA adeno-associated virus (AAV) significantly inhibited Akt-mTOR and Erk activation in murine retinal tissues and reduced retinal angiogenesis. Endothelial knockdown of Gαi1/3 significantly inhibited Netrin1-induced signaling and retinal angiogenesis in mice. Netrin-1 mRNA and protein expression were significantly elevated in retinal tissues of diabetic retinopathy (DR) mice. Importantly, silence of Netrin-1, by intravitreous Netrin-1 shRNA AAV injection, inhibited Akt-Erk activation, pathological retinal angiogenesis and retinal ganglion cells degeneration in DR mice. Lastly, Netrin-1 and CD146 expression is significantly increased in the proliferative retinal tissues of human proliferative diabetic retinopathy patients. Together, Netrin-1 induces CD146-Gαi1/3-Gab1 complex formation to mediate downstream Akt-mTOR and Erk activation, important for angiogenesis in vitro and in vivo.