GIT1Y321 phosphorylation is required for ERK1/2- and PDGF-dependent VEGF secretion from osteoblasts to promote angiogenesis and bone healing

Abstract

Bone healing depends on vascular endothelial growth factor (VEGF) secretion from osteoblasts to promote angiogenesis. We examined the influence of the tyrosine 321 site of G protein-coupled receptor kinase interacting protein 1 (GIT1) on platelet-derived growth factor (PDGF)-induced VEGF synthesis in vitro and on bone healing in vivo. Cultured osteoblasts were prepared from calvaria of 1-2-day-old rats. The phospho-activation of extracellular signal-regulated kinases 1/2 (ERK1/2), GIT1, the interaction between GIT1 and ERK1/2, and VEGF mRNA expression were measured in response to PDGF. In addition, PDGF was applied following pretreatment with the MEK1/2 inhibitor PD98059 or the Src inhibitor PP2. We mutated tyrosines 293 or 321 of GIT1 individually to phenylalanine (mutants GIT1Y293F and GIT1Y321F) and incorporated these mutants and native GIT1 into lentivirus vectors. The relationship between GIT1 and ERK1/2, and VEGF mRNA expression in cultured osteoblasts were detected after infection with GIT1WT-, GIT1Y293F- and GIT1Y321F-expressing lentivirus in response to PDGF. Bone healing and expression of VEGF and the angiogenic marker PECAM-1 were evaluated after infection at the fracture site. Activation of ERK1/2 by phosphorylation, GIT1 tyrosine phosphorylation, GIT1-ERK1/2 interaction, and VEGF mRNA expression were all significantly increased in osteoblasts after PDGF stimulation, but all responses were dramatically inhibited by pretreatment with PD98059. Tyrosine phosphorylation, GIT1 interaction with ERK1/2, and VEGF mRNA expression were dramatically inhibited by pretreatment with PP2 or infection with GIT1Y321F-expressing lentivirus. Expression of VEGF and PECAM-1 was significantly lower at the fracture sites infected with GIT1Y321F-expressing lentivirus and bone healing was significantly delayed compared to fracture sites infected with GIT1WT. In conclusion, tyrosine 321 of GIT1 is a critical phosphorylation site for GIT1 interaction with ERK1/2, regulation of ERK1/2 activation, VEGF expression and angiogenesis at the fracture site. Furthermore, GIT1 is a seminal signaling protein regulating bone healing.