Function And Mechnism Of ATF4in Regulation Of Bone Angiogenesis

Bone is a highly vascularized tissue. Accumulated evidence suggests that angiogenesis is essential for bone formation during skeletal development as well as bone regeneration in response to injury. However, the molecular mechanisms that regulate angiogenesis in bone are only partially elucidated.Transcription activation factor4(ATF4) is critical for bone remodeling during skeletal development and throughout life; however, its role in bone angiogenesis is unclear. We performed contrast-enhanced μCT imaging in Microfil-perfused femurs and showed that ATF4deficiency strikingly reduced vessel volume and vessel number in calvaria and femurs. Results from immunohistochemical staining of tibial sections using a specific antibody against CD31revealed that both the size and density of vessels within the metaphyseal periosteum and diaphyseal endosteum from Atf4-/-tibiae were dramatically decreased compared to those of WT tibiae. Quantitative Real-Time PCR analysis revealed that the mRNA levels of Vegf and its3isoforms (Vegf120, Vegf165, and Vegf188) were all also significantly decreased in Atf4-/-versus WT tibiae. And the expression of VEGF was dramatically reduced in Atf4-/-osteoblasts located on bone surfaces and in Atf4-/-bone matrix, either. Surprisingly, the level of serum VEGF was not different in mice of the two genotypes. In vitro angiogenesis assay showed that ablation of ATF4prevented endothelial sprouting from metatarsals, which was rescued by treatment with a recombinant VEGF. Lack of ATF4reduced HIF-la protein levels without altering the expression of pVHL in osteoblasts located on trabecular bone surfaces. Adenoviral overexpression of ATF4increased HIF-1α and VEGF expression levels in osteoblasts in a hypoxia-dependent manner.Mechanistically, loss of ATF4increased HIF-la ubiquitination and decreased HIF-la protein stability without affecting its mRNA stability and protein translation. In addition, loss of ATF4increased the binding of HIF-1α to prolyl hydroxylases (PHD1, PHD2, and PHD3), the enzymes that hydroxylate HIF-1α protein and promote its proteasomal degradation via the pVHL pathway. Interestingly, parathyroid hormone-related protein and receptor activator of NF-κB ligand, both well-known activators of osteoclasts, increased release of VEGF from the bone matrix and promoted angiogenesis through the protein kinase C-and ATF4-dependent activation of osteoclast differentiation and bone resorption. Meanwhile, our in vivo studies revealed that hypoxia/reoxygenation induction of HIF-1α and VEGF expression leading to bone angiogenesis, a key adaptive response to hypoxic condition, was severely compromised in mice lacking the Atf4gene.Taken together, our studies demonstrate that ATF4is a new key regulator of the HIF/VEGF axis in osteoblasts in response to hypoxia and of VEGF release from bone matrix, two critical steps for bone angiogenesis.