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5F�CI), TRAP activity (Fig. 5O�CR), and CTX (Fig. 5S) and VEGF (Fig. 5T) were abolished by the lack of ATF4. Thus, Atf4 deficiency decreases VEGF expression in osteoblasts as well as VEGF release from bone matrix owing to reduced osteoclast activity and bone resorption. To define signaling pathways that regulate bone angiogenesis, E17.5 metatarsals were treated with or without the indicated inhibitors or activator for various pathways. We observed that endothelial sprouting from WT metatarsal cultures was inhibited by GF109203X (PKC inhibitor) but not by U0126 (Erk1/2 inhibitor) (Fig. 6A�CC, L). In contrast, PMA (PKC activator) strikingly induced endothelial sprouting in WT metatarsal cultures in a dose- and time-dependent manner (Fig. 6D, L and Supplemental Fig. S5). The PMA-induced increase in endothelial sprouting was blocked by treatment with OPG (Fig. 6E, L), suggesting the involvement of osteoclast activation in this regulation. Indeed, PMA increased osteoclast differentiation in metatarsals (Supplemental Fig. S6). Further, RANKL- and PTHrP-induced increases in endothelial sprouting were completely suppressed by the PKC inhibition (Fig. 6F�CI, L). As shown in Fig. 6M, the level of VEGF in metatarsal cultures was decreased by GF109203X but increased by PMA. Further, RANKL- and PTHrP-induced increases in VEGF were blocked by the PKC inhibition. Importantly, PMA failed to induce VEGF and endothelial sprouting in the absence of ATF4 (Fig. 6J�CM). Collectively, these results suggest that PKC promotion of VEGF release from the bone matrix and bone angiogenesis is through ATF4-dependent osteoclast activation and bone resorption. To evaluate if ATF4 plays a role in regulation of bone angiogenesis under pathological conditions, we investigated whether ATF4 is required for hypoxia/reoxygenation induction of HIF-1�� and VEGF expression and angiogenesis in bone. One-month-old WT and Atf4?/? male mice were placed in a hypoxia chamber (Supplemental Fig. S7) (8% O2) 2 hours per day for 10 days. Results showed that in vivo hypoxia significantly increased the expression levels of HIF-1�� protein and Vegf and Glut1 mRNAs in WT tibias but not in Atf4?/? tibias (Fig. 7A�CC). In contrast, the levels of Hif-1�� and pVhl mRNAs were not altered by either hypoxia or ATF4 deficiency (Fig. 7D, E). CD31 staining of sections of the tibial chondro-osseous junction regions revealed that in vivo hypoxia/reoxygenation increased microvessel density by 1.8-fold in WT tibias (p?