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Increased physical loading of the skeleton activates new bone formation ensuring its ability to meet mechanical demands over time; however, the capacity of bone to respond to mechanical stimulation diminishes with age. Osteocytes, the cells embedded and dispersed throughout mineralized bone matrix, are master regulators of mechanoadaptation through recruitment of new bone-forming cells, the osteoblasts, via signaling to osteoprogenitors located on bone surfaces. We previously demonstrated that in vivo and in vitro mechanical stimulation significantly upregulated the chemokine C-X-C Motif Chemokine Ligand 12 (CXCL12) and its receptor, CXCR4, in osteocytes and bone lining cells, and that CXCR4 antagonism with AMD3100 attenuated in vivo load-induced bone formation. Here, we extended this work by showing that ablation of CXCL12+ cells and deletion of cxcl12 in late-stage osteoblasts and osteocytes significantly attenuated in vivo load-induced bone formation in the mouse tibia. This bone loading phenotype was rescued by treatment with recombinant CXCL12. To address mechanism, we showed that in vitro deletion of cxcl12 and cxcr4, separately, in bone marrow stromal cells resulted in significantly reduced osteogenic differentiation. Furthermore, CXCL12 treatment enhanced GSK-3b phosphorylation and β-catenin translocation to the nucleus, the former of which was partially blocked by AMD3100. Finally, CXCL12 synergized Wnt signaling leading to significantly increased total β-catenin protein and Axin2 expression, a Wnt signaling target gene. These findings together demonstrate that CXCL12 expression in late-stage osteoblasts and osteocytes is essential for load-induced bone formation, in part, by regulating osteogenic differentiation through activation of the Wnt signaling pathway.SignificanceSkeletal adaptation to mechanical loading is contingent on the recruitment of new osteoblasts to bone surfaces. CXCL12, a chemokine expressed by osteolineage cells, targets effector cells expressing its receptor CXCR4, including osteoprogenitors. Exogenous mechanical loading of mouse hind limbs upregulates CXCL12 in osteocytes, bone lining cells and marrow cells, while antagonizing CXCR4 led to significantly attenuated load-induced bone formation. Here, we show that CXCL12 expression in late-stage osteoblasts and osteocytes is required for load-induced bone formation. Treatment with recombinant CXCL12 rescued the bone loading phenotype suggesting that the CXCL12/CXCR4 signaling pathway may be a feasible drug target for promoting load-induced bone formation when exercise alone is insufficient to counteract low bone mass and osteoporosis. |
