Deletion of the auxiliary α2δ1 voltage sensitive calcium channel subunit in osteocytes and late-stage osteoblasts impairs femur strength and load-induced bone formation in male mice

Abstract

Osteocytes sense and respond to mechanical force by controlling the activity of other bone cells. However, the mechanisms by which osteocytes sense mechanical input and transmit biological signals remain unclear. Voltage-sensitive calcium channels (VSCCs) regulate calcium (Ca2+) influx in response to external stimuli. Inhibition or deletion of VSCCs impairs osteogenesis and skeletal responses to mechanical loading. VSCC activity is influenced by its auxiliary subunits, which bind the channel’s α1 pore-forming subunit to alter intracellular Ca2+ concentrations. The α2δ1 auxiliary subunit associates with the pore-forming subunit via a glycosylphosphatidylinositol (GPI) anchor and regulates the channel’s calcium-gating kinetics. Knockdown of α2δ1 in osteocytes impairs responses to membrane stretch, and global deletion of α2δ1 in mice results in osteopenia and impaired skeletal responses to loading in vivo. Therefore, we hypothesized that the α2δ1 subunit functions as a mechanotransducer, and its deletion in osteocytes would impair skeletal development and load-induced bone formation. Mice (C57BL/6) with LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, were crossed with mice expressing Cre under the control of the Dmp1 promoter (10 kb). Deletion of α2δ1 in osteocytes and late-stage osteoblasts decreased femoral bone quantity (p<0.05) by DXA, reduced relative osteoid surface (p<0.05), and altered osteoblast and osteocyte regulatory gene expression (p<0.01). Cacna2d1f/f, Cre+ male mice displayed decreased femoral strength and lower 10-wk cancellous bone in vivo μCT measurements at the proximal tibia (p<0.01) compared to controls, whereas Cacna2d1f/f, Cre+ female mice showed impaired 20-wk cancellous and cortical bone ex vivo μCT measurements (p<0.05) versus controls. Deletion of α2δ1 in osteocytes and late-stage osteoblasts suppressed load-induced calcium signaling in vivo and decreased anabolic responses to mechanical loading in male mice, demonstrating decreased mechanosensitivity. Collectively, the α2δ1 auxiliary subunit is essential for the regulation of osteoid-formation, femur strength, and load-induced bone formation in male mice.