Bone Abstracts

Using a CyPB-null mouse model, we previously demonstrated delayed folding, abnormal post-translational modification and altered crosslinking of type I collagen synthesized by osteoblasts. However, intracellular folding of collagen molecules was further delayed by CsA treatment of CyPB-null cells, suggesting involvement of additional PPIases in collagen folding. Since studies of CyPA functions in osteoblasts have not been reported, we investigated the role of this cytoplasmic PPIase in collagen synthesis and bone development using a CyPA knockout mouse model. CyPA^−/− mice exhibit moderate growth deficiency, with reduced weight (10%) and decreased (7–9%) femoral and tibial lengths versus WT at age 2 months. Impairment of endochondral bone formation was associated with decreased aBMD in male and female femora (12–14%) and L1/L2 vertebrae (6–12%), respectively. CyPA^−/− fibroblasts and osteoblasts secrete normal amounts of type I collagen, with normal electrophoretic mobility. In direct intracellular folding assays, the CyPA^−/− fibroblast collagen folding rate was equivalent to WT, and no further delay in folding was observed in CyPA/CyPB double knockout cells compared to CyPB^−/− cells alone. Interestingly, Ca²⁺ homeostasis was dysregulated in CyPA^−/− cells, resulting in decreased resting cytoplasmic [Ca²⁺]_i, and ATP- and ionomycin-induced Ca²⁺ release from intracellular stores. These findings extend data from platelets demonstrating that the interaction between CyPA and SERCA, the ER calcium replenishment pump, regulates SERCA function. Consequently, expression of both early and late osteoblast markers, including Rankl/Opg, Runx2, Sp7, Alpl, Ocn and Ibsp, was decreased 30–80% in differentiating CyPA^−/− osteoblasts, while osteoblast expression of adipogenic markers Cebpβ, Cebpδ, Pparg, and target genes Fabp4 and Lpl was increased 200–400% versus WT osteoblasts. Paradoxically, CyPA^−/− osteoblasts may activate Wnt signaling, with increased expression of Wnt5a (2-fold) and Axin2 (1.6-fold). Thus, although CyPA has no direct role related to type I collagen, its impact on Ca²⁺-dependent intracellular signaling pathways affects bone formation by altering osteoblast development.