Regulation of chondrocyte differentiation by phosphate

Objectives: Endochondral ossification is critical to the development and growth of mammalians. Growth retardation seen in X-linked hypophosphatemia (XLH) characterized by disrupted phosphate metabolism and high phosphate levels in cartilage suggest the relationship between phosphate and endochondral ossification. Since the endochondral ossification is largely dependent on cartilage differentiation, we studied the role of phosphate in chondrocyte differentiation. Methods: Hyp mice, which are the murine homologue of human XLH, were studied. Primary growth plate chondrocytes were isolated from the ribs of wild-type (WT) and Hyp mice by sequential enzymatic digestion. In some in vitro experiments, the N1511 mouse chondrogenic cell line was used. Results: Histological examination of Hyp mice showed disturbed endochondral ossification with widened and irregularly-arranged growth plate and decreased cartilage calcification compared with WT mice. Moreover, the number of apoptotic chondrocytes determined by TUNEL staining was also decreased in the Hyp growth plate.When Hyp mice were fed with high phosphate diet, the disturbed growth plate and diminished calcification was partially restored. Since phosphate is known to regulate cellular metabolism by entering cells through the sodium-dependent phosphate transporter (NPT), the expression of NPT was examined in WT and Hyp chondrocytes. We found NPT3 expression was decreased in Hyp chondrocytes compared with WT chondrocytes by RT-PCR, while NPT2a expression was not different. Consistent with this, 32P uptake by Hyp chondrocytes was reduced. Moreover, Hyp chondrocytes displayed reduced mineralization by alizarin red staining. Hyp chondrocytes transfected with NPT3 cDNA showed increased mineralization. The NPT competitive inhibitor phosphonoformic acid (PFA) decreased 32P uptake and mineralization in N1511 chondrocytes. Moreover, PFA administration caused hypophosphatemia and disturbed endochondral ossification with widened growth plate and diminished apoptosis in WT mice. Conclusion: Our results suggest phosphate regulates mineralization and apoptosis of chondrocytes by entering these cells through NPT3.