Phosphate Is a Specific Signal for ATDC5 Chondrocyte Maturation and Apoptosis-Associated Mineralization: Possible Implication of Apoptosis in the Regulation of Endochondral Ossification

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Abstract
Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis‐associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification. Introduction: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra‐ and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered. Materials and Methods: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase‐polymerase chain reaction [RT‐PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer‐1 (EthD‐1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl‐2/Bax mRNA ratio (RT‐PCR analysis). Results: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl‐2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase‐3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD‐fmk reduces π‐induced mineralization. Conclusions: These findings suggest that Pi regulates chondrocyte maturation and apoptosis‐associated mineralization, highlighting a possible role for Pi in the control of skeletal development.

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