Phosphate and Calcium are Required for TGFβ-Mediated Stimulation of ANK Expression and Function During Chondrogenesis

The expression of ANK, a key player in biomineralization, is stimulated by treatment with TGF beta. The purpose of this study was to determine whether TGF beta stimulation of ANK expression during chondrogenesis was dependent upon the influx of calcium and phosphate into cells. Treatment of ATDC5 cells with TGF beta increased ANK expression during all phases of chondrogenic differentiation, particularly at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. Phosphate uptake studies in the presence and absence of phosphonoformic acid (PFA), a competitive inhibitor of the type III Na+/Pi channels Pit-1 and Pit-2, indicated that the stimulation of ANK expression by TGF beta required the influx of phosphate, specifically by the Pit-1 transporter, at all phases of differentiation. At hypertrophy, when alkaline phosphatase is highly expressed, inhibition of its activity with levamisole also abrogated the stimulatory effect of TGF beta on ANK expression, further illustrating that Pi availability and uptake by the cells is necessary for stimulation of ANK expression in response to TGF beta. Since previous studies of endochondral ossification in the growth plate have shown that L-type calcium channels are essential for chondrogenesis, we investigated their role in the TGF beta-stimulated ANK response in ATDC5 cells. Treatment with nifedipine to inhibit calcium influx via the L-type channel Cav1.2 (alpha(1C)) inhibited the TGF beta stimulated increase in ANK expression at all phases of chondrogenesis. Our findings indicate that TGF beta stimulation of ANK expression is dependent upon the influx of phosphate and calcium into ATDC5 cells at all stages of differentiation. J. Cell. Physiol. 224: 540-548, 2010. (C) 2010 Wiley-Liss, Inc.