Induction of carbonic anhydrase II expression in osteoclast progenitors requires physical contact with stromal cells

Carbonic anhydrase II (CA II) expression is vital to normal osteoclast function. We and others have previously reported induction of CA II messenger RNA (mRNA) expression by 1,25(OH)(2)D-3 in myelomonocytic cells and marrow culture. However, since 1,25(OH)(2)D-3 stimulates osteoclast differentiation as well, we wished to separate direct effects of 1,25(OH)(2)D-3 on the CA II gene from the differentiating effects of the hormone. Using primary murine mixed marrow cultures, we measured CA II mRNA expression by RT-PCR. 10 nM 1,25(OH)(2)D-3 dose dependently induced expression of CA II mRNA (4.12 +/- 0.68-fold) at day 4 in culture compared with control with an ED50 of 0.25 nM. When nonadherent marrow cells containing osteoclast progenitors were depleted of stromal cells and exposed to 10 nM 1,25(OH)(2)D-3, CA II mRNA expression was decreased by more than 60%. Coculture of progenitors with ST-2 stromal cells for 3 days with 10 nM 1,25(OH)(2)D-3 stimulated CA II expression by 22 +/- 3.6-fold. 1,25(OH)(2)D-3 stimulated CA II mRNA expression in progenitors separated from ST-2 cells by transwells was insignificant demonstrating that the two cell types must be in physical contact. PTH also stimulated CA II mRNA expression (4.91 +/- 0.01-fold) to a similar degree as seen with 1,25(OH)(2)D-3 treatment. These results demonstrate that induction of CA II in osteoclast progenitors requires their physical communication with stromal cells and is inseparable from the osteoclast differentiation process.