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Induction of Carbonic Anhydrase II Expression in Osteoclast Progenitors Requires Physical Contact with Stromal Cells¹

Department of Medicine, Emory University School of Medicine and Veterans Administration Medical Center, Atlanta, Georgia 30033

Address all correspondence and requests for reprints to: Diane M. Biskobing, VA Medical Center - 111, 1670 Clairmont Road, Decatur, Georgia 30033. E-mail: dbiskob{at}emory.edu

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)₂D₃ in myelomonocytic cells and marrow culture. However, since 1,25(OH)₂D₃ stimulates osteoclast differentiation as well, we wished to separate direct effects of 1,25(OH)₂D₃ 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)₂D₃ 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)₂D₃, 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)₂D₃ stimulated CA II expression by 22 ± 3.6-fold. 1,25(OH)₂D₃ 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)₂D₃ 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.

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