Evidence that calcineurin is required for the genesis of bone-resorbing osteoclasts

Evidence that calcineurin is required for the genesis of bone-resorbing osteoclasts. Am J Physiol Renal Physiol 292:F285-F291, 2007. First published September 12, 2006; doi:10.1152/ajprenal.00415.2005.-Here, we demonstrate that the Ca2+/calmodulin-sensitive phosphatase calcineurin is a necessary downstream mediator for osteoclast differentiation. Using quantitative PCR, we detected the calcineurin isoforms A alpha, A beta, A gamma (catalytic), and B1 (regulatory) in osteoclast precursor RAW-C3 cells. We found that, although the expression of these isoforms remained relatively unchanged during osteoclast differentiation, there was a profound increase in the expression of their primary substrate for calcineurin, nuclear factor of activated T cells (NFAT)c1. For gain-of-function studies, we incubated osteoclast precursors for 10 min with a calcineurin fusion protein (TAT-calcineurin A alpha); this resulted in its receptorless influx into > 90% of the precursor cells. A marked increase in the expression of the osteoclast differentiation markers tartrate-resistant acid phosphatase (TRAP) and integrin beta(3) followed. In addition, the expression of NFATc1, as well is the alternative substrate for calcineurin, I kappa B alpha, was significantly enhanced. Likewise, transfection with constitutively active NFAT resulted in an increased expression of both TRAP and integrin beta(3). In parallel loss-of-function studies, transfection with dominant-negative NFAT not only inhibited osteoclast formation but also reversed the induction of NFATc1, TRAP, and integrin beta(3) by TAT-calcineurin A alpha. The expression of these markers was also inhibited by calcineurin A alpha U1 small nuclear RNA, which significantly reduced calcineurin A alpha mRNA and protein expression. Consistent with these observations, we observed a reduction in osteoclastogenesis in calcineurin A alpha mRNA cells and in osteoclast precursors treated with the calcineurin inhibitors cyclosporin A and FK506. Together, the gain- and loss-of-function experiments establish that calcineurin A alpha is necessary for osteoclast formation from its precursor and that this occurs via an NFATc1-dependent mechanism.