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Here, we demonstrate that the [Ca.sup.2+]/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)cl. 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].sub.3] followed. In addition, the expression of NFATc1, as well as 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].sub.3]. In parallel loss-of-function studies, transfection with dominant-negative NFAT not only inhibited osteoclast formation but also reversed the induction of NFATcl, TRAP, and integrin [[beta].sub.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].sup.-/-] cells and in osteoclast precursors treated with the calcineurin inhibitors cyclosporin A and FK506. Together, the gain- and loss-offunction experiments establish that calcineurin A[alpha] is necessary for osteoclast formation from its precursor and that this occurs via an NFATc1-dependent mechanism. osteoclastogenesis; cyclosporin; nuclear factor of activated T cells

This study explores the role of the calmodulin- and [Ca.sup.2+]-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin A[alpha] and A[beta] cDNA from a rabbit osteoclast library. Sequence analysis revealed an ~95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin A[alpha] mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin A[alpha] expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x [10.sup.-7] M) and FK506 (5 x [10.sup.-9] and 5 x [10.sup.-7] M). We then constructed a fusion protein comprising calcineurin A[alpha] and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin A[alpha] fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin A[alpha] and anti-TAT antibodies. Pit assays performed with TAT-calcineurin As-positive osteoclasts.revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin A[alpha] can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption. calcium channel; gene cloning; osteoclast; osteoporosis

We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a [Ca.sup.2+] influx channel and as a divalent cation ([Ca.sup.2+]) sensor. Cytosolic [Ca.sup.2+] measurements revealed [Ca.sup.2+] influx in osteoclasts at depolarized membrane potentials. The cytosolic [Ca.sup.2+] change was, as expected, not seen in [Ca.sup.2+]-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (~25 mV) ryanodine triggered extracellular [Ca.sup.2+] influx that was blocked by [Ni.sup.2+]. In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective ~60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential ([E.sub.rev]) ~0 mV]. In the presence of a [Ba.sup.2+] gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to ~120 pS and [E.sub.rev] shifted to 21 mV. The conductance was tentatively classified as a RyR-gated [Ca.sup.2+] channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, [Ab.sup.34]. To demonstrate that extracellular [Ca.sup.2+] sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic [Ca.sup.2+] signals triggered by either [Ni.sup.2+] (5 mM) or [Cd.sup.2+] (50 [micro]M). Finally, intracellular application of antiserum [Ab.sup.34] potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident [Ca.sup.2+] influx channel sensitive to RyR modulators; 2) an extracellular, as opposed to intracellular, divalent cation activation site; and 3) a cytosolic CaM-binding regulatory site for RyR. It is likely therefore that the surface RyR-2 not only gates [Ca.sup.2+] influx but also functions as a sensor for extracellular divalent cations. osteoclast; calcium receptor; calcium channels; osteoporosis; calcium