Cyclosporin A elicits dose-dependent biphasic effects on osteoblast differentiation and bone formation.

Cyclosporin A (CsA) is thought to prevent immune reactions after organ transplantation by inhibiting calcineurin (Cn) and its substrate, the Nuclear Factor of Activated T Cells (NFAT). A dichotomy exists in describing the effects of CsA on bone formation. The concept that the suppression of Cn/NFAT signaling by CsA inhibits bone formation is not entirely supported by many clinical reports and laboratory animal studies. Gender, dosage and basal inflammatory activity have all been suggested as explanations for these seemingly contradictory reports. Here we examine the effects of varying concentrations of CsA on bone formation and osteoblast differentiation and elucidate the role of NFATc1 in this response. We show that low concentrations of CsA (<1 microM in vitro and 35.5 nM in vivo) are anabolic as they increase bone formation, osteoblast differentiation, and bone mass, while high concentrations (>1 microM in vitro and in vivo) elicit an opposite and catabolic response. The overexpression of constitutively active NFATc1 inhibits osteoblast differentiation, and treatment with low concentrations of CsA does not ameliorate this inhibition. Treating osteoblasts with low concentrations of CsA (<1 microM) increases fra-2 gene expression and protein levels in a dose-dependent manner as well as AP-1 DNA-binding activity. Finally, NFATc1 silencing with siRNA increases Fra-2 expression, whereas NFATc1 overexpression inhibits Fra-2 expression. Therefore, NFATc1 negatively regulates osteoblast differentiation, and its specific inhibition may represent a viable anabolic therapy for osteoporosis.

Conditional disruption of calcineurin B1 in osteoblasts increases bone formation and reduces bone resorption.

We recently reported that the pharmacological inhibition of calcineurin (Cn) by low concentrations of cyclosporin A increases osteoblast differentiation in vitro and bone mass in vivo. To determine whether Cn exerts direct actions in osteoblasts, we generated mice lacking Cnb1 (Cn regulatory subunit) in osteoblasts (DeltaCnb1(OB)) using Cre-mediated recombination methods. Transgenic mice expressing Cre recombinase, driven by the human osteocalcin promoter, were crossed with homozygous mice that express loxP-flanked Cnb1 (Cnb1(f/f)). Microcomputed tomography analysis of tibiae at 3 months showed that DeltaCnb1(OB) mice had dramatic increases in bone mass compared with controls. Histomorphometric analyses showed significant increases in mineral apposition rate (67%), bone volume (32%), trabecular thickness (29%), and osteoblast numbers (68%) as well as a 40% decrease in osteoclast numbers as compared with the values from control mice. To delete Cnb1 in vitro, primary calvarial osteoblasts, harvested from Cnb1(f/f) mice, were infected with adenovirus expressing the Cre recombinase. Cre-expressing osteoblasts had a complete inhibition of Cnb1 protein levels but differentiated and mineralized more rapidly than control, green fluorescent protein-expressing cells. Deletion of Cnb1 increased expression of osteoprotegerin and decreased expression of RANKL. Co-culturing Cnb1-deficient osteoblasts with wild type osteoclasts demonstrated that osteoblasts lacking Cnb1 failed to support osteoclast differentiation in vitro. Taken together, our findings demonstrate that the inhibition of Cnb1 in osteoblasts increases bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis.