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

Abstract

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 (<1μM in vitro and 35.5 nM in vivo) are anabolic as they increase bone formation, osteoblast differentiation, and bone mass, while high concentrations (>1μM 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 (<1μM) 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

“…In contrast to the notion that Cn/NFAT is a positive regulator of bone formation, we discovered that the pharmacologic inhibition of Cn by low concentrations of CsA increased osteoblast differentiation in vitro and bone mass in vivo (5). The increase in bone mass was associated with enhanced bone formation and a decrease in bone resorption (5).…”

“…Also, bones were fixed, embedded in methyl methacrylate, sectioned, and stained with Goldner's Trichrome. A region of interest, an area at least 0.5 mm below the growth plate (excluding the primary spongiosa and trabecular-connected cortical bone), was selected and remained constant for all animals regardless of the shape of the section (5). Standard bone histomorphometry as described by Parfitt et al (20) was performed using the BioQuant image analysis software (R & M Biometrics, Nashville, TN) in the Histomorphometry and Molecular Analysis Core Laboratory of the University of Alabama at Birmingham Bone Center (5,21).…”

“…We also demonstrated that this response was associated with an increase in the expression and activation of Fra-2, an activator protein-1 family member that plays an important role in osteoblast differentiation and bone formation (5). Cn is a Ca 2ϩ /calmodulin-dependent serine/threonine-protein phosphatase that regulates several physiological processes (6).…”

“…We have recently shown that the pharmacologic inhibition of calcineurin (Cn) by low concentrations of cyclosporin A (CsA) increases osteoblast differentiation and bone formation (5). We also demonstrated that this response was associated with an increase in the expression and activation of Fra-2, an activator protein-1 family member that plays an important role in osteoblast differentiation and bone formation (5).…”

“…The increase in bone mass was associated with enhanced bone formation and a decrease in bone resorption (5). Therefore, we hypothesized that Cn signaling negatively regulates osteoblast differentiation and bone mass.…”