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.

We previously reported that the in vivo and in vitro suppression of Nuclear Factor of Activated T Cells (NFAT) signaling increases osteoblast differentiation and bone formation. To investigate the mechanism by which NFATc1 regulates osteoblast differentiation, we established an osteoblast cell line that overexpresses a constitutively active NFATc1 (ca-NFATc1). The activation of NFATc1 significantly inhibits osteoblast differentiation and function, demonstrated by inhibition of alkaline phosphatase activity and mineralization as well as a decrease in gene expression of early and late markers of osteoblast differentiation such as osterix and osteocalcin, respectively. By focusing on the specific role of NFATc1 during late differentiation, we discovered that the inhibition of osteocalcin gene expression by NFATc1 was associated with a repression of the osteocalcin promoter activity, and a decrease in TCF/LEF transactivation. Also, overexpression of NFATc1 completely blocked the decrease in total histone deacetylase (HDAC) activity during osteoblast differentiation and prevented the hyperacetylation of histones H3 and H4. Mechanistically, we show by Chromatin Immunoprecipitation (ChIP) assay that the overexpression of NFATc1 sustains the binding of HDAC3 on the proximal region of the osteocalcin promoter, resulting in complete hypoacetylation of histones H3 and H4 when compared to GFP-expressing osteoblasts. In contrast, the inhibition of NFATc1 nuclear translocation either by cyclosporin or by using primary mouse osteoblasts with deleted calcineurin b1 prevents HDAC3 from associating with the proximal regulatory site of the osteocalcin promoter. These preliminary results suggest that NFATc1 acts as a transcriptional co-repressor of osteocalcin promoter possibly in an HDAC-dependent manner.

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 (⌬Cnb1 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 ⌬Cnb1 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.Bone is a highly dynamic structure that is constantly renewing through a process called remodeling (1). This process is critical for maintaining healthy bones and is mainly controlled by the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The presence of these two opposing cell types with contrasting activities in close proximity requires tight regulation to maintain healthy and strong bones. Bone resorption is attained by the action of osteoclasts, which are specialized macrophages whose differentiation is primarily regulated by receptor activator of NF B ligand (RANKL) 2 and osteoprotegerin (OPG) (2). Osteoblasts originate from multipotent mesenchymal progenitors that replicate as undifferentiated cells but have the potential to differentiate into different lineages of mesenchymal tissues including bone, cartilage, fat, muscle, and marrow stroma (3, 4). Osteoblasts control bone formation not only by synthesizing bone matrix proteins and regulating mineralization but also by orchestrating the process of bone resorption through the modulation of RANKL and OPG expression (2, 4).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...

In the meta-analysis of public microarray databases for different skin diseases, we revealed seven commonly up-regulated genes, DSG3, KRT6, MAP17, PLSCR1, RPM2, SOD2 and SPRR2B. We postulated that the genes selected from the meta-analysis may be potentially associated with the abnormal keratinocyte differentiation. To demonstrate this postulation, we alternatively evaluated whether the genes of interest in the meta-analysis can be regulated by T-helper (Th) cell cytokines in normal human epidermal keratinocytes (NHEK). We found that MAP17 was significantly up-regulated in response to interferon-gamma, interleukin 4 (IL-4), IL-6, IL-17A or IL-22 in NHEK. Interestingly, MAP17 was originally reported to interact with PDZK1; in turn, the PDZK1 gene is localized within the atopic dermatitis-linked region on human chromosome 1q21. In an attempt to evaluate whether MAP17 regulates the expression of cornified envelope-associated genes at the 1q21 locus, such as filaggrin, loricrin and involucrin, we found that the over-expression of MAP17 in HaCaT keratinocytes significantly decreased the expression of filaggrin. Taken together, the Th cell cytokine-induced up-regulation of MAP17 expression may be linked to the down-regulation of filaggrin in NHEK, which may be associated with the abnormal epidermal differentiation observed in the dermatological diseases.

Promotion of osteoclast apoptosis is one therapeutic approach to osteoporosis. Calmodulin, the major intracellular Ca 2؉