NFATc1 mediates HDAC-dependent transcriptional repression of osteocalcin expression during osteoblast differentiation

Abstract

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.

Retinoic Acid-Induced CCR9 Expression Requires Transient TCR Stimulation and Cooperativity between NFATc2 and the Retinoic Acid Receptor/Retinoid X Receptor Complex

“…Indeed, although NFAT is an important mediator for the expression of various cytokines, evidence has accumulated that NFAT also represses the transcription of some genes. NFATc1 and NFATc2 repress the expression of osteocalcin and Cdk4, respectively, by increased recruitment of histone deacetylase (39,40). Thus, NFAT1 may inhibit CCR9 expression by recruiting histone deacetylase to the Ccr9 promoter or by preventing the complex formation of NFATc2 with RARa/RXRa and other transcriptional factors.…”

Histone deacetylases (HDAC) in physiological and pathological bone remodelling

“…Activation of NFATc1 has been previously shown to significantly inhibit osteoblast differentiation and activity via repression of osteocalcin. [76]. Thus HDACi that suppress NFATc1 may be able to both inhibit bone resorption and enhance osteoblast bone formation.…”

NFAT Signaling and Bone Homeostasis

“…These results suggest that the involvement of calcineurin-NFAT pathway is likely much more complex in osteoblasts with different isoform and possibly different NFAT protein exerting distinct function. In a separate study by the same lab, it was found that NFATc1 suppressed the promoter of osteocalcin during osteoblast differentiation possibly through a HDAC mediated mechanism [73]. Histone deacetylase (HDAC) 3 has been shown to interact with the amino terminus of Runx2 to repress osteocalcin promoter and osteoblast maturation [74].…”

“…This is fascinating and demonstrates that the skeletal system and the endocrine system reciprocally regulate each other. Since NFATc1 regulates the promoter of osteocalcin [70,73,79], one may suspect whether or not NFATc1 may also play an indirect role in regulating testosterone level and impacting bone health in males.…”