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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.