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.

Figure 1. Generation of MC3T3-E1 osteoblastic cell line overexpressing constitutively active NFATc1

MC3T3-E1 cells were transduced with GFP-empty vector (GFP) or constitutively active NFATc1 (ca-NFATc1) retrovirus. (A) Cells were immunostained with NFATc1 antibody followed by fluorescein isothiocyanate-conjugated secondary antibody. A nonspecific IgG was used for negative controls (insets). Microscopic fields were examined using ×100 objectives. (B) Nuclear proteins were used for immunoblotting with the antibodies against NFATc1 and Lamin B1. (C) Cells were transfected with pTA-NFAT luciferase construct. Forty eight hours after transfection, luciferase activity was measured. Data are expressed relative to CMV-β-galactosidase, and values represent the mean ± SE of 3 separate experiments each repeated in triplicate; *, p≤ 0.01.

Figure 2. NFATc1 negatively regulates osteoblast differentiation

GFP and ca-NFATc1 MC3T3-E1 osteoblasts were cultured for 14 days (A) or 21 days (B and C) in the presence of β-glycerophosphate and ascorbic acid-2-phosphate to induce osteoblast differentiation. Cells were stained for ALP activity (red) (A) or for mineralization by von Kossa (Brown) (B). Calcium deposition in the matrix was measured using a calcium detection kit (C). GFP, and ca-NFATc1 MC3T3-E1 osteoblasts were cultured for 4 days in proliferation medium (Pro) or 11 days in differentiation medium (Diff). RNA was extracted and Real time RT-PCR was performed for Runx2 (D), osterix (E) osteocalcin (F) and actin. Values, obtained from three independent experiments, represent the mean ± SE of mRNA expression relative to actin expression; **, p≤ 0.05, *, p≤ 0.01 compared to control.

Figure 3. NFATc1 decreases osteocalcin promoter activity

Schematic illustration of the osteocalcin promoter showing the relative positions of binding sites for the indicated transcription factors (A). GFP, and ca-NFATc1 MC3T3-E1 osteoblasts were transiently transfected with rat osteocalcin luciferase constructs, including full length osteocalcin promoter “OC-1050” (B) or proximal region (-285 to +1) “OC-285” (C), Runx2 luciferase construct (D) and TOP flash luciferase construct (E). Forty eight hours after transfection, luciferase activity was measured. Data are expressed relative to internal control (CMV-β-galactosidase) and values represent the mean ± SE of 3 separate experiments, each repeated in a triplicate; *, p≤ 0.01.

Figure 4. NFATc1 maintains elevated levels of HDAC activity and inhibits the acetylation of histones during osteoblast differentiation

GFP and ca-NFATc1 MC3T3-E1 osteoblasts were cultured for 4 days in proliferation medium (Pro) or 11 days in differentiation medium (Diff). (A) Nuclear proteins were used for immunoblotting with several antibodies. Data shown are representative of three separate experiments. (B) Nuclear proteins were collected on day 0, 3 and 11 after osteoblastic induction. Total histone deacetylase enzymatic activity was measured. Values were obtained from three independent experiments, each performed in triplicate and represent the mean ± SE of HDAC activity per 100 μg concentration of protein; *, p<0.05. (C) Nuclear extracts were used for immunoblotting with antibodies against total and acetylated histones 3 and 4. Shown is a representative of three separate experiments (left panel). Quantitative data were obtained as a ratio of acetylated histones/total histones by image analysis software (Image J:NIH). Values, obtained from three independent experiments, represent the mean ± SE (right panel).

Figure 5. NFATc1 forms a transcriptional repressor complex through the interaction with HDAC3 on the proximal regulatory region of the osteocalcin promoter

Schematic illustration of the osteocalcin proximal promoter showing the location of TCF and Runx2 sites and primers used in this study (A). GFP and ca-NFATc1 MC3T3-E1 osteoblasts were cultured for 4 days in proliferation medium (Pro) or 11 days in differentiation medium (Diff). Cellular DNA fragments were immunoprecipitated with antibodies against different proteins and normal rabbit or normal mouse IgG as controls. Immunoprecipitated DNA fragments were amplified by PCR using primers that encompass -236 bp to -1 bp or 3UTR site of the osteocalcin promoter. Quantitative data of protein binding to this site during osteoblast differentiation was obtained by image analysis software (Image J:NIH) and compared to that of proliferating GFP cells; set to 1.0 as a basal level. Values, obtained from three independent experiments, represent the mean of fold change (B). Nuclear proteins were immunoprecipitated with anti-NFATc1 antibody (IP:NFATc1) or normal mouse IgG as a control (IP:mIgG). Immunoblots were developed using antibodies against NFATc1 or HDAC3. Shown is a representative of three separate experiments (C).

Figure 6. Inhibition of calcineurin abrogates the recruitment of NFATc1 and HDAC3 to the proximal regulatory region of the osteocalcin promoter

MC3T3-E1 cells were treated with cyclosporine (500 nM) for 4 days in proliferation medium (Pro) or for 11 days in differentiation medium (Diff) (A). Primary osteoblasts from CnB1^f/f mice were transduced with adenovirus-GFP (Cre^-) or (Cre⁺) (B). Cellular DNA fragments were immunoprecipitated with antibodies NFATc1, HDAC3 and normal IgG. Immunoprecipitated DNA fragments were amplified by PCR using primers which encompass -236 bp to -1 bp or 3UTR site of the osteocalcin promoter. Quantitative data of protein binding to this site during osteoblast differentiation were obtained by image analysis software (Image J:NIH) and compared to proliferating GFP cells; set to 1.0 as a basal level. Values, obtained from three independent experiments, represent the mean of fold change.