Histone demethylase Jmjd3 regulates osteoblast differentiation via transcription factors Runx2 and osterix

Abstract

Post-translational modifications of histones including methylation play important roles in cell differentiation. Jumonji domain-containing 3 (Jmjd3) is a histone demethylase, which specifically catalyzes the removal of trimethylation of histone H3 at lysine 27 (H3K27me3). In this study, we examined the expression of Jmjd3 in osteoblasts and its roles in osteoblast differentiation. Jmjd3 expression in the nucleus was induced in response to the stimulation of osteoblast differentiation as well as treatment of bone morphogenetic protein-2 (BMP-2). Either treatment with Noggin, an inhibitor of BMP-2, or silencing of Smad1/5 suppressed Jmjd3 expression during osteoblast differentiation. Silencing of Jmjd3 expression suppressed osteoblast differentiation through the expression of bone-related genes including Runx2, osterix, osteopontin, bone sialoprotein (BSP), and osteocalcin (OCN). Silencing of Jmjd3 decreased the promoter activities of Runx2 and osterix and increased the level of H3K27me3 on the promoter regions of Runx2 and osterix. Introduction of the exogenous Runx2 and osterix partly rescued osteoblast differentiation in the shJmjd3 cells. The present results indicate that Jmjd3 plays important roles in osteoblast differentiation and regulates the expressions of BSP and OCN via transcription factors Runx2 and osterix.

Keywords: Bone; Bone morphogenetic protein (BMP); Differentiation; Histone methylation; Osteoblasts.

FIGURE 1.

The expression of Jmjd3 in osteoblasts. MC3T3-E1 cells were cultured in the osteoblast differentiation medium for the indicated periods. A, the expression of Jmjd3 was examined by real-time PCR. B, the protein expressions of Jmjd3 and H3K27me3 were examined by Western blot analysis. The relative expressions of Jmjd3/β-actin and H3K27me3/H3 were calculated by computer software (ImageJ). All the results in the real-time PCR are presented as means ± S.E. *, p < 0.05, **, p < 0.01

FIGURE 2.

BMP-2 induced Jmjd3 expression in osteoblasts. MC3T3-E1 cells were treated with 100 ng/ml BMP-2 for the indicated periods. A and B, the expression levels of Jmjd3 and H3K27me3 were examined by real-time PCR (A) and Western blot analysis (B). MC3T3-E1 cells were pretreated with 100 nm U0126, 50 μm LY294002, or 100 ng/ml Noggin for 3 h and cultured for another 72 h in the osteoblast differentiation medium. C and D, Jmjd3 expression was examined by real-time PCR (C) and Western blot analysis (D). MC3T3-E1 cells were transfected with siRNA for Smad1 or Smad5 and cultured in the medium containing 100 ng/ml BMP-2 for another 72 h. E–G, the expressions of Smad1 (E), Smad5 (F), and Jmjd3 (G) were examined by real-time PCR. The relative expressions of Jmjd3/β-actin and H3K27me3/H3 were calculated by computer software (ImageJ). All the data in the real-time PCR are presented as means ± S.E. *, p < 0.05, **, p < 0.01.

FIGURE 3.

Nuclear localization of Jmjd3. A, MC3T3-E1 cells were cultured for 7 days in the osteoblast differentiation medium or treated with 100 ng/ml BMP-2 for 72 h. The localization of Jmjd3 was determined by immunocytochemistry. B, MC3T3-E1 cells were treated without (Control) or with 100 ng/ml BMP-2 (BMP-2). Seventy-two hours later, the nuclear and cytoplasmic fractions were prepared. Western blot analysis was performed using the specific antibodies for Jmjd3, B23, and Eps15. The relative expression of Jmjd3/B23 or Eps15 was calculated by computer software (ImageJ). C, cytosolic fraction; N, nuclear fraction.

FIGURE 4.

Suppression of Runx2 and osterix expression in Jmjd3 transient silencing cells. MC3T3-E1 cells were transfected with nonspecific (siCont) or Jmjd3-specific (siJmjd3) siRNA and cultured for 3 days in the osteoblast differentiation medium. RNA was extracted, and real-time PCR was performed for Jmjd3, Runx2, and osterix. All the data are presented as means ± S.E. *, p < 0.05, **, p < 0.01.

FIGURE 5.

Establishment of stable Jmjd3 knockdown cells. MC3T3-E1 cells were transfected with nonspecific (shCont) or Jmjd3-specific (shJmjd3) shRNA and cultured for 3 days in the osteoblast differentiation medium. A, the expression levels of Jmjd3 mRNA were examined by real-time PCR. Data are presented as means ± S.E. **, p < 0.01. B, Western blot analysis was performed using the specific antibodies for Jmjd3, β-actin, H3K27me3, and H3. The relative expressions of Jmjd3/β-actin and H3K27me3/H3 were calculated by computer software (ImageJ).

FIGURE 6.

Inhibition of osteoblast differentiation by Jmjd3 silencing. A, the shCont and shJmjd3 cells were cultured in the osteoblast differentiation medium for 7 and 14 days, respectively. The cells were stained for ALP expression (left panel) or for mineralization by von Kossa (central panel) and Alizarin red (right panel). B, the shCont and shJmjd3 cells were cultured for 7 days in the osteoblast differentiation medium, and ALP activity was measured. C, the shCont and shJmjd3 cells were cultured for 21 days in the osteoblast differentiation medium, and the calcium levels in the cultured cells were quantified. D, the shCont and shJmjd3 cells were cultured in the osteoblast differentiation medium. The mRNA expressions of Runx2 and osterix were determined on day 3 and those of OPN, BSP, and OCN were determined on day 7 by real-time PCR with normalization by Gapdh expression. Each bar represents the mean ± S.E. **, p < 0.01.

FIGURE 7.

Local administration of siJmjd3 suppressed bone formation in mouse calvaria. A, three-dimensional μCT images of calvaria from mice (12-day-old, n = 4 for siJmjd3 injection, n = 4 for siCont injection) injected with siCont or siJmjd3. B, cortical bone mineral density (BMD) of calvaria was determined by μCT. Data are presented as means ± S.E. **, p < 0.01. C, upper, plastic sections of dissected mouse calvaria were examined under a fluorescence microscope. The distance between two calcein labeling layers reflects the bone mineralization rate. Middle, sections of dissected mice calvaria were stained with Goldner's trichrome to visualize bone thickness. Lower, sections of dissected mice calvaria were stained with von Kossa to visualize bone mineralization.

FIGURE 8.

Knockdown of Jmjd3 suppressed the transcriptional activity of Runx2 and osterix. A and B, schematic illustration of the promoter regions of Runx2 and osterix. The context of luciferase reporter vectors and the primers used for ChIP assay are indicated. C and D, the activity of Runx2 promoter, Runx2 3×binding site, and osterix promoter was decreased in shJmjd3 cells. E and F, knockdown of Jmjd3 increased the level of H3K27me3 on the Runx2 (E) and osterix (F) promoters. The shCont and shJmjd3 cells were cultured for 3 days in the osteoblast differentiation medium. Chromatin solution from the cells was subjected to ChIP analysis using anti-H3K27me3 and anti-IgG antibodies. PCR was performed with the primer pairs amplifying the promoter regions of Runx2 and osterix. IP, immunoprecipitate; C, shCont; J, shJmjd3.

FIGURE 9.

Overexpression of Runx2 partly rescued osteoblast differentiation in the shJmjd3 cells. The shJmjd3 cells were transfected with Runx2 expression vector. A, expressions of exogenous and endogenous Runx2 were examined by real-time PCR. B, cells were cultured in the osteoblast differentiation medium for 7 days for ALP staining (left panel) and 14 days for von Kossa (central panel) and Alizarin red staining (right panel). mock, mock-transfected cells. C, cells were cultured for 7 days in the osteoblast differentiation medium, and ALP activity was measured. D, the expressions of osterix, BSP, and OCN were determined by real-time PCR. All the data in the real-time PCR experiments are presented as means ± S.E. *, p < 0.05, **, p < 0.01.

FIGURE 10.

Overexpression of osterix partly rescued osteoblast differentiation in the shJmjd3 cells. The shJmjd3 cells were transfected with empty vector (Flag) or osterix expression vector (Flag-Osterix). A, transfected osterix was confirmed by immunocytochemistry with anti-FLAG antibody. B, the exogenous and endogenous osterix expressions were examined by real-time PCR. C, cells were cultured in the osteoblast differentiation medium for 7 and 14 days, respectively. The cells were stained for ALP expression (left panel) or for mineralization by von Kossa (central panel) and Alizarin red (right panel). D, cells were cultured for 7 days in the osteoblast differentiation medium, and ALP activity was measured. E, the expressions of BSP and OCN were determined by real-time PCR. All the data in the real-time PCR experiments are presented as means ± S.E. *, p < 0.05, **, p < 0.01.