Histone H3K9 demethylase JMJD2B/KDM4B promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells by regulating H3K9me2 on RUNX2

Abstract

Background: A variety of proteins including epigenetic factors are involved in the differentiation of human bone marrow mesenchymal stem cells. These cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment. Further in-depth study of their epigenetic alterations may make sense.

Methods: Chromatin Immunoprecipitation-PCR (ChIP-PCR) was used to detect the methylation enrichment status of H3K9me2 in the Runx2 promoter, alizarin red and alkaline phosphatase (ALP) staining were used to detect osteogenic differentiation and mineralization ability, western blot and quantitative RT-PCR were used to measure the differential expression of osteogenesis-related proteins and genes. Recombinant Lentivirus mediated gain-of-function and loss-of-function study. The scale of epigenetic modification was detected by laser confocal.

Results: Our results showed that compared with human bone marrow mesenchymal stem cells (hBMSCs) without osteogenic differentiation treatment, hBMSCs after osteogenic differentiation significantly promoted osteogenic differentiation and mRNA expression such as JMJD2B/KDM4B, osteogenesis-related genes like Runx2 and FAM210A in hBMSCs cells, suggesting that upregulation of JMJD2B/KDM4B is involved in the promoting effect of osteogenesis. After overexpression and silencing expression of JMJD2B, we found a completely opposite and significant difference in mRNA expression of osteogenesis-related genes and staining in hBMSCs. Overexpression of JMJD2B/KDM4B significantly promoted osteogenic differentiation, suggesting that JMJD2B/KDM4B could promote osteogenesis. In addition, ChIP-PCR showed that overexpression of JMJD2B/KDM4B significantly reversed the methylation enrichment status of H3K9me2 in Runx2 promoter. Furthermore, overexpression of JMJD2B/KDM4B significantly reverses the inhibitory effect of BIX01294 on H3K9me2, suggesting that JMJD2B/KDM4B regulates the osteogenic differentiation of hBMSCs by changing the methylation status of H3K9me2 at the Runx2 promoter.

Conclusions: Taken together, these results suggest that JMJD2B/ KDM4B may induce the osteogenic differentiation of hBMSCs by regulating the methylation level of H3K9me2 at the Runx2 promoter.

Keywords: Epigenetics; JMJD2B; KDM4B; Osteogenesis; hBMSCs.

Figure 1. The effects of osteogenic medium on osteogenic response of hBMSCs cells.

(A) hBMSCs cells were cultured in osteogenic medium as indicated. Quantitative real-time PCR (qRT-PCR) was performed at d0, d1, d3, d7, and d14 after treatment to measure mRNA expression of JMJD2B as well as osteogenesis- and myogenesis-related genes. GAPDH was used as an internal reference. Data are expressed as the mean ±standard deviation (SD). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. D0. All experiments were independently repeated at least three times. (B) Western blot analysis was conducted to measure protein expression of Runx2, JMJD2B, FAM210A protein levels. β-actin was used as an internal reference. (C) Immunofluorescence staining was performed based on JMJD2B and FAM210A in hBMSCs. Scale bar = 20 µm.

Figure 2. JMJD2B regulates the methylation enrichment status of H3K9me2 in RUNX2 promoter to influence the osteogenic differentiation of hBMSCs.

hBMSCs were transfected with empty or JMJD2B-overexpressing/silencing vectors and incubated in osteogenic medium or H3K9me2 inhibitor BIX01294/DMSO for 3, 7 or 14 days hBMSCs were transfected with empty or JMJD2B-overexpressing/silencing vectors and cultured in osteogenic medium (A) hBMSCs were treated for 14 days and the Runx2 promoter was ChIP-ed with anti-H3K9me2 or IgG control. Data are expressed as the mean ±SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001 vs. LV5-JMJD2B. (B) Alkaline phosphatase (ALP) staining and alizarin red staining were conducted to examine osteogenesis and mineralization, respectively. (C) Quantitative real-time PCR (qRT-PCR) was performed at 3 d after treatment to measure mRNA expression of Runx2 and JMJD2B genes. GAPDH was used as an internal reference. Data are expressed as the mean ±standard deviation (SD). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. All experiments were independently repeated at least three times. (D–E) Western blot analysis was conducted to measure protein expression of JMJD2B, Runx2, H3K9me2 protein levels. β-actin was used as an internal reference. (F) Quantitative real-time PCR (qRT-PCR) was performed at 3 d after treatment to measure mRNA expression of Runx2 and JMJD2B genes. GAPDH was used as an internal reference. Data are expressed as the mean ±standard deviation (SD). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. All experiments were independently repeated at least three times.

Figure 3. JMJD2B Is Required for Osteogenic Differentiation of hBMSCs.

hBMSCs were transfected with empty or JMJD2B-overexpressing/silencing vectors and incubated in osteogenic medium for 0, 1, 3, 7 or 14 days (A) Quantitative real-time PCR (qRT-PCR) was performed at 72 h after treatment to measure mRNA expression of JMJD2B as well as osteogenesis-related genes. GAPDH was used as an internal reference. Data are expressed as the mean ±standard deviation (SD). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 vs. 0 M. All experiments were independently repeated at least three times. ALP staining and alizarin red staining were performed to examine osteogenesis and mineralization, respectively (B) Western blot analysis was conducted to measure protein expression of JMJD2B protein levels. β-actin was used as an internal reference. (C) Alkaline phosphatase (ALP) staining and alizarin red staining were conducted to examine osteogenesis and mineralization, respectively. (D) Quantitative real-time PCR (qRT-PCR) was performed at 3 d after treatment to measure mRNA expression of osteogenesis- related genes. GAPDH was used as an internal reference. Data are expressed as the mean ±standard deviation (SD). ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. All experiments were independently repeated at least three times.