Dexamethasone shifts bone marrow stromal cells from osteoblasts to adipocytes by C/EBPalpha promoter methylation

Abstract

Dexamethasone (Dex)-induced osteoporosis has been described as the most severe side effect in long-term glucocorticoid therapy. The decreased bone mass and the increased marrow fat suggest that Dex possibly shifts the differentiation of bone marrow stromal cells (BMSCs) to favor adipocyte over osteoblast, but the underlying mechanisms are still unknown. In this paper, we established a Dex-induced osteoporotic mouse model, and found that BMSCs from Dex-treated mice are more likely to differentiate into adipocyte than those from control mice, even under the induction of bone morphogenetic protein-2 (BMP2). We also discovered both in vitro and in vivo that the expression level of adipocyte regulator CCAAT/enhancer-binding protein alpha (C/EBPalpha) is significantly upregulated in Dex-induced osteoporotic BMSCs during osteoblastogenesis by a mechanism that involves inhibited DNA hypermethylation of its promoter. Knockdown of C/EBPalpha in Dex-induced osteoporotic cells rescues their differentiation potential, suggesting that Dex shifts BMSC differentiation by inhibiting C/EBPalpha promoter methylation and upregulating its expression level. We further found that the Wnt/beta-catenin pathway is involved in Dex-induced osteoporosis and C/EBPalpha promoter methylation, and its activation by LiCl rescues the effect of Dex on C/EBPalpha promoter methylation and osteoblast/adipocyte balance. This study revealed the C/EBPalpha promoter methylation mechanism and evaluated the function of Wnt/beta-catenin pathway in Dex-induced osteoporosis, providing a useful therapeutic target for this type of osteoporosis.

Figure 1

Dex-induced osteoporosis and increased marrow fat. (a) Reduced trabecular bone in microCT images of the distal femur of Dex-administered mice compared with control mice. N=3. (b) MicroCT analysis indicated reduced femur BMD, trabecular bone volume as a percentage of total volume (BV/TV), trabecular bone numbers (Tb.N), trabecular bone thickness (Tb.Th), and increased trabecular spacing (Tb.Sp) in Dex-treated mice compared with control mice. Data are presented as mean±S.D. of three independent experiments. *P<0.05, N=3. (c) Reduced trabecular bone and increased adipocyte number in H&E sections from the distal femur of Dex-treated mice. N=4

Figure 2

Suppressed osteoblastogenesis and increased adipogenesis of Dex-induced osteoporotic BMSCs. (a) Proliferation of BMSCs from both Dex- and Saline-treated mice measured by MTT assay. (b) Apoptosis of BMSCs cultured in growth medium were detected by TUNEL staining. (c) Relative expressions of osteoblast and adipocyte markers in BMSCs from Dex- or saline-treated mice under the induction of BMP2 to indicated times were quantified by qRT-PCR. (d) Relative expressions of adipocyte genes in BMSCs from Dex- or saline-treated mice were quantified by qRT-PCR in transdifferentiation assay. (e) Oil red O staining of the transdifferentiated adipocytes from indicated cells showed higher adipocyte conversion potential of osteoporotic BMSCs. N=3. Respective cells cultured in the growth medium were used as control. (f) The expressions of osteoblast and adipocyte markers in C3H10T1/2 cells treated as indicated were quantified by qRT-PCR. (g) Relative expressions of adipocyte genes in transdifferentiated C3H10T1/2 cells that were treated with different doses of Dex. (h) Oil red O staining shows the transdifferentiated adipocytes from cells treated by 10^–6 M Dex. Cells cultured in the growth medium were used as control. N=3. All mRNA expression levels were normalized to beta-actin. Data are presented as mean±S.D. of three independent experiments. *P<0.05, **P<0.01, n.s. P>0.05, N=3

Figure 3

Dex upregulates C/EBPalpha during osteoblastogenesis by inhibiting DNA methylation of the C/EBPalpha promoter. (a) mRNA and protein levels of C/EBPalpha in indicated BMSCs cultured with 100 ng/ml BMP2 (above), as well as in C3H10T1/2 cells treated as indicated. Relative mRNA expressions were normalized to beta-actin. Data are presented as mean±S.D. of three independent experiments. *P<0.05, N=3. (b) The in vivo C/EBPalpha expression levels in the distal femur area of Dex- or saline-treated mice were shown by immunohistochemistry. N=4. (c) C/EBPalpha promoter methylation status in indicated BMSCs treated with BMP2, as well as in C3H10T1/2 cells treated as indicated for 21 days (open circle: unmethylated CpG; full circle: methylated CpG; 10 clones in each group were tested). N=3. (d) Protein levels of Dnmt 3a/3b in C3H10T1/2 cells treated as indicated for 21 days (above). N=3. (e) ChIP assay shows the binding of Dnmt 3a/3b to the C/EBPalpha promoter in C3H10T1/2 cells after 21 days of BMP2 treatment. N=3. Densitometric analysis was shown as fold changes relative to beta-actin (Western blot) or input (ChIP)

Figure 4

Knockdown of C/EBPalpha partly rescued the differentiation fate of Dex-induced osteoporotic BMSCs. (a) The knockdown efficiency of lentivirus encoding C/EBPalpha-targeting shRNA (shC/EBPalpha) was confirmed by comparison to control lentivirus (shControl). (b) Expression levels of both osteoblast genes and adipocyte genes in osteoporotic BMSCs treated by BMP2 after shC/EBPalpha or shControl infections at 10 MOI. (c) In the transdifferentiation assay, the expression levels of adipocyte genes, and the transdifferentiated adipocytes of osteoporotic BMSCs after indicated shRNA infection were shown by qRT-PCR and oil red O staining, respectively. All mRNA expression levels were normalized to beta-actin. Data are presented as mean±S.D. of three independent experiments. *P<0.05, **P<0.01, N≥3. Densitometric analysis was shown as fold changes relative to beta-actin

Figure 5

The Wnt/beta-catenin pathway is indispensible in BMP2-induced C/EBPalpha promoter methylation. (a) Co-IP assay shows interaction between Dnmt 3a and Dnmt 3b, but not with GR in C3H10T1/2 cells after 21 days of treatment by BMP2 and 10–6 M Dex. (b) ChIP assay shows GR did not bind to C/EBPalpha promoter in C3H10T1/2 cells after 21 days of treatment as indicated. (c) The in vivo protein level of active beta-catenin in the distal femur area of Dex- or saline-treated mice. N=4. (d) The protein levels of active beta-catenin in C3H10T1/2 cells treated as indicated. (e) Relative expressions of Wnt target gene Axin2 and CyclinD1, as well as Wnt inhibitor DKK1 in C3H10T1/2 cells treated as indicated. (f) C3H10T1/2 cells were treated by BMP2 with or without 10^–6 M IWR-1 for 7 days; the protein level of active beta-catenin and the mRNA level of Axin2 and CyclinD1 indicated the inhibition effect of IWR-1 on Wnt signaling. (g) The expression of osteoblast genes and adipocyte genes in C3H10T1/2 cells treated as indicated for 21 days. (h) In transdifferentiation assay, C3H10T1/2 cells were induced as indicated for 21 days before being switched to IFMD treatment. Adipocyte gene expression levels as well as transdifferentiated adipocytes were shown by qRT-PCR and oil red O staining, respectively. (i) DNA methylation status of C/EBPalpha promoter in C3H10T1/2 cells treated as indicated for 21 days (10 clones; Open circle: unmethylated CpG; full circle: methylated CpG, N=3). All mRNA expressions were normalized to beta-actin. Data are presented as mean±S.D. of three independent experiments. *P<0.05, **P<0.01, n.s. P>0.05, N=3. Densitometric analysis was shown as fold changes relative to beta-actin (western blot) or input (ChIP and Co-IP)

Figure 6

LiCl rescued the effects of Dex on osteoblast differentiation and C/EBPalpha promoter methylation. (a) Relative gene expression levels in osteoporotic BMSCs treated as indicated. (b) In transdifferentiation assay, the adipocyte gene expression levels and transdifferentiated adipocytes of osteoporotic BMSCs were shown by qRT-PCR and oil red O staining, respectively. (c) DNA methylation status of C/EBPalpha promoter in osteoporotic BMSCs treated with 100 ng/ml BMP2 plus 20 mM LiCl for 21 days (10 clones; open circle: unmethylated CpG; full circle: methylated CpG, N=3). (d) Relative gene expression levels in C3H10T1/2 cells treated as indicated. (e) Relative adipocyte gene expressions as well as adipocytes in transdifferentiated C3H10T1/2 cells were shown by qRT-PCR and oil red O staining, respectively. (f) DNA methylation status of C/EBPalpha promoter in C3H10T1/2 cells treated as indicated for 21 days (10 clones; open circle: unmethylated CpG; full circle: methylated CpG, N=3). (g) Protein level of Dnmt 3a/3b in C3H10T1/2 cells treated as indicated for 21 days (above). N=3. (h) ChIP assay shows the binding of Dnmt 3a/3b to C/EBPalpha promoter in C3H10T1/2 cells treated as indicated for 21 days. N=3. (i) A schematic diagram showing Dex blocks the signaling cascade activated by BMP2, and results in inhibition of C/EBPalpha promoter methylation and imbalanced osteoblast/adipocyte differentiation. All mRNA expression levels were normalized to beta-actin. Data are presented as mean±S.D. of three independent experiments. *P<0.05, **P<0.01, N≥3. Densitometric analysis was shown as fold changes relative to beta-actin (western blot) or input (ChIP)