Knocking down MITR inhibits osteogenesis and enhances adipogenesis. A, MSCs infected with MITR (shMITR-1 or -2) or control (shControl) shRNA lentiviral vectors were stained with Alizarin Red S, a measure for osteogenesis, after inducing osteoblast differentiation for 7 days. The quantification of staining was measured by spectrophotometer at 450 nm (A₄₅₀) and is presented as the mean ± S.D. B, expression of MITR, osteogenic markers RUNX2 and OPN, and adipogenic marker FABP4 in MSCs, osteoblasts (Ost.), adipocytes (Adi.) (lanes 1–3) or MSCs infected with indicated shRNAs viral vectors even days after inducing osteoblast differentiation (lanes 4 and 5) using Western blotting analysis. OPN, osteopontin; FABP4, fatty acid-binding protein 4. C, MSCs infected with indicated shRNA lentiviral vectors were stained with Oil Red O, a measure for adipogenesis, after inducing adipocytes differentiation for 7 days. Cells were stained to visualize intracellular lipid deposition by phase-contrast microscopy and photographed. The quantification of staining was measured by spectrophotometer at 510 nm (A₅₁₀) and presented as the mean ± S.D. p values were analyzed using Student's t test.

Knockdown of MITR inhibits osteogenesis in primary human MSCs. A, examination of MITR and osteogenic marker osteopontin (OPN) expression in primary human MSCs (pMSC-1) infected with MITR (shMITR) or control (shControl) shRNAs lentiviral vectors after inducing osteoblast differentiation for 7 days using Western blotting analysis. B, primary human MSCs treated as in A were stained with Alizarin Red S 7 days after induction of osteoblast differentiation. The quantification of staining was measured by spectrophotometer at 450 nm (A₄₅₀) and presented as the mean ± S.D. C, ectopic expression of MITR in primary human MSCs using adenovirus-based system. Cell extracts from pMSC-1 infected with MITR (AdMITR) or control (AdControl) adenovirus after inducing osteoblasts differentiation for 7 days were analyzed using Western blotting with antibodies as indicated. D, primary human MSCs treated as in C were stained with Alizarin Red S 7 days after induction of osteoblast differentiation. The quantification of staining was measured by spectrophotometer at 450 nm (A₄₅₀) and presented as the mean ± S.D. p values were analyzed using Student's t test.

MITR inhibits the transcriptional activity of PPARγ-2 via abrogating binding of PPARγ-2 to adipocyte marker gene FABP4 promoter. A, MSCs were differentiated into osteoblasts (Ost.) and adipocytes (Adi.) for 7 days and analyzed for FABP4 RNA expression using quantitative real time RT-PCR. FABP4 RNA expression was dramatically increased in adipocytes compared with MSCs and osteoblasts. B, MITR inhibits PPARγ-2-activated FABP4 promoter activity. MSCs and 293 cells were transfected with human FABP4 promoter-driven luciferase reporter plasmid (FABP4-P) together with internal control plasmid pRL-TK, PPARγ-2, or MITR plasmids as indicated. The FABP4 promoter-driven reporter luciferase activity was measured by Dual-Luciferase reporter assay system. Protein expression of PPARγ-2 or MITR was analyzed using Western blotting with antibodies as indicated. C, MITR blocks binding of PPARγ-2 to the human FABP4 promoter. MSCs were transfected with PPARγ-2, MITR, or vector control pcDNA3.1 plasmids as indicated. Cross-linked chromatin was immunoprecipitated using PPARγ-2 or IgG antibodies. The input and immunoprecipitated DNA were subjected to real time PCR using primers corresponding to the promoter region of FABP4 listed in supplemental Table 1. D, PPARγ-2 and RUNX2 specifically bind to FABP4 or OPN promoter in adipocytes and osteoblasts, respectively. Cross-linked chromatin from osteoblasts and adipocytes was immunoprecipitated using PPARγ-2, RUNX2, or IgG antibodies. The input and immunoprecipitated DNA were subjected to PCR or real time PCR using primers corresponding to promoter regions of FABP4 (top panel) or OPN (bottom panel) listed in supplemental Table 1. E, model for EZH2-MITR-mediated MSC differentiation into osteoblasts and adipocytes. EZH2 binds to the MITR promoter and inhibits MITR expression in adipocytes but not in osteoblasts. The presence of MITR in osteoblasts promotes MITR interacting with PPARγ-2 in the nucleus of osteoblasts, which interrupts PPARγ-2 transcriptional activity resulting in attenuation of adipogenesis and acceleration of osteogenesis. Values are represented as the mean ± S.D. p values were analyzed using Student's t test. FABP4, fatty acid-binding protein 4; OPN, osteopontin.

EZH2 binds to the MITR promoter and inhibits MITR expression in adipocytes but not in osteoblasts. A and B, human mesenchymal stem cell line 3A6 (MSC) was differentiated into osteoblasts (Ost.) and adipocytes (Adi.) for 7 days and analyzed for EZH2 expression by Western blotting (A) and EZH2-ChIP-on-chip assays (B). The total EZH2-targeted genes in undifferentiated MSCs, adipocytes, and osteoblasts are listed. C, EZH2-targeted genes were confirmed using real time RT-PCR. RNA expression in differentiated MSCs compared with parental MSCs is plotted. The data are expressed as mean ± S.D. CAST, calpastatin; PHTF1, putative homeodomain transcription factor 1; MITR, myocyte enhancer factor-2 interacting transcriptional repressor (also named histone deacetylase 9c, HDAC9c); NRG1, neuregulin; OSTF1, osteoclast stimulating factor 1; ZBTB10, zinc finger and BTB domain containing 10. D, protein expression levels of MITR in the indicated cells were analyzed using Western blotting analysis with indicated antibodies. E, protein expression levels of MITR in bone and adipose tissues from mice were examined using Western blotting analysis with indicated antibodies. F and G, binding of EZH2 and tri-methylated histone H3 at lysine 27 (H3K27me3) at MITR promoter during osteogenesis and adipogenesis were analyzed using quantitative ChIP assay. Cross-linked chromatin from MSC, osteoblasts (Ost.), and adipocytes (Adi.) was immunoprecipitated using EZH2 (F), H3K27me3 (G), or IgG antibodies. The input and immunoprecipitated DNA were subjected to PCR (upper panels) or real time PCR (lower panels) using primers corresponding to promoter region of MITR listed in supplemental Table 1. H, binding of EZH2 to MITR promoter during osteogenesis and adipogenesis was analyzed using EMSA. Biotin-labeled double-stranded oligonucleotides containing MITR promoter region was incubated with nuclear extracts harvested from differentiated osteoblasts and adipocytes (left panel). For the competition experiments, unlabeled cold competitive oligonucleotides were included in the reaction mixture. To verify the involvement of EZH2 in the complex, specific antibody against EZH2 was added to the reaction mixture (right panel). Oligonucleotides that contain EZH2-bound sequence of MITR promoter region were used for EMSA and are listed in supplemental Table 1.

Expression of MITR enhances osteogenesis and attenuates adipogenesis. A, expression of MITR in MSCs using the adenovirus-based system. Ectopic expression of MITR in MSCs infected with MITR (AdMITR) or control (AdControl) adenovirus was detected by Western blotting. B, MSCs infected with the indicated adenovirus were stained with Alizarin Red S after inducing osteoblast differentiation for 7 days. The quantitative results for the staining measured by A₄₅₀ are presented as the mean ± S.D. C, MSCs infected with indicated adenovirus were stained with Oil Red O 7 days after inducing adipocyte differentiation. Cells were stained to visualize intracellular lipid deposition by phase-contrast microscopy and photographed. The quantitative results for the staining measured by A₅₁₀ are presented as the mean ± S.D. p values were analyzed using Student's t test.

MITR interacts with PPARγ-2 in osteoblastic nucleus. A and B, cytosolic and nuclear cell extracts were harvested after inducing osteoblast differentiation of MSCs for 7 days and immunoprecipitated (IP) with MITR antibody (A) or PPARγ-2 antibody (B) and Western-blotted with antibodies as indicated. MITR and PPARγ-2 complex was detected in the nuclear fraction but not in the cytosolic fraction in osteoblasts (Ost.). C, MITR does not interact with Runx2 in the nuclear or cytoplasmic fraction of osteoblasts. Cytosolic and nuclear cells extracts from MSCs treated as in A were immunoprecipitated with MITR antibody and Western-blotted with antibodies as indicated.