Zinc finger protein 467 is a novel regulator of osteoblast and adipocyte commitment

Abstract

Osteoblasts and adipocytes are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. cDNA microarrays and quantitative real-time PCR (Q-PCR) were carried out in a differentiating mouse stromal osteoblastic cell line, Kusa 4b10, to identify gene targets of factors that stimulate osteoblast differentiation including parathyroid hormone (PTH) and gp130-binding cytokines, oncostatin M (OSM) and cardiotrophin-1 (CT-1). Zinc finger protein 467 (Zfp467) was rapidly down-regulated by PTH, OSM, and CT-1. Retroviral overexpression and RNA interference for Zfp467 in mouse stromal cells showed that this factor stimulated adipocyte formation and inhibited osteoblast commitment compared with controls. Regulation of adipocyte markers, including peroxisome proliferator-activated receptor (PPAR) γ, C/EBPα, adiponectin, and resistin, and late osteoblast/osteocyte markers (osteocalcin and sclerostin) by Zfp467 was confirmed by Q-PCR. Intra-tibial injection of calvarial cells transduced with retroviral Zfp467 doubled the number of marrow adipocytes in C57Bl/6 mice compared with vector control-transduced cells, providing in vivo confirmation of a pro-adipogenic role of Zfp467. Furthermore, Zfp467 transactivated a PPAR-response element reporter construct and recruited a histone deacetylase complex. Thus Zfp467 is a novel co-factor that promotes adipocyte differentiation and suppresses osteoblast differentiation. This has relevance to therapeutic interventions in osteoporosis, including PTH-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.

FIGURE 1.

Regulation of Zfp467 by PTH and gp130-binding cytokines and Zfp467 retroviral construction. Q-PCR for Zfp467 on (A) day 17 osteoblast-differentiated Kusa 4b10 cells and (B) day 9 differentiated primary calvarial osteoblasts (mPOB) treated without or with PTH(1–34) for 1, 6, and 24 h. C, Q-PCR for Zfp467 on Kusa 4b10 cells osteoblast differentiated for 17 days and treated with 50 ng/ml of oncostatin-M (OSM), cardiotrophin-1 (CT-1) or untreated for 1, 6, and 24 h. Results are mean fold-change ± S.E. of ≥3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with untreated control. Representation of Zfp467 retroviral constructs: D, MSCV-IRES-GFP vector control; E, MSCV-sense Zfp467-IRES-GFP vector; F, MSCV-antisense Zfp467-IRES-GFP vector. LTR, long terminal repeats; IRES, internal ribosomal entry site; GFP, enhanced green fluorescence protein reporter gene. G, Zfp467 mRNA levels during 21 days of osteoblast differentiation. Results are mean fold-change ± S.E. of 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with vector control. H, Western blot analysis of Kusa 4b10 cells untreated or transduced with MSCV-IRES-GFP vector, MSCV-sense Zfp467-IRES-GFP, and MSCV-antisense Zfp467-IRES-GFP probed with anti-Zfp467. Mouse Pan-actin was used as a loading control. Data in panel G are mean ± S.E. of 3 independent transductions. Symbols are defined in the figure. Standard error bars indicated, or within symbols.

FIGURE 2.

Zfp467 overexpression inhibits mineralization and expression of late osteoblast marker genes. A, representative von Kossa staining and quantification of nodule formation on days 13, 15, and 17 of osteoblast differentiating Kusa 4b10 cells infected with vector, sense, or antisense Zfp467 constructs. B, gene expression analysis for PTH receptor-1 (PTHR1), osteocalcin (OCN), and sclerostin (SOST) mRNA in osteoblast-differentiating Kusa 4b10 infected with vector, sense, or antisense Zfp467 constructs. Results are mean ± S.E. for 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with vector control. Symbols are defined in the figure.

FIGURE 3.

Zfp467 overexpression increased adipocyte formation during Kusa 4b10 differentiation under mineralizing conditions. A, representative images of Oil red O staining at days 9 and 11 of Kusa 4b10 differentiation. B, gene expression levels for adipocyte marker genes including PPARγ, C/EBPα, adiponectin, and resistin mRNA by Q-PCR. Results are mean fold-change ± S.E. of 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vector control. Symbols are defined in the figure. Standard error bars indicated, or within symbols.

FIGURE 4.

Zfp467 overexpression enhanced the osteoclast supporting ability of Kusa 4b10 cells. A, RANKL and OPG mRNA expression in differentiating Kusa 4b10 cells under mineralizing (A) or adipogenic conditions (B) by Q-PCR. Results are mean fold-change ± S.E. of 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with vector control. C, representative images of tartrate-resistant acid phosphatase positive (TRAP+) multinucleated cells (MNC) formed from bone marrow cells co-cultured with Zfp467 construct-transduced Kusa 4b10 cells and treated with 1,25-(OH)₂D₃ (10⁻⁸ m) for 7 days. D, quantification of TRAP⁺ multinucleated cells with 2–4 and ≥5 nuclei per cell after 7 days of treatment with 1,25-(OH)₂D₃ (10⁻⁸ m). Results are mean ± S.E. of ≥3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vector control. Symbols are defined in the figure. Standard error bars indicated, or within symbols.

FIGURE 5.

Zfp467 overexpression accelerates adipocyte formation in Kusa 4b10 cells under adipogenic conditions. A, representative Oil red O staining of adipocytes on days 4 and 7 of Kusa 4b10 cells; B, the number of Oil red O stained adipocytes per mm²; C, measured levels of solubilized Oil red O during adipogenic differentiation of Kusa 4b10 cells infected with vector, sense, and antisense Zfp467 constructs. Values are mean ± S.E. of ≥3 independent experiments; ***, p < 0.001 versus vector control completed in triplicate. D, the mRNA levels of adipocyte marker genes including PPARγ, C/EBPα, adiponectin, and resistin in Kusa 4b10 cells infected with vector, sense, and antisense Zfp467 constructs over 11 days of adipogenic differentiation analyzed by Q-PCR. Results are mean fold-changes ± S.E. of 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with vector control. Symbols are defined in the figure. Standard error bars indicated, or within symbols.

FIGURE 6.

Gene knockdown of Zfp467 by RNAi in differentiating Kusa 4b10 cells under adipogenic medium. A, Q-PCR analysis for Zfp467 mRNA levels during Kusa 4b10 differentiation. Results are mean fold-change ± S.E. of 3 independent experiments; ***, p < 0.001, compared with scrambled control. B, representative Western blot analysis of Kusa 4b10 cell lysates untreated or transfected with scrambled duplex control or Zfp467 interfering RNA (RNAi) and fold-change in Zfp467 band density normalized to Pan-actin. Results are mean fold-change ± S.E. of 3 independent experiments. Protein membranes were probed with anti-Zfp467 and mouse Pan-actin as a loading control. C, representative samples of Oil red O staining in Kusa 4b10 cells untreated and transfected with scrambled duplex control and Zfp467 RNAi and measured levels of solubilized Oil Red O over 11 days of culture; values are mean ± S.E. of ≥3 independent experiments; **, p < 0.01; ***, p < 0.001 versus vector control. D, Q-PCR for adipogenic marker genes PPARγ, C/EBPα, and adiponectin during Kusa 4b10 differentiation. Results are mean ± S.E. of 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with scrambled control. Symbols are defined in the figure. Standard error bars indicated, or within symbols.

FIGURE 7.

PPARγ-promoter transactivation and recruitment of HDAC complex by Zfp467. A, relative luciferase activity from ST2 cells transiently co-transfected with a reporter construct containing the PPAR-response element (PPRE), without or with pCDNA3-PPARγ, vector control, or Zfp467 cDNA. Results are mean ± S.E. of 3 independent experiments conducted in triplicate; **, p < 0.01 compared with reporter vectors and their respective controls. Firefly luciferase values are normalized for Renilla luciferase. B, deacetylation activity of Zfp467-FLAG immunoprecipitates from transient expression assays in HEK293T cells, and attenuation of this activity by the HDAC inhibitor, TSA. HeLa nuclear extracts were used as the positive control. Results are mean ± S.E. of 4 independent experiments; *, p < 0.05 compared with vector control. Standard error bars indicated.

FIGURE 8.

Intra-tibial injection of calvarial cells transduced with Zfp467 enhances adipocyte formation. A, gene expression analysis of RNA from tibiae injected with primary calvarial cells transduced with sense Zfp467, vector, or PBS vehicle using primers to the MigR1 vector sequence and Zfp467 transgene. B, representative hematoxylin and eosin-stained tibial sections and quantification of adipocyte number and volume, 2 weeks after tibiae were injected with calvarial cells transduced with sense Zfp467 cDNA compared with vector control cells. Data are mean ± S.E., n ≥ 8 animals per group. *, p < 0.05; **, p < 0.01 versus PBS and vector control. Standard error bars indicated.