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Overexpression of Runx2 and MKP-1 Stimulates Transdifferentiation of 3T3-L1 Preadipocytes into Bone-Forming Osteoblasts In Vitro

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Abstract

Runx2, a transcription factor, is essential for osteoblastic differentiation, bone formation, and maintenance. We examined the effect of Runx2 on transdifferentiation of 3T3-L1 preadipocytes into functional, mature osteoblasts. Forced expression of exogenous Runx2 using a retroviral gene-delivery system showed increases of alkaline phosphatase (ALP) activity and expression of the osteoblastic marker genes osteocalcin (OC), bone sialoprotein (BSP), and osterix (Osx), accompanied by low-level matrix mineralization. In contrast, adipocytic differentiation was completely blocked with downregulation of adipogenic transcription factors PPARγ2, C/EBPα, and C/EBPδ. Treatment of dexamethasone (Dex), a synthetic glucocorticoid, stimulated the formation of mineralized nodules in Runx2-overexpressing 3T3-L1 cells with increases of ALP, OC, BSP, and Osx expression. Here, we focused on a dual specific phosphatase, mitogen-activated protein kinase (MKP-1), since Dex significantly increased MKP-1 expression in Runx2-overexpressing 3T3-L1 cells. Forced expression of exogenous MKP-1 resulted in accumulation of robust matrix mineralization in parallel with induction of ALP activity and expression of OC, BSP, and Osx in Runx2-overexpressing 3T3-L1 cells. These results suggest that simultaneous overexpression of Runx2 and MKP-1 is effective for transdifferentiation of preadipocytes into fully differentiated bone-forming osteoblasts and provide a novel strategy for cell-based therapeutic applications requiring significant numbers of osteogenic cells to synthesize mineralized constructs for the treatment of large bone defects.

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Acknowledgments

We thank Prof. Gérard Karsenty (Department of Genetics and Development, Columbia University Medical Center) for Cbfa1-pCMV5 and 6 × OSE2/pGL3Basic, and Dr. Yoshikazu Mikami (Department of Anatomy, Nihon University School of Dentistry) for MKP-1/pcDNA3.1(+). This work was supported by grants from Dental Research Center, Nihon University School of Dentistry, Sato Fund, Nihon University School of Dentistry, and Graduate School of Dentistry, Nihon University.

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  1. Department of Anatomy, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan

    Tomihisa Takahashi

  2. Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-8310, Japan

    Tomihisa Takahashi

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Correspondence to Tomihisa Takahashi.

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Takahashi, T. Overexpression of Runx2 and MKP-1 Stimulates Transdifferentiation of 3T3-L1 Preadipocytes into Bone-Forming Osteoblasts In Vitro. Calcif Tissue Int 88, 336–347 (2011). https://doi.org/10.1007/s00223-011-9461-9

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