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FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation

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Abstract

To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK-dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1 −/− ; ERK2 flox/flox ; Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1 −/− ; ERK2 flox/flox ; Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation.

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Acknowledgments

We thank Lynda Bonewald for MLO-Y4 cells and James Martin for Prx1-Cre mice. We also thank Teresa Pizzuto and Ajay Singh for technical assistance and Valerie Schmedlen for editorial assistance. Work in the laboratory of the authors was supported by NIH grants R01AR055556 and R03DE019814 to S. Murakami. The Gene Expression and Genotyping Facility of the Case Comprehensive Cancer Center was supported by NIH grant P30CA43703.

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Authors and Affiliations

  1. Department of Orthopaedics, Case Western Reserve University, 2109 Adelbert Road BRB 329, Cleveland, OH, 44106, USA

    Ai Kyono, Zhufeng Ouyang & Shunichi Murakami

  2. Swagelok Center for Surface Analysis of Materials, Case School of Engineering, Case Western Reserve University, Cleveland, OH, 44106, USA

    Nanthawan Avishai

  3. Department of Neurosciences, Case Western Reserve University, Cleveland, OH, 44106, USA

    Gary E. Landreth

  4. Department of Genetics, Case Western Reserve University, Cleveland, OH, 44106, USA

    Shunichi Murakami

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  1. Ai Kyono
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Correspondence to Shunichi Murakami.

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Kyono, A., Avishai, N., Ouyang, Z. et al. FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation. J Bone Miner Metab 30, 19–30 (2012). https://doi.org/10.1007/s00774-011-0288-2

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  • DOI: https://doi.org/10.1007/s00774-011-0288-2

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