Dentin matrix protein 1 gene cis-regulation: Use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo

Wuchen Yang, Yongbo Lu, Ivo Kalajzic, Dayong Guo, Marie A. Harris, Jelica Gluhak-Heinrich, Shiva Kotha, Lynda F. Bonewald, Jian Q. Feng, David W. Rowe, Charles H. Turner, Alexander G. Robling, Stephen E. Harris

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Abstract

Dentin matrix protein 1 (DMP1) is highly expressed in osteocytes and is mechanically responsive. To study osteocyte-specific and mechanically regulated DMP1 gene expression, the transcriptional activity of three cis-regulatory regions was first examined in an osteoblast differentiation model in vitro using a green fluorescent protein (GFP) reporter. Expression of the -9624 to +1996 bp (10 kb) and -7892 to +4439 bp (8 kb) DMP1 cis-regulatory regions dramatically increased in areas of mineralized matrix, in dendritic, osteocyte-like cells. Mineralizing cultures expressing the 8-kb construct show dramatic GFP increases in response to loading in cells with a dendritic morphology. Transgenic mice expressing the 8-kb DMP1-GFP and -2433 to +4439 bp (2.5 kb) DMP1-LacZ were generated. Osteocyte-specific expression was found with the 8 kb but not with the 2.5 kb in postnatal animals. However, the 2.5 kb could support expression in rapidly forming osteoblasts and pre-osteocytes in the embryo. Primary calvarial osteoblast cultures demonstrated that the 2.5 kb supports weak expression in a subset of osteoblasts and pre-osteocytes, but not in mature osteocytes. However, the 8 kb supports robust expression in primary bone marrow cultures. Therefore the region -7892 to -2433 bp, termed a 5.5-kb "Osteocyte Enhancer Module," appears to be required for osteocyte specificity. Ulnae of mice with the 8-kb DMP1-GFP were subjected to mechanical loading where GFP expression increased selectively and locally in osteocytes, distal to the mid-shaft and near the surface of the bone. Thus, the 8-kb region of the DMP1 gene is a target for mechanotransduction in osteocytes, and its cis-regulatory activity may be correlated to local strain in bone.

Original languageEnglish (US)
Pages (from-to)20680-20690
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number21
DOIs
StatePublished - May 27 2005

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Yang, W., Lu, Y., Kalajzic, I., Guo, D., Harris, M. A., Gluhak-Heinrich, J., Kotha, S., Bonewald, L. F., Feng, J. Q., Rowe, D. W., Turner, C. H., Robling, A. G., & Harris, S. E. (2005). Dentin matrix protein 1 gene cis-regulation: Use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo. Journal of Biological Chemistry, 280(21), 20680-20690. https://doi.org/10.1074/jbc.M500104200