Cell-specific expression of GFP under the control of different lengths of the osteopontin promoter in transgenic mice identified the positive and negative regulatory regions for respective cell types. The results provide new insights for physiological and pathological expression of the osteopontin gene.
Introduction: Osteopontin (OPN) is a major non-collagenous bone matrix protein that is involved in normal and pathological calcification and is expressed in a tissue-specific manner. To investigate how such tissue-specific OPN gene expression is regulated in vivo, transgenic mice expressing the green fluorescent protein (GFP) reporter gene controlled by different lengths of the OPN promoter were generated.
Materials and methods: Cell- and developmental stage-specific osteopontin expression in transgenic mice was examined by Northern blotting, immunoblotting, fluorescence examination, and in situ hybridization and compared with those of OPN.
Results and conclusions: The line bearing the -5505 to +14 region of the OPN promoter was shown by Northern blotting and immunoblotting to express GFP in the same cells that express endogenous OPN (osteoblasts, hypertrophic chondrocytes, renal and mammary gland epithelial cells, and granulated metrial gland [GMG] placental cells) at the same stage in development. Thus, the 5.5-kb -5505 to +14 promoter region is sufficient for proper tissue-specific OPN expression. The lines carrying shorter segments of the OPN promoter showed different expression patterns. These patterns revealed a putative cis-acting element in the -5269 to -5263 region that restricts OPN expression to hypertrophic chondrocytes and a mammary gland-specific expressing element and a GMG cell-specific enhancing element in the -5505 to -3156 region. Furthermore, the -3155 to -1576 region seems to contain positive renal epithelial cell- and GMG cell-specific expression motif(s) as well as a negative regulatory element that prevents OPN expression in fibroblasts. Moreover, the -1576 to -910 region seems to contain a positive osteoblast-specific-expressing element. Thus, the 5.5-kb OPN promoter contains multiple cis-acting elements encoding positive and negative cell-specific regulatory systems.