Functional analysis of proteins belongs to SIBLING family in physiological and pathological calcification

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1. Identification of promoter region of Osteopontin in response to environmental change.Up-regulation of osteopontin was observed in transgenic mice carrying GFP cDNA ligated with distal promoter (-5505 bp to mRNA start site) (GFP5505 OPN) when mechanical stress was loaded to bone. Similar effect was observed in the ovaryextomized transgenic mice. Further investigation revealed that mechanical stress response element and estrogen respose element lies between -3156 and -5505 in the start site of transcription.2. Analysis of primary cultured cells derived from transgenic mice.Bone cells containing osteoblasts and osteocytes of the transgenic mice were cultured. Expression level of GFP was elevated when mechanical stress was loaded to cultured cells. Identification of mechanical stress-response element was performed by luciferase assay using various region of promoter region joined to luciferase cDNA. Finally, we identified the mechanical stress-response element between-3983 and -315G to the mRNA start site of osteopontin gene.3. Effects of truncated osteopontin on development of bone tissue.Transgenic mice carrying either calcium binding site or cell attachment site of osteopontin controlled -5505 promoter were generated. Transgenic mice were introduced to knockout mice of osteopontin gene. Generated mice showed abnormal structure in bone. The result indicate that calcium binding site and cell attachment site of osteopontin functioning in vivo.