The application of the transcription factor related to urinary stone formation to elucidation and gene therapy

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【Purpose】The formation of urinary tract stones is a self-defense reaction to eliminate oxalic acid. Does there exist an OPN-secreting system where the self-stimulation of OPN (adhesion) triggers the secretion of OPN? We speculated that the expression of the OPN protein may be self-regulated by autocrine mechanism. Therefore, we examined the expression of OPN with the phosphorylation of FAK using MDCK and NRK-52E. Introduction Focal adhesion kinase (FAK), a phosphoenzyme, is also a substrate for tyrosine phosphorylation by pp^<60V-Src>. In the renal tubule cells, signal transmission is considered to occur and the expression of various genes in the cell regulated by the tyrosine-phosphorylation of proteins such as FAK and cell structural proteins with the adherence of integrin and osteopontin (OPN). Recently, many results about OPN on stone formation have been reported as follows;1)Osteopontin (OPN) plays an important role in the formation of calcium stones.2)Integrin□V□3,the ligand of O … More PN is expressed in the canine distal renal tubule (MDCK) cells. We speculated that this phosphorylation pathway would be activated and OPN secretion is induced more and more in stone formation.【Methods】MTT assay;MDCK cells (cell density 1 x 10^7/ml) were cultured for 4 hours on a 96-well plate coated with OPN protein at a concentration of 1 -1000 ng/ml. Then, the number of adherent cells was examined by an MTT assay.Western Blotting, Northern Blotting;MDCK and NRK-52 cells (1 x 10^7/ml) stimulated for 4 hours with 1 -1000 ng/ml OPN protein. Then proteins and mRNA were extracted and Western blotting and Northern blotting were performed. (Before Western blotting, the cells were washed with PBS to remove the OPN protein).Immunoprecipitation;MDCK cells (cell density 1 x 10^7/ml) stimulated for 4 hours with OPN protein at a concentration of 1 -1000 ng/ml. Then, immunoprecipitation was performed using an FAK antibody.Phosphorylation Assay;The protein obtained by immunoprecipitation was subjected to Western blotting with anti-phosphotyrosine antibody (x1000).【Results】1)The number of MDCK cells adhering to the dish increased with the concentration of the OPN protein coating the plate.2)The phosphorylation of FAK was promoted by the cell (integrin) -extracellular matrix (OPN) adherence.3)The expression of OPN-mRNA and OPN protein increased with the concentration of OPN stimulating the MDCK cells and NRK-52 cells.【Discussion】The expression of OPN protein on the MDCK cells was increased by the stimulation by the adherence of OPN t6 the cells. An autocrine mechanism was found to exist, and the phosphorylation of FAK was suggested to be involved in the signal transmission in the cell. This autocrine mechanism is suspected to be involved in stone formation. Less