[Transfection of recombinant bone morphogenetic protein-7 expressing plasmid into cultured human renal tubular epithelial cells attenuates the extracellular matrix accumulation induced by transforming growth factor-beta].
ABSTRACT To investigate the effects of bone morphogenetic protein (BMP)-7 on the extracellular matrix (ECM) accumulation induced by transforming growth factor (TGF)-beta.
Mouse full length BMP-7 cDNA was ligated into a eukaryotic expression vector pcDNA3.1. Restriction enzymatic analyses and DNA sequencing were used to confirm the accuracy of the BMP-7 expressing plasmid thus constructed. The recombinant expression plasmid pcDNA3.1-BMP-7 was transfected into cultured human renal tubular epithelial cells of the line HK-2 mediated by liposome. Positive clones were selected so as to obtain the human renal epithelial cells with stable transfection. These HK-2 cells were cultured and divided into 5 groups to be treated with 5 ng/ml TGF-beta, blank plasmid pcDNA3.1, blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta, pcDNA3.1-BMP-7, pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta, and an additional grin the cells and the supernatant of the cell culture fluid were collected. The expression level of BMP-7 protein was determined by Western blotting. RT-PCR and ELISA were used to determine the mRNA and protein expression of collagen (Col) I and III, and fibronectin (FN) in the human renal tubular epithelial cells and supernatant of different groups.
The recombinant plasmid pcDNA3.1-BMP-7 was successfully constructed. The cell mRNA expression levels of Col I and III and FN of the 5 ng/ml TGF-beta group and blank plasmid pcDNA3.1 + 5 ng/ml TGF-beta group were all significantly higher than those of the blank plasmid pcDNA3.1 group, pcDNA3.1-BMP-7 group, and control group (all P < 0.05). The cell mRNA expression levels of Col I and FN of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta were all significantly lower than those of the TGF-beta group (all P < 0.05). The cell mRNA expression level of Col III of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta was lower, however, not significantly, than that of the TGF-beta group. The supernatant FN levels of the 5 ng/ml TGF-beta group and pcDNA3.1 + 5 ng/ml TGF-beta group were both significantly higher than that of the control group (both P < 0.05), and the supernatant FN level of the pcDNA3.1-BMP-7 + 5 ng/ml TGF-beta group was significantly lower that that of the 5 ng/ml TGF-beta group (P < 0.05).
Over-expression of BMP-7 significantly inhibits the increased syntheses of collagen I and III, and fibronectin induced by TGF-beta. BMP-7 exerts its antifibrotic effect partially through blocking the TGF-beta-induced accumulation of extracellular matrix in human renal tubular epithelial cells.