Abstract
Hyperphosphatemia in chronic kidney disease is highly associated with vascular calcification. Previous studies have shown that high phosphate-induced phenotypic switching of vascular smooth muscle cells (SMCs) into osteogenic cells plays an important role in the calcification process. In the present study, we determined whether Krüppel-like factor 4 (Klf4) and phosphorylated Elk-1, transcriptional repressors of SMC differentiation marker genes activated by intimal atherogenic stimuli, contributed to this process. Rat aortic SMCs were cultured in the medium with normal (0.9 mmol/liter) or high (4.5 mmol/liter) phosphate concentration. Results showed that high phosphate concentration induced SMC calcification. Moreover, high phosphate decreased expression of SMC differentiation marker genes including smooth muscle α-actin and SM22α, whereas it increased expression of osteogenic genes, such as Runx2 and osteopontin. High phosphate also induced Klf4 expression, although it did not phosphorylate Elk-1. In response to high phosphate, Klf4 selectively bound to the promoter regions of SMC differentiation marker genes. Of importance, siRNA-mediated knockdown of Klf4 blunted high phosphate-induced suppression of SMC differentiation marker genes, as well as increases in expression of osteogenic genes and calcium deposition. Klf4 was also induced markedly in the calcified aorta of adenine-induced uremic rats. Results provide novel evidence that Klf4 mediates high phosphate-induced conversion of SMCs into osteogenic cells.
Highlights
Elucidation of molecular mechanisms controlling vascular calcification is critical for chronic kidney disease patients
These results indicate that incubation with high phosphate concentration decreased expression of smooth muscle cells (SMCs) differentiation marker genes, whereas it increased expression of osteogenic genes and thereby induced calcification in our cultured SMCs
SiRNA-induced knockdown of Krüppel-like factor 4 (Klf4) and Runx2 did not exhibit additive effects on SMC calcification (Fig. 5), we examined whether Klf4 and Runx2, both of which were induced by high phosphate concentration in SMCs, acted additively or synergistically to repress SMC differentiation marker genes by co-transfection of these expression plasmids and smooth muscle (SM) ␣-actin promoter-luciferase construct into cultured aortic SMCs
Summary
Elucidation of molecular mechanisms controlling vascular calcification is critical for chronic kidney disease patients. Previous studies have shown that high phosphate-induced phenotypic switching of vascular smooth muscle cells (SMCs) into osteogenic cells plays an important role in the calcification process. Speer et al [20] showed that Runx, but not loss of myocardin, was required for high phosphate-induced vascular calcification These results suggest a critical role of Runx in high phosphateinduced SMC phenotypic switching into osteogenic cells. It remains unknown whether transcription factors required for repression of SMC differentiation marker genes by intimal atherogenic stimuli play important roles for medial calcification. The aims of the present studies were to determine whether Klf and phosphorylated Elk-1 contribute to high phosphate-induced SMC phenotypic switching into osteogenic cells, and if so, to determine their relationship with Runx
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