Kruppel-like factor 4 contributes to high phosphate-induced phenotypic switching of vascular smooth muscle cells into osteogenic cells

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.

FIGURE 1.

High phosphate concentration induced calcification of SMCs. A, rat aortic SMCs were cultured with normal (left) or high (right) phosphate concentration for 10 days. Representative pictures of von Kossa staining are shown (n = 3). Top, appearance of 60-mm dishes. Bottom, magnification x100. B–F, SMCs were incubated with normal (NP) or high (HP) phosphate concentration for 2, 4, 6, and 8 days. Expression of SM α-actin (B), SM22α (C), Runx2 (D), osteopontin (E), and ALP (F) mRNA was determined by real-time RT-PCR. Values represent the means ± S.E. *, p < 0.05 compared with SMCs with normal phosphate medium (n = 4).

FIGURE 2.

High phosphate induced Klf4 expression in cultured SMCs. SMCs were incubated with normal (NP) or high (HP) phosphate concentration for indicated times. Expression of Klf4 (A), Klf2 (C), and Klf5 (D) mRNA was determined by real-time RT-PCR. Values represent the means ± S.E. *, p < 0.05 compared with SMCs with normal phosphate medium (n = 4). B, expression of Klf4, SRF (nuclear protein), and vimentin (cytosolic protein) in SMCs with normal or high phosphate medium for 2 days was examined by Western blotting (n = 3). Cyto, cytosolic fraction; Nucl, nuclear fraction. E, expression of ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), Elk-1, phosphorylated Elk-1 (p-Elk-1), and GAPDH was examined by Western blotting (n = 4). P, POVPC treatment at 10 μg/ml for 6 h.

FIGURE 3.

Klf4 was induced in the aorta of adenine-induced uremic rats. The calcification status was examined by von Kossa staining in the thoracic aorta of adenine-induced uremic rats (B and D) and controls (A and C). Expression of SM α-actin (E and F) and Klf4 (G and H) was examined by immunohistochemistry in the thoracic aorta of adenine-induced uremic rats (F and H) and controls (E and G). Representative pictures are shown from 4 mice analyzed per group. Bars: 1 mm (A and B); 50 μm (C–H). Inset in H is a magnified image from white lined square. Black and white arrowheads indicate Klf4-positive and Klf4-negative cells, respectively.

FIGURE 4.

High phosphate concentration induced selectively Klf4 binding to the SMC promoters in SMCs. SMCs were cultured with normal (NP) or high (HP) phosphate concentration for 2 days, and the association of Klf4, SRF, and Runx2 with the CArGs/TCE-containing promoter regions of the SM α-actin gene (A) and the SM22α gene (B), or the intron 1 region of the SM22α gene (C) was determined by ChIP assays. Values represent the means ± S.E. *, p < 0.05 compared with SMCs with normal phosphate medium (n = 3).

FIGURE 5.

Knockdown of Klf4 attenuated high phosphate-induced phenotypic switching of SMCs. A, cultured SMCs were transfected with siRNA specific for Klf4 (siKlf4) or a scramble sequence (siScr) and incubated with normal (NP) or high (HP) phosphate concentration for 2 days. Expression of Klf4 and SRF in the nuclear fraction of SMCs was examined by Western blotting (n = 3). B, SMCs were transfected with siRNA for Runx2 (siRunx2) or siScr, followed by transfection of FLAG-Runx2 expression plasmid or control plasmid (Ct). Expression of Runx2 (anti-FLAG antibody) and GAPDH was examined by Western blotting (n = 3). C–G, cultured SMCs were transfected with siKlf4, siRunx2, and/or siScr and incubated with normal (NP) or high (HP) phosphate concentration for 6 days. Expression of SM α-actin (C), Runx2 (D), osteopontin (E), and ALP (F) mRNA was determined by real-time RT-PCR. Calcium deposition was measured and normalized by cellular protein content (G). Values represent the means ± S.E. *, p < 0.05 compared with SMCs with normal phosphate medium (n = 3).

FIGURE 6.

Transcriptional regulation of the SM α-actin gene and the osteopontin gene by Klf4 and Runx2 was not interdependent. Expression plasmids for Klf4 and Runx2 (0, 30, 100, or 300 ng) were co-transfected with wild-type (A) and CArGs/TCE-mutant (B) of the SM α-actin promoter-luciferase construct (900 ng), human osteopontin promoter-luciferase (hOPN −1500/+87 or hOPN −970/+87) construct, mouse osteopontin promoter-luciferase (mOPN −2287/+132) construct or pGL3-basic plasmid (900 ng) (D) into rat aortic SMCs. Luciferase activities were measured and normalized to protein content. An arbitrary value of 1 was assigned to the activity of cells transfected with the wild-type SM α-actin promoter-luciferase construct (A and B) or hOPN (−1500/+87) construct and empty expression plasmids. C, NIH/3T3 cells were co-transfected with pG5Luc reporter plasmid, expression plasmids for fusion proteins of GAL4-SRF and VP16-Klf4, and Runx2 expression plasmid. Luciferase activity was measured and normalized to protein content. An arbitrary value of 1 was assigned to the activity of cells transfected with pG5Luc reporter plasmid and empty vectors for GAL4 and VP16. Values represent the means ± S.E. (n = 3). RLU, relative luciferase units.