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Vascular calcification is coupled with phenotypic conversion of vascular smooth muscle cells through Klf5-mediated transactivation of the Runx2 promoter.

Zhang J, Zheng B, Zhou PP, Zhang RN, He M, Yang Z, Wen JK - Biosci. Rep. (2014)

Bottom Line: We found that high Pi (phosphate) increased the expression of Klf5, which is accompanied by loss of SM α-actin and SM22α (smooth muscle 22 α), as well as gain of Runx2 expression.Klf5 was also induced markedly in the calcified aorta of adenine-induced uremic rats.In conclusion, we demonstrate a critical role for Klf5-mediated induction of Runx2 in high Pi -induced VSMC calcification.

View Article: PubMed Central - PubMed

Affiliation: *Department of Biochemistry and Molecular Biology, the Key Laboratory of Neural and Vascular Biology, China Administration of Education, Hebei Medical University, No. 361, Zhongshan East Road, Shijiazhuang 050017, China.

ABSTRACT
Both Klf5 (Krüppel-like factor 5) and Runx2 are involved in phenotypic switching of VSMC (vascular smooth muscle cells). However, the potential link between Klf5 and Runx2 in mediating vascular calcification remains unclear. The aim of the present study was to elucidate the actual relationship between Klf5 and Runx2 in mediating VSMC calcification. We found that high Pi (phosphate) increased the expression of Klf5, which is accompanied by loss of SM α-actin and SM22α (smooth muscle 22 α), as well as gain of Runx2 expression. Overexpression of Klf5 increased, while knockdown of Klf5 decreased, Runx2 expression and calcification. Further study showed that Klf5 bound directly to the Runx2 promoter and activated its transcription. Klf5 was also induced markedly in the calcified aorta of adenine-induced uremic rats. In conclusion, we demonstrate a critical role for Klf5-mediated induction of Runx2 in high Pi -induced VSMC calcification.

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Related in: MedlinePlus

High Pi-induced Klf5 expression and VSMC calcification(A) Rat aortic VSMCs were cultured with control or high Pi (3.8 mM) medium for 12 days. Representative pictures of Alizarin red S staining are shown (n=3). Scale bar, 20 μm. (B) Quantitative analysis of calcium deposition. VSMCs cultured in control or high Pi medium for 12 days. VSMCs were lysed with 0.6N HCl overnight. Total calcium in the lysates was determined by O-cresolphthalein method and normalized by protein concentration. Values are expressed as means±S.E.M. from three independent experiments. *P<0.05 compared with VSMCs with control medium (n=3). (C) VSMCs were incubated with control or high Pi medium for 6 days. Expression of Klf5, Runx2, SM α-actin and SM22α mRNA was determined by real-time PCR. Values represent the means±S.E.M.. *P<0.05 compared with VSMCs incubated with control medium (n=3). (D) Expression of Runx2, Klf5, SM α-actin and SM22α in VSMCs incubated with control or high Pi medium for 4, 8 and 12 days was examined by Western blotting.
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Figure 1: High Pi-induced Klf5 expression and VSMC calcification(A) Rat aortic VSMCs were cultured with control or high Pi (3.8 mM) medium for 12 days. Representative pictures of Alizarin red S staining are shown (n=3). Scale bar, 20 μm. (B) Quantitative analysis of calcium deposition. VSMCs cultured in control or high Pi medium for 12 days. VSMCs were lysed with 0.6N HCl overnight. Total calcium in the lysates was determined by O-cresolphthalein method and normalized by protein concentration. Values are expressed as means±S.E.M. from three independent experiments. *P<0.05 compared with VSMCs with control medium (n=3). (C) VSMCs were incubated with control or high Pi medium for 6 days. Expression of Klf5, Runx2, SM α-actin and SM22α mRNA was determined by real-time PCR. Values represent the means±S.E.M.. *P<0.05 compared with VSMCs incubated with control medium (n=3). (D) Expression of Runx2, Klf5, SM α-actin and SM22α in VSMCs incubated with control or high Pi medium for 4, 8 and 12 days was examined by Western blotting.

Mentions: Previous studies have shown that the high Pi concentration induced VSMC calcification through increasing Runx2 expression [12]. To investigate the molecular mechanisms whereby high Pi-induced Runx2 expression, we first examined the effect of high Pi on pro-proliferation factor Klf5. We cultured VSMCs for 12 days in control or high (3.8 mM) Pi DMEM with 10% FBS, and the calcification status was determined by Alizarin red S staining. As shown in Figure 1(A), incubation of VSMCs with high Pi medium markedly increased calcification of cultured VSMCs, and total calcium in the cell lysates was enhanced ~100-fold (Figure 1B). Expression of VSMC differentiation- and proliferation-related genes as well as osteogenic gene was examined by real-time PCR and Western blotting. The results showed that incubation with the high Pi medium for 6 days increased Klf5 and Runx2 mRNA expression by 1.0-fold and 1.76-fold, respectively. In contrast, expression of VSMC differentiation marker genes, SM22α and SM α-actin, was significantly decreased by 43 or 38% with high Pi treatment (Figure 1C). Similarly, the protein levels of SM α-actin and SM22α were reduced in a time-dependent manner in VSMCs cultured with high Pi medium for 4, 8 and 12 days. Conversely, the levels of Runx2 and Klf5 time-dependently increased. These results indicate that high Pi promotes the expression of osteogenic gene Runx2 and VSMC calcification, and that Klf5 may play an important role in mediating Runx2 expression in VSMCs.


Vascular calcification is coupled with phenotypic conversion of vascular smooth muscle cells through Klf5-mediated transactivation of the Runx2 promoter.

Zhang J, Zheng B, Zhou PP, Zhang RN, He M, Yang Z, Wen JK - Biosci. Rep. (2014)

High Pi-induced Klf5 expression and VSMC calcification(A) Rat aortic VSMCs were cultured with control or high Pi (3.8 mM) medium for 12 days. Representative pictures of Alizarin red S staining are shown (n=3). Scale bar, 20 μm. (B) Quantitative analysis of calcium deposition. VSMCs cultured in control or high Pi medium for 12 days. VSMCs were lysed with 0.6N HCl overnight. Total calcium in the lysates was determined by O-cresolphthalein method and normalized by protein concentration. Values are expressed as means±S.E.M. from three independent experiments. *P<0.05 compared with VSMCs with control medium (n=3). (C) VSMCs were incubated with control or high Pi medium for 6 days. Expression of Klf5, Runx2, SM α-actin and SM22α mRNA was determined by real-time PCR. Values represent the means±S.E.M.. *P<0.05 compared with VSMCs incubated with control medium (n=3). (D) Expression of Runx2, Klf5, SM α-actin and SM22α in VSMCs incubated with control or high Pi medium for 4, 8 and 12 days was examined by Western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219426&req=5

Figure 1: High Pi-induced Klf5 expression and VSMC calcification(A) Rat aortic VSMCs were cultured with control or high Pi (3.8 mM) medium for 12 days. Representative pictures of Alizarin red S staining are shown (n=3). Scale bar, 20 μm. (B) Quantitative analysis of calcium deposition. VSMCs cultured in control or high Pi medium for 12 days. VSMCs were lysed with 0.6N HCl overnight. Total calcium in the lysates was determined by O-cresolphthalein method and normalized by protein concentration. Values are expressed as means±S.E.M. from three independent experiments. *P<0.05 compared with VSMCs with control medium (n=3). (C) VSMCs were incubated with control or high Pi medium for 6 days. Expression of Klf5, Runx2, SM α-actin and SM22α mRNA was determined by real-time PCR. Values represent the means±S.E.M.. *P<0.05 compared with VSMCs incubated with control medium (n=3). (D) Expression of Runx2, Klf5, SM α-actin and SM22α in VSMCs incubated with control or high Pi medium for 4, 8 and 12 days was examined by Western blotting.
Mentions: Previous studies have shown that the high Pi concentration induced VSMC calcification through increasing Runx2 expression [12]. To investigate the molecular mechanisms whereby high Pi-induced Runx2 expression, we first examined the effect of high Pi on pro-proliferation factor Klf5. We cultured VSMCs for 12 days in control or high (3.8 mM) Pi DMEM with 10% FBS, and the calcification status was determined by Alizarin red S staining. As shown in Figure 1(A), incubation of VSMCs with high Pi medium markedly increased calcification of cultured VSMCs, and total calcium in the cell lysates was enhanced ~100-fold (Figure 1B). Expression of VSMC differentiation- and proliferation-related genes as well as osteogenic gene was examined by real-time PCR and Western blotting. The results showed that incubation with the high Pi medium for 6 days increased Klf5 and Runx2 mRNA expression by 1.0-fold and 1.76-fold, respectively. In contrast, expression of VSMC differentiation marker genes, SM22α and SM α-actin, was significantly decreased by 43 or 38% with high Pi treatment (Figure 1C). Similarly, the protein levels of SM α-actin and SM22α were reduced in a time-dependent manner in VSMCs cultured with high Pi medium for 4, 8 and 12 days. Conversely, the levels of Runx2 and Klf5 time-dependently increased. These results indicate that high Pi promotes the expression of osteogenic gene Runx2 and VSMC calcification, and that Klf5 may play an important role in mediating Runx2 expression in VSMCs.

Bottom Line: We found that high Pi (phosphate) increased the expression of Klf5, which is accompanied by loss of SM α-actin and SM22α (smooth muscle 22 α), as well as gain of Runx2 expression.Klf5 was also induced markedly in the calcified aorta of adenine-induced uremic rats.In conclusion, we demonstrate a critical role for Klf5-mediated induction of Runx2 in high Pi -induced VSMC calcification.

View Article: PubMed Central - PubMed

Affiliation: *Department of Biochemistry and Molecular Biology, the Key Laboratory of Neural and Vascular Biology, China Administration of Education, Hebei Medical University, No. 361, Zhongshan East Road, Shijiazhuang 050017, China.

ABSTRACT
Both Klf5 (Krüppel-like factor 5) and Runx2 are involved in phenotypic switching of VSMC (vascular smooth muscle cells). However, the potential link between Klf5 and Runx2 in mediating vascular calcification remains unclear. The aim of the present study was to elucidate the actual relationship between Klf5 and Runx2 in mediating VSMC calcification. We found that high Pi (phosphate) increased the expression of Klf5, which is accompanied by loss of SM α-actin and SM22α (smooth muscle 22 α), as well as gain of Runx2 expression. Overexpression of Klf5 increased, while knockdown of Klf5 decreased, Runx2 expression and calcification. Further study showed that Klf5 bound directly to the Runx2 promoter and activated its transcription. Klf5 was also induced markedly in the calcified aorta of adenine-induced uremic rats. In conclusion, we demonstrate a critical role for Klf5-mediated induction of Runx2 in high Pi -induced VSMC calcification.

Show MeSH
Related in: MedlinePlus