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The Role and Mechanism of α-Klotho in the Calcification of Rat Aortic Vascular Smooth Muscle Cells.

Chen T, Mao H, Chen C, Wu L, Wang N, Zhao X, Qian J, Xing C - Biomed Res Int (2015)

Bottom Line: The calcium content increased, and the expression of α-SMA decreased.Alizarin Red S staining was positive under the high phosphorus conditions.BMP2, Runx2, and β-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; Department of Nephrology, The First People's Hospital of Changzhou, The Third Affiliated Hospital of Soochow University, Changzhou 213000, China.

ABSTRACT

Objective: To investigate the role and possible mechanism of α-Klotho in the calcification and the osteogenic transition of cultured VSMCs.

Methods: VSMCs were cultured in vitro and divided into 5 groups, each using a different medium: (1) control; (2) β-GP; (3) β-GP + Klotho; (4) β-GP + LiCl; (5) β-GP + Klotho + LiCl. Calcium deposits were visualized using Alizarin Red S staining. The calcium concentrations were determined by the o-cresolphthalein complexone method. BMP2, Runx2 and β-catenin levels were estimated by western blotting, and the level of α-SMA was determined by using immunofluorescence at day 12.

Results: β-GP induced an increase in the expression of BMP2, Runx2, and β-catenin. The calcium content increased, and the expression of α-SMA decreased. Alizarin Red S staining was positive under the high phosphorus conditions. BMP2, Runx2, and β-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl.

Conclusions: Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced by β-GP. The mechanism of Klotho in preventing calcification in VSMCs may be partially mediated by the inhibition of the Wnt/β-catenin signaling pathway.

No MeSH data available.


Related in: MedlinePlus

(a) Representative BMP2, Runx2, and β-catenin expression levels from VSMCs following treatment with the control, β-GP, β-GP + LiCl, β-GP + rmKlotho, or β-GP + rmKlotho + LiCl media for 12 d. BMP2, Runx2, and β-catenin expression was analyzed by western blotting using GAPDH as the loading control. The quantification of the expression levels of (b) β-catenin, (c) Runx2, and (d) BMP2 is represented as the mean with the SE (n = 5 per group) for each group in its respective column. ∗P < 0.01 versus control group, #P < 0.01 versus β-GP group, and &P < 0.01 versus β-GP + Klotho group.
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fig4: (a) Representative BMP2, Runx2, and β-catenin expression levels from VSMCs following treatment with the control, β-GP, β-GP + LiCl, β-GP + rmKlotho, or β-GP + rmKlotho + LiCl media for 12 d. BMP2, Runx2, and β-catenin expression was analyzed by western blotting using GAPDH as the loading control. The quantification of the expression levels of (b) β-catenin, (c) Runx2, and (d) BMP2 is represented as the mean with the SE (n = 5 per group) for each group in its respective column. ∗P < 0.01 versus control group, #P < 0.01 versus β-GP group, and &P < 0.01 versus β-GP + Klotho group.

Mentions: To further investigate the role of Klotho in the osteoblastic differentiation of VSMCs, the expression levels of α-SMA, BMP2, and Runx2 were determined in the control and treated groups. The immunofluorescence intensity of α-SMA markedly decreased in the β-GP treatment group compared with the intensity of the control group; however, the intensity was enhanced following treatment with rmKlotho at 50 ng/mL (Figure 3). Compared with the control group, the levels of BMP2 and Runx2 increased significantly in the β-GP-treated group, while rmKlotho treatment markedly reduced the expression of these two proteins (Figure 4).


The Role and Mechanism of α-Klotho in the Calcification of Rat Aortic Vascular Smooth Muscle Cells.

Chen T, Mao H, Chen C, Wu L, Wang N, Zhao X, Qian J, Xing C - Biomed Res Int (2015)

(a) Representative BMP2, Runx2, and β-catenin expression levels from VSMCs following treatment with the control, β-GP, β-GP + LiCl, β-GP + rmKlotho, or β-GP + rmKlotho + LiCl media for 12 d. BMP2, Runx2, and β-catenin expression was analyzed by western blotting using GAPDH as the loading control. The quantification of the expression levels of (b) β-catenin, (c) Runx2, and (d) BMP2 is represented as the mean with the SE (n = 5 per group) for each group in its respective column. ∗P < 0.01 versus control group, #P < 0.01 versus β-GP group, and &P < 0.01 versus β-GP + Klotho group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644825&req=5

fig4: (a) Representative BMP2, Runx2, and β-catenin expression levels from VSMCs following treatment with the control, β-GP, β-GP + LiCl, β-GP + rmKlotho, or β-GP + rmKlotho + LiCl media for 12 d. BMP2, Runx2, and β-catenin expression was analyzed by western blotting using GAPDH as the loading control. The quantification of the expression levels of (b) β-catenin, (c) Runx2, and (d) BMP2 is represented as the mean with the SE (n = 5 per group) for each group in its respective column. ∗P < 0.01 versus control group, #P < 0.01 versus β-GP group, and &P < 0.01 versus β-GP + Klotho group.
Mentions: To further investigate the role of Klotho in the osteoblastic differentiation of VSMCs, the expression levels of α-SMA, BMP2, and Runx2 were determined in the control and treated groups. The immunofluorescence intensity of α-SMA markedly decreased in the β-GP treatment group compared with the intensity of the control group; however, the intensity was enhanced following treatment with rmKlotho at 50 ng/mL (Figure 3). Compared with the control group, the levels of BMP2 and Runx2 increased significantly in the β-GP-treated group, while rmKlotho treatment markedly reduced the expression of these two proteins (Figure 4).

Bottom Line: The calcium content increased, and the expression of α-SMA decreased.Alizarin Red S staining was positive under the high phosphorus conditions.BMP2, Runx2, and β-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; Department of Nephrology, The First People's Hospital of Changzhou, The Third Affiliated Hospital of Soochow University, Changzhou 213000, China.

ABSTRACT

Objective: To investigate the role and possible mechanism of α-Klotho in the calcification and the osteogenic transition of cultured VSMCs.

Methods: VSMCs were cultured in vitro and divided into 5 groups, each using a different medium: (1) control; (2) β-GP; (3) β-GP + Klotho; (4) β-GP + LiCl; (5) β-GP + Klotho + LiCl. Calcium deposits were visualized using Alizarin Red S staining. The calcium concentrations were determined by the o-cresolphthalein complexone method. BMP2, Runx2 and β-catenin levels were estimated by western blotting, and the level of α-SMA was determined by using immunofluorescence at day 12.

Results: β-GP induced an increase in the expression of BMP2, Runx2, and β-catenin. The calcium content increased, and the expression of α-SMA decreased. Alizarin Red S staining was positive under the high phosphorus conditions. BMP2, Runx2, and β-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl.

Conclusions: Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced by β-GP. The mechanism of Klotho in preventing calcification in VSMCs may be partially mediated by the inhibition of the Wnt/β-catenin signaling pathway.

No MeSH data available.


Related in: MedlinePlus