Klotho基因超甲基化在硫酸吲哚酚诱导的血管平滑肌细胞成骨转化中的作用机制

Effect mechanism of Klotho gene hypermethylation in vascular SMC-osteoblast conversion induced by indoxyl sulfate

目的从细胞层面探讨Klotho基因超甲基化在硫酸吲哚酚(IS)诱导的血管平滑肌细胞成骨转化中的作用和相关机制。方法体外培养人主动脉血管平滑肌细胞(HASMC),以IS按0、200、500、1 000μmol/L的浓度梯度干预6 d,采用茜素红染色观察钙盐沉积,Real-time聚合酶链式反应(PCR)和Western印迹检测平滑肌细胞肌动蛋白α(α-SMA)、smoothin、骨桥蛋白(OPN)、碱性磷酸酶(ALP)、核结合因子α1(Cbfα1)、Klotho和不同DNA甲基化转移酶(DNMT)的m RNA和蛋白表达水平,同时利用焦磷酸测序方法检测HASMC的Klotho基因甲基化率;在IS 1 000μmol/L组培养基中加入不同浓度梯度的5-氮杂脱氧胞苷(5Aza-2dc)进行干预,观察茜素红染色、各基因表达水平和Klotho基因甲基化率的改变。组间均数比较采用独立样本t检验,组间率的比较采用卡方检验。结果IS 1 000μmol/L组HASMC细胞外基质呈红色,细胞结节处浓染,为钙盐沉积染色阳性。IS可以下调HASMC的α-SMAm RNA和蛋白及smoothin表达水平,上调OPN、ALP的m RNA和蛋白表达水平,使HASMC逐渐丧失平滑肌细胞表型而发生成骨转化。与对照组比较,IS 200、500、1 000μmol/L刺激6 d可显著升高HASMC的Klotho基因甲基化率(9±1%与4±0.7%,t=5.38,P<0.01;18±1.3与4±0.7%,t=6.92,P<0.01;41±2%与4±0.7%,t=9.26,P<0.001)。IS 500、1 000μmol/L可显著下调HASMC的Klotho m RNA和蛋白表达水平。同时IS 1 000μmol/L可显著上调HASMC的DNMT1 m RNA和蛋白表达水平。5Aza-2dc 10μmol/L干预可减轻IS诱导的细胞外钙盐沉积,上调α-SMA蛋白、smoothin表达水平并下调Cbfα1蛋白表达水平,同时减低HASMC的Klotho基因甲基化率(20±1%与41±2%,t=6.98,P<0.01)、上调HASMC的Klotho蛋白表达水平。结论 IS可通过上调血管平滑肌细胞DNMT表达,启动Klotho基因超甲基化,进而下调Klotho基因转录和表达,诱导血管平滑肌细胞成骨转化。

Objective This study aimed to investigate the effect and mechanism of Klotho gene hypermethylation in vascular SMC-osteoblast conversion induced by indoxyl sulfate （IS）. Methods Human aortic vascular smooth muscle cells （HASMC） were cultured. IS concentrations of 0, 200, 500, 1 000 μmol/L were used for 6 days. Alizarin red staining was used for observation of calcium salt deposits. Real-time polymerase chain reaction （PCR） and Western blotting were applied to detect mRNA and protein expressions of α- smooth muscle actin （ α- SMA）, smoothin, osteopontin （ OPN）, alkaline phosphatase （ALP）, nuclear binding factor alpha 1 （Cbf cd ）, Klotho, and different DNA methylation transferases （DNMT）. Klotho gene methylation level in HASMC was assessed by pyrosequencing assay. In IS 1 000 μmol/L group, different concentrations of 5-Aza-2-deoxycytidine （SAza-2dc） were added into the culture medium for interference experiment. Mean comparison between groups was done with independent sample t-test, while rate comparison between groups was done with chi-square test. Results In IS 1 000 μmol/L group, extracellular matrix of HASMC was stained red, which was concentrated in cell nodules, showing positive calcium salt deposition. IS decreased the expression of α-SMA mRNA and protein as well as smoothin, and increased the mRNA and protein expression of OPN and ALP, making the HASMC to gradually lose the SMC phenotype and transfer to osteoblast. Stimulation of HASMC by IS at the concentration of 200,500, and 1 000 p,mol/L for 6 days significantly induced Klotho gene hypermethylation ratio compareal with the control group （9 ± 1% vs 4 ±0. 7%, t =5.38, P 〈0. 01 ; 18± 1.3 vs 4 ±0. 7%, t=6.92, P〈0.01; 41 ±2% vs4±0.7%, t=9.26, P〈0.001）. IS at the concentration of 500 and 1 000 μmol/L also significantly decreased Klotho mRNA and protein levels in HASMC. At the same time, IS obviously increased the expression of DNMT1 mRNA and protein level. 5Aza-2dc 10 μmol/L reduced the calcium deposition induced by IS, upregulated ot-SMA protein and smoothin expression, down-regulated Cbf cd protein level, simultaneously decreased the Klotho gene methylation ratio （20 ± 1% vs 41± 2%, t = 6. 98, P 〈 0.01 ）, and increased the protein level of Klotho in HASMC. Conclusion IS induced the vascular SMC-osteoblast conversion. Through upregulating of DNMT1 activity, IS induced Klotho gene hypermethylation, and down-regulated Klotho protein expression. Block of Klotho gene hypermethylation reduced the vascular SMC-osteoblast conversion induced by IS.