KLF5介导高磷诱导的Runx2表达和血管平滑肌细胞成骨样分化

发布时间：2018-04-26 20:28

本文选题：KLF5 + Runx2　；参考：《河北医科大学》2014年博士论文

【摘要】：血管钙化是动脉粥样硬化、糖尿病、高血压、慢性肾脏病的主要并发症。血管钙化导致血管弹性和血管顺应性降低，使血管疾病突发事件增加，如心肌梗死，动脉粥样硬化斑块破裂等。很多因素，如矿物质代谢紊乱，慢性炎症，氧化应激等等和血管钙化的形成有关。越来越多的证据表明，血管钙化并非简单的钙磷被动沉积，而是受到精细调控的类似骨的骨化过程。其中包括血管平滑肌细胞（vascular smooth cell, VSMC）向成骨样细胞转化。体外和活体实验表明，在高磷刺激下，VSMC中骨相关蛋白，如碱性磷酸酶（alkaline phosphatase, ALP）、基质Gla蛋白（matrix Glaprotein, MGP）、骨桥蛋白（osteopontin, OPN），骨钙素（osteocalcin, OC）等表达增加，同时VSMC分化标志基因平滑肌肌动蛋白α（smooth muscleα-actin, SM α-actin）和平滑肌22α（smooth muscle22α, SM22α）表达减少。最近研究显示，在动脉粥样硬化患者的血管钙化组织和小鼠钙化的VSMC中检测到Runt相关转录因子2（Runt-related transcription factor2，Runx2，调节成骨和软骨分化的关键转录因子）的表达，而正常血管中没有检出。Runx2缺失能够抑制氧化应激诱导的VSMC标志基因的下调，降低成骨样细胞的形成。血管紧张素2通过激活Runx2和NF-кB加剧血管钙化，表明Runx2在血管钙化中具有关键作用。调控VSMC表型转变的转录因子也和血管钙化的发生发展有关。如高磷能够诱导Krüppel样因子4（Krüppel-like factor4，KLF4）的表达，KLF4敲低则可以减轻高磷诱导的VSMC向成骨样细胞转化，降低成骨标志基因的表达和钙沉积。KLF4在腺嘌呤诱导的尿毒症大鼠的钙化的主动脉中表达量也增加。我们课题组之前的研究发现，血管紧张素II（angiotensin II,Ang II）通过激活Krüppel样因子5（Krüppel-like factor5，KLF5）（促增殖转录因子）抑制p21的表达，促进VSMC向合成型细胞转化。KLF4和KLF5是KLF家族中紧密相关的转录因子，分别从正向和反向调节细胞增殖。然而，KLF5在VSMC钙化中是否发挥作用还不清楚。此外，KLF5和Runx2介导的VSMC钙化之间的内在关系也有待探讨。本文的目的是阐明KLF5在VSMC钙化中的作用，明确KLF5和Runx2在调制VSMC钙化中的相互关系。第一部分高磷促进血管平滑肌细胞的成骨样分化和KLF5的表达目的：探讨高磷诱导VSMC钙化与KLF5表达的关系。方法：以3.8mM无机磷处理大鼠VSMC，建立钙化模型。通过茜素红染色发现钙化结节、ALP活性升高、钙离子含量测定以确定VSMC成骨样转化。以real-time PCR和Western blotting检测KLF5、成骨标志基因Runx2、VSMC标志基因SM α-actin和SM22α表达。应用离体血管环验证高磷诱导的VSMC钙化及各基因的表达变化。结果：茜素红染色显示，VSMC在3.8mM无机磷诱导下，出现明显的钙化结节，碱性磷酸酶活性增强，钙离子含量增加，提示大鼠VSMC发生钙化。血管环实验Von kossa染色显示，钙化组出现明显的黑色钙沉积。细胞和离体血管环中KLF5、Runx2mRNA和蛋白均随钙化程度增加表达上升，SM α-actin和SM22α随钙化程度增加表达下降。小结：在高磷诱导下，VSMC向成骨样细胞转化，在VSMC向成骨样细胞转化和钙化过程中伴随着KLF5和Runx2表达上调，二者表达活性与VSMC钙化程度呈正相关。第二部分KLF5在血管平滑肌细胞成骨样分化中的作用及机制目的：探寻KLF5在高磷诱导VSMC向成骨样细胞分化过程中的具体机制。方法：分别应用腺病毒过表达KLF5及靶向KLF5的特异性siRNA敲低KLF5，用茜素红染色和钙离子含量测定检测VSMC中钙盐沉积程度，real-time PCR和Western blotting检测KLF5、Runx2、SM α-actin和SM22α表达。构建Runx2启动子指导的荧光素酶报告基因质粒，与KLF5表达质粒共转染血管平滑肌细胞，以荧光素酶报告基因检查KLF5对Runx2的调控作用。染色质免疫沉淀（chromatin immunoprecipitation, ChIP）分析和oligo pulldown方法分析KLF5在Runx2启动子上的结合位点。结果：钙离子浓度测定和茜素红染色发现，单纯过表达KLF5并不会使VSMC发生钙化。然而当给予VSMC高磷刺激时，与转染空病毒组相比，过表达KLF5组钙沉积是转染空病毒组的1.56倍，同时矿化结节的形成显著增加。高磷刺激后，Runx2表达量在转染空病毒组和过表达KLF5组分别增加1.2和2.3倍。给于高磷刺激后，SM α-actin和SM22α的表达活性在过表达KLF5组较转染空病毒的对照组分别下降40%和30%。离体动脉环实验得到相似的结果。与转染对照siRNA组相比，KLF5敲低之后，给予高磷刺激，VSMC中的钙沉积下降30%，矿化结节显著减少。Runx2表达活性下降了70%，SM α-actin和SM22α的表达水平较对照组明显增加。报告基因分析显示，不给于高磷刺激时，KLF5对Runx2基因启动子的活性无明显影响，高磷促进KLF5结合到Runx2启动子区并诱导其转录。ChIP分析结果显示，在高磷处理的VSMC中， KLF5可结合到Runx2启动子区的－185bp—－27bp，这个区段包含4个KLF5结合位点；oligopulldown分析结果显示，高磷促进KLF5与Runx2启动子区近端两个KLF5结合位点结合的作用大于远端的两个位点。小结：KLF5通过直接与Runx2启动子结合激活其转录，高磷通过促进KLF5与Runx2启动子的结合，进而发挥其促进VSMC向成骨细胞转化。第三部分KLF5在慢性肾衰竭血管钙化中的作用目的：进一步探讨KLF5在整体动物中调控慢性肾衰竭血管钙化中的作用及机制。方法：用含大鼠0.75%腺嘌呤的饲料饲喂大鼠，生化分析仪测定肾衰竭相关参数，超声心动图评估心脏功能；高分辨率超声、Von kossa染色和钙离子测定检测慢性肾衰竭大鼠主动脉的钙化情况；免疫组化方法检测KLF5、Runx2和SM α-actin在钙化动脉中的表达；Real-time PCR和Westernblotting检测KLF5和Runx2在主动脉中的表达结果：腺嘌呤成功诱导大鼠出现肾衰竭，肌酐、尿素氮、血磷和钙磷乘积增加；同时合并有心功能衰竭。超声显像和Von kossa染色显示主动脉出现明显的钙化，，钙化区域钙离子含量增加，并且血磷水平与血管钙化呈正相关；钙化区域KLF5和Runx2表达量增加， SM α-actin表达量降低，与细胞水平的结果相一致。小结：腺嘌呤饮食诱导的肾衰模型接近尿毒症血管钙化的发病机理，可作为研究慢性肾衰竭血管钙化的模型，在主动脉钙化区域的血管组织中，KLF5和Runx2同时高表达，提示在整体动物中KLF5表达上调与血管钙化相关。结论： 1.高磷诱导VSMC钙化和KLF5表达。 2. KLF5通过直接与Runx2启动子结合激活其转录，高磷通过促进KLF5与Runx2启动子的结合，进而发挥其促进VSMC向成骨样细胞转化的作用。 3.慢性肾衰竭大鼠钙化血管中KLF5表达增加，VSMC向成骨样细胞转化。 4.血管钙化伴随着VSMC表型转化，其表型转化与钙化的耦联是通过KLF5对Runx2启动子的反式激活而实现的。
[Abstract]:Vascular calcification is a major complication of atherosclerosis , diabetes , hypertension , and chronic renal disease . The vascular calcification results in decreased vascular elasticity and vascular compliance . There are many factors , such as myocardial infarction , atherosclerotic plaque rupture , etc .

In recent studies , the expression of Runt - related transcription factor 2 ( Runt - related transcription factor2 , Runx2 , the key transcription factor regulating the differentiation of bone and cartilage ) was detected in vascular calcification and calcification in atherosclerotic patients .

KLF4 and KLF5 are closely related transcription factors in KLF4 . KLF4 and KLF5 are closely related transcription factors in KLF4 .

The first part of high phosphorus promotes the osteoblast - like differentiation of vascular smooth muscle cells and the expression of KLF5

Objective : To investigate the relationship between high phosphorus - induced calcification and KLF5 expression .

Methods : The rats were treated with 3.8 mM inorganic phosphorus to establish a calcification model . The calcification nodules , ALP activity and calcium ion content were determined by alizarin red staining . The expression of KLF5 , KLF5 and SM22伪 were detected by real - time PCR and Western blotting .

Results : Alizarin red staining showed that , under the induction of 3.8 mM inorganic phosphorus , there appeared obvious calcification nodules , increased alkaline phosphatase activity and increased calcium ion content , which suggested that the calcium ion content increased and the calcium ion content increased . The expression of KLF5 , Runx2 mRNA and protein in calcification group increased with the degree of calcification . The expression of SM 伪 - actin and SM22伪 decreased with the degree of calcification .

Conclusion : The expression of KLF5 and Runx2 is up - regulated in the process of transforming and calcification into bone - like cells . Both of them are positively correlated with the degree of calcification .

The Role and Mechanism of the Second Part KLF5 in Osteoblast - like Differentiation of Vascular Smooth Muscle Cells

Objective : To explore the specific mechanism of KLF5 induced by KLF5 in the differentiation of bone - like cells .

Methods : The expression of KLF5 , Runx2 , SM 伪 - actin and SM22伪 were detected by using the specific siRNA targeting KLF5 and KLF5 respectively . The luciferase reporter plasmid was constructed by PCR and Western blotting . The luciferase reporter plasmid was co - transfected with KLF5 expression plasmid , and the regulatory action of KLF5 on Runx2 was examined by luciferase reporter gene . The binding sites of KLF5 on the Runx2 promoter were analyzed by chromatin immunoprecipitation ( ChIP ) analysis and oligo - assay .

Results : The calcium ion concentration and alizarin red staining showed that the simple overexpression of KLF5 did not lead to calcification . However , after high phosphorus stimulation , the expression of SM 伪 - actin and SM22伪 increased by 1.2 and 2.3 times , respectively . After high phosphorus stimulation , the expression of SM 伪 - actin and SM22伪 decreased by 40 % and 30 % respectively after high P stimulation .

Compared with control siRNA group transfected with KLF5 , after KLF5 was low , calcium deposition decreased by 30 % and mineralized nodules decreased significantly after KLF5 was knocked down . The expression of Runx2 decreased by 70 % , and the expression level of SM 伪 - actin and SM22伪 increased significantly compared with control group .

The results showed that KLF5 could bind to the promoter region of Runx2 and induce its transcription . The results of ChIP analysis showed that KLF5 could bind to - 185bp - -27bp in the promoter region of Runx2 , and the segment contained 4 KLF5 binding sites .
The results showed that the combination of KLF5 and KLF5 binding sites at the proximal end of the promoter region of Runx2 promoter region was greater than that at the distal end of KLF5 promoter region .

Summary : KLF5 activates transcription of KLF5 directly in combination with the Runx2 promoter to promote the binding of KLF5 to the Runx2 promoter , which in turn plays its role in promoting the transformation of the VV5 to the osteoblast .

Role of the third part KLF5 in vascular calcification of chronic renal failure

Objective : To investigate the role and mechanism of KLF5 in the regulation of vascular calcification of chronic renal failure .

Methods : Rats were fed with 0.75 % adenine in rats . The parameters of renal failure were measured by biochemistry analyzer , and the function of heart was evaluated by echocardiography .
High - resolution ultrasound , Von kossa staining and calcium ion assay were used to detect the calcification of aorta in rats with chronic renal failure .
Immunohistochemical method was used to detect the expression of KLF5 , Runx2 and SM 伪 - actin in calcified arteries .
Real - time PCR and Western blotting were used to detect the expression of KLF5 and Runx2 in aorta .
At the same time , heart failure was combined . Ultrasound imaging and Von kossa staining showed significant calcification in the aorta , the calcium ion content in calcified area increased , and the level of blood phosphorus was positively correlated with calcification of the vessel ;
The expression of KLF5 and Runx2 in calcified areas increased , and the expression of SM 伪 - actin decreased , which was consistent with the result of cell level .

Summary : The renal failure model induced by adenine diet is close to the pathogenesis of vascular calcification in uremia , and can be used as a model to study the vascular calcification of chronic renal failure . In the vascular tissue of the aortic calcification area , KLF5 and Runx2 are expressed simultaneously , suggesting that the up - regulation of KLF5 expression in the whole animal is related to calcification .

Conclusion :

1 . High phosphorus - induced calcification and KLF5 expression .

2 . KLF5 can activate the transcription of KLF5 and Runx2 promoter by directly binding to the promoter of Runx2 promoter .

3 . The expression of KLF5 in calcified vessels was increased in rats with chronic renal failure .

4 . The phenotypic transformation of vascular calcification is associated with the phenotypic transformation , and the coupling of phenotypic transformation and calcification is achieved by transactivation of the Runx2 promoter via KLF5 .

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.5

【共引文献】