Integrin mediated adhesion of osteoblasts to connective tissue growth factor (CTGF/CCN2) induces cytoskeleton reorganization and cell differentiation.
Integrin mediated adhesion of osteoblasts to connective tissue growth factor (CTGF/CCN2) induces cytoskeleton reorganization and cell differentiation.
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DOI：
10.1371/journal.pone.0115325
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Popoff SN
中科院分区：
文献类型：
--
作者：
Hendesi H;Barbe MF;Safadi FF;Monroy MA;Popoff SN
前成骨细胞粘附和细胞外基质（ECM）蛋白通过整合素受体的相互作用，导致激活的信号通路调节成骨细胞分化。结缔组织生长因子（CTGF/CCN 2）是分泌到ECM中的基质细胞蛋白。先前在各种细胞类型中的研究已经表明，细胞通过整联蛋白受体粘附到CTGF导致调节细胞功能（例如分化和细胞骨架重组）的特定信号传导途径的激活。到目前为止，还没有研究已经检查了CTGF是否可以作为成骨细胞的粘附基质。在这项研究中，我们使用MC 3 T3-E1细胞系证明CTGF作为成骨细胞的粘附基质。抗整合素阻断实验和免疫共沉淀实验表明，整合素αvβ1在成骨细胞与CTGF基质的粘附中起关键作用。CTGF基质上培养的成骨细胞的免疫荧光染色证实肌动蛋白细胞骨架重组，增强扩散，形成局灶性粘连，并激活Rac 1。碱性磷酸酶（ALP）染色和活性测定，以及茜素红染色表明，成骨细胞的CTGF基质附着增强成熟，骨结节形成和基质矿化。为了研究CTGF对成骨细胞分化的影响是否涉及整合素介导的特定信号通路的激活，我们进行了Western印迹，染色质免疫沉淀（ChIP）和qPCR测定。在CTGF基质上培养的成骨细胞显示出粘着斑激酶（FAK）和细胞外信号调节激酶（ERK）的总量和磷酸化（活化）形式增加。抑制ERK阻断CTGF基质上培养的细胞的成骨分化。与骨钙素基因启动子结合的侏儒相关转录因子2（Runx 2）增加，Runx 2调控的成骨标志物表达增加。总的来说，这项研究的结果首次证明CTGF作为一种合适的基质蛋白，增强成骨细胞粘附（通过αvβ1整联蛋白），并通过细胞骨架重组和Rac 1激活促进细胞扩散。此外，整联蛋白介导的ERK信号转导激活导致成骨细胞分化增加，伴随着Runx 2结合骨钙素启动子和成骨标志物表达的增加。
Pre-osteoblast adhesion and interaction with extracellular matrix (ECM) proteins through integrin receptors result in activation of signaling pathways regulating osteoblast differentiation. Connective tissue growth factor (CTGF/CCN2) is a matricellular protein secreted into the ECM. Prior studies in various cell types have shown that cell adhesion to CTGF via integrin receptors results in activation of specific signaling pathways that regulate cell functions, such as differentiation and cytoskeletal reorganization. To date, there are no studies that have examined whether CTGF can serve as an adhesive substrate for osteoblasts. In this study, we used the MC3T3-E1 cell line to demonstrate that CTGF serves as an adhesive matrix for osteoblasts. Anti-integrin blocking experiments and co-immunoprecipitation assays demonstrated that the integrin αvβ1 plays a key role in osteoblast adhesion to a CTGF matrix. Immunofluorescence staining of osteoblasts cultured on a CTGF matrix confirmed actin cytoskeletal reorganization, enhanced spreading, formation of focal adhesions, and activation of Rac1. Alkaline phosphatase (ALP) staining and activity assays, as well as Alizarin red staining demonstrated that osteoblast attachment to CTGF matrix enhanced maturation, bone nodule formation and matrix mineralization. To investigate whether the effect of CTGF on osteoblast differentiation involves integrin-mediated activation of specific signaling pathways, we performed Western blot, chromatin immunoprecipitation (ChIP) and qPCR assays. Osteoblasts cultured on a CTGF matrix showed increased total and phosphorylated (activated) forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Inhibition of ERK blocked osteogenic differentiation in cells cultured on a CTGF matrix. There was an increase in runt-related transcription factor 2 (Runx2) binding to the osteocalcin gene promoter, and in the expression of osteogenic markers regulated by Runx2. Collectively, the results of this study are the first to demonstrate CTGF serves as a suitable matrix protein, enhancing osteoblast adhesion (via αvβ1 integrin) and promoting cell spreading via cytoskeletal reorganization and Rac1 activation. Furthermore, integrin-mediated activation of ERK signaling resulted in increased osteoblast differentiation accompanied by an increase in Runx2 binding to the osteocalcin promoter and in the expression of osteogenic markers.
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4.8
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影响因子：
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作者：
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通讯作者：
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影响因子：
3.5
作者：
Hoshijima, M;Hattori, T;Takigawa, M
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