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.

Hendesi Honey; Barbe Mary F; Safadi Fayez F; Monroy M Alexandra; Popoff Steven N

PloS one

2015

2015

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 alphavbeta1 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 alphavbeta1 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.

Animals; Mice; Signal Transduction; Cell Line; Cell Adhesion; *Cell Differentiation; Connective Tissue Growth Factor/chemistry/*metabolism; Core Binding Factor Alpha 1 Subunit/metabolism; Cytoskeleton/*metabolism; Extracellular Signal-Regulated MAP Kinases/metabolism; Focal Adhesion Protein-Tyrosine Kinases/metabolism; Integrins/*metabolism; Osteoblasts/*cytology/*metabolism; rac GTP-Binding Proteins/metabolism; Transcriptional Activation; Receptors; Vitronectin/metabolism

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e0115325–e0115325

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