title: Integrin mediated adhesion of osteoblasts to connective tissue growth factor (CTGF/CCN2) induces cytoskeleton reorganization and cell differentiation.
abstract: 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.
authors.affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Honey
authors.initials: H
authors.lastname: Hendesi
authors.affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Barbe
authors.affiliation: Department of Anatomy and Neurobiology, Northeast Ohio Medical University (NEOMED), Rootstown, Ohio, United States of America.
authors.forename: Fayez F
authors.initials: FF
authors.lastname: Safadi
authors.affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America; Department of Surgery, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: M Alexandra
authors.initials: MA
authors.lastname: Monroy
authors.affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America; Department of Orthopedic Surgery, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Steven N
authors.initials: SN
authors.lastname: Popoff
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000737:chemistry / Q000378:metabolism*; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D019030:Receptors, Vitronectin / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D016023:Integrins; D019030:Receptors, Vitronectin; C094340:integrin alphavbeta1; D055513:Connective Tissue Growth Factor; D051416:Focal Adhesion Protein-Tyrosine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020744:rac GTP-Binding Proteins