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

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...

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Main Authors: Hendesi, Honey, Barbe, Mary F., Safadi, Fayez F., Monroy, M. Alexandra, Popoff, Steven N.
Format: Online
Language:English
Published: Public Library of Science 2015
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340870/
id pubmed-4340870
recordtype oai_dc
spelling pubmed-43408702015-03-04 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. Research Article 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. Public Library of Science 2015-02-25 /pmc/articles/PMC4340870/ /pubmed/25714841 http://dx.doi.org/10.1371/journal.pone.0115325 Text en © 2015 Hendesi et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Hendesi, Honey
Barbe, Mary F.
Safadi, Fayez F.
Monroy, M. Alexandra
Popoff, Steven N.
spellingShingle Hendesi, Honey
Barbe, Mary F.
Safadi, Fayez F.
Monroy, M. Alexandra
Popoff, Steven N.
Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation
author_facet Hendesi, Honey
Barbe, Mary F.
Safadi, Fayez F.
Monroy, M. Alexandra
Popoff, Steven N.
author_sort Hendesi, Honey
title Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation
title_short Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation
title_full Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation
title_fullStr Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation
title_full_unstemmed Integrin Mediated Adhesion of Osteoblasts to Connective Tissue Growth Factor (CTGF/CCN2) Induces Cytoskeleton Reorganization and Cell Differentiation
title_sort integrin mediated adhesion of osteoblasts to connective tissue growth factor (ctgf/ccn2) induces cytoskeleton reorganization and cell differentiation
description 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.
publisher Public Library of Science
publishDate 2015
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340870/
_version_ 1613192659109675008

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