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Integrin mediated adhesion of osteoblasts to connective tissue growth factor (CTGF/CCN2) induces cytoskeleton reorganization and cell differentiation.

Hendesi H, Barbe MF, Safadi FF, Monroy MA, Popoff SN - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

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.

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Inhibition of ERK prevents osteogenic differentiation in cells cultured on CTGF matrix.Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 1% BSA (A) or 2 μg/ml CTGF coated plates in the absence of ERK inhibitor (B) and in the presence of ERK inhibitor (C) for 14 days. Scale bar = 2 mm. (D) ALP activity of osteoblasts cultured on BSA or CTGF coated plates, quantified at day 14 of culture and normalized to total protein content; n = 9 wells.
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pone.0115325.g006: Inhibition of ERK prevents osteogenic differentiation in cells cultured on CTGF matrix.Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 1% BSA (A) or 2 μg/ml CTGF coated plates in the absence of ERK inhibitor (B) and in the presence of ERK inhibitor (C) for 14 days. Scale bar = 2 mm. (D) ALP activity of osteoblasts cultured on BSA or CTGF coated plates, quantified at day 14 of culture and normalized to total protein content; n = 9 wells.

Mentions: To confirm that activation of the ERK pathway plays a central role in CTGF induced osteoblast differentiation, we performed additional experiments using cells cultured on CTGF-coated plates with and without the ERK inhibitor, U0126. In these experiments, we assessed ALP staining and activity after 14 days in culture using BSA-coated plates as our negative control. Inhibition of ERK completely blocked the increased ALP staining and activity observed when cells are cultured on CTGF-coated plates (Fig. 6).


Integrin mediated adhesion of osteoblasts to connective tissue growth factor (CTGF/CCN2) induces cytoskeleton reorganization and cell differentiation.

Hendesi H, Barbe MF, Safadi FF, Monroy MA, Popoff SN - PLoS ONE (2015)

Inhibition of ERK prevents osteogenic differentiation in cells cultured on CTGF matrix.Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 1% BSA (A) or 2 μg/ml CTGF coated plates in the absence of ERK inhibitor (B) and in the presence of ERK inhibitor (C) for 14 days. Scale bar = 2 mm. (D) ALP activity of osteoblasts cultured on BSA or CTGF coated plates, quantified at day 14 of culture and normalized to total protein content; n = 9 wells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4340870&req=5

pone.0115325.g006: Inhibition of ERK prevents osteogenic differentiation in cells cultured on CTGF matrix.Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 1% BSA (A) or 2 μg/ml CTGF coated plates in the absence of ERK inhibitor (B) and in the presence of ERK inhibitor (C) for 14 days. Scale bar = 2 mm. (D) ALP activity of osteoblasts cultured on BSA or CTGF coated plates, quantified at day 14 of culture and normalized to total protein content; n = 9 wells.
Mentions: To confirm that activation of the ERK pathway plays a central role in CTGF induced osteoblast differentiation, we performed additional experiments using cells cultured on CTGF-coated plates with and without the ERK inhibitor, U0126. In these experiments, we assessed ALP staining and activity after 14 days in culture using BSA-coated plates as our negative control. Inhibition of ERK completely blocked the increased ALP staining and activity observed when cells are cultured on CTGF-coated plates (Fig. 6).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

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.

Show MeSH
Related in: MedlinePlus