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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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Osteoblast adhesion to CTGF is mediated by αvβ1 integrin receptor.(A) Adhesion assay of osteoblasts treated with different blocking integrin antibodies prior to culture on CTGF or BSA (negative control) coated wells. The number of adhered cells in each treatment group was compared to the adhesion level of untreated cells. Adhesion to CTGF matrix after treatment with β3 or β5 integrin antibodies was not significantly different from untreated cells. (B) Western blot analyzing CTGF levels following immunoprecipitation of different integrin heterodimers from osteoblast cell lysates and control blot analyzing β1 integrin levels to confirm that comparable amounts of integrin heterodimers were pulled down during the immunoprecipitation using their respective antibodies. IgG was used as negative control for integrin antibodies. (C) Immunofluorescence staining of osteoblasts cultured on CTGF coated slides for 24 hours at 37°C. Cells stained for αvβ1 (green), F-actin (red in left panel), or vinculin (red in right panel). Lower panel: Scale bar = 50 μm. Upper panel: Scale bar = 10 μm. n = 6, *p<0.05; **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.
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pone.0115325.g002: Osteoblast adhesion to CTGF is mediated by αvβ1 integrin receptor.(A) Adhesion assay of osteoblasts treated with different blocking integrin antibodies prior to culture on CTGF or BSA (negative control) coated wells. The number of adhered cells in each treatment group was compared to the adhesion level of untreated cells. Adhesion to CTGF matrix after treatment with β3 or β5 integrin antibodies was not significantly different from untreated cells. (B) Western blot analyzing CTGF levels following immunoprecipitation of different integrin heterodimers from osteoblast cell lysates and control blot analyzing β1 integrin levels to confirm that comparable amounts of integrin heterodimers were pulled down during the immunoprecipitation using their respective antibodies. IgG was used as negative control for integrin antibodies. (C) Immunofluorescence staining of osteoblasts cultured on CTGF coated slides for 24 hours at 37°C. Cells stained for αvβ1 (green), F-actin (red in left panel), or vinculin (red in right panel). Lower panel: Scale bar = 50 μm. Upper panel: Scale bar = 10 μm. n = 6, *p<0.05; **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.

Mentions: To determine which integrin mediates osteoblast adhesion to CTGF, we used blocking antibodies directed against the six primary integrin monomer subunits expressed on osteoblasts. Cells were incubated with 20 μg/ml of the appropriate blocking antibody for 30 minutes at 37°C prior to performing the adhesion assay. We observed a significant decrease in osteoblast adhesion to CTGF when αv, α2, α5, or β1 integrins were blocked, while blocking β3 or β5 integrins did not have any effect on osteoblast adhesion (Fig. 2A). To further examine the specificity of the CTGF-integrin interaction in osteoblasts, we immunoprecipitated different integrin heterodimers (αvβ1, α2β1, α5β1) from osteoblast cell lysates and examined CTGF levels in these integrin pull downs by Western blotting. These analyses confirmed that the most significant interaction occurs between the αvβ1 integrin and CTGF (Fig. 2B). In addition, we conducted flow cytometry which demonstrated highest levels of expression of the αv and β1 integrin subunits compared to the other α and β subunits (S1 Fig.). Immunofluorescent staining of osteoblasts cultured on recombinant CTGF demonstrated actin stress fibers, clustering of αvβ1 integrin receptors and vinculin staining in the areas of focal adhesions (Fig. 2C). The actin stress fibers converged on the sites of focal adhesions, and αvβ1 integrin and vinculin staining was co-localized in these areas (Fig. 2C, insets). Collectively, these data suggest that αvβ1 is the primary integrin involved in osteoblast adhesion to the CTGF matrix, but they do not preclude that α2β1 and α5β1 may also play a role, albeit to a lesser extent, in this process.


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)

Osteoblast adhesion to CTGF is mediated by αvβ1 integrin receptor.(A) Adhesion assay of osteoblasts treated with different blocking integrin antibodies prior to culture on CTGF or BSA (negative control) coated wells. The number of adhered cells in each treatment group was compared to the adhesion level of untreated cells. Adhesion to CTGF matrix after treatment with β3 or β5 integrin antibodies was not significantly different from untreated cells. (B) Western blot analyzing CTGF levels following immunoprecipitation of different integrin heterodimers from osteoblast cell lysates and control blot analyzing β1 integrin levels to confirm that comparable amounts of integrin heterodimers were pulled down during the immunoprecipitation using their respective antibodies. IgG was used as negative control for integrin antibodies. (C) Immunofluorescence staining of osteoblasts cultured on CTGF coated slides for 24 hours at 37°C. Cells stained for αvβ1 (green), F-actin (red in left panel), or vinculin (red in right panel). Lower panel: Scale bar = 50 μm. Upper panel: Scale bar = 10 μm. n = 6, *p<0.05; **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.
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pone.0115325.g002: Osteoblast adhesion to CTGF is mediated by αvβ1 integrin receptor.(A) Adhesion assay of osteoblasts treated with different blocking integrin antibodies prior to culture on CTGF or BSA (negative control) coated wells. The number of adhered cells in each treatment group was compared to the adhesion level of untreated cells. Adhesion to CTGF matrix after treatment with β3 or β5 integrin antibodies was not significantly different from untreated cells. (B) Western blot analyzing CTGF levels following immunoprecipitation of different integrin heterodimers from osteoblast cell lysates and control blot analyzing β1 integrin levels to confirm that comparable amounts of integrin heterodimers were pulled down during the immunoprecipitation using their respective antibodies. IgG was used as negative control for integrin antibodies. (C) Immunofluorescence staining of osteoblasts cultured on CTGF coated slides for 24 hours at 37°C. Cells stained for αvβ1 (green), F-actin (red in left panel), or vinculin (red in right panel). Lower panel: Scale bar = 50 μm. Upper panel: Scale bar = 10 μm. n = 6, *p<0.05; **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.
Mentions: To determine which integrin mediates osteoblast adhesion to CTGF, we used blocking antibodies directed against the six primary integrin monomer subunits expressed on osteoblasts. Cells were incubated with 20 μg/ml of the appropriate blocking antibody for 30 minutes at 37°C prior to performing the adhesion assay. We observed a significant decrease in osteoblast adhesion to CTGF when αv, α2, α5, or β1 integrins were blocked, while blocking β3 or β5 integrins did not have any effect on osteoblast adhesion (Fig. 2A). To further examine the specificity of the CTGF-integrin interaction in osteoblasts, we immunoprecipitated different integrin heterodimers (αvβ1, α2β1, α5β1) from osteoblast cell lysates and examined CTGF levels in these integrin pull downs by Western blotting. These analyses confirmed that the most significant interaction occurs between the αvβ1 integrin and CTGF (Fig. 2B). In addition, we conducted flow cytometry which demonstrated highest levels of expression of the αv and β1 integrin subunits compared to the other α and β subunits (S1 Fig.). Immunofluorescent staining of osteoblasts cultured on recombinant CTGF demonstrated actin stress fibers, clustering of αvβ1 integrin receptors and vinculin staining in the areas of focal adhesions (Fig. 2C). The actin stress fibers converged on the sites of focal adhesions, and αvβ1 integrin and vinculin staining was co-localized in these areas (Fig. 2C, insets). Collectively, these data suggest that αvβ1 is the primary integrin involved in osteoblast adhesion to the CTGF matrix, but they do not preclude that α2β1 and α5β1 may also play a role, albeit to a lesser extent, in this process.

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