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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 induces Runx2 transcriptional activation of osteogenic markers.(A) ChIP assay performed on osteoblasts cultured on CTGF coated, BSA coated or uncoated plates for 7 days while treated with osteogenic medium. Runx2 antibody or acetyl-Histone H4 antibody (positive control) used for chromatin immunoprecipitation. Quantitative PCR was performed using osteocalcin gene promoter primers. n = 3, ***p<0.001. ChIP assay repeated three times with similar results. (B) Quantitative PCR performed on osteoblasts cultured on CTGF or BSA coated plates for 7 days while treated with osteogenic media. OCN = osteocalcin, ALP = alkaline phosphatase. n = 3, **p<0.01; ***p<0.001. Experiments repeated three times with similar results.
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pone.0115325.g007: Osteoblast adhesion to CTGF induces Runx2 transcriptional activation of osteogenic markers.(A) ChIP assay performed on osteoblasts cultured on CTGF coated, BSA coated or uncoated plates for 7 days while treated with osteogenic medium. Runx2 antibody or acetyl-Histone H4 antibody (positive control) used for chromatin immunoprecipitation. Quantitative PCR was performed using osteocalcin gene promoter primers. n = 3, ***p<0.001. ChIP assay repeated three times with similar results. (B) Quantitative PCR performed on osteoblasts cultured on CTGF or BSA coated plates for 7 days while treated with osteogenic media. OCN = osteocalcin, ALP = alkaline phosphatase. n = 3, **p<0.01; ***p<0.001. Experiments repeated three times with similar results.

Mentions: Runx2 is a critical transcription factor for osteoblast differentiation that has been shown to regulate the transcription of other essential proteins such as osteocalcin [21,35]. To investigate if osteoblasts cultured on a CTGF matrix have an effect on Runx2 transcriptional activity, we performed chromatin immunoprecipitation (ChIP) assays to analyze Runx2 binding occupancy on the osteocalcin gene promoter (Fig. 7A). Cells cultured on CTGF for 7 days showed a significant increase in Runx2 binding occupancy compared to cells on BSA or uncoated plates (Fig. 7A). Next, we performed a quantitative PCR analysis to compare expression of osteogenic markers (Runx2, ALP and osteocalcin) in osteoblasts cultured on CTGF matrix for 7 days (the same condition as ChIP assay). We detected significant increases in the expression of each of these osteogenic markers in osteoblasts were cultured on CTGF-coated as compared to BSA-coated plates (Fig. 7B). Collectively, these data demonstrated that when osteoblasts are cultured on CTGF, there is a sustained up-regulation and activation of FAK and ERK, increased Runx2 binding occupancy of the osteocalcin gene promoter, and increased expression of osteogenic markers that are regulated by Runx2. These results also confirmed that CTGF induced activation of ERK signaling is necessary for osteoblast differentiation, consistent with previous reports regarding the central role of ERK signaling in osteoblast differentiation [20].


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 induces Runx2 transcriptional activation of osteogenic markers.(A) ChIP assay performed on osteoblasts cultured on CTGF coated, BSA coated or uncoated plates for 7 days while treated with osteogenic medium. Runx2 antibody or acetyl-Histone H4 antibody (positive control) used for chromatin immunoprecipitation. Quantitative PCR was performed using osteocalcin gene promoter primers. n = 3, ***p<0.001. ChIP assay repeated three times with similar results. (B) Quantitative PCR performed on osteoblasts cultured on CTGF or BSA coated plates for 7 days while treated with osteogenic media. OCN = osteocalcin, ALP = alkaline phosphatase. n = 3, **p<0.01; ***p<0.001. Experiments repeated three times with similar results.
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getmorefigures.php?uid=PMC4340870&req=5

pone.0115325.g007: Osteoblast adhesion to CTGF induces Runx2 transcriptional activation of osteogenic markers.(A) ChIP assay performed on osteoblasts cultured on CTGF coated, BSA coated or uncoated plates for 7 days while treated with osteogenic medium. Runx2 antibody or acetyl-Histone H4 antibody (positive control) used for chromatin immunoprecipitation. Quantitative PCR was performed using osteocalcin gene promoter primers. n = 3, ***p<0.001. ChIP assay repeated three times with similar results. (B) Quantitative PCR performed on osteoblasts cultured on CTGF or BSA coated plates for 7 days while treated with osteogenic media. OCN = osteocalcin, ALP = alkaline phosphatase. n = 3, **p<0.01; ***p<0.001. Experiments repeated three times with similar results.
Mentions: Runx2 is a critical transcription factor for osteoblast differentiation that has been shown to regulate the transcription of other essential proteins such as osteocalcin [21,35]. To investigate if osteoblasts cultured on a CTGF matrix have an effect on Runx2 transcriptional activity, we performed chromatin immunoprecipitation (ChIP) assays to analyze Runx2 binding occupancy on the osteocalcin gene promoter (Fig. 7A). Cells cultured on CTGF for 7 days showed a significant increase in Runx2 binding occupancy compared to cells on BSA or uncoated plates (Fig. 7A). Next, we performed a quantitative PCR analysis to compare expression of osteogenic markers (Runx2, ALP and osteocalcin) in osteoblasts cultured on CTGF matrix for 7 days (the same condition as ChIP assay). We detected significant increases in the expression of each of these osteogenic markers in osteoblasts were cultured on CTGF-coated as compared to BSA-coated plates (Fig. 7B). Collectively, these data demonstrated that when osteoblasts are cultured on CTGF, there is a sustained up-regulation and activation of FAK and ERK, increased Runx2 binding occupancy of the osteocalcin gene promoter, and increased expression of osteogenic markers that are regulated by Runx2. These results also confirmed that CTGF induced activation of ERK signaling is necessary for osteoblast differentiation, consistent with previous reports regarding the central role of ERK signaling in osteoblast differentiation [20].

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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Related in: MedlinePlus