Limits...
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.

Show MeSH

Related in: MedlinePlus

Osteoblast adhesion to CTGF is via integrin receptors and through fourth domain of CTGF.(A) Adhesion assay to analyze osteoblast adhesion to various concentrations of recombinant CTGF, used to coat the wells. The number of cells adhered to CTGF is compared to cell adhesion to 1% BSA (negative control). (B) Adhesion assay comparing osteoblast adhesion to 2 μg/ml CTGF, 2 μg/ml fibronectin, or 1% BSA (negative control) coated wells. (C) Adhesion assay comparing osteoblast adhesion to 2 μg/ml of full-length CTGF, third domain of CTGF, fourth domain of CTGF, or 1% BSA (negative control) coated wells. (D) Adhesion assay analyzing effect of osteoblast treatment with fourth domain of CTGF prior to culture on full-length CTGF coated wells. (E) Adhesion assay analyzing effect of divalent cation chelation by EDTA on osteoblast adhesion to full-length CTGF, and reversing this effect by adding excessive amount of divalent cations to the culture. (F) Adhesion assay studying the effect of blocking heparin binding site of CTGF molecule via adding heparin to osteoblast cell suspension prior to culture on CTGF coated wells. n = 6, *p<0.05; **p<0.01; ***p<0.001. Adhesion assays were repeated three times with similar results.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4340870&req=5

pone.0115325.g001: Osteoblast adhesion to CTGF is via integrin receptors and through fourth domain of CTGF.(A) Adhesion assay to analyze osteoblast adhesion to various concentrations of recombinant CTGF, used to coat the wells. The number of cells adhered to CTGF is compared to cell adhesion to 1% BSA (negative control). (B) Adhesion assay comparing osteoblast adhesion to 2 μg/ml CTGF, 2 μg/ml fibronectin, or 1% BSA (negative control) coated wells. (C) Adhesion assay comparing osteoblast adhesion to 2 μg/ml of full-length CTGF, third domain of CTGF, fourth domain of CTGF, or 1% BSA (negative control) coated wells. (D) Adhesion assay analyzing effect of osteoblast treatment with fourth domain of CTGF prior to culture on full-length CTGF coated wells. (E) Adhesion assay analyzing effect of divalent cation chelation by EDTA on osteoblast adhesion to full-length CTGF, and reversing this effect by adding excessive amount of divalent cations to the culture. (F) Adhesion assay studying the effect of blocking heparin binding site of CTGF molecule via adding heparin to osteoblast cell suspension prior to culture on CTGF coated wells. n = 6, *p<0.05; **p<0.01; ***p<0.001. Adhesion assays were repeated three times with similar results.

Mentions: To evaluate whether CTGF can serve as a matrix for osteoblast attachment, we conducted an adhesion assay using MC3T3-E1 osteoblasts that were seeded onto culture plates coated with various concentrations of CTGF. This assay demonstrated a significant, concentration-dependent increase in the attachment of cells plated onto the CTGF substrate (ranging from 0.25 to 2 μg/ml) compared to BSA (negative control), with cell attachment reaching a plateau at 2 μg/ml CTGF (Fig. 1A). Next, we compared the attachment of osteoblasts to CTGF and fibronectin, a well-documented bone matrix protein that promotes osteoblast adhesion [26–28]. While osteoblasts adhered to both fibronectin and CTGF (2 μg/ml), the number of cells that attached to fibronectin was greater than to CTGF under identical assay conditions (Fig. 1B). Next, we investigated which domain of CTGF provides the binding site for osteoblast adhesion. For these experiments, we coated wells with the third domain of CTGF, the fourth domain of CTGF, or the full length CTGF protein and performed adhesion assays. Osteoblast adhesion to the fourth domain was comparable to adhesion to the whole CTGF protein (Fig. 1C), while adhesion to the third domain was not significantly different from BSA (the negative control). To further determine if the fourth domain of CTGF plays an essential role for osteoblast adhesion to CTGF, we incubated osteoblasts with recombinant domain four for 30 minutes at room temperature prior to seeding cells in wells coated with full length CTGF. Pre-incubation of cells with the fourth domain of CTGF blocked adhesion to full length CTGF with levels comparable to the negative control (BSA) (Fig. 1D).


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 via integrin receptors and through fourth domain of CTGF.(A) Adhesion assay to analyze osteoblast adhesion to various concentrations of recombinant CTGF, used to coat the wells. The number of cells adhered to CTGF is compared to cell adhesion to 1% BSA (negative control). (B) Adhesion assay comparing osteoblast adhesion to 2 μg/ml CTGF, 2 μg/ml fibronectin, or 1% BSA (negative control) coated wells. (C) Adhesion assay comparing osteoblast adhesion to 2 μg/ml of full-length CTGF, third domain of CTGF, fourth domain of CTGF, or 1% BSA (negative control) coated wells. (D) Adhesion assay analyzing effect of osteoblast treatment with fourth domain of CTGF prior to culture on full-length CTGF coated wells. (E) Adhesion assay analyzing effect of divalent cation chelation by EDTA on osteoblast adhesion to full-length CTGF, and reversing this effect by adding excessive amount of divalent cations to the culture. (F) Adhesion assay studying the effect of blocking heparin binding site of CTGF molecule via adding heparin to osteoblast cell suspension prior to culture on CTGF coated wells. n = 6, *p<0.05; **p<0.01; ***p<0.001. Adhesion assays were repeated three times with similar results.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0115325.g001: Osteoblast adhesion to CTGF is via integrin receptors and through fourth domain of CTGF.(A) Adhesion assay to analyze osteoblast adhesion to various concentrations of recombinant CTGF, used to coat the wells. The number of cells adhered to CTGF is compared to cell adhesion to 1% BSA (negative control). (B) Adhesion assay comparing osteoblast adhesion to 2 μg/ml CTGF, 2 μg/ml fibronectin, or 1% BSA (negative control) coated wells. (C) Adhesion assay comparing osteoblast adhesion to 2 μg/ml of full-length CTGF, third domain of CTGF, fourth domain of CTGF, or 1% BSA (negative control) coated wells. (D) Adhesion assay analyzing effect of osteoblast treatment with fourth domain of CTGF prior to culture on full-length CTGF coated wells. (E) Adhesion assay analyzing effect of divalent cation chelation by EDTA on osteoblast adhesion to full-length CTGF, and reversing this effect by adding excessive amount of divalent cations to the culture. (F) Adhesion assay studying the effect of blocking heparin binding site of CTGF molecule via adding heparin to osteoblast cell suspension prior to culture on CTGF coated wells. n = 6, *p<0.05; **p<0.01; ***p<0.001. Adhesion assays were repeated three times with similar results.
Mentions: To evaluate whether CTGF can serve as a matrix for osteoblast attachment, we conducted an adhesion assay using MC3T3-E1 osteoblasts that were seeded onto culture plates coated with various concentrations of CTGF. This assay demonstrated a significant, concentration-dependent increase in the attachment of cells plated onto the CTGF substrate (ranging from 0.25 to 2 μg/ml) compared to BSA (negative control), with cell attachment reaching a plateau at 2 μg/ml CTGF (Fig. 1A). Next, we compared the attachment of osteoblasts to CTGF and fibronectin, a well-documented bone matrix protein that promotes osteoblast adhesion [26–28]. While osteoblasts adhered to both fibronectin and CTGF (2 μg/ml), the number of cells that attached to fibronectin was greater than to CTGF under identical assay conditions (Fig. 1B). Next, we investigated which domain of CTGF provides the binding site for osteoblast adhesion. For these experiments, we coated wells with the third domain of CTGF, the fourth domain of CTGF, or the full length CTGF protein and performed adhesion assays. Osteoblast adhesion to the fourth domain was comparable to adhesion to the whole CTGF protein (Fig. 1C), while adhesion to the third domain was not significantly different from BSA (the negative control). To further determine if the fourth domain of CTGF plays an essential role for osteoblast adhesion to CTGF, we incubated osteoblasts with recombinant domain four for 30 minutes at room temperature prior to seeding cells in wells coated with full length CTGF. Pre-incubation of cells with the fourth domain of CTGF blocked adhesion to full length CTGF with levels comparable to the negative control (BSA) (Fig. 1D).

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