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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 matrix induces Rac activation and cell spreading.(A) Immunofluorescence staining of F-actin in osteoblasts cultured on 1% BSA, 2 μg/ml of CTGF or fibronectin coated slides for 8 hours at 37°C. Scale bar = 50 μm. (B) Cell spreading area of osteoblasts cultured on BSA, CTGF or fibronectin for 8 hours and stained for actin were measured by ImageJ; n = 50. **p<0.01; ***p<0.001. (C) Western blot analysis of active Rac1, total Rac1 and actin to study Rac1 activation levels. Osteoblasts were cultured for 2 hours on uncoated plates or plates coated with BSA, CTGF or fibronectin. Abbreviations: fibronectin (FN) and negative control (Cont). Experiments were repeated three times with similar results.
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pone.0115325.g003: Osteoblast adhesion to CTGF matrix induces Rac activation and cell spreading.(A) Immunofluorescence staining of F-actin in osteoblasts cultured on 1% BSA, 2 μg/ml of CTGF or fibronectin coated slides for 8 hours at 37°C. Scale bar = 50 μm. (B) Cell spreading area of osteoblasts cultured on BSA, CTGF or fibronectin for 8 hours and stained for actin were measured by ImageJ; n = 50. **p<0.01; ***p<0.001. (C) Western blot analysis of active Rac1, total Rac1 and actin to study Rac1 activation levels. Osteoblasts were cultured for 2 hours on uncoated plates or plates coated with BSA, CTGF or fibronectin. Abbreviations: fibronectin (FN) and negative control (Cont). Experiments were repeated three times with similar results.

Mentions: Regulation of cytoskeletal reorganization is an important aspect of integrin dependent cell adhesion to extracellular matrix proteins [32]. To investigate the effects of osteoblast adhesion to CTGF on cytoskeletal reorganization and cell spreading, we compared cells that were cultured for eight hours on substrates of CTGF, BSA (negative control) or fibronectin (positive control) (Fig. 3A and B). Immunofluorescence staining of actin filaments and vinculin demonstrated that cells attached to CTGF had a more uniform, rounded shape, compared to cells attached to fibronectin, which exhibited a more polarized appearance (Fig. 3A). It is interesting to note that this difference in shape was more pronounced at earlier time points; many of the cells on CTGF had a more polarized appearance by 24 hours (see Fig. 2C). Quantification of cell area demonstrated that cells plated on CTGF spread well, compared to BSA, but not to the same extent as cells on fibronectin (Fig. 3B). We next examined if osteoblast adhesion/spreading on CTGF results in the activation of Rac1, a key signaling molecule in the regulation of cell spreading [33,34]. We performed Rac1 activation assays on cells cultured on dishes coated with BSA, fibronectin, or CTGF or on uncoated dishes for 2 hours. Activation of Rac1 occurred in cells cultured on fibronectin and CTGF compared to BSA or uncoated controls, with highest levels of active Rac1 observed in cells cultured on fibronectin (Fig. 3C). Total Rac levels were similar in under all conditions (Fig. 3C). Collectively, these data support a role for CTGF as matrix protein that promotes cytoskeletal reorganization, spreading and Rac activation in osteoblasts.


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 matrix induces Rac activation and cell spreading.(A) Immunofluorescence staining of F-actin in osteoblasts cultured on 1% BSA, 2 μg/ml of CTGF or fibronectin coated slides for 8 hours at 37°C. Scale bar = 50 μm. (B) Cell spreading area of osteoblasts cultured on BSA, CTGF or fibronectin for 8 hours and stained for actin were measured by ImageJ; n = 50. **p<0.01; ***p<0.001. (C) Western blot analysis of active Rac1, total Rac1 and actin to study Rac1 activation levels. Osteoblasts were cultured for 2 hours on uncoated plates or plates coated with BSA, CTGF or fibronectin. Abbreviations: fibronectin (FN) and negative control (Cont). Experiments were repeated three times with similar results.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4340870&req=5

pone.0115325.g003: Osteoblast adhesion to CTGF matrix induces Rac activation and cell spreading.(A) Immunofluorescence staining of F-actin in osteoblasts cultured on 1% BSA, 2 μg/ml of CTGF or fibronectin coated slides for 8 hours at 37°C. Scale bar = 50 μm. (B) Cell spreading area of osteoblasts cultured on BSA, CTGF or fibronectin for 8 hours and stained for actin were measured by ImageJ; n = 50. **p<0.01; ***p<0.001. (C) Western blot analysis of active Rac1, total Rac1 and actin to study Rac1 activation levels. Osteoblasts were cultured for 2 hours on uncoated plates or plates coated with BSA, CTGF or fibronectin. Abbreviations: fibronectin (FN) and negative control (Cont). Experiments were repeated three times with similar results.
Mentions: Regulation of cytoskeletal reorganization is an important aspect of integrin dependent cell adhesion to extracellular matrix proteins [32]. To investigate the effects of osteoblast adhesion to CTGF on cytoskeletal reorganization and cell spreading, we compared cells that were cultured for eight hours on substrates of CTGF, BSA (negative control) or fibronectin (positive control) (Fig. 3A and B). Immunofluorescence staining of actin filaments and vinculin demonstrated that cells attached to CTGF had a more uniform, rounded shape, compared to cells attached to fibronectin, which exhibited a more polarized appearance (Fig. 3A). It is interesting to note that this difference in shape was more pronounced at earlier time points; many of the cells on CTGF had a more polarized appearance by 24 hours (see Fig. 2C). Quantification of cell area demonstrated that cells plated on CTGF spread well, compared to BSA, but not to the same extent as cells on fibronectin (Fig. 3B). We next examined if osteoblast adhesion/spreading on CTGF results in the activation of Rac1, a key signaling molecule in the regulation of cell spreading [33,34]. We performed Rac1 activation assays on cells cultured on dishes coated with BSA, fibronectin, or CTGF or on uncoated dishes for 2 hours. Activation of Rac1 occurred in cells cultured on fibronectin and CTGF compared to BSA or uncoated controls, with highest levels of active Rac1 observed in cells cultured on fibronectin (Fig. 3C). Total Rac levels were similar in under all conditions (Fig. 3C). Collectively, these data support a role for CTGF as matrix protein that promotes cytoskeletal reorganization, spreading and Rac activation in osteoblasts.

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