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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 enhances osteoblast maturation and matrix mineralization.(A) Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 14 days. Scale bar = 2 mm. (B) ALP activity quantified at day 14 of culture and normalized to total protein content; n = 9 wells. (C) Alizarin red staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 35 days. Same magnification as in A. (D) Number of nodules formed after 35 days of culture; n = 9 wells. (E) Area of nodules measured by ImageJ software. **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.
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pone.0115325.g004: Osteoblast adhesion to CTGF matrix enhances osteoblast maturation and matrix mineralization.(A) Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 14 days. Scale bar = 2 mm. (B) ALP activity quantified at day 14 of culture and normalized to total protein content; n = 9 wells. (C) Alizarin red staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 35 days. Same magnification as in A. (D) Number of nodules formed after 35 days of culture; n = 9 wells. (E) Area of nodules measured by ImageJ software. **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.

Mentions: Previous in vivo and in vitro studies have demonstrated a role of CTGF in osteogenesis and early stages of osteoblast differentiation. To examine the effect of osteoblast adhesion to a CTGF matrix on subsequent differentiation, we cultured preosteoblastic cells (MC3T3-E1) on a matrix of CTGF or BSA (negative control) coated plates under osteogenic conditions (see Materials and Methods for details). After 14 days, we assessed osteoblast maturation by alkaline phosphatase (ALP) staining (Fig. 4A) and activity (Fig. 4B). The results demonstrated a more robust staining for ALP and significantly higher enzyme activity in cells cultured on CTGF compared to BSA (Fig. 4A and B). To address whether the increased differentiation observed when cells are cultured on a CTGF substrate could be an indirect effect of increased cell proliferation, we performed a proliferation assay. Cells cultured on CTGF-coated and BSA-coated plates did not exhibit a significant difference in cell number at days 7 or 14 (S2 Fig.). From these results we concluded that increased differentiation is not secondary to increased numbers of adhered/proliferating osteoblasts on CTGF substrate. Next, we evaluated bone nodule formation and matrix mineralization at day 35 of culture. Staining for Alizarin red showed large, well-formed nodules in cells cultured on the CTGF matrix compared to BSA (Fig. 4C). The number (Fig. 4D) and area (Fig. 4E) of nodules were also significantly greater on CTGF compared to BSA. Collectively, these data demonstrate that preosteoblasts grown on a CTGF matrix results in enhanced osteogenic differentiation, bone nodule formation and matrix mineralization.


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 enhances osteoblast maturation and matrix mineralization.(A) Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 14 days. Scale bar = 2 mm. (B) ALP activity quantified at day 14 of culture and normalized to total protein content; n = 9 wells. (C) Alizarin red staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 35 days. Same magnification as in A. (D) Number of nodules formed after 35 days of culture; n = 9 wells. (E) Area of nodules measured by ImageJ software. **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4340870&req=5

pone.0115325.g004: Osteoblast adhesion to CTGF matrix enhances osteoblast maturation and matrix mineralization.(A) Alkaline Phosphatase (ALP) staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 14 days. Scale bar = 2 mm. (B) ALP activity quantified at day 14 of culture and normalized to total protein content; n = 9 wells. (C) Alizarin red staining of osteoblasts cultured on 2 μg/ml CTGF or 1% BSA coated plates for 35 days. Same magnification as in A. (D) Number of nodules formed after 35 days of culture; n = 9 wells. (E) Area of nodules measured by ImageJ software. **p<0.01; ***p<0.001. Experiments were repeated three times with similar results.
Mentions: Previous in vivo and in vitro studies have demonstrated a role of CTGF in osteogenesis and early stages of osteoblast differentiation. To examine the effect of osteoblast adhesion to a CTGF matrix on subsequent differentiation, we cultured preosteoblastic cells (MC3T3-E1) on a matrix of CTGF or BSA (negative control) coated plates under osteogenic conditions (see Materials and Methods for details). After 14 days, we assessed osteoblast maturation by alkaline phosphatase (ALP) staining (Fig. 4A) and activity (Fig. 4B). The results demonstrated a more robust staining for ALP and significantly higher enzyme activity in cells cultured on CTGF compared to BSA (Fig. 4A and B). To address whether the increased differentiation observed when cells are cultured on a CTGF substrate could be an indirect effect of increased cell proliferation, we performed a proliferation assay. Cells cultured on CTGF-coated and BSA-coated plates did not exhibit a significant difference in cell number at days 7 or 14 (S2 Fig.). From these results we concluded that increased differentiation is not secondary to increased numbers of adhered/proliferating osteoblasts on CTGF substrate. Next, we evaluated bone nodule formation and matrix mineralization at day 35 of culture. Staining for Alizarin red showed large, well-formed nodules in cells cultured on the CTGF matrix compared to BSA (Fig. 4C). The number (Fig. 4D) and area (Fig. 4E) of nodules were also significantly greater on CTGF compared to BSA. Collectively, these data demonstrate that preosteoblasts grown on a CTGF matrix results in enhanced osteogenic differentiation, bone nodule formation and matrix mineralization.

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