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Involvement of the orphan nuclear estrogen receptor-related receptor α in osteoclast adhesion and transmigration.

Bonnelye E, Saltel F, Chabadel A, Zirngibl RA, Aubin JE, Jurdic P - J. Mol. Endocrinol. (2010)

Bottom Line: In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used.We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells.In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, 69007 Lyon, France. edith.bonnelye@inserm.fr

ABSTRACT
The orphan nuclear receptor, estrogen receptor-related receptor α (ERRα) is expressed in osteoblasts and osteoclasts (OCs) and has been proposed to be a modulator of estrogen signaling. To determine the role of ERRα in OC biology, we knocked down ERRα activity by transient transfection of an siRNA directed against ERRα in the RAW264.7 monocyte-macrophage cell line that differentiates into OCs in the presence of receptor activator of nuclear factor κB-ligands and macrophage colony-stimulating factor. In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used. Expression of OC markers was assessed by real-time PCR, and adhesion and transmigration tests were performed. Actin cytoskeletal organization was visualized using confocal microscopy. We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells. Decreased adhesion and the absence of podosome belts concomitant with abnormal localization of c-src were also observed in RAW-GFP-ERRαΔAF2-derived OCs. In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers. Our data show that the impairment of ERRα function does not alter OC precursor proliferation and differentiation but does alter the adhesion/spreading and migration capacities of mature OCs.

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Implication of ERRα in integrin β3 expression in GFP-ΔAF2-derived OCs (A) Total RNA was extracted from GFP(Ct)- and GFP-ΔAF2 (1, 2, and 3)-derived OCs after 6 days of culture, and real-time PCR was performed on triplicate samples by using primers specific for several genes involved in OCs spreading. Quantification shows a statistically significant decrease in β3 integrin and OPN in GFP-ΔAF2 (1, 2, and 3) (ANOVA, P=0·0002; ANOVA, P=0·0001, ΔAF2 (1, 2, and 3) versus Ct for OPN and β3 respectively). Markers for adhesion (integrin subunits αv, β1, and β5), OC cell–cell fusion (DC-STAMP) were not modified. (B) Overexpression of ERRα was induced by transient transfection of ERRαWT in RAW264.7 cells. Quantification shows a statistically significant increase in OPN and β3 integrin expression in ERRαWT-transfected cells (ANOVA, P=0·0001; ANOVA, P=0·0011, P=0·0023, for ERRα, OPN, and β3 respectively). (C) siRNA directed against ERRα or a control scrambled siRNA (Sc) was transiently transfected into RAW264.7 cells. As expected, ERRα expression was down-regulated after transfection with ERRα siRNA (ANOVA, P=0·0015). Concomitantly, OPN and integrin β3 expression was decreased (ANOVA, P=0·0053, P=0·077 for OPN and integrin β3 respectively). On the other hand, ERRγ was dramatically expressed in ERRα siRNA-transfected cells versus Sc (ANOVA, P=0·0004 for ERRγ).
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fig3: Implication of ERRα in integrin β3 expression in GFP-ΔAF2-derived OCs (A) Total RNA was extracted from GFP(Ct)- and GFP-ΔAF2 (1, 2, and 3)-derived OCs after 6 days of culture, and real-time PCR was performed on triplicate samples by using primers specific for several genes involved in OCs spreading. Quantification shows a statistically significant decrease in β3 integrin and OPN in GFP-ΔAF2 (1, 2, and 3) (ANOVA, P=0·0002; ANOVA, P=0·0001, ΔAF2 (1, 2, and 3) versus Ct for OPN and β3 respectively). Markers for adhesion (integrin subunits αv, β1, and β5), OC cell–cell fusion (DC-STAMP) were not modified. (B) Overexpression of ERRα was induced by transient transfection of ERRαWT in RAW264.7 cells. Quantification shows a statistically significant increase in OPN and β3 integrin expression in ERRαWT-transfected cells (ANOVA, P=0·0001; ANOVA, P=0·0011, P=0·0023, for ERRα, OPN, and β3 respectively). (C) siRNA directed against ERRα or a control scrambled siRNA (Sc) was transiently transfected into RAW264.7 cells. As expected, ERRα expression was down-regulated after transfection with ERRα siRNA (ANOVA, P=0·0015). Concomitantly, OPN and integrin β3 expression was decreased (ANOVA, P=0·0053, P=0·077 for OPN and integrin β3 respectively). On the other hand, ERRγ was dramatically expressed in ERRα siRNA-transfected cells versus Sc (ANOVA, P=0·0004 for ERRγ).

Mentions: To determine whether ERRα is involved in OC formation, GFP-ΔAF2 (1, 2, and 3) and GFP(Ct) cells were differentiated into mature multinucleated OCs in the presence of M-CSF and RANKL. Based on the cell counts after 3 days of culture, at the end of the proliferation stage, reduction of ERRα transactivation capacity had no effect on cell proliferation (data not shown). Concomitantly, the expression of early markers of OC differentiation, such as TRAP, c-fms, and RANKL (data not shown), and of the proliferation marker cyclin D1 was not affected (Fig. 2E and F). Later during differentiation, while the OPN expression was still down-regulated, we found that integrin β3 chain expression was also decreased in GFP-ΔAF2 (1, 2, and 3) compared with GFP(Ct) cells (Fig. 3A). On the other hand, the expression of αv, β1 and β5 integrin chain subunits, c-src, CD44, and Traf6 was not affected (Fig. 3A, and data not shown). Moreover, no differences were observed in the expression of OC factors involved in OC fusion (dendritic cell-specific transmembrane protein DC-STAMP (DCS)) or apoptosis (Bcl2 and Bax) in GFP-ΔAF2 (1, 2, and 3)- versus GFP(Ct)-derived OCs (Fig. 3A).


Involvement of the orphan nuclear estrogen receptor-related receptor α in osteoclast adhesion and transmigration.

Bonnelye E, Saltel F, Chabadel A, Zirngibl RA, Aubin JE, Jurdic P - J. Mol. Endocrinol. (2010)

Implication of ERRα in integrin β3 expression in GFP-ΔAF2-derived OCs (A) Total RNA was extracted from GFP(Ct)- and GFP-ΔAF2 (1, 2, and 3)-derived OCs after 6 days of culture, and real-time PCR was performed on triplicate samples by using primers specific for several genes involved in OCs spreading. Quantification shows a statistically significant decrease in β3 integrin and OPN in GFP-ΔAF2 (1, 2, and 3) (ANOVA, P=0·0002; ANOVA, P=0·0001, ΔAF2 (1, 2, and 3) versus Ct for OPN and β3 respectively). Markers for adhesion (integrin subunits αv, β1, and β5), OC cell–cell fusion (DC-STAMP) were not modified. (B) Overexpression of ERRα was induced by transient transfection of ERRαWT in RAW264.7 cells. Quantification shows a statistically significant increase in OPN and β3 integrin expression in ERRαWT-transfected cells (ANOVA, P=0·0001; ANOVA, P=0·0011, P=0·0023, for ERRα, OPN, and β3 respectively). (C) siRNA directed against ERRα or a control scrambled siRNA (Sc) was transiently transfected into RAW264.7 cells. As expected, ERRα expression was down-regulated after transfection with ERRα siRNA (ANOVA, P=0·0015). Concomitantly, OPN and integrin β3 expression was decreased (ANOVA, P=0·0053, P=0·077 for OPN and integrin β3 respectively). On the other hand, ERRγ was dramatically expressed in ERRα siRNA-transfected cells versus Sc (ANOVA, P=0·0004 for ERRγ).
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fig3: Implication of ERRα in integrin β3 expression in GFP-ΔAF2-derived OCs (A) Total RNA was extracted from GFP(Ct)- and GFP-ΔAF2 (1, 2, and 3)-derived OCs after 6 days of culture, and real-time PCR was performed on triplicate samples by using primers specific for several genes involved in OCs spreading. Quantification shows a statistically significant decrease in β3 integrin and OPN in GFP-ΔAF2 (1, 2, and 3) (ANOVA, P=0·0002; ANOVA, P=0·0001, ΔAF2 (1, 2, and 3) versus Ct for OPN and β3 respectively). Markers for adhesion (integrin subunits αv, β1, and β5), OC cell–cell fusion (DC-STAMP) were not modified. (B) Overexpression of ERRα was induced by transient transfection of ERRαWT in RAW264.7 cells. Quantification shows a statistically significant increase in OPN and β3 integrin expression in ERRαWT-transfected cells (ANOVA, P=0·0001; ANOVA, P=0·0011, P=0·0023, for ERRα, OPN, and β3 respectively). (C) siRNA directed against ERRα or a control scrambled siRNA (Sc) was transiently transfected into RAW264.7 cells. As expected, ERRα expression was down-regulated after transfection with ERRα siRNA (ANOVA, P=0·0015). Concomitantly, OPN and integrin β3 expression was decreased (ANOVA, P=0·0053, P=0·077 for OPN and integrin β3 respectively). On the other hand, ERRγ was dramatically expressed in ERRα siRNA-transfected cells versus Sc (ANOVA, P=0·0004 for ERRγ).
Mentions: To determine whether ERRα is involved in OC formation, GFP-ΔAF2 (1, 2, and 3) and GFP(Ct) cells were differentiated into mature multinucleated OCs in the presence of M-CSF and RANKL. Based on the cell counts after 3 days of culture, at the end of the proliferation stage, reduction of ERRα transactivation capacity had no effect on cell proliferation (data not shown). Concomitantly, the expression of early markers of OC differentiation, such as TRAP, c-fms, and RANKL (data not shown), and of the proliferation marker cyclin D1 was not affected (Fig. 2E and F). Later during differentiation, while the OPN expression was still down-regulated, we found that integrin β3 chain expression was also decreased in GFP-ΔAF2 (1, 2, and 3) compared with GFP(Ct) cells (Fig. 3A). On the other hand, the expression of αv, β1 and β5 integrin chain subunits, c-src, CD44, and Traf6 was not affected (Fig. 3A, and data not shown). Moreover, no differences were observed in the expression of OC factors involved in OC fusion (dendritic cell-specific transmembrane protein DC-STAMP (DCS)) or apoptosis (Bcl2 and Bax) in GFP-ΔAF2 (1, 2, and 3)- versus GFP(Ct)-derived OCs (Fig. 3A).

Bottom Line: In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used.We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells.In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, 69007 Lyon, France. edith.bonnelye@inserm.fr

ABSTRACT
The orphan nuclear receptor, estrogen receptor-related receptor α (ERRα) is expressed in osteoblasts and osteoclasts (OCs) and has been proposed to be a modulator of estrogen signaling. To determine the role of ERRα in OC biology, we knocked down ERRα activity by transient transfection of an siRNA directed against ERRα in the RAW264.7 monocyte-macrophage cell line that differentiates into OCs in the presence of receptor activator of nuclear factor κB-ligands and macrophage colony-stimulating factor. In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used. Expression of OC markers was assessed by real-time PCR, and adhesion and transmigration tests were performed. Actin cytoskeletal organization was visualized using confocal microscopy. We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells. Decreased adhesion and the absence of podosome belts concomitant with abnormal localization of c-src were also observed in RAW-GFP-ERRαΔAF2-derived OCs. In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers. Our data show that the impairment of ERRα function does not alter OC precursor proliferation and differentiation but does alter the adhesion/spreading and migration capacities of mature OCs.

Show MeSH
Related in: MedlinePlus