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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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ERRα is expressed during OC differentiation. (A and B) Real-time PCR showed that ERRα is expressed in leukocytes freshly isolated from murine spleen (d0), as well as during osteoclastogenesis (d6–d8). ERRα and ERRγ expression were normalized against that of the ribosomal protein gene L32. For comparison, the mRNA level for CTR is also shown. (C) Consistent with primary cells, ERRα is also expressed during OC formation from RAW264.7 cells. d, days in culture.
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fig1: ERRα is expressed during OC differentiation. (A and B) Real-time PCR showed that ERRα is expressed in leukocytes freshly isolated from murine spleen (d0), as well as during osteoclastogenesis (d6–d8). ERRα and ERRγ expression were normalized against that of the ribosomal protein gene L32. For comparison, the mRNA level for CTR is also shown. (C) Consistent with primary cells, ERRα is also expressed during OC formation from RAW264.7 cells. d, days in culture.

Mentions: Consistent with our previous findings on the presence of ERRα protein in multinucleated TRAP-positive cells in rat femur in vivo (Bonnelye et al. 2002), we found, by real-time PCR, ERRα mRNA expressed throughout all the stages of OC differentiation: in primary leukocytes extracted from spleen (day 0), proliferation (day 2), mononucleated progenitor fusion (day 4), and maturation (days 6–8) with significant increases on days 4–8, when multinucleated OCs were starting to form until they reached maturity (Fig. 1A and B). For comparison, mRNA levels for ERRβ, ERRγ, and the late OC differentiation marker CTR were also assessed (Fig. 1A and B). Interestingly and in contrast to ERRα, ERRγ mRNA expression decreased dramatically during OC differentiation, while ERRβ was not detected. As previously shown (Bonnelye et al. 2002), ERRα is also expressed during OC differentiation of the RAW264.7 macrophage cell line, although fold changes in the expression level are less than that observed in primary OC cultures, which is probably due to the fact that RAW264.7 cells are already committed into the macrophage lineage (Fig. 1C). Given its expression in mature OCs, we then checked whether ERRα was involved in their function.


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)

ERRα is expressed during OC differentiation. (A and B) Real-time PCR showed that ERRα is expressed in leukocytes freshly isolated from murine spleen (d0), as well as during osteoclastogenesis (d6–d8). ERRα and ERRγ expression were normalized against that of the ribosomal protein gene L32. For comparison, the mRNA level for CTR is also shown. (C) Consistent with primary cells, ERRα is also expressed during OC formation from RAW264.7 cells. d, days in culture.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2990392&req=5

fig1: ERRα is expressed during OC differentiation. (A and B) Real-time PCR showed that ERRα is expressed in leukocytes freshly isolated from murine spleen (d0), as well as during osteoclastogenesis (d6–d8). ERRα and ERRγ expression were normalized against that of the ribosomal protein gene L32. For comparison, the mRNA level for CTR is also shown. (C) Consistent with primary cells, ERRα is also expressed during OC formation from RAW264.7 cells. d, days in culture.
Mentions: Consistent with our previous findings on the presence of ERRα protein in multinucleated TRAP-positive cells in rat femur in vivo (Bonnelye et al. 2002), we found, by real-time PCR, ERRα mRNA expressed throughout all the stages of OC differentiation: in primary leukocytes extracted from spleen (day 0), proliferation (day 2), mononucleated progenitor fusion (day 4), and maturation (days 6–8) with significant increases on days 4–8, when multinucleated OCs were starting to form until they reached maturity (Fig. 1A and B). For comparison, mRNA levels for ERRβ, ERRγ, and the late OC differentiation marker CTR were also assessed (Fig. 1A and B). Interestingly and in contrast to ERRα, ERRγ mRNA expression decreased dramatically during OC differentiation, while ERRβ was not detected. As previously shown (Bonnelye et al. 2002), ERRα is also expressed during OC differentiation of the RAW264.7 macrophage cell line, although fold changes in the expression level are less than that observed in primary OC cultures, which is probably due to the fact that RAW264.7 cells are already committed into the macrophage lineage (Fig. 1C). Given its expression in mature OCs, we then checked whether ERRα was involved in their function.

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