Limits...
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.

Show MeSH

Related in: MedlinePlus

Integrin subunit β3 expression and localization are altered in GFP-ΔAF2-derived OCs. (A–L) β3 integrin subunit (A, D, C, and F in green), phosphorylated c-src (G, J, I, and L in green), and actin (B, C, E, F, H, I, K, and L in red) were immunostained in control (A–C; G–I) and in GFP-ΔAF2-2-derived OCs (D–F; J–L). Whereas control OC formed typical podosome belts with actin dots surrounded by β3 integrin and phosphorylated c-src (A–C; G–I), podosomes of GFP-ΔAF2-2-derived OCs were disorganized with mislocalized β3 integrin and phosphorylated c-src (D–F; J–L). Merged images of the closeup areas of actin, β3, and phosphorylated c-src respectively are also shown (C, F, I, and L). Bar=20 μm. (M) Adhesion ability was also evaluated (ANOVA, P=0·0001; ΔAF2 (1, 2, and 3)), while (N) OC viability was evaluated by counting the total number of OCs used in the adhesion test at T=0.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2990392&req=5

fig5: Integrin subunit β3 expression and localization are altered in GFP-ΔAF2-derived OCs. (A–L) β3 integrin subunit (A, D, C, and F in green), phosphorylated c-src (G, J, I, and L in green), and actin (B, C, E, F, H, I, K, and L in red) were immunostained in control (A–C; G–I) and in GFP-ΔAF2-2-derived OCs (D–F; J–L). Whereas control OC formed typical podosome belts with actin dots surrounded by β3 integrin and phosphorylated c-src (A–C; G–I), podosomes of GFP-ΔAF2-2-derived OCs were disorganized with mislocalized β3 integrin and phosphorylated c-src (D–F; J–L). Merged images of the closeup areas of actin, β3, and phosphorylated c-src respectively are also shown (C, F, I, and L). Bar=20 μm. (M) Adhesion ability was also evaluated (ANOVA, P=0·0001; ΔAF2 (1, 2, and 3)), while (N) OC viability was evaluated by counting the total number of OCs used in the adhesion test at T=0.

Mentions: Down-regulation of β3 expression in GFP-ΔAF2-2-derived OCs at day 6 (Fig. 3A) was confirmed by immunofluorescence using an antibody against integrin β3 (Fig. 5A and D). As expected, intense β3 integrin subunit staining was localized around the actin core of podosomes organized in a belt in GFP(Ct)-derived OCs at day 6 (Fig. 5A and B, see merged in Fig. 4C). In contrast, labeling for β3 integrin subunit protein was diffusely distributed within GFP-ΔAF2-2-derived OCs at day 6 (Fig. 5D and E, see merged in F). Since we, and others, have shown the importance of c-src in integrin signaling for actin organization, we looked for phosphorylated c-src localization in OCs expressing the dominant-negative form of ERRα. Whereas phosphorylated c-src was distributed around the podosome belt in GFP(Ct)-derived OCs on day 6 (Fig. 5G and H, see merged in I), it was diffusely localized in GFP-ΔAF2-2 cells (Fig. 5J and K, see merged in L). Similarly, vinculin localization was disrupted in GFP-ΔAF2 cells (data not shown) compared with GFP(Ct)-derived OCs at day 6. These results suggest that disturbing ERRα function impairs a signaling pathway linked to OPN, integrin β3, activated c-src, and actin organization. Moreover, and in agreement with the decrease in mature OC spreading, we observed a decrease in OC adhesion in GFP-ΔAF2 (1, 2, and 3)-derived OCs compared with GFP(Ct)-derived cells (by 40% in ΔAF2-1, 50% in ΔAF2-2, and 68% in ΔAF2-3 compared with GFP(Ct); Fig. 5M). It was observed that the differences in OC viability, as reflected by OC number, cannot account for the differences in spreading between GFP(Ct)- and GFP-ΔAF2 (1, 2, and 3)-derived OCs (T=0; Fig. 5N).


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)

Integrin subunit β3 expression and localization are altered in GFP-ΔAF2-derived OCs. (A–L) β3 integrin subunit (A, D, C, and F in green), phosphorylated c-src (G, J, I, and L in green), and actin (B, C, E, F, H, I, K, and L in red) were immunostained in control (A–C; G–I) and in GFP-ΔAF2-2-derived OCs (D–F; J–L). Whereas control OC formed typical podosome belts with actin dots surrounded by β3 integrin and phosphorylated c-src (A–C; G–I), podosomes of GFP-ΔAF2-2-derived OCs were disorganized with mislocalized β3 integrin and phosphorylated c-src (D–F; J–L). Merged images of the closeup areas of actin, β3, and phosphorylated c-src respectively are also shown (C, F, I, and L). Bar=20 μm. (M) Adhesion ability was also evaluated (ANOVA, P=0·0001; ΔAF2 (1, 2, and 3)), while (N) OC viability was evaluated by counting the total number of OCs used in the adhesion test at T=0.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Integrin subunit β3 expression and localization are altered in GFP-ΔAF2-derived OCs. (A–L) β3 integrin subunit (A, D, C, and F in green), phosphorylated c-src (G, J, I, and L in green), and actin (B, C, E, F, H, I, K, and L in red) were immunostained in control (A–C; G–I) and in GFP-ΔAF2-2-derived OCs (D–F; J–L). Whereas control OC formed typical podosome belts with actin dots surrounded by β3 integrin and phosphorylated c-src (A–C; G–I), podosomes of GFP-ΔAF2-2-derived OCs were disorganized with mislocalized β3 integrin and phosphorylated c-src (D–F; J–L). Merged images of the closeup areas of actin, β3, and phosphorylated c-src respectively are also shown (C, F, I, and L). Bar=20 μm. (M) Adhesion ability was also evaluated (ANOVA, P=0·0001; ΔAF2 (1, 2, and 3)), while (N) OC viability was evaluated by counting the total number of OCs used in the adhesion test at T=0.
Mentions: Down-regulation of β3 expression in GFP-ΔAF2-2-derived OCs at day 6 (Fig. 3A) was confirmed by immunofluorescence using an antibody against integrin β3 (Fig. 5A and D). As expected, intense β3 integrin subunit staining was localized around the actin core of podosomes organized in a belt in GFP(Ct)-derived OCs at day 6 (Fig. 5A and B, see merged in Fig. 4C). In contrast, labeling for β3 integrin subunit protein was diffusely distributed within GFP-ΔAF2-2-derived OCs at day 6 (Fig. 5D and E, see merged in F). Since we, and others, have shown the importance of c-src in integrin signaling for actin organization, we looked for phosphorylated c-src localization in OCs expressing the dominant-negative form of ERRα. Whereas phosphorylated c-src was distributed around the podosome belt in GFP(Ct)-derived OCs on day 6 (Fig. 5G and H, see merged in I), it was diffusely localized in GFP-ΔAF2-2 cells (Fig. 5J and K, see merged in L). Similarly, vinculin localization was disrupted in GFP-ΔAF2 cells (data not shown) compared with GFP(Ct)-derived OCs at day 6. These results suggest that disturbing ERRα function impairs a signaling pathway linked to OPN, integrin β3, activated c-src, and actin organization. Moreover, and in agreement with the decrease in mature OC spreading, we observed a decrease in OC adhesion in GFP-ΔAF2 (1, 2, and 3)-derived OCs compared with GFP(Ct)-derived cells (by 40% in ΔAF2-1, 50% in ΔAF2-2, and 68% in ΔAF2-3 compared with GFP(Ct); Fig. 5M). It was observed that the differences in OC viability, as reflected by OC number, cannot account for the differences in spreading between GFP(Ct)- and GFP-ΔAF2 (1, 2, and 3)-derived OCs (T=0; Fig. 5N).

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