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Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin.

Faccio R, Novack DV, Zallone A, Ross FP, Teitelbaum SL - J. Cell Biol. (2003)

Bottom Line: Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors.Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin.Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, 216 South Kingshighway, St. Louis, MO 63110, USA.

ABSTRACT
The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

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S752 in the β3 cytoplasmic domain regulates integrin localization. Mature β3−/− OCs transduced with the indicated β3 mutants were generated in the presence of RANKL and high dose M-CSF on glass coverslips (A) or dentin (B). Cells were stained with an anti–human β3 mAb (1A2) (red) and with FITC–phalloidin to detect actin distribution (green) and analyzed by confocal microscopy. Merged pseudocolored images obtained from the same confocal plane show the colocalization of β3 and F-actin in yellow. (A) OCs on coverslips bearing hβ3 WT or hβ3 Y747F/Y759F organize αvβ3 (a and j) in a donut-like structure, around the F-actin core (b and k) of podosomes (merged on c and l). β3-ΔC (d and e) or the S752P mutation (g and h) fail to organize around the F-actin core, but are diffuse on the cell surface (merged on f and i). (B) β3 WT or β3 Y747F/Y759F integrin (a and j) and actin (b and k) in OCs grown on dentin form well-defined, colocalizing rings (merged on c and l). In contrast, in β3-ΔC or β3 S752P mutants, the integrin (d and g) is diffusely distributed and fails to localize with the actin ring (e and h, merged in f and i). Bars, 5 μm.
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fig2: S752 in the β3 cytoplasmic domain regulates integrin localization. Mature β3−/− OCs transduced with the indicated β3 mutants were generated in the presence of RANKL and high dose M-CSF on glass coverslips (A) or dentin (B). Cells were stained with an anti–human β3 mAb (1A2) (red) and with FITC–phalloidin to detect actin distribution (green) and analyzed by confocal microscopy. Merged pseudocolored images obtained from the same confocal plane show the colocalization of β3 and F-actin in yellow. (A) OCs on coverslips bearing hβ3 WT or hβ3 Y747F/Y759F organize αvβ3 (a and j) in a donut-like structure, around the F-actin core (b and k) of podosomes (merged on c and l). β3-ΔC (d and e) or the S752P mutation (g and h) fail to organize around the F-actin core, but are diffuse on the cell surface (merged on f and i). (B) β3 WT or β3 Y747F/Y759F integrin (a and j) and actin (b and k) in OCs grown on dentin form well-defined, colocalizing rings (merged on c and l). In contrast, in β3-ΔC or β3 S752P mutants, the integrin (d and g) is diffusely distributed and fails to localize with the actin ring (e and h, merged in f and i). Bars, 5 μm.

Mentions: To delineate actin organization, OCs expressing the various mutants were immunostained with the anti–human β3 mAb 1A2 and costained with FITC–phalloidin (Fig. 2 A). The β3 integrin is organized in rosette-like structures, surrounding a core of F-actin bundles, in podosomes as previously described (Faccio et al., 2002). In nonresorbing OCs on glass, podosomes accumulate at high density at the periphery, yielding a row of actin dots flanked on each side by β3 integrin bands. This organization is present in β3−/− OCs bearing β3 WT or the β3 Y747F/Y759F mutation. Lack of functional β3, as in β3-ΔC and β3 S752P mutants, completely abrogates the distribution of the integrin around the peripheral ring of F-actin, despite the normal appearance of the actin cytoskeleton. Similar results were obtained with another anti–human β3 mAb, AP3 (unpublished data). Interestingly other cytoskeletal components, including talin and vinculin, remain normally distributed in the podosomes of all mutants (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200212082/DC1).


Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin.

Faccio R, Novack DV, Zallone A, Ross FP, Teitelbaum SL - J. Cell Biol. (2003)

S752 in the β3 cytoplasmic domain regulates integrin localization. Mature β3−/− OCs transduced with the indicated β3 mutants were generated in the presence of RANKL and high dose M-CSF on glass coverslips (A) or dentin (B). Cells were stained with an anti–human β3 mAb (1A2) (red) and with FITC–phalloidin to detect actin distribution (green) and analyzed by confocal microscopy. Merged pseudocolored images obtained from the same confocal plane show the colocalization of β3 and F-actin in yellow. (A) OCs on coverslips bearing hβ3 WT or hβ3 Y747F/Y759F organize αvβ3 (a and j) in a donut-like structure, around the F-actin core (b and k) of podosomes (merged on c and l). β3-ΔC (d and e) or the S752P mutation (g and h) fail to organize around the F-actin core, but are diffuse on the cell surface (merged on f and i). (B) β3 WT or β3 Y747F/Y759F integrin (a and j) and actin (b and k) in OCs grown on dentin form well-defined, colocalizing rings (merged on c and l). In contrast, in β3-ΔC or β3 S752P mutants, the integrin (d and g) is diffusely distributed and fails to localize with the actin ring (e and h, merged in f and i). Bars, 5 μm.
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Related In: Results  -  Collection

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fig2: S752 in the β3 cytoplasmic domain regulates integrin localization. Mature β3−/− OCs transduced with the indicated β3 mutants were generated in the presence of RANKL and high dose M-CSF on glass coverslips (A) or dentin (B). Cells were stained with an anti–human β3 mAb (1A2) (red) and with FITC–phalloidin to detect actin distribution (green) and analyzed by confocal microscopy. Merged pseudocolored images obtained from the same confocal plane show the colocalization of β3 and F-actin in yellow. (A) OCs on coverslips bearing hβ3 WT or hβ3 Y747F/Y759F organize αvβ3 (a and j) in a donut-like structure, around the F-actin core (b and k) of podosomes (merged on c and l). β3-ΔC (d and e) or the S752P mutation (g and h) fail to organize around the F-actin core, but are diffuse on the cell surface (merged on f and i). (B) β3 WT or β3 Y747F/Y759F integrin (a and j) and actin (b and k) in OCs grown on dentin form well-defined, colocalizing rings (merged on c and l). In contrast, in β3-ΔC or β3 S752P mutants, the integrin (d and g) is diffusely distributed and fails to localize with the actin ring (e and h, merged in f and i). Bars, 5 μm.
Mentions: To delineate actin organization, OCs expressing the various mutants were immunostained with the anti–human β3 mAb 1A2 and costained with FITC–phalloidin (Fig. 2 A). The β3 integrin is organized in rosette-like structures, surrounding a core of F-actin bundles, in podosomes as previously described (Faccio et al., 2002). In nonresorbing OCs on glass, podosomes accumulate at high density at the periphery, yielding a row of actin dots flanked on each side by β3 integrin bands. This organization is present in β3−/− OCs bearing β3 WT or the β3 Y747F/Y759F mutation. Lack of functional β3, as in β3-ΔC and β3 S752P mutants, completely abrogates the distribution of the integrin around the peripheral ring of F-actin, despite the normal appearance of the actin cytoskeleton. Similar results were obtained with another anti–human β3 mAb, AP3 (unpublished data). Interestingly other cytoskeletal components, including talin and vinculin, remain normally distributed in the podosomes of all mutants (see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200212082/DC1).

Bottom Line: Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors.Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin.Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, 216 South Kingshighway, St. Louis, MO 63110, USA.

ABSTRACT
The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

Show MeSH
Related in: MedlinePlus