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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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Pre-OC adhesion and migration require functional β3 cytoplasmic domain. (A) Adhesion assays to OPN were performed using β3−/− pre-OCs bearing the indicated β3 constructs. Adhesion to OPN is decreased threefold in cells carrying β3-ΔC or β3 S752P (CTR bars, dark gray) and is not affected by AP5 (light gray), HGF (black), or M-CSF (gray), which induce integrin activation. In contrast, the same factors increased adhesion of β3 WT– and β3 Y747F/Y759F–expressing cells by 40–50%. (B) Migration assay toward OPN was performed on the same cells as in A. Migration of untreated cells toward OPN (CTR, dark gray) is impaired in β3-ΔC and β3 S752P mutants. Moreover, AP5 (light gray), HGF (black), and M-CSF (gray) fail to rescue the directed mobility of these cells, whereas the same factors enhance the migratory capacity of β3 WT– and β3 Y747F/Y759F–expressing cells. *, P < 0.01 vs. CTR; **, P < 0.05 vs. CTR.
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fig6: Pre-OC adhesion and migration require functional β3 cytoplasmic domain. (A) Adhesion assays to OPN were performed using β3−/− pre-OCs bearing the indicated β3 constructs. Adhesion to OPN is decreased threefold in cells carrying β3-ΔC or β3 S752P (CTR bars, dark gray) and is not affected by AP5 (light gray), HGF (black), or M-CSF (gray), which induce integrin activation. In contrast, the same factors increased adhesion of β3 WT– and β3 Y747F/Y759F–expressing cells by 40–50%. (B) Migration assay toward OPN was performed on the same cells as in A. Migration of untreated cells toward OPN (CTR, dark gray) is impaired in β3-ΔC and β3 S752P mutants. Moreover, AP5 (light gray), HGF (black), and M-CSF (gray) fail to rescue the directed mobility of these cells, whereas the same factors enhance the migratory capacity of β3 WT– and β3 Y747F/Y759F–expressing cells. *, P < 0.01 vs. CTR; **, P < 0.05 vs. CTR.

Mentions: The fact that β3-ΔC– and β3 S752P–expressing OCs fail to spread in response to growth factors suggests that interaction of these mutant integrins with the extracellular matrix may be defective. To address this issue, we plated β3−/− pre-OCs bearing the various constructs on osteopontin (OPN), a substrate recognized by the αvβ3 integrin, or, as negative control, on BSA. Pre-OC adhesion to OPN is decreased threefold in cells carrying the β3-ΔC or S752P mutations, compared with β3 WT pre-OCs (Fig. 6 A), mirroring the defective adhesion of β3−/− cells (unpublished data). Furthermore, pretreatment with AP5, HGF, or M-CSF, all of which activate the integrin (Fig. 3), enhances the number of adherent β3 WT by 40–50%, and to a lesser extent β3 Y747F/Y759F–expressing cells (Fig. 6 A). On the other hand, these integrin-activating agents fail to impact the adhesion of β3-ΔC– and β3 S752P–bearing pre-OCs. Similar results were obtained using VN as a substrate (unpublished data). In contrast, adhesion to native collagen, a β3 integrin–independent substrate, was similar in all mutants and was not enhanced by growth factors (unpublished data).


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)

Pre-OC adhesion and migration require functional β3 cytoplasmic domain. (A) Adhesion assays to OPN were performed using β3−/− pre-OCs bearing the indicated β3 constructs. Adhesion to OPN is decreased threefold in cells carrying β3-ΔC or β3 S752P (CTR bars, dark gray) and is not affected by AP5 (light gray), HGF (black), or M-CSF (gray), which induce integrin activation. In contrast, the same factors increased adhesion of β3 WT– and β3 Y747F/Y759F–expressing cells by 40–50%. (B) Migration assay toward OPN was performed on the same cells as in A. Migration of untreated cells toward OPN (CTR, dark gray) is impaired in β3-ΔC and β3 S752P mutants. Moreover, AP5 (light gray), HGF (black), and M-CSF (gray) fail to rescue the directed mobility of these cells, whereas the same factors enhance the migratory capacity of β3 WT– and β3 Y747F/Y759F–expressing cells. *, P < 0.01 vs. CTR; **, P < 0.05 vs. CTR.
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Related In: Results  -  Collection

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fig6: Pre-OC adhesion and migration require functional β3 cytoplasmic domain. (A) Adhesion assays to OPN were performed using β3−/− pre-OCs bearing the indicated β3 constructs. Adhesion to OPN is decreased threefold in cells carrying β3-ΔC or β3 S752P (CTR bars, dark gray) and is not affected by AP5 (light gray), HGF (black), or M-CSF (gray), which induce integrin activation. In contrast, the same factors increased adhesion of β3 WT– and β3 Y747F/Y759F–expressing cells by 40–50%. (B) Migration assay toward OPN was performed on the same cells as in A. Migration of untreated cells toward OPN (CTR, dark gray) is impaired in β3-ΔC and β3 S752P mutants. Moreover, AP5 (light gray), HGF (black), and M-CSF (gray) fail to rescue the directed mobility of these cells, whereas the same factors enhance the migratory capacity of β3 WT– and β3 Y747F/Y759F–expressing cells. *, P < 0.01 vs. CTR; **, P < 0.05 vs. CTR.
Mentions: The fact that β3-ΔC– and β3 S752P–expressing OCs fail to spread in response to growth factors suggests that interaction of these mutant integrins with the extracellular matrix may be defective. To address this issue, we plated β3−/− pre-OCs bearing the various constructs on osteopontin (OPN), a substrate recognized by the αvβ3 integrin, or, as negative control, on BSA. Pre-OC adhesion to OPN is decreased threefold in cells carrying the β3-ΔC or S752P mutations, compared with β3 WT pre-OCs (Fig. 6 A), mirroring the defective adhesion of β3−/− cells (unpublished data). Furthermore, pretreatment with AP5, HGF, or M-CSF, all of which activate the integrin (Fig. 3), enhances the number of adherent β3 WT by 40–50%, and to a lesser extent β3 Y747F/Y759F–expressing cells (Fig. 6 A). On the other hand, these integrin-activating agents fail to impact the adhesion of β3-ΔC– and β3 S752P–bearing pre-OCs. Similar results were obtained using VN as a substrate (unpublished data). In contrast, adhesion to native collagen, a β3 integrin–independent substrate, was similar in all mutants and was not enhanced by growth factors (unpublished data).

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