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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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Growth factor activation of Rho GTPases requires functional β3 cytoplasmic domain. (A) OCs were exposed to HGF or M-CSF for 15 min, and Rho activation was assessed by pull-down binding assay. β3+/+ OCs respond to both growth factors with a sixfold increase in GTP-bound Rho, whereas β3−/− OCs do not respond to either growth factor. Immunoblot for total Rho in cell lysates is shown below. (B) β3+/+ and β3−/− cells were stimulated with M-CSF for the indicated times, and Rac activation was assessed by pull-down binding assay. As a loading control, a fraction of the cell lysates was immunoblotted with the Rac mAb. Rac activation occurs only in β3+/+ OCs, in a biphasic manner. (C) The experiment described in A was repeated with β3−/− OCs transfected with the indicated β3 constructs. Whereas β3 WT– and β3 Y747F/Y759F–bearing OCs respond like their β3+/+ counterparts, no such induction occurs in those expressing β3-ΔC or S752P.
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fig7: Growth factor activation of Rho GTPases requires functional β3 cytoplasmic domain. (A) OCs were exposed to HGF or M-CSF for 15 min, and Rho activation was assessed by pull-down binding assay. β3+/+ OCs respond to both growth factors with a sixfold increase in GTP-bound Rho, whereas β3−/− OCs do not respond to either growth factor. Immunoblot for total Rho in cell lysates is shown below. (B) β3+/+ and β3−/− cells were stimulated with M-CSF for the indicated times, and Rac activation was assessed by pull-down binding assay. As a loading control, a fraction of the cell lysates was immunoblotted with the Rac mAb. Rac activation occurs only in β3+/+ OCs, in a biphasic manner. (C) The experiment described in A was repeated with β3−/− OCs transfected with the indicated β3 constructs. Whereas β3 WT– and β3 Y747F/Y759F–bearing OCs respond like their β3+/+ counterparts, no such induction occurs in those expressing β3-ΔC or S752P.

Mentions: Numerous signal transduction molecules, including Rho GTPases, associate with integrin complexes in adherent cells and regulate adhesion-dependent morphological changes (Pavalko et al., 1991; Yamada and Miyamoto, 1995; Clark et al., 1998). Furthermore, distinct aspects of adhesion and migration are controlled by different Rho family members. For example, Rho, Rac, and Cdc42, respectively, regulate the formation of stress fibers, lamellipodia, and filopodia. As β3−/− OCs exhibit defective adhesion, spreading, and cytoskeletal rearrangement in response to growth factors, we asked if these phenomena reflect impaired activation of Rho GTPases. To this end, β3−/− and β3+/+ OCs were exposed to HGF or M-CSF, and Rho and Rac activation were assessed by pull-down binding assays. Mirroring the cytoskeletal defects, Rho activation in response to both growth factors is completely arrested in β3−/− OCs, compared with the sixfold increase seen in β3+/+ OCs (Fig. 7 A). Similarly, whereas Rac is activated in a biphasic manner in β3+/+ OCs exposed to M-CSF, no such activation occurs in β3−/− OCs (Fig. 7 B). To determine if integrin-mediated Rho activation is dependent on an intact β3 cytoplasmic domain, we repeated these experiments with β3−/− OCs transduced with the various β3 constructs. Whereas β3 WT– and β3 Y747F/Y759F–expressing OCs respond to growth factor with an increase in GTP-bound Rho, no induction occurs in those expressing β3-ΔC or β3 S752P (Fig. 7 C).


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)

Growth factor activation of Rho GTPases requires functional β3 cytoplasmic domain. (A) OCs were exposed to HGF or M-CSF for 15 min, and Rho activation was assessed by pull-down binding assay. β3+/+ OCs respond to both growth factors with a sixfold increase in GTP-bound Rho, whereas β3−/− OCs do not respond to either growth factor. Immunoblot for total Rho in cell lysates is shown below. (B) β3+/+ and β3−/− cells were stimulated with M-CSF for the indicated times, and Rac activation was assessed by pull-down binding assay. As a loading control, a fraction of the cell lysates was immunoblotted with the Rac mAb. Rac activation occurs only in β3+/+ OCs, in a biphasic manner. (C) The experiment described in A was repeated with β3−/− OCs transfected with the indicated β3 constructs. Whereas β3 WT– and β3 Y747F/Y759F–bearing OCs respond like their β3+/+ counterparts, no such induction occurs in those expressing β3-ΔC or S752P.
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Related In: Results  -  Collection

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fig7: Growth factor activation of Rho GTPases requires functional β3 cytoplasmic domain. (A) OCs were exposed to HGF or M-CSF for 15 min, and Rho activation was assessed by pull-down binding assay. β3+/+ OCs respond to both growth factors with a sixfold increase in GTP-bound Rho, whereas β3−/− OCs do not respond to either growth factor. Immunoblot for total Rho in cell lysates is shown below. (B) β3+/+ and β3−/− cells were stimulated with M-CSF for the indicated times, and Rac activation was assessed by pull-down binding assay. As a loading control, a fraction of the cell lysates was immunoblotted with the Rac mAb. Rac activation occurs only in β3+/+ OCs, in a biphasic manner. (C) The experiment described in A was repeated with β3−/− OCs transfected with the indicated β3 constructs. Whereas β3 WT– and β3 Y747F/Y759F–bearing OCs respond like their β3+/+ counterparts, no such induction occurs in those expressing β3-ΔC or S752P.
Mentions: Numerous signal transduction molecules, including Rho GTPases, associate with integrin complexes in adherent cells and regulate adhesion-dependent morphological changes (Pavalko et al., 1991; Yamada and Miyamoto, 1995; Clark et al., 1998). Furthermore, distinct aspects of adhesion and migration are controlled by different Rho family members. For example, Rho, Rac, and Cdc42, respectively, regulate the formation of stress fibers, lamellipodia, and filopodia. As β3−/− OCs exhibit defective adhesion, spreading, and cytoskeletal rearrangement in response to growth factors, we asked if these phenomena reflect impaired activation of Rho GTPases. To this end, β3−/− and β3+/+ OCs were exposed to HGF or M-CSF, and Rho and Rac activation were assessed by pull-down binding assays. Mirroring the cytoskeletal defects, Rho activation in response to both growth factors is completely arrested in β3−/− OCs, compared with the sixfold increase seen in β3+/+ OCs (Fig. 7 A). Similarly, whereas Rac is activated in a biphasic manner in β3+/+ OCs exposed to M-CSF, no such activation occurs in β3−/− OCs (Fig. 7 B). To determine if integrin-mediated Rho activation is dependent on an intact β3 cytoplasmic domain, we repeated these experiments with β3−/− OCs transduced with the various β3 constructs. Whereas β3 WT– and β3 Y747F/Y759F–expressing OCs respond to growth factor with an increase in GTP-bound Rho, no induction occurs in those expressing β3-ΔC or β3 S752P (Fig. 7 C).

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