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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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c-Src and c-Cbl, but not Pyk2, activation is impaired in β3−/− OCs. (A) Serum-starved pre-OCs were lifted and maintained in suspension (S) or replated on VN for 30 or 60 min. Equal amounts of proteins were immunoprecipitated with anti-Pyk2 mAb followed by immunoblot with antiphosphotyrosine mAb 4G10. Aliquots of the same lysate were immunoprecipitated with an antiphosphotyrosine mAb (PY99) followed by c-Src or c-Cbl immunoblot. Pyk2 phosphorylation, although not occurring in suspended cells, is indistinguishable in β3−/− and β3+/+ pre-OCs plated onto VN. In contrast, c-Cbl and c-Src phosphorylation occurs in a β3-dependent manner. (B) Lysates from β3+/+ or β3−/− pre-OCs, adherent with time on VN (30 or 60 min), were subjected to Pyk2 immunoprecipitation followed by c-Src or antiphosphotyrosine (PY99) immunoblot. In the absence of the β3 integrin, Pyk2 does not associate with c-Src. (C) β3−/− pre-OCs expressing the indicated β3 mutants were maintained in suspension (S) or were adherent (A) on VN for 30 min. Immunoprecipitation of lysates with antiphosphotyrosine (PY99) preceded immunoblot analysis of Pyk2, c-Cbl, and phospho-Src (Y416). β-Actin is the loading control. c-Src and c-Cbl phosphorylation are arrested in cells expressing β3-ΔC or β3 S752 P, but not β3 WT or β3 Y747F/Y759F. In all circumstances, Pyk2 phosphorylation remains intact. (D) Adherent OCs expressing the indicated β3 mutants were lysed, immunoprecipitated with Pyk2, and immunoblotted for β3 and paxillin. Association of Pyk2 with β3, but not with paxillin, requires intact β3 cytoplasmic tail and Ser752. Immunoblot for β3 shows similar expression level of the integrin in all mutant cells.
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fig8: c-Src and c-Cbl, but not Pyk2, activation is impaired in β3−/− OCs. (A) Serum-starved pre-OCs were lifted and maintained in suspension (S) or replated on VN for 30 or 60 min. Equal amounts of proteins were immunoprecipitated with anti-Pyk2 mAb followed by immunoblot with antiphosphotyrosine mAb 4G10. Aliquots of the same lysate were immunoprecipitated with an antiphosphotyrosine mAb (PY99) followed by c-Src or c-Cbl immunoblot. Pyk2 phosphorylation, although not occurring in suspended cells, is indistinguishable in β3−/− and β3+/+ pre-OCs plated onto VN. In contrast, c-Cbl and c-Src phosphorylation occurs in a β3-dependent manner. (B) Lysates from β3+/+ or β3−/− pre-OCs, adherent with time on VN (30 or 60 min), were subjected to Pyk2 immunoprecipitation followed by c-Src or antiphosphotyrosine (PY99) immunoblot. In the absence of the β3 integrin, Pyk2 does not associate with c-Src. (C) β3−/− pre-OCs expressing the indicated β3 mutants were maintained in suspension (S) or were adherent (A) on VN for 30 min. Immunoprecipitation of lysates with antiphosphotyrosine (PY99) preceded immunoblot analysis of Pyk2, c-Cbl, and phospho-Src (Y416). β-Actin is the loading control. c-Src and c-Cbl phosphorylation are arrested in cells expressing β3-ΔC or β3 S752 P, but not β3 WT or β3 Y747F/Y759F. In all circumstances, Pyk2 phosphorylation remains intact. (D) Adherent OCs expressing the indicated β3 mutants were lysed, immunoprecipitated with Pyk2, and immunoblotted for β3 and paxillin. Association of Pyk2 with β3, but not with paxillin, requires intact β3 cytoplasmic tail and Ser752. Immunoblot for β3 shows similar expression level of the integrin in all mutant cells.

Mentions: The data presented thus far establish that the β3 integrin plays an essential role in OC cytoskeletal function, a process mediated at least in part by Rho family GTPases. These observations prompted us to examine the more proximal events thought to mediate αvβ3 signal transduction. Pyk2, believed to be central to the mechanisms by which OCs resorb bone, is activated when these cells interact with αvβ3 ligands (Duong et al., 2000; Sanjay et al., 2001). Surprisingly, however, Pyk2 activation, as manifest by its phosphorylation, is indistinguishable in β3+/+ and β3−/− pre-OCs plated on VN for 30 and 60 min (Fig. 8 A, top). In contrast, c-Src phosphorylation occurs in a β3-dependent manner, and c-Cbl phosphorylation is markedly reduced in the absence of the integrin (Fig. 8 A). In keeping with this observation, Pyk2 and c-Src coprecipitate in VN-adherent β3+/+ cells but not those lacking the β3 integrin (Fig. 8 B). Furthermore, β3−/− pre-OCs expressing all β3 constructs show phosphorylation of Pyk2 upon adhesion, whereas c-Src and c-Cbl activation is defective in β3-ΔC and S752P (Fig. 8 C). Pfaff and Jurdic (2001) have recently shown direct interaction of both Pyk2 and paxillin, a structural component of podosomes, with the COOH-terminal region of the integrin β3 tail. Interestingly, we find that binding of Pyk2 to paxillin is identical in all β3 mutants, but its ability to bind the β3 receptor itself is dependent on an intact cytoplasmic tail and S752 (Fig. 8 D).


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)

c-Src and c-Cbl, but not Pyk2, activation is impaired in β3−/− OCs. (A) Serum-starved pre-OCs were lifted and maintained in suspension (S) or replated on VN for 30 or 60 min. Equal amounts of proteins were immunoprecipitated with anti-Pyk2 mAb followed by immunoblot with antiphosphotyrosine mAb 4G10. Aliquots of the same lysate were immunoprecipitated with an antiphosphotyrosine mAb (PY99) followed by c-Src or c-Cbl immunoblot. Pyk2 phosphorylation, although not occurring in suspended cells, is indistinguishable in β3−/− and β3+/+ pre-OCs plated onto VN. In contrast, c-Cbl and c-Src phosphorylation occurs in a β3-dependent manner. (B) Lysates from β3+/+ or β3−/− pre-OCs, adherent with time on VN (30 or 60 min), were subjected to Pyk2 immunoprecipitation followed by c-Src or antiphosphotyrosine (PY99) immunoblot. In the absence of the β3 integrin, Pyk2 does not associate with c-Src. (C) β3−/− pre-OCs expressing the indicated β3 mutants were maintained in suspension (S) or were adherent (A) on VN for 30 min. Immunoprecipitation of lysates with antiphosphotyrosine (PY99) preceded immunoblot analysis of Pyk2, c-Cbl, and phospho-Src (Y416). β-Actin is the loading control. c-Src and c-Cbl phosphorylation are arrested in cells expressing β3-ΔC or β3 S752 P, but not β3 WT or β3 Y747F/Y759F. In all circumstances, Pyk2 phosphorylation remains intact. (D) Adherent OCs expressing the indicated β3 mutants were lysed, immunoprecipitated with Pyk2, and immunoblotted for β3 and paxillin. Association of Pyk2 with β3, but not with paxillin, requires intact β3 cytoplasmic tail and Ser752. Immunoblot for β3 shows similar expression level of the integrin in all mutant cells.
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fig8: c-Src and c-Cbl, but not Pyk2, activation is impaired in β3−/− OCs. (A) Serum-starved pre-OCs were lifted and maintained in suspension (S) or replated on VN for 30 or 60 min. Equal amounts of proteins were immunoprecipitated with anti-Pyk2 mAb followed by immunoblot with antiphosphotyrosine mAb 4G10. Aliquots of the same lysate were immunoprecipitated with an antiphosphotyrosine mAb (PY99) followed by c-Src or c-Cbl immunoblot. Pyk2 phosphorylation, although not occurring in suspended cells, is indistinguishable in β3−/− and β3+/+ pre-OCs plated onto VN. In contrast, c-Cbl and c-Src phosphorylation occurs in a β3-dependent manner. (B) Lysates from β3+/+ or β3−/− pre-OCs, adherent with time on VN (30 or 60 min), were subjected to Pyk2 immunoprecipitation followed by c-Src or antiphosphotyrosine (PY99) immunoblot. In the absence of the β3 integrin, Pyk2 does not associate with c-Src. (C) β3−/− pre-OCs expressing the indicated β3 mutants were maintained in suspension (S) or were adherent (A) on VN for 30 min. Immunoprecipitation of lysates with antiphosphotyrosine (PY99) preceded immunoblot analysis of Pyk2, c-Cbl, and phospho-Src (Y416). β-Actin is the loading control. c-Src and c-Cbl phosphorylation are arrested in cells expressing β3-ΔC or β3 S752 P, but not β3 WT or β3 Y747F/Y759F. In all circumstances, Pyk2 phosphorylation remains intact. (D) Adherent OCs expressing the indicated β3 mutants were lysed, immunoprecipitated with Pyk2, and immunoblotted for β3 and paxillin. Association of Pyk2 with β3, but not with paxillin, requires intact β3 cytoplasmic tail and Ser752. Immunoblot for β3 shows similar expression level of the integrin in all mutant cells.
Mentions: The data presented thus far establish that the β3 integrin plays an essential role in OC cytoskeletal function, a process mediated at least in part by Rho family GTPases. These observations prompted us to examine the more proximal events thought to mediate αvβ3 signal transduction. Pyk2, believed to be central to the mechanisms by which OCs resorb bone, is activated when these cells interact with αvβ3 ligands (Duong et al., 2000; Sanjay et al., 2001). Surprisingly, however, Pyk2 activation, as manifest by its phosphorylation, is indistinguishable in β3+/+ and β3−/− pre-OCs plated on VN for 30 and 60 min (Fig. 8 A, top). In contrast, c-Src phosphorylation occurs in a β3-dependent manner, and c-Cbl phosphorylation is markedly reduced in the absence of the integrin (Fig. 8 A). In keeping with this observation, Pyk2 and c-Src coprecipitate in VN-adherent β3+/+ cells but not those lacking the β3 integrin (Fig. 8 B). Furthermore, β3−/− pre-OCs expressing all β3 constructs show phosphorylation of Pyk2 upon adhesion, whereas c-Src and c-Cbl activation is defective in β3-ΔC and S752P (Fig. 8 C). Pfaff and Jurdic (2001) have recently shown direct interaction of both Pyk2 and paxillin, a structural component of podosomes, with the COOH-terminal region of the integrin β3 tail. Interestingly, we find that binding of Pyk2 to paxillin is identical in all β3 mutants, but its ability to bind the β3 receptor itself is dependent on an intact cytoplasmic tail and S752 (Fig. 8 D).

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