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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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HGF- and M-CSF–induced cytoskeletal reorganization is αvβ3 dependent. (A) β3+/+ or β3−/− OCs, cultured on coverslips, were treated with 100 ng/ml M-CSF or 50 ng/ml HGF for 10 min. Cells were stained with FITC–phalloidin for actin (green) and anti–α-actinin mAb (red). Although the absence of αvβ3 does not alter the peripheral ring of actin in untreated cells (CTR), redistribution of podosomes, along with α-actinin, into membrane extensions in response to growth factors is observed only in β3+/+ OCs (pseudocolor overlays). High magnification images of the peripheral membrane (center) show the reorganization of actin (ACT) and α-actinin (α-Act) from podosomes to membrane ruffles in β3+/+ but not in β3−/− OCs. Bar, 10 μm. (B) β3+/+ or β3−/− OCs, treated as in A, were lysed in a Triton X-100 buffer, and soluble and insoluble fractions were separated by centrifugation. Immunoblot analysis was used to detect the distribution of α-actinin in the two fractions. (C) β3−/− OCs transduced with the indicated mutants were treated with M-CSF (A) stained for actin (green) or α-actinin (red). Overlay high magnification images show a redistribution of podosomes into newly formed membrane extensions only in β3 WT and β3 Y747F/Y759F mutants. (D) Transduced OCs, treated as in C were lysed in a Triton X-100 buffer, and α-actinin distribution in the soluble fraction was analyzed by Western blot. (E) Immunolocalization of c-Fms in mature OCs expressing the indicated β3 mutants. Cells were stained for c-Fms (green) and costained with TRITC–phalloidin for actin (red). Overlays show similar distribution of c-Fms in all β3 mutants. Bar, 10 μm.
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fig5: HGF- and M-CSF–induced cytoskeletal reorganization is αvβ3 dependent. (A) β3+/+ or β3−/− OCs, cultured on coverslips, were treated with 100 ng/ml M-CSF or 50 ng/ml HGF for 10 min. Cells were stained with FITC–phalloidin for actin (green) and anti–α-actinin mAb (red). Although the absence of αvβ3 does not alter the peripheral ring of actin in untreated cells (CTR), redistribution of podosomes, along with α-actinin, into membrane extensions in response to growth factors is observed only in β3+/+ OCs (pseudocolor overlays). High magnification images of the peripheral membrane (center) show the reorganization of actin (ACT) and α-actinin (α-Act) from podosomes to membrane ruffles in β3+/+ but not in β3−/− OCs. Bar, 10 μm. (B) β3+/+ or β3−/− OCs, treated as in A, were lysed in a Triton X-100 buffer, and soluble and insoluble fractions were separated by centrifugation. Immunoblot analysis was used to detect the distribution of α-actinin in the two fractions. (C) β3−/− OCs transduced with the indicated mutants were treated with M-CSF (A) stained for actin (green) or α-actinin (red). Overlay high magnification images show a redistribution of podosomes into newly formed membrane extensions only in β3 WT and β3 Y747F/Y759F mutants. (D) Transduced OCs, treated as in C were lysed in a Triton X-100 buffer, and α-actinin distribution in the soluble fraction was analyzed by Western blot. (E) Immunolocalization of c-Fms in mature OCs expressing the indicated β3 mutants. Cells were stained for c-Fms (green) and costained with TRITC–phalloidin for actin (red). Overlays show similar distribution of c-Fms in all β3 mutants. Bar, 10 μm.

Mentions: The observation detailed in Fig. 4 led us to hypothesize that the β3 integrin controls cytoskeletal changes induced by growth factors. To address this issue, we stained unstimulated and growth factor–treated β3+/+ and β3−/− OCs with FITC–phalloidin and analyzed actin organization by confocal microscopy. Once again, the absence of β3 integrin does not alter the peripheral podosomal distribution of F-actin in untreated OCs (Fig. 5 A, CTR). However, in the presence of HGF or M-CSF, F-actin moves from the podosomes to short filamentous protrusions, consistent with lamellipodia formation, only in β3+/+ OCs (Fig. 5 A, low and high magnification; Table I). To confirm that this observation is an integrin-dependent consequence of podosome reorganization, we examined the distribution of α-actinin, a cytoskeletal protein involved in the formation and stability of podosomes, and a link between actin and integrins (Pavalko et al., 1991). α-Actinin distribution in β3+/+ and β3−/− OCs mirrors that of actin, both in the presence and absence of growth factors (Fig. 5 A). Consistent with this finding, immunoblot analysis shows an increase in the pool of α-actinin present in the Triton X-100 soluble fraction exclusively in β3+/+ OCs treated with HGF and M-CSF (Fig. 5 B). In β3−/− cells, α-actinin remains in the insoluble fraction. Similar results were obtained using OCs transduced with the different β3 mutants. Dramatic changes in the peripheral ring of actin are seen in response to M-CSF (Fig. 5 C), and the content of α-actinin in the Triton X-100 soluble fraction increases in β3 WT and β3 Y747F/Y759F mutants, but not in those transduced with β3-ΔC or β3 S752P (Fig. 5 D). The failure of β3- cells to respond to M-CSF is not dependent on different expression levels of c-Fms (Fig. S1) or on its different localization among the various β3 mutants (Fig. 5 E). These observations suggest that, in OCs, growth factor–induced reorganization of podosomes, leading to the formation of new membrane ruffles, is a β3-dependent event.


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)

HGF- and M-CSF–induced cytoskeletal reorganization is αvβ3 dependent. (A) β3+/+ or β3−/− OCs, cultured on coverslips, were treated with 100 ng/ml M-CSF or 50 ng/ml HGF for 10 min. Cells were stained with FITC–phalloidin for actin (green) and anti–α-actinin mAb (red). Although the absence of αvβ3 does not alter the peripheral ring of actin in untreated cells (CTR), redistribution of podosomes, along with α-actinin, into membrane extensions in response to growth factors is observed only in β3+/+ OCs (pseudocolor overlays). High magnification images of the peripheral membrane (center) show the reorganization of actin (ACT) and α-actinin (α-Act) from podosomes to membrane ruffles in β3+/+ but not in β3−/− OCs. Bar, 10 μm. (B) β3+/+ or β3−/− OCs, treated as in A, were lysed in a Triton X-100 buffer, and soluble and insoluble fractions were separated by centrifugation. Immunoblot analysis was used to detect the distribution of α-actinin in the two fractions. (C) β3−/− OCs transduced with the indicated mutants were treated with M-CSF (A) stained for actin (green) or α-actinin (red). Overlay high magnification images show a redistribution of podosomes into newly formed membrane extensions only in β3 WT and β3 Y747F/Y759F mutants. (D) Transduced OCs, treated as in C were lysed in a Triton X-100 buffer, and α-actinin distribution in the soluble fraction was analyzed by Western blot. (E) Immunolocalization of c-Fms in mature OCs expressing the indicated β3 mutants. Cells were stained for c-Fms (green) and costained with TRITC–phalloidin for actin (red). Overlays show similar distribution of c-Fms in all β3 mutants. Bar, 10 μm.
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fig5: HGF- and M-CSF–induced cytoskeletal reorganization is αvβ3 dependent. (A) β3+/+ or β3−/− OCs, cultured on coverslips, were treated with 100 ng/ml M-CSF or 50 ng/ml HGF for 10 min. Cells were stained with FITC–phalloidin for actin (green) and anti–α-actinin mAb (red). Although the absence of αvβ3 does not alter the peripheral ring of actin in untreated cells (CTR), redistribution of podosomes, along with α-actinin, into membrane extensions in response to growth factors is observed only in β3+/+ OCs (pseudocolor overlays). High magnification images of the peripheral membrane (center) show the reorganization of actin (ACT) and α-actinin (α-Act) from podosomes to membrane ruffles in β3+/+ but not in β3−/− OCs. Bar, 10 μm. (B) β3+/+ or β3−/− OCs, treated as in A, were lysed in a Triton X-100 buffer, and soluble and insoluble fractions were separated by centrifugation. Immunoblot analysis was used to detect the distribution of α-actinin in the two fractions. (C) β3−/− OCs transduced with the indicated mutants were treated with M-CSF (A) stained for actin (green) or α-actinin (red). Overlay high magnification images show a redistribution of podosomes into newly formed membrane extensions only in β3 WT and β3 Y747F/Y759F mutants. (D) Transduced OCs, treated as in C were lysed in a Triton X-100 buffer, and α-actinin distribution in the soluble fraction was analyzed by Western blot. (E) Immunolocalization of c-Fms in mature OCs expressing the indicated β3 mutants. Cells were stained for c-Fms (green) and costained with TRITC–phalloidin for actin (red). Overlays show similar distribution of c-Fms in all β3 mutants. Bar, 10 μm.
Mentions: The observation detailed in Fig. 4 led us to hypothesize that the β3 integrin controls cytoskeletal changes induced by growth factors. To address this issue, we stained unstimulated and growth factor–treated β3+/+ and β3−/− OCs with FITC–phalloidin and analyzed actin organization by confocal microscopy. Once again, the absence of β3 integrin does not alter the peripheral podosomal distribution of F-actin in untreated OCs (Fig. 5 A, CTR). However, in the presence of HGF or M-CSF, F-actin moves from the podosomes to short filamentous protrusions, consistent with lamellipodia formation, only in β3+/+ OCs (Fig. 5 A, low and high magnification; Table I). To confirm that this observation is an integrin-dependent consequence of podosome reorganization, we examined the distribution of α-actinin, a cytoskeletal protein involved in the formation and stability of podosomes, and a link between actin and integrins (Pavalko et al., 1991). α-Actinin distribution in β3+/+ and β3−/− OCs mirrors that of actin, both in the presence and absence of growth factors (Fig. 5 A). Consistent with this finding, immunoblot analysis shows an increase in the pool of α-actinin present in the Triton X-100 soluble fraction exclusively in β3+/+ OCs treated with HGF and M-CSF (Fig. 5 B). In β3−/− cells, α-actinin remains in the insoluble fraction. Similar results were obtained using OCs transduced with the different β3 mutants. Dramatic changes in the peripheral ring of actin are seen in response to M-CSF (Fig. 5 C), and the content of α-actinin in the Triton X-100 soluble fraction increases in β3 WT and β3 Y747F/Y759F mutants, but not in those transduced with β3-ΔC or β3 S752P (Fig. 5 D). The failure of β3- cells to respond to M-CSF is not dependent on different expression levels of c-Fms (Fig. S1) or on its different localization among the various β3 mutants (Fig. 5 E). These observations suggest that, in OCs, growth factor–induced reorganization of podosomes, leading to the formation of new membrane ruffles, is a β3-dependent event.

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