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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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Localization of αvβ3 in lamellipodia and membrane extensions is regulated by functional β3 cytoplasmic domain. (A) Mature β3−/− OCs expressing the indicated β3 integrin constructs were either untreated (CTR) or stimulated with M-CSF or HGF to induce integrin activation, and stained with mAb AP5 (red), which detects only activated integrin. In control circumstances, cells expressing β3 WT (a) and β3 Y747F/Y759F (j) present the activated integrin in lamellipodia (arrows). In contrast, in OCs expressing β3-ΔC (d) and β3 S752P (g), the activated integrin is randomly distributed throughout the cytoplasm. M-CSF or HGF clusters activated β3 WT (b and c, arrows) and β3 Y747F/Y759F (k and l arrows) at the cell's edge, while β3-ΔC (e and f) and β3S752P (h and i) remain diffusely distributed. (B) β3−/− OCs expressing hβ3 WT or hβ3-ΔC cultured on dentin were stained with AP5 (red) and FITC–phalloidin (green). Shown are pseudocolored overlay images taken in the same confocal plane. β3 WT OCs stimulated with HGF or M-CSF form AP5-containing membrane extensions (arrows). In contrast, OCs expressing β3-ΔC show no response to the cytokines, despite their ability to form an actin ring. Bars, 10 μm.
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fig4: Localization of αvβ3 in lamellipodia and membrane extensions is regulated by functional β3 cytoplasmic domain. (A) Mature β3−/− OCs expressing the indicated β3 integrin constructs were either untreated (CTR) or stimulated with M-CSF or HGF to induce integrin activation, and stained with mAb AP5 (red), which detects only activated integrin. In control circumstances, cells expressing β3 WT (a) and β3 Y747F/Y759F (j) present the activated integrin in lamellipodia (arrows). In contrast, in OCs expressing β3-ΔC (d) and β3 S752P (g), the activated integrin is randomly distributed throughout the cytoplasm. M-CSF or HGF clusters activated β3 WT (b and c, arrows) and β3 Y747F/Y759F (k and l arrows) at the cell's edge, while β3-ΔC (e and f) and β3S752P (h and i) remain diffusely distributed. (B) β3−/− OCs expressing hβ3 WT or hβ3-ΔC cultured on dentin were stained with AP5 (red) and FITC–phalloidin (green). Shown are pseudocolored overlay images taken in the same confocal plane. β3 WT OCs stimulated with HGF or M-CSF form AP5-containing membrane extensions (arrows). In contrast, OCs expressing β3-ΔC show no response to the cytokines, despite their ability to form an actin ring. Bars, 10 μm.

Mentions: Upon growth factor activation, OC αvβ3 moves to the motile region of the cell membrane (Faccio et al., 2002). Having found that dysfunctional β3 mutants fail to properly localize in resting OCs, and do not become activated in response to growth factors, we determined if these mutants also fail to localize properly in the newly formed lamellipodia in response to growth factor stimulation. Thus, β3−/− OCs bearing WT β3, β3-ΔC, β3 Y747F/Y759F, or β3 S752P constructs grown on coverslips were exposed to HGF or M-CSF for 30 min and, after fixation, stained with AP5 (in high calcium) to detect localization of the activated form of αvβ3. Unstimulated OCs carrying β3 WT or the Y747F/Y759F mutation express the activated integrin along membrane ruffles and in lamellipodia (Fig. 4 A, CTR). Treatment with either growth factor induces AP5-positive membrane extensions (lamellipodia). This phenomenon is completely abrogated in β3-ΔC– and β3 S752P–bearing cells, which are unable to spread and form lamellipodia in response to growth factors (Fig. 4 A). These observations are confirmed by counting the percentage of cells with multiple lamellipodia extensions (Table I). These data show that M-CSF and HGF induce lamellipodia in cells expressing β3 WT and β3 Y747F/Y759F but not in those transduced with β3-ΔC or β3 S752P.


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)

Localization of αvβ3 in lamellipodia and membrane extensions is regulated by functional β3 cytoplasmic domain. (A) Mature β3−/− OCs expressing the indicated β3 integrin constructs were either untreated (CTR) or stimulated with M-CSF or HGF to induce integrin activation, and stained with mAb AP5 (red), which detects only activated integrin. In control circumstances, cells expressing β3 WT (a) and β3 Y747F/Y759F (j) present the activated integrin in lamellipodia (arrows). In contrast, in OCs expressing β3-ΔC (d) and β3 S752P (g), the activated integrin is randomly distributed throughout the cytoplasm. M-CSF or HGF clusters activated β3 WT (b and c, arrows) and β3 Y747F/Y759F (k and l arrows) at the cell's edge, while β3-ΔC (e and f) and β3S752P (h and i) remain diffusely distributed. (B) β3−/− OCs expressing hβ3 WT or hβ3-ΔC cultured on dentin were stained with AP5 (red) and FITC–phalloidin (green). Shown are pseudocolored overlay images taken in the same confocal plane. β3 WT OCs stimulated with HGF or M-CSF form AP5-containing membrane extensions (arrows). In contrast, OCs expressing β3-ΔC show no response to the cytokines, despite their ability to form an actin ring. Bars, 10 μm.
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Related In: Results  -  Collection

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fig4: Localization of αvβ3 in lamellipodia and membrane extensions is regulated by functional β3 cytoplasmic domain. (A) Mature β3−/− OCs expressing the indicated β3 integrin constructs were either untreated (CTR) or stimulated with M-CSF or HGF to induce integrin activation, and stained with mAb AP5 (red), which detects only activated integrin. In control circumstances, cells expressing β3 WT (a) and β3 Y747F/Y759F (j) present the activated integrin in lamellipodia (arrows). In contrast, in OCs expressing β3-ΔC (d) and β3 S752P (g), the activated integrin is randomly distributed throughout the cytoplasm. M-CSF or HGF clusters activated β3 WT (b and c, arrows) and β3 Y747F/Y759F (k and l arrows) at the cell's edge, while β3-ΔC (e and f) and β3S752P (h and i) remain diffusely distributed. (B) β3−/− OCs expressing hβ3 WT or hβ3-ΔC cultured on dentin were stained with AP5 (red) and FITC–phalloidin (green). Shown are pseudocolored overlay images taken in the same confocal plane. β3 WT OCs stimulated with HGF or M-CSF form AP5-containing membrane extensions (arrows). In contrast, OCs expressing β3-ΔC show no response to the cytokines, despite their ability to form an actin ring. Bars, 10 μm.
Mentions: Upon growth factor activation, OC αvβ3 moves to the motile region of the cell membrane (Faccio et al., 2002). Having found that dysfunctional β3 mutants fail to properly localize in resting OCs, and do not become activated in response to growth factors, we determined if these mutants also fail to localize properly in the newly formed lamellipodia in response to growth factor stimulation. Thus, β3−/− OCs bearing WT β3, β3-ΔC, β3 Y747F/Y759F, or β3 S752P constructs grown on coverslips were exposed to HGF or M-CSF for 30 min and, after fixation, stained with AP5 (in high calcium) to detect localization of the activated form of αvβ3. Unstimulated OCs carrying β3 WT or the Y747F/Y759F mutation express the activated integrin along membrane ruffles and in lamellipodia (Fig. 4 A, CTR). Treatment with either growth factor induces AP5-positive membrane extensions (lamellipodia). This phenomenon is completely abrogated in β3-ΔC– and β3 S752P–bearing cells, which are unable to spread and form lamellipodia in response to growth factors (Fig. 4 A). These observations are confirmed by counting the percentage of cells with multiple lamellipodia extensions (Table I). These data show that M-CSF and HGF induce lamellipodia in cells expressing β3 WT and β3 Y747F/Y759F but not in those transduced with β3-ΔC or β3 S752P.

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