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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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β3 S752 regulates inside-out, but not outside-in, activation of αvβ3. (A) β3−/− pre-OCs expressing the indicated mutants were incubated with AP5 (50 μg/ml) in low or high calcium and subjected to FACS® analysis. Results are presented as overlapping histograms for high (white) and low (gray) calcium treatments for each mutant. The tracings for high calcium buffer indicate basal activation of the integrin, as in this circumstance, AP5 binds previously activated αvβ3 integrin. A shift toward higher fluorescence intensity in low calcium buffer indicates that AP5 mediates integrin activation. In each group of transduced pre-OCs, incubation with AP5 in low calcium buffer yields a significant increase in mean fluorescence intensity, relative to high calcium buffer, indicating that activation by this anti-LIBS Ab from the cell's exterior does not depend on the β3 cytoplasmic domain. (B) Pre-OCs were preincubated with HGF (50 ng/ml, light gray bars) or M-CSF (100 ng/ml, dark gray bars) before staining with AP5 in high calcium buffer to determine the degree of growth factor–mediated integrin activation. Results are expressed as fold increase in the mean fluorescence intensity of AP5 staining compared with unstimulated control cells (CTR), arbitrarily designated as 1 for each untreated mutant.
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fig3: β3 S752 regulates inside-out, but not outside-in, activation of αvβ3. (A) β3−/− pre-OCs expressing the indicated mutants were incubated with AP5 (50 μg/ml) in low or high calcium and subjected to FACS® analysis. Results are presented as overlapping histograms for high (white) and low (gray) calcium treatments for each mutant. The tracings for high calcium buffer indicate basal activation of the integrin, as in this circumstance, AP5 binds previously activated αvβ3 integrin. A shift toward higher fluorescence intensity in low calcium buffer indicates that AP5 mediates integrin activation. In each group of transduced pre-OCs, incubation with AP5 in low calcium buffer yields a significant increase in mean fluorescence intensity, relative to high calcium buffer, indicating that activation by this anti-LIBS Ab from the cell's exterior does not depend on the β3 cytoplasmic domain. (B) Pre-OCs were preincubated with HGF (50 ng/ml, light gray bars) or M-CSF (100 ng/ml, dark gray bars) before staining with AP5 in high calcium buffer to determine the degree of growth factor–mediated integrin activation. Results are expressed as fold increase in the mean fluorescence intensity of AP5 staining compared with unstimulated control cells (CTR), arbitrarily designated as 1 for each untreated mutant.

Mentions: One possible explanation for the failure of β3-ΔC and β3 S752P to localize to podosomes is that the external domain of these mutants is not able to assume the activated, high-affinity conformation necessary for appropriate substrate interaction. One tool to assess the ability of β3 integrin to assume the activated conformation of the external domain is the anti–ligand-induced binding site (LIBS) antibody AP5. In low calcium buffer, this Ab binds all αvβ3 integrin on the cell surface, converting it to the active conformation. In high calcium buffer, AP5 binds only the integrin already in the activated state (Faccio et al., 2002). An increase in fluorescence intensity of αvβ3-expressing cells when AP5 binding is assessed in low calcium, relative to high calcium buffer, indicates that the external domain of the integrin can assume the activated conformation in response to the Ab. When pre-OCs transduced with β3-ΔC and β3 S752P, as well as with β3 WT and β3 Y747F/Y759F, are analyzed in this manner, there is an increase in AP5 binding in low calcium, indicating that each of these β3 constructs undergoes conformational change (Fig. 3 A). Thus, the failure of the β3-ΔC and β3 S752P mutants to properly localize in podosomes does not reflect an inability of these integrins to assume an activated conformation.


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)

β3 S752 regulates inside-out, but not outside-in, activation of αvβ3. (A) β3−/− pre-OCs expressing the indicated mutants were incubated with AP5 (50 μg/ml) in low or high calcium and subjected to FACS® analysis. Results are presented as overlapping histograms for high (white) and low (gray) calcium treatments for each mutant. The tracings for high calcium buffer indicate basal activation of the integrin, as in this circumstance, AP5 binds previously activated αvβ3 integrin. A shift toward higher fluorescence intensity in low calcium buffer indicates that AP5 mediates integrin activation. In each group of transduced pre-OCs, incubation with AP5 in low calcium buffer yields a significant increase in mean fluorescence intensity, relative to high calcium buffer, indicating that activation by this anti-LIBS Ab from the cell's exterior does not depend on the β3 cytoplasmic domain. (B) Pre-OCs were preincubated with HGF (50 ng/ml, light gray bars) or M-CSF (100 ng/ml, dark gray bars) before staining with AP5 in high calcium buffer to determine the degree of growth factor–mediated integrin activation. Results are expressed as fold increase in the mean fluorescence intensity of AP5 staining compared with unstimulated control cells (CTR), arbitrarily designated as 1 for each untreated mutant.
© Copyright Policy
Related In: Results  -  Collection

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fig3: β3 S752 regulates inside-out, but not outside-in, activation of αvβ3. (A) β3−/− pre-OCs expressing the indicated mutants were incubated with AP5 (50 μg/ml) in low or high calcium and subjected to FACS® analysis. Results are presented as overlapping histograms for high (white) and low (gray) calcium treatments for each mutant. The tracings for high calcium buffer indicate basal activation of the integrin, as in this circumstance, AP5 binds previously activated αvβ3 integrin. A shift toward higher fluorescence intensity in low calcium buffer indicates that AP5 mediates integrin activation. In each group of transduced pre-OCs, incubation with AP5 in low calcium buffer yields a significant increase in mean fluorescence intensity, relative to high calcium buffer, indicating that activation by this anti-LIBS Ab from the cell's exterior does not depend on the β3 cytoplasmic domain. (B) Pre-OCs were preincubated with HGF (50 ng/ml, light gray bars) or M-CSF (100 ng/ml, dark gray bars) before staining with AP5 in high calcium buffer to determine the degree of growth factor–mediated integrin activation. Results are expressed as fold increase in the mean fluorescence intensity of AP5 staining compared with unstimulated control cells (CTR), arbitrarily designated as 1 for each untreated mutant.
Mentions: One possible explanation for the failure of β3-ΔC and β3 S752P to localize to podosomes is that the external domain of these mutants is not able to assume the activated, high-affinity conformation necessary for appropriate substrate interaction. One tool to assess the ability of β3 integrin to assume the activated conformation of the external domain is the anti–ligand-induced binding site (LIBS) antibody AP5. In low calcium buffer, this Ab binds all αvβ3 integrin on the cell surface, converting it to the active conformation. In high calcium buffer, AP5 binds only the integrin already in the activated state (Faccio et al., 2002). An increase in fluorescence intensity of αvβ3-expressing cells when AP5 binding is assessed in low calcium, relative to high calcium buffer, indicates that the external domain of the integrin can assume the activated conformation in response to the Ab. When pre-OCs transduced with β3-ΔC and β3 S752P, as well as with β3 WT and β3 Y747F/Y759F, are analyzed in this manner, there is an increase in AP5 binding in low calcium, indicating that each of these β3 constructs undergoes conformational change (Fig. 3 A). Thus, the failure of the β3-ΔC and β3 S752P mutants to properly localize in podosomes does not reflect an inability of these integrins to assume an activated conformation.

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