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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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Expression levels of β3 mutants by flow cytometry and Western blot. (A) BMMs expressing hβ3 WT, hβ3-ΔC, hβ3 S752P, or hβ3 Y747F/Y759F were incubated with a mAb against β3 integrin (1A2), followed by FITC-conjugated secondary antibody (solid lines). Cells with secondary antibody alone were used as negative control (dotted lines). (B) Mature OCs expressing the indicated β3 mutants were subjected to Western blot analysis using 7G2, a mAb against hβ3. Equal loading was confirmed by actin.
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fig1: Expression levels of β3 mutants by flow cytometry and Western blot. (A) BMMs expressing hβ3 WT, hβ3-ΔC, hβ3 S752P, or hβ3 Y747F/Y759F were incubated with a mAb against β3 integrin (1A2), followed by FITC-conjugated secondary antibody (solid lines). Cells with secondary antibody alone were used as negative control (dotted lines). (B) Mature OCs expressing the indicated β3 mutants were subjected to Western blot analysis using 7G2, a mAb against hβ3. Equal loading was confirmed by actin.

Mentions: Mice deleted of the β3 integrin subunit become osteosclerotic due to dysfunctional OCs, which fail to efficiently resorb bone (McHugh et al., 2000). As expected, the function of β3−/− OCs is rescued by transduction with retrovirus expressing full-length β3 cDNA. On the other hand, β3 lacking its cytoplasmic domain, or carrying a S752P mutation, is incapable of rescuing OCs devoid of endogenous β3 (Feng et al., 2001). Furthermore β3−/− OC precursors cultured with RANKL and standard doses of M-CSF fail to differentiate fully (Faccio et al., 2003). This osteoclastogenic defect can be rescued by increasing M-CSF concentrations, but the ability of these cells to resorb bone requires the presence of the integrin (Faccio et al., 2003). To further define the role that the β3 integrin cytoplasmic domain plays in organizing the OC cytoskeleton, we studied the distribution of podosomes in β3−/− OCs transduced with different human β3 integrin mutants. Four constructs were used for this purpose: the β3 integrin lacking its cytoplasmic tail (β3-ΔC); a mutant carrying the S752P mutation or that bearing the OC nonsignificant double tyrosine mutation, Y747F/Y759F; and as positive control, the full-length human β3 (β3 WT). Equivalent levels of expression of all four constructs were confirmed by flow cytometry and by Western blot analysis (Fig. 1, A and B). Bone marrow macrophages (BMMs) expressing the indicated mutants were cultured on coverslips in the presence of RANKL (100 ng/ml) and a high dose of M-CSF (100 ng/ml), conditions that lead to the formation of completely spread OCs, even in nontransduced β3−/− cultures (Faccio et al., 2003). These cells cultured in high M-CSF are equivalent to their WT counterparts in terms of morphology, osteoclastogenic markers (Faccio et al., 2003), c-Fms (the receptor for M-CSF) levels, and pattern of integrin expression (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200212082/DC1).


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)

Expression levels of β3 mutants by flow cytometry and Western blot. (A) BMMs expressing hβ3 WT, hβ3-ΔC, hβ3 S752P, or hβ3 Y747F/Y759F were incubated with a mAb against β3 integrin (1A2), followed by FITC-conjugated secondary antibody (solid lines). Cells with secondary antibody alone were used as negative control (dotted lines). (B) Mature OCs expressing the indicated β3 mutants were subjected to Western blot analysis using 7G2, a mAb against hβ3. Equal loading was confirmed by actin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172699&req=5

fig1: Expression levels of β3 mutants by flow cytometry and Western blot. (A) BMMs expressing hβ3 WT, hβ3-ΔC, hβ3 S752P, or hβ3 Y747F/Y759F were incubated with a mAb against β3 integrin (1A2), followed by FITC-conjugated secondary antibody (solid lines). Cells with secondary antibody alone were used as negative control (dotted lines). (B) Mature OCs expressing the indicated β3 mutants were subjected to Western blot analysis using 7G2, a mAb against hβ3. Equal loading was confirmed by actin.
Mentions: Mice deleted of the β3 integrin subunit become osteosclerotic due to dysfunctional OCs, which fail to efficiently resorb bone (McHugh et al., 2000). As expected, the function of β3−/− OCs is rescued by transduction with retrovirus expressing full-length β3 cDNA. On the other hand, β3 lacking its cytoplasmic domain, or carrying a S752P mutation, is incapable of rescuing OCs devoid of endogenous β3 (Feng et al., 2001). Furthermore β3−/− OC precursors cultured with RANKL and standard doses of M-CSF fail to differentiate fully (Faccio et al., 2003). This osteoclastogenic defect can be rescued by increasing M-CSF concentrations, but the ability of these cells to resorb bone requires the presence of the integrin (Faccio et al., 2003). To further define the role that the β3 integrin cytoplasmic domain plays in organizing the OC cytoskeleton, we studied the distribution of podosomes in β3−/− OCs transduced with different human β3 integrin mutants. Four constructs were used for this purpose: the β3 integrin lacking its cytoplasmic tail (β3-ΔC); a mutant carrying the S752P mutation or that bearing the OC nonsignificant double tyrosine mutation, Y747F/Y759F; and as positive control, the full-length human β3 (β3 WT). Equivalent levels of expression of all four constructs were confirmed by flow cytometry and by Western blot analysis (Fig. 1, A and B). Bone marrow macrophages (BMMs) expressing the indicated mutants were cultured on coverslips in the presence of RANKL (100 ng/ml) and a high dose of M-CSF (100 ng/ml), conditions that lead to the formation of completely spread OCs, even in nontransduced β3−/− cultures (Faccio et al., 2003). These cells cultured in high M-CSF are equivalent to their WT counterparts in terms of morphology, osteoclastogenic markers (Faccio et al., 2003), c-Fms (the receptor for M-CSF) levels, and pattern of integrin expression (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200212082/DC1).

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