Limits...
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.

Show MeSH

Related in: MedlinePlus

Pyk2 activation in pre-OCs is dependent upon intracellular calcium and β2 integrin. (A) β3−/− and β3+/+ pre-OCs were preincubated with the calcium chelator BAPTA before plating on VN for 30 min. Cells were lysed and analyzed by Western blot for total Pyk2 and its Y402-phosphorylated species. BAPTA inhibits Pyk2 phosphorylation in both cell types. (B) β3+/+, β3−/−, and CD44−/− pre-OCs were lifted or replated onto OPN for 30 min, and lysates were immunoprecipitated for Pyk2 and analyzed by Western blot for phosphotyrosine (4G10) and paxillin. Cell adhesion leads to Pyk2 phosphorylation and increase in association with paxillin in all cells tested. (C) β3+/+, β3−/−, and β2−/− pre-OCs were maintained in suspension or plated on VN or plastic (PL) for 30 min. Adhesion on both VN and plastic induced Pyk2Y402 phosphorylation in β3+/+ and β3−/− pre-OCs. In contrast, Pyk2 phosphorylation on either VN or plastic is completely abrogated in cells lacking the β2−/− integrin. (D) RT-PCR for β3 integrin in β2−/− and β2+/+ pre-OCs cultured for 1, 2, and 3 d with RANKL and M-CSF.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172699&req=5

fig9: Pyk2 activation in pre-OCs is dependent upon intracellular calcium and β2 integrin. (A) β3−/− and β3+/+ pre-OCs were preincubated with the calcium chelator BAPTA before plating on VN for 30 min. Cells were lysed and analyzed by Western blot for total Pyk2 and its Y402-phosphorylated species. BAPTA inhibits Pyk2 phosphorylation in both cell types. (B) β3+/+, β3−/−, and CD44−/− pre-OCs were lifted or replated onto OPN for 30 min, and lysates were immunoprecipitated for Pyk2 and analyzed by Western blot for phosphotyrosine (4G10) and paxillin. Cell adhesion leads to Pyk2 phosphorylation and increase in association with paxillin in all cells tested. (C) β3+/+, β3−/−, and β2−/− pre-OCs were maintained in suspension or plated on VN or plastic (PL) for 30 min. Adhesion on both VN and plastic induced Pyk2Y402 phosphorylation in β3+/+ and β3−/− pre-OCs. In contrast, Pyk2 phosphorylation on either VN or plastic is completely abrogated in cells lacking the β2−/− integrin. (D) RT-PCR for β3 integrin in β2−/− and β2+/+ pre-OCs cultured for 1, 2, and 3 d with RANKL and M-CSF.

Mentions: Our data show that Pyk2 activation requires cell–matrix recognition but does not depend upon αvβ3 status. Previous studies have shown that Pyk2 phosphorylation is calcium dependent (Sanjay et al., 2001), and we find that in β3+/+ and β3−/− pre-OCs, the intracellular chelator, BAPTA, eliminates Pyk2 activation (Fig. 9 A). To determine if another OPN receptor or integrin might be responsible for Pyk2 phosphorylation, we assessed this parameter in cells deleted of CD44, which has been shown to mediate monocyte attachment to OPN (Weber et al., 1996), or in cells lacking the integrin α2β1, another adhesive receptor expressed on OCs (unpublished data). Attachment to OPN leads to Pyk2 phosphorylation and to an increase in its association with paxillin in all cells tested (Fig. 9 B). To find the receptor mediating Pyk2 phosphorylation in response to cell adhesion, we turned to the β2 integrin, which is expressed in pre-OCs (Fig. S1) and is known to be a plastic receptor (Hirano et al., 2002). Thus, β3+/+, β3−/−, and β2−/− pre-OCs were maintained in suspension or plated on VN or uncoated plastic for 30 min (Fig. 9 C). Plastic, like VN, induces Pyk2 phosphorylation in wild-type pre-OCs and those lacking αvβ3. However, Pyk2 phosphorylation of β2−/− pre-OCs is completely abrogated when these mutant cells are plated on plastic and is barely detectable on VN, an observation that does not reflect diminished β3 integrin expression (Fig. 9 D). Furthermore, these findings do not result from altered Pyk2 levels in β2−/− OCs (Fig. 9 C) or differing distribution (unpublished data). Thus, β2 integrin, but not β3 integrin or CD44, mediates Pyk2 activation in pre-OCs.


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)

Pyk2 activation in pre-OCs is dependent upon intracellular calcium and β2 integrin. (A) β3−/− and β3+/+ pre-OCs were preincubated with the calcium chelator BAPTA before plating on VN for 30 min. Cells were lysed and analyzed by Western blot for total Pyk2 and its Y402-phosphorylated species. BAPTA inhibits Pyk2 phosphorylation in both cell types. (B) β3+/+, β3−/−, and CD44−/− pre-OCs were lifted or replated onto OPN for 30 min, and lysates were immunoprecipitated for Pyk2 and analyzed by Western blot for phosphotyrosine (4G10) and paxillin. Cell adhesion leads to Pyk2 phosphorylation and increase in association with paxillin in all cells tested. (C) β3+/+, β3−/−, and β2−/− pre-OCs were maintained in suspension or plated on VN or plastic (PL) for 30 min. Adhesion on both VN and plastic induced Pyk2Y402 phosphorylation in β3+/+ and β3−/− pre-OCs. In contrast, Pyk2 phosphorylation on either VN or plastic is completely abrogated in cells lacking the β2−/− integrin. (D) RT-PCR for β3 integrin in β2−/− and β2+/+ pre-OCs cultured for 1, 2, and 3 d with RANKL and M-CSF.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Pyk2 activation in pre-OCs is dependent upon intracellular calcium and β2 integrin. (A) β3−/− and β3+/+ pre-OCs were preincubated with the calcium chelator BAPTA before plating on VN for 30 min. Cells were lysed and analyzed by Western blot for total Pyk2 and its Y402-phosphorylated species. BAPTA inhibits Pyk2 phosphorylation in both cell types. (B) β3+/+, β3−/−, and CD44−/− pre-OCs were lifted or replated onto OPN for 30 min, and lysates were immunoprecipitated for Pyk2 and analyzed by Western blot for phosphotyrosine (4G10) and paxillin. Cell adhesion leads to Pyk2 phosphorylation and increase in association with paxillin in all cells tested. (C) β3+/+, β3−/−, and β2−/− pre-OCs were maintained in suspension or plated on VN or plastic (PL) for 30 min. Adhesion on both VN and plastic induced Pyk2Y402 phosphorylation in β3+/+ and β3−/− pre-OCs. In contrast, Pyk2 phosphorylation on either VN or plastic is completely abrogated in cells lacking the β2−/− integrin. (D) RT-PCR for β3 integrin in β2−/− and β2+/+ pre-OCs cultured for 1, 2, and 3 d with RANKL and M-CSF.
Mentions: Our data show that Pyk2 activation requires cell–matrix recognition but does not depend upon αvβ3 status. Previous studies have shown that Pyk2 phosphorylation is calcium dependent (Sanjay et al., 2001), and we find that in β3+/+ and β3−/− pre-OCs, the intracellular chelator, BAPTA, eliminates Pyk2 activation (Fig. 9 A). To determine if another OPN receptor or integrin might be responsible for Pyk2 phosphorylation, we assessed this parameter in cells deleted of CD44, which has been shown to mediate monocyte attachment to OPN (Weber et al., 1996), or in cells lacking the integrin α2β1, another adhesive receptor expressed on OCs (unpublished data). Attachment to OPN leads to Pyk2 phosphorylation and to an increase in its association with paxillin in all cells tested (Fig. 9 B). To find the receptor mediating Pyk2 phosphorylation in response to cell adhesion, we turned to the β2 integrin, which is expressed in pre-OCs (Fig. S1) and is known to be a plastic receptor (Hirano et al., 2002). Thus, β3+/+, β3−/−, and β2−/− pre-OCs were maintained in suspension or plated on VN or uncoated plastic for 30 min (Fig. 9 C). Plastic, like VN, induces Pyk2 phosphorylation in wild-type pre-OCs and those lacking αvβ3. However, Pyk2 phosphorylation of β2−/− pre-OCs is completely abrogated when these mutant cells are plated on plastic and is barely detectable on VN, an observation that does not reflect diminished β3 integrin expression (Fig. 9 D). Furthermore, these findings do not result from altered Pyk2 levels in β2−/− OCs (Fig. 9 C) or differing distribution (unpublished data). Thus, β2 integrin, but not β3 integrin or CD44, mediates Pyk2 activation in pre-OCs.

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