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Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts.

Nakamura I, Lipfert L, Rodan GA - J. Cell Biol. (2001)

Bottom Line: The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function.However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner.M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

View Article: PubMed Central - PubMed

Affiliation: Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.

ABSTRACT
The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

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M-CSF–induced association of αvβ3 integrin with signaling molecules in Src−/− prefusion osteoclast-like cells. (A) Cell adhesion and M-CSF induce the association of β3 integrins with signaling molecules in pOCs. Src+/? and Src−/− pOCs (1.5 × 106 cells per condition) were plated on PL- or Vn-coated dishes. After culture for 60 min, Src−/− cells were treated with or without 5 nM M-CSF for 5 min. Total cell lysates were immunoprecipitated (IP) with anti–β3 integrin antibodies, followed by immunoblotting (IB) with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), anti–c-Src (lanes 13–16), and anti–β3 integrin (lanes 17–20). The molecular masses of marker proteins (in kD) are on the left. Positions of c-Src (arrowhead) and of p85 subunit of PI 3-kinase (asterisk) are as indicated. (B) PLC and PI 3-kinase inhibitors block M-CSF–induced association of αvβ3 integrin with signaling molecules in Src-deficient pOCs. Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn as described above, followed by incubation with either U73122 (1 μM) or LY294002 (50 μM) for 40 min, then with 5 nM M-CSF. Lysates were immunoprecipitated with hamster anti–murine β3 integrin antibodies (lanes 2–4, 6–8, 10–12, and 14–16) or control hamster IgG (lanes 1, 5, 9, and 13), followed by blotting with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), or anti–β3 integrin (lanes 13–16). p85 subunit of PI 3-kinase (small arrowheads). C, control hamster IgGs.
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Figure 8: M-CSF–induced association of αvβ3 integrin with signaling molecules in Src−/− prefusion osteoclast-like cells. (A) Cell adhesion and M-CSF induce the association of β3 integrins with signaling molecules in pOCs. Src+/? and Src−/− pOCs (1.5 × 106 cells per condition) were plated on PL- or Vn-coated dishes. After culture for 60 min, Src−/− cells were treated with or without 5 nM M-CSF for 5 min. Total cell lysates were immunoprecipitated (IP) with anti–β3 integrin antibodies, followed by immunoblotting (IB) with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), anti–c-Src (lanes 13–16), and anti–β3 integrin (lanes 17–20). The molecular masses of marker proteins (in kD) are on the left. Positions of c-Src (arrowhead) and of p85 subunit of PI 3-kinase (asterisk) are as indicated. (B) PLC and PI 3-kinase inhibitors block M-CSF–induced association of αvβ3 integrin with signaling molecules in Src-deficient pOCs. Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn as described above, followed by incubation with either U73122 (1 μM) or LY294002 (50 μM) for 40 min, then with 5 nM M-CSF. Lysates were immunoprecipitated with hamster anti–murine β3 integrin antibodies (lanes 2–4, 6–8, 10–12, and 14–16) or control hamster IgG (lanes 1, 5, 9, and 13), followed by blotting with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), or anti–β3 integrin (lanes 13–16). p85 subunit of PI 3-kinase (small arrowheads). C, control hamster IgGs.

Mentions: Since previous reports demonstrated the association of αvβ3 integrins with c-Src and PI 3-kinase in osteoclasts (Hruska et al. 1995; Lakkakorpi et al. 1997), we examined by coimmunoprecipitation with anti–β3 integrin antibodies the adhesion-dependent association of αvβ3 with PLC-γ, PI 3-kinase, c-Src, and PYK2. In pOCs, cell adhesion to Vn increased the association of β3 integrins with PLC-γ2, PI 3-kinase, PYK2, and c-Src (Fig. 8 A, lanes 1 and 2, 5 and 6, 9 and 10, and 13 and 14, respectively). These data suggest that integrin–ligand engagement induces not only tyrosine phosphorylation of these signaling molecules but also their association with the integrin receptor.


Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts.

Nakamura I, Lipfert L, Rodan GA - J. Cell Biol. (2001)

M-CSF–induced association of αvβ3 integrin with signaling molecules in Src−/− prefusion osteoclast-like cells. (A) Cell adhesion and M-CSF induce the association of β3 integrins with signaling molecules in pOCs. Src+/? and Src−/− pOCs (1.5 × 106 cells per condition) were plated on PL- or Vn-coated dishes. After culture for 60 min, Src−/− cells were treated with or without 5 nM M-CSF for 5 min. Total cell lysates were immunoprecipitated (IP) with anti–β3 integrin antibodies, followed by immunoblotting (IB) with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), anti–c-Src (lanes 13–16), and anti–β3 integrin (lanes 17–20). The molecular masses of marker proteins (in kD) are on the left. Positions of c-Src (arrowhead) and of p85 subunit of PI 3-kinase (asterisk) are as indicated. (B) PLC and PI 3-kinase inhibitors block M-CSF–induced association of αvβ3 integrin with signaling molecules in Src-deficient pOCs. Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn as described above, followed by incubation with either U73122 (1 μM) or LY294002 (50 μM) for 40 min, then with 5 nM M-CSF. Lysates were immunoprecipitated with hamster anti–murine β3 integrin antibodies (lanes 2–4, 6–8, 10–12, and 14–16) or control hamster IgG (lanes 1, 5, 9, and 13), followed by blotting with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), or anti–β3 integrin (lanes 13–16). p85 subunit of PI 3-kinase (small arrowheads). C, control hamster IgGs.
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Figure 8: M-CSF–induced association of αvβ3 integrin with signaling molecules in Src−/− prefusion osteoclast-like cells. (A) Cell adhesion and M-CSF induce the association of β3 integrins with signaling molecules in pOCs. Src+/? and Src−/− pOCs (1.5 × 106 cells per condition) were plated on PL- or Vn-coated dishes. After culture for 60 min, Src−/− cells were treated with or without 5 nM M-CSF for 5 min. Total cell lysates were immunoprecipitated (IP) with anti–β3 integrin antibodies, followed by immunoblotting (IB) with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), anti–c-Src (lanes 13–16), and anti–β3 integrin (lanes 17–20). The molecular masses of marker proteins (in kD) are on the left. Positions of c-Src (arrowhead) and of p85 subunit of PI 3-kinase (asterisk) are as indicated. (B) PLC and PI 3-kinase inhibitors block M-CSF–induced association of αvβ3 integrin with signaling molecules in Src-deficient pOCs. Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn as described above, followed by incubation with either U73122 (1 μM) or LY294002 (50 μM) for 40 min, then with 5 nM M-CSF. Lysates were immunoprecipitated with hamster anti–murine β3 integrin antibodies (lanes 2–4, 6–8, 10–12, and 14–16) or control hamster IgG (lanes 1, 5, 9, and 13), followed by blotting with anti–PLC-γ2 (lanes 1–4), anti-PYK2 (lanes 5–8), anti–PI 3-kinase (lanes 9–12), or anti–β3 integrin (lanes 13–16). p85 subunit of PI 3-kinase (small arrowheads). C, control hamster IgGs.
Mentions: Since previous reports demonstrated the association of αvβ3 integrins with c-Src and PI 3-kinase in osteoclasts (Hruska et al. 1995; Lakkakorpi et al. 1997), we examined by coimmunoprecipitation with anti–β3 integrin antibodies the adhesion-dependent association of αvβ3 with PLC-γ, PI 3-kinase, c-Src, and PYK2. In pOCs, cell adhesion to Vn increased the association of β3 integrins with PLC-γ2, PI 3-kinase, PYK2, and c-Src (Fig. 8 A, lanes 1 and 2, 5 and 6, 9 and 10, and 13 and 14, respectively). These data suggest that integrin–ligand engagement induces not only tyrosine phosphorylation of these signaling molecules but also their association with the integrin receptor.

Bottom Line: The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function.However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner.M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

View Article: PubMed Central - PubMed

Affiliation: Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.

ABSTRACT
The macrophage colony stimulating factor (M-CSF) and alpha(v)beta(3) integrins play critical roles in osteoclast function. This study examines M-CSF- and adhesion-induced signaling in prefusion osteoclasts (pOCs) derived from Src-deficient and wild-type mice. Src-deficient cells attach to but do not spread on vitronectin (Vn)-coated surfaces and, contrary to wild-type cells, their adhesion does not lead to tyrosine phosphorylation of molecules activated by adhesion, including PYK2, p130(Cas), paxillin, and PLC-gamma. However, in response to M-CSF, Src(-/-) pOCs spread and migrate on Vn in an alpha(v)beta(3)-dependent manner. Involvement of PLC-gamma activation is suggested by using a PLC inhibitor, U73122, which blocks both adhesion- and M-CSF-mediated cell spreading. Furthermore, in Src(-/-) pOCs M-CSF, together with filamentous actin, causes recruitment of beta(3) integrin and PLC-gamma to adhesion contacts and induces stable association of beta(3) integrin with PLC-gamma, phosphatidylinositol 3-kinase, and PYK2. Moreover, direct interaction of PYK2 and PLC-gamma can be induced by either adhesion or M-CSF, suggesting that this interaction may enable the formation of integrin-associated complexes. Furthermore, this study suggests that in pOCs PLC-gamma is a common downstream mediator for adhesion and growth factor signals. M-CSF-initiated signaling modulates the alpha(v)beta(3) integrin-mediated cytoskeletal reorganization in prefusion osteoclasts in the absence of c-Src, possibly via PLC-gamma.

Show MeSH
Related in: MedlinePlus