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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 intracellular signaling in Src−/− prefusion osteoclast-like cells. Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by treatment with 5 nM M-CSF for the indicated periods with or without PI 3-kinase inhibitors. (A) Total cell lysates were immunoprecipitated with anti-PYK2, anti-Cas, antipaxillin, and anti-Src, and blotted with antiphosphotyrosine (pTyr, left), anti-PYK2, anti-Cas, antipaxillin, and anti-Src antibodies (right). (B) Lysates were immunoprecipitated with anti–PLC-γ2, blotted with anti-pTyr (top), then with anti–PLC-γ2 antibodies (middle). Part of the total cell lysates were used for immunodetection of c-Src (bottom). (C) Lysates were immunoprecipitated with anti-Akt/PKB, blotted with anti–phospho-Akt/PKB, or anti-Akt/PKB antibodies. S, suspension; A, attached.
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Figure 7: M-CSF–induced intracellular signaling in Src−/− prefusion osteoclast-like cells. Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by treatment with 5 nM M-CSF for the indicated periods with or without PI 3-kinase inhibitors. (A) Total cell lysates were immunoprecipitated with anti-PYK2, anti-Cas, antipaxillin, and anti-Src, and blotted with antiphosphotyrosine (pTyr, left), anti-PYK2, anti-Cas, antipaxillin, and anti-Src antibodies (right). (B) Lysates were immunoprecipitated with anti–PLC-γ2, blotted with anti-pTyr (top), then with anti–PLC-γ2 antibodies (middle). Part of the total cell lysates were used for immunodetection of c-Src (bottom). (C) Lysates were immunoprecipitated with anti-Akt/PKB, blotted with anti–phospho-Akt/PKB, or anti-Akt/PKB antibodies. S, suspension; A, attached.

Mentions: As shown above, M-CSF induces spreading in Src−/− prefusion osteoclasts in an αvβ3-dependent manner. Therefore, we examined the downstream effectors involved in M-CSF–dependent signaling in the absence of c-Src. Wild-type and Src-deficient pOCs were plated on Vn-coated dishes for 1 h to achieve maximal activation of the adhesion-induced signals, and were then treated with M-CSF at the indicated time (Fig. 7). Although M-CSF appeared to further induce adhesion-dependent tyrosine phosphorylation of PYK2, p130Cas, and paxillin in wild-type cells, tyrosine phosphorylation of these molecules was not detected in M-CSF–treated Src-deficient pOCs (Fig. 7 A). In contrast, M-CSF rapidly induced tyrosine phosphorylation of PLC-γ2 (Fig. 7 A) and PLC-γ1 (data not shown) within 0.5 min in these cells, which gradually returned to basal levels after 60 min, suggesting that tyrosine phosphorylation of both PLC-γ isoforms by M-CSF is Src independent. The M-CSF-induced PLC phosphorylation was found to be transient, as compared to that of αvβ3-dependent PLC phosphorylation in Src+/? pOCs (Fig. 6 A).


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 intracellular signaling in Src−/− prefusion osteoclast-like cells. Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by treatment with 5 nM M-CSF for the indicated periods with or without PI 3-kinase inhibitors. (A) Total cell lysates were immunoprecipitated with anti-PYK2, anti-Cas, antipaxillin, and anti-Src, and blotted with antiphosphotyrosine (pTyr, left), anti-PYK2, anti-Cas, antipaxillin, and anti-Src antibodies (right). (B) Lysates were immunoprecipitated with anti–PLC-γ2, blotted with anti-pTyr (top), then with anti–PLC-γ2 antibodies (middle). Part of the total cell lysates were used for immunodetection of c-Src (bottom). (C) Lysates were immunoprecipitated with anti-Akt/PKB, blotted with anti–phospho-Akt/PKB, or anti-Akt/PKB antibodies. S, suspension; A, attached.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199610&req=5

Figure 7: M-CSF–induced intracellular signaling in Src−/− prefusion osteoclast-like cells. Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by treatment with 5 nM M-CSF for the indicated periods with or without PI 3-kinase inhibitors. (A) Total cell lysates were immunoprecipitated with anti-PYK2, anti-Cas, antipaxillin, and anti-Src, and blotted with antiphosphotyrosine (pTyr, left), anti-PYK2, anti-Cas, antipaxillin, and anti-Src antibodies (right). (B) Lysates were immunoprecipitated with anti–PLC-γ2, blotted with anti-pTyr (top), then with anti–PLC-γ2 antibodies (middle). Part of the total cell lysates were used for immunodetection of c-Src (bottom). (C) Lysates were immunoprecipitated with anti-Akt/PKB, blotted with anti–phospho-Akt/PKB, or anti-Akt/PKB antibodies. S, suspension; A, attached.
Mentions: As shown above, M-CSF induces spreading in Src−/− prefusion osteoclasts in an αvβ3-dependent manner. Therefore, we examined the downstream effectors involved in M-CSF–dependent signaling in the absence of c-Src. Wild-type and Src-deficient pOCs were plated on Vn-coated dishes for 1 h to achieve maximal activation of the adhesion-induced signals, and were then treated with M-CSF at the indicated time (Fig. 7). Although M-CSF appeared to further induce adhesion-dependent tyrosine phosphorylation of PYK2, p130Cas, and paxillin in wild-type cells, tyrosine phosphorylation of these molecules was not detected in M-CSF–treated Src-deficient pOCs (Fig. 7 A). In contrast, M-CSF rapidly induced tyrosine phosphorylation of PLC-γ2 (Fig. 7 A) and PLC-γ1 (data not shown) within 0.5 min in these cells, which gradually returned to basal levels after 60 min, suggesting that tyrosine phosphorylation of both PLC-γ isoforms by M-CSF is Src independent. The M-CSF-induced PLC phosphorylation was found to be transient, as compared to that of αvβ3-dependent PLC phosphorylation in Src+/? pOCs (Fig. 6 A).

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