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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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Adhesion-induced tyrosine phosphorylation of PYK2, Cas, paxillin, and PLC-γ in Src+/? and Src−/− prefusion osteoclast-like cells. (A) Src+/? pOCs were kept in suspension for 60 min or plated on Vn-coated dishes for the indicated periods in the absence of serum. Total cell lysates were immunoprecipitated (IP) with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies, followed by immunoblotting with anti–phosphotyrosine (pTyr) antibody (left). The same membranes were reblotted with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies (right). (B) Src+/? or Src−/− pOCs were kept in cell suspension or plated on Vn for 60 min. Total cell lysates were subjected to immunoprecipitation as described above. S, suspension; A, attached. (C) Src+/? or Src−/− pOCs (1.0 × 106 cells) were either plated on Vn-coated dishes for 60 min (lanes 1 and 3) or re-cultured with equal number of vitamin D3-treated MB1.8 cells on tissue culture dishes to generate OCLs (lanes 2 and 4). After 12 h, OCLs were purified as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-paxillin, followed by blotting with p-Tyr and anti-paxillin. Arrowhead shows the position of paxillin.
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Figure 6: Adhesion-induced tyrosine phosphorylation of PYK2, Cas, paxillin, and PLC-γ in Src+/? and Src−/− prefusion osteoclast-like cells. (A) Src+/? pOCs were kept in suspension for 60 min or plated on Vn-coated dishes for the indicated periods in the absence of serum. Total cell lysates were immunoprecipitated (IP) with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies, followed by immunoblotting with anti–phosphotyrosine (pTyr) antibody (left). The same membranes were reblotted with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies (right). (B) Src+/? or Src−/− pOCs were kept in cell suspension or plated on Vn for 60 min. Total cell lysates were subjected to immunoprecipitation as described above. S, suspension; A, attached. (C) Src+/? or Src−/− pOCs (1.0 × 106 cells) were either plated on Vn-coated dishes for 60 min (lanes 1 and 3) or re-cultured with equal number of vitamin D3-treated MB1.8 cells on tissue culture dishes to generate OCLs (lanes 2 and 4). After 12 h, OCLs were purified as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-paxillin, followed by blotting with p-Tyr and anti-paxillin. Arrowhead shows the position of paxillin.

Mentions: Initial events triggered by integrin engagement of ECM ligands include recruitment and phosphorylation of numerous signaling and cytoskeletal molecules, leading to cytoskeletal reorganization. We previously reported on the role of PYK2 and p130Cas in the αvβ3 integrin–mediated signaling pathways (Duong et al. 1998; Lakkakorpi et al. 1999) and on the involvement of PI 3-kinase in osteoclast adhesion and spreading (Lakkakorpi et al. 1997). Since U73122 blocked the adhesion- and M-CSF–dependent cell spreading of pOCs, we examined the tyrosine phosphorylation levels of PLC-γ1 and 2 in pOCs upon adhesion to Vn. Src+/? or Src−/− pOCs were either left in suspension or plated on Vn-coated dishes. Cell lysates were analyzed by Western blotting with antiphosphotyrosine antibodies after immunoprecipitation. In wild-type cells, PYK2, p130Cas, paxillin, and both PLC-γ1 and 2 became tyrosine phosphorylated paralleling the time course of cell spreading, i.e., peaking at 30 min after plating (Fig. 6 A). These data indicate that PLC-γ1 and 2 are downstream effectors of the integrin-mediated signaling pathway. Furthermore, tyrosine phosphorylation of these molecules after cell adhesion was absent in Src−/− pOCs (Fig. 6 B), supporting the morphological observations shown in Fig. 1. The data thus implicated Src tyrosine kinase as playing an essential role in osteoclast function by mediating integrin-dependent signaling triggered by ligand engagement.


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)

Adhesion-induced tyrosine phosphorylation of PYK2, Cas, paxillin, and PLC-γ in Src+/? and Src−/− prefusion osteoclast-like cells. (A) Src+/? pOCs were kept in suspension for 60 min or plated on Vn-coated dishes for the indicated periods in the absence of serum. Total cell lysates were immunoprecipitated (IP) with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies, followed by immunoblotting with anti–phosphotyrosine (pTyr) antibody (left). The same membranes were reblotted with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies (right). (B) Src+/? or Src−/− pOCs were kept in cell suspension or plated on Vn for 60 min. Total cell lysates were subjected to immunoprecipitation as described above. S, suspension; A, attached. (C) Src+/? or Src−/− pOCs (1.0 × 106 cells) were either plated on Vn-coated dishes for 60 min (lanes 1 and 3) or re-cultured with equal number of vitamin D3-treated MB1.8 cells on tissue culture dishes to generate OCLs (lanes 2 and 4). After 12 h, OCLs were purified as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-paxillin, followed by blotting with p-Tyr and anti-paxillin. Arrowhead shows the position of paxillin.
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Figure 6: Adhesion-induced tyrosine phosphorylation of PYK2, Cas, paxillin, and PLC-γ in Src+/? and Src−/− prefusion osteoclast-like cells. (A) Src+/? pOCs were kept in suspension for 60 min or plated on Vn-coated dishes for the indicated periods in the absence of serum. Total cell lysates were immunoprecipitated (IP) with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies, followed by immunoblotting with anti–phosphotyrosine (pTyr) antibody (left). The same membranes were reblotted with anti-PYK2, anti-Cas, anti–paxillin, anti–PLC-γ1 and 2, and anti-Src antibodies (right). (B) Src+/? or Src−/− pOCs were kept in cell suspension or plated on Vn for 60 min. Total cell lysates were subjected to immunoprecipitation as described above. S, suspension; A, attached. (C) Src+/? or Src−/− pOCs (1.0 × 106 cells) were either plated on Vn-coated dishes for 60 min (lanes 1 and 3) or re-cultured with equal number of vitamin D3-treated MB1.8 cells on tissue culture dishes to generate OCLs (lanes 2 and 4). After 12 h, OCLs were purified as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-paxillin, followed by blotting with p-Tyr and anti-paxillin. Arrowhead shows the position of paxillin.
Mentions: Initial events triggered by integrin engagement of ECM ligands include recruitment and phosphorylation of numerous signaling and cytoskeletal molecules, leading to cytoskeletal reorganization. We previously reported on the role of PYK2 and p130Cas in the αvβ3 integrin–mediated signaling pathways (Duong et al. 1998; Lakkakorpi et al. 1999) and on the involvement of PI 3-kinase in osteoclast adhesion and spreading (Lakkakorpi et al. 1997). Since U73122 blocked the adhesion- and M-CSF–dependent cell spreading of pOCs, we examined the tyrosine phosphorylation levels of PLC-γ1 and 2 in pOCs upon adhesion to Vn. Src+/? or Src−/− pOCs were either left in suspension or plated on Vn-coated dishes. Cell lysates were analyzed by Western blotting with antiphosphotyrosine antibodies after immunoprecipitation. In wild-type cells, PYK2, p130Cas, paxillin, and both PLC-γ1 and 2 became tyrosine phosphorylated paralleling the time course of cell spreading, i.e., peaking at 30 min after plating (Fig. 6 A). These data indicate that PLC-γ1 and 2 are downstream effectors of the integrin-mediated signaling pathway. Furthermore, tyrosine phosphorylation of these molecules after cell adhesion was absent in Src−/− pOCs (Fig. 6 B), supporting the morphological observations shown in Fig. 1. The data thus implicated Src tyrosine kinase as playing an essential role in osteoclast function by mediating integrin-dependent signaling triggered by ligand engagement.

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