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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–dependent association of PLC-γ2 and PYK2 in osteoclasts. Src+/? pOCs were cultured on Vn-coated dishes for 60 min in the absence of serum. (A) Total cell lysates were immunoprecipitated (IP) with anti–PLC-γ2 (lane 1) and anti–PLC-γ1 (lane 2), followed by blotting with anti-PYK2 (left), anti–PLC-γ2 (middle), or anti–PLC-γ1 (right) antibodies. (B) Lysates were immunoprecipitated with anti-PYK2 mAb 11 (lane 1) and anti-PYK2 N-19 antibodies (lane 2), followed by blotting with anti–PLC-γ2 (left) or anti-PYK2 (right). (C) Src+/? pOCs were cultured on PL or Vn for 60 min with or without 1 μM U73122. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2 (left), and anti–PLC-γ2 (right) antibodies. (D) Lysates of Src+/? OCLs were incubated with GST fusion proteins containing NH2- and COOH-terminal SH2 domains or SH3 domains of PLC-γ1 and blotted with anti-PYK2 antibodies (top) or incubated with GST fusion proteins of NH2- or COOH-terminal domains or kinase (K) domain of PYK2 and blotted with anti–PLC-γ2 antibodies (bottom). (E) Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn and treated without and with M-CSF. Adhesion of Src+/? pOCs on Vn was used as control. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2, followed by anti–PLC-γ2 antibodies (left) or immunoprecipitated with anti-N terminal PYK2 and blotted with anti–PLC-γ2, followed by anti-PYK2 antibodies (right). Molecular weight markers (in kD) are as indicated on the left. Positions of PYK2 (arrowhead) and of PLC-γ (asterisk) are as indicated.
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Figure 10: M-CSF–dependent association of PLC-γ2 and PYK2 in osteoclasts. Src+/? pOCs were cultured on Vn-coated dishes for 60 min in the absence of serum. (A) Total cell lysates were immunoprecipitated (IP) with anti–PLC-γ2 (lane 1) and anti–PLC-γ1 (lane 2), followed by blotting with anti-PYK2 (left), anti–PLC-γ2 (middle), or anti–PLC-γ1 (right) antibodies. (B) Lysates were immunoprecipitated with anti-PYK2 mAb 11 (lane 1) and anti-PYK2 N-19 antibodies (lane 2), followed by blotting with anti–PLC-γ2 (left) or anti-PYK2 (right). (C) Src+/? pOCs were cultured on PL or Vn for 60 min with or without 1 μM U73122. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2 (left), and anti–PLC-γ2 (right) antibodies. (D) Lysates of Src+/? OCLs were incubated with GST fusion proteins containing NH2- and COOH-terminal SH2 domains or SH3 domains of PLC-γ1 and blotted with anti-PYK2 antibodies (top) or incubated with GST fusion proteins of NH2- or COOH-terminal domains or kinase (K) domain of PYK2 and blotted with anti–PLC-γ2 antibodies (bottom). (E) Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn and treated without and with M-CSF. Adhesion of Src+/? pOCs on Vn was used as control. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2, followed by anti–PLC-γ2 antibodies (left) or immunoprecipitated with anti-N terminal PYK2 and blotted with anti–PLC-γ2, followed by anti-PYK2 antibodies (right). Molecular weight markers (in kD) are as indicated on the left. Positions of PYK2 (arrowhead) and of PLC-γ (asterisk) are as indicated.

Mentions: We next examined which molecular interactions are important for the convergence of the integrin- and M-CSF–dependent signals in prefusion osteoclasts in the absence of c-Src. Since we were previously unable to demonstrate stable interactions of PYK2 and PI 3-kinase in OCLs (Duong et al. 1998), we examined the association of PYK2 with PLC-γ in these cells. Both anti–PLC-γ1 and 2 antibodies coprecipitated PYK2 (Fig. 10 A) and anti-PYK2 antibodies pulled down PLC-γ2 (Fig. 10 B), supporting the in situ association of the two proteins in OCLs. Furthermore, upon adhesion to Vn a stronger association of PYK2 and PLC-γ was observed than in cells plated on PL (Fig. 10 C). Moreover, in the presence of U73122 the association of PYK2 and PLC-γ2 was reduced to the level observed in cells on PL (Fig. 10 C). These findings suggest that in osteoclasts, both integrin-dependent activation of PYK2 and PLC-γ2 and the phospholipase activity itself might be important for the stable interaction between these molecules.


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–dependent association of PLC-γ2 and PYK2 in osteoclasts. Src+/? pOCs were cultured on Vn-coated dishes for 60 min in the absence of serum. (A) Total cell lysates were immunoprecipitated (IP) with anti–PLC-γ2 (lane 1) and anti–PLC-γ1 (lane 2), followed by blotting with anti-PYK2 (left), anti–PLC-γ2 (middle), or anti–PLC-γ1 (right) antibodies. (B) Lysates were immunoprecipitated with anti-PYK2 mAb 11 (lane 1) and anti-PYK2 N-19 antibodies (lane 2), followed by blotting with anti–PLC-γ2 (left) or anti-PYK2 (right). (C) Src+/? pOCs were cultured on PL or Vn for 60 min with or without 1 μM U73122. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2 (left), and anti–PLC-γ2 (right) antibodies. (D) Lysates of Src+/? OCLs were incubated with GST fusion proteins containing NH2- and COOH-terminal SH2 domains or SH3 domains of PLC-γ1 and blotted with anti-PYK2 antibodies (top) or incubated with GST fusion proteins of NH2- or COOH-terminal domains or kinase (K) domain of PYK2 and blotted with anti–PLC-γ2 antibodies (bottom). (E) Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn and treated without and with M-CSF. Adhesion of Src+/? pOCs on Vn was used as control. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2, followed by anti–PLC-γ2 antibodies (left) or immunoprecipitated with anti-N terminal PYK2 and blotted with anti–PLC-γ2, followed by anti-PYK2 antibodies (right). Molecular weight markers (in kD) are as indicated on the left. Positions of PYK2 (arrowhead) and of PLC-γ (asterisk) are as indicated.
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Related In: Results  -  Collection

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Figure 10: M-CSF–dependent association of PLC-γ2 and PYK2 in osteoclasts. Src+/? pOCs were cultured on Vn-coated dishes for 60 min in the absence of serum. (A) Total cell lysates were immunoprecipitated (IP) with anti–PLC-γ2 (lane 1) and anti–PLC-γ1 (lane 2), followed by blotting with anti-PYK2 (left), anti–PLC-γ2 (middle), or anti–PLC-γ1 (right) antibodies. (B) Lysates were immunoprecipitated with anti-PYK2 mAb 11 (lane 1) and anti-PYK2 N-19 antibodies (lane 2), followed by blotting with anti–PLC-γ2 (left) or anti-PYK2 (right). (C) Src+/? pOCs were cultured on PL or Vn for 60 min with or without 1 μM U73122. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2 (left), and anti–PLC-γ2 (right) antibodies. (D) Lysates of Src+/? OCLs were incubated with GST fusion proteins containing NH2- and COOH-terminal SH2 domains or SH3 domains of PLC-γ1 and blotted with anti-PYK2 antibodies (top) or incubated with GST fusion proteins of NH2- or COOH-terminal domains or kinase (K) domain of PYK2 and blotted with anti–PLC-γ2 antibodies (bottom). (E) Src−/− pOCs (1.5 × 106 cells per condition) were plated on Vn and treated without and with M-CSF. Adhesion of Src+/? pOCs on Vn was used as control. Lysates were immunoprecipitated with anti–PLC-γ2 and blotted with anti-PYK2, followed by anti–PLC-γ2 antibodies (left) or immunoprecipitated with anti-N terminal PYK2 and blotted with anti–PLC-γ2, followed by anti-PYK2 antibodies (right). Molecular weight markers (in kD) are as indicated on the left. Positions of PYK2 (arrowhead) and of PLC-γ (asterisk) are as indicated.
Mentions: We next examined which molecular interactions are important for the convergence of the integrin- and M-CSF–dependent signals in prefusion osteoclasts in the absence of c-Src. Since we were previously unable to demonstrate stable interactions of PYK2 and PI 3-kinase in OCLs (Duong et al. 1998), we examined the association of PYK2 with PLC-γ in these cells. Both anti–PLC-γ1 and 2 antibodies coprecipitated PYK2 (Fig. 10 A) and anti-PYK2 antibodies pulled down PLC-γ2 (Fig. 10 B), supporting the in situ association of the two proteins in OCLs. Furthermore, upon adhesion to Vn a stronger association of PYK2 and PLC-γ was observed than in cells plated on PL (Fig. 10 C). Moreover, in the presence of U73122 the association of PYK2 and PLC-γ2 was reduced to the level observed in cells on PL (Fig. 10 C). These findings suggest that in osteoclasts, both integrin-dependent activation of PYK2 and PLC-γ2 and the phospholipase activity itself might be important for the stable interaction between these molecules.

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