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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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Colocalization of αvβ3 integrin and PLC-γ in M-CSF treated Src−/− prefusion osteoclasts. Src−/− pOCs were plated on Vn-coated glass coverslips for 1 hr, then treated with 5 nM M-CSF for 30 min. Cells were fixed double stained with polyclonal anti-β3 integrin and monoclonal anti-PLC-γ1 or PLC-γ2 antibodies. Pseudocolored confocal microscopic images of β3 integrins (a, d, in green) and double staining of PLC-γ1 (b, in red) or PLC-γ2 (e, in red). Colocalization (c, f, in yellow) appears to be more prominent in the adhesion contacts organized at the spreading edge of the cells. Images merged from optical sections of 4.7 μm thickness at the adhesion surface of the cells. Bar, 10 μm.
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Figure 9: Colocalization of αvβ3 integrin and PLC-γ in M-CSF treated Src−/− prefusion osteoclasts. Src−/− pOCs were plated on Vn-coated glass coverslips for 1 hr, then treated with 5 nM M-CSF for 30 min. Cells were fixed double stained with polyclonal anti-β3 integrin and monoclonal anti-PLC-γ1 or PLC-γ2 antibodies. Pseudocolored confocal microscopic images of β3 integrins (a, d, in green) and double staining of PLC-γ1 (b, in red) or PLC-γ2 (e, in red). Colocalization (c, f, in yellow) appears to be more prominent in the adhesion contacts organized at the spreading edge of the cells. Images merged from optical sections of 4.7 μm thickness at the adhesion surface of the cells. Bar, 10 μm.

Mentions: To further test the involvement of αvβ3 integrins in M-CSF–dependent spreading of Src−/− pOCs, we examined the localization of αvβ3 in M-CSF–treated cells. As shown in Fig. 9, β3 integrins (a and d, in green) colocalized with F-actin (Fig. 9 b, red) as well as PLC-γ2 (Fig. 9 e, red). Colocalization of β3 integrins and PLC-γ were found in adhesion contacts of the M-CSF-treated Src-deficient pOCs plated on Vn (Fig. 9 c and f, in yellow).


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)

Colocalization of αvβ3 integrin and PLC-γ in M-CSF treated Src−/− prefusion osteoclasts. Src−/− pOCs were plated on Vn-coated glass coverslips for 1 hr, then treated with 5 nM M-CSF for 30 min. Cells were fixed double stained with polyclonal anti-β3 integrin and monoclonal anti-PLC-γ1 or PLC-γ2 antibodies. Pseudocolored confocal microscopic images of β3 integrins (a, d, in green) and double staining of PLC-γ1 (b, in red) or PLC-γ2 (e, in red). Colocalization (c, f, in yellow) appears to be more prominent in the adhesion contacts organized at the spreading edge of the cells. Images merged from optical sections of 4.7 μm thickness at the adhesion surface of the cells. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Colocalization of αvβ3 integrin and PLC-γ in M-CSF treated Src−/− prefusion osteoclasts. Src−/− pOCs were plated on Vn-coated glass coverslips for 1 hr, then treated with 5 nM M-CSF for 30 min. Cells were fixed double stained with polyclonal anti-β3 integrin and monoclonal anti-PLC-γ1 or PLC-γ2 antibodies. Pseudocolored confocal microscopic images of β3 integrins (a, d, in green) and double staining of PLC-γ1 (b, in red) or PLC-γ2 (e, in red). Colocalization (c, f, in yellow) appears to be more prominent in the adhesion contacts organized at the spreading edge of the cells. Images merged from optical sections of 4.7 μm thickness at the adhesion surface of the cells. Bar, 10 μm.
Mentions: To further test the involvement of αvβ3 integrins in M-CSF–dependent spreading of Src−/− pOCs, we examined the localization of αvβ3 in M-CSF–treated cells. As shown in Fig. 9, β3 integrins (a and d, in green) colocalized with F-actin (Fig. 9 b, red) as well as PLC-γ2 (Fig. 9 e, red). Colocalization of β3 integrins and PLC-γ were found in adhesion contacts of the M-CSF-treated Src-deficient pOCs plated on Vn (Fig. 9 c and f, in yellow).

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