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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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Src−/− pOCs do not spread on Vn-coated dishes. Src+/? (A, C, E, and G) and Src−/− (B, D, F, and H) pOCs were plated on Vn (20 μg/ml). After culture for 5 (A and B), 15 (C and D), 30 (E and F), and 60 (G and H) min, cells were fixed and photographed. (I) To quantify cell area, the periphery of each cell was outlined and the total planar area was calculated, using an image analysis system (Empire Imaging Systems). Data are expressed as the means of ± SEM of >50 cells.
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Figure 1: Src−/− pOCs do not spread on Vn-coated dishes. Src+/? (A, C, E, and G) and Src−/− (B, D, F, and H) pOCs were plated on Vn (20 μg/ml). After culture for 5 (A and B), 15 (C and D), 30 (E and F), and 60 (G and H) min, cells were fixed and photographed. (I) To quantify cell area, the periphery of each cell was outlined and the total planar area was calculated, using an image analysis system (Empire Imaging Systems). Data are expressed as the means of ± SEM of >50 cells.

Mentions: Fibroblasts from Src-deficient mice were shown to have a reduced rate of spreading on fibronectin (Kaplan et al. 1995). We found that the pOCs derived from Src−/− mice exhibit a profound defect in cell spreading on Vn-coated surfaces (Fig. 1). Wild-type pOCs fully spread within 60 min of plating (Fig. 1, left), whereas Src−/− pOCs remained rounded at 60 min (Fig. 1, right), and up to 120 min (data not shown). Spreading area of wild-type and Src-deficient pOC on Vn were quantitated and are shown in Fig. 1 I. Initial attachment to Vn appeared to be normal in Src−/− pOCs; however, these cells were easily detached by shaking and tapping, indicating that the firm adhesion associated with cell spreading did not occur, although the expression level of αvβ3 integrins and their binding affinity were not altered in Src−/− pOCs (Lakkakorpi et al. 2000). The highest number of Src−/− pOCs attached to Vn-coated dishes was observed 60 min after seeding.


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)

Src−/− pOCs do not spread on Vn-coated dishes. Src+/? (A, C, E, and G) and Src−/− (B, D, F, and H) pOCs were plated on Vn (20 μg/ml). After culture for 5 (A and B), 15 (C and D), 30 (E and F), and 60 (G and H) min, cells were fixed and photographed. (I) To quantify cell area, the periphery of each cell was outlined and the total planar area was calculated, using an image analysis system (Empire Imaging Systems). Data are expressed as the means of ± SEM of >50 cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Src−/− pOCs do not spread on Vn-coated dishes. Src+/? (A, C, E, and G) and Src−/− (B, D, F, and H) pOCs were plated on Vn (20 μg/ml). After culture for 5 (A and B), 15 (C and D), 30 (E and F), and 60 (G and H) min, cells were fixed and photographed. (I) To quantify cell area, the periphery of each cell was outlined and the total planar area was calculated, using an image analysis system (Empire Imaging Systems). Data are expressed as the means of ± SEM of >50 cells.
Mentions: Fibroblasts from Src-deficient mice were shown to have a reduced rate of spreading on fibronectin (Kaplan et al. 1995). We found that the pOCs derived from Src−/− mice exhibit a profound defect in cell spreading on Vn-coated surfaces (Fig. 1). Wild-type pOCs fully spread within 60 min of plating (Fig. 1, left), whereas Src−/− pOCs remained rounded at 60 min (Fig. 1, right), and up to 120 min (data not shown). Spreading area of wild-type and Src-deficient pOC on Vn were quantitated and are shown in Fig. 1 I. Initial attachment to Vn appeared to be normal in Src−/− pOCs; however, these cells were easily detached by shaking and tapping, indicating that the firm adhesion associated with cell spreading did not occur, although the expression level of αvβ3 integrins and their binding affinity were not altered in Src−/− pOCs (Lakkakorpi et al. 2000). The highest number of Src−/− pOCs attached to Vn-coated dishes was observed 60 min after seeding.

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