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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 induces cell migration of Src−/− pOCs as well as wild-type cells. The motility of Src+/? and Src−/− pOCs was monitored using time-lapse video microscopy, as described in Materials and Methods. M-CSF (1 nM) was supplied through a micropipette to induce chemotaxis. Cells were observed using an inverted phase–contrast microscope coupled to a video camera and time-lapse video recorder. Images of cells were digitized using a computer-based image analysis system. Migration activity (net translocation of the cell center over a period of 4 h) was quantified (filled circles). 15 out of 26 (58%) Src+/? cells and 10 out of 19 (53%) Src−/− cells migrated towards the source of M-CSF. Means ± SEM (open circles) of migrating distance of wild-type and Src−/− cells are shown.
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Figure 4: M-CSF induces cell migration of Src−/− pOCs as well as wild-type cells. The motility of Src+/? and Src−/− pOCs was monitored using time-lapse video microscopy, as described in Materials and Methods. M-CSF (1 nM) was supplied through a micropipette to induce chemotaxis. Cells were observed using an inverted phase–contrast microscope coupled to a video camera and time-lapse video recorder. Images of cells were digitized using a computer-based image analysis system. Migration activity (net translocation of the cell center over a period of 4 h) was quantified (filled circles). 15 out of 26 (58%) Src+/? cells and 10 out of 19 (53%) Src−/− cells migrated towards the source of M-CSF. Means ± SEM (open circles) of migrating distance of wild-type and Src−/− cells are shown.

Mentions: Furthermore, M-CSF–stimulated osteoclast chemotaxis of Src-deficient cells was not different from wild-type cells (Fig. 4). In control cultures 15 out of 26 (58%) Src+/? pOCs migrated towards the source of M-CSF, and the net migration distance over a 4-h period was 25.5 ± 2.0 μm (means ± SEM). Similarly, 10 out of 19 (53%) Src−/− pOCs showed chemotactic migration, and the net distance was 24.5 ± 2.9 μm, not significantly different from wild-type. These observations suggest that Src function is not required for the M-CSF–induced cytoskeletal reorganization required for osteoclast spreading and migration.


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 induces cell migration of Src−/− pOCs as well as wild-type cells. The motility of Src+/? and Src−/− pOCs was monitored using time-lapse video microscopy, as described in Materials and Methods. M-CSF (1 nM) was supplied through a micropipette to induce chemotaxis. Cells were observed using an inverted phase–contrast microscope coupled to a video camera and time-lapse video recorder. Images of cells were digitized using a computer-based image analysis system. Migration activity (net translocation of the cell center over a period of 4 h) was quantified (filled circles). 15 out of 26 (58%) Src+/? cells and 10 out of 19 (53%) Src−/− cells migrated towards the source of M-CSF. Means ± SEM (open circles) of migrating distance of wild-type and Src−/− cells are shown.
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Related In: Results  -  Collection

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

Figure 4: M-CSF induces cell migration of Src−/− pOCs as well as wild-type cells. The motility of Src+/? and Src−/− pOCs was monitored using time-lapse video microscopy, as described in Materials and Methods. M-CSF (1 nM) was supplied through a micropipette to induce chemotaxis. Cells were observed using an inverted phase–contrast microscope coupled to a video camera and time-lapse video recorder. Images of cells were digitized using a computer-based image analysis system. Migration activity (net translocation of the cell center over a period of 4 h) was quantified (filled circles). 15 out of 26 (58%) Src+/? cells and 10 out of 19 (53%) Src−/− cells migrated towards the source of M-CSF. Means ± SEM (open circles) of migrating distance of wild-type and Src−/− cells are shown.
Mentions: Furthermore, M-CSF–stimulated osteoclast chemotaxis of Src-deficient cells was not different from wild-type cells (Fig. 4). In control cultures 15 out of 26 (58%) Src+/? pOCs migrated towards the source of M-CSF, and the net migration distance over a 4-h period was 25.5 ± 2.0 μm (means ± SEM). Similarly, 10 out of 19 (53%) Src−/− pOCs showed chemotactic migration, and the net distance was 24.5 ± 2.9 μm, not significantly different from wild-type. These observations suggest that Src function is not required for the M-CSF–induced cytoskeletal reorganization required for osteoclast spreading and migration.

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