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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–induced cell spreading of Src−/− pOCs is dependent on αvβ3 integrin. Src−/− pOCs were plated on Vn or PL in serum-free condition. After 60 min, cells were treated with 5 nM M-CSF for 30 min in the absence or presence of echistatin (1 nM). Cells were fixed and stained for TRAP activity, followed by quantitating cell area as described above. Data are presented as means ± SEM and n = 50 cells per group.
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Figure 3: M-CSF–induced cell spreading of Src−/− pOCs is dependent on αvβ3 integrin. Src−/− pOCs were plated on Vn or PL in serum-free condition. After 60 min, cells were treated with 5 nM M-CSF for 30 min in the absence or presence of echistatin (1 nM). Cells were fixed and stained for TRAP activity, followed by quantitating cell area as described above. Data are presented as means ± SEM and n = 50 cells per group.

Mentions: It has been shown that the M-CSF receptor, c-Fms, is expressed in mature osteoclasts and that M-CSF induces cell spreading and cell migration in rat primary osteoclasts and murine osteoclast-like cells (Felix et al. 1994). To determine whether c-Src function is required for M-CSF–induced cytoskeletal reorganization during cell spreading and migration, Src-deficient and wild-type pOCs were plated on Vn-coated dishes and treated with M-CSF. To obtain optimal numbers of attached cells, we first allowed Src−/− cells to adhere to Vn-coated surfaces for 60 min prior to M-CSF addition. Although Src−/− pOCs did not spread spontaneously on Vn, M-CSF rapidly induced Src−/− cell spreading (Fig. 2 A and 3, first and second bars). Moreover, M-CSF induced the formation of small punctate adhesion contacts in Src−/− pOCs, similar to podosomal adhesion structures found in wild-type cells (Fig. 3 B). Because a previous study found that 2.5 nM M-CSF did not induce cell spreading of nonpurified primary Src-deficient osteoclasts (Insogna et al. 1997), we examined cell spreading of wild-type and Src−/− pOCs at 0, 2.5 and 5.0 nM M-CSF (n = 50), to rule out a dose effect phenomenon. The cell area of untreated wild-type cells was 234 ± 41 μm2 and of Src−/− pOCs, 93 ± 15 μm2. M-CSF at 2.5 nM increased the cell area in wild-type to 279 ± 16 μm2 (119%) and in Src−/− pOC to 203 ± 22 μm2 (218%), respectively; while 5 nM M-CSF increased cell spreading area to 318 ± 25 μm2 (135%) in wild-type and 270 ± 27 μm2 (290%) in Src−/− pOC, respectively; i.e. at both doses, there was a pronounced effect on the spreading of Src−/− pOCs, and not of wild-type pOCs.


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–induced cell spreading of Src−/− pOCs is dependent on αvβ3 integrin. Src−/− pOCs were plated on Vn or PL in serum-free condition. After 60 min, cells were treated with 5 nM M-CSF for 30 min in the absence or presence of echistatin (1 nM). Cells were fixed and stained for TRAP activity, followed by quantitating cell area as described above. Data are presented as means ± SEM and n = 50 cells per group.
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Related In: Results  -  Collection

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

Figure 3: M-CSF–induced cell spreading of Src−/− pOCs is dependent on αvβ3 integrin. Src−/− pOCs were plated on Vn or PL in serum-free condition. After 60 min, cells were treated with 5 nM M-CSF for 30 min in the absence or presence of echistatin (1 nM). Cells were fixed and stained for TRAP activity, followed by quantitating cell area as described above. Data are presented as means ± SEM and n = 50 cells per group.
Mentions: It has been shown that the M-CSF receptor, c-Fms, is expressed in mature osteoclasts and that M-CSF induces cell spreading and cell migration in rat primary osteoclasts and murine osteoclast-like cells (Felix et al. 1994). To determine whether c-Src function is required for M-CSF–induced cytoskeletal reorganization during cell spreading and migration, Src-deficient and wild-type pOCs were plated on Vn-coated dishes and treated with M-CSF. To obtain optimal numbers of attached cells, we first allowed Src−/− cells to adhere to Vn-coated surfaces for 60 min prior to M-CSF addition. Although Src−/− pOCs did not spread spontaneously on Vn, M-CSF rapidly induced Src−/− cell spreading (Fig. 2 A and 3, first and second bars). Moreover, M-CSF induced the formation of small punctate adhesion contacts in Src−/− pOCs, similar to podosomal adhesion structures found in wild-type cells (Fig. 3 B). Because a previous study found that 2.5 nM M-CSF did not induce cell spreading of nonpurified primary Src-deficient osteoclasts (Insogna et al. 1997), we examined cell spreading of wild-type and Src−/− pOCs at 0, 2.5 and 5.0 nM M-CSF (n = 50), to rule out a dose effect phenomenon. The cell area of untreated wild-type cells was 234 ± 41 μm2 and of Src−/− pOCs, 93 ± 15 μm2. M-CSF at 2.5 nM increased the cell area in wild-type to 279 ± 16 μm2 (119%) and in Src−/− pOC to 203 ± 22 μm2 (218%), respectively; while 5 nM M-CSF increased cell spreading area to 318 ± 25 μm2 (135%) in wild-type and 270 ± 27 μm2 (290%) in Src−/− pOC, respectively; i.e. at both doses, there was a pronounced effect on the spreading of Src−/− pOCs, and not of wild-type pOCs.

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