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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 spreading of Src−/− pOCs on Vn-coated dishes. (A) Src−/− pOCs were plated on Vn-coated dishes in the absence of serum for 60 min, cells were then treated with 5 nM M-CSF for 0 (a), 2 (b), 5 (c), 15 (d), and 30 (e) min, without or with (f) 100 nM wortmannin. (B) Src+/? (a) and Src−/− (b and c) pOCs were plated on Vn for 60 min, untreated (a and b) or treated with 5 nM M-CSF treatment for additional 30 min (c). Cells were fixed and stained with rhodamine-conjugated phalloidin. Bars: (A) 10 μm; (B) 5 μm.
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Figure 2: M-CSF induces cell spreading of Src−/− pOCs on Vn-coated dishes. (A) Src−/− pOCs were plated on Vn-coated dishes in the absence of serum for 60 min, cells were then treated with 5 nM M-CSF for 0 (a), 2 (b), 5 (c), 15 (d), and 30 (e) min, without or with (f) 100 nM wortmannin. (B) Src+/? (a) and Src−/− (b and c) pOCs were plated on Vn for 60 min, untreated (a and b) or treated with 5 nM M-CSF treatment for additional 30 min (c). Cells were fixed and stained with rhodamine-conjugated phalloidin. Bars: (A) 10 μm; (B) 5 μm.

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 induces cell spreading of Src−/− pOCs on Vn-coated dishes. (A) Src−/− pOCs were plated on Vn-coated dishes in the absence of serum for 60 min, cells were then treated with 5 nM M-CSF for 0 (a), 2 (b), 5 (c), 15 (d), and 30 (e) min, without or with (f) 100 nM wortmannin. (B) Src+/? (a) and Src−/− (b and c) pOCs were plated on Vn for 60 min, untreated (a and b) or treated with 5 nM M-CSF treatment for additional 30 min (c). Cells were fixed and stained with rhodamine-conjugated phalloidin. Bars: (A) 10 μm; (B) 5 μm.
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Related In: Results  -  Collection

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Figure 2: M-CSF induces cell spreading of Src−/− pOCs on Vn-coated dishes. (A) Src−/− pOCs were plated on Vn-coated dishes in the absence of serum for 60 min, cells were then treated with 5 nM M-CSF for 0 (a), 2 (b), 5 (c), 15 (d), and 30 (e) min, without or with (f) 100 nM wortmannin. (B) Src+/? (a) and Src−/− (b and c) pOCs were plated on Vn for 60 min, untreated (a and b) or treated with 5 nM M-CSF treatment for additional 30 min (c). Cells were fixed and stained with rhodamine-conjugated phalloidin. Bars: (A) 10 μm; (B) 5 μm.
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