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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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PLC and PI 3-kinase inhibitors block adhesion-induced and M-CSF–induced cell spreading of prefusion osteoclast-like cells. (A) Src+/? pOCs (1, 2, and 4) and Src−/− pOCs (3 and 5–8) were plated on Vn under serum-free condition. After 60 min, cells were treated with (4–8) or without (1–3) M-CSF for 30 min. U73122 (2 and 6), wortmannin (7), or PD98059 (8) was preincubated for 40 min before M-CSF was added. Cells were fixed, stained for TRAP, and the total planar area was calculated as described in Materials and Methods. Data are presented as means ± SEM; n = 50 cells per group. (B) Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by the treatment with 5 nM M-CSF for indicated periods with or without 10 μM PD98059. Lysates were blotted with anti–phospho-ERK (top), followed by anti-ERK and anti-Src (bottom). Susp., suspension; Att., attachment.
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Figure 5: PLC and PI 3-kinase inhibitors block adhesion-induced and M-CSF–induced cell spreading of prefusion osteoclast-like cells. (A) Src+/? pOCs (1, 2, and 4) and Src−/− pOCs (3 and 5–8) were plated on Vn under serum-free condition. After 60 min, cells were treated with (4–8) or without (1–3) M-CSF for 30 min. U73122 (2 and 6), wortmannin (7), or PD98059 (8) was preincubated for 40 min before M-CSF was added. Cells were fixed, stained for TRAP, and the total planar area was calculated as described in Materials and Methods. Data are presented as means ± SEM; n = 50 cells per group. (B) Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by the treatment with 5 nM M-CSF for indicated periods with or without 10 μM PD98059. Lysates were blotted with anti–phospho-ERK (top), followed by anti-ERK and anti-Src (bottom). Susp., suspension; Att., attachment.

Mentions: We examined the involvement of PI 3-kinase and PLC-γ in M-CSF–mediated signaling in Src-deficient cells using either wortmannin (0.1 μM) or U73122 (1 μM). As shown in Fig. 5 A (bars 5–7) and Fig. 2 F, either inhibitor blocked M-CSF–induced cell spreading in Src−/− pOCs. Both inhibitors also blocked the migration of wild-type and Src-deficient cells assessed by time-lapse video microscopy (data not shown). Similarly, cell spreading of wild-type pOCs upon adhering to Vn was also inhibited by wortmannin (data not shown) and U73122 (Fig. 5 A, bars 1 and 2). Although U73122 is widely used as a PLC inhibitor, it has been shown to interfere with non PLC-dependent signals, usually at higher concentrations (>10 μM) (Walker et al. 1998). Nevertheless, our pharmacological findings suggest that PI 3-kinase and PLC-γ may take part in both adhesion-dependent and M-CSF–mediated signaling, which leads to cytoskeletal organization in prefusion osteoclasts.


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)

PLC and PI 3-kinase inhibitors block adhesion-induced and M-CSF–induced cell spreading of prefusion osteoclast-like cells. (A) Src+/? pOCs (1, 2, and 4) and Src−/− pOCs (3 and 5–8) were plated on Vn under serum-free condition. After 60 min, cells were treated with (4–8) or without (1–3) M-CSF for 30 min. U73122 (2 and 6), wortmannin (7), or PD98059 (8) was preincubated for 40 min before M-CSF was added. Cells were fixed, stained for TRAP, and the total planar area was calculated as described in Materials and Methods. Data are presented as means ± SEM; n = 50 cells per group. (B) Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by the treatment with 5 nM M-CSF for indicated periods with or without 10 μM PD98059. Lysates were blotted with anti–phospho-ERK (top), followed by anti-ERK and anti-Src (bottom). Susp., suspension; Att., attachment.
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Related In: Results  -  Collection

Show All Figures
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Figure 5: PLC and PI 3-kinase inhibitors block adhesion-induced and M-CSF–induced cell spreading of prefusion osteoclast-like cells. (A) Src+/? pOCs (1, 2, and 4) and Src−/− pOCs (3 and 5–8) were plated on Vn under serum-free condition. After 60 min, cells were treated with (4–8) or without (1–3) M-CSF for 30 min. U73122 (2 and 6), wortmannin (7), or PD98059 (8) was preincubated for 40 min before M-CSF was added. Cells were fixed, stained for TRAP, and the total planar area was calculated as described in Materials and Methods. Data are presented as means ± SEM; n = 50 cells per group. (B) Src+/? and Src−/− pOCs were kept in suspension or plated on Vn for 60 min in the absence of serum, followed by the treatment with 5 nM M-CSF for indicated periods with or without 10 μM PD98059. Lysates were blotted with anti–phospho-ERK (top), followed by anti-ERK and anti-Src (bottom). Susp., suspension; Att., attachment.
Mentions: We examined the involvement of PI 3-kinase and PLC-γ in M-CSF–mediated signaling in Src-deficient cells using either wortmannin (0.1 μM) or U73122 (1 μM). As shown in Fig. 5 A (bars 5–7) and Fig. 2 F, either inhibitor blocked M-CSF–induced cell spreading in Src−/− pOCs. Both inhibitors also blocked the migration of wild-type and Src-deficient cells assessed by time-lapse video microscopy (data not shown). Similarly, cell spreading of wild-type pOCs upon adhering to Vn was also inhibited by wortmannin (data not shown) and U73122 (Fig. 5 A, bars 1 and 2). Although U73122 is widely used as a PLC inhibitor, it has been shown to interfere with non PLC-dependent signals, usually at higher concentrations (>10 μM) (Walker et al. 1998). Nevertheless, our pharmacological findings suggest that PI 3-kinase and PLC-γ may take part in both adhesion-dependent and M-CSF–mediated signaling, which leads to cytoskeletal organization in prefusion osteoclasts.

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