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Oncogenic Ras-induced proliferation requires autocrine fibroblast growth factor 2 signaling in skeletal muscle cells.

Fedorov YV, Rosenthal RS, Olwin BB - J. Cell Biol. (2001)

Bottom Line: Oncogenic Ras does not appear to alter cellular export rates of FGF-2, which does not possess an NH(2)-terminal or internal signal peptide.Surprisingly, inhibiting the autocrine FGF-2 required for proliferation has no effect on oncogenic Ras-mediated repression of muscle-specific gene expression.We conclude that oncogenic Ras-induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from Ras-induced repression of terminal differentiation and involves activation of extracellularly localized, inactive FGF-2.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA.

ABSTRACT
Constitutively activated Ras proteins are associated with a large number of human cancers, including those originating from skeletal muscle tissue. In this study, we show that ectopic expression of oncogenic Ras stimulates proliferation of the MM14 skeletal muscle satellite cell line in the absence of exogenously added fibroblast growth factors (FGFs). MM14 cells express FGF-1, -2, -6, and -7 and produce FGF protein, yet they are dependent on exogenously supplied FGFs to both maintain proliferation and repress terminal differentiation. Thus, the FGFs produced by these cells are either inaccessible or inactive, since the endogenous FGFs elicit no detectable biological response. Oncogenic Ras-induced proliferation is abolished by addition of an anti-FGF-2 blocking antibody, suramin, or treatment with either sodium chlorate or heparitinase, demonstrating an autocrine requirement for FGF-2. Oncogenic Ras does not appear to alter cellular export rates of FGF-2, which does not possess an NH(2)-terminal or internal signal peptide. However, oncogenic Ras does appear to be involved in releasing or activating inactive, extracellularly sequestered FGF-2. Surprisingly, inhibiting the autocrine FGF-2 required for proliferation has no effect on oncogenic Ras-mediated repression of muscle-specific gene expression. We conclude that oncogenic Ras-induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from Ras-induced repression of terminal differentiation and involves activation of extracellularly localized, inactive FGF-2.

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Blocking FGF-2 signaling by suramin, anti–FGF-2 antibody, or sodium chlorate has no effect on inhibition of differentiation by Ras-G12V. MM14 cells were cotransfected with plasmids encoding CMV-LacZ, a luciferase reporter driven by α-cardiac actin promoter (1 μg each), and either RasG12V (▨) or pBSSK+ (_PCSTART_#pmc852_PCEND_) (1 μg each) plasmids. Cells were incubated in the presence or absence of FGF-2 and treated with suramin, anti–FGF-2 antibody, or sodium chlorate as indicated (all reagents were added 1 h after transfection). Cells were fixed and assayed 36 h after transfection. Data show fold induction of luciferase activity relative to cells incubated in the presence of FGF-2 (equal to 1). Each point represents three independent experiments, each conducted in triplicate, with the standard deviations indicated.
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Figure 5: Blocking FGF-2 signaling by suramin, anti–FGF-2 antibody, or sodium chlorate has no effect on inhibition of differentiation by Ras-G12V. MM14 cells were cotransfected with plasmids encoding CMV-LacZ, a luciferase reporter driven by α-cardiac actin promoter (1 μg each), and either RasG12V (▨) or pBSSK+ (_PCSTART_#pmc852_PCEND_) (1 μg each) plasmids. Cells were incubated in the presence or absence of FGF-2 and treated with suramin, anti–FGF-2 antibody, or sodium chlorate as indicated (all reagents were added 1 h after transfection). Cells were fixed and assayed 36 h after transfection. Data show fold induction of luciferase activity relative to cells incubated in the presence of FGF-2 (equal to 1). Each point represents three independent experiments, each conducted in triplicate, with the standard deviations indicated.

Mentions: The ability of oncogenic Ras to inhibit skeletal muscle differentiation has been well documented, but the Ras effector mediating repression of differentiation is not known (Ramocki et al. 1998). We wanted to determine if the ability of Ras to effectively inhibit MM14 differentiation was dependent on extracellular FGF-2, as is the proliferation response. Addition of suramin, NaClO3, or the neutralizing anti–FGF-2 antibody did not affect the ability of Ras-G12V to repress myogenesis (Fig. 5). These data are consistent with our results published previously, which demonstrate that independent FGF signaling events mediate repression of differentiation and proliferation (Kudla et al. 1998; Jones et al. 2000). Thus, similar to FGFR, Ha-Ras appears to utilize independent signaling mechanisms to repress terminal differentiation and promote proliferation.


Oncogenic Ras-induced proliferation requires autocrine fibroblast growth factor 2 signaling in skeletal muscle cells.

Fedorov YV, Rosenthal RS, Olwin BB - J. Cell Biol. (2001)

Blocking FGF-2 signaling by suramin, anti–FGF-2 antibody, or sodium chlorate has no effect on inhibition of differentiation by Ras-G12V. MM14 cells were cotransfected with plasmids encoding CMV-LacZ, a luciferase reporter driven by α-cardiac actin promoter (1 μg each), and either RasG12V (▨) or pBSSK+ (_PCSTART_#pmc852_PCEND_) (1 μg each) plasmids. Cells were incubated in the presence or absence of FGF-2 and treated with suramin, anti–FGF-2 antibody, or sodium chlorate as indicated (all reagents were added 1 h after transfection). Cells were fixed and assayed 36 h after transfection. Data show fold induction of luciferase activity relative to cells incubated in the presence of FGF-2 (equal to 1). Each point represents three independent experiments, each conducted in triplicate, with the standard deviations indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199216&req=5

Figure 5: Blocking FGF-2 signaling by suramin, anti–FGF-2 antibody, or sodium chlorate has no effect on inhibition of differentiation by Ras-G12V. MM14 cells were cotransfected with plasmids encoding CMV-LacZ, a luciferase reporter driven by α-cardiac actin promoter (1 μg each), and either RasG12V (▨) or pBSSK+ (_PCSTART_#pmc852_PCEND_) (1 μg each) plasmids. Cells were incubated in the presence or absence of FGF-2 and treated with suramin, anti–FGF-2 antibody, or sodium chlorate as indicated (all reagents were added 1 h after transfection). Cells were fixed and assayed 36 h after transfection. Data show fold induction of luciferase activity relative to cells incubated in the presence of FGF-2 (equal to 1). Each point represents three independent experiments, each conducted in triplicate, with the standard deviations indicated.
Mentions: The ability of oncogenic Ras to inhibit skeletal muscle differentiation has been well documented, but the Ras effector mediating repression of differentiation is not known (Ramocki et al. 1998). We wanted to determine if the ability of Ras to effectively inhibit MM14 differentiation was dependent on extracellular FGF-2, as is the proliferation response. Addition of suramin, NaClO3, or the neutralizing anti–FGF-2 antibody did not affect the ability of Ras-G12V to repress myogenesis (Fig. 5). These data are consistent with our results published previously, which demonstrate that independent FGF signaling events mediate repression of differentiation and proliferation (Kudla et al. 1998; Jones et al. 2000). Thus, similar to FGFR, Ha-Ras appears to utilize independent signaling mechanisms to repress terminal differentiation and promote proliferation.

Bottom Line: Oncogenic Ras does not appear to alter cellular export rates of FGF-2, which does not possess an NH(2)-terminal or internal signal peptide.Surprisingly, inhibiting the autocrine FGF-2 required for proliferation has no effect on oncogenic Ras-mediated repression of muscle-specific gene expression.We conclude that oncogenic Ras-induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from Ras-induced repression of terminal differentiation and involves activation of extracellularly localized, inactive FGF-2.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA.

ABSTRACT
Constitutively activated Ras proteins are associated with a large number of human cancers, including those originating from skeletal muscle tissue. In this study, we show that ectopic expression of oncogenic Ras stimulates proliferation of the MM14 skeletal muscle satellite cell line in the absence of exogenously added fibroblast growth factors (FGFs). MM14 cells express FGF-1, -2, -6, and -7 and produce FGF protein, yet they are dependent on exogenously supplied FGFs to both maintain proliferation and repress terminal differentiation. Thus, the FGFs produced by these cells are either inaccessible or inactive, since the endogenous FGFs elicit no detectable biological response. Oncogenic Ras-induced proliferation is abolished by addition of an anti-FGF-2 blocking antibody, suramin, or treatment with either sodium chlorate or heparitinase, demonstrating an autocrine requirement for FGF-2. Oncogenic Ras does not appear to alter cellular export rates of FGF-2, which does not possess an NH(2)-terminal or internal signal peptide. However, oncogenic Ras does appear to be involved in releasing or activating inactive, extracellularly sequestered FGF-2. Surprisingly, inhibiting the autocrine FGF-2 required for proliferation has no effect on oncogenic Ras-mediated repression of muscle-specific gene expression. We conclude that oncogenic Ras-induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from Ras-induced repression of terminal differentiation and involves activation of extracellularly localized, inactive FGF-2.

Show MeSH
Related in: MedlinePlus