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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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Inhibition of FGF-2 signaling by suramin or an anti–FGF-2 antibody blocks Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored as described in the legend to Fig. 1 (1 μg Ras-G12V or pBSSK+ per well). Suramin (A), an anti–FGF-2 antibody, and control antibody (B) were added to Ras-G12V–transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of three (A) or four (B) independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per each point.
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Figure 2: Inhibition of FGF-2 signaling by suramin or an anti–FGF-2 antibody blocks Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored as described in the legend to Fig. 1 (1 μg Ras-G12V or pBSSK+ per well). Suramin (A), an anti–FGF-2 antibody, and control antibody (B) were added to Ras-G12V–transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of three (A) or four (B) independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per each point.

Mentions: We hypothesized that Ha-Ras may induce FGF export or secretion of endogenously produced FGFs and therefore treated MM14 cells expressing RasG12V with agents that block FGF signaling. Addition of suramin, a negatively charged polysulfonated binaphthyl urea used as a general heparin-binding growth factor antagonist (Lozano et al. 1998), to cells ectopically expressing oncogenic Ras inhibited proliferation in a dose-dependent manner (Fig. 2 A). To test the involvement of specific FGFs, MM14 cells transiently expressing RasG12V were incubated with a neutralizing monoclonal anti–FGF-2 antibody specific for FGF-2 (Savage et al. 1993). Treatment with the anti–FGF-2 antibody completely abolished the capacity of Ha-Ras to stimulate proliferation (Fig. 2 A), whereas addition of a control monoclonal antibody had no effect (Fig. 2 B). Unexpectedly, we found that the ability of Ha-Ras to stimulate proliferation appears dependent on extracellularly supplied FGF-2.


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)

Inhibition of FGF-2 signaling by suramin or an anti–FGF-2 antibody blocks Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored as described in the legend to Fig. 1 (1 μg Ras-G12V or pBSSK+ per well). Suramin (A), an anti–FGF-2 antibody, and control antibody (B) were added to Ras-G12V–transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of three (A) or four (B) independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per each point.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199216&req=5

Figure 2: Inhibition of FGF-2 signaling by suramin or an anti–FGF-2 antibody blocks Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored as described in the legend to Fig. 1 (1 μg Ras-G12V or pBSSK+ per well). Suramin (A), an anti–FGF-2 antibody, and control antibody (B) were added to Ras-G12V–transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of three (A) or four (B) independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per each point.
Mentions: We hypothesized that Ha-Ras may induce FGF export or secretion of endogenously produced FGFs and therefore treated MM14 cells expressing RasG12V with agents that block FGF signaling. Addition of suramin, a negatively charged polysulfonated binaphthyl urea used as a general heparin-binding growth factor antagonist (Lozano et al. 1998), to cells ectopically expressing oncogenic Ras inhibited proliferation in a dose-dependent manner (Fig. 2 A). To test the involvement of specific FGFs, MM14 cells transiently expressing RasG12V were incubated with a neutralizing monoclonal anti–FGF-2 antibody specific for FGF-2 (Savage et al. 1993). Treatment with the anti–FGF-2 antibody completely abolished the capacity of Ha-Ras to stimulate proliferation (Fig. 2 A), whereas addition of a control monoclonal antibody had no effect (Fig. 2 B). Unexpectedly, we found that the ability of Ha-Ras to stimulate proliferation appears dependent on extracellularly supplied FGF-2.

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