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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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Ras-G12V induces MM14 cell proliferation in the absence of FGF. MM14 cells were cotransfected with plasmids encoding Ras-G12V (▨), pcDNA3 ( ˙), or pBSSK+ (_PCSTART_#pmc852_PCEND_), and 5 μg of CMV-LacZ as described in Materials and Methods. Cells were replated in clonal density 1 h after transfection, incubated with or without FGF-2 (300 pM), and then fixed and scored 36 h after transfection for LacZ-positive cells/clone. Confidence intervals (P = 0.05) of three independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per point.
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Figure 1: Ras-G12V induces MM14 cell proliferation in the absence of FGF. MM14 cells were cotransfected with plasmids encoding Ras-G12V (▨), pcDNA3 ( ˙), or pBSSK+ (_PCSTART_#pmc852_PCEND_), and 5 μg of CMV-LacZ as described in Materials and Methods. Cells were replated in clonal density 1 h after transfection, incubated with or without FGF-2 (300 pM), and then fixed and scored 36 h after transfection for LacZ-positive cells/clone. Confidence intervals (P = 0.05) of three independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per point.

Mentions: MM14 cells are absolutely dependent on exogenously supplied FGFs to repress myogenesis and promote proliferation, yet they express several FGFs (Hannon et al. 1996), suggesting that the endogenously produced FGFs are inaccessible to FGFR-1. In addition, we have established that distinct FGFR-1 signaling pathways mediate the proliferative and differentiation inhibitory responses in MM14 cells (Kudla et al. 1998; Jones et al. 2000). To test the involvement of Ras in both FGF-dependent pathways, MM14 cells were transiently transfected with the oncogenic Ras, RasG12V. Ectopic Ha-Ras expression stimulated proliferation and repressed differentiation of MM14 cells in the absence of exogenous FGF (Fig. 1). Activated Ras appears to replace only FGF-dependent signaling events since MM14 cells transfected with oncogenic Ras were unable to proliferate in growth medium with reduced (2.5%) serum (data not shown).


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)

Ras-G12V induces MM14 cell proliferation in the absence of FGF. MM14 cells were cotransfected with plasmids encoding Ras-G12V (▨), pcDNA3 ( ˙), or pBSSK+ (_PCSTART_#pmc852_PCEND_), and 5 μg of CMV-LacZ as described in Materials and Methods. Cells were replated in clonal density 1 h after transfection, incubated with or without FGF-2 (300 pM), and then fixed and scored 36 h after transfection for LacZ-positive cells/clone. Confidence intervals (P = 0.05) of three independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per point.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Ras-G12V induces MM14 cell proliferation in the absence of FGF. MM14 cells were cotransfected with plasmids encoding Ras-G12V (▨), pcDNA3 ( ˙), or pBSSK+ (_PCSTART_#pmc852_PCEND_), and 5 μg of CMV-LacZ as described in Materials and Methods. Cells were replated in clonal density 1 h after transfection, incubated with or without FGF-2 (300 pM), and then fixed and scored 36 h after transfection for LacZ-positive cells/clone. Confidence intervals (P = 0.05) of three independent experiments, each conducted in triplicate, are shown. No less than 100 clones were counted per point.
Mentions: MM14 cells are absolutely dependent on exogenously supplied FGFs to repress myogenesis and promote proliferation, yet they express several FGFs (Hannon et al. 1996), suggesting that the endogenously produced FGFs are inaccessible to FGFR-1. In addition, we have established that distinct FGFR-1 signaling pathways mediate the proliferative and differentiation inhibitory responses in MM14 cells (Kudla et al. 1998; Jones et al. 2000). To test the involvement of Ras in both FGF-dependent pathways, MM14 cells were transiently transfected with the oncogenic Ras, RasG12V. Ectopic Ha-Ras expression stimulated proliferation and repressed differentiation of MM14 cells in the absence of exogenous FGF (Fig. 1). Activated Ras appears to replace only FGF-dependent signaling events since MM14 cells transfected with oncogenic Ras were unable to proliferate in growth medium with reduced (2.5%) serum (data not shown).

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