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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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Transfection with Ras-G12V does not stimulate export of FGF-2 by MM14 cells. MM14 cells (105 cells per well in a 6-well plate) were cotransfected as described in the legend to Fig. 1 (1 μg Ras-G12V, pcDNA3, or pBSSK+ per well). Cells were washed once with BaF3/FR1 growth medium and incubated in the same medium containing 50 μg/ml heparin for 1 h at room temperature and then collected. BaF3/FR1 cells (104 cells per well in a 24-well plate) were grown in conditioned medium from Ras-G12V (CM-RasG12V; ▨), pcDNA3-transfected (CM-pcDNA3; ˙) MM14 cells, or in control conditions (no MM14-conditioned media; RPMI supported with 15% calf bovine serum; □) for 72 h. Viable cells were scored based on their ability to exclude trypan blue dye. Data and the standard deviation shown represent two independent experiments, each performed in triplicate.
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Figure 4: Transfection with Ras-G12V does not stimulate export of FGF-2 by MM14 cells. MM14 cells (105 cells per well in a 6-well plate) were cotransfected as described in the legend to Fig. 1 (1 μg Ras-G12V, pcDNA3, or pBSSK+ per well). Cells were washed once with BaF3/FR1 growth medium and incubated in the same medium containing 50 μg/ml heparin for 1 h at room temperature and then collected. BaF3/FR1 cells (104 cells per well in a 24-well plate) were grown in conditioned medium from Ras-G12V (CM-RasG12V; ▨), pcDNA3-transfected (CM-pcDNA3; ˙) MM14 cells, or in control conditions (no MM14-conditioned media; RPMI supported with 15% calf bovine serum; □) for 72 h. Viable cells were scored based on their ability to exclude trypan blue dye. Data and the standard deviation shown represent two independent experiments, each performed in triplicate.

Mentions: Little is known regarding the mechanisms involved in FGF-2 export since FGF-2 has no signal peptide sequence and is not secreted through the established Golgi-dependent secretory pathway (Mignatti et al. 1992). Instead, an unusual ATP-dependent pathway that includes the Na+/K+-ATPase appears to be involved (Florkiewicz et al. 1998). To determine whether oncogenic Ras is directly involved in regulating FGF-2 export, we asked if MM14 cells expressing Ha-Ras exhibited increased levels of extracellular FGF-2. Although transfection of MM14 cells with FGF-2 results in export of biologically active FGF-2 (Hannon et al. 1996), this extracellular FGF-2 cannot be detected in the tissue culture media, presumably due to its strong association with membrane-bound and extracellular matrix–associated heparan sulfate. Therefore, we have designed two assays to quantify FGF-2 export. One assay utilizes heparin treatment of MM14 cells to release bound FGF-2, which is then assayed on BaF3 cells expressing FGFR-1 (BaF3/FR1). BaF3 cells are pre-B cells that undergo apoptosis after interleukin 3 withdrawal and do not express either FGFRs or HSPGs. As such, these cells are unresponsive to FGFs, unless they ectopically express FGFRs and heparin is added as an HSPG substitute (Ornitz et al. 1992). We found that both Ras-G12V and control (pcDNA3)-transfected MM14 cells release similar levels of factor(s) that support BaF3/FR1 survival and promote BaF3/FR1 proliferation (Fig. 4). These activities are neutralized by a monoclonal anti–FGF-2 antibody, demonstrating that the released material is FGF-2 (Fig. 4). A second assay involves cotransfection of MM14 cells with a construct encoding an FGF-2–luciferase fusion protein and either Ras-G12V or a control vector. The exported FGF-2 is quantified using a luciferase assay after a heparin wash. The results from this assay are indistinguishable from the BaF3/FR1 cell assay, suggesting that similar levels of FGF-2 are exported by control and Ha-Ras–transfected cells (data not shown). We conclude that oncogenic Ras does not affect the level of FGF-2 export from MM14 cells. Although MM14 cells produce FGF-2 and export FGF-2 that can be recovered in an active form, this FGF-2 is not normally available to the cells (Hannon et al. 1996). Thus, our data suggest that exported FGF-2 is normally retained in an inactive form on the cell surface. We propose that Ha-Ras “activates” this inactive extracellular pool of FGF-2 either by promoting its release from HSPGs or by providing a mechanism for FGF-2 to gain access to cell surface FGFR-1. Although the mechanisms involved are not understood, the ability of the Ha-Ras mutant to promote proliferation is dependent on exogenous FGF-2 and subsequent FGF-2–mediated signaling events.


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)

Transfection with Ras-G12V does not stimulate export of FGF-2 by MM14 cells. MM14 cells (105 cells per well in a 6-well plate) were cotransfected as described in the legend to Fig. 1 (1 μg Ras-G12V, pcDNA3, or pBSSK+ per well). Cells were washed once with BaF3/FR1 growth medium and incubated in the same medium containing 50 μg/ml heparin for 1 h at room temperature and then collected. BaF3/FR1 cells (104 cells per well in a 24-well plate) were grown in conditioned medium from Ras-G12V (CM-RasG12V; ▨), pcDNA3-transfected (CM-pcDNA3; ˙) MM14 cells, or in control conditions (no MM14-conditioned media; RPMI supported with 15% calf bovine serum; □) for 72 h. Viable cells were scored based on their ability to exclude trypan blue dye. Data and the standard deviation shown represent two independent experiments, each performed in triplicate.
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Related In: Results  -  Collection

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Figure 4: Transfection with Ras-G12V does not stimulate export of FGF-2 by MM14 cells. MM14 cells (105 cells per well in a 6-well plate) were cotransfected as described in the legend to Fig. 1 (1 μg Ras-G12V, pcDNA3, or pBSSK+ per well). Cells were washed once with BaF3/FR1 growth medium and incubated in the same medium containing 50 μg/ml heparin for 1 h at room temperature and then collected. BaF3/FR1 cells (104 cells per well in a 24-well plate) were grown in conditioned medium from Ras-G12V (CM-RasG12V; ▨), pcDNA3-transfected (CM-pcDNA3; ˙) MM14 cells, or in control conditions (no MM14-conditioned media; RPMI supported with 15% calf bovine serum; □) for 72 h. Viable cells were scored based on their ability to exclude trypan blue dye. Data and the standard deviation shown represent two independent experiments, each performed in triplicate.
Mentions: Little is known regarding the mechanisms involved in FGF-2 export since FGF-2 has no signal peptide sequence and is not secreted through the established Golgi-dependent secretory pathway (Mignatti et al. 1992). Instead, an unusual ATP-dependent pathway that includes the Na+/K+-ATPase appears to be involved (Florkiewicz et al. 1998). To determine whether oncogenic Ras is directly involved in regulating FGF-2 export, we asked if MM14 cells expressing Ha-Ras exhibited increased levels of extracellular FGF-2. Although transfection of MM14 cells with FGF-2 results in export of biologically active FGF-2 (Hannon et al. 1996), this extracellular FGF-2 cannot be detected in the tissue culture media, presumably due to its strong association with membrane-bound and extracellular matrix–associated heparan sulfate. Therefore, we have designed two assays to quantify FGF-2 export. One assay utilizes heparin treatment of MM14 cells to release bound FGF-2, which is then assayed on BaF3 cells expressing FGFR-1 (BaF3/FR1). BaF3 cells are pre-B cells that undergo apoptosis after interleukin 3 withdrawal and do not express either FGFRs or HSPGs. As such, these cells are unresponsive to FGFs, unless they ectopically express FGFRs and heparin is added as an HSPG substitute (Ornitz et al. 1992). We found that both Ras-G12V and control (pcDNA3)-transfected MM14 cells release similar levels of factor(s) that support BaF3/FR1 survival and promote BaF3/FR1 proliferation (Fig. 4). These activities are neutralized by a monoclonal anti–FGF-2 antibody, demonstrating that the released material is FGF-2 (Fig. 4). A second assay involves cotransfection of MM14 cells with a construct encoding an FGF-2–luciferase fusion protein and either Ras-G12V or a control vector. The exported FGF-2 is quantified using a luciferase assay after a heparin wash. The results from this assay are indistinguishable from the BaF3/FR1 cell assay, suggesting that similar levels of FGF-2 are exported by control and Ha-Ras–transfected cells (data not shown). We conclude that oncogenic Ras does not affect the level of FGF-2 export from MM14 cells. Although MM14 cells produce FGF-2 and export FGF-2 that can be recovered in an active form, this FGF-2 is not normally available to the cells (Hannon et al. 1996). Thus, our data suggest that exported FGF-2 is normally retained in an inactive form on the cell surface. We propose that Ha-Ras “activates” this inactive extracellular pool of FGF-2 either by promoting its release from HSPGs or by providing a mechanism for FGF-2 to gain access to cell surface FGFR-1. Although the mechanisms involved are not understood, the ability of the Ha-Ras mutant to promote proliferation is dependent on exogenous FGF-2 and subsequent FGF-2–mediated signaling events.

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