Limits...
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.

Show MeSH

Related in: MedlinePlus

Heparan sulfate proteoglycans are required for Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored for β-galactosidase–positive clones as described in the legend to Fig. 1. NaCl (30 mM), NaClO3 (30 mM), or NaClO3 and heparin (50 μg/ml), in the presence or absence of FGF-2, were added to transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of four (A) and two (B) independent experiments, each conducted in triplicate, are shown for both A and B. No less than 100 clones were counted for each point.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199216&req=5

Figure 3: Heparan sulfate proteoglycans are required for Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored for β-galactosidase–positive clones as described in the legend to Fig. 1. NaCl (30 mM), NaClO3 (30 mM), or NaClO3 and heparin (50 μg/ml), in the presence or absence of FGF-2, were added to transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of four (A) and two (B) independent experiments, each conducted in triplicate, are shown for both A and B. No less than 100 clones were counted for each point.

Mentions: FGF signaling is dependent on heparan sulfate, which involves the interaction of heparan sulfate with both FGF and the FGFR tyrosine kinases (Rapraeger et al. 1991; Yayon et al. 1991; Plotnikov et al. 1999). In addition, heparan sulfate proteoglycans (HSPGs) participate in FGF storage, sequestration, and release (Rifkin and Moscatelli 1989). Treatment of MM14 cells with sodium chlorate, a reversible inhibitor of intracellular sulfation, prevents FGF binding and induces terminal differentiation (Rapraeger et al. 1991; Olwin and Rapraeger 1992). Incubation of oncogenic Ras-transfected MM14 cells with heparitinase (not shown) or sodium chlorate significantly decreases cell proliferation (Fig. 3 A). Both heparitinase (not shown) and chlorate-induced inhibition of MM14 cell proliferation was rescued by addition of heparin (50 μg/ml), indicating that the effect is heparan sulfate specific (Fig. 3 A). Surprisingly, addition of 600 pM FGF-2 to chlorate-treated Ha-Ras–transfected cells promoted proliferation, ameliorating the inhibitory chlorate effect (Fig. 3 A). This was unexpected since addition of FGF-2 had no effect on chlorate-treated parental MM14 cells or MM14 cells transfected with a pcDNA3 vector control (Fig. 3 B). The requirement for HSPGs and the ability to overcome this requirement with high concentrations of exogenously added FGF-2 suggests a more complicated role for HSPGs in addition to their known requirement for signaling. Taken together, our data demonstrate that RasG12V induces proliferation of skeletal muscle cells and that induction of proliferation requires an autocrine FGF-2 response.


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)

Heparan sulfate proteoglycans are required for Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored for β-galactosidase–positive clones as described in the legend to Fig. 1. NaCl (30 mM), NaClO3 (30 mM), or NaClO3 and heparin (50 μg/ml), in the presence or absence of FGF-2, were added to transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of four (A) and two (B) independent experiments, each conducted in triplicate, are shown for both A and B. No less than 100 clones were counted for each point.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Heparan sulfate proteoglycans are required for Ras-G12V–stimulated proliferation. MM14 cells were cotransfected with the indicated expression or control vectors, fixed, and scored for β-galactosidase–positive clones as described in the legend to Fig. 1. NaCl (30 mM), NaClO3 (30 mM), or NaClO3 and heparin (50 μg/ml), in the presence or absence of FGF-2, were added to transfected cells 1 h after replating. Data and confidence intervals (P = 0.05) of four (A) and two (B) independent experiments, each conducted in triplicate, are shown for both A and B. No less than 100 clones were counted for each point.
Mentions: FGF signaling is dependent on heparan sulfate, which involves the interaction of heparan sulfate with both FGF and the FGFR tyrosine kinases (Rapraeger et al. 1991; Yayon et al. 1991; Plotnikov et al. 1999). In addition, heparan sulfate proteoglycans (HSPGs) participate in FGF storage, sequestration, and release (Rifkin and Moscatelli 1989). Treatment of MM14 cells with sodium chlorate, a reversible inhibitor of intracellular sulfation, prevents FGF binding and induces terminal differentiation (Rapraeger et al. 1991; Olwin and Rapraeger 1992). Incubation of oncogenic Ras-transfected MM14 cells with heparitinase (not shown) or sodium chlorate significantly decreases cell proliferation (Fig. 3 A). Both heparitinase (not shown) and chlorate-induced inhibition of MM14 cell proliferation was rescued by addition of heparin (50 μg/ml), indicating that the effect is heparan sulfate specific (Fig. 3 A). Surprisingly, addition of 600 pM FGF-2 to chlorate-treated Ha-Ras–transfected cells promoted proliferation, ameliorating the inhibitory chlorate effect (Fig. 3 A). This was unexpected since addition of FGF-2 had no effect on chlorate-treated parental MM14 cells or MM14 cells transfected with a pcDNA3 vector control (Fig. 3 B). The requirement for HSPGs and the ability to overcome this requirement with high concentrations of exogenously added FGF-2 suggests a more complicated role for HSPGs in addition to their known requirement for signaling. Taken together, our data demonstrate that RasG12V induces proliferation of skeletal muscle cells and that induction of proliferation requires an autocrine FGF-2 response.

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