ras is a multifunctional signaling molecule acting as an essential component of signal transduction pathways that regulate cellular physiology ( campbell et al .
members of the ras family of proteins that are constitutively activated by point mutations play a major role in the onset of a large number of human cancers , including those originating from skeletal muscle tissue ( yoo et al .
these skeletal muscle tumors are incapable of differentiation and thus do not withdraw from the cell cycle . up to 35% of these tumors
contain activating ras point mutations , suggesting a major involvement of ras in rhabdomyosarcomas ( stratton et al .
constitutively active ras mutants stimulate secretion of growth and angiogenic factors ( rak et al .
1995 ) , potentially allowing neoplastic cells to overcome growth restrictions in their normal tissue environment . in skeletal muscle cells ,
activated ras mutants have been shown to promote secretion of an unidentified factor that can repress myogenic differentiation and may participate in the development of rhabdomyosarcomas ( weyman and wolfman 1997 ) . of particular interest is the observation that cultured human embryonal rhabdomyosarcoma cells express the fgf-2 gene and produce biologically active fgf-2 ( schweigerer et al .
release of fgf-2 may stimulate the growth and neovascularization of human rhabdomyosarcomas and contribute to tumor development .
although ectopic expression of oncogenic ha - ras in myogenic cell lines represses terminal differentiation , it is not reported to elicit a proliferative response ( olson et al .
1987 ; konieczny et al . 1989 ; weyman and wolfman 1997 ) . from these studies
, it has been concluded that ras inhibits muscle differentiation without affecting proliferative response pathways .
fgfs are likely candidates for such factors since they play critical roles in regulation of skeletal muscle differentiation in cultured cells ( linkhart et al .
1981 ; allen et al . 1985 ; kardami et al . 1985 ; seed and hauschka 1988 ; rando and blau 1994 ; flanagan - steet et al .
2000 ) , in skeletal muscle development in vivo ( flanagan - steet et al . 2000 ) , and in skeletal muscle regeneration ( anderson et al . 1995 ; floss et al .
mm14 myoblasts express fgf-1 , -2 , -6 , and -7 but are absolutely dependent on exogenously supplied fgfs to repress myogenesis and promote cell proliferation ( clegg et al .
fgf-2 is one of four fgfs that do not possess signal peptides and do not use the classical er / golgi - dependent secretory pathways for export from the cell ( florkiewicz et al .
since ectopically expressed ha - ras can repress differentiation of mm14 cells ( fedorov et al . 1998 ) , we asked if ha - ras was capable of stimulating proliferation in mm14 cells . here
we report that constitutively active ras stimulates mm14 myoblast proliferation via a novel mechanism that is dependent on export of endogenously produced fgf-2 and subsequent release or activation of the exported fgf-2 .
moreover , we also found that the signaling pathways used by oncogenic ras to stimulate proliferation and repress differentiation in myogenic cells are distinct and mediated independently .
mouse mm14 cells ( lim and hauschka 1984 ) were cultured as described previously ( kudla et al .
baf3/fr1 cells , a baf3 cell clone stably expressing fgf receptor ( fgfr)-1 , was cultured as described by ornitz et al . 1992 .
wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells ( ornitz et al .
human recombinant fgf-2 was purified from a yeast strain expressing this growth factor ( rapraeger et al .
heparin , nacl , and naclo3 were purchased from sigma - aldrich . a monoclonal anti 
1993 ) and anticysteine - rich fgfr control antibody ( zuber et al . 1997 ) were used as described previously ( hannon et al .
dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously ( kudla et al .
equivalent dna concentrations were maintained by the addition of a pbssk+ ( stratagene ) plasmid .
the pdcr - h - ras ( g12v ) expression vector encoding a constitutively active mutant of human ha - ras , rasg12v ( white et al .
mm14 cells were grown on 6-well plates to a density of 5  10 and transfected with the indicated expression vectors or control plasmids .
cells were trypsinized ( 0.05% trypsin , 0.53 mm edta ) and replated at clonal density ( 1,000 cells per 10-cm plate ) 1 h after transfection .
the cells were maintained in the presence or absence of fgf-2 ( 0.3 nm unless otherwise indicated ) , cultured for 36 h , then processed for -galactosidase histochemistry as described elsewhere ( sanes et al .
a differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection .
the reporter contained the firefly luciferase gene driven by a muscle - specific promoter ( msp ; human anti - cardiac actin promoter ) ( kudla et al .
transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf .
cells were then washed once with pbs ( ph 7.2 ) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin .
baf3/fr1 cells ( 10 cells per well in 24-well plates ) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue .
mouse mm14 cells ( lim and hauschka 1984 ) were cultured as described previously ( kudla et al .
baf3/fr1 cells , a baf3 cell clone stably expressing fgf receptor ( fgfr)-1 , was cultured as described by ornitz et al . 1992 .
wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells ( ornitz et al .
human recombinant fgf-2 was purified from a yeast strain expressing this growth factor ( rapraeger et al .
heparin , nacl , and naclo3 were purchased from sigma - aldrich . a monoclonal anti 
1993 ) and anticysteine - rich fgfr control antibody ( zuber et al . 1997 ) were used as described previously ( hannon et al .
dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously ( kudla et al .
equivalent dna concentrations were maintained by the addition of a pbssk+ ( stratagene ) plasmid .
the pdcr - h - ras ( g12v ) expression vector encoding a constitutively active mutant of human ha - ras , rasg12v ( white et al .
mm14 cells were grown on 6-well plates to a density of 5  10 and transfected with the indicated expression vectors or control plasmids .
cells were trypsinized ( 0.05% trypsin , 0.53 mm edta ) and replated at clonal density ( 1,000 cells per 10-cm plate ) 1 h after transfection .
the cells were maintained in the presence or absence of fgf-2 ( 0.3 nm unless otherwise indicated ) , cultured for 36 h , then processed for -galactosidase histochemistry as described elsewhere ( sanes et al .
a differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection .
the reporter contained the firefly luciferase gene driven by a muscle - specific promoter ( msp ; human anti - cardiac actin promoter ) ( kudla et al .
to determine their fgf-2 export capabilities , transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf .
cells were then washed once with pbs ( ph 7.2 ) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin . the medium was then collected and filtered through a 0.2-m filter .
baf3/fr1 cells ( 10 cells per well in 24-well plates ) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue .
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 .
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 ) .
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 .
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 .
unexpectedly , we found that the ability of ha - ras to stimulate proliferation appears dependent on extracellularly supplied fgf-2 .
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 .
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 .
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 g / 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 .
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 .
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 .
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 can not be detected in the tissue culture media , presumably due to its strong association with membrane - bound and extracellular matrix  associated heparan sulfate .
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 .
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 .
a second assay involves cotransfection of mm14 cells with a construct encoding an fgf-2luciferase fusion protein and either ras - g12v or a control vector .
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 .
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-2mediated signaling events .
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 .
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 .
fgf-2 antibody did not affect the ability of ras - g12v to repress myogenesis ( fig .
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 .
thus , similar to fgfr , ha - ras appears to utilize independent signaling mechanisms to repress terminal differentiation and promote proliferation . although the downstream signaling events that mediate the repression of myogenesis by ras are not understood , it is well documented that oncogenic ras stimulates secretion of growth factors and angiogenic factors ( rak et al .
moreover , ectopic expression of activated ras releases a factor that represses myogenic differentiation ( weyman and wolfman 1997 ) .
data presented in this report argue that oncogenic ras may be involved in the activation or release of extracellularly localized fgf-2 that is normally sequestered in a biologically inactive state .
it is noteworthy that all of the extracellular activity observed is neutralized by a specific blocking fgf-2 monoclonal antibody , since proliferating mm14 cells synthesize fgf-1 , -2 , -6 , and -7 ( hannon et al .
1996 ) . although detectable , fgf-2 mrna is present at extremely low concentrations in many adult tissues despite the presence of high levels of fgf-2 activity , suggesting a mechanism for retaining or storing fgf-2 in a biologically inert form ( baird et al .
oncogenic ras appears to be involved in activating or releasing inactive , extracellularly localized fgf-2 .
the fgf-2 produced by skeletal muscle cells can not be detected in the tissue culture medium , forming the basis for previous conclusions that the ras - secreted myogenic inhibitory factor was not an fgf ( weyman and wolfman 1997 ) .
the ras - dependent proliferation factor we identified is released by a heparin wash and its activity is abrogated by treatment with either chlorate , heparitinase , suramin , or a monoclonal anti  fgf-2 antibody , implying that the proliferation factor is fgf-2 .
together with the prevalence of oncogenic ras mutants in rhabdomyosarcomas and the involvement of fgfs in regulation of myogenesis , our data suggest that fgf-2 may be a critical factor for supporting ras - dependent growth of rhabdomyosarcomas .
 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 nh2-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 . 
Introduction
Materials and Methods
Cell Culture
DNA Transfection
Clonal Growth Assay
Muscle-specific Promoter Assay
FGF-2 Export Assay
Results and Discussion