Germ Line Gain of Function with SOS1 Mutation in Hereditary

Germ Line Gain of Function with SOS1 Mutation in HereditaryGingival Fibromatosis*□S

Received for publication, February 23, 2007, and in revised form, May 14, 2007 Published, JBC Papers in Press, May 17, 2007, DOI 10.1074/jbc.M701609200

Shyh-Ing Jang‡, Eun-Jin Lee‡, P. Suzanne Hart§, Mukundhan Ramaswami‡, Debora Pallos¶, and Thomas C. Hart‡1

From the ‡Section of Human and Craniofacial Genetics, NIDCR, National Institutes of Health (NIH), Bethesda, Maryland 20892,the §Office of the Clinical Director, NHGRI, NIH, Bethesda, Maryland 20892, and the ¶Periodontics Research and GraduateStudies Division, Department of Dentistry, University of Taubate, Sao Paulo 12020-270, Brazil

Mutation of human SOS1 is responsible for hereditary gingi-val fibromatosis type 1, a benign overgrowth condition of thegingiva. Here, we investigated molecular mechanisms responsi-ble for the increased rate of cell proliferation in gingival fibro-blasts caused bymutant SOS1 in vitro. Using ectopic expressionof wild-type and mutant SOS1 constructs, we found that trun-cated SOS1 could localize to the plasma membrane, withoutgrowth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in themagnitude and duration of ERK signaling in hereditary gingivalfibromatosis gingival fibroblasts that was associated with phos-phorylation of retinoblastoma tumor suppressor protein andthe up-regulation of cell cycle regulators, including cyclins C, D,and E and the E2F/DP transcription factors. These factors pro-mote cell cycle progression fromG1 to S phase, and their up-reg-ulationmay underlie the increased gingival fibroblast prolifera-tion observed. Selective depletion of wild-type and mutant SOS1through small interfering RNA demonstrates the link betweenmutation of SOS1, ERK signaling, cell proliferation rate, and theexpression levels of Egr-1 and proliferating cell nuclear antigen.These findings elucidate the mechanisms for gingival overgrowthmediated by SOS1 genemutation in humans.

Hereditary gingival fibromatosis (HGF)2 is a genetic condi-tion characterized by a slowly progressive, benign fibrousenlargement of keratinized gingiva (1–3). Genetic studies dem-onstrate locus heterogeneity for HGF, but etiologic mutationshave only been identified in the Son of Sevenless-1 gene (SOS1)

(4). An SOS1 g.126,142–126,143insC insertionmutation causesa frameshift and early termination of the protein, yielding achimeric 1,105-amino acid protein that consists of 1,083 SOS1N-terminal amino acids followed by 22 novel amino acids. Thistruncation abolishes four C-terminal proline-rich motifs,which are required for Grb2 binding. The form of HGF due toSOS1mutation is designated HGF1.SOS1 functions as a guanine nucleotide exchange factor

(GEF) that couples receptor tyrosine kinases to the Ras signal-ing pathway and controls cell proliferation, differentiation, ves-icle trafficking, and regulation of the actin cytoskeleton (5).Under the control of two classes of regulatory proteins, GEFand GTPase-activating proteins, Ras functions as a molecularswitch between GDP/GTP cycling (6). Three Ras-GEFs; SOS,guanine nucleotide-releasing factor, and guanyl nucleotide-re-leasing protein, have been characterized in controlling Ras acti-vation by catalyzing GDP release and association with GTP(7–11). Two regulatory regions govern theGEF activity of SOS1: acatalytic site that interacts with nucleotide-free Ras and an allo-steric site that enhances exchange activity with the binding ofnucleotide-Ras (12, 13). Upon activation of receptor tyrosinekinase, Grb2-SOS1 complexes are recruited to the plasma mem-brane leading to the exchange of GDP for GTP and Ras activation(14). Although the Grb2-SOS1 complex functions exclusively asanRas activator, SOS1 can also function as aGEF that is specific tothe GTPase Rac1. These two distinct catalytic functions of SOS1are mutually exclusive and reciprocally related (7).The activation of Ras signal stimulates downstream signaling

pathways, including the mitogen-activated protein kinase(MAPK) family, a ubiquitous signal transduction pathway (15,16). Differences in the duration, intensity, spatial distribution,and temporal qualities of ERK signaling govern distinct biolog-ical responses (17–19). For example, transient induction ofERK signaling by EGF leads to proliferation of PC12 cells,whereas sustained signaling induces differentiation (20–25). Inresponse to extracellular stimuli, MAPK signaling exerts con-trol of cell cycle progression. Translocation of activated ERKfrom the cytoplasm to the nucleus is necessary for activationand stabilization of Elk1, c-Jun, c-Myc, and c-Fos. These tran-scription factors then regulate the expression of genes, such ascyclin D1 and p21WAF1/CIP1, which are critical for the progres-sion from G1 to S phase (26, 27).Increased cell proliferation has been reported for HGF1 gin-

gival fibroblasts in both monolayer and three-dimensionalmatrix cultures (28). Herewe report thatmutant SOS1 contrib-utes an increased and sustained activation of ERK signaling in

* This work was supported from the intramural Research Program of theNIDCR, National Institutes of Health (to T. C. H.). The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. S1.

1 To whom correspondence should be addressed: Section of Human andCraniofacial Genetics, NIDCR, National Institutes of Health, Rm. 5-2531,Bldg. 10, 10 Center Drive, Bethesda, MD 20892. Tel.: 301-451-8994; Fax:301-480-4455; E-mail: [email protected].

2 The abbreviations used are: HGF, hereditary gingival fibromatosis; CCNE1,cyclin E1; CCNE2, cyclin E2; EGF, epidermal growth factor; EGFR, EGF recep-tor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activatedprotein kinase; PCNA, proliferation cell nuclear antigen; Rb, retinoblas-toma tumor suppressor protein; pRB, phospho-Rb; SOS1, Son of Seven-less-1; GEF, guanine nucleotide exchange factor; MEK, MAPK/ERK kinase;HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; TFDP1,-2, transcription factors DP1 and -2; TBP, TATA-box-binding protein; siRNA,small interference RNA.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 28, pp. 20245–20255, July 13, 2007Printed in the U.S.A.

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HGF1 fibroblasts under serum-starved conditions. SustainedERK signaling leads to increased expression of cell cycle regu-lators and transcription factors. RNA interference-mediatedSOS1 depletion was used to confirm the association betweenSOS1 mutation, ERK activation, and gingival fibroblast prolif-eration. These findings document a gain-of-function SOS1mutation and provide a molecular mechanism for gingivalovergrowth in HGF1.

EXPERIMENTAL PROCEDURES

Isolation of Fibroblasts and Cell Cultures—Human gingivaltissues were obtained with informed consent from three nor-mal subjects (control) and three patients (HGF1). All patientswere heterozygous carriers of the SOS1 g.126,142–126,143insCmutation. Three sets of gingival fibroblasts fromHGF1 patientsand age-matched normal controls (ages 20–24 years) were iso-lated and grown in Dulbecco’s modified Eagle’s medium con-taining 10% fetal bovine serum and 1� antibiotic-antimycoticsolution (growth medium) at 37 °C in a 5% CO2-humidifiedincubator and maintained up to 10 passages (28). HeLa cellswere obtained from ATCC (Manassas, VA).Plasmid Construction, Transfection, and Subcellular Frac-

tionation—The full-length human SOS1 expression plasmid(pCGN-HASOS1) was a gift from D. Bar-Sagi (State UniversityofNewYork, StonyBrook,NY). The vector control plasmidwasgenerated by HindIII digestion to release the HA-SOS1 insertand self-ligated. TheQuikChange site-directedmutagenesis kit(Stratagene, La Jolla, CA) was used with oligonucleotide (5�-AGCATCTGCACCAAATTCTTCCcAAGAACACCGTTA-ACACCTCC-3�, GenBankTM accession number L13857) togenerate the mutated pCGN-HASOS1 construct that carriedthe HGF1 mutation (small case and cytosine insertion). Themutation was confirmed by DNA sequencing. For transienttransfection, 15 � 104 cells/well were seeded in 6-well plates aday before experiments. Expression constructs (2 �g) weretransfected into primary gingival fibroblasts using JetPEI rea-gent (ISC Bioexperss, Kaysville, UT) or into HeLa cells by Lipo-fectamine 2000 (Invitrogen). Transfection was terminated48–72 h post-transfection, and total cellular lysates wereobtained (29). To study the subcellular distribution of SOS1,100 � 104 HeLa cells were plated in 10-cm dishes 24 h beforetransfection. Eight micrograms of indicated expression con-structs were used, and transfected cells were harvested 48 hpost-transfection. Cellular fractions were isolated using theProteoExtract Subcellular Proteome Extraction kit (EMD Bio-sciences, La Jolla, CA).Cell Proliferation and Ras Activation Assays—For prolifera-

tion assays, 1 � 104 cells/well of primary gingival fibroblastswere seeded in 48-well plates. After 24 h the growth mediumwas replaced with Dulbecco’s modified Eagle’s medium con-taining 20 mM HEPES (starving medium) for overnight. Themedium was then replaced with growth medium and main-tained for 9 days. Cells from triplicate wells were trypsinized ateach time point, and the total cell number was determined byusing a Coulter Counter (Beckman Z series). To monitor Rasactivation, cells were seeded in 150-mm culture dishes, grownto 85% confluence, and serum-starved for 16 h. After treatmentwith EGF (25 ng/ml) for 0.5–6.0 h at 37 °C, cellular extracts

were collected in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mMNaCl, 10 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40, 0.5%sodiumdeoxycholate, 10% glycerol, 2.0mMphenylmethanesul-fonyl fluoride, 20�MNa3VO4, 10�MNaF, 1�g/ml pepstatin A,and 10 �g/ml aprotinin plus protease inhibitor tablet (Com-plete tablet, Roche Applied Science). Ras activity wasmeasuredusing a Ras Activation kit (Upstate, Charlottesville, VA). Foreach reaction, 600 �g of whole cell lysate was incubated withRaf-1 Ras binding domain-agarose (15 �g) for 1 h at 4 °C. Thecomplexes were collected by centrifugation and washed fivetimes with buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10mM MgCl2, 1 mM EDTA, 10% glycerol, 20 �M NaF, and 1%Nonidet P-40). Protein complexes were releasedwith SDS sam-ple buffer, separated by 4–12% NuPAGE, and transferred to apolyvinylidene difluoridemembrane. Proteinswere detected bymouse anti-Ras antibody (0.05 �g/ml) and goat anti-mousehorseradish peroxidase-conjugated secondary antibody (Bio-Rad, 1:5000).Culture Treatment, Western Blot Analyses, and Indirect

Immunofluorescence Staining—To monitor the level of phos-phorylated ERK1/2, the cultures were either maintained ingrowthmediumor switched to starvingmedium for 16 h beforeaddition of EGF (Upstate) with indicated concentrations andincubation times. In some experiments, cultures were treatedwith AG1478 (EGFR inhibitor, 10 �M, EMD Biosciences) orPD98059 (MEK inhibitor, 10 �M, EMD Biosciences) 30 minprior to the addition of EGF as indicated (Fig. 2, legend). At eachtime point, cultures were washedwith cold phosphate-bufferedsaline, andwhole cellular extracts were prepared by adding lysisbuffer directly into monolayer cultures. Isolation of nuclearextracts and Western blotting were conducted as previouslydescribed (29). After washing with phosphate-buffered salinewith 0.1%Tween 20, the primary antibodies were detectedwitha polyclonal goat anti-rabbit or anti-mouse IgG coupled withhorseradish peroxidase. Primary antibodies included mouseanti-HA (16B12, 1:1500, Covance, Berkeley, CA), mouse anti-�-actin (1:800, Sigma), mouse anti-SOS1(N) (1:250, epitope atN-terminal, BD Bioscience Pharmingen), rabbit anti-SOS1(C)(1:500, epitope at C-terminal), rabbit anti-Egr-1 (1:300), rabbitanti-EGFR (1:300), rabbit anti-phospho-retinoblastoma (pRb,Ser-780), rabbit anti-retinoblastoma (Rb), and mouse anti-PCNA (1:500) from Santa Cruz Biotechnology, rabbit anti-ex-tracellular signal-regulated kinase (ERK1/2, 1:2000), and rabbitanti-phospho-ERK1/2 (1:2000) from Upstate. Signals weredetected by using ECL Western blotting Detection Reagents(Amersham Biosciences) and exposed to x-ray film (XAR,Kodak). All experiments were conducted at least three timesand quantitated using a FluorChem digital imaging system(Alpha Innotech, San Leandro, CA) and National Institutes ofHealth Image 1.63 software.Results were adjusted for loading controls. Immunofluores-

cent staining was conducted as previously described (30). Aftertransfection, gingival fibroblasts were maintained in eithergrowth or starving medium for 16 h and fixed with paraform-aldehyde (3.7%) for 15 min at room temperature. Cells wereincubated with anti-HA (1:1000) for 1 h at room temperature.After washing, the cells were incubated with goat anti-mouseconjugated with TRITC IgG antibody for 30 min. After wash-

SOS1 Mutation and Gingival Overgrowth

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ing, the cell nuclei were stained with 4�,6-diamidino-2-phenyl-indole, dihydrochloride (Sigma) for 5min at room temperatureand washed three additional times. Slides were examined withfluorescence microcopy (Olympus, IX71), and images wereprocessed using Adobe Photoshop CS.Real-time PCR—Total RNA from equivalent cell densities of

control and HGF1 fibroblast cultures maintained either instarving medium for 16 h or switched to growth medium for 1day were prepared by TRIzol solution (Invitrogen) and the“RNeasy Mini kit” (Qiagen). RNA quality and quantity weredetermined using a Bioanalyzer (Agilent 2100, Wilmington,DE). Reverse transcription-PCR experiments were conductedas described previously (32) withmodification. TaqMan probesand PCR primers were purchased fromABI Biosystems (FosterCity, CA). These included SOS1 (Hs00362308_m1), E2F1(Hs00153451_m1), E2F2 (Hs00 231667_m1), transcription fac-tor DP1 (TFDP1, Hs00955488_m1), transcription factor DP2(TFDP2,Hs00232366_m1), cyclin E1 (CCNE1,Hs00233356_m1),and cyclin E2 (CCNE2, Hs00180319_m1). The RNase P orTATA-box-binding protein (TBP) was used as endogenouscontrol for normalization. QPCRHumanReference Total RNA(Stratagene) was used as a calibrator in all quantitative reversetranscription-PCR experiments. Relative levels of the indicatedtranscripts in each sample were calculated as 2��Ct, where��Ct � [target gene’s Ct � TBP Ct]sample � [target gene’s Ct �TBP Ct]calibrator. Experiments were performed at least threetimes with triplicate samples.RNA InterferenceKnock-downandFocusOligoarrayAnalysis—

Duplex RNA of SOS1 target sequences flanking the cytosineinsertion were synthesized with the Silencer siRNA Construc-

tion Kit (Ambion). Transient trans-fection experiments were con-ducted as previously described (30).Briefly, 18 � 104 cells/well of gingi-val fibroblasts were plated in 6-wellplates 24 h before transfection.Double-stranded siRNA alone ortogether with SOS1 expression con-structs (1 �g/35-mm) was intro-duced into HeLa cells by Lipo-fectamine 2000 (Invitrogen). ThesiRNA for Luciferase (5�-CTTACG-CTGAGTACTTCGA) (31) wasused as a control. Total cellularlysates were collected after 48-htransfection and subjected toWest-ern blot analyses. To monitor theeffects of knock-down on prolifera-tion, fibroblasts were seeded at8000/well into 48-well plates. Cul-tures were transfected with indi-cated siRNA (50 nM) overnight andreplaced with fresh growth medium(day 0). At each time point, the totalnumber of cells was determined asdescribed above. Experiments wereperformed at least three times withtriplicate samples.

To monitor gene expression in response to EGF treatment,normal (control), and HGF1 fibroblast cultures were serum-starved for 16 h and treated with EGF (50 ng/ml) for 24 h. TotalRNA fromEGF-treated and untreated cultures was prepared asdescribed above. The complementary RNA probe was synthe-sized (TrueLabeling-Am kit, SuperArray Bioscience, Frederick,MD) together with biotin-16-UTP labeling. The complemen-tary RNA probes were incubated with cell cycle microarraymembranes, and the expression levels of each gene weredetectedwith chemiluminescence using a copalyl diphosphate-star substrate (SuperArray Bioscience). Membranes wereexposed to x-ray film (Kodak, XAR film) and signal analyzed bythe GEArray Expression Analysis Suite (SuperArray Bio-science) with subtraction of background and normalizationwith housekeeping genes of glyceraldehyde-3-phosphate dehy-drogenase and �-actin.Statistical Analyses—For comparisons of proliferation

assays, real-time PCR experiments and quantification of phos-pho-ERK levels, the Student’s t test was used to compare themean values between HGF1 and controls. Values of p � 0.05were considered significant.

RESULTS

Expression of Mutant SOS1 in Gingival Fibroblasts—Theinsertion mutation of SOS1 in HGF1 results in an early termi-nation as illustrated in Fig. 1A. To verify the presence of trun-cated SOS1 in HGF1 fibroblasts, antibody with the epitopeagainst theN-terminal region of SOS1 (SOS1(N)), detected oneband (�170 kDa) in controls and two bands (170 and 130 kDa)in HGF1 patients (Fig. 1B, upper panel). The 170-kDa band

FIGURE 1. Expression of mutant SOS1 in HGF1 fibroblasts. A, illustration of full-length and truncated SOS1with functional domains of histone folding (H), Dbl homology (DH), pleckstrin (PH), Ras exchanger motif (Rem),CDC25, and proline-rich binding domain (PRGB). The asterisk indicates the mutation site found in HGF1patients with insertion of cytosine (1083) followed by 22 residues (1083–1105) with frameshift region beforeearly termination. B, total cellular extracts (50 �g) of cultured fibroblasts from three control and three HGF1individuals were subjected to SDS-PAGE and blotted with SOS1(N) and �-actin antibody (upper panel). Controland HGF1 fibroblasts were transfected with indicated SOS1 constructs and probed with SOS1(N) and �-actinantibody (lower panel). The full-length (a) and truncated (b) SOS1 bands are indicated. C, total RNA fromfibroblasts of three controls and four HGF1 patients were subjected to reverse transcription-PCR analysis. Afternormalization against RNase P, the relative levels of SOS1 (solid bar) and TBP (striped bar) transcripts are pre-sented as mean � S.D. from three separate experiments with triplicate samples. The asterisk indicates signifi-cant difference between HGF1 and control (p � 0.05).




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corresponds to the full-length SOS1and the smaller �130-kDa bandapproximates the calculated molec-ular weight of the truncated mutantSOS1. To confirm this, expressionconstructs of wild-type and mutantSOS1 were transfected into primarygingival fibroblasts. As shown in Fig.1B (lower panel), the expressed wildtype (arrow a, lanes 2 and 5) andmutant (arrow b, lanes 3 and 6)bands were detected at the samepositions as the endogenous full-length (lanes 1 and 3) and truncatedSOS1 (lanes 4 and 5). Western blot-ting results revealed the intensity ofthe mutant (lower) band was 40%less than the full-length (upper)band in HGF1 samples (Fig. 1B).The lower amount of mutant SOS1could result from the instability ofeither its transcript or its proteinproduct. Real-time PCR experi-ments were conducted to monitorthe total levels, of both wild-typeand mutant SOS1 transcript inHGF1 fibroblasts. Overall levels ofSOS1 transcript were 40% lower inHGF1 fibroblasts than in controlfibroblasts (Fig. 1C). The TBP tran-script served as an internal control.The lower levels of mutant SOS1protein in HGF1 fibroblasts likelyreflect a less stable mutant SOS1transcript. Because all three sets ofcontrols andHGF1 patients showedsimilar results, only results fromoneset of control and HGF1 fibroblastsare presented.Sustained Activation of MAPK in

HGF1 Gingival Fibroblasts—Be-cause SOS1 plays a critical role inthe Ras/MAPK/ERK signaling path-way (22), the effect of the SOS1mutation on Ras signaling wasevaluated. Although Ras activitywas low in serum-starved controlfibroblasts, it increased rapidly afterEGF treatment and subsequentlydeceased, approaching basal levelsby 30 min (Fig. 2A). In contrast, Rasactivity was 5-fold higher in HGF1.Upon EGF stimulation, Ras activityin HGF1 fibroblasts showed higherlevels than control in all respectivetime intervals suggesting mutantSOS1 remained active even underserum-starved conditions, leading




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to higher Ras activity in response to growth factor stimuli. Wenext studied how the SOS1 mutation altered signal transduc-tion through the MAPK/ERK pathway. As shown in Fig. 2B,panel a (lanes 2–5), transient activation of ERK1/2 withincreasing concentrations of EGF resulted in a dose-relatedincrease in both control and HGF1 fibroblasts. The magnitudeof ERK1/2 activation was at least 30% greater in HGF1 than incontrol fibroblasts at each respective dosage of EGF treatment(Fig. 2B, panel b). In untreated cells, whereas the ERK1 signalwasweak but detectable only inHGF1 samples, the ERK2 signalalone showed�3-fold greater intensity inHGF1 than in controlfibroblasts (Fig. 2B, panel a, lane 1). These data suggest Rasremains active and leads to a sustained activation of ERK1/2signal in serum-starved HGF1 fibroblasts. Activation ofERK1/2 by EGF was evaluated in the presence of the selectivepharmacological inhibitors AG1478 and PD98059 (32, 33). Thepresence of PD98059 or AG1478 resulted in a similar degree ofreduction on phospho-ERK signal in both cell types (Fig. 2C,panel a, lanes 1–4). The overall phospho-ERK1/2 levelremained higher in HGF1 than in control fibroblasts (Fig. 2C,panel b). The duration of ERK activation was studied throughthe continued induction of EGF. In the presence of EGF, thephospho-ERK signal increased and peaked at the 1-h time pointin both cell types (Fig. 2D). The signal gradually returned tobasal levels after 6 h (Fig. 2D, panel a). Although the pattern ofphospho-ERK response to EGF induction was similar in bothcell types, the level was 50% higher in HGF1 than in controlfibroblasts (Fig. 2D, panel b).The effect of PD98059 on the duration of EGF-induced ERK

signaling is shown in Fig. 2E. Activation of ERK signaling wasreduced in the presence ofMEK inhibitor in both cell types (Fig.2E, panel a). However, the pattern of reduction was different inHGF1 compared with control cells (Fig. 2E, panel b). After20-min incubation with inhibitor, 30% of phospho-ERKremained in control fibroblasts, whereas 80% of phospho-ERK remained in HGF1 fibroblasts. The phospho-ERK signalwas sustained up to 2 h in HGF1 but not in control fibroblasts.At 4 h, phospho-ERKdecreased to basal levels in controls, how-ever, significant levels of active ERK signal remained in HGF1fibroblasts. These results demonstrate that, in the absence ofgrowth factors, Ras together with its downstream ERK1/2 sig-naling remained active and sustained in HGF1 fibroblasts.Transient induction by EGF elicited a stronger response, bothin magnitude and duration, indicating the functional conse-quence of the SOS1 mutation in HGF1 fibroblasts.

Subcellular Localization of Mutant SOS1 in GingivalFibroblasts—Because binding of growth factors to their receptorstriggers the recruitment of the SOS1-Grb2 complex from thecytoplasm to plasma membrane (34), we examined the distri-bution of SOS1 in subcellular fractions. In HGF1 fibroblasts,the endogenous full-length SOS1was found in both cytosol andmembrane fractions, with only trace amounts of mutant SOS1detected in the membrane fraction (data not shown). This isdue to the low expression level of the endogenousmutant SOS1in HGF1 fibroblasts (Fig. 1B). Therefore, the HA-tagged full-length and truncated SOS1 constructs were expressed in HeLacells, which have higher transfection efficiency than primarygingival fibroblasts (data not shown). Under starving condi-tions, �40% of full-length SOS1 was distributed in the mem-brane/organelle fraction (Fig. 3A, lane 4), whereas 60% oftruncated SOS1 was found in membrane/organelle fraction(lane 6). The SOS1(C) antibody, which only detects full-lengthSOS1, detected �30% of endogenous full-length SOS1 was dis-tributed in the membrane/organelle fraction (compare lanes 1and 2with lanes 5 and 6). The identity of the additional (upper)band found in the membrane/organelle fraction (lanes 2 and 6)remains unknown. These results demonstrate the relative ratioof distribution is higher for truncated SOS1 in the membrane/organelle fraction. Immunofluorescence staining revealed that,under growth conditions, both wild-type and mutant SOS1localized in the cytoplasm and cellularmembrane (Fig. 3B, pan-els a and b, arrows). However, in serum-starved fibroblasts,whereas wild-type SOS1 restricted in the cytoplasm (Fig. 3B,panel c), mutant SOS1 localized in both the cytoplasm andplasma membrane (Fig. 3B, panel d, arrows). These observationswere supported by the immunostaining on the distribution ofendogenous SOS1 in serum-starved fibroblasts (Fig. 3C). Theseresults suggest that, in the absence of growth factors, truncatedSOS1 localized in the plasma membrane even though it lacksthe carboxyl-proline-rich domains.Mutant SOS1 Leads to Stronger ERK Signaling in HGF1

Fibroblasts—To test if the presence of mutant SOS1 leads toincreased ERK signaling, SOS1 expression constructs weretransfected into gingival fibroblasts, and ERK was monitored.Transfection of increasing amounts of the mutant SOS1 con-struct into serum-starved control fibroblasts resulted in a dose-dependent increase of ERK signaling (Fig. 4, lanes 3–6). Levelsof phospho-ERK signal were 50% greater in fibroblasts trans-fected with truncated construct than those transfected withwild-type construct (lanes 2 and 6). Levels of phospho-ERK in

FIGURE 2. Ras activity and ERK signaling in HGF1 fibroblasts. Ras and ERK activation assays. A, serum-starved control and HGF1 fibroblasts were eitheruntreated (0 min) or treated for 5, 15, and 30 min with EGF (50 ng/ml). Ras activation was evaluated by the binding of Ras to Raf-Ras binding domain and blottedwith Ras antibody. Total Ras protein in whole cell lysate (WCL) is shown in the lower panel. The activation is expressed as -fold change over untreated normalcontrol. B–E, ERK activation assays. B and C, panel a, serum-starved fibroblasts were either treated with EGF (B, panel a, lanes 1–55: 0, 5, 10, 25, and 50 ng/ml) for10 min or preincubated with Me2SO (C, panel a, lane 2), PD98059 (lane 3), or AG1478 (lane 4) for 30 min before addition of EGF (50 ng/ml) for 10 min (lanes 2– 4).Cellular extracts were probed with antibodies of phospho-ERK1/2 and total ERK1/2 as a control. B and C, panel b, quantification of data from a. The data ofcontrol (solid bar) and HGF1 fibroblasts (striped bar) were normalized with respective ERK1/2 and expressed as the -fold changes over control (lane 1). D, a,cellular extracts of serum-starved fibroblasts treated with EGF (50 ng/ml) for indicated time intervals were probed with anti-phospho-ERK1/2 and �-actinantibodies. b, quantification of data from (a). The data of control (dashed line) and HGF1 fibroblasts (solid line) were normalized with each respective �-actin andexpressed as the -fold changes over control fibroblasts. E, panel a, serum-starved fibroblasts were treated with EGF (50 ng/ml) for 10 min, and medium wasreplaced with fresh medium containing PD 98059 (10 �M) (lanes 3– 6 and lanes 9 –12) and harvested at the indicated time points. Cellular extracts were probedwith antibodies of phospho-ERK1/2 and total ERK1/2 as a control. Panel b, quantification of data from a. The data of control (dashed line) and HGF1 fibroblasts(solid line) were normalized with each respective ��1/2 and expressed as the -fold changes over control (lane 1).




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wild-type-transfected fibroblasts were similar to fibroblaststransfected with three times less mutant construct (lanes 2 and4). These results provide evidence that the presence of mutantSOS1 leads to higher levels of phospho-ERK signal in gingivalfibroblasts.Selective Knock-down of Wild-type and Mutant SOS1 by

siRNA—RNA interference experiments were conducted to seeif eliminating the endogenous mutant SOS1 reduced ERK sig-naling and affected fibroblast proliferation. To achieve the spe-

cific knock-down on mutant tran-script, the siRNA target sequenceswere designed to flank themutationsite. Three sets of siRNAs weredesigned to include the extra cyto-sine base together with eitherupstream or downstream sequencesaround the mutation site (Fig. 5A,mS, mR, and mL). To evaluate thespecificity and effectiveness of eachsiRNA, the duplex RNAs were co-transfected with indicated SOS1expression constructs into HeLacells. Each siRNA showed differentdegrees of knock-down effect (Fig.5B). Almost all expressed full-lengthSOS1 was depleted in wS siRNA-treated samples (lane 3). Trace lev-els of SOS1 may represent endoge-nous SOS1, because it wasequivalent to that in the vector con-trol (lanes 1 and 3). Whereas wSshowed �100% depletion, mSexhibited �60% knock-down effectonwild-type SOS1 (lane 3 and 4). Asfor the other duplex RNAs, whereasmL reduced about half (lane 6), mRdid not have any effect on knock-down of wild-type SOS1 (lane 5). Inthe presence of expressed truncatedSOS1 transcript, the wS and mLsiRNAs showed slight or no knock-

down effect when compared with controls (lanes 7, 8, and 11).In contrast, both mS and mR siRNAs inhibited all of theexpressed truncated SOS1. The wS siRNA was therefore cho-sen to target the wild-type SOS1 transcript, andmR siRNAwaschosen to target mutant transcript due to its nominal effect onwild-type SOS1. To confirm the above observations, differentdosages ofmR siRNAwere co-transfectedwith either wild-typeor mutant SOS1 expression construct into HeLa cells. Fig. 5Cshows the level of wild-type SOS1 was gradually reduced as thedosage of mR increased, however, it was still detectable evenwhen 100 nM of mR was applied (lane 6). In contrast, mutantSOS1 transcript was depleted completelywhen as little as 25 nMof mR was used (lane 9). These results demonstrate that mRduplex RNA could specifically and effectively knock-down theexpressed truncated SOS1 with little effect on the expressedfull-length SOS1.The knock-down effects of wS and mR siRNA on endoge-

nous SOS1 and ERK signaling are shown in Fig. 5D. Wild-typeand mutant SOS1 were depleted 60 and 30%, respectively, bywS siRNA (lane 2). Although the mR siRNA did not affect thelevel of endogenouswild type, it showed a dose-response reduc-tion on the endogenous mutant SOS1 with almost completedepletion at 50 nM (lane 6). The functional effects of SOS1knock-down were demonstrated through the level of phospho-ERK signaling (Fig. 5, D and E). When transfected HGF1 fibro-blasts were maintained under serum-starved conditions, acti-

FIGURE 3. Subcellular distribution of full-length and truncated SOS1. A, HeLa cells were transfected withthe indicated constructs for 1 day and maintained in starving medium overnight before isolation of subcellularfractions. Equal amounts of cytosol (C; lanes 1, 3, and 5) and membrane/organelle (P; lanes 2, 4, and 6) fractionswere resolved in a NuPAGE gel and blotted with HA antibody. The blot was stripped and re-probed with SOS1(C) antibody. EGFR and �-actin were used as fraction marker and loading control. B, immunofluorescent stain-ing of transfected full-length (a and c) and truncated (b and d) SOS1 constructs in control gingival fibroblastsmaintained in either growth (a and b) or serum-starved (c and d) media. Cells were fixed and stained withanti-HA antibody and 4�,6-diamidino-2-phenylindole, dihydrochloride. The distribution of wild-type andmutant SOS1 on plasma membrane is indicated by an arrow (a, b, and d). Bars, 20 �m. C, serum-starved control(a) and HGF1 (b) fibroblasts were immunostained with SOS1(N) antibody. Arrows indicate endogenous SOS1distribution at cell peripheral. Bar, 5 �m.

FIGURE 4. Presence of mutant SOS1 results in an increase of ERK1/2 sig-nal. Control gingival fibroblasts were transfected with indicated SOS1expression constructs. The pCGN vector construct was added (lanes 3–5) toensure equal amounts of total plasmid were used for transfection. Culturemedium was replaced with serum-starved medium for 16 h before harvest.Cellular extracts were subjected to SDS-PAGE and probed with antibodiesagainst SOS1, phospho-ERK1/2, and total ERK1/2 as control.




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vated ERK signalingwas reduced�50% followingwS treatment(lane 2) and displayed a dose-dependent decrease in phospho-ERKwith increasing amounts ofmR siRNA (lanes 3–6). Similarreductions in phospho-ERK signal were observed when 50 nMwS or 5 nMmRwas applied; however, only 10% of the phospho-ERK signal remained compared with the control when 50 nMmR was used (Fig. 5E). Reduction in phospho-ERK by wS wasmost likely due to partial depletion of mutant SOS1 (Fig. 5D,lane 2). More importantly, although the mR siRNA showed

little effect on wild-type SOS1depletion, the phospho-ERK signaldecreased as the concentration ofmR increased (lanes 3–6) indicatingthat, under serum-starved cultureconditions, mutant SOS1 contrib-uted to sustained phospho-ERK sig-naling in HGF1 fibroblasts. Theseresults demonstrate that mR siRNAcan specifically target endogenousmutant transcript, with little if anyeffect on endogenous wild-typetranscript of SOS1.SOS1 Depletion Results in De-

creased Fibroblast Proliferation—To study the consequences of SOS1depletion on gingival fibroblast pro-liferation, fibroblasts were trans-fected with the indicated siRNAs,and cell proliferation was moni-tored. Proliferation of control fibro-blasts treated with wS (Fig. 6A,striped bar) decreased 20% on day 2compared with untreated controls(solid bar). On day 4, cell numberswere further decreased. Althoughcell numbers increased at both days6 and 8, the number of wS-treatedfibroblasts was still �30% lowerthan the untreated control fibro-blasts. As expected, mR siRNA didnot show any effect on the growth ofcontrol fibroblasts (unfilled bar). Asimilar trend appearedwhenwS andmR duplex RNAs were usedtogether (dotted bar), although thereduction in cell proliferation wasgreater compared with the culturestreated with wS alone (striped bar).Although depletion of wild-typeSOS1 reduced the proliferation ofcontrol fibroblasts, after day 4, cellproliferation started to recover,indicating the knock-down effectslast �4 days.When the same exper-iment was conducted for HGF1fibroblasts in the presence of eitherwS or mR siRNAs, cell proliferationalso decreased (Fig. 6B). Depletion

of wild-type SOS1 (striped bar) resulted in 35% decrease in cellproliferation compared with control HGF1 fibroblasts (solidbar) from day 2 to day 6; however, no significant difference wasobserved by day 8. Knock-down of mutant SOS1 (unfilled bar)resulted in a 50% reduction in cell proliferation comparedwith HGF1 controls; however, the cell growth remained lowand did not recover on day 8 as for the wild-type SOS1 (wS)knock-down. When HGF1 fibroblasts were treated with bothwS andmR siRNAs, cell proliferation decreased�70 and�80%

FIGURE 5. Selective knock-down of wild-type and mutant SOS1. A, sequences of siRNA designed to targeteither wild-type (wS) or mutant (mS, mR, and mL) SOS1 transcripts. The mutation site is indicated (underlined).B, HeLa cells were co-transfected with indicated SOS1 expression construct (1 �g) together with wS, mR, mS,and mL duplex siRNA (25 nM) for 48 h. The control samples were co-transfected with luciferase siRNA (lanes 2and 7). Cellular extracts were blotted with antibody of SOS1(N) and �-actin. C, HeLa cells were co-transfectedwith indicated SOS1 construct (pSOS1) and mR siRNA (0, 10, 25, 50, and 100 nM), and the cellular extracts wereblotted with SOS1(N) antibody and �-actin served as loading control. D, HGF1 fibroblasts were transfected withluciferase siRNA (control), wS (50 nM) or mR (lanes 3– 6: 5, 10, 25, and 50 nM) for 48 h, and cellular extracts wereresolved in SDS-PAGE and probed with antibodies of SOS1(N), phospho-ERK1/2, and total ERK1/2 as control.E, quantification of phospho-ERK1/2 from D. The data of phospho-ERK1/2 were normalized with each respec-tive ERK1/2 and expressed as -fold changes of mean � S.D. over normal control.

FIGURE 6. Effects of SOS1 depletion on gingival fibroblast proliferation. Gingival fibroblasts from normalcontrol subjects (A) or HGF1 patients (B) were transfected with siRNA (50 nM) of luciferase (control), wS, mR, orboth, incubated overnight, and the medium was replaced with fresh growth medium (day 0). Cell numberswere determined at indicated time points and presented as the average (�S.D.) -fold change over that of day2. Data are from three separate experiments with triplicate samples. The asterisk indicates statistical differencebetween control and siRNA duplex treated samples in HGF1 and normal control fibroblasts (p � 0.05).




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on days 2 and 4, respectively, compared with the HGF1 controlcells (dotted versus solid bars). These data suggest 1) targetedknock-down of the mutant SOS1 transcript reduced prolifera-tion of HGF1 fibroblasts and 2) knock-down of mutant SOS1had a greater effect on reduction of fibroblast proliferation thanthe knockdown of wild-type SOS1. These findings suggestmutant SOS1 is responsible for the increase of fibroblast pro-liferation seen in HGF1.Up-regulation of Cell Cycle Regulatory Proteins inHGF1Gin-

gival Fibroblasts—Sustained ERK signaling in fibroblasts isassociated with cell cycle control (35, 36). To test if prolongedERK signaling affects cell cycle progression in primary gingivalfibroblasts, we used a focused microarray containing 112 genesinvolved in cell cycle regulation. Under serum-starved condi-tions, HGF1- and EGF-treated control fibroblasts representsustained and transient ERK signaling, respectively. Tomonitorthe effects of either transient or sustained ERK signaling on theexpression level of cell cycle regulators, two experimentalgroups were studied: 1) EGF-treated versus untreated controlfibroblasts and 2) HGF1 versus control fibroblasts. As shown inTable 1, most transcripts showing a 2-fold increase in HGF1over control fibroblasts were involved in the cell cycle progres-sion and transition for G1 phase and from G1 into S phase. Inmost cases, the relative increase in differential expression wasgreater in the HGF1 versus control group than in the EGF-treated versus untreated control group, particularly, for cyclinsC, D1, D2, E1, and E2 and RBBP8 (CTIP) (Table 1). Althoughthe transcription factors E2F1 and E2F2 were increased�2-fold, their dimerization partners DP1 and DP2 were up-regulated 4- and 7-fold, respectively. These results revealthat, even under serum-starved conditions, key proteinsinvolved in cell cycle regulation continued to be expressed, sug-gesting that these genes may be downstream targets of the sus-tained ERK signaling in HGF1 fibroblasts.To validate themicroarray findings real-time PCR andWest-

ern blotting were performed. Cyclin E-Cdk2 and Rb-E2F-DPcomplexes are known to promote cell cycle transition from G1into S phase in mammalian cells (37, 38). When gingival fibro-blasts cultures were serum-starved overnight, more phospho-rylated Rb isoforms were detected in HGF1 fibroblasts than incontrols (Fig. 7A, lanes 1 and 3). After switching to growthmedium, a major phospho-Rb, most likely p130, was detected

in both cell types, and the overall phospho-Rb was higher inHGF1 fibroblasts (lanes 2 and 4) indicating sustained phospho-ERK signaling in HGF1 fibroblasts resulted in stronger Rbphosphorylation. Expression of cyclin E1 (CCNE1) was 3-foldgreater in HGF1 (Fig. 7B, unfilled bar) than in control fibro-blasts (solid bar) under serum-starved conditions, consistentwith microarray results. Similar increases were found in bothcontrol (striped bar) and HGF1 (dotted bar) fibroblasts grownin growth medium. Interestingly, cyclin E2 (CCNE2) expres-sion was 2.5-fold higher in HGF1 fibroblasts (unfilled bar) thanin control fibroblasts (solid bar) under serum-starved condi-tions. After switching to growth medium, levels of cyclin E2increased significantly: 15- and 46-fold higher in control(striped bar) andHGF1 (dotted bar) fibroblasts, respectively. Inaddition, cyclin E2 levels were 8-fold higher in HGF1 (dottedbar) than in control fibroblasts (striped bar) under growthmedium suggesting cyclin E2 plays a role in the overgrowth ofHGF1.Expressions of E2F1, E2F2, TFDP1, and TFDP2 were also

evaluated, because activation of the E2F family is the key stepfor cell cycle progression from G1 into S phase. Under serum-starved conditions, E2F1 transcript levels were similar for con-trol and HGF1 fibroblasts (Fig. 7C, solid and unfilled bars).After switching to growth medium, E2F1 expression increasedin control (10-fold) and HGF1 (40-fold) fibroblasts, respec-tively. Interestingly, the relative levels of E2F2 were 80-foldgreater than E2F1 in both types of fibroblasts under serum-starved conditions. TFDP1 expression levelswere 30%higher inHGF1 than in control fibroblasts in both serum-starved andgrowth conditions (Fig. 7D). No differences were found forTFDP2 expression between the two cells types regardless ofculture conditions; however, after switching from starving togrowth condition, it decreased 75% in both cell types. Takentogether, these data suggest that 1) cyclin E2, E2F1, and TFDP1play a major role in cell cycle regulation in gingival fibroblastsand 2) the cell cycle is more active in HGF1 fibroblasts than incontrol fibroblasts and may underlie the higher proliferationrates in HGF1 fibroblasts.One of the immediate-early gene products induced by ERK

signaling is the early growth response gene (Egr-1) (39). Underserum-starved conditions, trace amounts of Egr-1 weredetected in control fibroblasts (Fig. 8A, lane 1), whereas signif-

TABLE 1Comparison of sustained versus transient ERK signaling on the expression of cell cycle regulatorsGingival fibroblasts of normal and HGF1 were grown to 80% confluency, and the mediumwas switched to starving medium for 16 h before isolation of total RNA. For EGFtreatment, normal fibroblasts were serum-starved overnight and treated with EGF (50 ng/ml) for 24 h. The expression profile of cell cycle regulators was evaluated by CellCycle oligoarray.

Symbol GenBankTM Description (gene name) EGF-treated/untreateda HGF1/normal controla

CCNC NM_005190 Cyclin C 2.1 7.2bCCND1 NM_053056 Cyclin D1 1.3 4.1CCND2 NM_001759 Cyclin D2 1.8 4.6CCNE1 NM_001238 Cyclin E1 1.5 3.5CCNE2 NM_004702 Cyclin E2 2.4 5.2E2F1 NM_005225 E2F transcription factor 1 1.8 2.3E2F2 NM_004091 E2F transcription factor 2 2.0 2.9PCNA NM_182649 Proliferating cell nuclear antigen 2.6 3.6RB1 NM_000321 Retinoblastoma 1 (including osteosarcoma) (Rb) 5.1 3.8RBBP8 NM_002894 Retinoblastoma binding protein 8 (CTIP) 4.1 15.6TFDP1 NM_007111 Transcription factor DP-1 (DP1) 4.8 6.4TFDP2 NM_006286 Transcription factor DP-2 (DP2) 7.2 11.3

a The -fold change of “normal plus EGF versus untreated normal fibroblasts” and “HGF1 versus normal fibroblasts.”bA difference of 2-fold between two groups is shown in boldface.




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icant levels of Egr-1 were found in HGF1 fibroblasts (lane 2).After switching to growth medium, Egr-1 levels increasedslightly in both control and HGF1 fibroblasts (lanes 3 and 4),however, Egr-1 levels remained 5-fold higher in HGF1. Thepresence of wS, mR, or both siRNA duplexes reduced bothEgr-1 and PCNA, another marker for cell proliferation, inHGF1 fibroblasts compared with controls (Fig. 8B). Interest-

ingly, higher levels of Egr-1 andPCNA were detected in the pres-ence of wS compared with mR(lanes 2 and 3). Compared with thecontrol sample (lane 1) �10% ofEgr-1 and PCNA remained whenboth wS andmRwere used together(lane 4). These results are consistentwith the finding that greater ERKsignal was observed in the knock-down by wS than by mR siRNA inHGF1 fibroblasts (Fig. 5D). Theseobservations provide further evi-dence that sustained ERK signalingleads to the up-regulation of regula-tors that promote cell cycle progres-sion from G1 to S phase in HGF1fibroblasts.

DISCUSSION

In this study, we have elucidatedthe mechanism of gingival over-growth in HGF1 with SOS1 muta-tion. Our studies demonstrate thatthe overall level of SOS1 transcriptis lower in HGF1 fibroblasts sug-gesting the mutant transcript is sig-nificantly less than the wild-typetranscript. Although the relativelevel of wild-type SOS1 transcriptappears to be higher than themutant transcript in HGF1 fibro-blasts (Fig. 1B), targeting mutanttranscript by mR siRNA produced

more profound effects on reduction of cell proliferation thanwS (Fig. 6B), suggesting that the increased proliferation ofHGF1 fibroblasts was chiefly dependent upon the function ofmutant SOS1.Both the N- and the C-terminal domains are involved in

down-modulation of SOS activity (40). SOS1 lacking the C-ter-minal domains and targeted to the plasma membrane exhibitsincreased GEF activity and triggers the Ras signaling cascadewithout external stimuli (41–45). In HGF1 fibroblasts, withoutgrowth factors stimuli, we observed the truncated SOS1distrib-uted in themembrane/organelle faction.Although such subcel-lular fractionation analyses do not rule out the possibility thatmutant SOS1 could associate with endomembranes, immuno-staining of endogenous SOS1 and HA-tagged SOS1 showedthat mutant protein could distribute to the plasma membranein serum-starved fibroblasts. Themechanism for the transloca-tion remains unknown. It is unlikely that the novel 22 C-termi-nal residues resulting from the frameshift mutation play a roleinmembrane targeting, because it failed to direct green fluores-cent protein to the plasma membrane (data not shown).Recently, SOS1 substitution mutations found in some Noonansyndrome patients (46, 47) are critical in the maintenance ofSOS1 autoinhibition and lead to elevation of Ras activity. InHGF1, the truncation mutation abolishes the C-terminal pro-

FIGURE 7. Expression of cell cycle regulators in control and HGF1 fibroblasts. Fibroblast cultures weremaintained either in starving medium (S) overnight or switched to growth medium for 1 day (S G). A, nuclearextracts from control and HGF1 fibroblasts were resolved in NuPAGE and probed with phospho-Rb (pRb)antibody, and total Rb was used as control. B–D, total RNA from control and HGF1 fibroblasts were isolated, andthe expression levels of CCNE1 and CCNE2 (B), E2F1 and E2F2 (C), and TFDP1 and TFDP2 (D) were monitored byreverse transcription-PCR in control (solid and striped bars) and HGF1 (unfilled and dotted bars) fibroblast cul-tures maintained in serum-starved (S, solid and unfilled bars) or switched to growth medium (S G, striped anddotted bars). The data are from three separate experiments with triplicate samples and are presented as aver-ages (�S.D.) of the relative level over control fibroblasts in starving medium (control/S).

FIGURE 8. Expression of Egr-1 and PCNA in gingival fibroblasts. A, controland HGF1 fibroblasts were maintained in starving medium overnight (S) or instarving medium overnight and switched to growth medium for 1 day (S G).Cellular extracts were blotted with antibodies of Egr-1 and �-actin. B, HGF1fibroblasts were transfected with siRNA (50 nM) of luciferase (lane 1), wS, mR,or wS and mR together for 48 h, and cellular extracts were probed with anti-bodies of Egr-1, and PCNA, with total ERK1/2 as a control.




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line-rich domains and could also release the autoinhibitionstate of SOS1.Mutant SOS1 in HGF1 fibroblasts is capable of sustained

activation of Ras/MAPK signaling in the absence of growth fac-tors stimuli. Activation of ERK signaling was not blocked byAG1478 suggesting mutant SOS1 was already present in theplasma membrane, contributing to sustained activation of Rassignaling. The presence of PD98059 also reduced but did notcompletely block ERK signaling. These findings suggest thatmutant SOS1 may activate ERK signaling through other path-ways or that HGF1 cells reduced their sensitivity to the kinaseinhibitor through an unknown mechanism.Mutations in key components of the MAPK pathway that

result in sustained ERK activation correlate with tumor forma-tion (48, 49). Furthermore, high transformation activity inNIH3T3 fibroblasts and skin tumor formation in transgenicmice were shown by artificially constructed C-terminal trun-cated forms of SOS1 (43, 44, 50). It is believed that the strongpromoter activity used in these constructs produces high levelsof mutant protein expression, which leads to strong ERK sig-naling and transformation activity. Therefore, a threshold levelof expression might be necessary to transform cells. We specu-late that the benign form of overgrowth in HGF1 can beexplained as following: the low level of SOS1 mutant with partialrelease from its autoinhibition state could result in sustainedactivation with attenuated ERK signal, which is sufficient tolead to higher proliferation but not strong enough to causetransformation.In primary fibroblasts, overexpression of the key players in

the MAPK pathway such as Ras and MEK results in sustainedRas/ERK signaling and cell growth arrest (51, 52). When wecompared sustained (HGF1) versus transient (control treatedwith EGF) activated ERK signaling in gingival fibroblasts, thehigher expression of cyclin C, D, and E family members inHGF1 fibroblasts (Table 1) suggests these cyclin proteins arethe downstream targets of the sustained ERK signaling. Inter-estingly, the expression of cyclin E2 is low or undetectable innon-transformed cells and elevates significantly in tumor-de-rived cells (53).We found that levels of cyclin E2 increasemark-edly in both cell types after medium switch indicating cyclin E2plays a major role in transition from G1 to S phase in gingivalfibroblasts (Fig. 7B).E2F and TFDP also play a pivotal role in the control of cell

cycle progression (54, 55). Different E2F-DP complexes bind todifferent pRb family members, pRb, p107, and p130 (53). Uponhyperphosphorylation of Rb, the E2F-DP complex is releasedand activates their downstream target genes (56). We observedhigher levels of phospho-Rb in HGF1 fibroblasts under bothserum-starved and growth conditions. When serum-starvedfibroblasts were switched to growth conditions, E2F1 andTFDP1 levels, not E2F2 and TFDP2, were significantlyincreased (Fig. 7, C and D). It is likely that E2F1 plays a majorrole in cell cycle control in response to the sustainedERK signal.Both transient and sustained activation of ERK induce expres-sion of immediate-early genes such as fos, jun, and myc; how-ever, only sustained ERK signaling leads to phosphorylationand stabilization of immediate-early gene-encoded proteins(17, 18). We observed that the increase of Egr-1 expression in

HGF1, and the depletion of either wild-type or mutant SOS1,reduced the level of both Egr-1 and PCNA, one of the targetgenes of the E2F-DP complex (Fig. 8).The findings in this study reveal a Grb2-independent func-

tion of mutant SOS1 and suggest a mechanism for HGF1 fibro-blast proliferation (see supplemental Fig. S1). Unlike SOS1mutations in Noonan syndrome, where no gingival overgrowthwas reported (46, 47), the truncated SOS1 inHGF1 results clin-ically in gingival overgrowth. It is unclear why different SOS1mutations result in different clinical phenotypes. The SOS1insertion mutation in HGF1 results in a truncated, chimericprotein, whereas the Noonan syndrome mutations are substi-tutionmutations that alter a single amino acid. In addition, whythe overgrowth phenotype of HGF1 is restricted to gingival tis-sues remains unclear. At least four alternative isoforms of SOS1have been reported in different tissues and cell lines (49). Wehave observed two SOS1 isoforms (I and II) expressed in gingi-val fibroblasts, with themajority consisting of isoform II, whichcontains an extra 15 C-terminal residues that are not present inisoform I (57), (data not shown). It is unlikely these alternativeisoforms contribute to the tissue-specific phenotype observedin HGF1, because the early termination of SOS1 occursupstream of the 15-amino acid difference and would thereforeaffect both isoforms. It is possible that specific E2F-DP-Rbcomplexes together with their target genes may be present ingingival fibroblasts and contribute to the localized gingival phe-notype. In conclusion, the SOS1 mutation in HGF1 appears tobe a gain of function that drives an increase in gingival fibro-blast proliferation. Human HGF1 offers a unique model andopportunity to study the SOS1/Ras/MAPK signaling pathwayand its biological outcome in a disease state, and the appli-cation of RNA interference on allele-specific depletion ofSOS1 also provides a potential tool for controlling the gingi-val overgrowth.

Acknowledgments—We are grateful to Dafna Bar-Sagi, State Univer-sity of New York, Stony Brook, NY, for the humanHA-SOS1 construct.We thank Pamela G. Robey and Kenneth M. Yamada for criticalreading of the manuscript. We also thank Jung Eun Park for technicalassistance.

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and Thomas C. HartShyh-Ing Jang, Eun-Jin Lee, P. Suzanne Hart, Mukundhan Ramaswami, Debora Pallos

FibromatosisGerm Line Gain of Function with SOS1 Mutation in Hereditary Gingival

doi: 10.1074/jbc.M701609200 originally published online May 17, 20072007, 282:20245-20255.J. Biol. Chem.

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Germ Line Gain of Function with SOS1 Mutation in Hereditary