Molecular Cell, Vol. 1, 959–968, June, 1998, Copyright 1998 by Cell Press

The von Hippel-Lindau Tumor Suppressor ProteinIs Required for Proper Assemblyof an Extracellular Fibronectin Matrix

In keeping with Knudson’s two-hit model, inactivationor loss of both VHL alleles has been demonstrated inthe majority of sporadic clear cell renal carcinomas andcerebellar hemangioblastomas examined to date (Maherand Kaelin, 1997). Loss of heterozygosity at the VHL

Michael Ohh,* Robert L. Yauch,*Kim M. Lonergan,* Jean M. Whaley,†Anat O. Stemmer-Rachamimov,‡ David N. Louis,‡Brian J. Gavin,† Nikolai Kley,§William G. Kaelin, Jr.,*‖# and Othon Iliopoulos**Dana-Farber Cancer Institute and locus has been described in early premalignant lesions

of the kidney (Zhuang et al., 1995; Lubensky et al., 1996),Harvard Medical SchoolBoston, Massachusetts 02115 and reintroduction of wild-type pVHL into VHL2/2 renal

carcinoma cells suppresses their ability to form tumors†Bristol-Meyers Squibb Pharmaceutical ResearchInstitute in vivo (Iliopoulos et al., 1995; Gnarra et al., 1996). Thus,

inactivation of pVHL appears to be an early and possiblyPrinceton, New Jersey 08543‡Molecular Neuro-Oncology Laboratory requisite step in the development of clear cell carcinoma

of the kidney.Massachusetts General Hospital andHarvard Medical School pVHL is a 213-residue protein that resides primarily,Charlestown, Massachusetts 02129 but not exclusively, in the cytoplasm (Iliopoulos et al.,§Genome Therapeutics Corporation 1995; Lee et al., 1996; Los et al., 1996; Corless et al.,Waltham, Massachusetts 02154 1997). pVHL binds to elongin B and elongin C, two pro-‖Howard Hughes Medical Institute teins that were originally identified as components of aBoston, MA 02115 tripartite transcriptional elongation factor calledelongin,

or SIII (Aso et al., 1995; Duan et al., 1995b; Kibel et al.,1995; Kishida et al., 1995). Whether pVHL plays a rolein the control of transcriptional elongation in vivo is notSummaryknown. Cells lacking pVHL overproduce hypoxia-induc-ible mRNAs, such as the vascular endothelial growthFibronectin coimmunoprecipitated with wild-type vonfactor (VEGF) mRNA, under normoxic conditions (GnarraHippel-Lindau protein (pVHL) but not tumor-derivedet al., 1996; Iliopoulos et al., 1996; Siemeister et al.,pVHL mutants. Immunofluorescence and biochemical1996; Mukhopadhyay et al., 1997). The available data,fractionation experiments showed that fibronectinhowever, suggest that the effect of pVHL on hypoxia-colocalized with a fraction of pVHL associated withinducible mRNAs is mediated largely at the level ofthe endoplasmic reticulum, and cold competition ex-mRNA stability rather than at the level of transcriptionalperiments suggested that complexes between fibro-elongation (Gnarra et al., 1996; Iliopoulos et al., 1996).nectin and pVHL exist in intact cells. Assembly of an

The core elongin B/C binding domain within pVHLextracellular fibronectin matrix by VHL2/2 renal carci-(residues 157–172), at least as measured in vitro, is anoma cells, as determined by immunofluorescence andhotspot for mutations in VHL kindreds (Kibel et al., 1995;ELISA assays, was grossly defective compared withKishida et al., 1995; Zbar et al., 1996). Thus, binding toVHL1/1 renal carcinoma cells. Reintroduction of wild-elongin B and C likely contributes to tumor suppressiontype, but not mutant, pVHL into VHL2/2 renal carci-by pVHL. Recently, Hs-Cul2 has been shown to bind vianoma cells partially corrected this defect. Finally, ex-

tracellular fibronectin matrix assembly by VHL2/2 elongin C to pVHL (Pause et al., 1997; Lonergan et al.,mouse embryos and mouse embryo fibroblasts (MEFs), 1998). Binding to elongin B, elongin C, and Hs-Cul2 isunlike their VHL1/1 counterparts,was grossly impaired. necessary for pVHL to regulate the stabilty of hypoxia-These data support a direct role of pVHL in fibronectin inducible mRNAs (Lonergan et al., 1998). The primarymatrix assembly. sequences of Hs-Cul2 and elongin C are similar to those

of the Saccharomyces cerevisiae proteins Cdc53 andSkp1, respectively (Bai et al., 1996; Mathias et al., 1996;IntroductionPause et al., 1997; Lonergan et al., 1998). Furthermore,

von Hippel-Lindau disease is a hereditary cancer syn- elongin B contains an ubiquitin-like domain (Garrett etdrome caused by germline mutations of the von Hippel- al., 1995). In yeast, Cdc53 and Skp1 form componentsLindau tumor suppressor gene (Maher and Kaelin, 1997). of multiprotein complexes that target substrates forAffected individuals develop a variety of tumors, includ- ubiquitination (Jackson, 1996; Hopkin, 1997). Thus, oneing clear cell carcinomas of the kidney, pheochromo- model, which remains to be tested, is that inhibition ofcytomas, and vascular tumors of the central nervous hypoxia-inducible mRNAs and tumor suppression bysystem and retina (Maher and Kaelin, 1997). Tumor de- pVHL relates to its ability to regulate the covalent linkagevelopment in this setting is due to the absence of the of ubiquitin, or perhaps elongin B, to as yet undeter-wild-type VHL gene product (pVHL) in a susceptible cell mined effector molecules.as a consequence of mutation or loss of the remaining Nonetheless, many naturally occurring presumed loss-wild-type VHL allele (Maher and Kaelin, 1997). of-function VHL mutations map outside the elongin/Hs-

Cul2 binding domain (Duan et al., 1995a, 1995b; Kibelet al., 1995; Kishida et al., 1995; Zbar et al., 1996). Fur-# To whom correspondence should be addressed (e-mail: william_

kaelin@dfci.harvard.edu). thermore, striking genotype–phenotype correlations are

Molecular Cell960

Figure 1. p200 Specifically Coimmunopre-cipitates with pVHL

(A and B) 786-O renal carcinoma cells stablytransfected with a plasmid encoding HA-epi-tope-tagged wild-type pVHL ([A], lanes 6–10;[B], lane 2), pVHL Y98H ([B], lane 3), pVHLW117R ([B], lane 4), pVHL R167W ([B], lane5), or the backbone expression plasmid ([A],lanes 1–5; [B], lane 1) were labeled with 35S-methionine, lysed, and immunoprecipitatedwith the indicated antibodies (A) or anti-HAantibody (B). Bound proteins were resolvedby electrophoresis in a 7.5%–15% discontin-uous SDS–polyacrylamide gel and detectedby fluorography. Recovery of the pVHL spe-cies in (B) was confirmed by anti-HA immu-noblot analysis (lower). Asterisk indicatesbackground band (B).

emerging with respect to the risk of developing the vari- Resultsous tumors that comprise the VHL syndrome. For exam-ple, mutantVHL alleles can besegregated based ontheir 786-O renal carcinoma cells have undergone loss of

heterozygosity at the VHL locus, and the remaining VHLpropensity to cause pheochromocytomas (VHL type Ior II) or renal cell carcinomas (type IIA or IIB) (Zbar et allele contains a frameshift mutation (Gnarra et al., 1994).

Thus, these cells lack wild-type VHL protein (pVHL). Weal., 1996; Maher and Kaelin, 1997). The simplest interpre-tation of these findings is that biochemical functions previously generated 786-O subclones that were stably

transfected with a mammalian expression plasmid en-unrelated to binding to elongin B/C and Hs-Cul2 contrib-ute to tumor suppression by pVHL, and that the relative coding a hemagglutinin (HA)-tagged version of wild-type

pVHL (pRc-HA-VHL) or with the backbone expressioncontribution of these activities to tumor suppression bypVHL differs in different tissues. vector (pRc). Reintroduction of wild-type pVHL into

these cells did not grossly alter their ability to grow inFibronectin is an extracellular glycoprotein that bindsto and signals via heterodimeric cell surface receptors vitro but dramatically inhibited their ability to form tu-

mors in nude mice (Iliopoulos et al., 1995).known as integrins (Ruoslahti, 1991; Hynes, 1992). Lossof fibronectin matrix assembly has been recognized asa feature of cellular transformation for many years (Chenet al., 1976; Hynes and Destree, 1978; Hynes et al., 1978; pVHL Specifically Associates with a 200 kDa

Cellular ProteinLipkin et al., 1978; Vaheri et al., 1978; Ruoslahti, 1991).Furthermore, fibronectin can revert some aspects of the To look for cellular proteins that specifically associate

with pVHL, these cells were metabolically labeled withtransformed phenotype in vitro and malignant pheno-type in vivo (Giancotti and Ruoslahti, 1990; Pasqualini 35S-methionine, lysed, and immunoprecipitated with ei-

ther a polyclonal affinity-purified anti-VHL antibody (Fig-et al., 1996). Here, we show that pVHL and fibronectinbiochemically interact in intact cells. VHL2/2 renal carci- ure 1A, lanes 2 and 7), a monoclonal anti-HA antibody

(Figure 1A, lanes 3 and 8), a monoclonal anti-VHL anti-noma cells, unlike VHL1/1 renal carcinoma cells, weredefective for assembly of an extracellular fibronectin body (lanes 4 and 9), or control antibodies (lanes 1, 5,

6, and 10) under stringent (high-salt) conditions. Anti-matrix. VHL2/2 mouse embryos and mouse embryo fi-broblasts (MEFs), but not their VHL1/1 counterparts, VHL and anti-HA immunoprecipitates of HA-pVHL-pro-

ducing cells contained pVHL and five additional proteinswere likewise grossly defective in this regard. Reintro-duction of wild-type, but not mutant, pVHL into VHL2/2 with molecular weights of z200, 70, 68, 18, and 15 kDa

(compare lanes 7–9 to lanes 2–4). We and others haverenal carcinoma cells partially restored their ability toform a fibronectin matrix. Thus, pVHL and fibronectin previously shown that the 70, 18, and 15 kDa proteins

are Hs-Cul2 and elongin B and C, respectively (Duan etphysically interact in vivo, and this interaction affects theability of cells to assemble an extracellular fibronectin al., 1995b; Kibel et al., 1995; Pause et al., 1997; Lonergan

et al., 1998).matrix.

von Hippel-Lindau Protein and Fibronectin961

We next stably transfected 786-O cells with mamma-lian expression plasmids encoding HA-tagged versionsof pVHL missense mutants (Y98H, W117R, or R167W)corresponding to naturally occurring presumed loss-of-function VHL mutations (Figure 1B) (Zbar et al., 1996).These cells were metabolically labeled, lysed, and im-munoprecipitated with an anti-HA antibody as describedabove. All of these mutants measurably interacted, al-beit at reduced levels, with elongin B, elongin C, andHs-Cul2 under these assay conditions. In contrast, bind-ing to p200 and p68 was either absent or barely detect-able, raising the possibility that binding to p200 or p68or both contributes to tumor suppression by pVHL.

p200 Is Fibronectinp200 was next purified by preparative anti-HA immuno-electrophoresis of HA-pVHL-producing cells, and twotryptic peptides were isolated and sequenced. Eachrevealed a sequence present in fibronectin. To test thepossibility that p200 was indeed fibronectin, 786-Ocells stably transfected with pRc-HA-VHL, pRc-HA-VHL(R167W) or the backbone vector were metabolically la-beled with 35S-methionine, lysed, and immunoprecipi-tated with a control antibody, an anti-HA antibody, oran antifibronectin antibody (Figure 2A). Under these

Figure 2. p200 Is Fibronectinelectrophoretic conditions, p200 resolved into a thinlyspaced doublet (p200 upper and p200 lower; compare (A) 786-O renal carcinoma cells stably transfected with a plasmid

encoding HA-epitope-tagged wild-type pVHL (lanes 4–6), pVHLlane 5 with lanes 2 or 8). A similar doublet was observedR167W (lanes 7–9), or the backbone expression plasmid (lanes 1–3)in antifibronectin immunopreciptates (compare lanes 5were labeled with 35S-methionine, lysed, and immunoprecipitatedand 6). Partial proteolytic peptide mapping confirmedwith the indicated antibodies. Bound proteins were resolved by

that the p200 proteins present in the anti-VHL immuno- electrophoresis in a 7.5% SDS–polyacrylamide gel and detected byprecipitates were highly similar to those present in the fluorography.

(B) Partial proteolytic peptide mapping of the upper and lower radio-antifibronectin immunoprecipitates (Figure 2B). Finally,labeled p200 bands in (A) lane 5 (anti-HA) and lane 6 (anti-fibronec-the p200 proteins present in anti-HA immunoprecipi-tin), followingdigestion with 50 or 500 ng of V8protease as indicated.tates reacted with antifibronectin antibodies in Western(C) 786-O renal carcinoma cells stably transfected with a plasmidblot assays (Figure 2C, compare lane 5 with lanes 6encoding HA-epitope-tagged wild-type pVHL (lanes 4–6) or the

and 2). Conversely, pVHL was present in antifibronectin backbone expression plasmid (lanes 1–3) were lysed and immuno-immunoprecipitates as determined by anti-HA Western precipitated with the indicated antibodies. Bound proteins were

resolved by electrophoresis in either 7.5% or 15% SDS–poly-blot analysis (compare lane 6 with lanes 5 and 3). Takenacrylamide gels and detected by anti-fibronectin or anti-HA immu-together, these results confirmed that p200 was fibro-noblot analysis, respectively.nectin.

A protein with an apparent molecular weight of z68kDa was detected in antifibronectin immunoprecipitatesin the absence of pVHL (data not shown). This protein antibody (Figure 3A center, red). In either case, stainingcomigrated with p68 present in anti-VHL immunoprecip- of the cytoplasm, with a particulate pattern suggestiveitates, and the partial proteolytic peptide maps of these of endoplasmic reticulum/Golgi, was observed. Super-two proteins were identical (data not shown). Thus, the imposing both images suggested significant colocaliza-presence of p68 in anti-pVHL immunoprecipitates can tion of both pVHL and fibronectin in these cells (Figurebe explained by virtue of its ability to bind to fibronectin. 3A right, yellow). In the second set of experiments, these

cells were biochemically fractionated into nuclear, cyto-plasmic, and membrane fractions (Figure 3B). TheseA Fraction of Cellular pVHL Colocalizes

with Fibronectin fractions were immunoprecipitated with anti-HA anti-body (lanes 2–5), anti-fibronectin (lanes 6–9), or controlTo ask whether pVHL and fibronectin might form com-

plexes in intact cells, several approaches were used. antibody (lanes 1 and 10). The immunoprecipitates werethen resolved by SDS–polyacrylamide gel electrophore-Biochemical fractionation and immunofluorescence stud-

ies suggest that pVHL resides largely in the cytoplasm sis and immunoblotted with anti-fibronectin (top) andanti-HA (lower) antibodies. As expected, pVHL was pres-and, to a lesser extent, in the nuclear and cell membrane

compartments (Iliopoulos et al., 1995; Lee et al., 1996; ent in anti-fibronectin immunoprecipitates prepared fromthe membrane fraction (lane 6) but not in the nuclear orLos et al., 1996; Corless et al.,1997; Pause et al.,1997). In

the first set of experiments, 786-O cells stably producing cytoplasmic fractions (lanes 8 and 7). Similarly, fibronec-tin was found exclusively in the anti-HA immunoprecipi-HA-pVHL were immunostained with either an anti-HA

antibody (Figure 3A left, green) or an anti-fibronectin tates prepared from the membrane fractions (lane 5).

Molecular Cell962

Figure 4. pVHL and Fibronectin Form Complexes Prior to Cell Lysis

(A) Unlabeled 786-O renal carcinoma cells ectopically producingHA-pVHL W117R (lane 1) or HA- pVHL (lanes 2–6) were lysed andimmunoprecipitated with anti-HA antibody. In parallel, 786-O cellsthat did not ectopically produce pVHL were radiolabeled with 35S-methionine and lysed. Aliquots of the radiolabeled extract were thenadded to the unlabeled anti-HA immunoprecipitates in the absence(lanes 1 and 2) or presence of increasing amounts of unlabeledpurified fibronectin (lanes 3–6). After an additional 1 hr incubation,the immune complexes were then washed, resolved by SDS–polyacrylamide gel electrophoresis, and detected by fluorography.Both the unlabeled immunoprecipitates and the aliquots of radiola-beled cell extact were derived from a comparable number of cells.(B) 786-O renal carcinoma cells ectopically producing HA-pVHLW117R (lane 1) or HA-pVHL (lanes 2–6) were radiolabeled with 35S-methionine, lysed in the absence (lanes 1 and 2) or presence ofincreasing amounts of unlabeled purified fibronectin (lanes 3–6; finalfibronectin concentration 5 28, 56, 83, and 111 nM, respectively),and immunoprecipitated with anti-HA antibody. Bound proteins

Figure 3. Colocalization of pVHL and Fibronectin in Cells were detected as in (A).In (A) and (B), lane 6 contained an z10-fold molar excess of exoge-(A) Immunofluorescence staining of 786-O renal carcinoma cellsnous unlabeled fibronectin, relative to the amount present in thestably producing HA-wild-type pVHL using mouse anti-HA mono-labeled cell extract, as determined by anti-fibronectin Western blotclonal antibody (left) and rabbit anti-fibronectin sera (middle). Boundanalysis (data not shown). (NS) indicates nonspecific bands that doantibody was detected using FITC-conjugated anti-mouse and rho-not react with anti-fibronectin antibodies in Western blot assays.damine-conjugated anti-rabbit antibodies, respectively, and a con-

focal microscope. Overlay of VHL and fibronectin images is shownin the right panel.(B) 786-O renal carcinoma cells ectopically producing HA-wild-type pVHL Physically Interacts with FibronectinpVHL were disrupted by hypotonic lysis and dounce homogeniza-

in Intact Cellstion. Nuclear (N), cytoplasmic (C), and membrane (M) fractions wereTaken together, these experiments suggested that fibro-prepared and immunoprecipitated with anti-HA (lanes 3–5) and anti-nectin and a fraction of pVHL might colocalize in thefibronectin (lanes 6–8) antibodies. In parallel, whole cell extracts

were prepared and similarly immunoprecipitated with control (lanes endoplasmic reticulum/Golgi. They left open the possi-1 and 10), anti-HA (lane 2), and anti-fibronectin (lane 9) antibodies. bility, however, that complex formation between pVHLSpecifically bound proteins were detected by anti-fibronectin (up- and fibronectin occurred only after cell lysis. To addressper) or anti-HA (lower) immunoblot analysis.

this possibility, cold competition experiments were per-(C) 293 human embryonic kidney cells were fractionated as in (B) andformed with purified fibronectin (Figure 4). 786-O cellsimmunoprecipitated with an anti-VHL monoclonal antibody (IG32)producing HA-pVHL W117R (Figure 4B, lane 1) or HA-(lanes 1–4) or a control antibody (PAb419) (lane 5). Bound proteins

were detected by anti-fibronectin immunoblot analysis. wild-type pVHL (Figure4B, lanes 2–6) weremetabolicallylabeled with 35S-methionine and then lysed in the pres-ence of excess purified unlabeled fibronectin (Figure4B, lanes 3–6). Under these conditions, unlabeled fibro-In parallel, 293 human embryonic kidney cells, which

contain wild-type pVHL (Iliopoulos et al., 1995; Kibel et nectin had no effect on the recovery of radiolabeledfibronectin in the anti-HA immunoprecipitates (Figureal., 1995), were similarly fractionated and immunopre-

cipitated with an anti-VHL monoclonal antibody (IG32) 4B, compare lane 2 to lanes 3–6), suggesting that pVHL/fibronectin complexes exist prior to cell lysis.(Figure 3C, lanes 1–4) or a control antibody (lane 5). Anti-

fibronectin immunoblot analysis of the immunoprecipi- As a control for this experiment, similar cold competi-tion experiments were conducted under conditionstates again confirmed the presence of fibronectin speci-

fically in the anti-VHL immunoprecipitates prepared where radiolabeled fibronectin could only bind to pVHLafter cell lysis. Unlabeled 786-O cells producing HA-from the membrane fraction. Thus, complexes con-

taining fibronectin and pVHL can be detected in cells pVHL W117R (Figure 4A, lane 1) or HA-pVHL (Figure 4A,lanes 2–6) were lysed and immunoprecipitated with anproducing physiological amounts of pVHL.

von Hippel-Lindau Protein and Fibronectin963

anti-HA antibody. In parallel, parental 786-O cells weremetabolically labeled with 35S-methionine, lysed, andaliquoted.These aliquots were added to the immunopre-cipitated HA-pVHL species in the absence (Figure 4A,lanes 1 and 2) or presence of increasing amounts ofpurified fibronectin (lanes 3–6). After an additional incu-bation period, the immune complexes were washed andanalyzed as above. p200/fibronectin bound to wild-typepVHL (lane 2) but not to pVHL W117R (lane 1) in thisassay. Thus, the failure of fibronectin to coimmunopre-cipitate with pVHL W117R (Figure 1B) appears to be anintrinsic defect of this molecule and not a result of al-tered subcellular localization. Furthermore, purified un-labeled fibronectin now significantly and specificallycompeted with radiolabeled fibronectin for binding topVHL. Thus, excess cold fibronectin blocked the interac-tion between pVHL and radiolabeled fibronectin underexperimental conditions where complex formation oc-curred exclusively after cell lysis. In summary, while fi-bronectin is capable of binding to pVHL after cell lysis(Figure 4A, lane 2), the recovery of fibronectin in anti-VHL immunoprecipitates of VHL1/1 extracts, such as inFigure 1, is due primarily to fibronectin/pVHL complexesthat exist prior to cell lysis.

Renal Carcinoma Cells Lacking pVHL Exhibita Defect in Fibronectin Matrix AssemblyTo begin to understand the functional significance ofpVHL binding to fibronectin, anti-fibronectin immuno-fluorescence staining of VHL2/2 and VHL1/1 renal carci-noma cells was performed (Figure 5 and data notshown). VHL2/2 renal carcinoma cells (A498 [A] and786-O [C]) exhibited intense fibronectin staining in apattern most consistent with localization in the endo-

Figure 5. Renal Carcinoma Cells Display Alterations in Fibronectinplasmic reticulum/Golgi and scant fibronectin stainingMatrix Assembly

in the extracellular space. In contrast, VHL1/1 renal car-(A–F) Anti-fibronectin immunofluorescence. A498 (VHL2/2) (A),

cinoma cells (CAKI-1 [B] and ACHN [data not shown]) CAKI-1 (VHL1/1) (B), and 786-O (VHL2/2) (C) renal carcinoma cells,produced abundant extracellular fibronectin in charac- as well as 786-O renal carcinoma cells stably transfected with ateristic fibrillar arrays. 786-O cells ectopically producing plasmid encoding HA-epitope-tagged wild-type pVHL (F), pVHL

W117R (E), or the backbone expression plasmid (D), were grownwild-type pVHL (WT-8 [F]), unlike cells producing pVHLon coverslips for 6 days. Fibronectin deposition was detected byW117R (E) or cells stably transfected with an emptyindirect immunofluorescence using a rabbit anti-fibronectin an-expression plasmid (D), now produced a recognizabletisera.

extracellular fibronectin matrix. In contrast, the distribu- (G and H) Fibronectin ELISA. A498 (VHL2/2), CAKI-1 (VHL1/1), andtion of laminin, as determined by immunofluorescence 786-O (VHL2/2) renal carcinoma cells, as well as 786-O renal carci-staining, did not measurably differ between VHL1/1 and noma cells stably transfected with a plasmid encoding HA-epitope-

tagged wild-type pVHL, pVHL W117R, or the backbone expressionVHL2/2 renal carcinoma cells (data not shown).plasmid (pRc) were grown on a plastic surface for 6 days in 96-wellWe noted, however, that the fibronectin staining ofplates. Fibronectin deposition was measured by ELISA as described786-O cells ectopically producing wild-type pVHL wasin the Experimental Procedures. The amount of fibronectin depos-

heterogeneous. This, coupled with the subjective nature ited was quantitated from a standard fibronectin curve, and the dataof the immunofluorescence assay, led us to develop a is representative of five individual experiments. Error bars indicatequantitative assay for fibronectin deposition. To this one standard deviation. (p , .01 comparing VHL1/1 and VHL2/2

cells).end, equal numbers of cells were plated in 96-well plas-tic dishes. Six days later, cells were removed by treat-ment with EDTA. Complete removal of the cells wasconfirmed by microscopy. The wells were then incu- vastly exceeded that of A498 (VHL2/2) renal carcinoma

cells (Figure 5G). Similarly, fibronectin deposition bybated with a polyclonal anti-fibronectin antibody fol-lowed by an alkaline phosphatase–conjugated goat 786-O cells ectopically producing wild-type pVHL greatly

exceeded that of cells ectopically producing pVHLanti-rabbit antibody. Bound antibody was assayed col-orimetrically. In keeping with the immunofluorescence W117R or pVHL C162F, or of empty vector transfectants

(pRc) (Figure 5H). Neither pVHL W117R nor pVHL C162Fdata cited above, the ELISA showed that fibronectindeposition by CAKI-1 (VHL1/1) renal carcinoma cells bind to fibronectin (Figure 1B and data not shown). In

Molecular Cell964

contrast, the presence or absence of pVHL had no effecton the ability of renal carcinoma cells tobind to fibronec-tin-coated surfaces in quantitative adhesion assays(data not shown). Thus, the failure of VHL2/2 cells toproperly assemble a fibronectin matrix cannot be as-cribed to their lacking integrins capable of binding tofibronectin (see also Discussion).

These results left open the possibility that the ob-served differences in fibronectin matrix assembly werean indirect consequence of the above-mentioned abilityof pVHL to bind to elongin B, elongin C, and Cul2. Thislatter activity has been linked to the ability of pVHL toregulate hypoxia-inducible mRNA accumulation (Loner-gan et al., 1998) and, under certain experimental condi-tions, the ability to exit the cell cycle (Pause et al., 1998).To this end, we also tested 786-O cells ectopically pro-ducing N-terminal (N72) and C-terminal pVHL (C197)truncation mutants that bind to elongin B, elongin C,and Cul2 (Lonergan et al., 1998) but are incapable ofbinding to fibronectin (data not shown). These mutants,despite the ability to regulate hypoxia-inducible mRNAs(Lonergan et al., 1998), did not restore the ability of786-O cells to deposit an extracellular fibronectin matrix(Figure 5H). Thus, fibronectin binding is necessary, andbinding to elongin B, elongin C, and Cul2 is not sufficientfor pVHL to promote fibronectin matrix assembly.

Defective Fibronectin Matrix Formationin VHL Nullizygous Mouse EmbryosTaken together, the immunofluorescence and ELISAdata supported a role of pVHL in fibronectin matrix as-sembly. To test this further, day 9–10 VHL1/1 and VHL2/2

embryos were stained with a polyclonal anti-fibronectin Figure 6. Diminished Fibronectin Staining in VHL Nullizygoussera (Figure 6A, bottom) or, as a control, a polyclonal Mouse Embryosanti-actin serum (Figure 6A, top). Fibronectin staining (A) Sagittal sections from VHL1/1 (left) and VHL2/2 (right) mouse

embryos were stained with either anti-actin (top) or anti-fibronectinwas more intense in the VHL1/1 embryos than in the(bottom) polyclonal antibodies. Magnification 5 403.VHL2/2 embryos (Figure 6A). At higher power magnifica-(B) Sagittal sections from VHL1/1 (left) and VHL2/2 (right) mousetion, linear deposits of fibronectin were readily appreci-embryos were stained with anti-fibronectin antisera. Shown are

ated in the basement membrane regions of VHL1/1 em- higher power magnification (10003) views of comparable regions.bryos but were virtually absent in VHL2/2 embryos Note the presence of extracellular fibronectin fibrillar arrays in base-(Figure 6B). Finally, VHL1/1, VHL1/2, and VHL2/2 MEFs ment membrane regions (arrows).were grown on coverslips and stained for fibronectin(Figure 7). As expected, VHL1/1 and VHL1/2 MEFs pro-duced an abundant fibronectin matrix (A and C). In con- data that suggest that pVHL interacts with fibronectin.trast, virtually no fibronectin matrix was produced by Specifically, we found that fibronectincoimmunoprecip-VHL2/2 MEFs (E). Comparable numbers of MEFs were itated with wild-type pVHL but not various pVHL mis-present in these fields as determined by DAPI staining sense mutants under stringent conditions, and cold(B, D, and F). Thus, the results obtained with otherwise competition experiments strongly suggested that theisogenic mouse embryos and mouse embryo fibroblasts pVHL/fibronectin complexes detected existed prior tocorroborated the findings obtained with renal carcinoma cell lysis. Furthermore, biochemical fractionation andcell lines. immunofluorescence studies showed that a significant

fraction of pVHL colocalized with fibronectin. Finally,examination of VHL1/1 and VHL2/2 renal carcinoma cellDiscussionlines, as well as VHL1/1 and VHL2/2 mouse embryos andmouse embryo fibroblasts, revealed that assembly ofThe identification of interacting proteins has helped toan extracellular fibronectin matrix required an intact VHLelucidate the functions of tumor suppressor proteins.allele. Taken together, these results suggest that pVHLFor example, the function of the retinoblastoma proteinphysically interacts with fibronectin invivo underphysio-has been linked to its ability to bind to members of thelogical conditions, and that this interaction affects theE2F transcription factor family (Kaelin, 1997). Likewise,ability of cells to assemble an extracellular fibronectinthe function of the APC tumor suppressor protein hasmatrix.been linked to its ability to bind to b-catenin (Nakamura,

1997). In this report, we provide biochemical and genetic The mechanism by which physical association of

von Hippel-Lindau Protein and Fibronectin965

elongin C and Cul2. These two proteins are suspectedof playing a role in protein ubiquination based on theirsimilarity to Skp1 and Cdc53, respectively (Bai et al.,1996; Mathias et al., 1996; Pause et al., 1997; Lonerganet al., 1998). Thus, one model, which remains to betested, is that the absence of functional pVHL leads toan accumulation of malfolded fibronectin species thatinterfere with the assembly of macroscopic fibronectinfibrillar arrays.

In metabolic labeling studies, we detect five cellularproteins that specifically coimmunoprecipitate with thevon Hippel-Lindau protein under stringent conditions.Three of these proteins, elongin B, elongin C, and Hs-Cul2, interact with a colinear domain present within thepVHL C terminus (Lonergan et al., 1998). Many naturallyoccurring VHL mutations map outside of this domainand, as shown here and elsewhere, give rise to proteinsthat measurably interact with elongin B, elongin C, andCul2 (Duan et al., 1995; Kibel et al., 1995; Kishida et al.,1995; Zbar et al., 1996). In contrast, every pVHL mutantwe have examined to date has lost the ability to bindto fibronectin, strongly suggesting that binding to fibro-

Figure 7. Defective Extracellular Fibronectin Matrix Assembly by nectin contributes to tumor suppression by pVHL. Con-VHL2/2 Mouse Embryo Fibroblasts

ceivably, the clinical phenotype associated with a givenVHL1/1 (A and B), VHL1/2 (C and D), and VHL2/2 (E and F) were

pVHL mutant is determined largely by the degree togrown on coverslips. Fibronectin was detected by indirect immuno-which these various biochemical activities are quantita-fluorescence using a rabbit anti-fibronectin antisera (left). Cell num-tively or qualitatively altered.ber was visualized by DAPI staining.

VHL-associated tumors are typically hypervascularand overproduce angiogenic peptides such as vascular

pVHL with fibronectin affects the formation of an extra- endothelial growth factor (Maher and Kaelin, 1997). Re-cellular fibronectin matrix is not yet clear. Pulse-chase cent studiessuggest that pVHL plays a role in the regula-experiments suggest that pVHL does not grossly affect tion of hypoxia-inducible mRNAs such as the VEGFthe rate at which newly synthesized fibronectin is se- mRNA (Maher and Kaelin, 1997). Our data raise the pos-creted into tissue culture supernatant (data not shown). sibility that the regulation of angiogenesis by pVHL in-This might suggest that physical association with pVHL volves not only alterations in the production of angio-is required for a later step in fibronectinmatrix formation, genic peptides but also changes in the composition ofsuch as the subsequent assembly of fibronectin into the extracellular matrix.fibrillar arrays visualizable by fluorescence microscopy. The majority of clear cell renal carcinomas have eitherFibronectin assembly is a complex process that involves mutated or transcriptionally silenced the VHL gene.the activation of integrin receptors capable of inter- Thus, renal carcinoma cell lines such as CAKI-1 andacting with the cell cytoskeleton (Wu et al., 1995). One ACHN are unusual insofar as they retain a wild-type VHLmight predict that the effect of pVHL would be catalytic allele and produce a wild-type pVHL protein (Gnarrarather than stoichiometric given that fibronectin appears et al., 1994; Iliopoulos et al., 1995; Kibel et al., 1995).to be in vast molar excess of pVHL. Kochevar et al. (1992) reported that ACHN cells harbor

How does pVHL, a cytoplasmic protein, interact with a frameshift mutation of the fibronectin gene and pro-a secreted protein such as fibronectin? We and others duce a truncated fibronectin molecule capable of stimu-find that a fraction of the total cellular pVHL copurifies lating cell growth in vitro. This observation may under-with cell membranes (Iliopoulos et al., 1995; Pause et al., score a potential role of fibronectin in the control of1997; this paper). Furthermore, our confocal microscopy renal carcinoma cell growth and raises the possibilitydata suggest that pVHL can associate with the endo- that mutationof fibronectin might decrease theselectionplasmic reticulum and Golgi apparatus. Thus, one can pressure to inactivate pVHL in renal carcinoma cells.envision at least three possibilities. The first is that some Patients with von Hippel-Lindau disease, like autoso-pVHL, despite the absence of a recognizable leader mal dominant polycystic kidney disease (PKD) patients,sequence, is secreted. The second possibility is that the develop cysts in multiple organs, most notably the kid-interaction between fibronectin and pVHL is indirect and ney. Cyst development in the setting of von Hippel-is mediated by a protein spanning the ER membrane. Lindau disease appears to involve loss of the remainingThe identification of p68 may be informative in this re- wild-type VHL allele (Zhuang et al., 1995; Lubensky etgard. Finally, recent evidence suggests that malfolded al., 1996). The PKD1 protein is a transmembrane proteinor malprocessed proteins within the ER can undergo thought to play a role in cell–matrix and cell–cell interac-retrograde transport to the cytoplasmic surface of the tions (Carone et al., 1994; Harris et al., 1995). Thus, cystER membrane, where they areubiquitinated by multipro- formation in VHL disease and PKD disease may sharetein complexes (reviewed by Kopito, 1997). This latter a common etiology, namely, an inability of epithelial cells

to interact properly with the extracellular matrix.model is provocative given the association of pVHL with

Molecular Cell966

of gestation. The embryos were dissected free of decidua in D-PBS,VHL patients develop clear cell renal carcinomas,and extraembryonic membranes were removed. Individual embryospheochromocytomas, and hemangioblastomas. Clearwere then digested in 200 ml of 0.05% trypsin for 5–10 min at 378Ccell tumors are thought to arise from proximal renalwith agitation. Trypsinization was terminated by addition of com-

tubular epithelial cells. During embryogenesis, mesen- plete medium (DMEM with 10% fetal bovine serum and penicillin/chymal cells within the kidney are induced to become streptomycin [GIBCO-BRL]), and cells were plated into wells of 48

(for small embryos)- or 24-well dishes (Falcon) coated with collagenglomeruli or tubular epithelial cells by signals sent by(Collaborative Biomedical Products). Cell cultures were fed dailythe ureteric bud (Bard, 1992; Ekblom, 1992; Davies,with complete medium and passaged when confluent. Fibroblasts1993; Hay, 1995). Cell–cell and cell–matrix adhesivecultured from between passages 1 to 4 were plated onto cover slipsevents appear to play an important role in this process.(Corning), coated with collagen, and grown for an additional 24–48

The renal mesenchyme and ureteric bud are mesoder- hr prior to fixation in 95% ethanol. A 40 ml aliquot of each trypsinizedmal derivatives, whereas pheochromocytomas arise embryo was removed prior to plating. These cells and the extraem-

bryonic membranes of each embryo were used for the preparationfrom cells embryologically derived from the neural crest.of genomic DNA using standard procedures. Genotyping of eachIn sum, VHL patients are at risk of developing kidneysample was then performed using a PCR assay for both the targetedtumors derived from mesodermal cells, pheochromocy-and wild-type VHL allele as described elsewhere (Gavin et al., sub-tomas derived from neural crest cells, and hemangio-mitted).

blastomas that are characterized by marked vascularproliferation.

AntibodiesFibronectin is expressed throughout embyrogenesis.Anti-HA mouse monoclonal antibody (clone 12CA5) was obtained

Fibronectin nullizygous embryos initially develop nor- from Boehringer Mannheim (Indianapolis, IN). Anti-fibronectin rabbitmally but by day eight begin to develop defects in the IgG Ab was obtained from Collaborative Biomedical Products. Anti-neural tube (neural crest) and in mesodermally derived VHL Abs, IG32 (mouse IgG monoclonal) and R98 (rabbit IgG poly-

clonal), are described elsewhere (Iliopoulos et al., 1995; Kibel et al.,tissues (George et al., 1993; Watt and Hodivala, 1994).1995). Anti-actin rabbit sera was obtained from Sigma (St. Louis,Defects also develop in the heart and vasculature, andMO) and anti-collagen IV rabbit sera was a gift of Dr. Martin Hemlerthe embryos begin to regress at about day 10.5 (George(Dana-Farber Cancer Institute). Secondary Abs used were affinity

et al., 1993). Thus, fibronectin appears to play an impor- pure goat-anti-rabbit-lissamine (LRSC) and affinity pure goat-anti-tant role in the development of tissues that give rise to mouse-fluorescein isothiocyanate (FITC) from Jackson ImmunoRe-tumors in VHL disease. Furthermore, VHL nullizygous search Laboratories Inc. (West Grove, PA).embryos likewise die between days 9 and 11 and exhibitsimilarities to fibronectin nullizgyous embryos, including Plasmidsan abnormal extraembryonic vasculature (Gnarra et al., Mammalian expression vector pRc/CMV was obtained from In-

Vitrogen. pRc-HA-VHL (wild-type) and pRc-HA-R167W, both of1997). One hypothesis, which remains to be proven, iswhich introduce an N-terminal HA-epitope tag, were described pre-that abnormal fibronectin deposition contributes to theviously (Iliopoulos et al., 1995). pRc-HA-Y98H and pRc-HA-W117Rdemise of VHL nullizygous embryos.were generated by replacing the NotI–BglII fragment in the pRc-Fibronectin has profound effects on cellular behaviorHA-VHL (wild-type) with the NotI–BglII fragments from pSX-VHL

in vitro and in vivo. Loss of fibronectin deposition has Y98H and W117R, respectively (Duan et al., 1995b) (kindly providedbeen correlated with cellular transformation in vitro. Fur- by Dr. Richard Klausner, National Institutes of Health).thermore, fibronectin in model systems can suppresscellular properties associated with malignancy (Gian- Metabolic Labelingcotti and Ruoslahti, 1990; Pasqualini et al., 1996). Taken Radioisotopic labeling was performed by methionine starvation fortogether, these earlier observations established the 30 min followed by growth in 5 ml of methionine-free DMEM supple-

mented with [35S]-methionine [200 mCi/ml medium; EXPRESS35Splausibility that defects in fibronectin matrix assemblyprotein-labeling mix (New England Nuclear)] and 2% dialyzed fetalmight contribute to carcinogenesis. This study providesbovine serum for 4 hr at 378C in a humidified 10% CO2 atmosphere.a direct link between a human cancer susceptibility gene

and regulation of fibronectin matrix assembly.Immunoprecipitation and ImmunoblottingImmunoprecipitation and Western blotting were performed as de-Experimental Proceduresscribed previously (Iliopoulos et al., 1995; Kibel et al., 1995). In brief,cells were lysed in EBC buffer (50 mM Tris [pH 8.0], 120 mM NaCl,Cell Culture0.5% Nonidet P [NP]-40) supplemented with protease and phospha-Renal carcinoma cell lines 786-O, A498, ACHN-1, and CAKI-1, ob-tase inhibitors. Immunoprecipitates were washed four times withtained from the American Type Culture Collection (Rockville, Mary-NETN-900 (20 mM Tris [pH 8.0], 900 mM NaCl, 1 mM EDTA, andland), and 293 cells were grown in Dulbecco’s modified Eagle’s0.5% NP-40) and once with NETN prior to boiling in SDS-containingmedium (DMEM) containing 10% heat-inactivated defined/supple-sample buffer.mented bovine calf serum (Hyclone) at 378C in a humidified, 10%

For mixing studies, cells were grown to z90% confluence in 100CO2 atmosphere. 786-O subclones ectopically producing HA-mm plastic dishes and lysed in 1 ml of EBC. For Figure 4B, EBCtagged wild-type pVHL, pVHL C162F, pVHL R167W, pVHL C197was supplemented where indicated with purified fibronectin (GIBCO)(containing residues 1–197), and pVHL N72 (containing residuesprior to cell lysis, and each immunoprecipitation reaction contained72–213) are described elsewhere and were maintained in DMEM200 ml of radiolabeled cell extract from the indicated cell line. Forcontaining 10% heat-inactivated defined/supplemented bovine calfFigure 4A, 200 ml of unlabeled cell extract from the indicated cellserum supplemented with G418 (1 mg/ml) (Iliopoulos et al., 1995;line was immunoprecipitated with an anti-HA antibody. The immuno-Kibel et al., 1995; Lonergan et al., 1998). 786-O subclones producingprecipitates, immobilized on protein A–Sepharose, were washedpVHL W117R and pVHL Y98H were selected and maintained in thefive times with NETN and then incubated with 200 ml of radiolabeledsame manner (Iliopoulos et al., 1995; Lonergan et al., 1998).786-O cell extract for 1 hr with rocking. Where indicated, purifiedfibronectin was added prior to the addition of the labeled extract.Mouse Embryo Fibroblast HarvestsThe immunoprecipitates were washed five times with NETN andEmbryos from matings of VHL1/2 mice generated by gene targeting

(Gavin et al., submitted) were harvested between 9.5 and 10.5 days eluted by boiling in SDS-containing sample buffer.

von Hippel-Lindau Protein and Fibronectin967

Peptide Microsequence Analysis Kit, Vector Laboratories, Bulingame, CA) for 45 min at RT. Followingthree additional washes with PBS, 3,39-diaminobenzidine (DAB Per-Isolation of p200 by preparative SDS-PAGE, digestion with trypsin,

and sequencing of tryptic peptides were performed as described oxidase Substrate Kit, Vector Laboratories, Bulingame, CA) wasadded until the sections were properly stained, as visualized underpreviously (Kibel et al., 1995).the microscope, at which point the reaction was stopped in dH2O.Sections were counterstained in hematoxillin for 1 min, washedPartial Proteolytic Peptide Mappingthree times in dH2O, placed in 100% ethanol for 3 min and in xylenePartial proteolytic peptide mapping of [35S]-methionine radiolabeledfor 15 min, and then mounted.p200 was performed as described elsewhere (Harlow and Lane,

1988).

AcknowledgmentsSubcellular FractionationBiochemical fractionation of cells was performed as described else- We thank Drs. Lan Bo Chen, Martin E. Hemler, Thomas Rapoport,where (Radke et al., 1983; Lee et al., 1987). The nuclear and mem- and Melissa Roles for useful discussions and for critical reading ofbrane pellets were then lysed in EBC buffer. Sufficient Tris (pH 8), this manuscript; Drs. Stine-Kathrein Kraeft and Lan Bo Chen forNaCl, and NP-40 were added to the cytoplasmic fraction such that help with the confocal microscopy; Dr. Hemler for help establishingtheir final concentrations were equivalent EBC buffer. the fibronectin ELISA; Dr. William Lane andcoworkers at the Harvard

Microchemistry Facility for help with protein microsequencing; andImmunofluorescence Staining our colleagues in the Kaelin Laboratory for continued support. ThisCells (5 3 105) were grown on coverslips for 6 days, washed three work was supported by grants from the National Institutes of Healthtimes with PBS, and fixed/permeabilized in prechilled 95% EtOH at (W. G. K. and D. N. L.), the Medical Research Council of Canada2708C for 30 min. EtOH was then aspirated, and the residual EtOH (M. O.), the von Hippel-Lindau Family Alliance (O. I.), the Murraywas allowed to air dry at 48C. Cells were stained with either mono- Foundation (O. I. and W. G. K.), the National Kidney Cancer Associa-clonal anti-HA antibody (4 mg/ml) or polyclonal anti-fibronectin anti- tion (O. I.), and the Novartis Research Institute (W. G. K.). W. G. K.body (5 mg/ml) overnight at 48C. The coverslips were then incubated is a Howard Hughes Medical Institute Assistant Investigator. Thiswith FITC-conjugated anti-mouse or rhodamine-conjugated anti- paper is dedicated to the memory of Michael Murray.rabbit antibodies (5 mg/ml), respectively, for 1 hr at 378C followedby two washes with PBS. The coverslips were then stained with 4’- Received February 27, 1998; revised March 19, 1998.6-diamidino-2-phenylindole hydrochloride (DAPI) (1 mg/ml), washedonce with PBS, once with ddH2O, and then mounted with cytoseal

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