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[Cancer Research 66, 1553-1560, February 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

von Hippel-Lindau Tumor Suppressor Protein Regulates the Assembly of Intercellular Junctions in Renal Cancer Cells through Hypoxia-Inducible Factor–Independent Mechanisms

Maria J. Calzada1, Miguel A. Esteban1, Monica Feijoo-Cuaresma1, Maria C. Castellanos1, Salvador Naranjo-Suárez1, Elisa Temes1, Fernando Méndez1, Maria Yánez-Mo1, Michael Ohh2 and Manuel O. Landázuri1

1 Servicio de Inmunología, Hospital de la Princesa, Departamento de Medicina, Universidad Autónoma de Madrid, Madrid, Spain and 2 Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada

Requests for reprints: Manuel O. Landázuri, Hospital de la Princesa, Diego de León 62, 28006 Madrid, Spain. Phone: 34-91-5202-662; Fax: 34-91-5202-374; E-mail: mortiz.hlpr{at}saludmadrid.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2{alpha} did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF. (Cancer Res 2006; 66(3): 1553-60)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Germ line mutations of the von Hippel-Lindau (VHL) gene cause the VHL syndrome, a rare inherited disorder characterized mainly by renal cell cancers (RCC), hemangioblastomas of the central nervous system, and phaeocromocytomas (1). Remarkably, VHL is also inactivated in >80% of sporadic RCC (the most frequent form of renal malignancy; ref. 1), and reexpression of VHL prevents the tumorigenic effects in severe combined immunodeficient mice of cell lines obtained from sporadic RCC (2–4). The latter shows that inactivation of VHL is responsible for developing RCC rather than being a concomitant alteration.

The best known function of the VHL protein (VHL) is to act as the recognition subunit of a complex with ubiquitin ligase activity, which targets hypoxia-inducible factor-{alpha} (HIF-{alpha}) subunits for proteolytic degradation (5–8). The molecular basis for VHL recognition and degradation of HIF is the hydroxylation, by a family of 2-oxoglutarate–dependent dioxygenases termed PHDs (for prolyl hydroxylase domain containing proteins; ref. 9), of two key proline residues (Pro402 and Pro564 in HIF-1; Pro405 and Pro531 in HIF-2; refs. 10, 11). In VHL-negative cells, as well as in VHL expressing cells exposed to hypoxia, HIF becomes stabilized and promotes the transcription of multiple target genes (12). However, it is not yet clear how VHL inactivation ultimately leads to the clinical manifestation of VHL disease or to sporadic RCCs.

Previous studies using VHL-positive and VHL-negative cells reveal the existence of HIF-dependent and HIF-independent gene induction patterns (13). Additionally, specific suppression of HIF-2{alpha} in VHL-negative RCC cells by a small interfering RNA approach, or overexpression of a constitutively mutant active form of HIF-2{alpha} (carrying substitutions in the two prolines essential for VHL recognition) into wild-type VHL stably transfected RCC cells, has clearly established a direct link between HIF-2{alpha} and RCC tumors (14–16). On the other hand, it has been reported that the lack of a specific type of covalent modification of VHL (termed neddylation) by a ubiquitin-like protein called NEDD8 does not impair the regulation of HIF but abolishes the tumor suppressor effect of VHL in RCC cell lines (17). Mutations in the COOH-terminal acidic domain of VHL have also been described to behave similarly (18). Interestingly, other authors have shown that expression of a VHL-resistant HIF-1{alpha} protein is not sufficient to promote tumorigenesis (19). These latter findings indicate that both HIF-dependent and HIF-independent mechanisms seem essential for VHL-mediated tumor suppressor effects.

There are several VHL-regulated cell functions for which the link to HIF remains unclear. These include events that may be important for RCC tumor progression/initiation, such as the assembly of ß1 integrin-fibrillar adhesions and extracellular fibronectin fibers (20, 21), control of cell motility (22, 23), stability of microtubules (24, 25), and the maintenance of an epithelial-like cell shape and monolayer organization (20, 26, 27). An experimental approach that tests the dependence or independence of HIF on VHL-mediated function is the stable transfection into VHL-negative RCC cells of mutant forms of VHL that retain the ability to regulate HIF normally. In this regard, a neddylation-defective form of VHL, or a naturally occurring type 2C VHL mutant have been used to show that the lack of a fibronectin extracellular matrix in VHL-negative RCC cells does not depend on HIF (17, 28).

The appropriate formation of intercellular junctional structures is essential for the maintenance of a regular cell shape and a polarized epithelial cytoarchitecture. Multiple studies have revealed a correlation between the reduction of their integrity and tumor initiation and progression (29–33). Adherens junctions and tight junctions are two of the most relevant junctional complexes in the kidney epithelium. Adherens junctions are mediated by a family of transmembrane glycoproteins called cadherins, which associate intracellularly with the actin cytoskeleton and other signaling proteins through the catenin family of proteins (30). Tight junctions consist of transmembrane components, such as the claudins and occludin, which interact with the actin cytoskeleton through the ZO family of proteins (34). Our results showed that multiple VHL-negative RCC cell lines display very abnormal adherens junction and tight junction structures (which are associated with an elongated cell shape, disrupted cell polarity, and increased paracellular permeability), and that reexpression of wild-type VHL into these cell lines reverted all these phenotypic alterations. We propose that this novel function of VHL may be important for its normalizing effects on cell shape and epithelial cytoarchitecture. We have also shown that in the VHL-defective 786-O cell line, neither the disruption of intercellular junctions nor the changes in cell shape characteristic of VHL-negative RCC cells are mediated via HIF.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents. 786-O cells and subclones stably producing wild-type VHL (WT8 and WT10), a truncated form of VHL (1-115) or empty vector (PRC3; ref. 2), VHL L188V (35), VHL(RRR) and VHL(KRR) (17), parental RCC4 and RCC10, and subclones stably producing wild-type VHL (36) were maintained in RPMI 1640 with GLUTAMAX-I (Invitrogen, San Diego, CA) supplemented with 10% FCS and grown at 37°C with 5% CO2.

Anti-ß1 integrin monoclonal antibodies TS2/16 and VJ1/14 have been previously described (20, 37). Anti-VHL (PharMingen, San Diego, CA), anti-ZO1 (Zymed Laboratories, San Francisco, CA), anti-ß-catenin (BD Biosciences, San Jose, CA), and anti-fibronectin (Calbiochem, La Jolla, CA).

For experiments in hypoxia, cells were incubated overnight to 1% oxygen in an in vivo 400 hypoxia workstation (Ruskinn Technology, West Yorkshire, United Kingdom).

Immunofluorescence microscopy. These experiments were done as described elsewhere (20). Coverslips were incubated with the primary antibody and washed. Secondary antibodies (Alexa 488 goat anti-mouse IgG or Rhodamine Red-X goat anti-rabbit IgG; Molecular Probes, Inc., Eugene, OR) were added and incubated for 30 minutes at 37°C and washed with TBS-T. Finally, cells were washed with distilled water and mounted with Mowiol. For double-label immunofluorescence studies, primary and secondary antibodies (Alexa 488 goat anti-mouse IgG or Rhodamine Red-X goat anti-rabbit IgG; Molecular Probes, Inc.) were incubated separately, and mouse serum was used to prevent cross-reactions. For filamentous-actin double immunostaining, phalloidin coupled to Alexa 488, was added to the secondary antibody. Samples were analyzed with a Leica TCS-SP (Leica Microsystems, Heidelberg, Germany) confocal microscope or a conventional Leica DMR photomicroscope equipped with QFISH software.

Detergent extraction assay. 786-O cells (4.8 x 105) were grown for 5 to 6 days in six-well culture dishes. Cell monolayers were washed and scraped in PBS. After centrifugation, cell pellets were extracted in mild detergent conditions [1% Triton X-100, 150 mmol/L NaCl, 50 mmol/L Tris (pH 6.8), 0.01 % NaN3, 2 mmol/L EDTA, 1 mmol/L sodium orthovanadate, and protease inhibitors] for 30 minutes on ice. Samples were centrifuged at 16,000 x g for 30 minutes, and Triton-soluble fraction was eliminated. Triton-insoluble fractions were triturated in the same buffer supplemented with 0.5% deoxycholate and 0.1% SDS and kept on ice for 30 minutes; samples were centrifuged at 16,000 x g for 30 minutes and supernatants were collected. To obtain total cell lysates, cell monolayers were lysed with radioimmunoprecipitation assay buffer, centrifuged at 18,000 x g, and the supernatants collected. Immunodetection after SDS-PAGE electrophoresis was done using enhanced chemiluminescence (Amersham, Arlington Heights, IL).

Measure of paracellular permeability. Permeability across confluent RCC monolayers was measured in Transwell inserts (polycarbonate filter, 6.5 µm diameter and 0.4 µm pore size; Costar, Cambridge MA). RCC cell lines were cultured in 150 µL of culture medium for several days, and the assay was done as described elsewhere (32). The initial number of cells plated was carefully adjusted to each particular cell type.

Quantitative real-time PCR analysis. Equal numbers of cells were grown to confluence in 100-mm culture dishes. Quantitative RNA analysis protocol and primers pairs used have been described elsewhere (38).

Retroviral infection. Retroviral vectors encoding VHL, HIF-2{alpha} P405A;P531A [HIF-2{alpha} (P->A)2], HIF-2{alpha} P405A;P531A;bHLH* [HIF-2{alpha} (P->A)2*], or the empty vector pBabe have been described before (14). GP2-293 cells (Clontech, Palo Alto, CA) were transfected in 60-mm dishes using calcium phosphate precipitation. We used 3.5 µg of each retroviral vector, 4 µg of MLV gag-pol expression vector (pNGLV-MLV-gag-pol), and 2.5 µg of amphotropic envelope expression vector (pVSV-G). Medium was changed 24 hours after transfection, and cell culture supernatants were harvested 24 hours later, filtered with 0.22 µm filter, and diluted (1:2) with fresh medium. Diluted viral supernatants containing 6 µg/mL polybrene were then added to the cells and incubated overnight at 37°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VHL is required for the proper assembly of intercellular junctions in RCC cells. Adherens junctions and tight junctions are specialized cell-cell adhesion structures essential for keeping a regular architecture and normal monolayer organization in epithelial cells and are commonly disrupted in most cancers of epithelial origin (30, 33, 34). VHL-negative RCC cells have an elongated cell shape and grow as disorganized monolayers, and reexpression of pVHL restores an epithelial-like phenotype (refs. 20, 26; Fig. 1A). These phenotypic alterations suggested to us that VHL might be necessary for the assembly of intercellular junctions in RCC cells. To test our hypothesis, we did double-color immunofluorescence of essential constituents of adherens junctions (ß-catenin) and tight junctions (ZO1) in several RCC cell lines. In wild-type VHL-transfected 786-O cells, horizontal confocal captures of ß-catenin label across the whole monolayer, displayed a clear-cut distribution at cell-cell contact areas (Fig. 1B, a and c, top). Likewise, vertical computer reconstructed images of the same field showed the classic distribution along the basolateral cell membrane (Fig. 1B, a and c, bottom). Horizontal captures of ZO1 also outlined well-defined cell boundaries in VHL-transfected 786-O cells (Fig. 1B, b and c, top), with the corresponding vertical reconstructions exclusively localized at the most apical region of the lateral membrane (Fig. 1B, b and c, bottom). In contrast, VHL-negative 786-O cells showed a very irregular and discontinuous pattern of ß-catenin and especially ZO1, which was better appreciated in the vertical computer-reconstructed sections (Fig. 1B, d-f, top and bottom).


Figure 1
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  		Figure 1. Organization of adherens junctions and tight junctions in VHL-positive and VHL-negative 786-O, RCC4, and RCC10 cells. A, reexpression of pVHL induces an epithelial-like cobblestone morphology in VHL-negative RCC cell lines. VHL-transfected (a-c) and VHL-negative (d-f) 786-O and RCC10 and VHL-positive and VHL-negative RCC4 cells were plated on 24-well dishes and grown to confluence. Pictures were taken using a phase-contrast microscope. 786-O (B), RCC10 (C), and RCC4 (D) cells were grown to confluence on coverslips for 3 to 5 days, fixed, and double stained with anti-ß-catenin and anti-ZO1 antibodies. Samples were then analyzed with a confocal microscope. Horizontal sections and vertical computer-reconstructed sections (bottom, in 786-O and RCC10 cells) of the same field (at least six different fields were evaluated). One representative experiment.

 
Remarkably, differences in the distribution of ß-catenin and ZO1 were not restricted to the 786-O cell line, and similar reproducible results were also observed between wild-type VHL-transfected and VHL-negative RCC10 and RCC4 cell lines (Fig. 1C and D). We next did double immunofluorescence of ß1 integrins (green) and ZO1 (red) in 786-O cells, because ß1 integrins distribute over the entire surface of epithelial cells and thus give a better indication of cell boundaries and monolayer thickness than does ß-catenin. Direct vertical captures of ß1 integrins across the whole monolayer showed thick and regular wild-type VHL-transfected cells (Fig. 2A). On the contrary, we regularly observed that VHL-negative cells were very flat and elongated and sometimes grew as two or three superimposed layers (Fig. 2B). Moreover, empty spaces were frequently found at the most basal level between VHL-negative 786-O cells (Fig. 2B; see white arrow), further showing the incapability of these cells to properly interact with each other. As expected, direct vertical captures of ZO1 showed well-defined apical dots at the interface between VHL expressing 786-O cells (Fig. 2A, see pink arrow) and a less defined and irregular distribution in VHL-negative cells (Fig. 2B). Notably, the abnormal cell to cell interactions between pVHL-negative cells became more evident when we obtained horizontal captures (only anti-ß1 integrin is shown) and focused on the most basal cell-substrate contact plane in both VHL-transfected and VHL-negative 786-O cells (Fig. 2C and D; see white arrows).


Figure 2
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  		Figure 2. VHL regulates epithelial cytoarchitecture in RCC cells. VHL-positive and VHL-negative 786-O cells were grown to confluence on coverslips for 3 to 5 days, fixed, double stained with anti-ß1 integrin and anti-ZO1 and analyzed with a confocal microscope. Direct vertical captures (showing ß1 integrin in green and ZO1 in red; A and B) and horizontal sections corresponding to the most basal level (showing exclusively ß1 integrin; C and D). Mean thickness of cell monolayers as the mean of 10 different fields. One representative experiment.

 
Inactivation of VHL in RCC cells disrupts the association of ZO1 with the actin cytoskeleton and increases paracellular permeability. Anchorage of junctional proteins to the cytoskeleton is essential for their function and stability at zones of cell-cell contact (29, 30, 34). We therefore studied whether the interaction of ß-catenin and ZO1 with the actin cytoskeleton in 786-O cells was influenced by their VHL status. Cell monolayers were extracted under mild detergent conditions, which were sufficient to solubilize cytoskeleton-unbound molecules. Insoluble fractions and total lysates were analyzed by Western blot with the corresponding antibodies. Similar expression levels of ß-catenin and ZO1 were found in the total lysates of wild-type VHL and VHL-negative 786-O cells (Fig. 3A). Likewise, the amount of nonsoluble cytoskeleton associated ß-catenin did not greatly differ between either group of cells. However, cytoskeleton associated ZO1 was clearly diminished in VHL-negative cells (Fig. 3A). Using double immunofluorescence of actin and ZO1, we evaluated the formation of an apical actin ring, a classic feature of well-polarized cells and basically reflects an adequate interaction between the actin cytoskeleton and junctional proteins. Because wild-type VHL 786-O monolayers are thick (see Fig. 2), two planes of actin distribution were typically observed: the top corresponded to the perijunctional cortical actin ring, which colocalized with ZO1 (Fig. 3B); and the bottom corresponded to abundant and well-organized actin stress fibers. In pVHL-negative 786-O cells, no actin ring was observed, and only one plane of minimally organized actin fibers was appreciated (Fig. 3B); a disrupted ZO1 organization and occasional empty spaces were also observed in VHL-negative 786-O cells (Fig. 3B, see arrows).


Figure 3
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  		Figure 3. Inactivation of VHL in RCC cells diminishes the association of ZO1 with the actin cytoskeleton. A, total lysates and cytoskeleton-associated fractions of VHL-positive and VHL-negative 786-O cells were analyzed by Western blotting with the corresponding antibodies. B, 786-O cells grown to confluence on coverslips (4-6 days), double stained with anti-ZO1 and green fluorescent phalloidin, and analyzed by conventional immunofluorescence microscopy. C, comparative analysis of paracellular permeability between VHL-positive and VHL-negative RCC cell lines. RCC cells (between 104 and 1.5 x 104, depending on the cell line) were grown for 2 to 4 days on Transwell filters. The ability of FITC-dextran to diffuse through the monolayers was measured at the indicated times. One representative experiment of three. Points, mean of three independent wells. MIF, mean fluoresce intensity.

 
Tight junctions control the diffusion of solutes and liquid through neighboring epithelial cells. Increased paracellular permeability is therefore a logical consequence of tight junction dysfunction (32). The ability of a fluorescein-labeled dextran solution to move across confluent cell monolayers cultured on Transwell filters is a widely accepted method to evaluate the integrity of tight junctions (32). As expected, VHL-negative 786-O and also VHL-negative RCC4 and RCC10 cell lines were more permeable in this assay than their wild-type VHL stably transfected counterparts (Fig. 3C).

Breakage of intercellular junctions and altered cell shape of VHL-negative RCC cells are independent of HIF. Our results above show that the effect of VHL on renal epithelial cell differentiation is associated with the restoration of normal intercellular junctions, strongly suggesting that both phenomena are linked. However, one important remaining question is how VHL is affecting both cell shape and junctional organization. Given that HIF is a known target of VHL and a direct link between HIF and RCC tumors has been established (see introduction), we first studied whether these effects were mediated via HIF. To this aim, we used several different approaches. First, we used 786-O sublines stably transfected with well-characterized mutant forms of VHL that have been previously reported to regulate HIF normally: the naturally occurring type 2C VHL mutant L188V and a nonneddylateable version of VHL termed RRR (17). Notably, reexpression of VHL type 2C mutations into VHL-negative cells allows normal regulation of HIF in normoxia (28, 39). Stable transfectants of 786-O cells with a mutant version of VHL(RRR), in which lysines (K) 159, 171, and 196, have been substituted by an arginine (R), with K159 being an essential residue for neddylation of VHL, also regulates HIF normally. As a control, we used 786-O cells transfected with another version of a VHL(KRR) that carries mutations only at Lys171 and Lys196 (which do not interfere with the normal VHL function). The expression of the VHL transgene was verified in all three lines (L188V, RRR, and KRR) by Western blotting (Fig. 4A). Likewise, Western blotting for HIF-2{alpha} showed that HIF-2{alpha} levels were normalized in all three cell lines when compared with VHL-negative 786-O cells (Fig. 4A). Moreover, quantitative Real Time PCR for PHD3 (a target gene of HIF-2{alpha}; ref. 40) showed an absent or extremely reduced HIF transcriptional activity in L188V, RRR, and KRR, when compared with VHL-negative 786-O control cells (Fig. 4B). Interestingly, phase-contrast captures of L188V mutants presented an undifferentiated shape compared with wild-type VHL, but RRR cells showed quite disorganized monolayers, in contrast to the more regular and differentiated shape of wild-type VHL and KRR transfectants (Fig. 4C, top). Moreover, double-immunofluorescence confocal microscopy analysis of ß-catenin and ZO1 (only horizontal captures representing the sum of sections of the whole monolayer are presented) showed discontinuous and poorly defined staining in L188V transfectants compared with the neat and well defined staining of the wild-type VHL. Staining in RRR transfectants was not as deficient as in VHL-negative cells but more discontinuous and diffuse if compared with KRR sublines (Fig. 4C, bottom). These results show that normalization of HIF-2{alpha} in 786-O RCC cells is not sufficient to restore neither a differentiated epithelial phenotype nor proper intercellular junction assembly. They also show that neddylation of VHL seems to be important for its normalizing effects on cell shape and intercellular junctions.


Figure 4
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  		Figure 4. Overexpression of a nonneddylateable form of VHL and a type 2C mutant VHL does not restore tight junction and adherens junction organization in RCC cells. A, HIF2{alpha} and VHL protein levels in lysates from VHL-negative 786-O cells and 786-O subclones stably producing wild-type VHL or different mutant versions of VHL (L188V, KRR, and RRR) were analyzed by Western blot. B, PHD3 gene transcription levels in these cell lines were determined by quantitative real-time PCR. C, the same cell lines were grown to confluence on coverslips for 3 to 6 days and fixed, and captures were taken with a phase-contrast microscope (top). These coverslips were then double stained with anti-ß-catenin (green) and ZO1 (red). Horizontal sections of confocal microscopy analysis (bottom).

 
To further verify the independence on HIF of these phenotypic alterations, we next infected wild-type VHL-transfected 786-O cells, with a retrovirus encoding a constitutively active form of HIF-2{alpha} (HIF-2{alpha} P405A;P531A), in which the two key prolines (P405 and P531) required for VHL recognition have been changed to alanine (P405A and P531A; ref. 15). As controls, we used both the empty vector (pBabe) and a version of HIF-2{alpha} (HIF-2{alpha} P405A;P531A;bHLH*) that contains the P405A and P531A substitutions but also a third amino acid substitution in the basic helix-loop-helix domain of HIF-2{alpha}, which results in the loss of DNA-binding capability and thus transcriptional activity. Western blotting showed higher expression of HIF-2{alpha} protein levels in both HIF-2{alpha} P405A;P531A and HIF-2{alpha} P405A;P531A;bHLH* VHL-positive 786-O cells when compared with VHL-negative cells (Fig. 5A). Quantitative RT-PCR for PHD3 showed a potent increase in HIF-mediated transcriptional activity only in HIF-2{alpha} P405A;P531A VHL-positive 786-O cells but not in VHL-positive cells infected with either the empty vector or the HIF-2{alpha} P405A;P531A;bHLH* construct (Fig. 5B). Remarkably, phase-contrast captures and double-immunofluorescence confocal analysis of ß-catenin and ZO1 showed that despite bearing very high levels of a transcriptionally active HIF-2{alpha}, VHL-positive 786-O cells infected with HIF-2{alpha} P405A;P531A presented a very regular cell shape and well-organized intercellular junctions (Fig. 5C). These results supported that HIF is not involved in either cell shape or intercellular junction formation.


Figure 5
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  		Figure 5. Overexpression of a constitutively active form of HIF-2{alpha} in VHL-positive 786-O cells does not affect adherens junction and tight junction organization. A, lysates corresponding to 786-O subclones transfected with wild-type VHL (WT8) or with an empty plasmid (PRC3), as well as WT8 cells infected with either an empty retrovirus (pBabe) or retrovirus encoding for HIF-2{alpha} P405A;P531A [HIF-2{alpha} (P->A)2] and HIF-2{alpha} P405A;P531A;bHLH* [HIF2{alpha} (P->A)2*] were immunoblotted with the indicated antibodies. B, PHD3 gene transcription levels in the same cell lines were determined by quantitative real-time PCR. C, phase-contrast captures (top) and double-immunofluorescence analysis (bottom) of ß-catenin (green) and ZO1 (red) in the indicated cell lines. Horizontal sections obtained with a confocal microscope.

 
Blockade of fibronectin matrix assembly does not alter epithelial cytoarchitecture in wild-type VHL stably transfected 786-O cells. As we have described above, both L188V and RRR 786-O cell lines in spite of regulating HIF normally still display an elongated cell shape and disrupted organization of intercellular junctions. Interestingly, both cell lines have been reported to show a disruption of the extracellular fibronectin matrix (17, 28). Because the extracellular matrix controls multiple aspects of cell behavior, including cell proliferation, susceptibility to apoptosis, and the maintenance of tissue cytoarchitecture (41), we hypothesized that in RCC cells, the breakage of the fibronectin matrix might be disturbing the junctional organization and cell shape. To evaluate this, we cultured wild-type VHL-transfected 786-O cells in the presence of a ß1 integrin-blocking antibody (VJ1/14); this antibody block matrix assembly by preventing binding of soluble fibronectin to surface integrins. However, although the assembly of extracellular fibronectin in VHL-transfected 786-O cells was dramatically suppressed, their monolayer organization, tight junction assembly (Fig. 6), and paracellular permeability3 remained largely unaffected. These results suggest that the absence of a fibronectin matrix alone does not explain the cytoarchitectural alterations of VHL-negative cells.


Figure 6
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  		Figure 6. Effect of blocking fibronectin (FN) matrix assembly on the epithelial-like phenotype of VHL-positive 786-O RCC cells. Confluent VHL-positive 786-O cells or VHL-positive 786-O cells treated with affinity purified ß1 integrin–blocking monoclonal antibody (VJ1/14, 20 µg/mL) were cultured on coverslips for 4 to 5 days. Samples were fixed and photographed with a phase-contrast microscope, then stained with polyclonal antibodies anti-fibronectin and/or anti-ZO1, and analyzed with a conventional immunofluorescence microscope.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of functions have been ascribed to VHL; however, the underlying mechanisms remain yet to be defined. Two clear examples are the role of VHL in the organization of an extracellular fibronectin matrix and the maintenance of a differentiated phenotype (20, 21, 26, 42). With regard to the latter, several groups have shown that reexpression of VHL induces a cobblestone epithelial-like organization in otherwise elongated VHL-negative RCC cell lines (20, 26, 42). Moreover, VHL-transfected RCC cells grown as spheroids develop compacted and dense structures, whereas VHL-negative cells remain loosely attached to each other (43). Given the essential role of intercellular junctions in regulating epithelial cytoarchitecture (29–33), a plausible explanation for these alterations would be that the presence of VHL in RCC cells is necessary for the assembly of intercellular junctions. Related to this, we have shown in this report that VHL-negative RCC cell lines display abnormal intercellular adherens junction and tight junctions structures and have alterations in cell polarity, and that reexpression of VHL corrects both phenotypic alterations. The integrity of intercellular junctions has been studied not only by confocal immunofluorescence microscopy of two essential adherens junction and tight junction constituents (ß-catenin and ZO1) but also by using functional paracellular permeability assays. Moreover, biochemical fractionation and subsequent Western blotting has revealed that the major determinant in the failure to form proper adherens junction and tight junction is not a reduced expression of ß-catenin and ZO1 but rather a diminished interaction with the actin cytoskeleton.

To date, the best characterized function of VHL is to recognize hydroxylated HIF-{alpha} subunits in normoxia and target them for proteasomal-mediated degradation (5–8). To ascertain whether the effects of VHL loss on intercellular junctions and epithelial cytoarchitecture are directly or indirectly related to HIF is an important step towards understanding the consequences of VHL inactivation. In this regard, several studies have shown that hypoxia is able to promote changes in cell shape in different cell types, although a formal link between HIF and the induction of a differentiated phenotype has not been provided (44, 45). Interestingly, our experiments show that transfecting VHL-negative 786-O RCC cells with VHL mutant forms that regulate HIF-{alpha} subunits normally did not improve either their epithelial characteristics or intercellular junctions. Moreover, overexpressing a constitutively active form of HIF-2{alpha} in VHL-transfected 786-O cells did not alter their regular shape and differentiated phenotype. On the one side, this reinforces the emerging concept of existing VHL-mediated functions, which are independent of HIF. Related to this, an elegant recent study in worms showed that ancestral genes involved in the control of cell-matrix interactions are regulated by VHL in a HIF-independent manner (46). Moreover, the lack of fibronectin matrix in VHL-negative RCC cells has been reported to be independent of HIF (47). On the other side, our results may help to understand how VHL regulates RCC tumorigenesis through HIF-independent mechanisms. In this regard, activation of HIF-2{alpha} and not HIF-1{alpha} has been reported to be essential for the tumorigenesis of VHL-negative RCC lines (14–16). However, two recent articles also show that certain mutant forms of VHL (including the non-neddylateable form of VHL used in this article) that retain the ability to regulate HIF normally do not prevent RCC tumorigenesis (17, 18). In agreement with these results, other authors have shown that a VHL-resistant HIF-1{alpha} protein expressed in renal cell lines is not sufficient to promote tumorigenesis (19). Given this complex picture, it is likely that the sum of both HIF-dependent and HIF-independent pathways is important for the progression of RCC and that suppressing either of the two aspects reduces the tumorigenesis of VHL-negative RCC cell lines. We suggest that the inability to form proper intercellular junction structures might be another way by which VHL inactivation can promote tumor growth in a HIF-independent fashion.

If HIF is not involved, how then does VHL regulate intercellular junctions and cell polarity? It is well established that VHL-negative cells lack formation of fibronectin fibers (21). Given that epithelial cytoarchitecture is regulated by the fibronectin matrix (41), we then considered that the lack of a fibronectin matrix could underlie the disruption of intercellular junctions in VHL-negative RCC cells. However, blockade of fibronectin matrix assembly in VHL-transfected RCC cells did not minimally alter their epithelial-like phenotype, showing that the breakage of fibronectin fibers alone is not responsible for the junctional alterations of VHL-negative cells. An alternative possibility would be that VHL is controlling the expression of key junctional constituents (either transmembrane, cytoskeletal, or signaling proteins). Interestingly, neither VHL-positive nor VHL-negative 786-O cells express the essential adherens junction transmembrane molecule E-cadherin (26), indicating that at least in this cell background, other explanations are needed. Moreover, because the subcellular distribution and function of junctional molecules can be regulated by more subtle changes, such as phosphorylation of specific residues (30), it is feasible that VHL is controlling intercellular junctions through such mechanism.

In addition to the abovementioned possibilities, several recent studies have shown a link between VHL and two additional cell functions that could explain the altered cell polarity and cell-cell junctions of VHL-negative RCC cells (7). These VHL functions are the regulation of microtubule stability and the regulation of some atypical protein kinase C (PKC) isoforms. VHL colocalizes with the microtubule network and promotes microtubule stability (24, 25). Importantly, disrupting the microtubule cytoskeleton alters cell polarity in multiple cell models (48, 49). To clarify whether diminished microtubule stability is responsible for the cytoarchitectural alterations of VHL-negative RCC cells, it will be important to understand in the first instance how VHL binds to microtubules and regulates them. VHL has also been reported to bind {lambda} and {theta} isoforms of atypical PKCs (50). Moreover, in the case of PKC lambda, VHL regulates the ubiquitination of the activated forms of the protein, leading to its elimination and thus limiting its total cellular activity (51). Atypical PKCs control cell polarity and intercellular junctions, at least in part by regulating the activity of a family of master cytoskeletal regulators: the Rho family of small GTPases (52, 53). As a first approach, it would be important to study whether the mutations of VHL (non-nonneddylateable VHL and type 2C VHL) that are associated with alterations in intercellular junctions are also associated with dysregulated atypical PKC activity.

In conclusion, we believe that our findings described herein may help to understand how VHL acts as a gatekeeper gene in the kidney and also provide an insight into the existence of VHL-regulated functions through HIF-independent mechanisms.


   Acknowledgments
 
Grant support: "Ministerio de Ciencia y Tecnología" grant SAF 2004-00824, "Red Temática de Enfermedades Cardiovasculares, RECAVA" grant CO3/01, and "Comunidad Autónoma de Madrid" grant GRSALO789-2004.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. W.G. Kaelin (Medical Oncology/Molecular and Cellular Department, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) for providing the vectors encoding EPAS P405A;P531A and EPAS P405A;P531A;bHLH* and Dr. Luis del Peso for critical reading of this article.


   Footnotes
 
Note: M.J. Calzada and M.A. Esteban contributed equally to this work.

3 Unpublished results. Back

Received 9/ 8/05. Revised 11/ 4/05. Accepted 11/22/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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