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[Cancer Research 66, 6903-6907, July 15, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Primary Cilium Formation Requires von Hippel-Lindau Gene Function in Renal-Derived Cells

Mallory S. Lutz1 and Robert D. Burk1,2,3

Departments of 1 Microbiology and Immunology, 2 Pediatrics, and 3 Epidemiology and Social Medicine, Marion Bessin Liver Research Center and Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York

Requests for reprints: Robert D. Burk, Ullmann Building, Room 515, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-3720; Fax: 718-430-8975; E-mail: burk{at}aecom.yu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, occurs in the majority of renal clear cell carcinomas (RCC). VHL's function, regulating the degradation of hypoxia-inducible factor {alpha} (HIF{alpha}) subunits, explains the angiogenic nature of these tumors, but not tumor initiation. Because the development of renal cysts precedes tumor formation, and because the dysfunction of primary cilium is a common pathogenic mechanism in polycystic kidney diseases, we determined whether kidney-derived VHL– cells required VHL for the generation of cilium. Ectopic expression of VHL in RCC(VHL–) cells induced increased polarization and primary cilium formation. Cilium formation correlated directly with the expression of both wild-type VHL isoforms and a VHL mutant not associated with RCC development, whereas expression of RCC-associated VHL mutants did not support ciliogenesis. Requirement of VHL for ciliogenesis was independent of HIF{alpha} abundance. These data indicate separable independent functions for VHL (HIF{alpha} degradation and differentiation) and suggest a mechanism whereby disruption of both functions is required for renal carcinogenesis. (Cancer Res 2006; 66(14): 6903-07)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Germ line mutations in the von Hippel-Lindau tumor suppressor gene (VHL) lead to a hereditary cancer syndrome that manifests as hemangioblastomas of the retina and central nervous system, pheochromocytomas, and renal cysts and renal clear cell carcinomas (RCC). Inactivation of the VHL locus also occurs in 70% to 80% of sporadic RCC, resulting in ~2% of cancer deaths worldwide (1). Loss of heterozygosity at the VHL locus in microscopic renal cysts from patients with inherited VHL disease established cyst formation as an early step in the pathogenesis of RCC (2). However, the development of RCC requires additional mutations at non–VHL loci (1).

The best characterized function of pVHL is as an F-box-like component of an E3 ubiquitin ligase complex (1), targeting {alpha} subunits of hypoxia-inducible transcription factor (HIF{alpha}) for ubiquitin-mediated proteasomal degradation in an oxygen-dependent manner (1). Renal cysts and RCC from patients with VHL disease show increased concentrations of both HIF1{alpha} and HIF2{alpha} (3, 4). Resultant transcriptional activation of hypoxia response genes, including vascular endothelial growth factor, is reflected in the highly vascular nature of VHL tumor types. Reduction of HIF2{alpha} abundance leads to the suppression of tumor growth in heterotransplants, similar to the reintroduction of wild-type VHL in RCC(VHL–) cells (5, 6).

Genotype-phenotype correlations in VHL disease lead to the classification of VHL syndrome subtypes. Dysregulation of VHL-dependent degradation of HIF{alpha} is observed in subtypes 1, 2A, and 2B. Whereas VHL-dependent HIF{alpha} degradation is observed in type 2C VHL mutants, although fibronectin matrix assembly is abnormal (1). In addition, decreased microtubule stability has been associated with VHL subtype 2A mutations (1). Current research suggests that pVHL is a multifunctional protein with disease pathology dependent on the relationship between mutations, function affected, and anatomic site of VHL loss (1). VHL syndrome subtypes 1 and 2B show increased incidence of RCC compared with subtypes 2A and 2C, which are weakly associated with RCC.

Differences in cell differentiation are observed between RCC(VHL+) and RCC(VHL–) cells. In vitro observations showed the importance of cell-cell and cell-extracellular matrix interactions in VHL-dependent differentiation. Decreased cellular proliferation was observed in RCC(VHL+) cells grown as three-dimensional aggregate monolayers on type 1 collagen as compared with RCC(VHL–) counterparts (7, 8).

Breakthroughs in understanding the pathogenesis of renal cystic diseases, including autosomal-dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, nephronophthisis, and Bardet-Biedl syndrome show a strong correlation between cyst development and primary cilium biogenesis or dysfunction. Inactivation of critical components of intraflagellar trafficking results in an absence of ciliogenesis and renal cyst formation similar to that observed in polycystic kidney diseases (9).

We sought to investigate the mechanism of VHL dysfunction and renal cyst development by asking whether VHL-dependent differentiation differences relate to epithelial cell polarity and primary cilium formation. Here, we show that VHL mutations commonly associated with the development of RCC did not support ciliogenesis. In contrast, a VHL mutation, Y98H, which is not associated with RCC (1, 10), supported cilium formation. Our results bring VHL syndrome into the fold of renal cystic syndromes associated with primary cilium dysfunction.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell culture and primary cilium formation. The 786-0 and A498 renal clear cell carcinoma, RCC(VHL–), parental cell lines were purchased from the American Type Culture Collection (Manassas, VA). All cells were grown on type 1 collagen (BD Bioscience, Bedford, MA) coated surfaces in DMEM containing 10% fetal bovine serum, except where specifically noted for cilium formation. Stably transfected cultures of 786-0 cells expressing either empty vector pCR3 (Invitrogen, Carlsbad, CA), pCR3-VHLp (VHLp24-MPR or VHLp18-MEA), or FLAG-tagged versions of VHLp and VHLp-missense mutants have been previously described (8). No differences were observed between cells expressing long (VHLp24-MPR) or short (VHLp18-MEA) forms of VHLp. Retroviral supernatants were used to infect A498 cells for stable transfectants containing either empty retroviral expression vector pLNCX2 (Clontech Laboratories, Mountain View, CA) or pLNCX2-VHLp18-MEA. Polarization studies were done on cultures 3 days postconfluence. Primary cilium formation was induced by the maintenance of confluent cultures for 7 days in serum-free medium. Hypoxia mimetics, deferoxamine mesylate (Calbiochem, La Jolla, CA) and CoCl2 (Sigma, St. Louis, MO), were added directly to the culture medium 18 hours prior to fixation at 200 and 250 µmol/L final concentrations, respectively. Statistical analyses were done using STATA/SE 8.2. The {chi}2 test was used to examine the significance of differences in proportions.

Fixation and immunofluorescence microscopy. Cells were fixed in 2% paraformaldehyde and extracted with Triton X-100 following the methods of Pazour et al. (11). Primary antibodies included rabbit anti-ß-catenin (1:1,000; Sigma); mouse anti-human CD26, clone M-A261 (1:50; Serotec, Raleigh, NC); mouse anti-acetylated tubulin, clone 611B-1 (1:10,000; Sigma); and rabbit anti-human polycystin-2, YCC2 (1:2,000; a generous gift from Drs. Steve Somlo and Y. Cai, Yale University Medical School, New Haven, CT). Alexa Fluor 488- and 568-conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (Molecular Probes, Carlsbad, CA) were used at 1:500.

Cells were visualized by wide-field, fluorescence microscopy using a Zeiss Axiovert 200M with Apotome for optical sectioning (Zeiss, Thornwood, NY) and a 63x 1.4 n.a. oil objective. Images were collected using a high-resolution AxioCam MRm digital camera and Zeiss Axiovision software. NIH Image J software was used to reorient Z-merged image projections of individual cilium. Confocal light microscopy was done using a Leica TCS SP2 with a 63x 1.4 n.a. oil objective. Serial sections were collected in x-y and x-z planes.

Western blotting. Cell lysates were prepared as previously described (8). Briefly, cells were washed twice with PBS, lysed in ELB buffer [50 mmol/L Hepes (pH 7.6), 250 mmol/L NaCl, 0.1% Igepal CA 630 (Fluka, Buchs SG, Switzerland), 5 mmol/L EDTA, 1x Complete mini protease inhibitor cocktail (Roche Diagnostics GmbH, Basel, Switzerland)], and supernatants were collected at 4°C. SDS-PAGE gels were blotted to polyvinylidene difluoride membrane overnight and probed using mouse anti-VHL monoclonal antibody 11E12, rabbit anti-HIF 2{alpha} (Novus Biologicals, Littleton, CO), rabbit anti-ß-catenin (Sigma), and mouse anti-human CD26, clone M-A261 (Serotec). Cells used for cell lysis were grown in parallel cultures under the same conditions as cells fixed for polarization and primary cilium analysis.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
We previously showed that ectopic expression of VHL in RCC(VHL–) cells induced differentiation (8). To determine whether VHL also lead to cell polarization, we examined confluent monolayers using apical and lateral epithelial polarization markers. Observation of the apical membrane marker, CD26 (dipeptidyl peptidase IV), showed CD26 in a fine punctate pattern localized to the apical surface (arrows) with enhanced fluorescence at cell edges (arrowhead) in RCC(VHL+) cells with minimal signal in RCC(VHL–) cells (Fig. 1A, top ). Serial x-z axis sections emphasize CD26 protein in apical caps (arrow) in RCC(VHL+) cells and no specific staining pattern in RCC(VHL–) cells (Fig. 1A, bottom). Western blot analysis confirmed reduced expression of CD26 in RCC(VHL–) cells (Fig. 1C). To examine the pattern of intercellular adherans junctions, we labeled cells with antibodies against ß-catenin. Intense, tight staining was observed along lateral junctions in RCC(VHL+) cells compared with a less focused staining pattern in RCC(VHL–) cells (Fig. 1B, top). Consistent with this, x-z sections revealed ß-catenin restricted to the lateral surfaces between RCC(VHL+) cells (arrowheads), whereas in RCC(VHL–) cells, ß-catenin was more randomly dispersed at apical and lateral surfaces, and throughout the cytosol (Fig. 1B, bottom).


Figure 1
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  		Figure 1. Increased polarization by ectopic expression of VHL in RCC(VHL–) parental cells. A and B, immunofluorescent x-y and x-z confocal optical sections of RCC(VHL–) and RCC(VHL+) monolayers maintained for 3 days and then probed with apical marker, CD26 (A) or basolateral marker, ß-catenin (B); bar, 20 µm. Arrows, apical cell staining; arrowheads, lateral cell-cell staining. C, Western blot of CD26 and ß-Catenin proteins from RCC(VHL–) and RCC(VHL+) cell lines used in (A and B). ß-Tubulin loading control.

 
Recent work links proteins involved in regulating the establishment of apico-basal cell polarity with the formation of primary cilium (12). Primary cilium formation occurs in cells reaching quiescence (13). Based on VHL function required for cell cycle exit (1), and the above observations that the presence of pVHL influences epithelial cell polarity, we tested whether VHL is also required for primary cilium formation. Confluent monolayers of 786-0 and A498 RCC cells, stably expressing VHL or plasmid alone, were probed with antibodies against acetylated tubulin to visualize ciliary axonemes. Primary cilium (arrowheads) formed in RCC(VHL+) cells independent of RCC cell line background (Fig. 2A, panels a and c ). In contrast, there was an absence of primary cilium formation in RCC(VHL–) cells (Fig. 2A, panels b and d). Interestingly, the pattern of acetylated tubulin in the cytoplasm differed between ciliated and nonciliated cells (Fig. 2A-C). Cells with a primary cilium showed a decreased presence of microtubules organized around the centrosomal microtubule organizing center, whereas nonciliated cells retained a microtubule cytoskeleton radiating from a centrosomal microtubule organizing center, as seen in Fig. 2A, panel b. This is consistent with changes in microtubule organization described for polarized versus nonpolarized epithelial cells, respectively (14).


Figure 2
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  		Figure 2. Primary cilium formation is inversely correlated with VHL genotypes associated with RCC. Immunofluorescent, wide-field z-stack merged image planes of confluent monolayers of RCC cells with plasmid alone or stably expressing VHL or indicated VHL mutants. Cells were costained for acetylated-tubulin (green) and 4',6-diamidino-2-phenylindole (blue) to mark primary cilium and nuclei, respectively. Arrowheads, cilium; arrow, a more fibroblastic array of microtubules (b). Extension of cilium above the monolayer (z-direction) can be seen along top- and right-hand edges. A, stable transfectants expressing wild-type VHL (a and c) form primary cilium in two different RCC(VHL–) cell lines, 786-0 and A498, in contrast to stable transfectants with plasmid alone (b and d). B, cilium formation in low incidence of RCC stable transfectant, 786-0(VHL:Y98H). C, no cilia are observed in high incidence of RCC stable transfectant, 786-0(VHL:W117R). Bars, 10 µm.

 
At present, the majority of protein products derived from genes involved in the development of polycystic kidney diseases have been structurally or functionally related to the primary cilium (9). We employed an antibody against polycystin-2 (PKD2) as a specific marker for primary cilium. Figure 3A and B shows a punctate distribution pattern of PKD2 along the length of the primary cilium from RCC(VHL+) cultures, consistent with PKD2 distribution in renal epithelial monolayers (11).


Figure 3
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  		Figure 3. Characterization of primary cilium in RCC(VHL+) and RCC(VHL:Y98H) low risk RCC mutant cells. Immunofluorescent, wide-field z-stack merged image planes containing representative primary cilium. Monolayers were costained for acetylated-tubulin (green) and PKD2 (red). Bar, 5 µm.

 
VHL syndrome subtypes are associated with specific disease mutations within pVHL. The risk of RCC is high for VHL subtypes 1 and 2B, whereas risk is low for subtype 2A (1). We tested cells expressing VHL mutants from each of these subtypes for primary cilium formation to determine if ciliogenesis was associated with RCC risk. Cilium formation exhibited a genotype-phenotype relationship associated with different VHL clinical mutations. Cilia were only observed in cells expressing VHL subtype 2A (low-risk RCC) VHL:Y98H mutants (Fig. 2B, arrowheads), but not in cells with the VHL subtype 1 mutants (high-risk RCC) 786-0 and A498 cell lines (Fig. 2A, panels b and d) nor in cells expressing a subtype 2B (W117R high-risk RCC) mutant VHL (Fig. 2C). The cilium formed in RCC(VHL:Y98H) cells showed colocalization of acetylated-tubulin and PKD2 (Fig. 3C) comparable with the colocalization observed in RCC(VHL+) cells (Fig. 3A and B). We consistently observed fewer ciliated cells in the low-risk RCC subtype 2A VHL:Y98H mutant cells (average, 9% ciliated cells/field; range, 3-22%) compared with RCC(VHL+) (average, 38% ciliated cells/field; range, 24-61%; P < 0.01). Cilium were never observed in RCC(VHL–) or RCC(VHL:W117R) cells. These observations suggest that VHL mutations that do not restore cilium formation are associated with the development of RCC.

Loss of VHL function leads to the increased abundance of HIF{alpha} subunits under normoxic conditions (1). To investigate if the dysregulation of HIF{alpha} degradation in VHL mutant cell lines was also involved in primary cilium formation, we analyzed the effect of increased HIF2{alpha} abundance on primary cilium. RCC 786-0 cells do not express HIF1{alpha} (15). Our results showed that increased abundance of HIF2{alpha} in RCC(VHL+) confluent cells did not affect primary ciliogenesis. Confluent cells routinely formed cilium between days 6 and 7 of growth in serum-free medium. Therefore, cultures were treated with hypoxia mimetics, CoCl2 or deferoxamine mesylate, on day 6 to inhibit HIF2{alpha} degradation. Western blot analysis confirmed the increased abundance of HIF2{alpha} in response to deferoxamine mesylate or CoCl treatment (Fig. 4A ). Deferoxamine and mock-treated RCC(VHL+) monolayers (Fig. 4B) showed equivalent proportions of ciliated cells per field (24% average ciliated cells/field; range, 10-50%; 29% average ciliated cells/field; range, 16-44%, respectively; P = 0.21) indicating that ciliogenesis was independent of HIF{alpha} abundance. Similar cilium formation was observed with CoCl2 treatment (data not shown).


Figure 4
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  		Figure 4. Increased abundance of HIF2{alpha} in VHL+ cells does not inhibit primary cilium formation. A, Western blot of 786-0(VHL+) cell lysates, from monolayers grown under conditions for primary cilium formation and treated with and without hypoxia mimetics, 200 µmol/L of deferoxamine mesylate (DFO), or 250 µmol/L of CoCl2. Blots were probed for HIF2{alpha} protein. Equivalent amounts of total protein were loaded onto each lane. B, wide-field immunofluorescent, z-stack merged image planes of confluent monolayers of RCC(VHL+) cells treated overnight with or without 200 µmol/L of deferoxamine mesylate. Bar, 20 µm.

 
The VHL-dependent, HIF-independent response of cilium formation parallels other HIF-independent functions of VHL (1, 16, 17). Phosphorylation of the pVHL acidic NH2 terminus affects pVHL association with fibronectin but not HIF{alpha} degradation (16). Neddylation defective pVHL is unable to properly assemble a fibronectin extracellular matrix, although HIF ubiquitylation is intact (17). Because fibronectin-induced Ca2+ signaling is dependent on the presence of primary cilium (18), these HIF-independent properties of VHL may be related. It is interesting to note that the cell lines carrying type 2A VHL subtype mutant, Y98H, showed cilium formation and retained the ability to degrade HIF (data not shown; ref. 10). Thus, although there is a correlation between wild-type and mutant VHL that retain HIF{alpha} degradation function with cilium formation, experiments using hypoxia mimetics indicated that ciliogenesis is separable from HIF regulation.

Loss of heterozygosity of the VHL locus, followed by an independent, second locus mutation is believed to be the trigger event in the development of RCCs in patients with VHL syndrome and most sporadic cases of clear cell renal carcinomas (1). Based on our results showing that VHL is required for epithelial polarization and primary cilium formation in cultured renal epithelial cells, we hypothesize that early loss of pVHL leads to dedifferentiation of renal epithelial cells, followed by cyst development. Loss of VHL functions disrupt two cellular pathways within a single cell (i.e., differentiation/primary cilium formation and hypoxia-related gene expression). Precancerous regions of VHL syndrome kidneys show increased expression of the HIF1{alpha} downstream target, carbonic anhydrase, whereas the earliest detection of HIF2{alpha} is in distorted tubular structures, with increased expression observed in cysts and cancerous lesions (3, 4). A mechanistic pathway of tumorigenesis, based on our results, begins with the initial loss of VHL, which results in the loss of primary cilium and dedifferentiation of an epithelial cell within the kidney tubule epithelium. Resulting from this event is the accumulation of HIF{alpha} and activation of hypoxia-inducible target genes which stimulate increased growth and cell division of dedifferentiated kidney epithelial cells and increased angiogenesis (19, 20). This might explain the pathogenic differences between VHL-associated lesions of the kidney and elsewhere (i.e., kidney lesions do not exhibit a paracrine effect in contrast to hemangioblastomas that do; ref. 1). Loss of primary cilium and associated dedifferentiation caused by loss of VHL function are necessary prerequisites to cyst formation, and ultimately, RCC. Thus, it is during the time of epithelial cell growth and cell division that non-VHL second-site genomic mutations are likely to occur, resulting in the development of clear cell renal carcinomas.


   Acknowledgments
 
Grant support: CA85412.

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. Anne Muesch for assistance with microscopy techniques, Andrew Prior for assistance with cell lysis and Western blots, Drs. Steve Somlo and Y. Cai for the gift of rabbit anti-human polycystin-2 (YCC2), and Dr. Alan Shoenfeld for critical review of this manuscript.

Received 2/ 8/06. Revised 5/15/06. Accepted 6/ 5/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Kim WY, Kaelin WG. Role of VHL gene mutation in human cancer. J Clin Oncol 2004;22:4991–5004.[Abstract/Free Full Text]
  2. Lubensky IA, Gnarra JR, Bertheau P, Walther MM, Linehan WM, Zhuang Z. Allelic deletions of the VHL gene detected in multiple microscopic clear cell renal lesions in von Hippel-Lindau disease patients. Am J Pathol 1996;149:2089–94.[Abstract]
  3. Raval RR, Lau KW, Tran MG, et al. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol 2005;25:5675–86.[Abstract/Free Full Text]
  4. Mandriota SJ, Turner KJ, Davies DR, et al. HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron. Cancer Cell 2002;1:459–68.[CrossRef][Medline]
  5. Kondo K, Kim WY, Lechpammer M, Kaelin WG, Jr. Inhibition of HIF2{alpha} is sufficient to suppress pVHL-defective tumor growth. PLoS Biol 2003;1:E83.[Medline]
  6. Zimmer M, Doucette D, Siddiqui N, Iliopoulos O. Inhibition of hypoxia-inducible factor is sufficient for growth suppression of VHL–/– tumors. Mol Cancer Res 2004;2:89–95.[Abstract/Free Full Text]
  7. Lieubeau-Teillet B, Rak J, Jothy S, Iliopoulos O, Kaelin W, Kerbel RS. von Hippel-Lindau gene-mediated growth suppression and induction of differentiation in renal cell carcinoma cells grown as multicellular tumor spheroids. Cancer Res 1998;58:4957–62.[Abstract/Free Full Text]
  8. Davidowitz EJ, Schoenfeld AR, Burk RD. VHL induces renal cell differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signaling. Mol Cell Biol 2001;21:865–74.[Abstract/Free Full Text]
  9. Pazour GJ. Intraflagellar transport and cilia-dependent renal disease: the ciliary hypothesis of polycystic kidney disease. J Am Soc Nephrol 2004;15:2528–36.[Abstract/Free Full Text]
  10. Clifford SC, Cockman ME, Smallwood AC, et al. Contrasting effects on HIF-1{alpha} regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease. Hum Mol Genet 2001;10:1029–38.[Abstract/Free Full Text]
  11. Pazour GJ, San Agustin JT, Follit JA, Rosenbaum JL, Witman GB. Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease. Curr Biol 2002;12:R378–80.[CrossRef][Medline]
  12. Fan S, Hurd TW, Liu CJ, et al. Polarity proteins control ciliogenesis via kinesin motor interactions. Curr Biol 2004;14:1451–61.[CrossRef][Medline]
  13. Tucker RW, Pardee AB, Fujiwara K. Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell 1979;17:527–35.[CrossRef][Medline]
  14. Rodriguez-Boulan E, Kreitzer G, Musch A. Organization of vesicular trafficking in epithelia. Nat Rev Mol Cell Biol 2005;6:233–47.[CrossRef][Medline]
  15. Maxwell PH, Wiesener MS, Chang GW, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999;399:271–5.[CrossRef][Medline]
  16. Lolkema MP, Gervais ML, Snijckers CM, et al. Tumor suppression by the von Hippel-Lindau protein requires phosphorylation of the acidic domain. J Biol Chem 2005;280:22205–11.[Abstract/Free Full Text]
  17. Stickle NH, Chung J, Klco JM, Hill RP, Kaelin WG, Jr., Ohh M. pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development. Mol Cell Biol 2004;24:3251–61.[Abstract/Free Full Text]
  18. Praetorius HA, Praetorius J, Nielsen S, Frokiaer J, Spring KR. ß1-integrins in the primary cilium of MDCK cells potentiate fibronectin-induced Ca2+ signaling. Am J Physiol Renal Physiol 2004;287:F969–78.[Abstract/Free Full Text]
  19. de Paulsen N, Brychzy A, Fournier MC, et al. Role of transforming growth factor-{alpha} in von Hippel-Lindau (VHL)(–/–) clear cell renal carcinoma cell proliferation: a possible mechanism coupling VHL tumor suppressor inactivation and tumorigenesis. Proc Natl Acad Sci U S A 2001;98:1387–92.[Abstract/Free Full Text]
  20. Smith K, Gunaratnam L, Morley M, Franovic A, Mekhail K, Lee S. Silencing of epidermal growth factor receptor suppresses hypoxia-inducible factor-2-driven VHL–/– renal cancer. Cancer Res 2005;65:5221–30.[Abstract/Free Full Text]



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