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Formation of Primary Cilia in the Renal Is Regulated by the von Hippel-Lindau Tumor Suppressor Protein

Miguel A. Esteban, Sarah K. Harten, Maxine G. Tran, and Patrick H. Maxwell Renal Laboratory, Imperial College London, Hammersmith Campus, London, United Kingdom

Growing evidence points to defects in the primary as a critical mechanism underlying renal development. Inactivation of the VHL gene is responsible for the autosomal dominant condition von Hippel-Lindau (VHL) disease and is implicated in most sporadic clear renal . Manifestations of VHL disease include in several organs, particularly in the kidney. Here it is shown that VHL inactivation is associated with abrogation of the primary cilium in renal cysts of patients with VHL disease and in VHL-defective cell lines. Complementation of VHL-defective clear cell renal cell lines with wild-type VHL restored primary cilia. Moreover, it is shown that the effects of VHL on the primary cilium are mediated substantially via hypoxia-inducible factor. The effect of VHL status on the primary cilium provides a potential mechanism for renal cyst development in VHL disease and may help in the understanding of how VHL acts as a tumor suppressor. J Am Soc Nephrol 17: 1801–1806, 2006. doi: 10.1681/ASN.2006020181

any different hereditary conditions are associated re-expression of VHL in cell lines that are derived from CCRCC with development of renal cysts, often with other suppresses their tumorigenicity in nude mice (11). In view of M clinical manifestations. These include autosomal the proposed role of the primary cilium in other kidney cystic dominant polycystic kidney disease, Bardet-Biedl syndrome, diseases, we hypothesized that the VHL protein (pVHL) may nephronophthisis, and oral-facial-digital type 1 syndrome. Re- influence the formation, maintenance, and/or function of the markably, a common link has emerged in that mutations in the primary cilium. genes underlying these cystic conditions alter the structure or function of the primary cilium (1–7), a luminal hair-like extra- Materials and Methods cellular appendage that transmits calcium-mediated intracellu- Imaging Techniques lar signals after mechanical bending (8,9). These calcium signals Immunohistochemistry and immunofluorescence microscopy were are thought to regulate cytoarchitecture and cellular prolifera- performed as described previously (12,13). For immunofluorescence, tion of renal tubular cells in response to flow. we also used a laser scanning confocal microscope (Zeiss LSM 5 PAS- von Hippel-Lindau (VHL) disease is an uncommon autoso- CAL, Carl Zeiss, Oberkochen, Germany) equipped with Zeiss LSM mal dominant condition that is caused by inheritance of a image browser version Scanning electron microscopy was mutant VHL allele; the main renal manifestations are a very performed using a Jeol microscope (Akishima, Japan) at University College London, courtesy of Prof. K. Matter. high risk for developing clear cell renal carcinomas (CCRCC) and renal cysts. Other manifestations include pheochromocy- toma; hemangioblastomas in the retina, cerebellum, and spinal Cells and Antibodies cord; endolymphatic sac tumors; and epididymal cysts. The RCC4, RCC10, and sublines were described previously (13,14). For imaging experiments, cells were plated to confluence and studied after VHL gene is situated at 3p25, and a large number of different 3or4d. mutations have been identified in kindreds with VHL disease. Anti–hypoxia-inducible factor 1␣ (anti–HIF-1␣) was purchased from VHL behaves as a classic two-hit tumor suppressor gene that Transduction Labs (Lexington, KY), anti–HIF-2␣ was purchased from conforms to Knudson’s model (10), with the clinical manifesta- Research UK (London, UK), anti–␣-tubulin and antiacetylated tions (including renal cysts and tumors) invariably involving ␣-tubulin were purchased from Sigma (St. Louis, MO), and anti–car- somatic inactivation of the remaining wild-type VHL allele. bonic anhydrase IX (anti-CAIX) was a gift from S. Pastorekova (Insti- VHL also is inactivated in the majority of sporadic CCRCC, tute of Virology, Bratislava, Slovak Republic). which is the most common type of renal cancer. Importantly, Retroviral and Plasmids pCMVR-VHL N78S was constructed by transferring an insert from a Published online ahead of print. Publication date available at pcDNA3 plasmid (gift of W. Krek [Institute of Cell Biology, Zurich, Switzerland] and A. Hergovich [Friedrich Miescher Institute, Basel, Address correspondence to: Dr. Patrick H. Maxwell, Renal Laboratory, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. Switzerland]). Other retroviral vectors and procedures for infection Phone: ϩ44-20-8383-8594; Fax: ϩ44-20-8383-2062; E-mail: [email protected] were described previously (13,15).

Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1707-1801 1802 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1801–1806, 2006

Quantitative Real-Time Reverse Transcription–PCR those seen in RCC4/VHL and RCC10/VHL. The presence of PCR analysis using SYBR Green (AB gene) was performed as de- primary cilia in RCC4/VHL and RCC10/VHL also was verified scribed previously (13). All real-time reverse transcription–PCR data using confocal microscopy (Figure 2B). Similar results were ␤ are given as a value normalized to the level of - expression in the obtained using scanning electron microscopy (Figure 2C). Con- same retrotranscription. Sequences for primers are available on request. sistent with the immunofluorescence, pVHL-defective RCC4 and RCC10 cells displayed very few or no cilia on confocal siRNA Transfection Transfection procedures and oligo sequences were described previ- microscopy (Figure 2B) and scanning electron microscopy (data ously (13,16). not shown). pVHL has been reported to have a number of biochemical Results and Discussion functions, including regulation of stability, cell To study whether VHL status may influence the primary differentiation, cell , assembly, cilium, we first performed immunohistochemistry for acety- JunB, and atypical isoforms of protein kinase C (15,17–22). lated ␣-tubulin (an essential constituent of the primary cilium) However, the best characterized function of pVHL is to act as on paraffin-embedded material that contained cysts from the an essential component in the degradation of HIF-␣ subunits kidneys of two different patients with VHL disease. In the same (10,14). In the presence of oxygen, pVHL captures ␣ subunits of section, normal kidney tubules showed primary cilia protrud- the transcription factor HIF, resulting in their degradation by ing from the luminal side of epithelial cells (Figure 1, left), but the proteasome. The molecular signal for pVHL-mediated cap- these structures could not be identified in cysts (Figure 1, right). ture is the hydroxylation of two prolyl residues in the central Adjacent sections of the cyst wall showed strong positive la- part of HIF-␣ subunits, by a family of oxygen-dependent di- beling for CAIX (Figure 1, right), consistent with biallelic inac- oxygenases, PHD1-3 (prolyl hydroxylase domain–containing tivation of VHL in the cells that line the cyst (12). proteins) (23). When oxygenation is reduced or pVHL is absent, Next, we used two different pVHL-negative CCRCC cell HIF becomes stabilized and promotes the transcription of mul- lines (RCC4 and RCC10) and corresponding isogenic sublines tiple target genes that are involved in diverse pathways, exam- that express pVHL (hereafter referred to as RCC4/VHL and ples of which are erythropoiesis, angiogenesis, uptake, RCC10/VHL). Cells were studied 3 to 4 d after reaching con- and glycolysis (24). Broadly, these actions can be seen as adapt- fluence, which also is necessary for the development of cilia in ing the cell, , or whole to low oxygen. There are MDCK cells (7). Remarkably, immunofluorescence microscopy three different HIF-␣ isoforms (HIF-1␣, HIF-2␣, and HIF-3␣), for acetylated ␣-tubulin readily detected abundant primary ␣ ␣ cilia only in the CCRCC cells that expressed pVHL (Figure 2A). the best characterized of which are HIF-1 and HIF-2 (10). ␣ ␣ In all four cell lines, labeling for acetylated ␣-tubulin also was HIF-1 and HIF-2 are not redundant, based on genetic inac- present in the cytoplasm, with pVHL-negative cells showing tivation experiments in mice. However, their relative roles in somewhat more cytoplasmic signal. When pVHL-negative responses to hypoxia are not yet completely understood. It is ␣ RCC4 and RCC10 cultures were studied at longer intervals after interesting that in CCRCC cell lines, HIF-2 is necessary for reaching confluence, some rudimentary cilia were visible, but tumorigenesis in xenograft models and selectively increases these were always sparse and much less well developed than Cyclin D1 expression (25,26), whereas HIF-1␣ selectively in-

Figure 1. Primary cilia are lost in renal cysts from patients with von Hippel-Lindau (VHL) disease. Serial sections from a single block that contained a cyst and normal renal tubules were labeled for acetylated ␣-tubulin (top) and carbonic anhydrase IX (CAIX; a marker of biallelic inactivation of VHL; bottom). Middle panels show sections with low magnification; left and right panels show the indicated areas in the central panels at increased magnification. In the normal tubules (left), primary cilia (indicated with arrowheads) are visible, and CAIX is not expressed. In the cyst (right), primary cilia are not seen, and the cells express CAIX. J Am Soc Nephrol 17: 1801–1806, 2006 Regulation of the Primary Cilium by VHL 1803

Figure 2. Re-expression of VHL protein (pVHL) in clear cell renal carcinoma (CCRCC) cells restores cilia. (A) Immunofluorescence for acetylated ␣-tubulin in RCC4 and RCC10 cells. Arrow in the bottom panel indicates primary cilia in RCC4/VHL cells. Bars ϭ 20 ␮m. (B) Vertical computer reconstruction (using a confocal immunofluorescence microscope) of RCC4 and RCC10 cells and stable transfectants that expressed pVHL. *Primary cilia. Bars ϭ 20 ␮m. (C) Scanning electron microscopy of CCRCC cells complemented with pVHL. Bars ϭ 5 and 10 ␮m.

creases expression of CAIX and the proapoptotic gene BNIP3 formation of the primary cilium was restored in RCC4 and (25). RCC10 cells that were infected with pVHL19, pVHL30, and also We hypothesized that constitutive activation of HIF may be the two pVHL 2C mutations (Figure 3C). Infection of pVHL- the mechanism underlying altered ciliogenesis when VHL is negative cells with a mutant VHL gene encoding a different inactivated. To dissect this, we first used retroviral gene trans- missense substitution that abolishes the ability to regulate HIF fer to express several different pVHL molecules. These in- (VHL N78S) (27) was used as an additional control and did not cluded the full-length p30 isoform (pVHL30) and the p19 iso- result in formation of cilia (Figure 3D). These experiments show form (pVHL19). The latter isoform also is able to regulate HIF that restoration of the primary cilium shown in stably trans- and arises from an alternative translation initiation site at fected RCC4/VHL and RCC10/VHL cells (see Figure 2A) is not amino acid 54 (10). We also tested two disease-associated mis- due to clone-specific effects, because it also was seen in these mutations in pVHL, resulting in single amino acid sub- freshly prepared heterogeneous pooled populations. stitutions, pVHL-V84L and pVHL-L188V. These mutations are The concordance between the ability of pVHL molecules to associated with type 2C VHL disease, in which patients de- suppress HIF and restore the primary cilium supported the velop pheochromocytoma without other clinical manifestations notion that the mechanism involved activation of HIF. To test (10). pVHL type 2C mutations retain the ability to regulate HIF this directly, we then performed genetic knockdown of HIF-1␣ normally (27,28), thereby providing a useful tool to identify or HIF-2␣ using siRNA treatment of pVHL-negative RCC10 specific consequences of pVHL loss of function that are inde- cells. Specificity was confirmed by Western blotting (Figure pendent of HIF (29,30). As expected, expression of pVHL19, 3D); siRNA knockdown of HIF-2␣ resulted in a reproducible pVHL30, or either of the two pVHL type 2C mutants (but not increase in HIF-1␣ as reported previously (13,25). It is interest- the empty vector) comparably reduced protein levels of HIF-␣ ing that immunofluorescence microscopy for acetylated ␣-tu- subunits in infected pools of RCC4 and RCC10 cells (Figure bulin showed that only knockdown of HIF-1␣ significantly 3A); quantitative real-time reverse transcription–PCR analysis restored cilium formation (Figure 3E). To demonstrate further also showed comparable reduction in glucose transporter 1 (a the role of HIF-1␣ in preventing renal ciliogenesis, we also well-characterized HIF target) mRNA levels (Figure 3B). Nota- infected RCC10/VHL with retroviruses that encode constitu- bly, immunofluorescence for acetylated ␣-tubulin showed that tively active forms of HIF-1␣; infection with the empty vector 1804 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1801–1806, 2006

Figure 3. Activation of hypoxia-inducible factor ␣ (HIF-1␣) underlies the abrogation of primary cilia in CCRCC cells. (A) Western blotting shows comparable suppression of HIF-1␣ and HIF-2␣ in cells infected with wild-type pVHL or the type 2C mutations. Expression of wild-type and mutant pVHL proteins was confirmed by Western blot analysis (data not shown) (13). (B) Quantitative real-time reverse transcription–PCR (RT-PCR) of glucose transporter 1 (GLUT1) mRNA. (C) Immunofluorescence for acetylated ␣-tubulin. Bars ϭ 20 ␮m. (D) Western blotting (HIF-1␣, HIF-2␣, and ␣-tubulin), quantitative real-time RT-PCR of GLUT1 mRNA, and immunofluorescence for acetylated ␣-tubulin (bars ϭ 20 ␮m) in RCC4 cells infected with empty vector, pVHL30, or pVHL N78S. (E) siRNA treatment of pVHL-negative RCC10 cells with specific oligos directed against HIF-1␣, HIF-2␣, or firefly luciferase (control). Western blotting shows specific inhibition of HIF-1␣ and HIF-2␣. Immunofluorescence microscopy for acetylated ␣-tubulin shows restoration of primary cilia on knocking down HIF-1␣. Bars ϭ 20 ␮m. (F) pVHL stably transfected RCC10 cells infected with retrovirus that encodes a constitutively active form of HIF-1␣ or empty vector. Western blotting verified the corresponding presence of HIF-1␣. Immunofluorescence microscopy for acetylated ␣-tubulin in the same cells. Bars ϭ 20 ␮m. J Am Soc Nephrol 17: 1801–1806, 2006 Regulation of the Primary Cilium by VHL 1805 was used as a control. The corresponding presence of HIF-1␣ Sayer JA, Lillo C, Jimeno D, Coucke P, De Paepe A, Rein- was verified by Western blotting (Figure 3F). Notably, immu- hardt R, Klages S, Tsuda M, Kawakami I, Kusakabe T, nofluorescence staining showed that compared with control Omran H, Imm A, Tippens M, Raymond PA, Hill J, Beales cells, active HIF-1␣ altered the formation of the cilia (Figure 3F). P, He S, Kispert A, Margolis B, Williams DS, Swaroop A, Therefore, suppression of HIF-1␣ restores cilium formation in Hildebrandt F: Nephrocystin-5, a ciliary IQ domain pro- tein, is mutated in Senior-Loken syndrome and interacts pVHL-negative CCRCC cells, and activation of HIF-1␣ in with RPGR and calmodulin. Nat Genet 37: 282–288, 2005 CCRCC cells that re-express pVHL abolishes it. 3. Ansley SJ, Badano JL, Blacque OE, Hill J, Hoskins BE, Understanding how pVHL regulates the primary cilium via Leitch CC, Kim JC, Ross AJ, Eichers ER, Teslovich TM, ␣ HIF-1 will require further work, but it is intriguing to specu- Mah AK, Johnsen RC, Cavender JC, Lewis RA, Leroux MR, late how this may occur. It is possible that the effect on cilia is Beales PL, Katsanis N: Basal body dysfunction is a likely because pVHL is required for normal interactions between cause of pleiotropic Bardet-Biedl syndrome. 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