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

Fast Track Formation of Primary Cilia in the Renal Epithelium 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 cilium as a critical mechanism underlying renal cyst 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 cell renal carcinomas. Manifestations of VHL disease include cysts 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 carcinoma 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 urine 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 3.2.0.115. 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- Cancer 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 Infection and Plasmids pCMVR-VHL N78S was constructed by transferring an insert from a Published online ahead of print. Publication date available at www.jasn.org. 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 -actin 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 microtubule stability, cell To study whether VHL status may influence the primary differentiation, cell motility, extracellular matrix 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, glucose 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, tissue, or whole organism 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),

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