HIF-2α downregulation in the absence of functional VHL is not sufficient for renal cell differentiation
© Hughes et al. 2007
Received: 07 April 2007
Accepted: 28 June 2007
Published: 28 June 2007
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© Hughes et al. 2007
Received: 07 April 2007
Accepted: 28 June 2007
Published: 28 June 2007
Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene has been linked to hereditary as well as sporadic clear cell renal carcinomas. The product of the VHL gene, pVHL, acts to target hypoxia-inducible factor alpha (HIF-α) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2α in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2α toward cellular pVHL activities.
Knockdown of HIF-2α resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2α regulation. However, cellular morphological changes and downregulation of integrins α5 and β1, which were seen upon pVHL replacement, were not faithfully phenocopied by HIF-2α reduction. Moreover, fibronectin deposition and expression of renal cell differentiation markers were observed in cells containing replaced pVHL, but not in HIF-2α knockdown cells, indicating that these pVHL functions may occur independently of HIF-2α downregulation.
These results indicate that HIF-2α regulation is not sufficient for pVHL-induced renal cell differentiation. We hypothesize that in addition to HIF-2α dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.
von Hippel-Lindau (VHL) disease arises from heterozygous germline mutations in the VHL tumor suppressor gene, which resides on chromosome 3p25, and is characterized by clear cell renal carcinomas, hemangiomas, pheochromocytomas, as well as other tumor types [1, 2]. The development of tumors in VHL disease results from loss or inactivation of the remaining wild type allele, leading to an absence of functional VHL protein . Somatic VHL mutations are also common in sporadic clear cell renal carcinoma and hemangioblastomas (reviewed in ). Restoration of VHL function is sufficient to suppress in vivo tumor formation in VHL-defective renal carcinoma cells [3, 4].
The VHL gene produces two protein products due to an internal translation initiation start site at codon 54 [3, 5, 6]. The larger protein produced from the normal start site is a 213 amino acid protein approximately 24–30 kDa, (VHLp30), and the shorter produced from the internal start site is an 18–19 kDa isoform (VHLp19) of 160 amino acids. The shorter form has been shown to contain full tumor suppressor function [3, 5, 6]. Both protein products of the VHL gene (collectively called pVHL) contain an alpha and beta domain . The alpha domain associates with elongin B, elongin C, Cul2 and Rbx1 and this pVHL complex acts as a ubiquitin E3 ligase [8–13]. HIF-α, the alpha subunit of the heterodimeric transcription factor hypoxia inducible factor (HIF), binds to pVHL's beta domain and is the best-known substrate of the pVHL E3 ligase complex [14, 15].
One of the conditions for pVHL to successfully bind to the HIF-α subunit is the presence of oxygen . The HIF-α subunit has an oxygen dependent domain that contains conserved proline residues that are hydroxylated in the presence of oxygen [17, 18]. This serves as a signal for the beta domain of pVHL to bind effectively to HIF-α, resulting in its polyubiquination and proteosomal degradation. In hypoxic or anoxic conditions, HIF-α does not get hydroxylated and is unable to bind pVHL and its levels remain elevated. Deletion mutations in VHL or point mutations in the elongin binding domain that result in loss of functional VHL result in high HIF-α levels . Likewise, point mutations in the beta domain of pVHL cause a similar effect. VHL disease has been shown to display a complex genotype-phenotype correlation wherein specific VHL mutations are associated with a higher risk for a subset of VHL-associated tumor types [20–22]. The type 2A disease phenotype is marked by mutations in pVHL beta domain that hinder its ability to bind HIF-α. Interestingly, although these mutations lead to higher HIF-α levels, families with these mutations show a low incidence of renal cell carcinoma (RCC) . This finding suggests the possibility of VHL activities other than HIF-α regulation, the loss of which leads to RCC.
Studies to date have shown that downregulation of HIF-α is necessary for VHL dependent tumor suppression in some model systems . However, other model systems have shown that high levels of HIF-α alone are insufficient for malignant transformation . Therefore, it is currently unclear what role HIF-α plays in VHL-associated tumors, especially with regard to initiation of renal carcinomas. In this report, we have examined the role of HIF-2α regulation in specific cellular phenotypes that have been previously associated with VHL. Notably, VHL replacement in VHL-null cells has been shown to regulate cellular morphology and organization, levels of cell surface integrins, fibronectin matrix assembly, and to direct growth arrest and cellular differentiation of cells grown on extracellular matrices [26–28]. Using an RNA interference approach to lower HIF-2α levels in VHL-null renal cell lines that express this HIF-α isoform only, we have investigated whether lower levels of HIF-2α can mimic the effects of VHL replacement in these cells. Here, we report that HIF-2α regulation plays a role in certain, but not all VHL-associated cellular phenotypes. These results suggest that pVHL has other important biochemical and cellular functions in addition to the ubiquitination of HIF-α that may be important for tumor suppression.
786-O and A498 VHL-null renal cell lines both contain high levels of HIF-2α, but not the other isoforms of HIF-α . In order to investigate whether downregulation of HIF-2α is sufficient to mimic VHL replacement in these cells, we lowered the levels of HIF-2α using two retroviral vectors that direct the expression of short hairpin RNA (shRNA) directed against HIF-2α. These constructs are based on the pSuperRetro shRNA system  and have been previously shown to specifically inhibit HIF-2α in the 786-O cell line . The empty vector, pSuperRetro, was used as a control.
VHL has been linked to a number of phenotypes at the cellular level that may be important for tumor suppression. In this study, we have analyzed whether certain cellular VHL functions are dependent on pVHL's ability to regulate HIF-2α. We chose cell lines lacking HIF-1α to specifically examine the isolated role of HIF-2α, and used retroviral infection to obtain multiclonal pools, limiting the potential of specific clonal populations. We report that HIF-2α downregulation was sufficient to cause cell cycle arrest, whereas VHL-associated cellular morphological changes and downregulation of α5 and β1 integrins were minimally dependent on HIF-2α regulation. Notably, HIF-2α knockdown was not sufficient to bring about proper fibronectin matrix assembly and differentiation of renal cells, both of which are normally mediated by VHL.
Downregulation of HIF-2α levels by shRNA has been shown to be sufficient to suppress tumor growth of VHL-defective renal carcinoma cells in nude mice [24, 30]. In the present study, decreased levels of HIF-2α led to cell cycle arrest of cells grown on collagen I, suggesting a mechanism for the in vivo growth arrest. Additionally, knockdown of HIF-2α abrogated aneuploidy in the A498 cell line. These findings reinforce the notion that HIF-2α is important in the progression of VHL-associated tumors. This role in renal cells may be specific to HIF-2α (rather than HIF-1α), since HIF-2α preferentially transactivates protumorigenic genes, including cyclin D1 and vascular endothelial growth factor (VEGF) . However, while many of the clinical manifestations of VHL inactivation, including tumors with a high degree of vascularization, might be explained by upregulated hypoxia-inducible genes, it is doubtful whether HIF-α dysregulation (or HIF-2α dysregulation) alone can initiate renal tumorigenesis. In support of this notion, common mutations in the HIF-α binding domain of pVHL that fall within the type 2A disease phenotype result in higher levels of HIF-α, yet are associated with a low incidence of RCC . Additionally, constitutive expression HIF-2α was seen to be less invasive in Matrigel than loss of VHL . Overexpression of HIF-2α was also seen to reduce the growth of rat glioma tumors and cause apoptosis . Collectively, these findings suggest that HIF-2α overexpression contributes to tumorigenesis, but is insufficient for the initiation of renal carcinomas. Thus, loss of some other VHL-associated function is required for initiation of renal carcinomas.
Recent studies have focused on a VHL role in earlier stages of renal tumor development, namely renal cyst formation [33, 39, 40]. These studies have suggested that renal cyst formation may rely more on disruption of VHL functions such as cell polarity and cilia formation, although the importance of HIF-α regulation for these phenomena was not in agreement [33, 39]. The role of HIF-2α on other aspects of cellular morphology is also currently unclear. In the present studies, HIF-2α knockdown had only a modest effect on cell morphology and on expression of α5 and β1 integrins. In other studies assessing various parameters of cell morphology and cell-to-cell interaction, overexpression of a constitutively active HIF-2α was seen to have no effect on adherens junctions (as assayed by β-catenin) and tight junction organization . On the other hand, HIF-2α has been seen to regulate the expression of another adherens junction protein, E-cadherin [39, 41]. A more precise determination of the contribution of HIF-2α toward adherens junction activities as well the exact relationship of these markers to VHL-mediated renal tumorigenesis would clarify these findings.
Fibronectin matrix assembly has been suggested to be a key VHL function for VHL tumor suppression . In the present studies, fibronectin deposition was shown to be unaffected by HIF-2α knockdown. Similarly, cells with constitutive expression of HIF-2α have been shown to have intact assembly of fibronectin and collagen IV extracellular matrices  and conversely, a pVHL mutant that retained its ability to ubiquitinate HIF-α failed to promote fibronectin matrix assembly . These findings all support the notion that this VHL function is an HIF-α independent phenotype. However, it is unlikely that loss of normal fibronectin assembly is responsible for the initiation of renal tumors, since VHL proteins harboring type 2C disease mutations are defective in fibronectin assembly, yet these mutations are associated with a low risk of RCC. Also, the differences observed in fibronectin deposition between 786-O cell lines expressing either of the two isoforms of VHL in the present studies might indicate a more subtle role of fibronectin, since these cell lines have been shown to have equal tumor suppression ability .
Another non-HIF-2α associated phenotype observed in the present studies was VHL-dependent expression of renal differentiation markers. It is likely that dedifferentiation or failure to differentiate is a very early step in renal tumorigenesis. Notably, dedifferentiation has been observed in renal cysts following inactivation of VHL and changes in a number of renal cell differentiation markers have been observed in renal cysts [40, 42]. Differentiation may also underlie the formation of primary cilium that is seen upon VHL replacement in VHL-null cells [33, 39]. Interestingly, a type 2A VHL mutant (low risk of RCC) was seen to support ciliogenesis , suggesting that with type 2A VHL mutations, renal cells remain differentiated and are therefore not capable of initiating a tumor. In contrast to type 2A VHL disease, specific mutants within the elongin binding site (type 2B phenotype) correlate with a high incidence of renal cell carcinoma . Since these mutations disrupt VHL-mediated ubiquitination as a whole, it is possible that there is another substrate that the pVHL complex targets, which is dysregulated in type 2B, but not type 2A VHL disease. This alternative substrate of pVHL may be more directly linked to cell differentiation than HIF-2α and may be more relevant to initiation of renal carcinomas. However, non-ubiquitination functions of VHL, which have yet to be fully elucidated, may prove to be more crucial for VHL-mediated differentiation. In either case, it is likely that the loss of VHL-mediated differentiation synergizes with the effects of high HIF-α levels to lead to the development of renal carcinomas.
HIF-2α regulation plays a role in certain, but not all VHL-associated cellular phenotypes. Notably, HIF-2α regulation is insufficient for pVHL-associated renal cell differentiation. These results suggest that pVHL has other important biochemical and cellular functions in addition to the ubiquitination of HIF-α that may be important for tumor suppression.
All renal cell lines (293T, 786-O and A498) were obtained from the American Type Culture Collection. All cells were grown in Dulbecco's modified Eagle's medium containing 10% Serum Supreme (BioWhitaker) and penicillin-streptomycin (100 U/ml and 10 μg/ml, respectively). The 786-O and A498 renal carcinoma cell lines both contain a single VHL allele harboring a frameshift mutation (at codons 104 and 142, respectively) [1, 43]. 786-O cells stably transfected with VHLp19 and VHLp30 have been described previously . A498 cells stably transfected with VHLp19 plasmid  were isolated following selection with media containing 600 μg/ml of G418 . To condition cells at high density, cells were allowed to grow for 1–2 weeks post-confluency with media replenishment every 48 h. For culture on collagen I, cells were either grown on purchased plates coated with a thin layer of collagen I (Becton Dickinson) or on an approximate 2–3 mm thick layer of rat collagen I (Becton Dickinson), prepared as previously described . FACS analysis of cells grown 2 weeks post-confluency on thick layers of collagen I was performed as previously described . Analysis of DNA histograms was performed using ModFitLT 3.1 software (Verity Software House). The first major G1 peak with a corresponding G2 peak (containing DNA content double that of G1) was classified as the diploid cell population, whereas cell populations displaying alternative G1 peaks with corresponding G2 peaks were classified as anueploid.
Two retroviral plasmids directing expression of short hairpin RNA's (shRNA) containing unique HIF-2α 19mer sequences  were generously provided by Dr. William Kaelin (Dana Farber Cancer Center). To produce retroviral supernatants, each retroviral plasmid was transfected into 293T cells plated in duplicate on 35 mm dishes using Lipofectamine Plus reagent (Invitrogen) as directed by the manufacturer. Each transfection mix also received the retroviral packing plasmid, pCL-Ampho . Following transfection, 293T cells from duplicate transfections were pooled and replated into 100 ml dishes. Medium containing retroviruses was collected at 72 h post-transfection and filtered through a 45 μm pore-size filter. To create stable pools of retrovirally infected cells, 786-O and A498 cells were incubated overnight in a mixture (1:1) of retroviral supernatant and fresh medium supplemented with polybrene (10 μg/ml). Two days later, cells were selected with puromycin (1 μg/ml) for 10 to 14 days.
For all immunoblots, cells were grown to 100% confluency. Culture plates were rinsed with phosphate buffer saline (PBS) and cells were lysed using 500 μl of lysis buffer (50 mM HEPES (pH 7.6), 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na2VO3 and 2 μg/ml each of aprotinin, bestatin, and leupeptin), incubating at 4°C for 30 minutes. To prepare lysates of cells grown at high density, the lysis buffer was supplemented with 0.5% Triton X-100 and 0.5% Nonidet P-40 to ensure solubilization of proteins. Lysed cells were scraped with a plastic scraper, resuspended by pipetting, collected and then microcentrifuged for 15 minutes. Supernatant containing clarified protein lysate was removed and protein levels were determined by Bradford assay (Bio-Rad). Equal amounts (25 μg) of protein lysates were added to an appropriate amount of 3.5× SDS PAGE loading buffer (175 mM Tris (pH 6.8), 0.75 mM DTT, 7% SDS, 35% glycerol, 0.35% bromophenol blue) and resolved by SDS-polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane overnight. Rabbit polyclonal anti-HIF-2α antibody was from Novus Biological. Rabbit polyclonal anti-Glut-1 antibody was from Alpha Diagnostics. Anti-VHL mAb 11E12 has been previously described . Anti-leucine aminopeptidase mAb was obtained from Lab Vision/NeoMarkers. Integrin and HNF-1α mAbs were obtained from BD Transduction Laboratories. Rabbit anti-fibronectin antibody, anti-alpha tubulin mAb, as well as all secondary rabbit and mouse antibodies used were from Sigma.
Cells (2 × 104) were plated in triplicate in a 96-well tissue culture plate and grown for six days. Cell removal was performed with Phosphate Buffered Saline (PBS) containing 2 mM EDTA, incubating at 37°C for 5–10 minutes. Complete removal of cells was confirmed by microscopy. Plates were blocked using TBS-Tween/0.1% BSA (Tris buffered saline containing 0.5% Tween-20 and 0.1% heat-inactivated Bovine Serum Albumin) for 45 minutes at 37°C and subsequently incubated with 2 μg/ml of rabbit anti-fibronectin antibody in TBS-Tween/0.1% BSA for one hour. Plates were washed three times with TBS-Tween and then incubated for 45 minutes at 37°C with anti-rabbit secondary antibody, diluted to a final concentration of 0.1 μg/ml with TBS-Tween/0.1% BSA. Plates were again washed three times with TBS-Tween and color was developed with 200 μl of p-nitrophenyl phosphate (Sigma) in ELISA substrate buffer (1 mg/ml in 50 mM NaCO3, 1 mM MgCl2, pH 9.8). Reactions were stopped with 50 μl of 3 M NaOH, and the OD at a wavelength of 405 nm was determined for each well in a microplate reader (BioRad).
renal cell carcinoma
short hairpin RNA
This work was supported by National Institutes of Health grants CA121992 (to A.R.S.) and CA85412 (to R.D.B) and by an Adelphi University Faculty Development Award (to A.R.S.).
We thank Leiping Fu for assistance with the FACS analysis and Mallory Lutz for critical review of the manuscript.
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