Protective Function of von Hippel-Lindau Protein against Impaired Protein Processing in Renal Carcinoma Cells

The absence of functional von Hippel-Lindau (VHL) tumor suppressor gene leads to the development of neoplasias characteristic of VHL disease, including renal cell carcinoma (RCC). Here, we compared the sensitivity of RCC cells lacking VHL gene function with that of RCC cells expressing the wild-type VHL gene (wtVHL) after exposure to various stresses. While the response to most treatments was not affected by the VHL gene status, glucose deprivation was found to be much more cytotoxic for RCC cells lacking VHL gene function than for wtVHL-expressing cells. The heightened sensitivity of VHL-deficient cells was not attributed to dissimilar energy requirements or to differences in glucose uptake, but more likely reflects a lesser ability of VHL-deficient cells to handle abnormally processed proteins arising from impaired glycosylation. In support of this hypothesis, other treatments which act through different mechanisms to interfere with protein processing (i.e., tunicamycin, brefeldin A, and azetidine) were also found to be much more toxic for VHL-deficient cells. Furthermore, ubiquitination of cellular proteins was elevated in VHL-deficient cells, particularly after glucose deprivation, supporting a role for the VHL gene in ubiquitin-mediated proteolysis. Accordingly, the rate of elimination of abnormal proteins was lower in cells lacking a functional VHL gene than in wtVHL-expressing cells. Thus, pVHL appears to participate in the elimination of misprocessed proteins, such as those arising in the cell due to the unavailability of glucose or to other stresses.     

Ectopic expression of von Hippel-Lindau tumor suppressor induces apoptosis in 786-O renal cell carcinoma cells and regresses tumor growth of 786-O cells in nude mouse

The von Hippel-Lindau (VHL) is a known tumor suppressor that binds to alpha-subunits of hypoxia-inducible factors and induces ubiquitin-mediated degradation of the protein in an oxygen-dependent manner. VHL is also involved in the regulation of tumor angiogenesis, glycolysis, cell cycle regulation, and apoptosis. In the present study, we showed that ectopic expression of VHL induces apoptosis in renal cell carcinoma 786-O cells which contain only the mutant VHL, evidenced by TUNEL assay and DAPI staining. Furthermore, biochemical studies indicated that expression of VHL in 786-O cells results in both PARP and CPP32 cleavage, suggesting that VHL-induced apoptosis in 786-O cells is caspase dependent. Moreover, we also observed that apoptosis induced by ectopic VHL expression was associated with up-regulation of p27 as well as Bax, implicating the roles of these two proteins in VHL-induced apoptosis. The up-regulation of p27 and Bax by VHL was specific since we did not detect any changes in the level of other apoptotic factors including Fas and Bcl2 by the expression of VHL. We next examined the effect of VHL expression on the tumor growth of 786-O renal cell carcinoma cells in nude mouse. The results showed that injection of Ad.VHL adenovirus regresses the tumor growth of 786-O cells in nude mouse. The analysis by TUNEL assay as well as DAPI staining of 786-O tumors injected with Ad.VHL showed clear evidence of apoptosis. These results suggest that ectopic VHL expression induces apoptotic response in 786-O VHL mutant cells both in vitro and in vivo.     

Identification of the RNA polymerase II subunit hsRPB7 as a novel target of the von Hippel-Lindau protein

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the hereditary VHL disease and sporadic clear cell renal cell carcinomas (CCRCC). VHL-associated tumors are highly vascularized, a characteristic associated with overproduction of vascular endothelial growth factor (VEGF). The VHL protein (pVHL) is a component of the ubiquitin ligase E3 complex, targeting substrate proteins for ubiquitylation and subsequent proteasomic degradation. Here, we report that the pVHL can directly bind to the human RNA polymerase II seventh subunit (hsRPB7) through its beta-domain, and naturally occurring beta-domain mutations can decrease the binding of pVHL to hsRPB7. Introducing wild-type pVHL into human kidney tumor cell lines carrying endogenous mutant non-functional pVHL facilitates ubiquitylation and proteasomal degradation of hsRPB7, and decreases its nuclear accumulation. pVHL can also suppress hsRPB7-induced VEGF promoter transactivation, mRNA expression and VEGF protein secretion. Together, our results suggest that hsRPB7 is a downstream target of the VHL ubiquitylating complex and pVHL may regulate angiogenesis by targeting hsRPB7 for degradation via the ubiquitylation pathway and preventing VEGF expression.     

Folliculin Contributes to VHL Tumor Suppressing Activity in Renal Cancer through Regulation of Autophagy

Von Hippel-Lindau tumor suppressor (VHL) is lost in the majority of clear cell renal cell carcinomas (ccRCC). Folliculin (FLCN) is a tumor suppressor whose function is lost in Birt-Hogg-Dubé syndrome (BHD), a disorder characterized by renal cancer of multiple histological types including clear cell carcinoma, cutaneous fibrofolliculoma, and pneumothorax. Here we explored whether there is connection between VHL and FLCN in clear cell renal carcinoma cell lines and tumors. We demonstrate that VHL regulates expression of FLCN at the mRNA and protein levels in RCC cell lines, and that FLCN protein expression is decreased in human ccRCC tumors with VHL loss, as compared with matched normal kidney tissue. Knockdown of FLCN results in increased formation of tumors by RCC cells with wild-type VHL in orthotopic xenografts in nude mice, an indication that FLCN plays a role in the tumor-suppressing activity of VHL. Interestingly, FLCN, similarly to VHL, is necessary for the activity of LC3C-mediated autophagic program that we have previously characterized as contributing to the tumor suppressing activity of VHL. The results show the existence of functional crosstalk between two major tumor suppressors in renal cancer, VHL and FLCN, converging on regulation of autophagy.     

Von Hippel-Lindau disease (VHL): a need for a murine model with retinal hemangioblastoma

Von Hippel-Lindau (VHL) disease is a highly penetrant autosomal dominant systemic malignancy that gives rise to cystic and highly vascularized tumors in a constellation of organs. Patients with VHL disease commonly present with hemangioblastomas in the central nervous system and the eye while other manifestations include pheochromocytoma, clear cell renal cell carcinoma, endolymphatic sac tumors of the middle ear, pancreatic cystadenomas, epididymal and broad ligament cystadenomas. Animal models inactivating the VHL gene product in various organ tissues have been constructed over the past 15 years to parse its HIF-associated mechanisms and its link to tumorigenesis. These models, despite advancing our understanding the molecular role of VHL, are by and large unable to recapitulate the more common features of human VHL disease. Up to date, no model exists that develop retinal hemangioblastomas, the most common clinical manifestation. The purpose of this review is: (1) to discuss the need for an ocular VHL model, (2) to review the animal models that recapitulate clinical VHL disease and (3) to propose potential mechanisms of tumorigenesis for the development of ocular VHL.     

Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel‐Lindau transfectant cell line model

The von Hippel‐Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL‐defective RCC cell line UMRC2 by transfection with vector control or VHL (?/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin‐affinity chromatography and analysis by GeLC‐MS/MS, VHL‐associated changes in plasma membrane proteins were analysed. Comparative analysis of ‐/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the α3 and β1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL‐defective cells. Western blotting confirmed these changes and also revealed VHL‐dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient‐matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL‐dependent proteins that may be important in tumorigenesis.     

Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility

The tumor suppressor VHL (von Hippel-Lindau protein) serves as a negative regulator of hypoxia-inducible factor-alpha subunits. However, accumulated evidence indicates that VHL may play additional roles in other cellular functions. We report here a novel hypoxia-inducible factor-independent function of VHL in cell motility control via regulation of fibroblast growth factor receptor 1 (FGFR1) endocytosis. In VHL null tumor cells or VHL knock-down cells, FGFR1 internalization is defective, leading to surface accumulation and abnormal activation of FGFR1. The enhanced FGFR1 activity directly correlates with increased cell migration. VHL disease mutants, in two of the mutation hot spots favoring development of renal cell carcinoma, failed to rescue the above phenotype. Interestingly, surface accumulation of the chemotactic receptor appears to be selective in VHL mutant cells, since other surface proteins such as epidermal growth factor receptor, platelet-derived growth factor receptor, IGFR1, and c-Met are not affected. We demonstrate that 1) FGFR1 endocytosis is defective in the VHL mutant and is rescued by reexpression of wild-type VHL, 2) VHL is recruited to FGFR1-containing, but not EGFR-containing, endosomal vesicles, 3) VHL exhibits a functional relationship with Rab5a and dynamin 2 in FGFR1 internalization, and 4) the endocytic function of VHL is mediated through the metastasis suppressor Nm23, a protein known to regulate dynamin-dependent endocytosis.     

Dbp5 — From nuclear export to translation

The DEAD-box RNA helicase Dbp5 is an essential and conserved mRNA export factor which functions in the ATP dependent remodeling of RNA/protein complexes. As such it displaces mRNA bound proteins at the cytoplasmic site of the nuclear pore complex. For the regulation of its RNA-dependent ATPase activity during late steps of nuclear transport, Dbp5 requires the nucleoporin Nup159 and its cofactors Gle1 and IP₆. In addition to its role in mRNA export, a second important function of Dbp5 was identified in translation termination, where it acts together with eRF1 once the translation machinery has reached the stop codon. Similar to mRNA export, this function also requires Gle1–IP₆, however, the counterpart of Nup159 is still missing. Potential other functions of the nucleo-cytoplasmic protein Dbp5 are discussed as well as its substrate specificity and details in its regulatory cycle that are based on recent biochemical and structural characterization. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

猪VHL基因克隆及敲低克隆胚胎的构建

通过在细胞和胚胎水平对猪Von Hippel-Lindau(VHL)基因进行了基因敲低研究,以便为建立VHL疾病模型猪奠定基础.研究首先通过 3'RACE和5'RACE克隆得到猪VHL基因cDNA全长序列(2 725 bp),Real-time PCR结果表明VHL基因广泛表达于猪的各种组织器官,其中在肾上腺、肝脏、胰腺、心脏和睾丸等组织器官高量表达.进一步在猪iPS细胞中对5条干扰片段进行筛选,获得2条高效的干扰片段,干扰效率分别达到72％(P=0.0012)和64％ (P＜ 0.01).以稳定干扰VHL基因的猪胎儿成纤维细胞为核供体,构建克隆胚胎,结果表明,克隆胚胎的发育能力与对照组相比没有明显差异,而且在克隆囊胚中VHL基因的干扰效率达到71％ (P＜ 0.01).综上所述,文中获得了猪VHL基因全长序列并获得该基因稳定敲低的猪细胞和胚胎,从而为VHL疾病模型猪的构建奠定了良好的基础.     

The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.     

基于数据挖掘分析ATP1A1基因在肾透明细胞癌中的表达及意义

目的:通过数据挖掘分析ATP1A1在肾透明细胞癌中的表达及意义。方法:通过Oncomine数据库检索关于ATP1A1的mRNA信息,采用The Human Protein Atlas分析ATP1A1蛋白在正常肾组织和肾透明细胞癌中表达情况,GEPIA网站中TCGA数据对ATP1A1低表达的肾透明细胞癌患者进行生存分析,Meth HC数据库分析ATP1A1甲基化水平和蛋白相互作用,利用String-DB数据分析ATP1A1与上下游蛋白的相互作用。结果:肾透明细胞癌(Clear cell Renal Cell Carcinoma,cc RCC)组织中ATP1A1的mRNA表达水平较正常对照组明显降低。免疫组化结果证实ATP1A1蛋白质表达变化与mRNA相似。TCGA数据中得出ATP1A1低表达患者的总体生存期明显短于高表达组患者。此外,ATP1A1基因启动子区在肾透明细胞癌中的甲基化水平明显高于正常肾组织中。同时,ATP1A1与ATP1B1、FXYD2、ATP1B2等蛋白可能存在相互作用。结论:大数据分析结果表明ATP1A1在肾透明细胞癌中低表达,并与其发生发展相关,可能作为其潜在的治疗靶点。     

Inhibition of insulin-like growth factor-I-mediated cell signaling by the von Hippel-Lindau gene product in renal cancer

Insulin-like growth factor-I (IGF-I)-mediated signaling is thought to be involved in the regulation of multiple cellular functions in different tumors including renal cell carcinoma (RCC). Blocking IGF-I signaling by any of the several strategies abolishes or delays the progression of a variety of tumors in animal models. Herein, we demonstrate that in RCC cell lines, IGF-I-mediated signaling is found to be inhibited in the presence of wild type von Hippel-Lindau (VHL) tumor suppresser gene. Moreover, molecular modeling and biochemical approaches have revealed that beta-domain of the VHL gene product by interacting directly with protein kinase Cdelta inhibits its association with IGF-IR for downstream signaling. We also demonstrated that RCC has IGF-I-mediated invasive activity where protein kinase Cdelta is an important downstream molecule, and this invasiveness can be blocked by wild type VHL. These experiments thus elucidate a novel tumor suppresser function of VHL with its unique kinase inhibitory domain.