Clusterin Is a Secreted Marker for a Hypoxia-Inducible Factor-Independent Function of the von Hippel-Lindau Tumor Suppressor Protein

Abstract

Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene predispose people to renal cancer, hemangioblastomas, and pheochromocytomas in an allele-specific manner. The best documented function of the VHL gene product (pVHL) relates to its ability to polyubiquitinate, and hence target for destruction, the ␣ subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF). pVHL mutants linked to familial pheochromocyctoma (type 2C VHL disease), in contrast to classical VHL disease, appear to be normal with respect to HIF regulation. Using a simple method for identifying proteins that are differentially secreted by isogenic cell line pairs, we confirmed that the HIF targets IGBP3 and PAI-1 are overproduced by pVHLdefective renal carcinoma cells. In addition, cells lacking wild-type pVHL, including cells producing type 2C pVHL mutants, were defective with respect to expression and secretion of clusterin, which does not behave like a HIF target. Decreased clusterin secretion by pVHL-defective tumors was confirmed in vivo by immunohistochemistry. Therefore, clusterin is a secreted marker for a HIF-independent pVHL function that might be especially important in pheochromocytoma development. von Hippel-Lindau disease, caused by heterozygous germline inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, presents clinically as a hereditary cancer syndrome.1,2 Tumor development in this setting is due to somatic inactivation of the remaining wild-type VHL allele in a susceptible cell.3 The classical tumors observed in VHL disease are retinal and central nervous system (usually within the cerebellum or spinal cord) blood vessel proliferations called hemangioblastomas. These tumors, although benign, cause significant morbidity and mortality because of mass effect. Some VHL families also exhibit an increased risk of clear cell renal cell carcinoma or pheochromocytoma. In keeping with the Knudson 2-hit model, many sporadic hemangioblastomas and renal cell carcinomas are also due to VHL inactivation, as a result of either somatic mutations or hypermethylation.3,4 For reasons that are still unclear, somatic VHL mutations are rare in sporadic pheochromocytomas absent an occult germline VHL mutation, despite the fact that certain germline VHL mutations confer an increased risk of this tumor.Genotype-phenotype correlations have emerged in VHL disease. VHL families can be subdivided into type 1 (low risk of pheochromocytoma) and type 2 (high risk of pheochromocytoma). Type 2 disease can be subdivided into type 2A (low risk of renal cell carcinoma) and type 2B (high risk of renal cell carcinoma).1 Some VHL families exhibit an increased risk of pheochromocytoma without Supported in part by grants from the National Institutes of Health (to W.G.K.), the Kurozumi Medical Foundation (to E.N.), the Japanese Clinical Oncology Fund (to E.N.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (to E.N. and O.O.), and the Murray Foundation (to W.G.K.

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“…As protein-rich FCS could easily mask proteins of interest in this analysis, we used serum-free medium as described previously (11,12) and excluded contamination as far as possible by removing chromatogram fractions with peaks originating from FCS. As shown in Fig.…”

“…Recently, we established a new method for analyzing the proteins in cell culture supernatants by using serum-free medium. Using this method, we identified clusterin as a marker for the hypoxia-inducible factor -independent function of von Hippel-Lindau protein and revealed its role in the development of familial pheochromocytoma (11,12). In these studies, secreted proteins in the serum-free medium were precipitated with trichloroacetic acid and analyzed by two-dimensional PAGE.…”