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

Abstract

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.Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene predispose individuals to the development of tumors characteristic of VHL disease, including retinal angiomas, hemangioblastomas, pheochromocytomas, and renal cell carcinomas (6,12,20,21,23). In humans, the product of the VHL gene is a 213-amino-acid protein that is localized primarily in the cytoplasm (31). Although highly conserved across species, pVHL bears little similarity to other known proteins. Most studies to elucidate its function have focused on analysis of the interaction of pVHL with other cellular proteins. Thus, pVHL was found to interact with elongins B and C, the regulatory subunits of the elongin-SIII complex. Binding of elongins B and C to elongin A, the catalytic subunit, is necessary for elongin-SIII function and leads to enhanced transcriptional activity by RNA polymerase II (1); binding of pVHL to elongins B and C results in inhibition of elongin function and decreased RNA polymerase II activity in vitro (7,26,29). Elongin A and pVHL have not been found in the same complex, suggesting that their association with elongins B and C may be mutually exclusive. More recently, another pVHLinteracting protein, Hs-CUL-2 was identified; yeast Cul2 (also termed cdc53) is found in multiprotein complexes also, including Skp1 and cdc34, which have been proposed to participate in targeting specific proteins for ubiquitin-mediated proteolysis (24,28,38,44). Based on the associatio...

Up-regulation of hypoxia-inducible factors HIF-1α and HIF-2α under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function

“…Maxwell et al (1999) proposed a model in which VHL forms complexes with the HIF-a subunits under normal cellular O 2 tension, thereby targeting the HIF-a subunits for destruction. In addition, ubiquitination of cellular proteins in VHL de®cient cells was elevated after glucose deprivation supporting an involvement of VHL in ubiquitin dependent degradation of proteins (Gorospe et al, 1999). Recent ®ndings point to an involvement of the HIF-1 complex in tumor angiogenesis (Maxwell et al, 1997;Carmeliet et al, 1998).…”

The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway

“…This suggests that TRC8 may be somewhat analogous to the Hrd1/der3p protein complex in the ER, perhaps targeting a subset of proteins for degradation in a VHL-dependent manner. Interestingly, Gorospe et al (1999) reported that VHL mutant RCC cell lines were more sensitive to agents that a ect protein folding (and the unfolded protein response) than wild type VHL cells. Thus, TRC8 might conceivably be involved in this aspect of VHL function.…”

Serial analysis of gene expression in renal carcinoma cells reveals VHL-dependent sensitivity to TNFα cytotoxicity

“…Each pair was composed of one cell line with null VHL status (parental) and one expressing wild-type VHL (wtVHL) (described in Gorospe et al, 1999). Cultures were exponentially growing, with a density at the time of collection of approximately 60 ± 70%.…”

“…Matched pairs of the human RCC lines 786 ± 0, UMRC6 and UOK-121 were obtained that either expressed no functional VHL (parental) or expressed a wild-type VHL (wtVHL) allele through stable transfection; both 786 ± 0 parental and UOK-121 parental cells were transfected with corresponding empty vectors and selected accordingly, while UMRC6 parental cells did not carry an empty vector (Gorospe et al, 1999). All cells were cultured in Dulbecco's modi®ed essential medium (Gibco-BRL) supplemented with 10% fetal bovine serum.…”