Type 2B von Hippel-Lindau disease: molecular biology, tumor growth, and development

Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma, and pheochromocytoma. Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-α regulation. Interaction analysis in VHL –transgenic murine embryonic stem (ES) and renal cell carcionoma-derived cell lines supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF-α, most likely via a remnant complex containing ROC1 and Cullin-2 and reduced or absent Elongin C. Murine embryonic stem (ES) cells in which the endogenous wild-type Vhl gene was replaced with the representative Type 2B VHL hotspot mutation R167Q (Vhl2B/2B) likewise displayed preserved physiologic regulation of both HIF factors with slightly more normoxic dysregulation of HIF-2α. Differentiated Vhl2B/2B-derived teratomas overexpressed the joint HIF targets Vegf and EglN3 but not the HIF-1α-specific target Pfk1 and displayed a growth advantage over Vhl-/--derived teratomas, suggestive of a tight connection between perturbations in the degree and ratio of HIF-1α and HIF-2α stabilization and cell growth. Vhl2B/2B mice displayed mid-gestational embryonic lethality, while adult Vhl2B/+ mice exhibited susceptibility to carcinogenpromoted renal neoplasia compared with wild-type littermates at twelve months. Our experiments support a model in which the representative Type 2B R167Q mutant pVhl retains intermediate HIF-α suppression via formation of a remnant ubiquitin ligase complex, thereby producing a unique profile of HIF-α dysregulation with tissue-specific effects on cell growth, development, and tumor predisposition

VHL Type 2B Mutations Retain VBC Complex Form and Function

Background: von Hippel-Lindau disease is characterized by a spectrum of hypervascular tumors, including renal cell carcinoma, hemangioblastoma, and pheochromocytoma, which occur with VHL genotype-specific differences in penetrance. VHL loss causes a failure to regulate the hypoxia inducible factors (HIF-1a and HIF-2a), resulting in accumulation of both factors to high levels. Although HIF dysregulation is critical to VHL disease-associated renal tumorigenesis, increasing evidence points toward gradations of HIF dysregulation contributing to the degree of predisposition to renal cell carcinoma and other manifestations of the disease. Methodology/Principal Findings: This investigation examined the ability of disease-specific VHL missense mutations to support the assembly of the VBC complex and to promote the ubiquitylation of HIF. Our interaction analysis supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF. We additionally demonstrated that Type 2B mutant pVHL forms a remnant VBC complex containing the active members ROC1 and Cullin-2 which retains the ability to ubiquitylate HIF-1a. Conclusions: Our results suggest that subtypes of VHL mutations support an intermediate level of HIF regulation via a remnant VBC complex. These findings provide a mechanism for the graded HIF dysregulation and genetic predisposition fo

Burnt and Blossoming: Material Mysticism in Trilogy and Four Quartets

This paper brings two WWII poems into dialogue: H.D.'s Trilogy and Eliot's Four Quartets. Both poems express a creative response to the destruction of war. My reading of Trilogy suggests a material mysticism in which vision and renewal are situated within the natural world, rituals and bodily experience. Bringing this understanding of mysticism to bear on Four Quartets reveals tension between transcendence and materiality. For Eliot, redemption comes through time and location, while for H.D., redemption lies within material particularity. Four Quartets oscillates between an apophatic discourse that seeks to transcend desire and history and an emphasis on material particularities

VHL disease associated mutations demonstrate a graded amount of HIF Regulation.

<p>A. Anti-HA immunoblot for expression of HA-tagged human pVHL in transgenic 786-0 clones. Whole-cell protein extracts were prepared from 786-0 clones deficient for <i>VHL</i> expression (Vector) or modestly expressing wild-type (WT) or missense (Y112H, R167Q) mutant HA-tagged human pVHL. B. Anti-HIF-2α immunoblot for HIF stabilization in 786-0 clones. Whole-cell protein extracts were prepared from <i>VHL</i>-deficient 786-0 cells or WT or mutant HA-pVHL-rescued 786-0 cells incubated in the presence or absence of the hypoxia mimetic CoCl₂ for 24 hours. Ku80 immunoblot was used as a control for equal loading.</p

Reverse co-immunoprecipitation confirms CUL2-pVHL protein interaction in R167Q mutation.

<p>Anti-myc immunoprecipitation of myc-CUL2 and associated HA-pVHL in 786-0 clones. Stable 786-0 clones expressing vector-only (ST) or WT or mutant HA-pVHL were transiently transfected with wild-type myc-tagged CUL2 and subjected to myc-IP. Upper panel, IP of Myc-tagged Cul2 detected by anti-CUL2 immunoblot. Lower panel, co-IP of WT and mutant HA-pVHL detected by anti-HA immunoblot.</p

Co-immunoprecipitation of VBC complex proteins with type 2B mutant pVHL.

<p>A. Anti-HA immunoprecipitation of HA-pVHL and associated VBC complex members in transgenic ES cell clones. Anti-HA IP products were probed for successful pull-down of WT or mutant HA-pVHL and for co-IP of the indicated VBC complex members in stably-transfected <i>Vhl^−/−</i> murine ES cells. B. and C. Anti-HA immunoprecipitation of HA-pVHL from transgenic 786-0 clones. Anti-HA IP products were probed for successful pull-down of WT or mutant HA-pVHL by anti-pVHL (B) or anti-HA (C) immunoblot and for co-IP of the indicated VBC complex members in stably-transfected 786-0 cells.</p

pVHL-dependent HIF-1α ubiquitylation in 786-0 cell lines.

<p>Anti-myc immunoblot to visualize migration shift of myc-tagged HIF-1α <i>in vitro</i>. Cell extracts from 786-0 clones expressing vector alone or WT or mutant HA-pVHL were incubated in the presence or absence of ubiquitylation reaction solution and TNT HIF-1α-myc and subjected to anti-myc immunoblot. A band representing unmodified TNT HIF-1α-myc, as depicted in the TNT lane, runs just below the ladder marker at 113.9 kDa. Upward shift of myc-HIF-1α, as seen in complete reaction lanes for WT and R167Q, indicates poly-ubiquitylation. The TNT HIF-1α-myc migration pattern for each complete reaction lane is graphically summarized to the right of the immunoblot.</p