VHL Deficiency Research Articles

Von Hippel-Lindau–Coupled and Transcription-Coupled Nucleotide Excision Repair–Dependent Degradation of RNA Polymerase II in Response to Trabectedin

Ecteinascidin 743 (Et743; trabectedin, Yondelis) has recently been approved in Europe for the treatment of soft tissue sarcomas and is undergoing clinical trials for other solid tumors. Et743 selectively targets cells proficient for TC-NER, which sets it apart from other DNA alkylating agents. In the present study, we examined the effects of Et743 on RNA Pol II. We report that Et743 induces the rapid and massive degradation of transcribing Pol II in various cancer cell lines and normal fibroblasts. Pol II degradation was abrogated by the proteasome inhibitor MG132 and was dependent on TC-NER. Cockayne syndrome (CS) cells and xeroderma pigmentosum (XP) cells (XPD, XPA, XPG, and XPF) were defective in Pol II degradation, whereas XPC cells whose defect is limited to global genome NER in nontranscribing regions were proficient for Pol II degradation. Complementation of the CSB and XPD cells restored Pol II degradation. We also show that cells defective for the VHL complex were defective in Pol II degradation and that complementation of those cells restores Pol II degradation. Moreover, VHL deficiency rendered cells resistant to Et743-induced cell death, a similar effect to that of TC-NER deficiency. These results suggest that both TC-NER-induced and VHL-mediated Pol II degradation play a role in cell killing by Et743.

Loss of PL6 protein expression in renal clear cell carcinomas and other VHL‐deficient tumours

Mutations in the von Hippel-Lindau tumour suppressor gene (VHL) cause the VHL hereditary cancer syndrome and occur in most sporadic clear cell renal cell cancers (CC-RCCs). The mechanisms by which VHL loss of function promotes tumour development in the kidney are not fully elucidated. Here, we analyse expression of PL6, one of the potential tumour suppressor genes from the critical 3p21.3 region involved in multiple common cancers. We classify PL6 as a Golgi-resident protein based on its perinuclear co-localization with GPP130 in all cells and tissues analysed. We show that PL6 RNA and protein expression is completely or partially lost in all analysed CC-RCCs and other VHL-deficient tumours studied, including the early precancerous lesions in VHL disease. The restoration of VHL function in vitro in the VHL-deficient CC-RCC cell lines was found to reinstate PL6 expression, thus establishing a direct link between VHL and PL6. Insensitivity of PL6 to hypoxia suggested that PL6 is regulated by VHL via a HIF-1-independent pathway. We ruled out mutations and promoter methylation as possible causes of PL6 down-regulation in CC-RCC. We hypothesize that loss of a putative PL6 secretory function due to VHL deficiency is an early and important event that may promote tumour initiation and growth.

Hypoxia and podocyte-specificVhlhdeletion confer risk of glomerular disease

Hypoxia is a potent regulator of a multitude of cellular processes, including metabolism and cell survival. The transcriptional response to oxygen deprivation is mainly mediated by hypoxia-inducible factors (HIFs), which are targeted for proteasomal degradation by the von Hippel-Lindau tumor suppressor protein (pVHL) under normoxia. Podocytes, as part of the glomerular filtration barrier, are prone to hypoxic injury during diseases affecting the glomerulus. VHL and HIF1 were functional in mature murine podocytes in vivo and in vitro, with HIF1 protein stabilization and target gene transcription under both hypoxia and VHL deficiency. Podocyte-specific Vhlh gene loss, mimicking podocyte hypoxia, in young mice of mixed background led to glomerulomegaly and occasional glomerulosclerosis, despite preserved glomerular development. In parallel, hypoxia effects on podocytes in cell culture included increased susceptibility to apoptosis, associated with nuclear translocation of apoptosis-inducing factor (AIF). Similarly, Vhlh gene inactivation in podocytes in vitro resulted in a significant survival disadvantage, particularly in conjunction with additional proapoptotic stimuli. Evaluation of the global transcriptional response to hypoxia in podocytes by microarray analysis revealed a typical upregulation of HIF target genes as well as the induction of genes relevant for stress response, cell-cell, and cell-extracellular matrix interaction. While the lack of a prominent phenotype in young mice with VHL-deficient podocytes is consistent with the absence of specific glomerular manifestations in human VHL disease, a low-oxygen environment of podocytes may contribute to the progression of glomerular disease by altering cellular metabolism and survival.

Hypoxic repression of STAT1 and its downstream genes by a pVHL/HIF-1 target DEC1/STRA13

DEC1/STRA13 is a bHLH type transcriptional regulator involved with immune regulation, hypoxia response and carcinogenesis. We recently demonstrated that STRA13 interacts with STAT3 in the transcriptional activation of STAT-dependent promoters. Here, we pursue STRA13 involvement in the JAK/STAT pathway by studying its role in STAT1 expression. First, we showed that VHL deficiency or HIF-1 activation resulted in the repression of endogenous STAT1 mediated by STRA13. We then characterized the STAT1 proximal promoter to assess its response to STRA13 by transient coexpression in a luciferase reporter assay. Using sequential truncation and site-directed mutagenesis of the STAT1 promoter with STRA13 deletion constructs, we showed that the STRA13 C-terminal trans-activation domain, which is known to bind HDAC1, mostly determines the repressive activity. Involvement of HDAC activity in STAT1 regulation was validated by TSA inhibition and chromatin immunoprecipitation (ChIP) assay. Thus, we demonstrate that STRA13-mediated repression of STAT1 transcription utilizes an HDAC1-dependent mechanism. Furthermore, we show that targets of unphosphorylated STAT1, such as antigen presenting genes and CASP1, are also repressed by hypoxia possibly through the same STRA13-mediated mechanism. Thus, the newly discovered link between HIF-1 and STAT1 reveals a previously unknown role of STRA13 in hypoxia and carcinogenesis.

Different subtypes of pseudohypoparathyroidism in the same family with an unusual psychiatric presentation of the index case.

A 13 year old Asian girl presenting with apparent hysterical paralysis and subsequent rapid cycling bipolar mood disorder was found to have biochemical evidence of pseudohypoparathyroidism type II. The mood disorder responded to treatment of the pseudohypoparathyroidism with a vitamin D analogue. Investigation of her parents and siblings showed phenotypes consistent with two distinct types of pseudohypoparathyroidism (type I and type II) in different family members.