Functional analysis of the von Hippel-Lindau tumour suppressor in mice

Abstract

Von Hippel-Lindau (VHL) disease is a dominantly inherited cancer syndrome characterized by the development of multiple tumours, among which the most common are tumours of blood vessels called hemangioblastomas (HB) that can be found in the retina and the central nervous system (CNS). The disease is caused by germline mutations in the VHL tumour suppressor gene and tumour development is linked to somatic inactivation of the remaining wild-type allele. The best documented role of pVHL is as the substrate recognition component of a Skp1/cullin/F-box (SCF)-like E3 ubiquitin protein ligase complex that ubiquitinates the oxygen-sensitive α-subunit of hypoxia inducible factors HIF-1 and HIF-2 under normoxia and targets them for proteasomal degradation, thereby inactivating HIF. Loss of VHL function leads to the constitutive activation of HIF and subsequent up-regulation of hypoxia-inducible mRNAs encoding angiogenic growth factors such as vascular endothelial growth factor (VEGF), Erythropoietin (EPO) and glucose transporter 1 (GLUT1). Up-regulation of these factors characterizes VHL-associated tumours at the molecular level. VHL gene expression studies on human fetal and adult tissues showed VHL mRNA to be ubiquitously detectable. Expression was not restricted to specific areas known to undergo abnormal differentiation as part of the VHL syndrome such as kidney, cerebellum and pancreas, but it was also present, among others, in the heart, lung and prostate. In addition VHL gene expression was evident in all derivatives of the three germ cell layers also during mouse embryogenesis, being most prominent in epithelial components of the lung, kidney and eye. Expression studies of the VHL protein (pVHL) utilizing poly- and monoclonal antibodies against human VHL revealed wide cytoplasmic expression in human adult tissues. However, little is known about VHL expression patterns during development and in particular, the detailed distribution of VHL within specific tissues. To investigate pVHL expression during murine development and adulthood a mouse pVHL-specific antibody was raised and utilized in a detailed immunohistochemical study focusing on the development of two tissues that play a very important role in the course of VHL disease, namely the retina and cerebellum. Studying VHL disease, as any other disease, in humans is a difficult task that can be circumvented by the usage of genetically engineered mice that phenotypically mimic the disease. The complete knockout of VHL unfortunately didn’t provide a mouse model in which to investigate molecular pathology, gene-gene or protein-tissue interactions or even therapeutic intervention as the animals died early during gestation. In an attempt to create a mouse model that circumvented the block encountered in the complete knockout, we used Cre/loxP technology to design a strategy for creating a conditional VHL knockout, i.e. mice having exon 1 flanked by loxP sites (floxed). As our intent was not crowned with success and the floxed VHL mice were published in the meantime by another laboratory we could benefit by using these mice to specifically knock-out VHL in tissues we found to be interesting due to expression studies that had been undertaken as part of this thesis. In the thesis presented herein the expression of pVHL in the retina and the cerebellum is described and the potential value of localizing VHL to previously unidentified cells is discussed (chapter 8). Moreover, an outline of an unsuccessful endeavour to create a conditional knockout is provided (chapter 7). Nevertheless, given the availability of such mice from a different laboratory, we undertook a cell-specific deletion approach to substantiate our immunohistochemical observations in vivo as presented in chapter 9.