Shukla, D.H.; (2011) Manipulation of the VHL/HIF pathway in mouse kidney epithelia and pancreatic β-cells. Doctoral thesis , UCL (University College London).

Abstract

Biallelic inactivation of the von Hippel-Lindau tumour suppressor gene (VHL) underlies both, sporadic clear cell renal cell carcinoma (ccRCC) that arises as part of an inherited multi-cancer VHL syndrome. Pancreatic tumours also occur in VHL disease and are thought to be of neuroendocrine origin. The most extensively studied role of VHL is hypoxia-inducible-factor (HIF) regulation. The HIF pathway is known operates in all cell types examined to date and plays a central role in many physiological responses to oxygen concentration. This thesis investigates the role of the Vhlh tumour suppressor protein in the kidney and pancreatic β-cells of mice. Cell-specific inactivation of Vhlh in mice was obtained using the Cre/loxP system. Analysis from mice constitutively expressing an active form of the HIF-2α transgene (KsptmHIF2α) was also performed. Furthermore, I have also used a doxycycline controlled Cre transgene system to allow temporal control of Vhlh ablation in the kidney. The main results arising from this thesis are: Vhlh inactivation in the distal tubular segment of the nephron led to an increase in both HIF isoforms (HIF-1α and HIF-2α) accompanied with upregulation of downstream gene expression. Constitutive expression of HIF-2α in the distal tubular segment upregulated downstream gene expression but did not lead to cyst or tumour formation; interestingly, inactivating another tumour suppressor gene, fumarate hydratase (Fh1) in the mouse kidney, resulted in cyst formation accompanied by HIF activation. Taken together, these findings suggest that HIF is not solely responsible for cyst formation in the Fh1 knockout mice, as cysts were not seen with the Vhlh knockout mice. The other major finding arising from this thesis is that, intact Vhlh is necessary for glucosestimulated- insulin secretion. I show that this is mediated via HIF transactivation and involves a glucose transporter switch, from glucose transporter–2 (GLUT-2) to glucose transporter–1 (GLUT-1). In addition, I have found that the expression of microRNA is altered in a murine pancreatic β-cell line, MIN6 in response to hypoxia. Therefore, the work arising from this thesis provides significant insights into understanding how the VHL/HIF pathway can be used to identify early morphological alterations, leading to a more aggressive phenotype or tumourigenesis. This pathway also plays a crucial role in glucose homeostasis, and interference in this pathway causes defects in β-cell function and insulin secretion.

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