Volker H. Haase - US grants

The von Hippel-Lindau (VHL) tumor suppressor gene plays a critical role in the pathogenesis of clear-cell renal carcinoma and CNS hemangioblastomas. The functional role of the VHL gene in cell growth, cell differentiation, kidney and brain development is not well understood. VHL-deficient mice generated by conventional gene targeting in embryonic stem (ES) cells die in utero at 10.5 to 12.5 days of gestation and therefore can not provide the mouse model needed for the analysis of the developmental and long term effects of VHL deficiency in kidney and brain. In this project a conditional gene targeting strategy based on the Cre/loxP system will be used to generate mice in which a temporally controlled and tissue-specific inactivation of VHL can be accomplished. Such a mouse model will permit the study of VHL function in specific tissues at specific times of development. The work described will be performed in the outstanding environment of the Whitehead Institute for Biomedical Research in the laboratory of Dr. Rudolf Jaenisch, a world-renowned expert in the field of mouse developmental biology and pioneer in transgenic and gene targeting technology. Collaborators include Dr. Vikas Sukhatme, Renal Division Chief at Beth Israel Deaconess Medical Center, who will provide reagents and technical expertise for the analysis of VHL-deficient mice in the later stages of the project, as well as Dr. Helmut G. Rennke, Associate Professor of Pathology at Brigham and Women's Hospital and internationally renowned expert in renal pathology who will help with histological analysis of VHL deficient mice. The PI is currently a second year renal fellow at Beth Israel Deaconess Medical Center and trainee in Dr. Jaenisch's laboratory as an integral part of his renal fellowship. He has three years of experience in basic molecular biology methods from his work as a Research Fellow at Massachusetts General Hospital. The new methodologies the applicant will learn include all aspects of cutting edge gene knockout technology, such as targeting vector design, genetic manipulations of ES cells in culture, genetic recombination mediated by Cre/loxP, and generation of mice by blastocyst injection, furthermore he will learn in situ-hybridization and advanced PCR techniques. He is absolutely committed to a career centered on basic research in an academic Nephrology division applying the techniques learned during the award period.

DESCRIPTION (provided by applicant): Patients with mutations in the von Hippel-Lindau gene (VHL) develop a pleomorphic familial tumor syndrome that is characterized by the development of highly vascularized tumors with different biological behaviors such as CNS hemangioblastomas and clear cell renal cell cancers (RCC) which are often preceded by renal cysts. Recently the VHL protein (pVHL) has been found to regulate the stability of hypoxia-inducible-factor (HIF). However not all aspects of this disease, such as the malignant phenotype of RCCs, can be easily explained by this interaction. A VHL mouse model would provide a powerful tool for the identification of molecular factors that are critical for the pathogenesis of the VHL phenotype. However, the development of such a model was hampered by the fact that conventional VHL knockout mice died during midgestation from placental failure, which initially precluded a functional analysis of this gene in the adult. In order to overcome this problem with embryonic lethality, the Principal Investigator has generated mice that allow tissue-specific deletions of Vhl in the adult through Cre/loxP-mediated recombination. Several conditional mouse mutants have been generated. Inactivation of Vhl in the liver for example leads to the accumulation of neutral lipids in hepatocytes, proliferation of endothelial cells, as well as erythrocytosis, all of which is consistent with pVHL's role in the regulation of HIF. At the moment it remains unclear whether the phenotypes that were observed in conditional VHL knockout mice can be solely attributed to the up-regulation of HIF-1 dependent target genes and whether pVHL controlled molecular pathways exist that are not regulated by HIF. The studies proposed here will address these questions and aim at defining the critical molecular mediators of the VHL phenotype by generating several tissue-specific conditional mouse mutants that are double deficient in Vhl and Hif-1. The molecular analysis of these mutants with microarrays might provide important insights into the regulation of growth and apoptosis of renal tubule cells. The proposed investigations represent a continuation and expansion of research that was initially described in the applicant's K08 and is based on data that were generated under this grant. They will be carried out in the laboratory of Dr. Volker H. Haase, who is an Assistant Professor of Medicine in the University of Pennsylvania's Renal Division. Dr. Michael Madaio, Professor of Medicine, will function as Dr. Haase's mentor for this grant.

DESCRIPTION (provided by applicant): Clear cell carcinoma of the kidney (RCC), the most common form of kidney cancer, is associated with the inactivation of the von HippeI-Lindau (VHL) tumor suppressor. Mutations in the VHL gene can be found in approximately 70% of sporadic RCCs. One of the major functions of the VHL gene product, pVHL, is the targeting of the oxygen sensitive alpha-subunit of hypoxia-inducible factor (HIF) for ubiquitination and subsequent proteasomal degradation. Inactivation of VHL is felt to be an early event during RCC tumorigenesis and results in constitutive expression of two major HIF isoforms, HIF-1 and HIF-2. This results in increased transcription of genes that regulate glycolysis, angiogenesis and erythropoiesis. HIF-1 has furthermore been shown to up-regulate factors that promote growth arrest and apoptosis. The role of increased HIF expression in VHL associated tumorigenesis remains controversial. The hypothesis that the ratio of HIF-1 to HIF-2 levels is important for VHL associated renal tumor development will be investigated. Conditional gene targeting technology based on Cre-loxP mediated recombination as well as targeted transgenesis will be used to manipulate the expression levels of both pVHL and HIF in vivo and in vitro. This system enables the study of the functional relationship between VHL deficiency and HIF activation in regard to renal cell growth and viability in primary renal epithelial cells of different histogenetic origin. Specifically, the proposed investigations will examine the effects of increased or absent HIF-1 and increased HIF-2 expression in VHL deficient and wild type backgrounds. Studies will be performed in primary cell culture and in vivo with kidney specific conditional knock out mice. Gene expression studies will provide information regarding differential HIF-1 and HIF-2 target gene expression in different nephron segments with a special emphasis on genes involved in the regulation of cell survival. Taken all together, the proposed in vivo and in vitro studies will not only provide novel insights into the early events of renal oncogenesis and the histogenetic origin of RCC, but also examine fundamental aspects of HIF-1 and HIF-2 function in different nephron segments. Ultimately they have the potential to create a murine model of VHL associated renal tumors.

[unreadable] DESCRIPTION (provided by applicant): Cellular hypoxia is not only a common clinical problem which occurs, for example, as a result of acute and chronic pulmonary and cardiovascular conditions, but is also an important micro-environmental factor that influences normal embryonic development. Hepatocytes, like other mammalian cell types, are critically dependent on adequate oxygen tissue levels for development, survival and normal function. Hypoxia Inducible Factor-1 and -2 (HIF-1 and HIF-2) are heterodimeric basic-loop-helix transcription factors and are key mediators of cellular adaptation to diminished oxygen supply. Previously we have shown that increased expression of HIF in hepatocytes results in the development of cavernous hemangiomas, polycythemia and hepatocellular steatosis. We hypothesize that HIF-2 is the key regulator in the hypoxic induction of liver erythropoietin and angiogenic gene expression and plays a key role in liver lip genesis. We furthermore hypothesize that glycolytic gene expression in hepatocytes is mainly controlled by HIF-1. Studies are proposed that investigate the specific roles of HIF-1 and HIF-2 in the regulation of angiogenic and metabolic gene expression by use of transgenic and compound conditional knock-out mice that either lack or over- express HIF-1 or HIF-2 as a result of VHL gene deletion. We also propose studies that aim at the identification of novel HIF-2 specific targets using a combination of genome scale expression profiling and promoter microarrays. Our investigations will help to understand the unique roles of hypoxic signaling through HIF-1 and HIF-2 with regard to liver angiogenesis, hepatic glucose and fat metabolism, as well as the regulation of liver EPO. The data generated from this grant application will be used as basis for a five year NIH grant application that investigates HIF-dependent and HIF-independent hypoxic signaling in the context of liver disease. [unreadable] [unreadable] [unreadable]