Inactivation of the Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Suppresses von Hippel-Lindau Disease-Associated Vascular Tumors in Mice

Abstract

Germ line mutations in the VHL tumor suppressor gene result in von Hippel-Lindau (VHL) disease, a familial tumor syndrome that predisposes affected patients to the development of highly vascularized neoplasms. These include hemangioblastomas of the retina and central nervous system (CNS), renal-cell carcinomas (RCC) of the clear-cell type, and endocrine and exocrine pancreatic tumors, as well as pheochromocytomas (34). In addition, VHL has also been found to be inactivated in the majority of sporadic RCC (8, 13).VHL deficiency leads to constitutive activation of hypoxiainducible factor (HIF) and increased expression of its target genes irrespective of the oxygen concentration (38). The VHL gene product (pVHL), together with elongins B and C (6, 25), Cullin-2 (43), and Rbx1 (23), forms an E3 ubiquitin ligase (19), which targets the hydroxylated, oxygen-sensitive ␣ subunits of HIF-1, -2, and -3 for ubiquitination and subsequent degradation by the 26S proteasome (18,20,39). As a normal physiological response to hypoxia, HIF-1 and HIF-2 facilitate both oxygen delivery and adaptation to oxygen deprivation by regulating genes that are involved in glucose uptake and metabolism, angiogenesis, erythropoiesis, cell proliferation, and apoptosis (50, 62). HIFs belong to the PAS (Per-Arnt-Sim) family of basic helix-loop-helix (bHLH) transcription factors that bind to DNA as heterodimers composed of an oxygensensitive ␣ subunit and a constitutively expressed ␤ subunit, also known as the arylhydrocarbon receptor nuclear translocator (ARNT). ARNT is the general binding partner for the bHLH/PAS domain-containing proteins. In addition to forming heterocomplexes with HIF, ARNT also heterodimerizes with single minded (SIM), which is involved in neural development, and with the arylhydrocarbon receptor (AhR), which is involved in the xenobiotic response to environmental toxins (for a review, see reference 24).The expression patterns of the HIF subunits differ within embryonic and adult tissues. In the adult, Hif-1␣ mRNA is ubiquitously expressed, and Hif-1␣ protein can be detected at baseline levels within multiple cell types in various tissues under normoxia and is significantly enhanced under conditions of hypoxia (55). In contrast, while Hif-2␣ mRNA expression has been detected within many tissues, Hif-2␣ protein has been found to be restricted to specific cell types within various tissues. In addition to being expressed in endothelial cells, Hif-2␣ is also expressed in glial cells of the brain, type II pneumocytes of the lung, cardiomyocytes, fibroblasts of the kidney, intersitial cells of the pancreas and duodenum, and hepatocytes (26,63). Arnt expression is ubiquitous, and it seems to be the only Hif-␤ subunit present in the liver (21).pVHL appears to have multiple functions besides regulating HIF, and its contributions to the development of VHL-associated tumors are presently subject to intense investigations. As a result of these efforts, it has been shown that pVHL plays an important role in fibronectin extracellular-matrix...

The VHL tumor suppressor and HIF: insights from genetic studies in mice

“…131 Whatever the underlying mechanisms, clinical studies have also demonstrated a correlation between HIF-2a expression and the development of VHL-associated angiogenic lesions, 132 suggesting that pharmacological targeting of HIF-2 may by an effective therapy for the treatment of these tumors. pVHL-and pVHL/ARNT-deficient liver tissues are histologically similar and direct comparison of gene expression changes has not revealed major differences 31 (also VH Haase et al, unpublished observation), suggesting that the effects of pVHL gene deletion in the liver are largely HIF mediated. Indeed, forced expression of non-degradable HIF-1a and HIF-2a phenocopied the VHL phenotype in the liver and skin 133 (Table 2a).…”

“…Renal cysts at low frequency; inactivation of ARNT, but not HIF-1a suppressed the development of renal cysts Liver Haase et al 76 Rankin et al 31 Rankin et al 32 …”

“…For example, genes encoding glycolytic enzymes appear to be predominantly controlled by HIF-1, 29 whereas HIF-2 appears to be the main regulator of VEGF and EPO in tissues that express both HIF-1 and HIF-2. [30][31][32][33] Target gene preference may be the result of tissue-specific interactions with other nuclear factors, differential interactions with transcriptional cofactors or a reflection of tissue-and celltype-dependent differences in the ratios of HIF-a protein levels (for a review on this topic see Wenger et al 28 ).…”

Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response: a role for IL-6

“…per ml) were incubated with 30 mg of proteins for 20 min at room temperature in binding buffer, and the complex were separated in a 6% electrophoretic gel at 200 V for 2 h, as previously described. 22 The supershift analysis was performed by incubating the protein extracts with 5 mg of anti-HIF-1a and of anti-HIF-2a (Novus Biologicals) 30 min before the reaction with binding buffer.…”

Vascular biology and pathobiology of the liver: Report of a single-topic symposium

“…For example, whereas HIF target genes involved in glycolysis, such as PGK, are regulated by the HIF-1 isoform, the hypoxic induction of VEGF and liver angiogenesis appear to be predominantly dependent on the HIF-2 isoform. [58][59][60][61] Thus, targeting HIF could be an important pathway by which to regulate VEGF and angiogenesis in liver.…”