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The von Hippel-Lindau tumor suppressor pVHL regulates the stability of Hypoxia-Inducible Factors (HIF) -1 and –2, oxygen-sensitive basic helix-loop-helix transcription factors, which mediate the hypoxic induction of angiogenic growth factors such as vascular endothelial growth factor (VEGF). Loss of VHL function results in constitutive activation of HIF-1 and HIF-2 and is associated with the development of highly vascularized tumors in multiple organs. We have used a conditional gene targeting approach to investigate the relative contributions of HIF-1 and HIF-2 to VHL-associated vascular tumorigenesis in a mouse model of liver hemangiomas. Here we demonstrate genetically that conditional inactivation of HIF-2α suppressed the development of VHL-associated liver hemangiomas and that angiogenic gene expression in hepatocytes is predominantly regulated by HIF-2 and not by HIF-1. These findings suggest that HIF-2 is the dominant HIF in the pathogenesis of VHL-associated vascular tumors and that pharmacologic targeting of HIF-2 may be an effective strategy for their treatment.
Patients with germ-line mutations in the von Hippel-Lindau tumor suppressor, pVHL, develop a familial tumor syndrome characterized by the development of highly vascularized tumors. The most common clinical manifestation of VHL disease are hemangioblastomas, which typically develop in the CNS and retina, but can also manifest in other organs such as the liver (Maher & Kaelin, 1997; McGrath et al., 1992). Although not malignant, hemangioblastomas are clinically devastating vascular tumors and consist of pericytes, endothelial, stromal, and mast cells. They are thought to originate from pVHL-deficient stromal cells, which express high levels of vascular endothelial growth factor (VEGF) and the transcription factor HIF-2α (Flamme et al., 1998; Vortmeyer et al., 1997) resulting in the proliferation of neighboring endothelial cells. pVHL is the substrate recognition component of an E3-ubiquitin ligase, which targets the oxygen-sensitive α-subunit of Hypoxia-Inducible Factors (HIF) for ubiquitination and proteasomal degradation under normoxia and thus plays a critical role in the regulation of molecular oxygen sensing. Loss of pVHL function results in constitutive activation of HIF-1 and HIF-2, whose individual contributions to VHL-associated tumorigenesis are currently under intense investigation (Kapitsinou & Haase, 2008).
VHL-associated vascular tumorigenesis can be modeled in mice by conditional inactivation of Vhlh (murine VHL) in hepatocytes, resulting in constitutive activation of Hif-1 and Hif-2, and the development of cavernous liver hemangiomas (Haase et al., 2001; Rankin et al., 2005). Although VHL-associated liver hemangiomas and CNS hemangioblastomas are distinct with regard to their cellular origin, they share common histological features, such as endothelial cell proliferation, angiectasis and lipid droplet accumulation in neoplastic cells (Haase et al., 2001). We have used this mouse model as a genetic tool to dissect the relative contributions of individual HIF transcription factors to VHL-associated vascular tumor development. We have previously shown that liver hemangioma formation does not require HIF-1α, but is dependent on the HIF-β subunit, also known as the Arylhydrocarbon receptor nuclear translocator (ARNT), suggesting that HIF-2α may play an essential role in VHL-associated vascular tumorigenesis (Rankin et al., 2005).
To investigate the role of HIF-2 in this process, we have used Cre-loxP recombination based gene targeting to inactivate Hif-2α in Vhlh-deficient mice that are prone to the development of cavernous liver hemangiomas (Rankin et al., 2005). In this model, Vhlh is targeted in approximately 20 to 30% of hepatocytes by Cre-recombinase under control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter (Rankin et al., 2005). Recombination of Vhlh and Hif-2α conditional alleles occurred with similar efficiency in PEPCK-Cre expressing mice that were homozygous for both alleles, which from hereon are referred to as PEPCK-Vhlh/Hif-2α mutants (Figure 1A).
Cavernous hemangiomas were observed in ~35% of PEPCK-Vhlh mutants 6 months of age or older (4/11), while ~80% of mutants developed microscopic vascular lesions (9/11), which were characterized by angiectasis, proliferating endothelial cells and the presence of lipid droplet containing hepatocytes (Figure 1B and C, and Rankin et al., 2005). Similarly, 5 of 10 PEPCK-Vhlh/Hif-1α mutant mice developed cavernous hemangiomas and 7 of 10 developed microscopic vascular lesions that were histologically identical to those in PEPCK-Vhlh mutant mice (Figure 1B and C). In contrast, PEPCK-Vhlh/Hif-2α double mutant livers were microscopically and macroscopically similar to control mice with the exception of one case (1/13) in which a small area of angiectasis was detected by macroscopic inspection (Figure 1B and C). Strikingly, however, cavernous hemangiomas were not observed in PEPCK-Vhlh/Hif-2α mutants at the ages of 8 months or older (Figure 1B). These results indicate that Hif-2 is required for the development of VHL-associated vascular tumors in the liver. Immunohistochemical analysis of Hif-α expression revealed that both Hif-1α and Hif-2α were detectable in hepatocytes that neighbored vascular lesions, suggesting a paracrine mechanism by which a HIF-induced excessive production of hepatocyte-derived angiogenic growth factors results in endothelial cell proliferation and vascular tumor development (PEPCK-Cre targets hepatocytes and not endothelial cells, Figure 1D). This is mechanistically consistent with hemangioblastoma development in the CNS, where pVHL-deficient stromal cells are the source of HIF-2 induced vascular growth factor production. Interestingly, the absence of Hif-1α did not change the histological appearance of vascular networks and lesions, nor did it affect lipid droplet accumulation in hepatocytes (Figure 1B and C), suggesting that Hif-1 is dispensable for hemangioma development in this model. Aside from its role in the pathogenesis of VHL-associated hemangiomas and hemangioblastomas, HIF-2 expression has been associated with poor clinical outcome in a variety of other tumor types, which include renal cell and hepatocellular cancer, melanoma and neuroblastoma (Holmquist-Mengelbier et al., 2006; Rankin & Giaccia, 2008). These observations, together with our findings suggest that pharmacological targeting of HIF-2 rather than HIF-1 may be a more effective strategy in the treatment of certain tumor types.
Since the development of VHL tumors is associated with increased angiogenic growth factor production, in particular VEGF (Flamme et al., 1998), we examined the role of Hif-2 in the regulation of Vegf in Vhlh knockout livers. Real time PCR analysis demonstrated that inactivation of Hif-2, but not Hif-1, was sufficient to suppress Vegf despite increased Hif-1 transcriptional activity, suggesting that Hif-2 is the dominant HIF for the regulation of hepatic Vegf (Figure 2A). We next sought to identify and characterize additional changes in angiogenic gene expression by cDNA microarray analysis. Due to limited Cre-recombinase expression in PEPCK-Cre livers, we utilized Albumin-Cre transgenic mice to inactivate Vhlh, Hif-1α, and Hif-2α in a larger percentage (>90%) of hepatocytes. Genes that were differentially expressed (> 1.5 fold) between Vhlh/Hif-1α/Hif-2α (mice that lack both Hif-1 and Hif-2) and Vhlh/Hif-1α or Vhlh/Hif-2α mutant livers were sorted according to functional categories using the NIH David gene ontology program. Using this approach, we identified 17 additional HIF-regulated genes that are involved in blood vessel morphogenesis and development, angiogenesis, and vasculature development, 12 of which were up-regulated in Vhlh or Vhlh/Hif-1α mutant livers (Figure 2B). Using real time PCR analysis, we determined that inactivation of Hif-2α suppressed the majority of up-regulated genes (12 of 13) more efficiently (> 75% reduction) than Hif-1α inactivation, suggesting Hif-2 dominance in the transcriptional regulation of angiogenic genes (Figure 2B). Among the genes identified was Hey1, a transcriptional target of Notch, which is involved in cardiovascular development (Figure 2C, (Fischer et al., 2004)). Recent reports demonstrated that Notch signaling is enhanced during hypoxia through functional interaction of HIF-1α with the Notch intracellular domain, providing a possible explanation for increased Notch target gene expression under conditions of HIF activation (Gustafsson et al., 2005). Other genes induced in a Hif-2 dependent manner included Bmp4, a TGF-β super family member previously shown to regulate tumor angiogenesis (Figure 2C, (Rothhammer et al., 2007)), Klf5, Nr2f2, Angpt13, Anxa2, and Cdh5 (Figure 2C). Whether increased expression of these genes resulted from direct transcriptional regulation by Hif-2 or was a consequence of increased angiogenesis is unclear and requires further analysis.
The expression of other HIF-2 targets that are commonly associated with pVHL-defective tumors, such as cyclin D1 (CCND1) in renal cancer cells (Bindra et al., 2002; Raval et al., 2005; Zatyka et al., 2002), did not change in the liver (data not shown), indicating that HIF-2 mediated induction of gene expression is tissue- and context-dependent. Although a specific polymorphism in the CCND1 gene was found to be associated with increased susceptibility to retinal and CNS hemangioblastomas in one study (Zatyka et al., 2002), no clear correlation between CCND1 genotype and expression was found in another clinical report (Gijtenbeek et al., 2005).
Collectively, our findings demonstrate that HIF-2 is the dominant HIF for the regulation of VEGF and other angiogenic factors in hepatocytes. A role for HIF-2 in the regulation of VEGF has recently been observed in human renal cell cancer and neuroblastoma cell lines, while in other cell types such as thymocytes and astrocytes Vegf expression was found to be Hif-1 dependent (Biju et al., 2004; Chavez et al., 2006; Holmquist-Mengelbier et al., 2006). Taken together these data suggest that both HIF-1 and HIF-2 have the ability to activate VEGF, however, they appear to preferentially regulate its expression within individual cell types. This may be attributable to different HIF-1 and HIF-2 expression levels in individual cell types or may be due to the presence of cell-specific transcriptional co-factors that modulate HIF activity. In the context of human VHL-associated hemangioblastomas, high levels of HIF-2 are associated with increased VEGF mRNA levels, while correlation of HIF-1 expression with tumor VEGF levels was poor (Flamme et al., 1998), which is consistent with our data in mice.
In summary, our studies provide genetic evidence that HIF-2 is the critical regulator of angiogenic gene expression and vascular tumorigenesis in pVHL-deficient livers and may therefore represent a therapeutic target for the treatment of VHL-associated vascular tumors.
Primer sequences used for quantitative real time PCR analysis of angiogenic gene expression in Vhlh mutant livers. Shown are the sequences of forward and reverse primers used to detect changes in gene expression with SYBR green on an Applied Biosystems platform. All genes were normalised to 18S levels using the 18S Taqman set from Applied Biosystems.
This work was supported by NIH grant DK073467 and CA100787 (both to VHH), the Center for Molecular Studies in Digestive and Liver Disease (P30-DK50306) and the Penn Diabetes and Endocrinology Research Center Functional Genomics Core (P30-DK19525). E.B.R. was supported by a fellowship grant from the American Heart Association.