The VHL tumor suppressor in development and disease: functional studies in mice by conditional gene targeting

Abstract

The von Hippel-Lindau tumor suppressor pVHL plays a critical role in the pathogenesis of familial and sporadic clear cell carcinomas of the kidney and hemangioblastomas of the retina and central nervous system. pVHL targets the oxygen sensitive alpha subunit of hypoxia-inducible factor (HIF) for proteasomal degradation, thus providing a direct link between tumorigenesis and molecular pathways critical for cellular adaptation to hypoxia. Cell type specific gene targeting of VHL in mice has demonstrated that proper pVHL mediated HIF proteolysis is fundamentally important for survival, proliferation and differentiation of many cell types and furthermore, that inactivation of pVHL may, unexpectedly, inhibit tumor growth under certain conditions. Mouse knock out studies have provided novel mechanistic insights into VHL associated tumorigenesis and established a central role for HIF in the development of the VHL phenotype.

Fig. 1

Hepatic vascular tumors in mice with hepatocyte specific inactivation of Vhlh. (A and B) Gross photography of formalin fixed livers from hepatocyte-specific Vhlh knock out mice. Arrows in (A) point to cavernous hemangiomas that appear deflated after resection of the liver. The gallbladder is labeled by a star. (B) shows a section of the same liver. Arrows point to blood filled cavities containing fibrin clots. (C and D) Paraffin sections of livers with cavernous hemagiomas stained with H&E. (C) Large endothelial cell lined, blood filled cavities are depicted by stars. Arrow depicts an area of hemorrhage. (D) Cavernous hemangiomas are typically associated with macro- and microvesicular hepatocellular steatosis, which results from the accumulation of neutral lipid [64], here depicted by arrows. Lipid accumulation is also a feature of stromal cells, the neoplastic component of VHL associated central nervous system hemangioblastomas [69,70]. Magnification in (C) is ×100 and in (D) is ×400.

Fig. 2

Genomic structure of the Vhlh conditional allele. Shown are the genomic structures of Vhlh in wild type (wt) configuration, the Vhlh 2-lox allele and the recombined Vhlh 1-lox allele. LoxP sites are depicted as red triangles, Vhlh exons are shown as black boxes. Exon 1 and the Vhlh promoter are deleted by Cre-mediated recombination, deleted area is shown in red. This allele was generated in 129 J-1 ES cells by homologous recombination with a targeting vector containing a “floxed” hygromycin-thymidine-kinase selection cassette, which was removed after transient transfection with Cre-recombinase [64]. Abbreviations: H, HindIII; N, NcoI.

Fig. 3

Inactivation of Vhlh in proximal renal tubule (PRT) cells results in renal cysts at low frequency. (A) Gross in situ photograph of the right kidney from a conditional knock out mouse with inactivation of Vhlh in the PRT. Arrows point to two superficial cysts. Evidence of renal cell cancer or epithelial dysplasia was not detected (Rankin et al., unpublished observation). (B) Paraffin section of kidney with renal cysts stained with H&E. Shown is a cluster of smaller cysts lined with renal epithelial cells (arrows). Magnification is ×200.

Fig. 4

HIF plays a central role in VHL associated renal cystogenesis and tumorigenesis. Above: proposed sites of origin of VHL renal cycts and tumors. A schematic representation of a nephron is shown above. The area shaded in blue represents the proximal renal tubule, the area shaded in red represents the THP expressing medullary thick ascending limb of Henle and early distal tubule. Below: overview of selected hypoxia/HIF dependent and independent pVHL targets with potential importance for renal cystogenesis and tumorigenesis. Ovals identify transcriptionally regulated targets, rectangles identify targets, which are regulated by non-transcriptional mechanisms; vertical arrows indicate up- or down regulation. HIF-1α counteracts Myc activity through direct protein-protein interaction and has been shown to induce cell cycle arrest in colon cancer cells [104], what this interaction means for renal tumorigenesis is unclear. BNIP-3 is a pro-apoptotic factor and is directly up-regulated by HIF-1 [–118]. BCL-2, an anti-apoptotic protein, has been shown to be HIF-1 dependent in ES cell derived teratomas [105]. Up-regulation of cyclin D1 (CCND1), an important regulator of cell cycle progression, was found in VHL-deficient renal cancer cell lines and shown to be hypoxia-dependent in a cell type specific manner [–122]. Chemokine receptor 4 (CXCR4) is a direct HIF target and has been shown to correlate with poor survival and metastatic ability of CC-RCC [100]. Jade 1 is a novel pVHL interacting protein that may play a role in renal cytogenesis through interaction with polycystin-1 [123]. Abbreviations: gl, glomerulus; cd, collecting duct.