Collagen matrix assembly is driven by the interaction of von Hippel–Lindau tumor suppressor protein with hydroxylated collagen IV alpha 2

Oncogene (2008) 27, 1004–1012 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Collagen matrix assembly is driven by the interaction of von Hippel–Lindau tumor suppressor protein with hydroxylated collagen IV alpha 2 G Kurban1, E Duplan1, N Ramlal1, V Hudon1, Y Sado2, Y Ninomiya3 and A Pause1 1 McGill Cancer Center, Department of Biochemistry, McGill University, Montreal, Quebec, Canada; 2Division of Immunology, Shigei Medical Research Institute, Okayama, Japan and 3Department of Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan Inactivation of the von Hippel–Lindau (VHL) tumor suppressor gene predisposes to vascular tumor formation in several organs. VHL regulates two evolutionary conserved pathways: the targeting of hydroxylated hypoxia-inducible factor-a (HIF-a) for proteasomal degradation and the remodeling of extracellular matrix (ECM). The biochemical mechanisms of the ECM assembly pathway remain poorly defined. Here, we provide evidence supporting a biochemical role for VHL in ECM assembly. We show that VHL directly binds to the collagen IV alpha 2 (COL4A2) chain and that this interaction is necessary for its assembly into the ECM. The VHL–COL4A2 interaction is dependent on endoplasmic reticulum (ER)mediated COL4A2 hydroxylation and independent of cytosolic, hypoxia regulated HIF-a-modifying enzymes. We find that the N-terminal tail of COL4A2 protrudes from the ER lumen into the cytosol where it is bound by VHL. Failure of VHL to interact with COL4A2 correlates with loss of collagen IV network formation in vitro and collagen IV remodeling in vivo. Our data suggest a HIF-a-independent role for the VHL–COL4A2 interaction in suppression of angiogenic tumor formation through collagen IV network assembly. Oncogene (2008) 27, 1004–1012; doi:10.1038/sj.onc.1210709; published online 13 August 2007 Keywords: VHL; collagen IV; extracellular matrix; renal cell carcinoma Introduction von Hippel–Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL tumor suppressor gene (Kaelin, 2002; Semenza, 2003; Barry and Krek, 2004). The VHL syndrome is the most common syndrome of hereditary kidney cancer whereby affected individuals develop vascular tumors including Correspondence: Dr A Pause, McGill Cancer Center, Department of Biochemistry, McIntyre Building, 3655 Sir William Osler Promenade, Room 716, McGill University, Montreal, Quebec, Canada H3G 1Y6. E-mail: Received 21 May 2007; revised 22 June 2007; accepted 1 July 2007; published online 13 August 2007 clear cell renal cell carcinoma (RCC), together with hemangioblastomas of the central nervous system and retina, as well as pheochromocytomas (Kaelin, 2002; Semenza, 2003; Barry and Krek, 2004). Somatic mutations of the VHL gene have also been observed in sporadic forms of most VHL-associated tumors, including hemangioblastomas and clear cell RCC (Kaelin, 2002; Semenza, 2003; Barry and Krek, 2004). Two major conserved pathways have been described defining the role of VHL as a tumor suppressor: the hypoxia-inducible factor-a (HIF-a) pathway and the extracellular matrix (ECM) pathway. The HIF-a pathway involves the VHL protein as part of an E3 ubiquitin ligase complex that binds HIF-a leading to its polyubiquitination and proteasomal degradation. The VHL– HIF-a interaction is mediated by HIF-a hydroxylation at two proline residues by cytosolic prolyl hydroxylases (PHDs) that require oxygen, iron and the cofactor 2-oxoglutarate (Kaelin, 2002; Semenza, 2003; Barry and Krek, 2004). In cells that have lost the VHL ligase function, HIF-a accumulates leading to activation of HIF-a target genes including VEGF and transforming growth factor-a, thereby mimicking the cellular response to hypoxia. Genetic studies have identified an important role for the HIF-2a isoform in RCC tumorigenesis (Kondo et al., 2002; Zimmer et al., 2004; Raval et al., 2005). Hypoxia-mimetics, including iron chelators, cobalt chloride (CoCl2) and dimethyl-oxallylglycine (DMOG) inhibit PHD activity thus stabilizing HIF-a and resulting in induction of HIF-a target genes (Kaelin, 2002; Semenza, 2003; Barry and Krek, 2004). Genotype/phenotype correlations suggested the existence of other HIF-a-independent, tumor suppressor mechanisms for VHL. This activity became apparent when VHL tumor-associated mutants, which maintain HIF-a binding/degradation, were reintroduced into VHL-negative 786-0 cells (Clifford et al., 2001; Hoffman et al., 2001). All VHL mutants tested so far failed to assemble an ECM in cell culture and could only be rescued by reintroduction of wild-type VHL (Ohh et al., 1998; Esteban-Barragan et al., 2002). In Caenorhabditis elegans, VHL was shown to function along a HIF-adependent pathway and an ECM pathway independent of HIF-a suggesting evolutionary conservation of both pathways (Bishop et al., 2004). The role of VHL in ECM assembly and tumor suppression correlated VHL–COL4A2 interaction and ECM assembly G Kurban et al 1005 ∗ 170 - ∗ ∗ Results Identification of COL4A2 as a VHL interacting protein We previously demonstrated that VHL co-immunoprecipitates and interacts directly with a 200 kDa protein (Iwai et al., 1999). In our current study, we used the VHL-negative RCC cell line 786-0, either stably expressing wild-type HA–VHL (WT8, WT7 and WTE), or wild-type HA–VHL along with a non-degradable form of HIF-a (PA) or mutant forms of HA–VHL (L188V and R64P) that are functional in the HIF-a interaction/ degradation pathway but defective in the ECM assembly pathway (Hoffman et al., 2001; Kondo et al., 2002; Kurban et al., 2006). We performed 35S metabolic labeling followed by HA immunoprecipitation (IP) using total cell extracts (Figure 1a). An interaction between VHL and p200 was observed in cells expressing wild-type VHL (WT8, WT7, WTE and PA) but not tumor-associated VHL mutants (L188V and R64P). 786-0 and pRC cell lines, which lack VHL, were used as control and showed no interaction with p200 (Figure 1a). P200 was identified by mass spectrometry that revealed five peptides whose sequences matched that of COL4A2 (Figure 1b). To confirm the mass spectrometry results, VHL was immunoprecipitated from total extracts of the various 786-0 cell lines and analysed by western blot using anti-COL4A2 (H22) (Figure 1c). COL4A2 binding was observed in cells expressing wild type (WT8, WT7, WTE and PA) but not mutant VHL protein (L188V and R64P). We observed R64P L188V PA WTE WT7 WT8 pRC 786-0 with VHL neddylation and its interaction with fibronectin. Therefore, the ECM assembly activity of VHL is considered essential for the VHL tumor suppressor function (Ohh et al., 1998; Stickle et al., 2004; Kurban et al., 2006). Recently, we have shown that inactivation of the VHL–ECM assembly pathway results in highly vascular tumors with remodeled ECM and increased matrix metalloprotease-2 (MMP-2) activity. (...truncated)