VHL Induces Renal Cell Differentiation and Growth Arrest through Integration of Cell-Cell and Cell-Extracellular Matrix Signaling

Abstract

Mutations in the vonMutations of the von Hippel-Lindau (VHL) gene are involved in the family cancer syndrome for which it is named and the development of sporadic renal cancer and renal cystic disease (for review, see reference 15). VHL has no significant homology to previously identified proteins (21). Insights into the biochemistry of VHL have come predominantly from the identification of proteins that associate with VHL products (9,17,19,26,29,34). These include elongins B and C (9, 12, 19, 37), cul-2 (26, 34), and Rbx1 (17), which are similar to components of a yeast E3 ubiquitin ligase complex (2,16,26,34). A current hypothesis for VHL activity is that it functions as an F-box protein, directing specific substrates for ubiquitination. Indeed, VHL has been shown to have in vitro ubiquitin ligase activity (13,25) and to target HIF-1␣, a hypoxia inducible transcription factor, for proteasomal degradation (7,18,28,38). However, the exact biochemical function(s) of VHL that is disrupted in VHL disease and which results in susceptibility to clear renal cell cancer (RCC) remains elusive.Alterations of cell-extracellular matrix (ECM) interactions are associated with renal cystic disease (for review, see references 4, 10, and 40). A role for VHL in the synthesis and degradation of ECM has begun to emerge. VHL was found to associate with intracellular fibronectin and was required for assembly of extracellular fibronectin (29). VHL also controls matrix degradation by regulating both matrix metalloproteinases 2 and 9 and their inhibitors (20), as well as the urokinasetype plasminogen activator system (27). (11,33,37). In addition, VHLdeficient RCC cells ectopically expressing VHL demonstrated morphological differentiation and growth arrest when grown as multicellular tumor spheroids, but not under standard culture conditions (23). These studies suggest the importance of the extracellular milieu to elicit biological functions of VHL. Previous studies indicated that reintroduction of VHL into carcinoma cells lacking functional VHL [VHL(Ϫ)] leads to growth suppression in nude mice but not in cells grown under standard culture conditionsIn this report, we describe VHL function in the context of cell-cell and cell-ECM interactions. These studies demonstrate that VHL-dependent morphological and biochemical differentiation requires the establishment of high-density cell-cell contact and, in combination with cell-ECM interactions, results in VHL-dependent growth arrest. MATERIALS AND METHODSCell lines and culture. The VHL(Ϫ) cell lines consist of the parental renal carcinoma cell line, 786-O, and its derivative lines containing either the empty expression vector pCR3 (Invitrogen, Carlsbad, Calif.) or a nonfunctional VHL deletion construct, VHL(MPR)del(114-178) (37). The VHL(ϩ) cell lines consist of 786-O derivative lines stably expressing the VHLp24(MPR) or the VHLp18 (MEA) constructs as previously described (37). At least two independent clones were analyzed for each construct. Cells were grown in 10-cm-diameter dishes in a h...

The von Hippel–Lindau tumor suppressor gene product represses oncogenic β-catenin signaling in renal carcinoma cells

“…3). Consistent with prior observations (29), phase contrast microscopy revealed that untreated VHL-negative cells were less well organized as an epithelial monolayer than their VHL-expressing counterparts (data not shown). Immunofluorescence with anti-␤-catenin showed that intercellular contacts were less extensive and ␤-catenin was more randomly distributed in the absence of VHL expression than in its presence (Fig.…”

Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1α eradicates gliomas

“…33 The present study reveals that VHL-mediated apoptosis of C6 glioma cells is accompanied by downregulation of Bcl-2. VHL is a multifunctional tumor suppressor protein exerting effects on hormone levels, 34 the extracellular matrix, [35][36][37] cell differentiation 38 and growth, 39 protease and protease inhibitor secretion, 40 the cytoskeleton and cell motility and invasiveness, mRNA stability, 41 the cell cycle, 42 and cell signaling, 43 all of which may play a role in the antitumour activity of VHL.…”

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

“…Aside from mediating HIF-a proteolysis, pVHL is involved in extracellular matrix (ECM) assembly matrix turnover, [42][43][44][45][46][47] the regulation of intracellular junctions, 48 NF-kB signaling, 49 the regulation of c-Met receptor responsiveness to hepatocyte growth factor (HGF) involving b-catenin 45,50 and the regulation of p53 transcriptional activity by suppressing Mdm2-mediated ubiquitination and nuclear export. 51 Furthermore, pVHL has been shown to regulate microtubule stability and cilia maintenance [52][53][54][55] and controls the activity of plant homeodomain protein Jade-1, 56,57 and atypical protein kinase C isoforms.…”

“…Similarly, Evans et al 107 demonstrated that knockdown of pVHL resulted in E-cadherin suppression via HIF-dependent induction of E2 box-dependent transcriptional repressors Snail and SIP1, and Krishnamachary et al 108 reported that HIF-1 activation in VHL-deficient cells downregulated Ecadherin, led to the loss of cell-cell adhesion and promoted epithelial to mesenchymal transition through the induction of transcriptional repressors TCF3, ZFHX1A and ZFHX1B/SIP1. Therefore, cellular changes, such as loss of intercellular junctions and epithelial de-differentiation involving HIFdependent as well as HIF-independent molecular pathways 48,[106][107][108] in addition to HIF-dependent and -independent alterations in p53 or NF-kB activity, 34,35,49,51 HGF signaling, 45,50,109 and modifications in ECM turnover and re-modeling [42][43][44][45][46][47] create the molecular environment for the development CC-RCC, which most likely requires additional genetic events. The importance of HIF activation in CC-RCC pathogenesis and growth is furthermore underscored by experimental and clinical studies, which demonstrated that inhibition of HIF-a translation by pharmacological targeting of mTOR correlated with reduced tumor growth, 110 and that increased expression of certain HIF target genes, such as CXCR4, as well as E-cadherin suppression was associated with disease progression.…”