Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through Hypoxia-inducible factor

Abstract

Loss of the von Hippel–Lindau gene (VHL) expression ca-uses deregulation of contact inhibition of cell growth, which might be one of the bases of the tumor suppressor function of VHL. Here we show that this function of the VHL gene product (pVHL) depends on cell autonomous events. To identify the target gene of pVHL, which is directly involved in the contact inhibition, we compared the gene expression profile between VHL-deficient renal carcinoma 786-O cells and those infected with an adenovirus vector encoding VHL. In addition to known pVHL-regulated genes, such as vascular endothelial growth factor and carbonic anhydrase, we found cyclinD1 as a new target of pVHL at a high cell density. In VHL-expressing cells (VHL (+) cells), the cyclinD1 mRNA expression level diminishes at a high cell density, while it remains at a relatively high level in VHL-deficient cells (VHL (−) cells). The cyclinD1 expression level was also abnormally high in VHL (−) cells at a high cell density. Consequently, the phosporylation level of the retinoblastoma (Rb) protein remained high in these cells, whereas there was no phosporylated Rb in VHL (+) cells under the contact inhibition. The abnormal expression of cyclinD1 at a high cell density was observed even in VHL (+) cells under the hypoxic state. Moreover, ectopic expression of a HIF mutant resistant to pVHL-mediated proteolysis causes the abnormal cyclinD1 expression in VHL (+) cells. Taken together, these observations indicate that VHL is required for the downregulation of cyclinD1 at a high cell density through HIF.

Introduction

Inactivation of the von Hippel–Lindau gene (VHL) is associated with the development of VHL disease, which is characterized by a predisposition to develop tumors or cysts in many tissues including the eyes, central nervous system, kidneys, adrenal glands and pancreas (Latif et al., 1993; Maher and Kaelin, 1997). The product of this tumor suppressor gene (pVHL) forms a stable complex called the VCB–Cul2 complex with elongin C, elongin B, Cul2 and Rbx1 (also called ROC1 or Hrt1) (Pause et al., 1997), which functions as ubiquitin ligase E3 (Iwai et al., 1999; Kamura et al., 1999). A pVHL-containing E3 enzyme ubiquitinates HIFα, which is an α subunit of the hypoxia-inducible factor (HIF) transcription factor, in the presence of oxygen (Maxwell et al., 1999). Recently, the interaction of pVHL with HIFα has been proved to depend on oxygen-dependent hydroxylation of a conserved proline residue in HIFα (Ivan et al., 2001; Jaakkola et al., 2001). Loss of pVHL causes accumulation of HIFα irrespective of the oxygen concentration and the deregulated activation of HIF target genes including vascular endothelial growth factor (VEGF), TGFα, erythropoietin, glucose transporter-1 (Glut-1) and other hypoxia-inducible genes (Iliopoulos et al., 1996; Knebelmann et al., 1998; Semenza, 2001). Among them, VEGF is regarded as one of the most important target of HIF in tumorigenesis because of its fundamental roles in tumor angiogenesis (Ferrara, 2002). However, VEGF may not be the sole HIF target in relation to tumor formation, since angiogenesis is not likely involved in the transformation of epithelial cells, an initial event of tumorigenesis. It should be noted that HIF induction is found upon the inactivation of VHL in small foci representing an initial step of tumorigenesis (Mandriota et al., 2002). However, how the loss of VHL triggers carcinogenesis and how HIF mediates it remain to be clarified.

CyclinD1 belongs to the G1 cyclin family and plays a key role in cell cycle regulation during the G1/S transition in cooperation with its catalytic partners CDK4/CDK6 (Sherr, 1995). CyclinD1–CDK4/CDK6 complexes promote G1 progression, at least in part, by phosphorylating the retinoblastoma (Rb) protein, thereby inactivating its ability to bind to proteins of the E2F family and to suppresses their transcriptional activity (Weinberg, 1995; Sellers and Kaelin, 1996). There are many reports suggesting that cyclinD1 can function as an oncogene: the overexpression of which may lead to growth advantage for culture cells through cell cycle progression (Jiang et al., 1993; Quelle et al., 1993; Hinds et al., 1994). In various human cancers, translocation or amplification of the cyclinD1 gene and its subsequent overexpression have frequently been observed (Hunter and Pines, 1991). Moreover, oncogenic ras-mediated skin tumorigenesis is substantially reduced in cyclinD1-deficient mice (Robles et al., 1998). Taken together, these observations indicate the fundamental roles of cyclinD1 in tumorigenesis. As for the regulatory mechanism for cyclinD1 expression, there are many reports on the induction of cyclinD1 via the Ras-ERK pathway (Roovers and Assoian, 2000). However, the molecular mechanism that induces deregulated cyclinD1 expression at the initial step of tumorigenesis has not been fully understood.

We have reported that pVHL is required for the establishment and maintenance of contact inhibition of cell growth in cultured renal epithelial cells (Baba et al., 2001). Since the loss of contact inhibition in epithelial cells should occur at the initial stage of tumorigenesis, the requirement for VHL explains the function of VHL as a gate keeper. To address the molecular mechanism underlying the role of VHL as a gate keeper, we attempted to identify genes responsible for the VHL-dependent growth inhibition at a high cell density by comparing the expression profiles of 12 000 genes using 786-O cells infected with an empty adenovirus vector (Ax-W1) or a VHL-encoding adenovirus vector (Ax-VHL) as mRNA sources. We report here a deregulated cyclinD1 expression in VHL-deficient cells at a high cell density and the possible involvement of the HIF signaling pathway in this irregular cyclinD1 expression.

Results

Growth suppression by pVHL at high cell density is not influenced by soluble factors

To understand the molecular mechanism underlying the suppression of cell proliferation at a high cell density by pVHL, we first tested the contribution of soluble factors such as VEGF, TGFα and PDGFβ, that may be released by VHL-deficient cells (Kondo and Kaelin, 2001). The renal cell carcinoma cell line 786-O lacking an intact VHL gene, and its stable transformants Wt8 expressing exogenous VHL were cocultured separately on the Transwell, which enabled these cells to share the culture medium with released soluble factors if any. If the accelerated growth of 786-O at a high cell density is caused by the production of some growth factors, the growth rate of Wt8 cocultured with 786-O should be accelerated. However, the growth rate of Wt8 remains constant regardless of its coculture with 786-O (Figure 1). These observations show that the deregulated growth of VHL-deficient cells at a high cell density cannot be explained solely by the overproduction of some growth factors if any. Therefore, there must be a cell autonomous signaling mechanism for the pVHL-dependent regulation of contact inhibition, which leads to the induction or suppression of genes directly involved in the regulation of cell proliferation.

Figure 1
figure1

Growth curve of VHL-positive cells is not affected by cocultivation with VHL-deficient cells. Renal cell carcinoma 786-O cells lacking the intact VHL gene or their stable transformant expressing wild-type pVHL (Wt8) were seeded at a density of 0.5 × 104 cells per well in 24-well plates. On the TranswellR membrane, 786-O or Wt8 cells were seeded at the same density in the combination indicated in the figure. For cell counting, the cells on the 24-well plates were stained with Hoechst 33258, and the fluorescent signal was measured by a plate reader. The cell growth profile was determined from day 1 to day 11 after seeding

Loss of VHL causes deregulated cyclinD1 mRNA expression only at a high cell density

To identify genes whose expression is deregulated in the absence of pVHL, we examined the expression profiles of 12 000 genes using GeneChip (Affymetrix) with 786-O cells infected by an Ax-W1 or an Ax-VHL as mRNA sources. Since the VHL-dependent change in growth rate depends on cell density, mRNA was prepared from cells cultured at different densities, namely subconfluent, confluent and overconfluent. The signal intensities of each mRNA were plotted on the graph, whose horizontal axis represents the relative expression level in 786-O cells infected by Ax-W1 (VHL (−)) and whose vertical axis represents that in 786-O cells infected by Ax-VHL (VHL (+)) (Figure 2a). There were no statistically significant differences in the total mRNA expression profile between VHL (−) and VHL (+) cells at any of the cell densities. However, we found some genes whose expression level differs significantly in the presence or absence of VHL, particularly for overconfluent cells. For example, the expression level of genes (e.g. VEGF, carbonic anhydrase), which are known to be regulated by VHL (Siemeister et al., 1996; Ivanov et al., 1998) were elevated in VHL (−) cells (Figure 2b). Focusing our attention to the genes involved in cell proliferation, we found that the cyclinD1 expression level was elevated in VHL (−) cells and was reduced by introducing VHL at the high cell density (Figure 2b). When we compared the cyclinD1 mRNA level at different cell densities in VHL (−) cells, the cyclinD1 expression level was constantly high regardless of the cell density. On the other hand, in VHL (+) cells, the cyclinD1 mRNA expression level decreased as the cell density increased (Figure 2c). Such suppression of cyclinD1 mRNA expression at the high cell density was also observed for 786-O-derived cell lines (Wt8, AC6) stably transfected with the wild-type VHL gene (Figure 3a). Since the change in the cyclinD1 mRNA level correlates well with the VHL-dependent differences in the cell growth rate at the high cell density (see Figure 1), this cell cycle regulator may mediate the function of pVHL in contact inhibition of cell growth.

Figure 2
figure2

Gene expression profiling of VHL-positive or VHL-deficient cells at different cell densities. 786-O cells were infected with Ax-W1 or Ax-VHL and seeded at different cell densities, namely subconfluent (3 × 106 cells/15-cm dish), confluent (6 × 106 cells/15-cm dish) or overconfluent (6 × 106 cells/10-cm dish). cRNA probes were synthesized from cDNA reverse transcribed from mRNA of the infected cells and hybridized with oligonucleotide arrays (Affymetrix). Hybridized probes were quantified by a laser scanner and the levels of expression of each gene (average difference) were calculated with Affymetrix Microarray Suite ver.4.0 software. (a) Each dot indicates the relative expression level of one gene, X-axis shows the relative expression level in 786-O cells infected by Ax-W1 (VHL (−)) and Y-axis shows that in 786-O cells infected by Ax-VHL (VHL (+)). 1, VEGF; 2, CA12; 3, cyclinD1. (b) Relative expression levels of representative genes upregulated by VHL deficiency are shown. (c) Relative expression levels of cyclinD1 at different cell densities are shown

Figure 3
figure3

The expression of cyclinD1 is not downregulated in VHL-deficient cells at the high cell density. VHL-deficient 786-O cell and its stable transformants of wild-type VHL (Wt8 and Ac6) were seeded at different cell densities (S: sparse, 1 × 106 cells/10-cm dish; C: overconfluent, 2 × 106 cells/6-cm dish). After 48 h, cells were harvested for RNA and protein extraction. (a) Total RNA (20 μg) was analysed by Northern blotting using cyclinD1 cDNA as a probe (upper panel). The ethidiumbromide staining patterns for 28S and 18S rRNAs are also shown (lower panel). (b) Western blot analysis using antibodies against cyclinD1, Rb, HIF2α and actin. Note the well-correlated expression pattern of the cyclinD1 protein and its mRNA shown in (a). The decreased expression level of the HIF2α protein in Wt8 and Ac6 indicates the expression of the functional pVHL in these cells. The same expression level of actin in each lanes indicates the equality of protein amount analysed. pRb, phosphorylated form of Rb protein. (c) The change in expression pattern of cell-cycle-related proteins after seeding at high cell densities. 786-O cells were infected with Ax-W1 or Ax-VHL. After 24 h cells were seeded at high cell densities (2 × 106 cells/6-cm dish) and harvested at the each time point after seeding. The cell lysates containing equal amounts of protein (20 μg) were used for Western blot analysis using antibodies against cyclinA, cyclinD1, cyclinE, p130, Rb, phosho-Erk, p27, HIF2α and actin. The lanes in left half (lanes 1–5) are protein samples from 786-O cells with Ax-W1 (VHL (−)), and lanes in right half (lanes 6–10) are from 786-O cells with Ax-VHL (VHL (+)). Decreased HIF2α expression level in VHL (+) cells and nearly equal quantity of actin in all the lanes shows the appropriateness of experimental conditions. The difference in the expression level of cyclinA, cyclinE, p27 and phospho-Erk is not obvious. CyclinD1 expression level decreased in VHL (+) cells, while it remained high in VHL (−) cells 48 h after seeding. (d) The signal intensities of cyclinD1 bands at the time point of 48 h in VHL (−) and (+) cells were quantified. Results from three independent experiments are shown in a graph with error bars representing standard deviations. (e) The ratios of the amount of pRb to that of non-pRb at the time point of 48 h were calculated for VHL (−) and VHL (+) cells. Results from three independent experiments are shown in a graph with error bars representing standard deviations

Protein level and activity of cyclinD1 remain high even at high cell density in VHL-deficient cells

To confirm the difference in cyclinD1 expression at the protein level, we analysed protein samples prepared from VHL-deficient cells and VHL-positive cells cultured at different cell densities by Western blotting using an antibody against cyclinD1. Consistent with Northern blotting and GeneChip analysis data, the cyclinD1 protein level in VHL (−) 786-O cells was constantly high regardless of the cell density, while it declined at the high cell density in VHL (+) cells, Wt8 and Ac6 (Figure 3b). Since the half-life of the cyclinD1 protein did not differ with the VHL status (data not shown), pVHL may regulate cyclinD1 expression mainly at the level of transcription or RNA processing. The difference in the cyclinD1 protein expression level at the high cell density accounts for the difference in the phosphorylation level of Rb. Specifically, while there were no phosphorylated Rb bands for VHL (+), Wt8 and AC6 cells at the high cell density because of contact inhibition, the phosporylated band still remains obvious for VHL (−) 786-O cells at a high cell density (confluent).

Next, we examined quantitative or qualitative changes of various cell-cycle-related proteins in more detail. 786-O cells in the logarithmic growth phase were infected with the Ax-W1 or the Ax-VHL, and seeded at the high cell density after 24 h. Figure 3c shows changes in the expression level of the cell-cycle-related proteins at different time points after cells were seeded. The HIF2α protein level was low in 786-O cells infected by Ax-VHL (VHL (+)), and high in 786-O cells infected by Ax-W1 (VHL (−)) at all time points, which is consistent with the proposed function of pVHL as ubiquitin ligase E3 for HIFα. The amount of cyclinA and cyclinE decreased gradually and that of p27 increased with time regardless of the VHL status. The cyclinD1 protein expression level in both VHL (+) and VHL (−) cells decreased until 24 h after seeding. However, it was upregulated after 36 h or later in VHL (−) cells, while such upregulation at the late phase was not observed in VHL (+) cells. The phosphorylation levels of Rb and p130 changed in accordance with the cyclinD1 expression level in each type of cell; in VHL (−) cells, it remained high 48 h after seeding, while it greatly decreased in VHL (+) cells. Figure 3d shows the quantitative analysis of the cyclinD1 protein and the ratio of the intensity of phosphorylated bands to that of dephosphorylated bands for Rb at 48 h after seeding. There were statistically significant differences in the cyclinD1 protein level (P=0.001, Student's t-test) and the phosphorylation ratio of Rb (P=0.028, Student's t-test) between VHL (−) and VHL (+) cells. These observations strongly support the notion that cyclinD1 is involved in pVHL-dependent deregulation of cell growth observed at the high cell density. Note, that the expression level of active, phosporylated, ERK was equally suppressed within a relatively early time point, that is, 12 h after seeding, under this high cell density condition regardless of the presence or absence of VHL. Since ERK is one of the activators for cyclinD1 expression, this observation can explain at least in part the temporal decrease in the cyclinD1 expression level in 786-O cells (Figure 3c, 12 and 24 h).

Hypoxic stimuli as well as the loss of VHL cause deregulated cyclinD1 expression at high cell density

VHL protein mediates hypoxia-dependent gene expression, and the loss of VHL induces a set of genes whose expression becomes prominent only under hypoxic conditions. We then tested if cyclinD1 is one of such hypoxia-inducible genes or not. Protein samples were prepared from 786-O, Wt8 and Ac6 cells cultured under hypoxic conditions or using a medium containing DFO or CoCl2, which stimulates Fe-dependent hypoxia signaling. The HIF2α protein induction under hypoxic conditions proved that these culture conditions were appropriate (Figure 4, a panel next to the bottom). The high expression level of cyclinD1 in 786-O cells (VHL (−)) was observed consistently under all culture conditions (Figure 4). In Wt8 and AC6 cells (VHL (+)), the cyclinD1 expression level decreased under normoxic conditions, while it was elevated under hypoxic conditions, showing unusual cyclinD1 expression even though they were at a high cell density, the overconfluent state. The phosphorylation level of Rb changed in accordance with the cyclinD1 expression level. These data show that the deregulated expression of cyclinD1 at a high cell density can be induced by the activation of hypoxic signals caused by the loss of VHL.

Figure 4
figure4

Deregulated cyclinD1 expression at high cell density induced by hypoxic signals. VHL-deficient 786-O and its VHL-stable transformants (Wt8 and Ac6) were seeded at the high cell density (2 × 106 cells/6-cm dish). After 48 h of culture, cells were treated for 12 h under different culture conditions (N: normoxia, H: hypoxia, DFO: normoxia with 100 μM desferrioxamine, CoCl2: normoxia with 100 μ M CoCl2). Cell lysates containing 20 μg of protein were used for Western blot analysis using antibodies against cyclinD1, HIF2:Rb and actin. The low-level expression of HIF2α in VHL (+) cells (Wt8 and Ac6) at normoxia and the equal intensity of actin signals show the appropriateness of experimental conditions. Note the presence of p-Rb in cells expressing a high level of cyclinD1

Expression of pVHL-resistant HIF2α mutant in VHL-positive cells upregulates cyclinD1 expression at high cell density

Given the results that hypoxic signals can induce unusual cyclinD1 expression in VHL-positive cells at the high cell density, it is conceivable that cyclinD1 expression is regulated by HIF. To test this possibility, we introduced an adenovirus vector for HIF together with that for VHL into 786-O cells and examined the expression of endogenous proteins, and compared the cyclinD1 protein expression level at the high cell density with that in cells introduced only the Ax-VHL. The mutant mouse-HIF2α, HIF2αPA (P530 A) without pVHL binding ability was used in this experiment, which was stable even in VHL-positive cells under normoxic conditions (Ivan et al., 2001; Jaakkola et al., 2001). We have confirmed that the mutant HIF2α had a proper transcription activity using the VEGF promoter reporter gene (data not shown). The cyclinD1 expression level was decreased by introducing VHL into 786-O cells at the high cell density (Figure 5, lane 2) as also shown in Figure 3c. When HIF2αPA was coexpressed with pVHL, the cyclinD1 expression level was restored (Figure 5, lanes 3 and 4). The expression level of Glut-1, another target gene of HIF, was also decreased by introducing VHL and restored by the coexpression of HIF2αPA. These results suggest that HIF is responsible for the deregulated expression of cyclinD1 in VHL-deficient cells at the high cell density.

Figure 5
figure5

Deregulated cyclinD1 expression in VHL-positive cells induced by the expression of pVHL-resistant HIF2α mutant. 786-O cells were seeded at high cell density (1 × 106 cells/3.5-cm dish). After 12 h, cells were infected with Ax-VHL and Ax-mouse-HIF2αPA (pVHL-resistant HIF2α mutant, P530A) as described above. Total MOI of the adenovirus vector was adjusted to 40 by adding Ax-W1. After 48 h, cell lysates were prepared and 20 μg of protein was used for the Western blotting using antibodies against HIF2α, cyclinD1, Glut-1, pVHL and actin. The expressions of actin and pVHL show the appropriateness of experimental conditions. The endogenous HIF2α expression level decreased when pVHL is expressed (lane 2). The pVHL-resistant HIF2α mutant (HIF2αPA) was expressed depending on MOI regardless of the pVHL expression (lane 3: MOI=15, lane 4: MOI=30). The induction of Glut-1 by HIF2αPA indicates the sufficient transcription activity of HIF2αPA (lanes 3 and 4)

Relatively high level of cyclinD1 expression in VHL-altered sporadic clear cell renal cell carcinoma

The results shown above strongly support the notion that the continuous upregulation of cyclinD1 expression might be the basis of carcinogenesis caused by the loss of VHL. To confirm this, we examined the correlation between cyclinD1 expression level and the VHL gene alteration in surgically removed clear cell renal cell carcinomas (CCRCC). Total RNA was extracted from 98 sporadic CCRCC samples and the expression levels of cyclinD1 in tumor tissue relative to that in the normal part of the kidney was estimated by Northern blotting. Figure 6 shows representative results of Northern blotting. The cyclinD1 expression level tends to be higher in tumor tissue than in normal kidney. We also analysed the VHL gene alteration in cells of these tumor tissue and found the alteration in 53 of 98 tumors. The average relative expression levels of cyclinD1 (tumor tissue/normal tissue) were 1.772 (s.d.=0.907) in VHL gene-altered tumors and 1.391 (s.d.=0.775) in VHL-nonaltered tumors. There is a statistical significance (t<0.02) in the relative expression level of cyclinD1 between VHL altered and nonaltered tumors (Table 1). This result indicates that the cyclinD1 expression level is relatively high in VHL altered tumors, and this observation supports the notion that the disruption of contact inhibition of cell growth by the upregulation of cyclinD1 is a basis of carcinogenesis induced by VHL loss.

Figure 6
figure6

CyclinD1 expression in CCRCCs. Total RNA and genomic DNA were extracted from 98 surgically removed CCRCCs and normal part of kidney. CyclinD1 mRNA expression levels were measured by Northern blot analysis. The presence of VHL alteration was determined by SSCP/heteroduplex analysis and direct sequencing. The methylation status of the VHL promoter region was examined by methylation-specific PCR assay. Representative results of the Northern blot analysis are shown (N: normal tissue, T: tumor)

Table 1 Correlation between cyclinD1 expression and VHL alteration in CCRCCs

Discussion

In the present study, we demonstrate that the tumor suppressor protein pVHL regulates the expression of cyclinD1, which plays a key role in cell cycle regulation and carcinogenesis. Importantly, the suppression of cyclinD1 expression at the high cell density is impaired by the loss of functional pVHL, and the deregulated cyclinD1 expression causes the high-level phosphorylation of pRb even at the high cell density. These findings indicate that cyclinD1 is a target of pVHL, which supports the notion that pVHL functions in the regulation of contact inhibition of cell growth. Moreover, we show that hypoxic culture conditions or the expression of the pVHL-resistant form of HIFα can induce the deregulated cyclinD1 expression at the high cell density in VHL-positive cells. Therefore, this transcription factor may induce directly or indirectly the expression of cyclinD1. During the course of preparing this paper, two different groups reported cyclinD1 as the target of pVHL (Bindra et al., 2002; Zatyka et al., 2002). Although they have not clearly referred to contact inhibition or regulation by HIF, their results are consistent with our findings in the respect that VHL regulates the cyclinD1.

Deregulated cyclinD1 expression in VHL-deficient cells

What is the molecular mechanism underlying the deregulated cyclinD1 expression owing to the loss of VHL? The results of GeneChip and Northern blot analyses and the half-life of cyclinD1 protein, which is the same for VHL (+) cells and VHL (−) cells (MB, unpublished data), indicate that cyclinD1 expression at the high cell density is regulated at the mRNA level. Although the molecular mechanism for the regulation of the cyclinD1 mRNA expression level by pVHL is still obscure, it is likely that HIF is a direct target of pVHL, which is responsible for the regulation of cyclinD1 expression, based on the following reasons: (1) The hypoxic culture condition, which can induce endogenous HIF, can induce the deregulated cyclinD1 expression even in VHL-positive cells. (2) The expression of a HIF-point mutant, which cannot bind to pVHL (Ivan et al., 2001; Jaakkola et al., 2001), can induce cyclinD1 expression in VHL-positive cells at a high cell density. (3) The endogenous HIF protein level is high in VHL-deficient cells but not in VHL-positive cells at a high cell density. It is worth noting that there are three HIF-binding consensus sequences (HRE) in the cyclinD1 promoter region (within-1745 bp), which can bind to HIF2α at least in vitro (MB, unpublished data). With this observation only, we cannot conclude whether or not HIF induces cyclinD1 mRNA transcription directly. Further analysis is required to clarify the mechanism of cyclinD1 regulation by HIF. In any case, the observations reported here suggest that the aberrant expression of HIF because of the loss of VHL at the high cell density induces the expression of cyclinD1 and abolishes the contact inhibition of cell growth. These findings suggest the presence of a novel signal transduction pathway linking hypoxia signal and cell proliferation.

Effect of the VHL failure becomes notable only at high cell density

pVHL does not affect the growth rate of cells growing logarithmically at a low cell density (Iliopoulos et al., 1995; Baba et al., 2001), and the expression level of cyclinD1 remains at a high level regardless of the presence or absence of pVHL (Figure 3a, b). In such cells, extracellular growth signals may induce cyclinD1 expression (Matsushime et al., 1991; Won et al., 1992; Lavoie et al., 1996) via pVHL- and HIF-independent pathways (Figure 7 I, III), which may be sufficient for obtaining the maximum rate of cell cycle progression. At a high cell density, cell–cell contact inhibits the growth signal pathway including ERK (Figure 3c), and cyclinD1 expression is suppressed in normal cells (Vinals and Pouyssegur, 1999), in which HIF is degraded by pVHL and loses its function. As a result, the cyclinD1 expression level remains low and cell growth is suppressed (Figure 7 (I)). In VHL (−) cells, the growth signal pathway including ERK is also suppressed by cell–cell contact (Figure 3c). However, the expression level of the HIF protein induced by the loss of VHL is not reduced even at the high cell density (Figure 3b, c), and this protein now plays an essential role in the induction of cyclinD1 expression (Figure 7 (IV)).

Figure 7
figure7

Model of cyclinD1 regulation by pVHL at different cell densities. (I) VHL-positive normal cells growing in logarithmic phase. In these cells, extracellular growth signal induces cyclinD1 expression via the activation of ERK. HIFα is degraded by pVHL. (II) Contact inhibited VHL-positive normal cells. The cell–cell contact inhibits the growth signal pathway including ERK, and cyclinD1 expression is suppressed. Since HIFα degradation by pVHL occurs regardless of cell density, the cyclinD1 level remains at a low level. (III) VHL-deficient cells in logarithmically growing phase. The main signal that upregulates cyclinD1 is extracellular growth signal via ERK. HIFα can also contribute to a certain extent. (IV) VHL-deficient cells at high cell density. While the growth signal via ERK is suppressed, accumulating HIFα keeps the expression of cyclinD1 at a high level

Molecular mechanism of carcinogenesis induced by loss of VHL

Among many reported molecular mechanisms that explain the deregulated cell growth caused by the inactivation of VHL, the most practical one is that the deregulation of HIF (Kondo et al., 2002a) followed by overproduction of HIF target genes. A representative target, VEGF (Iliopoulos et al., 1996; Siemeister et al., 1996), causes hypervascularization and was proved to contribute to tumor growth (Ferrara, 2002). However, at the primary phase of tumorigenesis, neovascularization may not be critical. As one of other targets of pVHL, TGFα was reported (Knebelmann et al., 1998), which forms a TGFα/EGFR autocrine loop to accelerate the proliferation of renal cell carcinoma cells (de Paulsen et al., 2001). However, our data obtained from the coculture assay indicate that the some autocrine/paracrine loops if any may not be sufficient to disrupt contact inhibition in VHL-deficient cells (Figure 1). Therefore, we suppose that pVHL is involved in the cell-autonomous regulation of epithelial cell proliferation. The present finding that pVHL regulates cyclinD1 expression at a high cell density can explain such cell-autonomous regulation and tumor suppressor function of VHL at the initial step of tumorigenesis, the so-called carcinogenesis.

Whether or not the upregulation of cyclinD1 alone following the loss of VHL is sufficient for carcinogenesis remains to be elucidated. CyclinD1 plays an important role in the regulation of G1/S transition (Sherr, 1995; Weinberg, 1995), which is a critical event for cells in which the cell cycle may proceed or be arrested by contact inhibition. In fact, there is much evidence supporting the involvement of cyclinD1 in carcinogenesis and cell transformation (Jiang et al., 1993; Hinds et al., 1994). Recently, one group reports that Ras-induced focus-forming activity correlates with sustained cyclinD1 expression at a high cell density and the activation of Ras is sufficient to relieve cells from contact inhibition (Jacobsen et al., 2002). However, cyclinD1 overexpression alone cannot induce the focus formation, an indicator of the loss of contact inhibition, in dish culture cells (Hinds et al., 1994). Therefore, unknown factors upregulated by oncogenic ras in addition to cyclinD1 might be required for the focus formation. The contribution of such additional factors may also be the case of the crisis of contact inhibition by the loss of VHL. Even though our data shown in Figure 1 indicate that the release of growth factors such as TGFα is not sufficient for the acceleration of cell growth at a high cell density, we cannot rule out the possibility that the production of growth factors work together with cyclinD1 in deregulated cell growth at a high cell density.

Two types of CCRCC

CCRCCs are classified into the two subgroups, VHL alteration related and nonrelated. In our recent study, alteration of the VHL gene occurs in 58% of sporadic CCRCCs and alteration of VHL gene is an independent determination factor for the good prognosis of CCRCCs (Yao et al., 2002). Here we reported the correlation between the elevated expression level of cyclinD1 and alteration of the VHL gene in surgical specimens of sporadic CCRCCs. This observation in vivo supports the notion that pVHL inactivation leads to deregulated expression of cyclinD1 and suggests that the high level of expression of cyclinD1 is a factor in the generation of VHL alteration-related CCRCCs. Why is the prognosis better for patients with an altered VHL gene? In VHL nonaltered CCRCCs, there might be unknown trigger(s) other than VHL alteration, that are associated with not only carcinogenesis (loss of contact inhibition) and the clear cell phenotype, but also with metastatic activity or another malignant phenotype. In VHL alterated CCRCCs, VHL alteration is the trigger of carcinogenesis and the expression of the clear cell phenotype but not of the metastatic activity or another malignant phenotype, which grows worse the prognosis of patients. CCRCC is resistant to radiation therapy and anticancer agent, and operation only enables radical cure. Our new findings can contribute to clearer understanding of CCRCC incidence and to the development of new drugs and therapy for CCRCCs.

Materials and methods

Cell culture

The renal cell carcinoma cell line lacking the wild-type VHL, 786-O, and its stable transfectants (Wt8: HA-VHL1-213 a.a.-expressing 786-O) were a gift from Dr William G Kaelin (Iliopoulos et al., 1995). AC6 was established by the stable transfection of 786-O cells with an expression vector for pVHL1-213 a.a. and pSV2-Neo followed by their culture in a selection medium containing 1 mg/ml G-418. These cell lines were maintained in 10% FCS/DMEM at 37°C in a humidified, 5% CO2 atmosphere. Hypoxic incubation was in an atmosphere of 1.5% oxygen/5% CO2/balance nitrogen in an incubator (MCO-175M Sanyo, Japan). The inhibitor of prolyl hydroxylases (DFO: desferrioxamine or CoCl2) were added to the culture medium at a final concentration of 100 μ M.

For the coculture experiment, TranswellR membranes, 16.2 mm in diameter, 0.4 μm pore size (Corning, USA) were used. The cells for counting were seeded on the bottom of 24-well plates and the other cells were seeded on a TranswellR membrane separately. One-half of the culture medium was changed to a fresh one every 24 h during cell growth in the log phase. After the cells reached confluency, the entire amount of medium was changed to a fresh one every 24 h. At each time point, the culture medium was removed and plates were washed with ice-cold PBS three times for cell counting using Papadimitriou's method (Papadimitriou and Lelkes, 1993). Briefly, after 1-h incubation at 37°C with 555 μl of water in each well, cells were frozen at −80°C. Then cells were thawed at room temperature and homogenized by pipetting. Hoechst 33258 (555 μl) diluted in TNT buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA; 2 M NaCl) at the concentration of 20 μg/ml was added to each well. Then fluorescence intensity was measured by a plate reader, SPECTRA Fluor Plus (TECAN, Austria).

Adenovirus vector transfections

Adenovirus vectors for human pVHL and mouse HIF2αPA were generated using a cosmid vector pAXCAwt (Miyake et al., 1996). HIF2αPA cDNA encoding alanine instead of proline 530 was generated by introducing a point mutation to mouse HIF2α cDNA using an ExSite™ PCR-Based Site-Directed Mutagenesis kit (STRATAGENE, USA) according to the manufacturer's instruction. The primer set for mutagenesis were as follows: IndexTermGGAGACCTTGGCAGCCTACATCC CTATGG and IndexTermCCATAGGGATGTAGGCTGCCA AGGTC TCC.

786-O cells were infected with Ax-W1 (empty vector) or Ax-VHL and or Ax-HIF2αPA at MOI=20, a condition sufficient for nearly 100% infection of the cells.

RNA extraction and GeneChip

786-O cells were infected with Ax-W1 or Ax-VHL at MOI=20. After 12 h, cells were trypsinized and reseeded at different cell densities (subconfluent: 3 × 106 cells/15-cm dish, confluent: 6 × 106 cells/15-cm dish, overconfluent: 6 × 106 cells/10-cm dish). After 48 h of culture under normal conditions, the culture medium was removed and Sepasol-RNAI (Nacalai tesque, Japan) was directly added to the dishes. The cell lysate was extracted with chloroform and total RNA was precipitated in EtOH and purified with a RNeasy Mini kit (QIAGEN, Germany). cDNA was synthesized from the total RNA. And labeled cRNA was synthesized with Bio-11-CTP and Bio-16-UTP (Roche Biochemicals, Germany). Thereafter, the cRNA was purified by column chromatography. HuU95A array (Affymetrix, USA) was used for high-density oligonucleotide arrays. The hybridization was performed as previously described (Iizuka et al., 2002). Each pixel level was collected by a laser scanner (Affymetrix, USA) and the levels of the expression of each cDNA (average difference) and reliability (present/absent call) were calculated with the Affymetrix Microarray Suite ver.4.0 software.

Northern blot analysis

Surgically removed tissues were sampled from the normal part of the kidney and from the tumor, and were immediately frozen by liquid nitrogen. Total RNA extraction was performed by the conventional method using Trizol reagent. Total RNA from cultured cells was prepared using a Quick Prep total RNA Extraction kit (Amersham Biosciences, USA). cDNA fragments corresponding to the whole ORF of human cyclinD1 and human GAPDH were radio labeled with [α-32P]dCTP and used to probe membranes onto which the RNAs were blotted as described previously (Izumi et al., 1997). Hybridization was performed using ExpressHyb™ hybridization solution (Clontech, USA). The signal intensities were directly measured by the BAS2000 system (Fuji Film, Japan). The experiments using human tissues were approved by the ethics committee of Yokohama City University School of Medicine, and informed consent of the person concerned was obtained.

Western blot analysis

Cell extracts were separated by SDS–PAGE on polyacrylamide gel of an appropriate concentration. After electrophoresis, the separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane, Immobilon™-p (Millipore, USA). The blotted membrane was soaked in PBS containing 5% skim milk overnight at 4°C. The membrane was then soaked in 5% calf serum in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h, and then incubated with the antibody appropriately diluted in TBST containing 0.1% bovine serum albumin for 1 h at 37°C. After washing with TBST, the membrane was incubated with the peroxidase-conjugated rabbit or mouse IgG antibody (Amersham Biosciences, USA) in TBST containing 5% skim milk. The membrane was washed again and the immuno reactions were visualized with an ECL or ECL Plus chemiluminescence system (Amersham Biosciences, USA). The intensity of the luminescence was directly quantified using a CCD camera combined with an image analysing system LAS-1000 (Fuji Film, Japan). Affinity-purified rabbit polyclonal anti-pVHL antibody was generated as previously described (Baba et al., 2001). The antiactin antibody (BT560, rabbit polyclonal) was from Biomedical Technologies (USA). The anti p-27, cyclinA, cyclinE, p130 antibodies (rabbit polyclonal) were from Santacruz (USA). The anti-Rb antibody (BD-554136, mouse monoclonal) was from PharMingen (USA). The anticyclinD1 antibody (72-13G, mouse monoclonal) was from Santacruz (USA). The anti-HIF2α antibody (NB100-122, rabbit polyclonal) was from Novus (USA). The antiphospho Erk antibody (V803A, rabbit polyclonal) was from Promega (USA). The anti-Glut-1 antibody (rabbit polyclonal) was from Alpha Diagnostic Inc. (USA).

Mutation analysis and methylation-specific PCR assay

High-molecular-weight genomic DNA was prepared from tumor and normal kidney samples by proteinase K/phenol chloroform extraction using standard protocols. SSCP/heteroduplex analysis and direct sequencing for detecting intragenic mutation of the VHL-coding region were performed essentially as previously described (Shuin et al., 1994). The methylation status of the VHL promoter region was examined by methylation-specific PCR assay as previously described (Kondo et al., 2002b).

Abbreviations

VHL:

von Hippel–Lindau

CCRCC:

clear cell renal cell carcinoma

VEGF:

vascular endothelial growth factor

SDS–PAGE:

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

HIF:

hypoxia-inducible-factor

Glut-1:

glucose transporter-1

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Acknowledgements

We thank Dr William G Kaelin for 786-O cells and their VHL-expressing subclones. We also thank Y Amano for the excellent technical support.

This work was supported in part by Grants in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and Grants from the Japan Society for the Promotion of Science. MB received support from the Yokohama Foundation for Advancement of Medical Science, Yokohama Academic Foundation and Public Trust Haraguchi Memorial Cancer Research Fund.

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Correspondence to Syuiohi Hirai or Shigeo Ohno.

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Baba, M., Hirai, S., Yamada-Okabe, H. et al. Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through Hypoxia-inducible factor. Oncogene 22, 2728–2738 (2003). https://doi.org/10.1038/sj.onc.1206373

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Keywords

  • VHL
  • tumor suppressor
  • contact inhibition
  • renal cell carcinoma
  • cyclinD1
  • HIF

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