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Tumor Biology |
Department of Urology, Kochi Medical School, Nankoku, 783-8505, Japan [M. K., H. O., T. S.]; Laboratory of Radiation and Life Science, School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852-8521, Japan [K. S.]; Kanagawa Dental College, Department of Biochemistry, Yokosuka, 238-8580, Japan [Y. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(8)
, the GLUT-1 glucose transporter (9)
, and VEGF (10, 11, 12, 13, 14)
. It has been shown that VHL-associated tumors are highly vascularized and overproduce VEGF. Recently, it was demonstrated that hypoxia inducible factor, which is required for the transcription for VEGF, interacts with the ß-domain of pVHL to cause its degradation (15
, 16)
. Roles for pVHL in fibronectin matrix assembly and reactivity to hepatocyte growth factor have been reported (17)
. Fibronectin is an extracellular matrix component and functions as a tumor suppressor (18
, 19)
. These studies have shown a specific relationship between pVHL and cytoskeleton-associated proteins. Thus, it is likely that pVHL regulates cytoskeletal organization. However, there is as yet no direct evidence for this. Changes in cellular morphology occur as a result of cell adhesion, cell motility, progression of the cell cycle, cell differentiation, and neoplastic conversion. Actin plays a major role in such morphological changes, and its reorganization should occur simultaneously with changes in cellular morphogenesis. The processes of the cellular morphogenesis pathway are signaled through various components such as growth factors, growth factor receptors, integrins, G proteins, and PTKs (20) . However, little is known about the cytoskeletal reorganization and the morphological changes induced by pVHL expression.
This study indicates that cytoskeletal organization, focal adhesion formation, and cell motility are significantly affected by pVHL in the RCC cell line UMRC-6 and lung carcinoma cell line H1299. Actin filaments are stabilized as stress fibers through the development of focal adhesions by pVHL, and cell motility is significantly inhibited by the stabilization of cytoskeletal proteins. Here, we provide evidence that pVHL induces dynamic changes in cell shape with actin reorganization and acts as a unique tumor suppressor protein.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids and Transfections.
To express pVHL in the cells, we used an ecdysone-inducible expression vector. The pVgRXR, pIND, and pIND/lacZ plasmids were obtained from Invitrogen (San Diego, CA). pIND/VHL was prepared by cloning the HindIII/XbaI fragment of human wild-type-VHL cDNA (1.3 kbp) into pIND. H1299 cells were transfected with the plasmids using the electroporation method. Clone selection was performed with zeocin (100 µg/ml) for pVgRXR and with G418 (400 µg/ml) for pIND/VHL. To induce pVHL expression, ponasterone A (a synthetic analogue of ecdysone; Invitrogen, San Diego, CA) was added to the culture medium (final concentration, 2 µM) for 0–96 h. A stable clone obtained by double selection, 99VHL, was screened by immunoblotting using an anti-pVHL monoclonal antibody.
Attachment and Cell Spreading Assays.
We measured three parameters of cell morphology: attachment to glass, cell spreading, and cell area. Cells (1x104) were seeded onto chamber slides and incubated at 37°C for periods from 10 min to 3 h. Cell attachment was performed as reported previously (21)
and measured as the number of cells remaining in each microscope field (expressed as the percentage of cells/field). The number of attached cells was counted in 10 randomly selected fields of phase-contrast microscopy at a magnification of x200. The results are presented as means ± SD (in triplicate assay from three independent experiments). Statistical analysis was performed using Student’s t test.
Cell spreading was measured as the proportion of cells with a spread morphology among 100 cells (expressed as the percentage of cells). Cell morphology was assigned to one of three types (round, partially spreading, or fully spreading; 22 ). These experiments were performed in triplicate and the results presented as means ± SD. Statistical analysis was performed using Student’s t test or the Mann-Whitney U test.
The extent of cell spreading was examined by a cell area assay. The area of each cell was measured using NIH Image 1.62 (NIH, Bethesda, MD). One hundred cells were measured and the results presented as means ± SD. The significance of the differences was determined by Student’s t test.
Quantification of Focal Contact and Stress Fibers.
Focal adhesion formation was measured by a modification of the method of Woods and Couchman (23)
. After cells were cultured for 48 h, they were harvested by trypsinization and inoculated onto chamber slides. Cells were allowed to adhere to a normal chamber glass for 3 h, processed for immunofluorescence, and then stained for vinculin or F-actin. Preparations were examined using a fluorescence microscope (Laser Scan Microscope LSM410 Invert; Carl Zeiss, Jena, Germany). In each microscope field at a magnification of x200, the number of cells that had a typical pattern of vinculin-rich focal adhesions or stress fibers was counted and expressed as a proportion of the total number of cells in the field. Ten fields were examined, and data are presented as means ± SD (in triplicate assay from three independent experiments). The significance of the differences was analyzed by Student’s t test.
Membrane Fractionation.
Cells were washed in ice-cold PBS and then scraped off with a rubber policeman into a buffer containing protease inhibitors [10 mM Tris-HCl (pH 7.4), 1% NP-40, 0.15 M NaCl, 1 mM EDTA, 1 mM Na3VO4, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 mM PMSF]. The cell suspensions were lysed on ice with 100 strokes of a homogenizer, and centrifuged at 1,200 x g at 4°C for 30 min. The supernatants were additionally centrifuged at 100,000 x g at 4°C for 30 min, after which the pellet was collected as the membrane fraction.
Western Blotting Analysis.
Cell lysates were prepared by standard methods (24)
. Briefly, the blot was incubated with primary antibody overnight at 4°C, and then incubated with horseradish peroxidase-conjugated antimouse IgG for 1 h at room temperature. The primary antibodies used were mouse monoclonal antihuman VHL antibody (BD PharMingen, San Diego, CA) and mouse monoclonal anti-vinculin antibody (Sigma Chemical Co., St. Louis, MO). The experiments were performed in triplicate. Densitometric analysis was performed with NIH Image 1.62.
Immunofluorescence.
Cells (1 x 104) were seeded onto a chamber slide and left to grow for varying periods (10 min to 3 h). The cells were fixed for 20 min with 1% paraformaldehyde and permeabilized with 0.5% Triton X-100 in Tris-buffered saline for 10 min. For F-actin detection, the cells were stained for 30 min with rhodamine-phalloidin (Sigma Chemical Co.). For vinculin detection, the cells were incubated with a monoclonal anti-vinculin antibody (Sigma Chemical Co.) for 2 h. After incubation with the primary antibodies, a secondary FITC-labeled goat antimouse IgG antibody (Sigma Chemical Co.) was applied for 30 min. Coverslips were applied and observations were made using a confocal laser scanning microscope (LSM410).
Cell Migration Assay.
The cell migration assay was carried out using Boyden chambers and type IV collagen-coated polyvinylpyrrolidone-free polycarbonate Nuclepore porous filters (diameter, 13 mm; pore size, 8 µm; Corning, Corning, NY) as described previously (25
, 26)
. Cells (1.2–1.5 x 105) were placed onto type IV collagen-coated filters. After the indicated incubation time (1.5–3 h) in a CO2 incubator, the migrated cells were fixed with methanol, stained with Giemsa solution, and counted in eight randomly selected light microscope fields at a magnification of x200. The results were expressed as means ± SD from triplicate assays.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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pVHL Expression in H1299, 99VHL, and UMRC-6 Cells.
The level of expression of pVHL was examined by Western blotting analysis. pVHL was not detected in H1299 and UMRC-6 cells. Ponasterone A (ecdysone)-inducible clones of 99VHL cells expressed pVHL after treatment with ponasterone A. The maximum level of expression of pVHL in 99VHL cells was obtained at 48 h after induction. This indicated that we needed to replenish the ponasterone A every 2 days to maintain adequate pVHL-expression. pVHL was also detected in UMRC-6-pVHL cells (Fig. 1)
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, a' and b'). In contrast, many rounded or only partially spreading cells were found among non-pVHL-expressing cells (Fig. 2
, a and b). Change in cell shape in H1299 cells was not induced by treatment with ponasterone A (Fig. 2
, c and c').
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. All of the incubated cells were stained with rhodamine-phalloidin. Morphological changes were unremarkable for 10–30 min after seeding (Fig. 3A
; a, a', d, and d'), and the shape of all cells was round after 10 min in culture. At 1–3 h, the shape of both types of pVHL-expressing cells (99VHL(+) and UMRC-6-pVHL cells; Fig. 3A
; b', c', e', and f’) was significantly different from that of non-pVHL-expressing cells [99VHL(-) and UMRC-6 cells; Fig. 3A
, b, c, e, and f], and the size of the pVHL-expressing cells was dramatically increased at 1 and 3 h compared with non-pVHL-expressing cells. After 3 h, a large number of pVHL-expressing cells had actin stress fibers (Fig. 3A
, c' and f'). However, stress fibers were detected in only a few non-pVHL-expressing cells (Fig. 3A
, c and f).
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. The mean areas of 99VHL(-), 99VHL(+), UMRC-6, and UMRC-6-pVHL cells were 0.28 ± 0.075, 1.03 ± 0.74, 0.23 ± 0.12, and 0.36 ± 0.15 units (means ± SD), respectively. Areas of pVHL-expressing cells were significantly increased compared with those of non-pVHL-expressing cells (P < 0.001). In particular, the mean area of 99VHL(+) cells was three times larger than that of 99VHL(-) cells. No increase in cell area was induced by treatment with ponasterone A.
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, c and f; P < 0.05). The proportion of fully spreading cells among pVHL-expressing cells was significantly increased compared with that for non-pVHL-expressing cells at 1 and 3 h (Fig. 3B
; b, c, e, and f; P < 0.05). Cell spreading in H1299 cells was not affected by treatment with ponasterone A.
Because morphological change in cells usually depends on alterations of focal adhesion and actin stress fiber formation, the degree of cell attachment was estimated by a cell attachment assay. pVHL-expressing cells attached to glass more rapidly than non-pVHL-expressing cells, and the number of attached pVHL-expressing cells was significantly larger than the number of attached non-pVHL-expressing cells at all incubation times (10 min to 3 h; Fig. 3C
, a and b; P < 0.05). Similar results were observed on the fibronectin- and type IV collagen-coated slide glasses: the number of attached cells was significantly increased in the pVHL-expressing lines, and their morphology was spread on such substrates (data not shown). The number of attached H1299 and H1299v cells was not increased by treatment with ponasterone A.
pVHL Promoted Vinculin Assembly on the Cell Membrane.
To analyze the focal adhesion formation associated with cell spreading or attachment, vinculin expression was examined by Western blot analysis. All cell lines were found to express vinculin at high levels from 20 min to 3 h after seeding (Fig. 4A
, a). The data from 30 min and 1 h are not shown; however, the amount of vinculin did not change during this time course. To determine the amount of vinculin contained in focal adhesions, membrane fraction lysates were obtained from both pVHL-expressing and nonexpressing cells. Vinculin in the membrane fraction was remarkably higher in the 99VHL(+) cells than in 99VHL(-) cells, suggesting that pVHL contributed to the assembly of vinculin in the membrane rather than to the induction of its expression (Fig. 4A
, b). Similarly, vinculin was detected more in the membrane fraction of UMRC-6-pVHL cells than of UMRC-6 cells (Fig. 4A
, c). For quantification of the amount of vinculin, densitometric analyses were performed, and relative amounts of vinculin were determined. The amount of vinculin in the membrane fractions increased with time in pVHL-expressing cells, and the differences in vinculin content between pVHL-expressing cells and non-pVHL-expressing cells were particularly marked: at 3 h incubation, a 3-fold difference was observed between 99VHL(+) and 99VHL(-) cells (Fig. 4B
, a; P < 0.05), and a 2-fold increase was seen in UMRC-6-pVHL cells compared with UMRC-6 cells (Fig. 4B
, b; P < 0.05).
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a). The proportion of cells displaying the typical vinculin-rich pattern was significantly increased in pVHL-expressing cells: 99VHL(+) and UMRC-6-pVHL cells with typical vinculin patterns were encountered five and two times more frequently than those in 99VHL(-) and UMRC-6 cells, respectively (Fig. 5
a'; P < 0.05). Cells with a typical pattern of actin stress fiber formation were investigated by staining for F-actin (Fig. 5
b). pVHL-expressing cells with a rich stress fiber pattern were seen in significantly higher numbers than non-pVHL-expressing cells with the typical pattern (Fig. 5
b'; P < 0.05).
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; P < 0.05). In particular, the motility of UMRC-6-pVHL cells was one-fourth that of UMRC-6 cells. However, the number of attached cells on type IV-collagen was increased by pVHL expression as mentioned above. These results suggest that pVHL caused inhibition of cell motility through the stabilization of actin stress fibers, which in turn occurred by way of vinculin localized in the cell membrane.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell subsequent to the initial attachment of cells to substrates involves a rearrangement of cytoskeletal and membrane components that can result in the formation of focal adhesions and the reorganization of stress fibers (27
, 28)
. The well-characterized cytoplasmic proteins vinculin, talin, and -actinin accumulate at focal contacts and form part of adhesion plaques (29)
. We showed that pVHL expression stimulated the translocation of vinculin from the cytoplasm to focal adhesions on the cell membrane. Ben-Ze’ev et al. (30)
similarly reported that induction of vinculin in 3T3 fibroblasts stimulated by serum and several growth factors caused the formation of a dotted distribution of adhesion plaques. Because loss of focal adhesion is thought to be able to facilitate cell migration, it was suggested that pVHL inhibits cell migration through vinculin translocation to focal adhesions.
Focal adhesion formation is believed to be regulated by many proteins linked to the small GTP-binding proteins Rho, Rac, and cdc42 (31, 32, 33) . Although stress fiber formation is observed along with focal adhesion, it is now believed that stress fiber and focal adhesion formation are controlled by independent cascades. Rho mediates actin stress fiber formation, whereas Rac and cdc42 induce focal adhesion with lamellipodia and filopodia, respectively. Because the morphological changes induced by pVHL expression are characterized by full spreading with an abundance of focal adhesions and stress fibers, pVHL may be involved in the Rho activation cascades.
We have already reported the binding of pVHL to PKC isoforms (34) . Vinculin phosphorylation by PKC was found to be a crucial step in the assembly of epithelial junctional complexes (35) . PKC is a link to the PTK-mediated signaling pathways and regulates focal adhesion formation (20) . pVHL also binds to fibronectin and promotes extracellular fibronectin matrix assembly (17 , 18) . These studies indicate that pVHL plays an important role in both cell adhesion to a substrate and cell-cell adhesion. Our data suggest that pVHL regulates not only the assembly of a fibronectin matrix, but also the adhesion to the extracellular matrix through focal adhesion formation. Recently, the PTK family associated with focal adhesion has been observed to react to growth factors and to be involved in motility (20) . FAK have been shown to be an important mediator between growth factor receptors and integrin signaling pathways (36) .
Cell motility activity is regulated by a collaboration between Rho and Rab family members which control the assembly and disassembly of actin and focal adhesion (37) . Actin polymerization and depolymerization is regulated by many cytoskeleton-associated proteins. Our results suggest that a shift to actin polymerization occurs as a result of pVHL expression. Thus, pVHL causes inhibition of cell motility through a stabilization of actin stress fibers mediated by vinculin.
In summary, we have demonstrated significant effects of pVHL on the stabilization of actin assembly, focal adhesion formation, and cell motility activity. Although recent research on pVHL has focused on hypoxia inducible factor -mediated VEGF up-regulation, our observations suggest that the processes of growth factor-mediated Rho-PTK signaling and actin fiber reorganization may also be modulated by pVHL. pVHL is an important suppressor factor in the multistep process of invasion or metastasis of cancer. Our results suggest that it acts by limiting the ability of cells to move, through the formation of focal adhesions, and by encouraging the adhesion of a tissue to its substrate. From these findings, we conclude that pVHL is a multifunctional and unique tumor suppressor protein.
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FOOTNOTES |
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1 Supported by a grant from the Ministry of Education, Science, Sports, and Culture of Japan. 
2 To whom requests for reprints should be addressed, at Department of Urology, Kochi Medical School, Kohasu, Okoh-cho, Nankoku, Kochi 783-8505, Japan. Phone: 81-88-880-2401; Fax: 81-88-880-2404; E-mail: shuint{at}kochi-ms.ac.jp
3 The abbereviations used are: VHL, von Hippel-Lindau; RCC, renal cell carcinoma; VEGF, vascular endothelial growth factor; PTK, focal adhesion protein tyrosine kinase; PKC, protein kinase C.
Received 8/14/00. Accepted 3/16/01.
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REFERENCES |
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is a target for the von Hippel-Lindau tumor suppressor. Cancer Res., 58: 226-231, 1998. binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. J. Biol. Chem., 275: 25733-25741, 2000.-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium. J. Cell Sci., : 3563-3571, 1998.
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