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*
Cell Signalling Group, Department of Renal Medicine, Guy's, King's College
and St. Thomas' School of Medicine, King's College London, London, United
Kingdom
Department of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad,
California.
Correspondence to Dr. Bruce M. Hendry, Department of Renal Medicine, King's College London, Bessemer Road, London SE5 9PJ, United Kingdom. Phone: 44-171-346-3741; Fax: 44-171-346-3742; E-mail: bruce.hendry{at}kcl.ac.uk
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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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A host of cytokines, growth factors, and integrins have been implicated in
this pathogenic process, including transforming growth factor ß1,
fibroblast growth factor, epidermal growth factor (EGF), platelet-derived
growth factor, thrombin, angiotensin II, interleukins 1, 6, and 8,
endothelin-1, and 
1, 2, 5, v, and ß1
integrins
(2,3,4,5,6,7,8).
Although each of these factors activates a specific cell surface receptor,
many of their intracellular transduction pathways have been demonstrated to
converge on the Ras monomeric GTPases, which act as molecular switches
transducing signals to downstream effector cascades
(9,10,11).
For this reason, we chose to investigate strategies designed to inhibit the
function of Ras in stimulated renal fibroblasts.
Three closely related isoforms of Ras, known as Harvey (Ha)-, Kirsten (Ki)-, and neural (N)-Ras, are expressed in mammalian cells. Ki-Ras may be expressed in two splice variants, Ki(4A) and Ki(4B), which differ in their terminal fourth exon. The specific functions of these isoforms have not yet been fully elucidated. We previously presented evidence that Ki-Ras is the predominantly expressed isoform in a primate renal fibroblast cell line (Vero), where it is the only isoform involved in EGF-, fibroblast growth factor-, and serum-stimulated proliferation (12).
This work is a study of Ras isoforms in adult human renal fibroblasts in primary culture and was designed to establish which isoforms are expressed and involved in the stimulation of proliferation. Reverse transcription (RT)-PCR and Ras isoform-specific monoclonal antibodies were used to define expression. 2'-O-Methoxyethyl-modified chimeric phosphorothioate antisense oligonucleotides were used to block translation of specific Ras isoforms during serum- and EGF-stimulated cell proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary Fibroblast Culture
Tumor-free renal cortex was obtained from four native kidneys, all of which
had been removed because of renal cell carcinoma. No other renal condition had
been diagnosed for any of the patients. Ethical permission for this use of
renal tissue was obtained from the King's College Hospital ethics committee.
After removal of the capsule, the cortex was minced and digested in a 1 mg/ml
solution of collagenase type IV (Sigma Chemical Co., Poole, Dorset, UK). The
digest was passed through a 70-µm mesh, to remove contaminating glomeruli
and tubules, and then separated on a 50% Percoll density gradient (containing
0.84 g of mannitol and 100 µl of 1 M HCl in 30 ml) by centrifugation at
30,000 x g. The top band (F1) was seeded onto 10-cm culture
dishes (90 mg/dish) in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12
Nut-mix (Gibco BRL, Life Technologies Ltd., Paisley, UK) with 10% fetal calf
serum (FCS), allowed to grow to confluence, and then passaged twice in the
presence of serum, to specifically select for fibroblast growth. The cells
were typed by immunocytochemical methods and stained positively for vimentin
and negatively for cytokeratin. Approximately 95% of cells were phenotypically
fibroblastic after two passages.
Oligonucleotides
The antisense oligonucleotides used were single-stranded, phosphorothioate,
20-mer, 2'-methoxyethyl-modified oligonucleotides
(13) targeted to
Ha-ras (ISIS 13920; sequence,
5'-TCCGT-CATCGCTCCTCAGGG-3'; binds to Ha-ras mRNA at the
initiation start site codon, AUG), Ki-ras (ISIS 15170; sequence,
5'-CAGTGCCTGCGCCGCGCTCG-3'; binds to the 5' untranslated
region of Ki-ras mRNA), and N-ras (ISIS 16737; sequence,
5'-CCATACAACCCTGAGTCCCA-3'; binds to the 3' untranslated
region of N-ras mRNA). An oligonucleotide complementary to a part of
the HIV promoter sequence was used as a control (ISIS 15167; sequence,
5'-TCAGTAATAGCCCCACATGG-3'). This control sequence is not
complementary to any known part of the human genome. The Ki-Ras
oligonucleotide, which is complementary to a region close to the initiation
codon, would be expected to affect both splice variants (4A and 4B).
The oligonucleotides were transfected into the cells using Lipofectin (Life Technologies BRL), with 6- to 18-h incubations at oligonucleotide concentrations of 100 to 200 nM. An initial solution of Lipofectin at 6 to 12 µl/ml OptiMEM (Life Technologies) was vortex-mixed and incubated at room temperature for 45 min. Oligonucleotide was then added, and the vortex-mixed solution was maintained for an additional 15 min before being added to the cells. The Lipofectin/oligonucleotide ratio was 0.3 µl/10 pmol in each case.
RT-PCR
To determine which isoforms of ras are transcribed in the primary
culture cells, isoform-specific primers were designed for use in RT-PCR. These
were as follows: N-ras: forward,
5'-GAAAAGCGCACTGACAATCC-3'; reverse,
5'-CACCACACATGGCAATCCC-3'; Ki-ras: forward,
5'-AGTGCCTTGACGATACAG-3'; reverse, Ki(4B),
5'-GCATCATCAACACCCTGTCTT-3' (spanning the exon 3-exon 4B
junction); Ki(4A), 5'-AAGAAGAAAAGACTCCTGG-3' (in exon 4A);
Ha-ras: forward, 5'-CAAGAGTGCGCTGACCATCC-3'; reverse,
5'-CCGGATCTCACGCACCAAC-3'. Cells were grown to 80% confluence in
DMEM/Ham's F-12 Nut-mix with 10% FCS, in 75-cm2 flasks, and washed
once with phosphate-buffered saline, and total RNA was extracted using the
Qiagen RNeasy mini-kit (Qiagen, Crawley, UK). To assess the effects of the
oligonucleotides on mRNA levels, the cells were transfected with 200 nM
oligonucleotide for 16 h, followed by immediate RNA extraction, as described
above. The RT and amplification reactions were performed using a Promega
Access RT-PCR kit (Promega, Southampton, UK), following the instructions
provided by the manufacturer, with 0.5 µg of total RNA. The annealing
temperatures used were 63°C for Ha- and N-ras and 59°C for
Ki(4A)- and Ki(4B)-ras. As an internal standard, 18S ribosomal RNA
primers combined with primer competimers in a 3:7 ratio were used. These were
supplied in the Ambion Quantum RNA 18S internal standards kit (Ambion,
Whitney, Oxfordshire, UK). The optimal primer/competimer ratio was determined
by serial alterations, following the instructions provided by the
manufacturer. Ten percent of the reaction products were analyzed on a 1.5%
agarose gel and stained with ethidium bromide.
For initial detection of ras isoform mRNA, 35 cycles of PCR were used. The linear phase of the amplification reaction was identified for each primer pair by varying the cycle number from 10 to 40. Ten percent of the reaction mixture was observed on an agarose gel, and the bands were analyzed using densitometry. For each pair, a cycle number in the linear phase of amplification was used in experiments to determine the effects of the antisense oligonucleotides on mRNA levels for that isoform.
Western Blotting
For Western blotting experiments, human fibroblasts were grown to 50 to 80%
confluence in 35- or 100-mm plates, in DMEM/Ham's F-12 Nut-mix containing 10%
FCS. Cell lysis detergent medium was composed of 1 x phosphate-buffered
saline, 1% Triton X-100, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, 0.5
µg/ml leupeptin, 1.0 µg/ml pepstatin, 1.0 mM
ethylenediaminetetraacetate, and 0.2 mM phenylmethylsulfonyl fluoride. At this
point, cells were either lysed or, for experiments involving the
oligonucleotides, transfected for 16 h with 200 nM oligonucleotide in OptiMEM.
After transfection, the medium was removed and replaced with DMEM/Ham's F-12
Nut-mix containing 10% FCS. Twenty-four hours later, the cells were washed
with phosphate-buffered saline and lysed. Nuclear debris was removed by
centrifugation at 2300 x g for 15 min at 4°C. Cell lysates
were assayed for protein content, and equal amounts of protein were made up to
a uniform volume. Immunoprecipitation was performed by adding 15 µl of rat
anti-pan-Ras antibody-agarose conjugate (clone Y13-259)/ml lysate and shaking
the mixture overnight at 4°C. Pellets were then resuspended in 40 µl of
Western sample buffer containing 5% 2-mercaptoethanol and were heated to
100°C for 3 min. The agarose was removed by pelleting for 10 s, and the
supernatants were collected, cooled, and loaded on a 4%/15% discontinuous
polyacrylamide gel. After blotting, detection was performed using either an
anti-pan-Ras monoclonal antibody (mouse anti-pan-Ras antibody clone Ras 10,
1.0 µg/ml) or the isoform-specific monoclonal antibodies. The dilutions of
the isoform-specific antibodies used were determined using slot-blot analysis
with the recombinant proteins. The concentrations chosen yielded equivalent
densitometric signals for 200 ng of the specific isoforms, without
cross-reaction with the other isoforms. This method was previously described
(12).
Cell Proliferation
To study the actions of oligonucleotides on proliferation, human
fibroblasts were trypsinized, resuspended in OptiMEM, and placed in triplicate
in 96-well plates (5000 cells/well). A 200 to 400 nM
oligonucleotide-Lipofectin suspension was added, to yield a final
oligonucleotide concentration of 100 to 200 nM, and cells were incubated for
14 to 18 h. Extracellular oligonucleotide was then removed, and cells were
grown in minimum essential medium with either 1000 ng/ml EGF or 10% FCS. The
growth incubation was performed at 37°C in 95% O2/5%
CO2, and cell numbers were determined at 0 and 48 h after
transfection. The plating densities determined from the time 0 measurements
demonstrated <5% variability between wells in individual experiments. Cell
numbers were determined in MTS assays (Cell Titer 96; Promega), with
measurement of absorbance at 490 nm. The MTS assay is a tetrazolium salt-based
assay of viable cell numbers, and the correlation between cell number and
absorbance at 490 nm is linear
(14).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 2 illustrates the RT-PCR detection of mRNA for the various ras isoforms, as well as the 18S ribosomal subunit, which acts as an internal control. Figure 2A demonstrates the presence of mRNA for Ha- and N-ras and the Ki(4B) splice variant of Ki-ras. Ki(4A)-ras mRNA could not be detected.
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Figure 2 (B to D) shows experiments designed to examine the activity and specificity of the antisense oligonucleotides, using linear-range RT-PCR. Figure 2B presents RT-PCR products obtained using Ki(4B)-ras primers with mRNA from cells treated with each of the four oligonucleotides. Incubation with the Ki-Ras antisense oligonucleotide at 200 nM for 16 h abolished the Ki(4B)-ras RT-PCR product; the Ha-Ras and N-Ras antisense oligonucleotides were inactive, compared with the control oligonucleotide. Similarly, Figure 2C was obtained using the Ha-ras primers and demonstrates depletion of Ha-ras mRNA by the Ha-Ras antisense oligonucleotide alone. Figure 2D demonstrates that the N-Ras oligonucleotide is equally specific in the depletion of mRNA for its target isoform. These results demonstrate specific effects of the three Ras antisense oligonucleotides on the levels of cellular mRNA for each isoform.
Western blot analysis was performed to assess which Ras protein isoforms were present and in what relative quantities. Cells grown in 10% FCS were lysed, either immediately or after 30 h in serum-free medium, immunoprecipitated with an anti-pan-Ras monoclonal antibody, and immunodetected with isoform-specific monoclonal antibodies, as shown in Figure 3A. The predominant isoform detected was Ki-Ras (representing >95% of the total Ras protein detected). Small quantities of N-Ras were detected when levels were analyzed using densitometry. Ha-Ras either was not detected or was detected in only very small quantities. The removal of serum for 30 h had no effect on either total Ras expression or the pattern of expression of the individual isoforms.
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The effects of the antisense oligonucleotides on the total cellular expression of Ras were studied. Cells were transfected with antisense oligonucleotides at 200 nM or treated with Lipofectin alone, and protein was extracted 24 h later. Figure 3B presents a Western blot analysis of the expression of total Ras protein after these treatments; the immunodetection was performed using an anti-pan-Ras monoclonal antibody. The Ki-Ras antisense oligonucleotide markedly reduced total cellular Ras levels (by 70 to 85%), compared with the Ha-Ras or control oligonucleotides (which were inactive, compared with cells undergoing mock transfection with Lipofectin alone). The N-Ras oligonucleotide also had no effect on total cellular Ras levels (data not shown).
Figure 4 demonstrates the effects of Ras antisense oligonucleotides on the serum-stimulated proliferation of human renal fibroblasts in primary culture. Cell numbers were measured after a 48-h incubation with 10% serum after transfection with oligonucleotides. The Ki-Ras oligonucleotide at 100 nM reduced serum-stimulated proliferation by >50%, compared with data obtained with the control oligonucleotide (P < 0.01). The N-Ras oligonucleotide was not active, compared with the control oligonucleotide. The Ha-Ras oligonucleotide was not significantly active at 100 nM, but it reduced serum-stimulated proliferation by 13% at 200 nM (P < 0.01).
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Figure 5 demonstrates the results of experiments on EGF-stimulated proliferation. The Ki-Ras oligonucleotide at 100 nM reduced EGF-stimulated proliferation by 25%, compared with the control oligonucleotide (P < 0.01). The N-Ras oligonucleotide was not active, compared with the control oligonucleotide. The Ha-Ras oligonucleotide was not significantly active at 100 nM but it reduced EGF-stimulated growth by 40% at 200 nM (P < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Relative quantification of mRNA for each ras isoform during the linear phase of amplification was achieved by comparing the abundance of the product obtained with the relevant primer pair with the abundance of the 18S ribosomal subunit RT-PCR product, as demonstrated in Figure 2. The transfection efficiency for the oligonucleotides used was high (>90%) (Figure 1). This is consistent with the data in Figure 2, showing that the antisense oligonucleotides were both highly effective and specific in reducing the levels of their target mRNA. Additional evidence for the effectiveness of the Ki-Ras antisense oligonucleotide is provided in Figure 4, in which the Ki-Ras antisense oligonucleotide clearly reduced total cellular Ras levels and the other oligonucleotides tested exhibited no action.
Ki-Ras not only is the predominantly expressed isoform but also is of functional significance in the control of proliferation, as demonstrated in Figures 4 and 5. In both serum- and EGF-stimulated growth assays, the Ki-Ras antisense oligonucleotide was able to significantly reduce cell proliferation, even at the lower oligonucleotide concentration. Although apparently present in low abundance, Ha-Ras also seems to play a distinct role in the stimulation of proliferation. The antiproliferative effect of the Ha-Ras antisense oligonucleotide at 200 nM in serum-stimulated growth assays was not as large as the effect of the Ki-Ras oligonucleotide (13 versus 23%). In EGF-stimulated growth assays, however, the Ki- and Ha-Ras oligonucleotides reduced proliferation equally, by approximately 40%. These results suggest that Ki- and Ha-Ras have distinct roles in the control of proliferation and that both are independently necessary for EGF-stimulated growth. The partial suppression of proliferation by the Ki- and Ha-Ras oligonucleotides does not seem to be the result of inefficient transfection, with a complete inhibitory action on only a subpopulation of cells. It is likely that Ras-independent pathways for the stimulation of proliferation also exist in these cells. Although the relative effects of different Ras isoforms may vary under different growth conditions, this does not seem to be attributable to changes in either total cellular Ras levels or the relative quantities of the isoforms. Serum starvation had no effect on Ras expression (normalized to total cellular protein levels), compared with growth in the presence of 10% FCS (Figure 3A), suggesting that the overall pattern of Ras expression in these cells remains stable.
Although little is known regarding the specific function of each of the Ras isoforms, there is increasing evidence that the isoforms do participate in distinct signal transduction pathways, which would enable them to have individual biologic roles. Many of the cell surface receptors originally identified as acting through Ras were receptor tyrosine kinases; more recently, G-protein-coupled receptors and integrins have been demonstrated to activate Ras-dependent pathways (9,10,11,16). These different cell surface receptors (directly or indirectly) influence the activation state of Ras via a group of guanine nucleotide exchange factors. These factors bind to Ras and cause it to disassociate from GDP and bind to GTP, thereby activating it. An opposing group of GTPase-activating proteins regulate this process by enhancing the intrinsic ability of Ras to hydrolyze GTP to GDP, thereby turning off the signal (17). Many guanine nucleotide exchange factors have been identified, some of which have been shown to be receptor type- and Ras isoform-specific (18,19), suggesting that individual growth factors and hormones may be able to activate individual isoforms of Ras.
Downstream, Ras is known to bind many effectors with different functional end points, although the relative affinities of the isoforms for the various effectors may differ. Voice et al. (20) recently demonstrated that the four human Ras isoforms differ in their abilities to activate Raf-1, with Ki(4B)-Ras being the most efficient, followed by Ki(4A)-, N-, and Ha-Ras (in descending order). Hamilton and Wolfman (21) demonstrated in murine fibroblasts that N-Ras had a higher affinity for Raf-1 than did Ha-Ras, although both were able to transform cells when constitutively active. The transforming ability of Ha-Ras was abrogated when N-Ras was removed, suggesting that this second isoform was required for effective Ha-Ras transformation. A study by Yan et al. (22) that compared Ki- and Ha-Ras suggested that Ki-Ras recruits Raf-1 to the plasma membrane more efficiently than does Ha-Ras but Ha-Ras is a more potent activator of phosphoinositide 3-kinase. One possible origin of these differences involves the different orientations of Ras isoforms at the inner leaflet of the cell membrane, which arise from the presence of a polybasic region near the carboxylterminus of Ki(4B)-Ras, compared with an S-acyl group for Ha-Ras (23).
When these data are considered together with the results presented in this
report, it seems likely that the different isoforms of Ras regulate
proliferation via different downstream cascades, and more than one isoform may
be necessary for sustained growth. Seufferlein et al.
(24) recently demonstrated
that transforming growth factor -stimulated cell growth in two
pancreatic cancer cell lines possessing oncogenic Ki-ras mutations is
dependent on the activation of Ha-Ras. This unexpected result supports the
concept that Ki-Ras and Ha-Ras play distinct roles in the control of cell
proliferation. In our previous work, we demonstrated the predominant role of
Ki-Ras in the stimulated proliferation of the Vero primate renal fibroblast
cell line. This predominant role was also demonstrated by Chen et al.
(15) in a human lung
fibroblast cell line. The work on primary human cultures presented here
demonstrates that Ha-Ras may also play an important role in renal fibroblast
proliferation, and it is this result that is more likely to reflect the
isoform-specific expression and function of Ras in renal fibroblasts in
vivo. Whether this Ras expression pattern is maintained for fibroblasts
in both the quiescent and stimulated states in vivo needs to be
examined in both normal and early fibrosing renal tissue. This could be
achieved most effectively using enhanced in situ hybridization
techniques to study both cell type- and condition-dependent Ras expression.
When this issue has been clarified, it will be possible to assess whether a
specific Ras isoform, such as Ki- or Ha-Ras, might be suitable as a
therapeutic target in renal fibrosis.
Ras has been a target for therapeutic intervention since it was discovered to be mutated in 30% of human cancers (25,26,27). Many of the agents investigated were designed to inhibit post-translational modification of Ras. To become membrane-bound (enabling activation), Ras is prenylated, usually by the addition of a farnesyl group (23,26). The enzyme that catalyzes this process, farnesyltransferase, is the target of highly specific inhibitors, which are currently undergoing trials among patients with cancer (26). These inhibitors may be useful future tools for the prevention of renal fibrosis, but they offer little Ras isoform specificity. To obtain this specificity, antisense oligonucleotides could themselves be used for therapeutic intervention. There is some evidence that oligonucleotides can be targeted to different cell types within the kidney, thereby increasing the specificity of the system and reducing unwanted side effects. One possible strategy uses liposome vectors modified from the hemagglutinating virus of Japan. These are able to deliver DNA to either tubular cells or interstitial fibroblasts, depending on the lipid composition of the vector (28). By targeting the Ras isoforms in this way, it may be possible to directly influence renal interstitial fibroblast proliferation and circumvent the problem of redundancy of the numerous cytokines and growth factors that have been implicated in this pathologic process.
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