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| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
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| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
Medizinische Klinik IV, Universität Erlangen-Nürnberg, Erlangen, Germany.
Correspondence to Dr. Harald O. Schöcklmann, Medizinische Klinik IV, Universität Erlangen-Nürnberg, Nephrologisches Forschungslabor, Loschgestrasse 8, D-91054 Erlangen, Germany. Phone: 49-9131-853-9206; Fax: 49-9131-853-9202; E-mail: Harald.Schoecklmann{at}t-online.de
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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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Numerous factors that stimulate or inhibit MC proliferation, including various cytokines, autacoids, and hormones, have been identified. Many of these factors possess autocrine growth-modulating activities; one example is platelet-derived growth factor (PDGF), which is a potent MC mitogen in culture (5) and in vivo (6). PDGF and its receptor ß-subunit are induced during mesangioproliferative glomerular changes, supporting the role of PDGF as an autocrine growth factor in glomerulonephritis (7). Moreover, blockade of PDGF activity, by infusion of neutralizing antibodies against PDGF, in anti-Thy1.1-treated nephritic rats prevents MC hyperplasia and improves renal function (2). Among the autocrine inhibitors of MC proliferation, transforming growth factor-ß (TGF-ß) has been demonstrated to play a major role in vitro and in vivo (8,9). Although TGF-ß has profibrogenic effects in inflammatory glomerular diseases, it might also exert beneficial effects through its growth-inhibiting properties. Several studies have confirmed the antimitogenic potential of the TGF-ß1 isoform (10,11). Moreover, Kitamura et al. (12) demonstrated that MC overexpression of active TGF-ß1 in anti-Thy 1.1-induced nephritis inhibits glomerular cell proliferation.
Cell cycle progression is controlled by cyclin-dependent kinases (cdk), whose catalytic activity is modulated by association with different cyclins, functioning as regulatory subunits. In mammalian cells, several classes of cyclins have been identified (13) and can associate with different cdk catalytic subunits (14). Three different D-type cyclins (D1, D2, and D3) and cyclin E are involved in controlling G1 phase progression and entry into S phase. D-type cyclins are growth factor-regulated genes. Depending on the cell type, D-type cyclins associate with either cdk4 or cdk6 to form active kinase complexes (15). Cyclin D activates cdk4 during G1 phase (16), which leads to phosphorylation of the retinoblastoma tumor suppressor protein (pRb) (17). This phosphorylation results in the inactivation of pRb and plays an essential role in the progression of cells from G1v to S, mainly through regulation of the E2F family of transcription factors (18). Because cdk targets such as pRb are localized to the nucleus, active cdk complexes must have access to the nuclear compartment. It has been observed that the activation of cyclin D1-cdk4 complexes that occurs in response to extracellular signals involves translocation of the complexes into the nucleus (19). Because no sequence identifiable as a basic nuclear localization sequence (NLS) is present in the primary sequence of vertebrate cdk or cyclins, it has been suggested that cyclin-cdk complexes bind substrates or regulators that contain recognizable NLS (20). Indeed, the cyclin kinase inhibitor (CKI) p21Waf-1, which contains diverse NLS motifs, was recently demonstrated to be important for the translocation of cdk4 and cyclin D1 to the nucleus (21).
Both serum and PDGF are known to induce cyclin D1 mRNA and protein expression in fibroblasts (22,23), and overexpression of the gene for cyclin D1 has been observed in some tumors (24). Terada et al. (25) recently reported that cyclin D1 is the predominant cyclin D isoform in cultured MC. Protein expression of cyclin D1 in rat MC can be induced by mitogens such as endothelin-1 (25) and PDGF (26). The two other cyclin D isoforms, D2 and D3, are expressed in low abundance in cultured rat MC (25).
CKI, such as p21Waf-1, p27Kip-1, and members of the INK4 family of cdk inhibitors (p15, p16, p18, and p19), negatively regulate the cell cycle by inhibiting the formation or activation of cyclin-cdk complexes (27). The cdk inhibitor p21Waf-1, a 21-kD product of the WAF1/CIP1/MDA6 gene, can be induced by p53-dependent or -independent pathways (28). p21Waf-1 was originally identified as an inhibitor of cyclin-cdk complexes. However, it was also observed that p21Waf-1-containing cyclin-cdk complexes retained kinase activity, which was abolished by the addition of more p21Waf-1 molecules, indicating that p21Waf-1 can exert different functional activities (29). LaBaer et al. (30) reported that in vivo, endogenous cyclin D1 and p21Waf-1 bind concomitantly to cdk4 after release of the cells from G0 arrest. This complex formation occurs in parallel with an increase in cyclin D1-associated kinase activity. LaBaer et al. (30) also demonstrated that at lower concentrations, p21Waf-1 promotes the assembly of cdk4 with D-type cyclins, whereas at higher concentrations it inhibits cdk4 activity. Interestingly, a recent study indicated that the expression of p21Waf-1 mRNA is under the control of the transcription factor E2F (31). In glomeruli of normal rats, the CKI p27Kip-1 is highly expressed, whereas the levels of p21Waf-1 are low (32). The proliferation of MC in rat anti-Thy 1.1-induced glomerulonephritis was associated with transient reduction of p27Kip-1 abundance, whereas the expression of p21Waf-1 was substantially increased. The glomerular abundance of p21Waf-1 remained elevated after the resolution of proliferation (32). However, the role of p21Waf-1 in the regulation of glomerular cell mitogenesis is presently unclear.
Gene therapy is emerging as a potential strategy for the treatment of proliferative disorders. It was demonstrated that a single intraluminal administration of antisense oligonucleotides (ODN) against cdk2 resulted in sustained inhibition of neointima formation after balloon angioplasty in a rat carotid injury model (33). In cultured MC, proliferation could be inhibited by using antisense ODN against the transcription factor early growth response gene 1 (34) or other cell cycle-associated proteins, such as proliferating cell nuclear antigen or Ki-67 (35). Recent findings by Terada et al. (25) suggested that transfection of antisense ODN against cyclin D1 reduced the proliferative responses of MC to endothelin-1, indicating that cyclin D1 plays a role in MC mitogenic signaling elicited by this vasopressor.
In this study, we investigated the expression of cyclin D1 protein during MC hyperplasia in rat glomerulonephritis and its involvement in cell cycle regulation of cultured rat MC. Specifically, we examined the effects of promitogenic and antimitogenic factors on the regulation of protein expression of cyclin D1 and cyclin D1-associated proteins and on the nuclear translocation of cyclin D1. In addition, we tested the hypothesis that antisense ODN against cyclin D1 inhibit MC proliferation induced by the potent MC mitogen PDGF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunohistochemistry
After overnight fixation in methyl Carnoy's solution, tissues were
dehydrated by bathing with increasing concentrations of methanol, followed by
100% isopropanol. After embedding in paraffin, 4-mm sections were cut with a
Leitz SM 2000 R microtome (Leica Instruments, Nussloch, Germany). After
deparaffinization, endogenous peroxidase activity was blocked with 3%
H2O2 in methanol for 20 min at room temperature.
Sections were then layered with the primary antibody and incubated overnight
at 4°C. After addition of the secondary antibody (biotin-conjugated goat
anti-rabbit IgG, diluted 1:500), the sections were incubated with
avidin-biotinylated horseradish peroxidase complex (Vectastain DAB kit; Vector
Laboratories, Burlingame, CA) and exposed to 0.1% diaminobenzidine
tetrahydrochloride and 0.02% H2O2 (as a source of
peroxidase substrate). Each slide was counterstained with hematoxylin. As
negative controls, equimolar concentrations of preimmune rabbit or goat IgG
were used. Double immunostaining for cyclin D1 and Thy1.1 was detected using
immunofluorescence microscopy. Primary antibodies were applied simultaneously,
overnight at 4°C. After washing, sections were incubated with secondary
antibodies (CY2-labeled goat anti-mouse IgG and CY3-labeled goat anti-rabbit
IgG; both from Dianova, Hamburg, Germany) concurrently for 2 h. Washed
sections were then covered with Tris-buffered mowiol, pH 8.6 (Hoechst,
Frankfurt, Germany). Antibodies to cyclin D1 (diluted 1:50) and Thy1.1
(diluted 1:250) were purchased from Santa Cruz Biotechnology (Heidelberg,
Germany) and from Serotec (Biozol, Eching, Germany), respectively. Cyclin
D1-positive nuclei were enumerated in glomerular profiles in renal sections
from two rats for each time point. The total number of nuclei and the number
of nuclei that were positive for cyclin D1 staining in each glomerular tuft
were determined in a minimum of 30 glomeruli per animal.
MC Isolation and Culture
Glomeruli from rat kidneys were isolated, and glomerular outgrowth and
subsequent subculturing of MC were performed as described previously
(37). MC were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated
(50°C for 30 min) fetal calf serum (FCS), 50 U/ml penicillin, 50 µg/ml
streptomycin, 2 mM glutamine, and 5 µg/ml insulin in a 95% air/5%
CO2 humidified atmosphere at 37°C. MC between passages 12 and
20 were used for experiments. Growth factors were used in the following
concentrations: PDGF-BB (Sigma, Deisenhofen, Germany), 20 ng/ml; TGF-ß1
(Life Technologies, Eggenstein, Germany), 5 ng/ml.
Protein Extraction and Western Blot Analysis
MC were grown to subconfluence in medium supplemented with 10% FCS and were
growth-arrested for 72 h in medium containing 0.2% FCS. At the indicated time
points, MC were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM
ethylenediaminetetra-acetic acid (EDTA), pH 7.5, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 10 µg/ml aprotinin,
and 2 µg/ml leupeptin. MC lysates were incubated for 10 min on ice, and
cellular debris was cleared by centrifugation (15,000 x g for
15 min at 4°C).
For detection of nuclear cyclin D1, MC nuclei were isolated. MC were washed twice with ice-cold phosphate-buffered saline (PBS) containing 1 mM sodium vanadate and 1 mM sodium fluoride and were collected with a rubber policeman in 50 mM Tris, pH 7.5, 10 mM EDTA, pH 7.5, 1 mM sodium vanadate, 1 mM sodium fluoride. Cells were collected by centrifugation (1000 x g for 10 min at 4°C) and resuspended in 5 times the packed cell volume of ice-cold hypotonic buffer [10 mM Hepes, pH 7.5, 10 mM KCl, 0.1 mM EDTA, pH 7.5, 0.1 mM ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetra-acetic acid (EGTA), pH 7.5, 300 mM sucrose, 1 mM dithiothreitol (DTT), 1 mM sodium vanadate, 2.5 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride]. After swelling on ice for 10 min, cells were disrupted by repeated aspiration through a 22-gauge needle. Cell disruption was confirmed by microscopic observation. Nuclei were separated from the cytosolic fraction by centrifugation (960 x g for 10 min at 4°C), washed once with hypotonic buffer, and solubilized in lysis buffer, as described above. Debris was cleared by centrifugation (15,000 x g for 15 min at 4°C). Equal amounts of either total MC protein or nuclear proteins (15 µg) were separated by SDS-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose filters (Amersham Pharmacia, Freiburg, Germany); transfer was controlled by Ponceau-S staining. Membranes were blocked for 12 h at 4°C in PBS containing 0.06% Tween 20 and 5% nonfat dried milk. Proteins were observed using the ECL detection system (Amersham Pharmacia, Freiburg, Germany) after incubation for 2 h at room temperature with the indicated primary antibodies. Applied primary antibodies were directed against cyclin D1 (1:5000), cdk4 (1:10,000), p21Waf-1 (1:5000), p27Kip-1 (1:1000), and cdk2 (1:5000) (all purchased from Santa Cruz Biotechnology, Santa Cruz, CA). The applied secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Dianova).
Immunocytochemistry
For cyclin D1 staining, MC grown on 8-well glass chamber slides, with or
without stimulation with soluble agonists, were fixed in 3%
paraformaldehyde/PBS and permeabilized for 10 min at 4°C with 1% Triton
X-100 in PBS. Nonspecific binding was blocked using 100% FCS. Primary antibody
against cyclin D1 (diluted 1:50; Santa Cruz Biotechnology) was applied
overnight at 4°C. As a negative control, equal concentrations of
polyclonal rabbit IgG were used. After washing, slides were incubated for 2 h
with CY3-labeled goat anti-rabbit IgG secondary antibody (Dianova). Washed
slides were then covered with Tris-buffered mowiol, pH 8.6 (Hoechst).
Transfection of Antisense ODN
Phosphothioate-modified, 20-nucleotide, antisense, sense control, or
mismatch control ODN were purchased from MWG Biotech (Ebersberg, Germany) and
dissolved in 10 mM Tris-HCl, pH 8, 1 mM EDTA, pH 8. The ODN had the following
sequences and positions, as derived from the rat cyclin D1 sequence
(38): AS2 (positions 234 to
253), 5'-GTCTTAAGCATGGCTCGCAG-3'; S2,
5'-CTGCGAGCCATGCTTAAGAC-3'; AS3 (positions 148 to 167),
5'-GAGCTGGTGTTCCATGCGCG-3'; MM3,
5'-GGTGGTTCTGCG-3'.
For assessment of transfection efficiency, fluorescein-labeled ODN AS2 was used (MWG Biotech). For transfection of ODN, two different methods were used (1). Polycationic transfection reagent (Superfect; Quiagen, Hilden, Germany) was used to facilitate uptake of ODN, according to the protocol recommended by the manufacturers. A mixture of ODN with polycationic transfection reagent was preincubated for 3 h before mitogenic stimulation of MC. At that time, ODN and polycationic transfection reagent were removed by washing with PBS, and Dulbecco's modified Eagle's medium with 2% FCS was added (2). When no polycationic transfection reagent was used, MC were preincubated with ODN for 16 h. After preincubation, medium was removed and MC were exposed to fresh medium containing ODN (at the same concentration as during the preincubation) and mitogenic factors as indicated. To achieve equal transfection efficiencies with the two methods, different concentrations of ODN had to be used. Tenfold higher concentrations of ODN were used for treatment without polycationic transfection reagent, compared to treatment with polycationic transfection reagent.
Determination of [3H]Thymidine Uptake and Cell
Numbers
In 96-well plates, MC were subcultured until subconfluent in medium
supplemented with 10% FCS and were growth-arrested for 48 to 72 h in medium
containing 0.2% FCS. During serum deprivation, MC were exposed for 16 h to
fresh medium without FCS, containing ODN in the indicated concentrations;
cells were then stimulated with 2% FCS or PDGF (20 ng/ml). For determination
of [3H]thymidine uptake, MC were pulsed with 1 µCi/ml
[methyl-3H]thymidine (specific activity, 5 mCi/mmol;
Amersham, Braunschweig, Germany) for 24 h. MC were then washed twice with PBS,
trypsinized, and collected onto filter paper using an automated cell harvester
(Bibby Dunn, Asbach, Germany). Incorporated counts were measured in a liquid
scintillation counter (Beckmann, Munich, Germany). Cell numbers were
determined 72 h after growth stimulation. Monolayers of MC were washed twice
in PBS, and MC were trypsinized and transferred into 10 ml of Isoton solution
(Coulter, Luton, United Kingdom) for counting in a cell counter (Coulter).
Assay of Cdk4-Associated Kinase Activity
MC were lysed in cdk4 immunoprecipitation buffer (50 mM Hepes, pH 7.5, 150
mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 1 mM DTT, 0.1% Tween 20, 10 mM
ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, 2 µg/ml
aprotinin, 5 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Cellular
debris was removed by centrifugation (16,000 x g for 10 min at
4°C). Five hundred micrograms of total extracted protein was precleared
with 10 µl of protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) for
1 h at 4°C. Cdk4 complexes were precipitated for 6 h at 4°C with 10
µl of protein A-Sepharose precoated with 10 µg of a mouse monoclonal
antibody against cdk4 (Ab-1, clone DCS-35; Neomarkers, Fremont, CA).
Precipitates were washed four times with immunoprecipitation buffer and twice
with 50 mM Hepes, pH 7.5, containing 1 mM DTT. Next, the precipitates were
resuspended in kinase assay buffer (50 mM Hepes, pH 7.5, 10 mM
MgCl2, 1 mM DTT, 2.5 mM EGTA, 10 mM ß-glycerophosphate)
containing 1 µg/assay of the substrate protein glutathione
S-transferase-pRb (Santa Cruz Biotechnology). ATP was added to a final
concentration of 50 µM, in addition to 10 µCi of
[
-32P]ATP (6000 Ci/mmol). The kinase assay was performed at
30°C for 30 min. The reaction was stopped by adding Laemmli sample buffer
and boiling for 5 min. Finally, the samples were separated by
SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography.
Statistical Analyses
Statistical analyses were performed using t tests for unpaired
valuses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TGF-ß1 Reduces the PDGF-Mediated Increases in Cyclin D1 and
p21Waf-1 Protein Abundance
To examine the effects of the well characterized MC mitogen PDGF and the
antimitogenic factor TGF-ß1 on the regulation of cyclin D1 protein
abundance, Western blot analyses were performed
(Figure 2). Incubation of MC
with PDGF (20 ng/ml) markedly upregulated the protein levels of cyclin D1
after 12 and 24 h. Coincubation of TGF-ß1 (5 ng/ml) and PDGF reduced the
PDGF-induced increases in cyclin D1 protein levels at 12 and 24 h, whereas
TGF-ß1 alone had no significant effect on cyclin D1 protein levels,
compared with growth-arrested control MC. Next, we investigated whether PDGF
and TGF-ß1 affected the expression of the cyclin D1-associated proteins
cdk4, p21Waf-1, and p27Kip-1
(Figure 2). The cdk inhibitor
protein p21Waf-1 was markedly upregulated by stimulation with PDGF
for 12 and 24 h, whereas TGF-ß1 alone had no effect on
p21Waf-1 protein levels. Coincubation with TGF-ß1 slightly
diminished the PDGF-induced increase in p21Waf-1 protein levels at
24 h. In contrast, protein levels of the CKI p27Kip-1 were reduced
by PDGF at 12 and 24 h with or without coincubation with TGF-ß1.
Incubation with TGF-ß1 alone for 12 or 24 h led to an increase in
p27Kip-1 expression. Protein levels of cdk4 were not notably
affected by PDGF or TGF-ß1 at the time points examined
(Figure 2).
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TGF-ß1 Blocks the Nuclear Translocation of Cyclin D1 Induced by
PDGF
In addition, we investigated how PDGF and TGF-ß1 regulate the
subcellular localization of cyclin D1. Immunocytochemical analyses revealed
that stimulation of growtharrested MC with PDGF led to nuclear accumulation of
cyclin D1 after 6 h. This phenomenon was markedly diminished by coincubation
with TGF-ß1 and PDGF for 6 h (Figure
3). The negative control, with irrelevant polyclonal rabbit IgG,
revealed no detectable staining pattern (data not shown). These results were
confirmed when the cyclin D1 protein abundance in nuclear extracts was
analyzed by Western blotting (Figure
4, bottom row). Whereas TGF-ß1 inhibited PDGF-induced
accumulation of cyclin D1 protein in nuclear extracts, no notable alteration
of total cellular cyclin D1 protein levels by TGF-ß1 was observed at this
time point in PDGF-stimulated MC (Figure
4, top row).
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Antisense ODN Directed Against Cyclin D1 Inhibit MC DNA Synthesis
Induced by FCS
To investigate whether suppression of cyclin D1 protein induction was
sufficient to inhibit MC mitogenesis, we transfected antisense ODN against
cyclin D1 mRNA regions located close to the translation start site. As shown
in Figure 5, the antisense ODN
AS2 and AS3 inhibited [3H]thymidine uptake by MC induced by FCS
(2%) in a dose-dependent manner. The antisense ODN AS2 and AS3 (20 µM)
diminished the FCS-induced increase in [3H]thymidine uptake to 35
± 6.7% (P < 0.001) and 64 ± 2.6% (P <
0.01), respectively. The corresponding sense (S2) and mismatch (MM3) control
ODN did not significantly affect [3H]thymidine incorporation. The
transfection efficiencies of both methods (with or without transfection
reagent) were assessed by microscopic detection of fluorescein-labeled ODN in
MC. Transfection of 1 µM fluorescein-labeled ODN without transfection
reagent or of 0.1 µM fluorescein-labeled ODN in the presence of
transfection reagent led to cellular and nuclear uptake of ODN in 100% of MC
after 24 h (Figure 6).
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Antisense ODN Directed Against Cyclin D1 Inhibit MC Proliferation
Induced by PDGF
In dose-response studies, we observed that antisense ODN concentrations as
low as 0.1 µM were sufficient to reduce MC proliferation induced by PDGF
(data not shown). Transfection with 0.1 µM concentrations of the antisense
ODN AS2 led to reduction of PDGF-stimulated [3H]thymidine uptake to
36% (P < 0.001), whereas PDGF stimulation of MC treated with 0.1
µM levels of the sense control ODN S2 reached 76% of PDGF-stimulated
control MC values (Figure 7A).
Figure 7B depicts ODN-induced
changes in MC growth, as indicated by [3H]thymidine uptake and MC
counts 24 and 72 h after stimulation with PDGF, respectively. Transfection
with 0.1 µM levels of the antisense ODN AS2 reduced the PDGF-stimulated
increase in cell counts to 35% (P < 0.01). The increase in cell
number for MC treated with 0.1 µM levels of the corresponding sense control
ODN S2 was 80% of the PDGF-stimulated control MC value. In addition, we
examined whether reduction of cyclin D1 protein levels by transfection with
antisense ODN against cyclin D1 led to induction of apoptosis. MC nuclei were
observed by staining with Hoechst dye, and apoptotic MC were determined on the
basis of nuclear morphologic features 24 h after transfection with 10 µM
ODN. On average, 3% of the nuclei of untransfected control MC demonstrated an
apoptotic phenotype. This percentage of apoptotic MC nuclei was not notably
altered in MC transfected with either antisense ODN or sense control ODN (data
not shown).
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Transfection of Antisense ODN AS2 Against Cyclin D1 Inhibits Cyclin
D1 Protein Expression Induced by FCS or PDGF
To confirm that the antimitogenic effects of antisense ODN against cyclin
D1 are indeed attributable to reduced cyclin D1 protein levels, Western blot
analyses of ODN-transfected MC were performed. The antisense ODN AS2 markedly
decreased cyclin D1 protein levels induced by 2% FCS
(Figure 8). Densitometric
analyses revealed reductions of cyclin D1 protein levels to 42 and 27% by 0.05
and 0.1 µM concentrations of antisense ODN AS2, respectively. Application
of 0.1 µM sense control ODN S2 reduced cyclin D1 protein levels to 68% of
control levels. To assess nonspecific effects of ODN on overall MC protein
synthesis, we also performed Western blot analyses for the G1 phase
protein cdk2. Neither antisense nor sense ODN altered cdk2 protein expression
levels in these experiments (Figure
8). PDGF-induced cyclin D1 protein expression was reduced by the
antisense ODN AS2 (Figure 9).
Densitometric analyses revealed reductions of cyclin D1 protein levels to 45
and 10% by 0.1 and 1 µM antisense ODN AS2, respectively. Control ODN S2 had
no significant effect on cyclin D1 protein levels
(Figure 9).
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Antisense ODN Against Cyclin D1 Inhibit Cdk4-Associated Kinase
Activity
Mitogenic stimulation of serum-starved MC with 2% FCS led to a maximal
1.9-fold increase in cdk4-associated kinase activity after 4 h, as assessed by
pRb-kinase assays (Figure 10)
and densitometric analyses. Transfection with 0.1 µM antisense ODN AS2
prevented the FCS-induced increase in cdk4-associated kinase activity,
resulting in a relative activity that was only 37% of the sense ODN control
value.
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Antisense ODN Against Cyclin D1 Inhibit p21Waf-1 Protein
Expression Induced by PDGF
Finally, we investigated whether the expression of the E2F-dependent gene
product p21Waf-1
(31) was affected by the
reduction in cyclin D1 protein levels. As depicted in
Figure 11, the abundance of
p21Waf-1 protein was markedly decreased by MC transfection with
antisense ODN against cyclin D1. Densitometric analyses revealed that 0.1 and
1 µM concentrations of antisense ODN AS2 diminished PDGF-stimulated
p21Waf-1 protein levels to 29 and 19%, respectively. Similar
results were obtained when MC were transfected with antisense ODN and
stimulated with 2% FCS (data not shown). Sense control ODN had no significant
effects on PDGF-related p21Waf-1 increases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further examine the relevance of the in vivo findings of enhanced cyclin D1 expression in MC hyperplasia, we studied the effects of soluble regulators of MC growth on cyclin D1 expression and cellular localization. TGF-ß1 causes growth arrest in G1 phase in several cell types and is a potent inhibitor of MC proliferation. Previously, we showed that TGF-ß1 almost completely prevented the PDGF-driven transition of MC from G0/G1 phase into S phase, when TGF-ß1 was coincubated with PDGF within the first 6 h (11). TGF-ß1-induced growth arrest attributable to reductions in cyclin D1 mRNA and protein levels was demonstrated in rat intestinal epithelial cells (42). However, in other cell types (e.g., mouse keratinocytes [(43)] or human breast adenocarcinoma cells [(10)], TGF-ß1 caused growth inhibition without altering cyclin D1 protein levels. In rat MC, entry into S phase occurred 14 h after PDGF stimulation. Previously, we noted that coincubation with TGF-ß1 did not affect the PDGF-induced abundance of cyclin D1 protein up to 10 h after mitogenic stimulation (11). The data presented here demonstrate that PDGF caused marked increases in cyclin D1 protein expression even at later time points, i.e., 24 h after mitogenic stimulation (after completion of G1 phase) (11). Moreover, PDGF markedly increased the translocation of cyclin D1 to the nucleus after 6 h, i.e., during the progression of MC through G1 phase (11). At late time points, TGF-ß1 diminished PDGF-induced cyclin D1 protein expression, suggesting that downergulation of cyclin D1 protein abundance by TGF-ß1 is a late mechanism of its growthinhibiting action. In addition, TGF-ß1 inhibited the PDGF-induced nuclear localization of cyclin D1 during G1 phase, which may reflect an early mechanism of the growth-inhibiting action of TGF-ß1 in MC. Although the molecular mechanisms of TGF-ß1-mediated effects on the subcellular localization of cyclin D1 are presently unknown, TGF-ß1 could possibly inhibit the association of cyclin D1 with proteins that contain recognizable NLS or otherwise mediate nuclear importing. Alternatively, TGF-ß1 could affect posttranslational modifications of cyclin D1 or associated proteins important for retention of cyclin D1 in the nucleus. In this context, TGF-ß1 was recently found to facilitate increased nuclear exporting of Smad7 (44). In renal epithelial normal rat kidney 49F cells, intracellular localization of cyclin D1-cdk4 complexes was demonstrated to be involved in the regulation of their activity (19). Calmodulin seems to be essential for the nuclear accumulation of cyclin D1 and cdk4 (19), as well as for the nuclear translocation of p21Waf-1 (45). Also, p21Waf-1 was demonstrated to be important for the translocation of cdk4 and cyclin D1 to the nucleus (21). At present, little is known regarding the effects of soluble growth factors on the nuclear translocation of cyclin-cdk complexes. It remains to be determined whether the inhibitory effect of TGF-ß1 on the nuclear translocation of cyclin D1 is critical for the antimitogenic action of TGF-ß1 in MC.
Because cyclin D1 is a key regulator of early G1 phase progression, we examined whether reduction of cyclin D1 protein abundance by transfection of antisense ODN is sufficient to inhibit cdk4 kinase activity and MC proliferation. Several studies successfully used transfection of antisense ODN to examine the roles of MC growth regulators and to block MC replication. In cultured MC, proliferation could be inhibited by using antisense ODN against the early growth response gene EGR1 (34) or other cell cycle-associated proteins, such as proliferating cell nuclear antigen or Ki-67 (35). The results presented here demonstrated that transfection of antisense ODN against cyclin D1 very effectively reduced MC proliferation. Antisense ODN led to reductions in cyclin D1 protein levels and profoundly inhibited both FCS- and PDGF-induced DNA synthesis and replication of rat MC. Furthermore, we demonstrated that transfection with antisense ODN against cyclin D1 reduced the FCS-induced increase in cdk4-associated kinase activity. Because the arrest of cell cycle progression in G1 phase can lead to the induction of apoptosis under certain conditions (46), we investigated whether the growth arrest mediated by antisense ODN against cyclin D1 might cause apoptosis in MC. We observed no increase in the percentages of apoptotic MC in antisense ODN-transfected MC, compared with sense ODN-transfected or untransfected MC. However, overexpression of cyclin D1 has been reported to lead to increases in apoptosis for fibroblasts and epithelial cells (47,48).
One of the cyclin D1/cdk4-associated proteins is p21Waf-1, which can act as an assembly factor or as an inhibitor of cdk-cyclin complexes (29,30). Shankland et al. (32) reported de novo expression of p21Waf-1 in anti-Thy1.1-induced glomerulonephritis that coincided with the reduction in MC hyperplasia, suggesting that p21Waf-1 may be an important factor in the resolution of MC proliferation. In investigation how soluble growth regulators and antisense ODN against cyclin D1 affect p21Waf-1 expression in cultured MC, we observed that PDGF caused marked increases in p21Waf-1 protein levels in MC. Furthermore, we transfected MC with a reporter plasmid containing the chloramphenicol acetyltransferase gene under the control of the p21Waf-1 promotor. Our preliminary data indicated that p21Waf-1 gene expression in MC was transcriptionally induced by PDGF (data not shown). In contrast, protein levels of the CKI p27Kip-1 were significantly suppressed by incubation with PDGF for 12 h. Induction of p21Waf-1 protein by PDGF was also observed in p53-deficient and normal mouse fibroblasts, supporting the interpretation of p53-independent upregulation of p21Waf-1 by PDGF (28). In other cell types, TGF-ß1 was observed to enhance p21Waf-1 expression by p53-independent mechanisms (49,50). However, our experiments showed that in cultured rat MC, TGF-ß1 led to a slight reduction in the p21Waf-1 expression induced by PDGF, suggesting that the patterns of TGF-ß1-mediated effects on cell cycle regulatory proteins can differ considerably among various cell types. The expression of p21Waf-1 was also reduced by transfection with antisense ODN against cyclin D1. Hiyama et al. (51) recently demonstrated that ectopic expression of cyclin D1 in human glioma and rodent fibroblast cell lines induced p21Waf-1 gene expression and that coexpression of the transcription factors E2F-1 and DP-1 could specifically transactivate the p21Waf-1 promotor. Furthermore, an active E2F binding site was identified within the p21Waf-1 promotor (31). Therefore, downregulation of p21Waf-1 by antisense ODN against cyclin D1 might be attributable to an E2F-dependent mechanism. Conceivably, other cell cycle regulatory proteins that are involved in the activation of G1 phase cdk (e.g., cyclin E/cdk2) might also promote p21Waf-1 gene expression by enhancing the release of E2F.
The results of in vitro studies with rat MC demonstrated that mitogenic PDGF strongly induced protein expression and nuclear translocation of cyclin D1 in cultured MC. Both effects were inhibited by antimitogenic TGF-ß1. Reduction of cyclin D1 protein levels by transfection with antisense ODN against cyclin D1 resulted in marked inhibition of MC proliferation, without changes in the percentage of apoptotic cells. Furthermore, cdk4-associated kinase activity and expression of the E2F-dependent protein p21Waf-1 were negatively affected by antisense ODN against cyclin D1. Antisense ODN against cyclin D1 have also been shown to diminish the endothelin-1-induced increases in DNA synthesis in rat MC (25). Therefore, strategies aiming to reduce cyclin D1 expression, e.g., by transfer of antisense ODN or with specific cyclin antagonists, might represent effective methods to inhibit MC proliferation in vivo.
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