Renal tubular hypertrophy is an adaptive process that occurs in diabetes mellitus, when there is a loss of renal mass, during protein feeding, and a number of other conditions1,2. In many of these conditions, this hypertrophy has been postulated to cause a progressive reduction in renal function3. In spite of the potential importance of hypertrophy in renal disease, the cellular and molecular mechanisms leading to its development are not well understood. A clear understanding of these mechanisms may provide a basis for novel therapies in the treatment of hypertrophic renal disorders.
One possible mechanism is that hypertrophy represents an aborted cell cycle, with cells entering the G1 phase and initiating protein synthesis and growth, but failing to progress into the S phase. The proliferation of eukaryotic cells is tightly regulated through a precious balance of positive and negative regulatory proteins that exert their effects during the first gap phase (G1) of the cell cycle4,5,6. Stimulation of quiescent cells to re-enter the cell cycle induces the accumulation of G1 cyclins (three types of cyclin D and cyclin E), thereby activating associated cyclin-dependent kinases (CDKs). This leads to the phosphorylation of downstream targets and an eventual entry into the S phase. The activity of these G1 cyclin kinases is modulated by a family of proteins, including p21CIP1, p16INK4, and p27Kip15,7,8,9,10,11,12,13,14, and this modulation defines a part of the negative control of the G1 phase. There are at least two distinct families of CDK inhibitors in mammalian cells: the p21/p27 family and the p15/p16 family 5,6. p21CIP1 and p27Kip1 proteins share considerable sequence homology and have been shown to potently inhibit almost all cyclin-CDK enzymes 5,1314. In contrast, the p16 family specifically interacts with CDK4 and CDK65,10,11. We reported recently that the overexpression of p16INK4 and p21CIP1 inhibited the proliferation of mesangial cells induced by growth-promoting factors15,16.
Angiotensin II (Ang II) and transforming growth factor-
(TGF-) have recently been reported to cause hypertrophy in renal tubules17,18. Preisig et al have shown that there are two mechanisms of hypertrophy in renal epithelial cells: a cell-cycle–dependent form and an alkalinization-induced hypertrophy form1,18,19. Wolf and Stahl showed that Ang II stimulated the expression of p27Kip1 and that this p27Kip1 induction was closely related to hypertrophy2,17. These studies have suggested that the mechanisms of hypertrophy may be closely related to cell-cycle regulation, especially CDK activity and CDK inhibitors. However, there has been no direct evidence confirming a relationship between CDK inhibitors and hypertrophy.
The purpose of this study is to determine whether CDK inhibitors play key roles in the mechanisms of renal tubular hypertrophy. We investigated how overexpression of the CDK inhibitors (p27Kip1, p21CIP1, and p16INK4) in LLC-PK1 cells affected the hypertrophic changes. As a result, we found that overexpression of p27Kip1 and p21CIP1 caused hypertrophy in LLC-PK1 cells but that p16INK4 had no such effect.
METHODS
Recombinant adenoviruses
Replication-defective, recombinant adenoviruses AxCAp16, AxCAp21, and AxCAp27 were constructed by homologous recombination between the expression cosmid cassette and the parental virus genome using the same method described previously15,20,21. Briefly, the expression cosmid cassette was constructed by inserting an expression unit comprised of the polyA sequence together with the CAG (chicken -actin promoter + cytomegalovirus enhancer) promoter21, p16INK4, p21CIP1, p27Kip1, or LacZ coding sequence into the SwaI site of Adex1w. The expression cosmid cassette and adenovirus DNA-terminal protein complex (DNA-TPC) were cotransfected into 293 cells by the calcium phosphate precipitation method as described previously15,20,21. Adex1w and DNA-TPC were kindly provided by Dr. I. Saito20,21. The full-length rat p21CIP1 cDNA (2.1 kb; kindly provided by Dr. A. Noda)7, human p27Kip1 cDNA (2.0 kb; kindly provided by Dr. T. Hunter)14, and human p16INK4 cDNA (1.8 kb; kindly provided by Dr. E. Hawlow)22 were isolated by restriction enzyme, digested, and ligated into the SmaI site of the expression cosmid cassette with T4 DNA ligase (Takara, Tokyo, Japan) using essentially the same method reported previously15,20,21.
A recombinant adenovirus expressing the LacZ gene (AxCALacZ) was provided by Dr. I. Saito20,21. Each adenovirus preparation was titrated by plaque assay on 293 cells. Viral stocks [1011 plaque-forming units (pfu)/ml] were stored at -80°C and were thawed on ice for five minutes before use.
Cell culture
LLC-PK1 is a well-characterized renal tubular epithelial cell line (American Type Culture Collection, Rockville, MD, USA). The cells were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO, Eggenstein, Germany) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 
g/ml streptomycin at 37°C in a 5% CO2 incubator.
The cells were subcultured in 10 cm dishes or 24-well dishes at a density of 5 
104 cells/ml and were then incubated in medium plus 10% fetal calf serum (FCS) until approximately 70% confluence. Before adenovirus infection, the cells were incubated in medium without FCS for 24 hours. After removal of the medium, viral solutions were added to the culture dishes for one hour with shaking every 20 minutes. The cells were incubated in a medium containing 10-8M epidermal growth factor (EGF; Boehringer, Mannheim, Germany) or 10% FCS for indicated times.
Detection of 
-galactosidase activity
Histological staining of -galactosidase was conducted as previously described15. In brief, the cells were washed twice with phosphate-buffered saline (PBS), fixed with 80% ethanol for 10 minutes at 4°C, washed another four times with PBS, and incubated in staining solution for four hours at 37°C. The staining solution was comprised of 1 mg/ml 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal), 2 mM MgCl2, 5 mM K4Fe (CN)6, and 5mM K4Fe (CN)6 in 100 mM sodium phosphate buffer (pH 7.4). -galactosidase activity was detected as the development of blue pigmentation due to the enzymatic cleavage of X-galactosidase.
Antibodies
Antibodies against antimouse-p27Kip1, antimouse-cyclin D1, and antimouse-cyclin E were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against antihuman-p21CIP1 and antihuman-p16INK4 were purchased from Pharmigen (San Diego, CA, USA).
Western blot analysis
The proteins were extracted from mesangial cells at 12, 24, and 48 hours after adenovirus infection using Tri-Reagent. Pellets were solubilized in sodium dodecyl sulfate (SDS) sample buffer. Fifty micrograms of each protein were resolved in SDS-polyacrylamide gel electrophoresis, and the proteins were then transferred to an Immobilon P membrane (Daiichikagaku, Tokyo, Japan). To detect p16INK4, p21CIP1, p27Kip1, cyclin D1, and cyclin E, the membrane was incubated with antibody at a dilution of 1:500 in TBS containing 0.1% Tween 20 for two hours at 37°C. The primary antibodies (diluted 1/1000) were detected using horseradish peroxidase (HRP)-conjugated rabbit antimouse IgG or goat antirabbit IgG and were visualized by the Amersham ECL system (Amersham Corp., Arlington Heights, IL, USA) after extensive washing of the membranes.
[3H]-leucine and [3H]-thymidine incorporation
LLC-PK1 cells were plated in 24-well plates, incubated in medium without FCS for 24 hours, and then infected with various concentrations of adenovirus for one hour. Next, the cells were incubated for 48 hours in medium containing Ang II (10-7 to 10-9M) or 10-8M EGF, with pulsing with 1 Ci [3H]-leucine or [3H]-thymidine for the last six or four hours, respectively (Amersham). After the cells were washed three times in ice-cold PBS, they were precipitated twice with 10% trichloroacetic acid (TCA), redissolved in 0.5 M NaOH with 0.1% Triton X-100, and counted in Aquasol-2 scintillation cocktail (NEN Research Products, Boston, MA, USA). To correct the [3H]-thymidine incorporation values by protein contents, we measured the protein contents in each well using a protein assay kit (Bio-Rad, Tokyo, Japan).
Cell cycle analysis by fluorescence-activated cell sorter analysis
LLC-PK1 cells were cultured in a 10 cm dish and incubated in medium without FCS for 24 hours before adenovirus infection. After removal of the medium, viral solutions were added to the culture dishes for one hour, and the cells were then incubated in a second medium containing 10-8M EGF for 48 hours. The samples to be analyzed for DNA content were harvested by trypsinization and centrifugation, washed twice with PBS, and resuspended in 70% ethanol for at least 12 hours at 4°C. After the fixed and permeabilized cells were collected by centrifugation and washed with PBS, they were stained with propidium iodide, analyzed by a flow cytometer, and investigated by forward-angle light scatter to determine relative cell size18,23. All experiments were repeated at least three times.
Measurement of cell protein and DNA
LLC-PK1 cells were grown in six-well dishes and washed with PBS, harvested with 0.25% trypsin and 0.5 mM ethylenediaminetetraacetic acid (EDTA) for five minutes, and pelleted at 1500 g for five minutes. Each pellet was resuspended in 1 ml lysis buffer (50 mM Na2PO4, pH 7.4), and the cells were lyzed on ice by repeated passage through a 27-gauge needle. Next, the DNA and protein contents were determined as previously reported18,19.
CDK4 and CDK2 kinase assay
Immune complex kinase assay was performed by essentially the same methods described by Matsushime et al24. The cells were suspended at 1 106 to 5 106/ml in IP buffer [50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM ethylene glycol-bis (-aminoethyl-ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 0.1% Tween-20] containing 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 10 g of leupeptine per ml, 20 U of aprotinin per ml, 10 mM-glycerophosphate, 1 mM NaF, and 0.1 mM sodium orthovanadate (Sigma Chemicals, St. Louis, MO, USA) and were sonicated at 4°C (Sonicator; Daiichikagaku, Tokyo, Japan). Lysates were clarified by centrifugation at 10,000 g for five minutes. The supernatants were incubated for two hours at 4°C with 20 l of protein G-plus agarose (Oncogene Science, Uniondale, NY, USA) and 10 l of antimouse-CDK4 antibody or antimouse-CDK2 antibody, and immune complexes were recovered by centrifugation. Immunoprecipitated proteins on beads were washed three times with 1 ml of IP buffer, and the beads were suspended in 30 l of kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT) containing substrate (0.2 g of soluble glutathione S-transferase-pRb fusion protein, which is discussed later in this article, or 0.2 g of Histon H1; Sigma), 2.5 mM EGTA, 10 mM-glycerophosphate, 1 mM NaF, 20 M ATP, and 10 Ci of [
-32P] ATP (6000 Ci/mmol; NEN, Dupont). After incubation for 30 minutes at 30°C with occasional mixing, the samples were boiled in polyacrylamide gel sample buffer containing SDS and were separated by electrophoresis. Phosphorylated proteins were visualized by autoradiography.
pRb substrate was prepared using essentially the same method described by Matsushime et al24. The concentration and purity of soluble pRb were estimated by Coomassie blue staining of electrophoretically separated proteins on denaturing polyacrylamide gels and comparisons with protein standards of known concentrations.
Statistics
The results were given as means 
SEM. The differences were tested using analysis of variance. A P < 0.05 was considered significant.
RESULTS
Ang II causes hypertrophy and regulates p21CIP1 and p27Kip1 protein expression of LLC-PK1 cells
We first tested whether the treatment of LLC-PK1 cells with Ang II would stimulate hypertrophy. As shown in Figure 1, treatment with 10-7 and 10-8 Ang II for 48 hours caused significant increases in [3H]-leucine incorporation and significant decreases in [3H]-thymidine incorporation. As shown in Figure 1c, Ang II caused a significant increase in the protein:DNA ratio. These results indicate that Ang II caused hypertrophy of LLC-PK1 cells.
Figure 1.
Effect of angiotensin II (Ang II) on [3H]-leucine and [3H]-thymidine incorporation and the protein:DNA ratio in LLC-PK1 cells. (A) LLC-PK1 cells incubated for 48 hours in serum-free medium. The cells were incubated in mediums with various concentrations of Ang II for 48 hours. For the last four hours, the cells were pulsed with 1 Ci [3H]-thymidine. (B) LLC-PK1 cells incubated for 48 hours in serum-free medium. The cells were incubated in mediums with various concentrations of Ang II for 48 hours. For the last six hours, the cells were pulsed with 1 Ci [3H]-leucine. (C) LLC-PK1 cells incubated for 48 hours in serum-free medium. The cells were incubated in mediums with various concentrations of Ang II for 48 hours. The ratio of protein:DNA is plotted on the y axis as a percentage of control values (cells treated with vehicles only; N = 5, mean SEM). *Statistically significant difference (P < 0.05) compared with control data obtained from LLC-PK1 cells incubated without Ang II.
We examined how Ang II regulated the protein expressions of p21CIP1, p27Kip1, p16INK4, cyclin D1, and cyclin E. As shown in Figure 2, Western blots for p27Kip1 and p21CIP1 revealed dose-dependent increases in the expression of both proteins in the presence of Ang II. The expression of p16INK4 protein is generally very low, and it did not significantly change in the presence of Ang II in our experimental condition. Cyclin D1 expression showed a significant dose-dependent increase in the presence of Ang II, and cyclin E increased slightly.
Figure 2.
Effect of Ang II on protein expressions of p21C1P1, p27Kip1, p16INK4, cyclin D1, and cyclin E. LLC-PK1 cells incubated for 48 hours in mediums with or without Ang II (10-7 to 10-9M). A total of 50 g of protein from each cellular lysate was separated by sodium dodecyl sulfate-electrophoresis. The blots represent four independent experiments with qualitatively similar changes.
Overexpression of p21CIP1, p27Kip1, and p16INK4 using adenovirus vector
To further investigate whether this induction of CDK inhibitors may contribute to hypertrophy of LLC-PK1 cells, we used adenovirus-mediated gene transfer of p21CIP1, p27Kip1, and p16INK4 to LLC-PK1 cells. Specifically, we tested whether the AxCAp21, AxCAp27, and AxCAp16 adenoviruses express the p21CIP1, p27Kip1, and p16INK4 proteins in LLC-PK1 cells. The protein expression was detected at 12 to 48 hours after infection, which was in conformance with our previous report (data not shown)15,16. A dose-dependent increase of the protein expression was observed for each CDK inhibitor at 107, 108, and 109 pfu/ml, as shown in Figure 3. Judging from the histologic staining of -galactosidase, over 90% of the cells were transfected with adenoviral vector. As reported previously, the LDH release from the cells to the medium gave no indication of cytotoxic effects in the concentration of 109 pfu/ml15,16. In the following studies, we used the AxCAp16, AxCAp21, and AxCAp27 adenoviruses at concentrations of 107, 108, and 109 pfu/ml.
Figure 3.
Dose-dependent expressions of p21C1P1, p27Kip1, and p16INK4 using adenovirus vector. LLC-PK1 cells were infected with various concentrations of AxCAp16, AxCAp21, AxCAp27, or vehicle control for one hour. Total protein was harvested at 48 hours after infection. Fifty micrograms of protein were used for Western blot analysis. The 21 kDa for p21CIP1 protein, the 27 kDa for p27Kip1 protein, and the 16 kDa for p16INK4 protein are observed. These blots represent four independent experiments with qualitatively similar changes.
Full figure and legend (11K)Overexpression of p21CIP1 and p27Kip1 caused hypertrophy in LLC-PK1 cells
We next investigated the effects of p21CIP1, p27Kip1, and p16INK4 overexpression on cell size under stimulation of EGF using mean forward light scatter, an index of cell size. Figure 4a illustrates a representative experiment plotting cell number as a function of forward light scatter, and Figure 4b summarizes the data. The overexpression of p21CIP1 and p27Kip1 significantly increased the mean forward light scatter, but overexpression of p16INK4 did not. We also used mean forward light scatter to compare cell size in the presence and absence of EGF. As shown in Figure 4a and b, there were no differences between the two groups in cell size (mean forward light scatter).
Figure 4.
Cell size analysis by forward light scatter of AxCAp16-, AxCAp21-, AxCAp27-, and AxCALacZ-infected LLC-PK1 cells. Effects of overexpression of p21CIP1, p27Kip1, and p16INK4 on the cell size of LLC-PK1 cells were examined. (A) LLC-PK1 cells were incubated with 109 pfu/ml of AxCAp16, AxCAp21, AxCAp27, or AxCALacZ for 1 hour and with 10-8M EGF or without EGF for 48 hours. Representative tracing of relative cell number (plotted on the y axis) as a function of forward light scatter (plotted on the x axis) is shown. AxCAp16-, AxCAp21-, AxCAp27-, AxCALacZ-, and AxCALacZ (without EGF)-infected LLC-PK1 cells were represented as blue line, orange line, red line, green line, and black line, respectively. (B) The mean forward light scatter is plotted on the y axis as a percentage of AxCALacZ-treated cells after 48 hours of incubation with or without 10-8M EGF (N = 5, mean SEM). *Statistically significant difference (P < 0.05) compared with value of AxCALacZ (with EGF)-infected LLC-PK1 cells.
To examine whether the increase in cell size was associated with an increase in protein synthesis, we next examined the effects of overexpression of p21CIP1, p27Kip1, and p16INK4 on the protein synthesis. Cells were infected with 109 pfu/ml concentrations of adenovirus for one hour and were then incubated with 10-8M EGF or without EGF for 48 hours. As shown in Figure 5, 109 pfu/ml concentrations of AxCAp21 and AxCAp27 infection significantly increased [3H]-leucine incorporation, whereas AxCAp16 infection did not effect any significant change. In contrast to [3H]-leucine incorporation, [3H]-thymidine incorporation was significantly decreased by the overexpression of p16INK4 and was slightly decreased by the overexpression of p21CIP1 and p27Kip1Figure 5b. In the absence of EGF, both [3H]-leucine and [3H]-thymidine incorporation were significantly decreased. AxCAp21 and AxCAp27 infection significantly increased the protein:DNA ratio, and AxCAp16 did not. There was no significant change in the protein:DNA ratio between the EGF-treated and untreated cells.
Figure 5.
Effects of gene transfer of p21CIP1, p27KIP1, and 16INK4 on [3H]-leucine and [3H]-thymidine incorporation and the protein:DNA ratio in LLC-PK1 cells. (A) LLC-PK1 cells were infected with 109 pfu/ml of AxCAp16, AxCAp21, AxCAp27, or AxCALacZ for 1 hour and incubated with 10-8M EGF or without EGF for 48 hours. For the last four hours, the cells were pulsed with 1 Ci [3H]-leucine. Incorporation values were adjusted by measured protein values. (B) LLC-PK1 cells were infected with 109 pfu/ml of AxCAp16, AxCAp21, AxCAp27, or AxCALacZ for 1 hour and incubated with 10-8M EGF or without EGF for 48 hours. For the last six hours, the cells were pulsed with 1 Ci [3H]-thymidine. Incorporation values were adjusted by measured protein values. (C) LLC-PK1 cells were infected with 109 pfu/ml of AxCAp16, AxCAp21, AxCAp27, or AxCALacZ for 1 hour and incubated with 10-8M EGF or without EGF for 48 hours. The ratio of protein:DNA is plotted on the y axis as a percentage of control values (cells treated with EGF and AxCALacZ; (N = 5, mean SEM). *Statistically significant difference (P < 0.05) compared with value of AxCALacZ-infected LLC-PK1 cells.
Effects of p21CIP1, p27Kip1, and p16INK4 overexpression on CDK4 and CDK2 activity
To investigate further whether the overexpression of p21CIP1, p27Kip1, and p16INK4 may functionally inhibit CDK2 and CDK4 enzyme activity, kinase activities were measured using Histon H1 or pRb as a substrate. Overexpression of p21CIP1 significantly inhibited 10-8M EGF-stimulated CDK2 and CDK4 enzyme activity to 15.1 4.5% and 14.1 4.7% of overexpression of LacZ, respectively Figure 6. The overexpression of p27Kip1 also significantly inhibited 10-8M EGF-stimulated CDK2 and CDK4 enzyme activity to 17.2 5.2% and 15.6 5.7%, respectively. Overexpression of p16INK4 significantly inhibited 10-8M EGF-stimulated CDK4 enzyme activity to 21.9 6.7%, but it did not significantly change CDK2 enzyme activity. We observed marked decrease in CDK2 and CDK4 activity in the absence of EGF Figure 6.
Figure 6.
Effect of gene transfer of p21CIP1, p27KIP1, and 16INK4 on CDK2 kinase and CDK4 kinase activities of LLC-PK1 cells. LLC-PK1 cells were infected with 109 pfu/ml of AxCAp16, AxCAp21, AxCAp27, or AxCALacZ for 1 hour and were incubated for 48 hours with 10-8M EGF or without EGF. Cell lysates were immunoprecipitated with anti-CDK2 antibody. Immune complexes were assayed using Histon H1 as a substrate (upper panel). LLC-PK1 cells were infected with 109 pfu/ml of AxCAp16, AxCAp21, AxCAp27, or AxCALacZ for 1 hour and were incubated with 10-8M EGF or without EGF for 48 hours. Cell lysates were immunoprecipitated with anti-CDK4 antibody. Immune complexes were assayed using GST-pRb as a substrate (lower panel). The blots represent four independent experiments with qualitatively similar changes.
Full figure and legend (7K)DISCUSSION
This study demonstrates that Ang II caused hypertrophy of LLC-PK1 cells accompanying the increases of p27Kip1 and p21CIP1 protein levels, and that overexpression of p27Kip1 and p21CIP1 also caused hypertrophy in LLC-PK1 cells. On the other hand, the overexpression of p16INK4 did not cause hypertrophy.
Recently, a number of major advances in molecular biology have led to the identification of what are called cyclins, a group of critical genetic and enzymatic pathways that control cell cycling. Cyclins act in concert with the CDKs to phosphorylate key substrates that facilitate the passage of the cell through each phase of the cell cycle4,5,6. A critical target of cyclin-CDK enzymes is the retinoblastoma tumor suppresser protein, and phosphorylation of this protein inhibits its ability to restrain the activity of a family of transcription factors (E2F family) that induces expression of genes important for cell proliferation4,5,6.
Cyclin-dependent kinases are negatively controlled by two distinct groups of inhibitory proteins, the CDK inhibitors (CKIs). The first group, inhibitors of the CDK4 (INK4) family (including p16INK4a, p15INK4b, p18INK4c, and p19INK4d), is specific for the G1 phase CDKs, CDK4 and CDK6, and inhibits the kinase activity of the cyclin D/CDK4-CDK6 complexes on pRb5,10,11,22. p16INK4 was first discovered as a 16 kDa protein that associates with CDK4. By binding to CDK4 or CDK6 but not to cyclins or other known CDKs, recombinant p16INK4 forms a binary complex lacking kinase activity5,22.
The second CKIs family comprises p21CIP1, p27Kip1, and p57Kip2 (Kip family). Their inhibitory action affects a large range of cyclin/CDK complexes involved in the G1 and S phases5,7,8,9,12,13,14. p21CIP1, a protein induced in part by p53, is up-regulated by senescence, DNA damage, and cellular differentiation. p21CIP1 forms quaternary complexes with CDKs, cyclins, and PCNA7,8,9,12.
Several recent reports have demonstrated that cell cycle regulators, especially CDK and CDK inhibitors, are closely related to hypertrophy in renal tubular cells1,2,17,18. Renal tubular hypertrophy occurs in a number of clinical conditions such as diabetes mellitus, chronic metabolic acidosis, and potassium depletion1,2. In this study, we demonstrated clearly that p21CIP1 and p27Kip1 overexpression directly converted hyperplasia to hypertrophy in renal tubular cells. Surprisingly, however, overexpression of p16INK4 did not cause hypertrophy. One possible mechanism of hypertrophy by the p21/p27 KIP family is the pattern of cell cycle entering into the growth phase from the G0 to G1 phase without subsequent progress into the S phase. This may result in the synthesis of cell proteins but can be assumed to prevent DNA synthesis and subsequent cell division. The commitment of cells to enter the S phase, the phase in which DNA synthesis occurs, depends on the passage beyond a distinct restriction (R) point late in the G1 phase4,25. After passage through the R point, mitogenic growth factors are no longer required, and cells progress to complete mitosis. If the cells do not pass the R point and remain arrested in the late G1 phase, they may undergo cellular hypertrophy.
The levels of p27Kip1 and p21CIP1 protein expression are high in cells arrested in the G1 phase, and their abundance declines when the cells progress toward the S phase of the cell cycle. p27Kip1 has been implicated in G1 phase arrest induced by TGF-, contact inhibition, or deprivation of growth factors9,14,26,27,28. Thus, overexpression or accumulation of p27Kip1 or p21CIP1 may cause cell cycle arrest in the late G1 phase and may induce increases in the protein:DNA ratio and hypertrophic change. On the other hand, overexpression of p16INK4 decreased [3H]-thymidine incorporation and [3H]-leucine incorporation in our experiment. p16INK4 significantly inhibited CDK4 activity but did not change CDK2 activity5,22. These findings offer one possible explanation for the failure of p16INK4 to induce hypertrophy. Overexpression of p16INK4 may cause cell cycle arrest at the early G1 phase by strong inhibition of CDK4. In this state, very few cells may be retained at the late G1 phase because p16INK4 does not suppress CDK2. The possible mechanism of hypertrophy is the incomplete inhibition of CDK4 kinase by p21CIP1/p27Kip, and the progress of cells goes into the late G1 phase. The difference of two groups, that is, p16INK4-overexpressed cells and p21CIP1/p27Kip1-overexpressed cells, might have occurred at the late G1 phase at the step of CDK2 activity. The cells overexpressing p21CIP1 or p27Kip1 were blocked not only at the early phase, but also at the late G1 via inhibition of CDK2 activity. We postulated that these cells retained at the late G1 phase may cause hypertrophy. On the other hand, the cells that overexpressed p16INK4 were not retained at the late G1 phase at the step of CDK2 activity. These cells were presumed not to show hypertrophic changes. These differences between the p21/p27 KIP family and p16INK4 may support the idea of "cell-cycle–dependent hypertrophy" in renal tubular cells.
Preisig et al reported that there are two mechanisms of hypertrophy1,18,19. The first, hypertrophy associated with alkalinization, is unrelated to increases in protein synthesis and occurs in the absence of active pRb. The second is a "cell-cycle–dependent form of hypertrophy" that requires the presence of active pRb or a related family member. Wolf and Stahl reported that Ang II stimulated hypertrophy and increased the p27KIP1 protein level and that antisense oligonucleotide against p27KIP1 inhibited Ang II-induced hypertrophy17. As with our results, their report strongly suggested that p27KIP1 mediates hypertrophy of LLC-PK1 cells.
High glucose inhibits mesangial proliferation in vitro and induces hypertrophy in mesangial cells in culture and in experimental diabetic nephropathy29,30,31,32. In this condition, Kuan et al reported that the expression of p21CIP1 was increased, whereas the levels of p27KIP1 remained unchanged30. On the other hand, Wolf et al reported that the expression of p27KIP1 was increased in hypertrophic glomeruli of db/db mouse31. Both groups suggested the importance of the p21/p27 KIP family in the pathogenesis of hypertrophy, but there are several discrepancies in their results. These differences may stem from differences in their experimental conditions or the diabetic states of the mice. To confirm that this p21/p27 KIP family could induce hypertrophy directly, we used adenovirus to overexpress these CDK inhibitors, taking advantage of its high transfection efficiency. Through this means, we showed clearly that overexpression of p27Kip1 and p21CIP1 caused hypertrophy in renal tubular cells, but that p16INK4 had no such effect.
In summary, we demonstrated that p27Kip1 and p21CIP1 may play an important role in hypertrophy of LLC-PK1 cells, but that p16INK4 did not cause any hypertrophic changes in these cells. The complexity of the regulatory pathways of hypertrophic change has only begun to emerge, and the results of this study provide a basis for further investigation for basic and therapeutic approaches toward hypertrophic changes in renal diseases.
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Acknowledgments
We thank Dr. I. Saito (University of Tokyo) for kindly providing the Adex1w, DNA-TPC, and AxCALacZ used in this study. We also thank Dr. H. Matsushime (University of Tokyo), Dr. E. Hawlow (Massachusetts General Hospital Cancer Center), Dr. T. Hunter (Salk Institute), and Dr. A. Noda (Meiji Cell Technology Center) for kindly providing the cDNAs.
