TGF-beta 1-mediated hypertrophy involves inhibiting pRB phosphorylation by blocking activation of cyclin E kinase

Baolian Liu and Patricia Preisig

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8856


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

When renal epithelial cells are exposed to epidermal growth factor-transforming growth factor-beta 1 (EGF-TGF-beta 1) the typical EGF-mediated hyperplastic growth response is converted to a hypertrophic growth response. Hypertrophy in this setting involves cell entrance into G1, but arrest of cell cycle progression at the G1/S interface. Late G1 arrest is mediated by retaining retinoblastoma protein (pRB) in its active, hypophosphorylated state. The present studies examine the mechanism by which pRB is retained in its active state. The results demonstrate that TGF-beta 1-mediated conversion of hyperplasia to hypertrophy involves preventing activation of cdk2/cyclin E kinase but has no effect on cdk4(6)/cyclin D kinase activity. Preventing activation of cyclin E kinase is associated with 1) decreased abundance of cdk2/cyclin E complexes and 2) retention of p57Kip2 in formed cdk2/cyclin E complexes. The development of hypertrophy does not involve regulation of either cdk2, cyclin E, or cdc25A protein abundances, or the abundance of p27Kip1 or p21 in formed complexes.

kidney; cell cycle; cell growth; cyclin kinase inhibitors; G1 kinases


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RENAL EPITHELIAL CELL hypertrophy occurs in a number of conditions, including diabetes mellitus, loss of renal mass, protein feeding, chronic metabolic acidosis, and chronic potassium deficiency (5, 23, 30). In the short term, hypertrophy may serve to augment renal function. However, in the long term, hypertrophy is associated with sclerosis and the progressive loss of renal function. Despite its importance, the mechanism by which renal tubule epithelial cell hypertrophy occurs is largely unknown. We have previously reported an in vitro model of cell hypertrophy that involves modulation of cell cycle processes (8, 25). In this model, transforming growth factor-beta 1 (TGF-beta 1) converts epidermal growth factor (EGF)-induced hyperplasia to hypertrophy. The conversion of hyperplasia to hypertrophy is due to persistent hypophosphorylation of a member of the retinoblastoma (pRB) family, resulting in cell cycle arrest in late G1.

Phosphorylation of pRB proteins, and thus regulation of their activity, is governed by two G1 kinases, cdk4(6)/cyclin D (cyclin D) and cdk2/cyclin E (cyclin E). The present studies examine the regulation of these two kinases during the development of hypertrophy. As would be expected for a mitogen-stimulated cell, exposure to EGF increases the activity of both cyclin D and cyclin E kinases. TGF-beta 1 converts hyperplasia to hypertrophy by blocking activation of cyclin E kinase but has no effect on EGF-induced cyclin D kinase activation.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Chemicals were purchased from Sigma Chemical (St. Louis, MO), except as noted: DMEM-Ham's F-12 culture media, fetal bovine serum, and trypsin/EDTA were from GIBCO-BRL (Life Technologies, Gaithersburg, MD); penicillin and streptomycin were from BioWhitaker, M.A. Bio-products (Walkersville, MD); culture dishes were from Corning Glassworks (Corning, NY); recombinant human TGF-beta 1 and EGF were from R&D Systems (Minneapolis, MN); [3H]thymidine and ECL kit were from Amersham (Arlington Heights, IL); anti-cell cycle protein antibodies and GST-pRB [pRB amino acids 769-921 fused to glutathione S-transferase (GST)] fusion protein were from Santa Cruz Biotechnology (Santa Cruz, CA); [gamma -32P]ATP was from DuPont New England Nuclear (Boston, MA); and histone H1 was from Boehringer-Mannheim Biochemica (Indianapolis, IN).

Cell culture. NRK-52E cells (a rat kidney epithelial cell line) were obtained from the American Type Culture Collection (Rockville, MD) at passage 15 and passaged and grown in low-glucose DMEM with 5% FCS. Cells were grown to confluence in 100-mm dishes, rendered quiescent by the removal of serum for 48 h, and then exposed to the EGF, TGF-beta 1, or vehicles for the indicated time periods. In general, all studies compared four experimental groups: 1) control, with both EGF and TGF-beta 1 vehicle; 2) EGF, with TGF-beta 1 vehicle; 3) TGF-beta 1, with EGF vehicle; and 4) the combination of EGF and TGF-beta 1. Recombinant human TGF-beta 1 was reconstituted in 4 mM HCl containing 0.1% heat-treated BSA. Recombinant human EGF was reconstituted in sterile 10 mM acetic acid containing 0.1% human serum albumin. Media were changed daily. In all studies, TGF-beta 1 was used at 10-10 M and EGF at 10-8 M, based on previously defined dose-response curves (8).

Measurement of [3H]thymidine incorporation. Rates of DNA synthesis were measured as rates of [3H]thymidine incorporation. For these studies, 1 µCi/well of [3H]thymidine was added 6 h before harvest of cells grown in a 96-well tissue culture plate. Cells were harvested onto filter paper using a cell harvester (model PHD; Cambridge Technologies, Cambridge, MA), and filters counted in a scintillation counter. Results are expressed as counts per minute per well.

Immunoprecipitation and immunoblotting of whole cell lysates. Cells were washed with ice-cold PBS (twice), harvested in 1 ml ice-cold PBS containing 1 mM dithiothreitol (DTT), 1 mM NaVO3, 1 mM NaF, 0.1 mg/ml phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml leupeptin, 0.234 TIU/ml aprotinin, and 1 µg/ml pepstatin A, and centrifuged at 20,000 g for 20 min to pellet the cells. The resulting pellet was resuspended in 500 µl lysis buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 25 mM EDTA, 5 mM EGTA, 0.25% sodium deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaVO3, 1 mM NaF, 0.1 mg/ml PMSF, 2 µg/ml leupeptin, 0.234 TIU/ml aprotinin, and 1 µg/ml pepstatin A), then the cells were lysed by repeated passage through a 27-gauge needle, further broken apart by incubating on a rocking aliquot shaker for 1 h at 4°C, and centrifuged at 4,000 g for 10-15 min at 4°C to pellet debris, and the supernatant was saved. Following measurement of the protein concentration by the Bradford assay (1), samples were aliquoted for immunoblotting or immunoprecipitation. Samples were size fractionated by SDS-PAGE, and immunoblotting was performed using appropriate antibodies, as previously reported (8). Bands were detected using ECL, and the abundance quantitated by scanning densitometry.

For immunoprecipitation, aliquots of protein prepared as above were incubated overnight with the appropriate antibody at a dilution of 1 µg antibody/1,000 µg cell protein and then with 200 µl of protein G-Sepharose beads/1,000 µg cell protein for 2 h. The beads were then collected by centrifugation at 15,000 g for 15 s, the supernatant was discarded, and the beads were washed three times with 1 ml lysis buffer. For immunoblotting of precipitated proteins, the pellet was mixed with Laemmli sample buffer and processed as described above. To measure kinase activity, beads were washed an additional two times using kinase reaction buffer that does not contain either cold or hot ATP or kinase substrate (see below). Following the second wash, the pellet was resuspended in kinase reaction buffer, as described in the next section.

To assay cdk4(6)/cyclin D kinase activity, the lysis buffer contained 50 mM HEPES (pH 7.5), 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 0.1% Tween-20, 1 mM DTT, 1 mM NaVO3, 1 mM NaF, 0.1 mg/ml PMSF, 2 µg/ml leupeptin, 0.234 TIU/ml aprotinin, and 1 µg/ml pepstatin A.

In vitro kinase assays. Kinase activity was measured on immunoprecipitates from whole cell lysates. To assay cdk4(6)/cyclin D kinase activity, the pellet was resuspended in 25 µl of kinase reaction buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 2.5 mM EGTA, 1 mM DTT, 10 mM beta -glycerophosphate, 0.1 mM NaVO3, 1 mM NaF, 20 mM ATP, 1 µg GST-pRB, and 0.83 µCi of [gamma -32P]ATP (6,600 Ci/mmol)] and incubated in a shaking water bath for 30 min at 30°C. The kinase reaction was stopped by the addition of Laemmli sample buffer, and the samples were boiled for 5 min and size separated by SDS-PAGE on a 12% gel. The gel was stained with Coomassie blue stain to confirm equal amounts of GST-pRB in each sample, destained, and dried. Phosphorylated pRB was visualized by autoradiography and quantitated by densitometry.

To assay cdk2/cyclin E kinase activity, the same procedure was used, except that the kinase reaction buffer contained 50 mM Tris · HCl (pH 7.5), 10 mM MgCl2, 1 µM DTT, 70 mM NaCl, 0.1 mM ATP, 0.2 µg/µl histone H1, and 0.01 µCi/µl [gamma -32P]ATP (6,600 Ci/mmol).

Statistics. Statistical significance was determined by ANOVA or paired t-test as appropriate. Differences between means were considered significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TGF-beta 1 converts EGF-induced hyperplasia to hypertrophy by allowing cells to enter G1 but then causing arrest of cell cycle progression at the G1/S transition (8). This latter effect is mediated by inhibiting phosphorylation of pRB or a related family member (8). The present studies examine the mechanism by which TGF-beta 1 modulates phosphorylation of pRB or a family member in this growth model.

Figure 1 shows the time course of the effects of EGF and TGF-beta 1 on thymidine incorporation. After exposure to EGF, thymidine incorporation increases significantly at 18 h. TGF-beta 1 causes an inhibition of thymidine incorporation, first seen at 12 h. The effect of EGF-TGF-beta 1 is identical to that of TGF-beta 1 alone. Since the focus of the remaining studies is the activity of the G1 kinases that regulate progression into S phase, all studies were done at time points in late G1, a few hours prior to initiation of DNA synthesis.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on [3H]thymidine incorporation. NRK-52E cells were grown to confluence, rendered quiescent by removal of serum for 48 h, and then treated with EGF (), TGF-beta 1 (), or EGF-TGF-beta 1 (black-triangle) for the indicated amounts of time. [3H]thymidine was added to the media for 6 h prior to harvest. Cells were harvested as described in METHODS; n = 12-24 for each time point. * P < 0.05 vs. control. At 12 h all 3 data points are significantly different from control.

TGF-beta 1 does not affect EGF-induced activation of cdk4(6)/cyclin D kinase. Figure 2 shows cdk4(6)/cyclin D kinase activity following exposure to EGF and/or TGF-beta 1. Irrespective of the immunoprecipitating antibody, cyclin D-associated kinase activity is increased at 12 and 15 h. TGF-beta 1 has no effect on the EGF-induced activation of cdk4(6)/cyclin D kinase activity at either time point.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of EGF and TGF-beta 1 on cdk4(6)/cyclin D kinase activity. Following exposure to EGF and/or TGF-beta 1 for the indicated times, kinase activity was measured in vitro on immunoprecipitates from the indicated antibodies and with GST-pRB [pRB amino acids 769-921 fused to glutathione S-transferase (GST)] as the substrate, as described in METHODS; n = 2-5 for each study. A: representative blots using cdk4 immunoprecipitates. B: summary of all experiments. Con, control; IP, immunoprecipitating antibody; n = 2-5 for each study. * P < 0.05 vs. control.

TGF-beta 1 inhibits activation of cdk2/cyclin E kinase. Figure 3 shows a time course of cdk2/cyclin E kinase activity assayed in cyclin E immunoprecipitates. In EGF-treated cells, cyclin E kinase activity is increased at 12 h (although it did not reach statistical significance), peaks at 15 h, and then decreases back to baseline values by 18 h. The return to baseline values by 18 h is consistent with the lack of cdk2/cyclin E kinase activity after the cell has crossed the Restriction Point near the G1/S interface. At 15 h, which represents the late G1 phase, TGF-beta 1 blocks 76% of the EGF-induced increase in cyclin E kinase activity.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of EGF and TGF-beta 1 on cyclin E kinase activity. Following exposure to EGF and/or TGF-beta 1 for the indicated times, kinase activity was measured in vitro on cyclin E immunoprecipitates using histone H1 as the substrate, as described in METHODS; n = 3-6 for each study. A: representative blots. B: summary of all experiments. * P < 0.05 vs. control. # P < 0.05 for EGF-TGF-beta 1 vs. EGF alone.

When cdk2 is the immunoprecipitating antibody, EGF-induced kinase activity progressively increases between 12 and 18 h (Fig. 4). Again, at 15 h, TGF-beta 1 inhibits EGF-induced kinase activity. Thus TGF-beta 1 prevents activation of cdk2/cyclin E kinase in late G1. The only notable difference between kinase activities measured on cyclin E and cdk2 immunoprecipitates occurs with EGF alone at 18 h. The persistent kinase activity in cdk2 immunoprecipitates probably represents cdk2/cyclin A kinase activity, which peaks in early S phase (24).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of EGF and TGF-beta 1 on cdk2 kinase activity. Following exposure to EGF and/or TGF-beta 1 for the indicated times, kinase activity was measured in vitro on cdk2 immunoprecipitates using histone H1 as the substrate, as described in METHODS; n = 4-6 for each study. A: representative blots. B: summary of all experiments. * P < 0.05 vs. control. # P < 0.05 for EGF-TGF-beta 1 vs. EGF alone.

TGF-beta 1 inhibits cdk2/cyclin E kinase complex formation. cdk2/cyclin E kinase activity can be regulated by the abundance of the complex. To measure complex abundance, cyclin E immunoprecipitates were subjected to immunoblotting with anti-cdk2 antibodies. Since cdk2/cyclin E kinase activity peaks at 15 h in mitogen-stimulated cells, these studies were performed at 15 h. As shown in Fig. 5, EGF alone increases the abundance of cdk2 in cyclin E immunoprecipitates to 324% of control. TGF-beta 1 inhibits the EGF-induced increase by 63%. Thus the TGF-beta 1-induced decrease in cdk2/cyclin E kinase activity is due at least in part to a decreased number of cdk2/cyclin E complexes.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of EGF and TGF-beta 1 on cdk2 abundance in cyclin E immunoprecipitates. Following immunoprecipitation with anti-cyclin E antibodies, immunoblotting using anti-cdk2 antibodies was done to determine the abundance of cdk2 in the immunoprecipitates following 15-h exposure to EGF and/or TGF-beta 1; n = 7 for each group. A: representative blots. B: summary of all experiments. * P < 0.05 vs. control. # P < 0.05 for EGF-TGF-beta 1 vs. EGF alone.

The effect of TGF-beta 1 to decrease cdk2/cyclin E complex abundance may be due to decreased abundance of one of the components needed for complex formation. To examine this, cdk2 and cyclin E abundances were examined in whole cell lysates (Fig. 6). As shown, EGF alone increases the abundance of both proteins, with peak increases at 15 h consistent with the peak in cdk2/cyclin E kinase activity. TGF-beta 1 alone has a slight inhibitory effect on the abundance of both proteins, but in combination with EGF does not affect EGF-induced increases in the abundance of either protein. Thus regulation of subunit abundance does not explain the decreased number of complexes.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of EGF and TGF-beta 1 on cdk2 and cyclin E protein abundances. Immunoblotting was used to determine the abundance of cyclin E (A) and cdk2 (B) in whole cell lysates following exposure to EGF and/or TGF-beta 1. Mean percent change in protein abundance is plotted on y-axis; time is on x-axis; n = 4-7 per study. * P < 0.05 vs. control. # Not significant (NS) for EGF-TGF-beta 1 vs. EGF alone.

TGF-beta 1 regulates p57Kip2 but not p21 or p27Kip1 abundance in the cdk2/cyclin E complexes. To determine whether the TGF-beta 1-induced decrease in cdk2/cyclin E kinase activity also involves preventing activation of formed cdk2/cyclin E complexes, we examined the abundance of cyclin kinase inhibitors (CKIs) associated with cdk2/cyclin E complexes. Figure 7 shows the ratio of CKI to cdk2 in cyclin E immunoprecipitates for the three CKIs that associate with cdk2/cyclin E complexes. The abundance of p27Kip1 in the complexes is similar in EGF, TGF-beta 1, and EGF-TGF-beta 1 groups. p57Kip2 abundance is decreased in EGF-treated cells, consistent with release of an inhibitory protein from the complex and activation of the kinase. TGF-beta 1 alone has very little effect on p57Kip2 abundance in the complexes. However, TGF-beta 1 blocks the EGF-induced decrease in p57Kip2 abundance in the complexes. The retention of p57Kip2 in the complexes likely contributes to TGF-beta 1-mediated inhibition of kinase activity.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of EGF and TGF-beta 1 on the ratio of cyclin kinase inhibitor (CKI) to cdk2 in cyclin E immunoprecipitates. Immunoblotting was performed on cyclin E immunoprecipitates following 15-h exposure to EGF and/or TGF-beta 1 using the indicated anti-CKI and anti-cdk2 antibodies. Ratio of CKI to cdk2 in cyclin E immunoprecipitates is plotted on y-axis; n = 6-8 for each group. * P < 0.05 vs. control. # NS for EGF-TGF-beta 1 vs. EGF alone.

The profile of p21Cip1,Waf1,Sdi1 (p21) abundance in the cdk2/cyclin E complexes is different from either p27Kip1 or p57Kip2 (Fig. 7). In EGF-treated cells, p21 abundance is increased. TGF-beta 1 alone has no significant effect on p21 abundance in the complexes and does not prevent the EGF-induced increase. The EGF-induced increase in p21 associated with cdk/cyclin complexes is consistent with studies in both fibroblasts and U20S cells showing that p21 promotes cdk/cyclin complex assembly (18, 32). As p21 abundance progressively increases in the complex, there is an increase in kinase activity (18, 32).

TGF-beta 1 does not affect cdc25A abundance. Activation of formed cdk/cyclin complexes also involves both phosphorylation of a conserved threonine residue in the COOH-terminal end and dephosphorylation of conserved threonine and tyrosine residues in the NH2-terminal end of the cdk molecule. Dephosphorylation of the NH2-terminal residues in the G1 kinases is mediated by the dual-specificity phosphatase, cdc25A, whose mRNA and protein abundances are increased by mitogenic stimuli and suppressed by antiproliferative agents, such as TGF-beta 1 and interferon-alpha in numerous cell types (9, 11, 12, 14, 15, 29).

To determine whether cdc25A abundance is regulated during TGF-beta 1-mediated hypertrophy, Western blotting was performed on whole cell lysates using an anti-cdc25A antibody. Figure 8 shows that EGF increases the abundance of cdc25A, consistent with increased phosphatase activity in G1 in mitogen-stimulated cells. TGF-beta 1 has no effect on EGF-induced increases in cdc25A abundance at any time point. Thus regulation of cdc25A abundance does not contribute to TGF-beta 1-induced inhibition of cdk2/cyclin E kinase activity.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of EGF and TGF-beta 1 on cdc25A abundance. Immunoblotting was used to determine the abundance of cdc25A in whole cell lysates following exposure to EGF and/or TGF-beta 1. Mean percent change in protein abundance is plotted on y-axis; time is on x-axis; n = 5 for each group. A: representative blots. B: summary of all experiments. * P < 0.05 vs. control. # NS for EGF-TGF-beta vs. EGF alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Renal hypertrophy occurs in a number of conditions, including chronic metabolic acidosis and potassium deficiency, following the loss of renal mass, in pregnancy, and in diabetes mellitus (5, 23, 30). Using in vitro (cell culture) models of renal epithelial cell growth, we characterized two mechanisms by which the development of hypertrophy can be mediated (7, 8, 25). One mechanism is independent of the cell cycle and involves alkalinization of intravesicular compartments. The second mechanism, elicited by EGF-TGF-beta 1 involves modulation of cell cycle processes. EGF causes cells to enter the cell cycle, grow, and then divide, whereas TGF-beta 1 blocks progression to S phase and DNA synthesis and leads to the development of hypertrophy.

Blockade at the G1/S transition by TGF-beta 1 is mediated by inhibiting phosphorylation of the retinoblastoma protein, pRB, or a related family member, which maintains these proteins in their active state (8). Inactivation of these pRB family proteins by expressing either SV40 large T antigen or the human papilloma virus 16-E7 protein prevents TGF-beta 1-induced hypertrophy, demonstrating a role for active pRB in the development of hypertrophy (8). pRB activity is regulated by the G1 kinases, cdk4(6)/cyclin D and cdk2/cyclin E. Although controversy remains about the exact function of each kinase in G1 progression and the regulation of pRB activity, recent studies have begun to elucidate a specific role for each. Cyclin D and E kinases phosphorylate different sites on the pRB molecule (2, 16, 21). Phosphorylation by cyclin D converts pRB from the unphosphorylated state (present in newly synthesized pRB or pRB residing in quiescent cells) to the hypophosphorylated state (4, 21). The hypophosphorylated form of pRB binds E2F and causes it to negatively regulate transcription of S phase-required genes. This ensures that the cell is not equipped to move into S phase and begin DNA synthesis, and is consistent with studies suggesting that cyclin D plays a specific role in the G0-to-G1 transition (26). In late G1, pRB is further phosphorylated by cyclin E, which leads to the protein being in the hyperphosphorylated, inactive state (21). In the hyperphosphorylated state, pRB can no longer bind E2F, leading to transcription of S phase-required genes and the initiation of DNA synthesis. Activation of cyclin E appears to demonstrate a threshold phenomenon, meaning that if sufficient kinase activity is present, then movement into S phase is an all-or-nothing phenomena. If the kinase is not activated or insufficient kinase activity is present, then cells are arrested in the late G1 phase. Recent studies have shown that cyclin D cannot fully phosphorylate pRB, and cyclin E cannot phosphorylate the unphosphorylated form of pRB (21). Thus progression through G1 requires the coordinated effects of both kinases.

The present studies sought to elucidate the mechanism by which TGF-beta 1 inhibits phosphorylation of pRB family proteins. The results show that activation of cdk4(6)/cyclin D is not affected by TGF-beta 1. The studies also demonstrate that renal epithelial cells express both catalytic subunits (cdk4 and cdk6) that associate with the cyclin D family of regulatory subunits and that both catalytic subunits support cyclin D kinase activity. In addition, these cells express at least two of the three members of the cyclin D family, both of which can support kinase activity. (We were unable to determine whether cyclin D2 is expressed in these cells because the available rodent anti-cyclin D2 antibodies cross-react with cyclin D1.)

The main effect of TGF-beta 1 is to inhibit cdk2/cyclin E kinase. Activation of the cell cycle kinases is a multistep process. Increases in kinase activity are often associated with increased abundance of the regulatory (cyclin) and/or catalytic (cdk) subunits. Complexes form between one catalytic and one regulatory subunit, with additional proteins, such as proliferating cell nuclear antigen, associated with the complex (25). Activation of the formed complex then requires phosphorylation of a conserved threonine residue in the COOH-terminal region of the cdk molecule by cyclin-dependent kinase activating kinase (CAK) and, probably for most cell cycle kinases, dephosphorylation of conserved threonine and tyrosine residues in the NH2-terminal region of the cdk molecule by a dual-specificity phosphatase (3, 6, 19, 22). For activation of the G1 kinases, cdc25A is the dual-specificity phosphatase that is responsible for dephosphorylating the NH2-terminal residues (12, 15).

In this model of hypertrophy, TGF-beta 1-induced inhibition of cdk2/cyclin E kinase is associated with decreased formation of stable cdk2/cyclin E complexes. The lack of effect of TGF-beta 1 on EGF-induced increases in either cdk2 or cyclin E protein abundances demonstrates that the decrease in complex formation is not due to insufficient subunits, but rather suggests regulation of a step critical to complex formation and/or stability.

Induction of TGF-beta 1-mediated G1 arrest by inhibition of cdk2/cyclin E but not cdk4(6)/cyclin D kinase activity has been observed in other cell types. In HepG2 cells, TGF-beta 1 causes G1 arrest by a mechanism that retains cdk6/cyclin D kinase activity but negatively regulates cdk2 kinase activity and also retains pRB in the hypophosphorylated state. This observation, along with the present studies, supports the hypothesis that cyclin D kinase plays a role in hypophosphorylating pRB but not in hyperphosphorylating and thus inactivating the protein (4). In Mv1Lu cells, TGF-beta 1-mediated growth arrest is also associated with inhibition of cyclin E kinase activity (17). In addition, these studies showed, like the present studies, that inhibition of kinase activity was not associated with a decrease in either cdk2 or cyclin E protein abundance, but was associated with a decrease in the formation of stable complexes between cdk2 and cyclin E.

The most common mechanism by which activation of formed complexes is negatively regulated is by the association of a CKI with the complex. The CKIs are small-molecular-weight proteins that belong to one of two families, grouped by sequence homology and the mechanism by which they prevent kinase activation (28). Only one family, made up of p21, p27Kip1, and p57Kip2, binds formed cdk2/cyclin E complexes and prevents their activation (28). In the present studies, the abundance of p57Kip2 in cyclin E immunoprecipitates is decreased by EGF. TGF-beta 1 blocks the EGF-induced decrease, retaining p57Kip2 in the cdk2/cyclin E complexes. Thus the decrease in kinase activity likely involves a combined effect of decreased complex formation and p57Kip2-induced blockade of kinase activation in formed complexes.

p27Kip1 is expressed, but not regulated by either EGF or TGF-beta 1, in NRK-52E cells. This observation is in contrast to studies in other renal cells. In cultured mesangial cells in vitro and intact glomeruli in vivo, p27Kip1 abundance decreases in cytokine-induced mesangial cell proliferation in vitro and during the reparative phase of anti-Thy-1-induced glomerulonephritis (27). In cultured mesangial cells in which p27Kip1 expression has been knocked out by the use of antisense oligodeoxynucleotides, TGF-beta 1 is still able to reduce proliferation induced by either platelet-derived growth factor or basic fibroblast growth factor, suggesting that the inhibitory effect of TGF-beta 1 is not mediated solely by p27Kip1 despite regulation of protein expression (27). The present studies demonstrate that p27Kip1 is not responsible for TGF-beta 1-mediated growth arrest in cultured renal epithelial cells either, despite expression of the protein. In contrast, p27Kip1 protein abundance and association with G1 kinases is upregulated in angiotensin II-induced hypertrophy in LLC-PK1 cells (31).

p21 serves not only as a kinase inhibitor, but also as an activator of the G1 kinases (32). In the absence of p21, the kinase is inactive. As the concentration of p21 in the complex increases up to a point, kinase activity increases and then abruptly decreases with higher p21 concentrations. In NRK-52E cells, EGF increases the abundance of p21 in the complex, and TGF-beta 1 does not modify this effect. Thus regulation of p21 abundance in the complexes is not involved in the conversion of hyperplasia to hypertrophy.

The last step of the process involved in kinase activation that was examined in the present studies was the abundance of cdc25A, the dual-specificity phosphatase involved in dephosphorylating the NH2-terminal residues of the catalytic subunit. EGF increases the abundance of cdc25A, and TGF-beta 1 does not alter this induced increase. Since cdc25A activity is regulated by regulating its protein abundance in several cell types, these studies suggest that prevention of cdk2 dephosphorylation is not responsible for the low levels of kinase activity (9, 11, 12, 14, 15, 29).

Thus cell cycle-dependent hypertrophy is a mitogen-mediated process in which progression through the cell cycle is arrested at the G1/S interface. Blocking progression into S phase is mediated by preventing sufficient pRB phosphorylation to inactivate the protein. In NRK-52E cells, TGF-beta 1 inhibits pRB phosphorylation by preventing activation of cdk2/cyclin E kinase by: 1) decreasing the formation of stable cdk2/cyclin E complexes and 2) increasing p57Kip2 abundance in formed cdk2/cyclin E complexes.

On the basis of these observations, we propose the following model for cell cycle-dependent hypertrophy. After either a hyperplastic or hypertrophic growth stimulus that causes cells to enter G1, there is activation of cyclin D kinase. Since this kinase is activated in both hyperplasia and cell cycle-dependent hypertrophy, we propose that it is involved in initiating the physical growth of the cell, a part of both growth patterns. If the cells progress to a point where cyclin E kinase is activated sufficiently to reach threshold, then pRB is hyperphosphorylated, cells move into S phase, and hyperplasia is the resulting growth pattern. However, if cyclin E kinase is either not activated at all or is insufficiently activated to reach the threshold, then pRB remains in its hypophosphorylated state, cells arrest in G1, and the result is hypertrophy. Thus cyclin D governs the physical growth of the cell, whereas cyclin E defines the growth pattern as either hyperplasia or hypertrophy.

Recently we have found that in the in vivo setting regulation of the G1 kinases is associated with the development of hypertrophy following uninephrectomy and in streptozotocin-induced diabetes mellitus (10, 13, 20). In both conditions there is a correlation between kinase activities and hyperplasia and hypertrophy that parallels the observations with EGF and EGF-TGF-beta in cultured cells. Thus it is likely that both diabetes-induced and compensatory renal hypertrophy are mediated by a cell cycle-dependent process and that the EGF and EGF-TGF-beta 1 cell culture models of hyperplasia and hypertrophy, respectively, are relevant to renal tubule epithelial cell growth in vivo.


    ACKNOWLEDGEMENTS

We appreciate the technical assistance of M. Ferguson and E. Abdel-Salam. We thank Bob Alpern for helpful discussions and critical reading of the manuscript.


    FOOTNOTES

This work was supported by American Heart Association Grant-In-Aid 94017970.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Preisig, Dept. of Internal Medicine, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Rm. H5.112, Dallas, TX 75235-8856 (E-mail: patricia.preisig{at}emailswmed.edu).

Received 17 November 1998; accepted in final form 7 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 243-245, 1976.

2.   Connell-Crowley, L., J. W. Harper, and D. W. Goodrich. Cyclin D1/cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation. Mol. Biol. Cell 8: 287-301, 1997[Abstract].

3.   Deshaies, R. J. Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast. Curr. Opin. Genet. Dev. 7: 7-16, 1997[Medline].

4.   Ezhevsky, S. A., H. Nagahara, A. M. Vocero-Akbani, D. R. Gius, M. C. Wei, and S. F. Dowdy. Hypo-phosphorylation of the retinoblastoma protein (pRB) by cyclin D: cdk4/6 complexes results in active pRb. Proc. Natl. Acad. Sci. USA 94: 10699-10704, 1997[Abstract/Free Full Text].

5.   Fine, L. G., J. T. Norman, D. A. Kujubu, and A. Knecht. Renal hypertrophy. In: The Kidney: Physiology and Pathophysiology, edited by D. W. Selden, and G. Giebish. New York: Raven, 1992, p. 3113-3133.

6.   Fisher, R. P. CDKs and cyclins in transition(s). Curr. Opin. Genet. Dev. 7: 32-38, 1997[Medline].

7.   Franch, H. A., and P. A. Preisig. NH4Cl-induced hypertrophy is mediated by weak base effects and is independent of cell cycle processes. Am. J. Physiol. 270 (Cell Physiol. 39): C932-C938, 1996[Abstract/Free Full Text].

8.   Franch, H. A., J. W. Shay, R. J. Alpern, and P. A. Preisig. Involvement of pRB family in TGFB-dependent epithelial cell hypertrophy. J. Cell Biol. 129: 245-254, 1995[Abstract].

9.   Galaktionov, K., X. Chen, and D. Beach. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 382: 511-517, 1996[Medline].

10.   Ginsberg, D., G. Vairo, T. Chittenden, Z. X. Xiao, G. Xu, K. L. Wydner, J. A. DeCaprio, J. B. Lawrence, and D. M. Livingston. E2F-4, a new member of the E2F transcription factor family, interacts with p107. Genes Dev. 8: 2665-2679, 1994[Abstract].

11.   Gu, Y., J. Rosenblatt, and D. O. Morgan. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 11: 3995-4005, 1992[Abstract].

12.   Hoffmann, I., G. Draetta, and E. Karsenti. Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J. 13: 4302-4310, 1994[Abstract].

13.   Huang, H.-C., and P. A. Preisig. Cyclin D kinase is activated in all diabetic renal growth, while cyclin E kinase determines whether the growth pattern will be hyperplasia or hypertrophy. (Abstract). J. Am Soc. Nephrol. 9: 440A, 1998.

14.   Iavarone, A., and J. Massague. Repression of the CDK activator Cdc25A and cell-cycle arrest by cytokine TGF-beta in cells lacking the CDK inhibitor p15. Nature 387: 417-422, 1997[Medline].

15.   Jinno, S., K. Suto, A. Nagata, M. Igarashi, Y. Kanaoka, H. Nojima, and H. Okayama. Cdc25A is a novel phosphatase functioning early in the cell cycle. EMBO J. 13: 1549-1556, 1994[Abstract].

16.   Kitagawa, M., H. Higashi, H.-K. Jung, I. Suzukitakahashi, M. Ikeda, K. Tamai, J. Kato, K. Segawa, E. Yoshida, S. Nishimura, and Y. Taya. The consensus motif for phosphorylation by cyclin D1-cdk4 is different from that for phosphorylation by cyclin A/E-cdk2. EMBO J. 15: 7060-7069, 1996[Abstract].

17.   Koff, A., M. Ohtsuki, K. Polak, J. M. Roberts, and J. Massague. Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-beta . Science 260: 536-539, 1993[Medline].

18.   LaBaer, J., M. D. Garrett, L. F. Stevenson, J. M. Slingerland, C. Sandhu, H. S. Chou, A. Fattaey, and E. Harlow. New functional activities for the p21 family of CDK inhibitors. Genes Dev. 11: 847-862, 1997[Abstract].

19.   Lew, D. J., and S. Kornbluth. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr. Opin. Cell Biol. 8: 795-804, 1996[Medline].

20.   Liu, B., and P. A. Preisig. Compensatory renal hypertrophy (CRH) is mediated by both cell cycle-dependent and -independent growth processes (Abstract). J. Am. Soc. Nephrol. 9: 444A, 1998.

21.   Lundberg, A. S., and R. A. Weinberg. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell. Biol. 18: 753-761, 1998[Abstract/Free Full Text].

22.   Morgan, D. O. The dynamics of cyclin dependent kinase structure. Curr. Opin. Cell Biol. 8: 767-772, 1996[Medline].

23.   Norman, J. T., and L. G. Fine. Renal growth and hypertrophy. In: Textbook of Nephrology, edited by S. G. Massry, and R. J. Glassock. Baltimore: Williams & Wilkins, 1995, p. 146-158.

24.   Pagano, M., R. Pepperkok, F. Verde, W. Ansorge, and G. Draetta. Cyclin A is required at two points in the human cell cycle. EMBO J. 11: 961-971, 1992[Abstract].

25.   Preisig, P. A., and H. A. Franch. Renal epithelial cell hyperplasia and hypertrophy. Semin. Nephrol. 15: 327-340, 1995[Medline].

26.   Resnitzky, D., M. Gossen, H. Bujard, and S. I. Reed. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol. Cell. Biol. 14: 1669-1679, 1994[Abstract].

27.   Shankland, S. J. Cell-cycle control and renal disease. Kidney Int. 52: 294-308, 1997[Medline].

28.   Sherr, C. J., and J. M. Roberts. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9: 1149-1163, 1995[Medline].

29.   Tiefenbrun, N., D. Melamed, N. Levy, D. Resnitzky, I. Hoffmann, S. I. Reed, and A. Kimchi. Alpha interferon suppresses the cyclin D3 and cdc25A genes, leading to a reversible Go-like arrest. Mol. Cell. Biol. 16: 3934-3944, 1996[Abstract].

30.   Wolf, G., and E. G. Neilson. Molecular mechanisms of tubulointerstitial hypertrophy and hyperplasia. Kidney Int. 39: 401-420, 1991[Medline].

31.   Wolf, G., and R. A. K. Stahl. Angiotensin II-stimulated hypertrophy of LLC-PK1 cells depends on the induction of the cyclin-dependent kinase inhibitor p27Kip1. Kidney Int. 50: 2112-2119, 1996[Medline].

32.   Zhang, H., G. J. Hannon, and D. Beach. p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 8: 1750-1758, 1994[Abstract].


Am J Physiol Renal Physiol 277(2):F186-F194
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society