Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8856
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ABSTRACT |
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When renal epithelial cells are exposed to epidermal growth
factor-transforming growth factor-1 (EGF-TGF-
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-
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
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INTRODUCTION |
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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-1
(TGF-
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-1 converts hyperplasia to
hypertrophy by blocking activation of cyclin E kinase but has no effect
on EGF-induced cyclin D kinase activation.
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METHODS |
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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-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);
[
-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-1, or vehicles for
the indicated time periods. In general, all studies compared four
experimental groups: 1) control, with both EGF and TGF-
1 vehicle;
2) EGF, with TGF-
1 vehicle; 3) TGF-
1, with EGF vehicle; and
4) the combination of EGF and TGF-
1. Recombinant human TGF-
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-
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 ![]() |
RESULTS |
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TGF-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-
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-1 on thymidine incorporation. After exposure
to EGF, thymidine incorporation increases significantly at 18 h.
TGF-
1 causes an inhibition of thymidine incorporation, first seen at
12 h. The effect of EGF-TGF-
1 is identical to that of TGF-
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.
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TGF-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-
1. Irrespective of the
immunoprecipitating antibody, cyclin D-associated kinase activity is
increased at 12 and 15 h. TGF-
1 has no effect on the
EGF-induced activation of cdk4(6)/cyclin D kinase activity at either
time point.
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TGF-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-
1 blocks 76% of
the EGF-induced increase in cyclin E kinase activity.
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TGF-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-
1
inhibits the EGF-induced increase by 63%. Thus the TGF-
1-induced
decrease in cdk2/cyclin E kinase activity is due at least in part to a
decreased number of cdk2/cyclin E complexes.
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TGF-1 regulates
p57Kip2 but not p21 or
p27Kip1 abundance in the cdk2/cyclin E
complexes.
To determine whether the TGF-
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-
1, and EGF-TGF-
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-
1 alone has very
little effect on p57Kip2 abundance
in the complexes. However, TGF-
1 blocks the EGF-induced decrease in
p57Kip2 abundance in the
complexes. The retention of
p57Kip2 in the complexes likely
contributes to TGF-
1-mediated inhibition of kinase activity.
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TGF-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-
1 and interferon-
in numerous cell types (9, 11, 12, 14, 15, 29).
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DISCUSSION |
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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-1 involves
modulation of cell cycle processes. EGF causes cells to enter the cell
cycle, grow, and then divide, whereas TGF-
1 blocks progression to S
phase and DNA synthesis and leads to the development of hypertrophy.
Blockade at the G1/S transition by
TGF-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-
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-1
inhibits phosphorylation of pRB family proteins. The results show that
activation of cdk4(6)/cyclin D is not affected by TGF-
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-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-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-
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-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-
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-
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-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-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-
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-
1 is not mediated solely by
p27Kip1 despite regulation of
protein expression (27). The present studies demonstrate that
p27Kip1 is not responsible for
TGF-
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-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-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-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- 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-
1 cell
culture models of hyperplasia and hypertrophy, respectively, are
relevant to renal tubule epithelial cell growth in vivo.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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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
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
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- 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-.
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
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].