(Received for publication, July 8, 1996, and in revised form, January 22, 1997)
From the Departments of Experimental Therapeutics and
Molecular Medicine, Roswell Park Cancer Institute,
Buffalo, New York 14263
The molecular mechanisms underlying protein
kinase C (PKC) isozyme-mediated control of cell growth and cell cycle
progression are poorly understood. Our previous analysis of PKC isozyme
regulation in the intestinal epithelium in situ revealed
that multiple members of the PKC family undergo changes in expression
and subcellular distribution precisely as the cells cease proliferating
in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in
this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC
isozyme(s) in control of intestinal epithelial cell growth and cell
cycle progression was examined directly using the IEC-18 immature crypt
cell line as a model system. Treatment of IEC-18 cells with PKC
agonists resulted in translocation of PKC ,
, and
from the
soluble to the particulate subcellular fraction, cell cycle arrest in
G1 phase, and delayed transit through S and/or G2/M phases. PKC-mediated cell cycle arrest in
G1 was accompanied by accumulation of the
hypophosphorylated, growth-suppressive form of the retinoblastoma
protein and induction of the cyclin-dependent kinase
inhibitors p21waf1/cip1 and p27kip1. Reversal of these
cell cycle regulatory effects was coincident with activator-induced
down-regulation of PKC
,
, and
. Differential down-regulation
of individual PKC isozymes revealed that PKC
in particular is
sufficient to mediate cell cycle arrest by PKC agonists in this system.
Taken together, the data implicate PKC
in negative regulation of
intestinal epithelial cell growth both in vitro and
in situ via pathways which involve modulation of Cip/Kip
family cyclin-dependent kinase inhibitors and the
retinoblastoma growth suppressor protein.
Protein kinase C (PKC)1 is a family of
serine/threonine kinases which play a central role in signal
transduction and have been widely implicated in control of cell growth,
differentiation, and transformation (1-4). The primary physiological
activator of PKC is diacylglycerol (DAG), which is transiently
generated by agonist-induced hydrolysis of phosphoinositides and other
membrane phospholipids (1, 5). PKC activation, often accompanied by
increased association of the enzyme with cellular membranes and/or
cytoskeletal elements (6-9), initiates a signaling cascade leading to
alterations in gene expression and modulation of a variety of cellular
functions. Initial interest in PKC stemmed from its identification as
the major cellular receptor for tumor-promoting phorbol esters (10),
suggesting a role in stimulation of cell growth and transformation.
While studies in a number of tissues have implicated PKC in positive
control of cell growth (11-15) and transformation (16, 17),
accumulating evidence also points to its involvement in cell growth
inhibition and differentiation (14, 15, 18-21). A key to understanding
these diverse responses is the observation that PKC represents a
multigene family of 11 closely related enzymes with varying structures
and enzymological characteristics (1, 2): the conventional PKC isozymes
,
I,
II, and
, which require Ca2+ for activity
and respond to phorbol esters; the novel isozymes
,
,
,
,
and µ, which do not require Ca2+, and the atypical
isoforms
and
, which neither require Ca2+ nor
respond to phorbol esters. Individual PKC isozymes also exhibit varying
substrate specificity, tissue distribution, and subcellular localization (1, 2, 22, 23); these differences, together with the
varied consequences of PKC activation in the same cell (e.g.
Refs. 24-26), the expression of more than one isozyme in most cell
types (1, 2, 21, 27), and conservation of the isozymes in higher
organisms (2), argue that individual isozymes play specific,
specialized roles in cell signaling. However, understanding of the
biological functions of individual isozymes and of the molecular
regulatory pathways in which they participate remains limited.
The growth-regulatory consequences of PKC activation suggest a link
between PKC signaling and control of the cell cycle machinery. Activation of PKC has been shown to result in alterations in cell cycle
progression in either stimulatory or inhibitory directions in several
systems (14, 19, 20, 28-30). Moreover, limited evidence supports a
role for individual isozymes in modulation of major cell cycle
transitions. For example, PKC /
(29) and
(31) have been
implicated in control of the G1
S transition in
vascular smooth muscle cells and NIH 3T3 fibroblasts, respectively, while PKC
II has been shown to play a requisite role in progression from G2 into M phase in HL-60 cells (30), and PKC
has
been associated with control of M phase in Chinese hamster ovary cells (32). Orderly progression through the cell cycle is now known to be
dependent on the coordinated interaction between key cell cycle
regulatory molecules including cyclins, cyclin-dependent kinases (cdks), and cdk inhibitory proteins such as
p21waf1/cip1 and p27kip1 (33-35). Together, these
molecules control the activity of a number of important substrates
including the retinoblastoma gene product (Rb), a critical regulator of
the G1
S transition (36, 37). The interplay between
specific PKC isozyme signaling and control of the cell cycle machinery
is an important question that remains to be addressed at the molecular
level.
Previous studies in this laboratory have identified the mammalian
intestinal epithelium as a useful model system in which to define the
physiological role(s) of individual PKC isozyme(s) in control of cell
growth and cell cycle progression (27). This dynamic and complex tissue
system undergoes continuous and rapid renewal (38); its polarized
architecture, with well-defined regions of cell proliferation, growth
arrest, and differentiation, allows correlation of the expression and
activation of PKC isozymes with specific stages of cell development
(27). Using a combined biochemical and morphological approach to detect
changes in PKC isozyme expression and activation status at the
individual cell level, our previous studies revealed that multiple PKC
isozymes are expressed in the intestinal epithelium and that they are
differentially regulated with respect to cell growth and
differentiation (27). Of particular note was the finding that four
members of the PKC family, PKC ,
II,
, and
, undergo marked
changes in expression and subcellular distribution indicative of
activation precisely at the point in the mid-crypt at which cells cease
dividing, suggesting that one or more of these molecules are involved
in negative growth-regulatory signaling pathways in this tissue.
In the present study, we have extended the characterization of PKC
isozyme expression and activation in the rat intestinal epithelium
in situ and, based on this analysis, have explored the role
of PKC isozyme(s) in control of cell growth and cell cycle progression
using the non-transformed IEC-18 immature crypt cell line as a
complementary in vitro model system. In keeping with the
finding that activation of specific isozymes coincides with cell growth
arrest in the intestinal crypt, PKC agonists were found to block IEC-18
cell cycle progression in G1 and delay transit through S
and/or G2/M phases. Analysis of the mechanism(s) underlying
PKC-mediated regulation of the G1 S transition revealed that PKC agonist-induced G1 arrest is accompanied by Rb
hypophosphorylation and rapid induction of the Cip/Kip family cdk
inhibitors p21waf1/cip1 and p27kip1. Taking advantage
of differential down-modulation of individual PKC isozymes under
different PKC agonist treatment conditions, PKC
was identified as
sufficient to mediate these negative growth-regulatory effects. Taken
together, the data presented provide evidence for control of the cell
cycle machinery in the intestinal epithelium by PKC-mediated pathways
and demonstrate a mechanism for the integration of environmental
anti-mitogenic stimuli with regulation of cell division in this tissue.
Furthermore, they indicate that PKC
, in particular, is linked to a
pathway which negatively modulates cell growth and cell cycle
progression in intestinal epithelial cells both in vitro and
in situ.
Monoclonal antibody specific for the catalytic
domain of PKC was obtained from Upstate Biotechnology, Inc. (Lake
Placid, NY). Polyclonal rabbit anti-PKC
,
II,
,
, and
were purchased from Life Technologies, Inc. and Santa Cruz
Biotechnology (Santa Cruz, CA). Antibodies specific for PKC
,
,
, and
were purchased from Transduction Laboratories, Inc.
(Lexington, KY) and Santa Cruz Biotechnology. The antibodies used in
this study have been extensively characterized for the absence of
cross-reactivity with other PKC isozymes (27). To ensure reliability of
the data obtained, at least two antibodies were used for each isozyme
studied. Polyclonal antibody specificity was confirmed by competition
assays with the appropriate antigenic peptide as described previously (27). Polyclonal anti-Rb and anti-p21waf1/cip1 antibodies were
obtained from Santa Cruz Biotechnology. Monoclonal antibody specific
for p27kip1 was obtained from Transduction Laboratories.
Horseradish peroxidase-conjugated rat anti-mouse and goat anti-rabbit
secondary antibodies were purchased from Boehringer Mannheim.
Immunofluorescence analysis of PKC isozyme expression and subcellular distribution in rat intestinal epithelial tissue was performed on 4-6-µm cryosections as described previously (27).
Isolation of Rat Intestinal Epithelial Cells and Preparation of Membrane FractionsSequential release of epithelial cell populations from rat small intestine was performed as described previously (27, 39). Briefly, populations of cells at different developmental stages were isolated using timed incubations in a calcium-chelating buffer. Cells were washed in PBS, and fractions were pooled into crypt (proliferating), lower villus (differentiating), and upper villus (functional) populations. Membranes were prepared from isolated crypt or villus cells as described previously (27).
IEC-18 Cells and PKC Activation ProtocolsThe IEC-18 cell line (ATCC CRL-1589) is an immature, non-transformed cell line derived from rat ileal epithelium which maintains many characteristics of proliferating crypt cells (27, 40, 41). IEC-18 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 4 mM glutamine, 10 µg/ml insulin, and 5% fetal calf serum (FCS).
PKC isozymes were activated in IEC-18 cells by treatment with either
100 nM phorbol 12-myristate 13-acetate (PMA;
Sigma), 100 nM phorbol 12,13-dibutyrate
(PDBu; Sigma), or 20 µg/ml 1,2-dioctanoyl-sn-glycerol (DiC8; Sigma) for various times. PMA and PDBu were
dissolved in ethanol, with a final vehicle concentration in the medium
of <0.1%; DiC8 was dissolved in acetone, with a final
vehicle concentration of <0.2%. Control cells were treated with the
appropriate vehicle alone. PMA was administered either as a 15-min
pulse, followed by two washes in PBS and a return to fresh medium for
various times, or in continuous exposure. DiC8 treatments
required addition of fresh drug every 6 h, as DiC8 is
rapidly metabolized by the cell (42, 43). Depletion of PKC ,
,
and
from IEC-18 cells was accomplished by treatment with 1 µM PDBu for 24 h.
IEC-18 cells were synchronized in G0/G1 by serum deprivation. Briefly, subconfluent cells growing in complete medium were washed twice in DMEM with no FCS and incubated with DMEM containing 0.5% FCS and 4 mM glutamine (no insulin) for 72 h. More than 90% of cells were arrested in G0/G1 by this method, as determined by flow cytometric analysis. Cells were released from G0/G1 arrest by addition of complete growth medium containing 5% FCS and 10 µg/ml insulin.
IEC-18 cells were synchronized in G1/S phase by incubation with 1 µg/ml aphidicolin for 24 h. Cells were released from G1/S arrest by removal of aphidicolin and incubation in complete growth medium.
Flow Cytometric Analysis of IEC-18 Cell Cycle DistributionSubconfluent IEC-18 cells were briefly washed in PBS, harvested by trypsinization, fixed in 70% ethanol, and treated with 0.04 mg/ml RNase A (Sigma) in 20 mM Tris, pH 7.5, 250 mM sucrose, 5 mM MgCl2, and 0.37% Nonidet P-40 (Sigma). Cellular DNA was stained with 25 µg/ml propidium iodide (Sigma) in 0.05% sodium citrate and quantified by flow cytometry. Cell cycle analysis was performed using the Winlist and Modfit programs (Verity Software House, Topsham, ME).
Subcellular FractionationIEC-18 cells were partitioned into soluble (cytosolic) and particulate fractions essentially as described previously (27). Briefly, cells were washed twice in cold PBS and scraped in an extraction buffer containing 20 mM Tris, pH 7.5, 2 mM EGTA, 2 mM EDTA, 0.5 mg/ml digitonin, 10 mM NaF, 4 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin (digitonin buffer). Digitonin-soluble (cytosolic) and -insoluble (particulate) fractions were separated by ultracentrifugation at 100,000 × g for 40 min at 4 °C. The cytosolic protein in the supernatant was precipitated with 10% trichloroacetic acid for 10 min on ice, pelleted, washed in acetone, solubilized in 100 mM NaOH, and neutralized by the addition of 100 mM HCl. The particulate pellet was incubated on ice for 30 min in digitonin buffer containing 1% Triton X-100 (Triton buffer). The membrane sample was then cleared by centrifugation (10,000 × g) for 30 min at 4 °C. Cytosolic and membrane fractions were boiled in Laemmli sample buffer (44) for 5 min before being subjected to SDS-PAGE and Western blot analysis.
Whole Cell ExtractsSubconfluent IEC-18 cells were rapidly washed twice in PBS at 4 °C and incubated for 5-15 min on ice in 20 mM Tris, pH 7.6, 120 mM NaCl, 100 mM NaF, 200 µM Na3V04, 4 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.5% Nonidet P-40 (Sigma). Cell extracts were cleared by a 30-min centrifugation (10,000 × g) at 4 °C and boiled in Laemmli sample buffer (44) for 5 min before being subjected to SDS-PAGE and Western blot analysis.
Western Blot AnalysisCell lysates (30 µg) were subjected to SDS-PAGE (44), using 7.5% (Rb), 10% (PKC isozymes), or 15% (p21waf1/cip1, p27kip1) polyacrylamide gels. Protein was electrophoretically transferred to nitrocellulose membrane, and membranes were blocked in Tris-buffered saline (TBS; 20 mM Tris-HCl, 137 mM NaCl, pH 7.6) containing 5% non-fat dried milk (TBS/milk) for 30 min at 37 °C. Membranes were incubated for 2 h at room temperature or overnight at 4 °C with primary antibody in TBS/milk with 0.1% Tween 20 (TBSt/milk; Sigma), followed by six 5-min washes in TBSt/milk. Blots were then incubated for 1 h at room temperature in secondary HRP-conjugated antibody in TBSt/milk, followed by six 5-min washes in TBSt/milk and three 5-min washes in TBS. Bound horseradish peroxidase was then detected using the SuperSignal CL system (Pierce). Specificity of the antibodies was determined using the relevant antigenic peptide in competition experiments as recommended by the manufacturer. All data presented are representative of at least three independent experiments.
IEC-18 cells
are non-transformed, immature intestinal epithelial cells derived from
rat ileum, which retain many phenotypic characteristics of
proliferating intestinal crypt cells (40, 41). To evaluate this cell
line as an in vitro model system in which to examine
directly the involvement of PKC isozymes in intestinal epithelial cell
growth regulation, we compared PKC isozyme profiles in proliferating
crypt cells in situ and cultured IEC-18 cells. Previous
morphological and biochemical analysis of the expression and
subcellular distribution of individual PKC isozymes in the rat
intestinal epithelium revealed that PKC ,
II,
,
, and
,
but not PKC
or
I, are present in cells of the crypt-villus unit
and are differentially regulated with respect to cell growth and
differentiation (27). In situ immunofluorescence analysis
demonstrated that marked changes in the expression and subcellular
distribution of several PKC isozymes coincides precisely with cell
growth arrest in the mid-crypt region. Representative data are shown
for PKC
in Fig. 1A: proliferating lower
crypt cells exhibit diffuse cytosolic immunostaining for this isozyme (arrowhead), presumably reflecting its presence in an
inactive conformation. At cell position 14-18 from the crypt base
(i.e. the mid-crypt), the region in which cells cease
division and commit to differentiation (38), levels of PKC
expression markedly increase, and PKC
immunostaining becomes
clearly detectable in the lateral membrane domains and in the
developing brush-border microvilli. Similar changes, confirmed by
biochemical analysis (27), were observed for PKC
II,
, and
(27), suggesting that one or more of these PKC isozymes play a
role in signaling pathway(s) related to negative control of cell growth
in the intestinal epithelium in situ.
Further analysis has revealed the presence of three additional
isozymes, PKC ,
, and
, in this tissue (Fig. 1B).
Based on the premise that association of PKC isozymes with the
particulate fraction may be indicative of activation (6), the pattern
of regulation of these PKC isozymes along the crypt-to-villus axis was
examined by comparing the level of membrane-associated expression in
isolated proliferating (crypt), differentiating (lower villus), and
functional (upper villus) intestinal epithelial cells. As shown in Fig.
1B, crypt cell membranes express low levels of PKC
, and
only trace amounts of PKC
and
. Since PKC
and
were also
essentially undetectable in whole cell extracts of intestinal crypt
cells (data not shown), these isozymes appear to be absent from
proliferating cells. Thus, taken together with our previous findings
(27), these data demonstrate that in situ proliferating cells of the intestinal crypts express PKC
,
II,
,
,
,
and
. Interestingly, in contrast to cells of the proliferation zone, differentiating and functional cells of the villus express readily detectable levels of membrane-associated PKC
,
, and
(Fig. 1B), indicating that increased expression/activation of
these isozymes is associated with post-mitotic events in intestinal epithelial cells in situ. Immunofluorescence localization of
PKC
revealed that this isozyme is expressed mainly in cells of the mid- to upper villus, in association with the endoplasmic reticulum (data not shown), suggesting a role in enterocyte mature function (45).
Due to a lack of suitable reagents, we were unable to perform
morphological analysis of the distribution of PKC
and
in tissue
enterocytes.
As shown in Fig. 1C, Western blot analysis revealed that
IEC-18 cells express the same panel of PKC isozymes as tissue crypt cells. While PKC ,
II,
,
,
, and
were readily
detectable in IEC-18 whole cell extracts, PKC
and
, which are
expressed only in villus cell populations in situ, were
found to be absent from these cells. In the present study, the IEC-18
cell line was used as an in vitro model system in which to
address the hypothesis that one or more PKC isozymes play a role in
negative regulation of cell growth and cell cycle progression in the
intestinal epithelium in situ.
To determine the effects of PKC
activation on IEC-18 cell cycle progression, asynchronously growing
IEC-18 cell populations were treated with a panel of PKC agonists to
activate PKC isozyme(s) directly, and perturbations in cell cycle
distribution were determined by flow cytometric analysis after various
times over a 24-h period. Three PKC agonists were used for this
analysis: the phorbol esters PMA (100 nM) and PDBu (100 nM) and the DAG analogue, DiC8 (20 µg/ml).
Phorbol esters are potent activators of most members of the PKC family,
with the exception of PKC and
(1, 10, 22), and can be used to
mimic the effects of DAG and to bypass normal agonist-mediated control
of the enzyme. The use of two phorbol esters in this study was based on
evidence that different members of this class of agents can induce
distinct cellular responses (22). The membrane-permeant DAG analogue
DiC8, a less potent but more physiological PKC agonist, was
used to confirm the involvement of a PKC-mediated pathway in observed
phorbol ester cell cycle-specific effects. PMA was administered either
as a 15-min pulse or in continuous exposure, while PDBu and
DiC8 were added only in continuous exposure.
As shown in Fig. 2A.i, treatment of IEC-18
cells with PMA, added as a 15-min pulse or in continuous exposure,
resulted in a marked reduction (70-75%, n = 6) in the
relative number of cells in S phase by 6 h. This decrease was
accompanied by a 20-30% (n = 6) increase in the
relative number of cells in G1, indicating that PKC
activation negatively regulates transit through this phase of the cell
cycle. Although quantitative analysis revealed no significant change in
cell cycle distribution at 3 h following addition of PMA, the
number of cells with DNA content corresponding to early S phase was
markedly reduced by this time (arrows), indicating that
entry into S phase had been restricted; thus, the G1 S block was evident by 3 h. Pulse or continuous treatment of IEC-18 cells with PMA for 6 h also resulted in a 70-80%
(n = 6) increase in the proportion of cells with
apparent 4n DNA content, consistent with an accumulation of cells in
G2/M and/or possibly late S phase of the cell cycle. This
increase could reflect a PMA-mediated delay in transit of IEC-18 cells
through G2/M and/or delayed exit from S phase. Inhibition
of cell cycle progression was transient; reversal of the
G2/M and/or S phase effects occurred prior to release of
the G1/S block, resulting in accumulation of the majority (>80%) of the population in G1 by 12 h. The
G1/S block in cell cycle progression was consistently
released 12-14 h following addition of PMA. By 24 h, cells could
be seen in all phases of the cell cycle. Treatment of IEC-18 cells with
the phorbol ester PDBu produced similar results (Fig.
2A.ii); an accumulation of cells in G1 phase as
well as in G2/M and/or late S phase of the cell cycle was
seen by 6 h of continuous exposure to this PKC agonist. However,
release of the G1 block was evident by 9-10 h, indicating
that the kinetics of the effect were slightly accelerated in
PDBu-treated cells.
PKC agonists inhibit cell cycle progression in IEC-18 cells. A, subconfluent cultures of asynchronously growing IEC-18 cells were treated with PKC agonists for various times over a 24-h period, and cell cycle distribution was determined by flow cytometric analysis of DNA content in propidium iodide-stained cells as described under "Experimental Procedures." i, IEC-18 cells were treated with vehicle alone (Untreated) or 100 nM PMA (either as a 15-min pulse or in continuous exposure), and relative DNA content was determined after 3, 6, 12, 14, and 24 h. ii, IEC-18 cells were treated continuously with 100 nM PDBu, and cell cycle distribution was analyzed at 6, 9, 10, and 24 h. iii, IEC-18 cells were treated with 20 µg/ml DiC8, and cell cycle distribution was determined at 6 and 24 h. Numbers indicate percentage of cells in G0/G1, S, and G2/M phases. Arrows indicate initial restriction of entry into S phase at 3 h. Data are representative of three or more independent experiments. B, subconfluent cultures of IEC-18 cells synchronized in G0/G1 phase by serum withdrawal for 72 h were released from growth arrest by addition of 5% FCS (time 0) and treated with 100 nM PMA or 20 µg/ml DiC8 8 h later; cell cycle distribution was determined at 14, 18, and 22 h after serum stimulation (i.e. 6, 10, and 14 h of PKC agonist treatment). Data are representative of three independent experiments. C, subconfluent cultures of IEC-18 cells synchronized in G1/S phase with aphidicolin were released from cell growth arrest by removal of the drug and treated with PKC agonists 4 h later; cell cycle distribution was determined at 6 and 8 h after removal of aphidicolin (i.e. 2 and 4 h of PKC agonist treatment). Data are representative of three independent experiments.
In contrast to the transient cell cycle arrest resulting from phorbol ester treatment, DiC8 produced a sustained inhibition of cell cycle progression in IEC-18 cells over the 24-h experimental period (Fig. 2A.iii). As with phorbol ester treatment, a significant reduction (~60%, n = 4) in the relative number of cells in S phase was consistently evident by 6 h following administration of the DAG analogue; this reduction was accompanied by a 20-30% (n = 4) increase in the proportion of cells in G1 and a 30-40% (n = 4) increase in the proportion of cells with apparent 4n DNA content. The G1 effects were maintained over the 24-h period, as indicated by a sustained >30% (n = 3) increase in the proportion of G1 cells relative to control cell populations. The persistence of a significant population of cells with apparent 4n DNA content at the 24-h time point despite a sustained G1/S block suggests that a delay in G2/M progression and/or exit from S phase is also sustained over the 24-h experimental period. Taken together, these data demonstrate that activation of PKC by three different PKC agonists results in inhibition of IEC-18 cell cycle progression at two (or more) points in the cell cycle: one in G1 and the other in G2/M and/or possibly S phase. The duration of these effects was found to differ with the PKC agonist used; while phorbol esters produced transient cell cycle arrest in IEC-cells, DiC8 treatment resulted in a sustained inhibition of cell cycle progression over the 24-h experimental period.
To determine if PKC-mediated cell cycle arrest is associated with
differentiation of IEC-18 cells into absorptive enterocytes, we
examined PKC agonist-treated cells for expression of the
differentiation marker, alkaline phosphatase (39), using an in
situ enzyme cytochemical approach. Neither control nor PKC
agonist-treated cells were found to express the differentiation marker
(data not shown). Furthermore, PKC agonists did not induce the
expression of PKC or
, isozymes associated with differentiated
cells of the villus in situ (data not shown). Taken
together, the data indicate that PKC-mediated withdrawal from the cell
cycle is not associated with differentiation in this system.
To confirm and further investigate PKC-mediated regulation of cell cycle progression in IEC-18 cells, experiments were conducted using synchronized cell populations. IEC-18 cells were synchronized in G0/G1 by serum deprivation for 72 h and released from growth arrest by addition of 5% FCS (time 0). Entry of cells into S phase was evident by 12-14 h following serum stimulation, and by 22 h, cells had progressed through G2/M and begun to re-enter G0/G1 (Fig. 2B). Treatment of IEC-18 cells with either PMA or DiC8 in mid-G1 (6-8 h following serum stimulation) resulted in inhibition of cell cycle progression into S phase. PMA treatment resulted in transient G1 arrest, with release kinetics similar to those seen in unsynchronized cells; i.e. arrest was maintained for ~12 h following addition of phorbol ester. DiC8 treatment, on the other hand, resulted in sustained inhibition of cell cycle progression into S phase. At 22 h after serum stimulation (14 h after addition of DiC8), DiC8-treated cells were still accumulated in G1, in sharp contrast to both PMA-treated and control cells.
To examine PKC-mediated effects on IEC-18 cell cycle progression through S and G2/M phases, cells were synchronized in G1/S phase by treatment with 1 µg/ml aphidicolin for 24 h. Following removal of aphidicolin, cells progressed through S and G2/M phases, returning to G1 by 6 h (Fig. 2C). Consistent with the increase in the proportion of cells with apparent 4n DNA content observed following treatment of asynchronously growing IEC-18 cells with PKC agonists (Fig. 2, A.i, A.ii, and A.iii), the addition of PMA or DiC8 4 h following removal of aphidicolin markedly retarded progression of IEC-18 cells into G1 phase, likely reflecting delayed transit through G2/M and/or S phase of the cell cycle. Progression of cells into G1 phase was delayed for at least 6 h following addition of either PKC agonist (data not shown).
Phorbol Esters and the DAG Analogue DiC8 Differentially Modulate PKC Isozyme Expression and Subcellular Distribution in IEC-18 CellsTo investigate the mechanism(s) underlying phorbol ester-
and DiC8-mediated inhibition of cell cycle progression in
IEC-18 cells and to examine the basis for the differential responses to
these agents, the effects of each agonist on PKC isozyme expression and
subcellular distribution were examined at various times during the 24-h
treatment period. In the absence of known specific in vivo
substrates for the individual PKC isozymes examined in this system, PKC
isozyme translocation (i.e. association with the particulate fraction) and down-regulation were used as measures of agonist-induced isozyme-specific effects and possible indicators of PKC isozyme activation (6, 22). IEC-18 cells were treated with 100 nM PMA (administered as a 15-min pulse or in continuous exposure), 100 nM PDBu (in continuous exposure), or 20 µg/ml
DiC8 (in continuous exposure), harvested at various times
(i.e. 15 min, 2 h, 6 h, 12 h, or 24 h),
and partitioned into soluble and particulate fractions; PKC isozyme
levels in each fraction were determined by Western blot analysis.
Treatment with PMA or PDBu resulted in essentially complete
translocation of PKC ,
, and
to the particulate subcellular fraction within 15 min (Fig. 3). In cells exposed
continuously to either PMA or PDBu (Fig. 3, A and
B), down-regulation of PKC
,
, and
was evident by
6 h, and these isozymes were essentially depleted from the cells
by 12 h. PKC
was more resistant to PMA-mediated down-regulation than PKC
or
, with significant levels persisting in the particulate fraction for longer than 6 h. PMA pulse
treatment produced similar effects on PKC isozyme subcellular
distribution and expression, with a notable difference in the
regulation of PKC
. As shown in Fig. 3C, PMA pulse
treatment also resulted in marked down-regulation of PKC
and
over the 24-h period. PKC
and cytosolic PKC
were depleted from
the cells by 12 h, and membrane-associated PKC
was
significantly down-regulated under these treatment conditions. Low
levels of down-regulation-resistant PKC
were frequently observed in
the particulate fraction following either pulse or continuous treatment
with PMA for 24 h (see Fig. 3, A and C). PKC
, on the other hand, showed a unique response to pulse treatment
with PMA. While down-regulation of PKC
was evident in the
particulate subcellular fraction by 6 h, this effect was
accompanied by reappearance of the isozyme in the soluble fraction. By
12 h, control patterns of PKC
expression and subcellular distribution had been re-established in PMA pulse-treated cells (Fig.
3C). Phorbol ester treatment did not affect the expression or subcellular distribution of the atypical PKC isozymes
and
(data shown only for PKC
, Fig. 3D). Interestingly, these
PKC agonists also had no effect on the expression or subcellular
distribution of PKC
II.
DiC8, in contrast to phorbol esters, induced translocation
but did not significantly down-regulate PKC ,
, and
over the 24-h experimental period (Fig. 4). Interestingly, while
DiC8 treatment resulted in complete translocation of PKC
and
to the particulate fraction, PKC
was only partially
translocated by this DAG analogue. PKC
II,
, and
were neither
translocated to the particulate fraction nor down-regulated by this
agent (data not shown). Taken together, the data demonstrate that
induction and maintenance of PKC-mediated inhibition of cell cycle
progression in IEC-18 cells is coincident with the presence of PKC
,
, and/or
in the particulate subcellular fraction. Thus, phorbol
ester treatment results in transient growth arrest and transient
compartmentalization of these PKC isozymes in the particulate fraction.
In contrast, DiC8 produces sustained inhibition of cell
cycle progression and sustained presence of PKC
,
, and
in
this subcellular compartment. Therefore, the data support a role for
PKC
,
, and/or
in negative growth regulatory signaling in
IEC-18 intestinal epithelial cells. This notion is further supported by
the observation that release of the PMA-induced G1/S block
in cell cycle progression in synchronized IEC-18 cells is also
coincident with down-regulation of PKC
,
, and
(data not
shown).
PKC Activation in IEC-18 Cells Results in Rb Hypophosphorylation
To explore the molecular pathways by which PKC interacts with the intestinal epithelial cell cycle and induces G1 arrest, the effects of phorbol esters or DiC8 on the expression and activity of key G1 control molecules was investigated. The retinoblastoma gene product (Rb) is a major regulator of the G1/S transition (36, 37). In its underphosphorylated, active form, assumed during late mitosis, Rb binds the transcription factor E2F and prevents it from participating in expression of genes required for DNA synthesis. During the course of G1, Rb is functionally inactivated by sequential phosphorylation by several cyclin·cdk complexes, resulting in release of E2F and allowing transcription of S phase genes.
To examine the effects of PKC activation on the functional state of Rb,
asynchronously growing IEC-18 cells were exposed to PKC agonists for
various times, and Rb phosphorylation state was examined by Western
blot analysis. This analysis revealed that PMA (administered as a
15-min pulse or in continuous exposure) or PDBu produced a marked
increase in the levels of hypophosphorylated, active Rb in IEC-18 cells
(Fig. 5, A and B). Rb
hypophosphorylation was evident at both 2 h and 6 h following
addition of phorbol ester. However, increased levels of
hypophosphorylated Rb were no longer detectable at 24 h,
indicating that the effect was transient. More detailed analysis of the
time course of the effect revealed a significant loss of
hyperphosphorylated, inactive Rb, with concurrent appearance of the
hypophosphorylated, active form of the protein, by 60-90 min following
addition of these agents (Fig. 5C). Hypophosphorylation of
Rb was maintained for 8-10 h and was undetectable by 12 h (Fig. 5D). Thus, PKC agonist-induced translocation of PKC ,
, and/or
in these cells was associated with a shift in Rb
phosphorylation favoring the hypophosphorylated, transcription
factor-binding form which retards cell cycle progression.
Down-regulation of PKC
,
, and
, on the other hand, was
accompanied by a shift favoring the hyperphosphorylated,
growth-permissive form of Rb and coincided with release of phorbol
ester-mediated cell cycle arrest in IEC-18 cells.
DiC8 treatment also produced an accumulation of the
hypophosphorylated, growth-suppressive form of Rb by 2 h,
confirming the involvement of a PKC-mediated signal transduction
pathway in modulation of Rb phosphorylation state in IEC-18 cells (Fig.
5E). In contrast to the effects seen with PMA and PDBu,
hypophosphorylation of Rb was maintained over the 24-h experimental
period. The sustained change in Rb phosphorylation state is consistent
with the sustained presence of PKC ,
, and
in the particulate
subcellular fraction and sustained cell growth arrest, supporting a
role for one or more of these isozymes in negative growth-regulatory
pathways involving modulation of the Rb growth suppressor protein in
IEC-18 cells.
To examine the mechanism(s) underlying PKC-mediated
modulation of Rb phosphorylation state, the effects of phorbol esters and DiC8 on the expression of the Cip/Kip family cdk
inhibitors p21waf1/cip1 and p27kip1 were examined by
Western blot analysis. p21waf1/cip1 and p27kip1
represent a family of molecules which block the activity of the cyclin·cdk complexes responsible for phosphorylation of Rb (46). Treatment of IEC-18 cells with 100 nM PMA (administered as
a 15-min pulse or in continuous exposure) resulted in a marked increase in levels of p21waf1/cip1 by 2 h; levels of this cdk
inhibitor remained elevated at 6 h but were indistinguishable from
those in control cells by 24 h (Fig.
6A). Strong induction of p21waf1/cip1
expression was seen within 60 min of PMA addition and reached a maximum
at 2 h (Fig. 6B). The level of p21waf1/cip1
induction by PMA was comparable with that observed 2 h following UV irradiation, a strong inducer of the protein (47). As with other
effects of phorbol ester treatment, the increase in
p21waf1/cip1 expression was transient. Elevated levels were
maintained for at least 6 h, but were found to diminish
significantly thereafter (Fig. 6, A and C).
DiC8 (20 µg/ml) treatment also produced an increase in
p21waf1/cip1 expression; in this case, however, increased
levels were sustained over the 24-h treatment period (Fig.
6D). Taken together, the data indicate that a PKC-mediated
signaling cascade modulates p21waf1/cip1 expression in IEC-18
cells; reversal of the effect in phorbol ester-treated cells correlates
with down-regulation of PKC ,
, and
, reappearance of the
hyperphosphorylated form of Rb, and release of the block in cell cycle
progression. Treatment with DiC8, which resulted in
sustained translocation of PKC
,
and
, produced a sustained
elevation of p21waf1/cip1 levels in IEC-18 cells. Analysis of
the levels of the related cdk inhibitor p27kip1 consistently
revealed similar, but more modest, changes in response to continuous
PMA treatment for 2 or 4 h (Fig. 7). A reproducible ~1.5-fold increase in p27kip1 levels was seen at these times
following PKC agonist treatment.
PKC Agonists Modulate Rb Phosphorylation State and cdk Inhibitor Levels in Synchronized IEC-18 Cells
To determine if PKC agonists
induce changes in cell cycle control molecules when applied
specifically in the G1 phase, the effects of PKC agonists
on Rb phosphorylation state and on the expression of
p21waf1/cip1 and p27kip1 were examined in
G1-synchronized cells. IEC-18 cells synchronized by serum
deprivation were released from growth arrest by serum stimulation and
treated with PMA 8 h later; control and treated cells were
collected at 10 and 16 h following release from growth arrest, and
lysates were subjected to Western blot analysis. PMA treatment in
mid-G1 resulted in hypophosphorylation of Rb and induction
of p21waf1/cip1 and p27kip1 within 2 h of PKC
agonist treatment (Fig. 8). Hypophosphorylation of Rb
was maintained in PMA-treated cells at 16 h after serum stimulation, a point at which control cells express fully
hyperphosphorylated Rb.
PKC
To confirm the requirement
for PKC ,
, and/or
in mediating growth-inhibitory effects in
IEC-18 cells, we examined the ability of PKC agonists to induce cell
cycle arrest, Rb hypophosphorylation, and p21waf1/cip1
expression in IEC-18 cells depleted of these isozymes. Treatment of
IEC-18 cells with 1 µM PDBu for 24 h resulted in
complete down-regulation of PKC
,
, and
(Fig.
9A), without affecting levels of PKC
II,
, or
(data not shown). These results are consistent with reported evidence that long term treatment with high concentrations of
phorbol ester promotes proteolytic degradation and thus depletion of
PKC isozymes in many biological systems (48), allowing assessment of
PKC agonist effects in the absence of specific members of this enzyme
family. Following PDBu treatment, the cells were washed extensively to
remove the agonist. Subsequent retreatment of these PKC
-,
-, and
-depleted cells with 100 nM PMA failed to produce the
cell cycle arrest, Rb hypophosphorylation, or induction of p21waf1/cip1 which were evident in cells in which PKC isozymes
had not been depleted by previous exposure to PDBu (Fig. 9,
B-D). These data provide further support for the
requirement for one or more of these isozymes in mediating PKC
agonist-induced growth-inhibitory effects. They also indicate that PKC
II,
, and
are not involved in PKC agonist-induced negative
growth regulation in this system.
PKC
As shown in
Fig. 3C, pulse treatment (15 min) of IEC-18 cells with 100 nM PMA results in translocation followed by eventual depletion of cytosolic PKC and
by 24 h. In contrast,
following initial translocation, PKC
disappears from the
particulate fraction and begins to re-emerge in the cytosolic
compartment by 6 h. Thus, restimulation of IEC-18 cells with PKC
agonists 24 h after a PMA pulse treatment addresses the response
of these cells under conditions in which the major PKC
agonist-responsive isozyme is PKC
. The small amount of
membrane-associated, down-regulation-resistant PKC
expressed by
these cells is unlikely to play a role in negative growth-regulatory
signaling events in this system, since the presence of comparable
levels of this isozyme was unable to sustain growth arrest in cells
exposed continuously to PMA over a 24-h period. Retreatment of these
PKC
/
-deficient cells with 100 nM PMA resulted in
complete translocation of PKC
to the particulate fraction (Fig.
10A), inhibition of IEC-18 cell cycle
progression (Fig. 10B), hypophosphorylation of Rb (Fig.
10C), and accumulation of p21waf1/cip1 (Fig.
10D). Taken together, these data support the ability of PKC
alone to mediate negative growth-regulatory signaling in IEC-18
cells, involving pathways controlling Rb phosphorylation and cdk
inhibitor expression. Moreover, they indicate that PKC
and
do
not play an obligate role in mediating growth arrest in intestinal
epithelial cells.
Despite extensive evidence for a role of the PKC family of signal
transduction molecules in positive or negative control of cell growth
(3), understanding of the underlying mechanisms involved and of the
specific growth-regulatory function(s) of individual isozymes remains
limited. We have previously shown a strong correlation between
activation of PKC family members and control of cell growth in
intestinal crypts in situ (27). In the present study, the
role of PKC isozymes in growth-inhibitory signaling was established
directly in the IEC-18 crypt cell line, using a panel of PKC activators
to bypass normal agonist-mediated control of PKC isozyme expression and
activation state. The data presented provide strong support for the
involvement of one or more specific PKC isozymes in negative regulation
of cell cycle progression in G1 phase and demonstrate that
PKC alone is sufficient to mediate this growth-regulatory effect.
PKC-mediated G1 arrest was found to involve induction of
Cip/Kip family cdk inhibitors and activation of the growth-suppressive
function of the Rb protein, thus linking PKC
to control of cdk
inhibitor expression and Rb phosphorylation state in this system. The
data also demonstrate a secondary cell cycle effect of PKC agonists in
G2/M and/or possibly S phase, indicating that PKC
isozyme(s) can act at multiple stages to inhibit cell cycle progression
in IEC-18 cells.
Phorbol ester- or DiC8-mediated inhibition of cell cycle
progression was shown to correlate directly with translocation of three
members of the PKC family, PKC ,
, and
, to the particulate subcellular fraction in IEC-18 cells. The involvement of these isozyme(s) in PKC agonist-induced cell cycle-specific effects was
further indicated by the finding that the different abilities of
phorbol esters and DAG analogues to sustain cell cycle-inhibitory effects in this system correlated with differential modulation of the
expression of these molecules. Phorbol esters produced rapid
down-regulation of these isozymes which coincided temporally with
release of the block in cell cycle progression. In contrast, DiC8 maintained significant levels of PKC
,
, and
in the particulate fraction over the 24-h experimental period,
consistent with the sustained inhibition of cell cycle progression
produced by this agent. Furthermore, depletion of PKC
,
, and
from IEC-18 cells abrogated PKC agonist-induced cell cycle effects in
this system. Involvement of PKC
II,
, and
in these
growth-regulatory effects was excluded by the findings that
(a) IEC-18 cells expressing only PKC
II,
, and
failed to undergo growth arrest in response to PKC agonists (Fig. 9),
and (b) neither the expression nor the subcellular
distribution of these isozymes was affected by treatment with phorbol
esters or DiC8. Thus, of the six PKC isozymes expressed in
the IEC-18 cell line, one classical (PKC
) and two novel (PKC
and
) isozymes were implicated in the observed growth-inhibitory response.
To gain insight into the individual contribution of PKC ,
,
and/or
to negative growth regulation in IEC-18 cells, we used a
pharmacological approach to manipulate specific PKC isozyme expression
levels and subcellular distribution in this system. These studies
implicated PKC
, in particular, in mediating negative cell cycle
regulatory events in IEC-18 cells. Conditions were established
(i.e. a 15-min pulse treatment with 100 nM PMA
followed by incubation in fresh medium for 24 h) which produced a
population of IEC-18 cells in which the major PKC agonist-responsive
isozyme present was PKC
. Stimulation of these PKC
- and
-deficient cells with phorbol esters (Fig. 10) or DiC8
(data not shown) resulted in translocation of PKC
to the
particulate subcellular fraction and recapitulated the inhibition of
cell cycle progression observed in cells expressing the full profile of
PKC isozymes. As mentioned above, this effect was abrogated in IEC-18
cells also depleted of PKC
as a result of long term treatment with
1 µM PDBu (Fig. 9). Taken together with the finding that,
in phorbol-ester treated cells, release of the block in cell cycle
progression at 12 h correlated temporally with depletion of PKC
, while PKC
and
exhibited different kinetics of
down-regulation (Figs. 2 and 3), the data demonstrate that PKC
alone is sufficient to mediate PKC agonist-induced negative
growth-regulatory signals in IEC-18 cells and that PKC
and
do
not play a requisite role in these responses. Although the ability of
PKC
and
to mediate growth-inhibitory signals has not been ruled
out in this study, the notion that these isozymes do not participate in
negative growth regulation in this system is consistent with the
demonstration that the expression and subcellular distribution of PKC
do not change with cell growth arrest in the intestinal crypt
in situ, and that the most pronounced changes in PKC
occur in association with enterocyte mature function on the villus
(27). The ability of PKC
to mediate PKC agonist-induced
growth-regulatory effects in IEC-18 cells correlated directly with its
presence in the particulate subcellular fraction; i.e.
disappearance of membrane-associated PKC
coincided with reversal of
the cell cycle effects, even when accompanied by its reappearance in
the cytosol. Based on evidence from a number of studies supporting a
link between PKC
translocation and enzymic activation, the data
point to a role for PKC
kinase activity in mediating negative
growth-regulatory signaling in intestinal epithelial cells, a notion
that will be addressed directly in future studies.
The data presented in this report implicating PKC isozyme(s) in
negative growth-regulatory signaling pathways and inhibition of cell
cycle progression are consistent with increasing evidence from studies
in a number of cellular systems, including vascular smooth muscle cells
(29), vascular endothelial cells (14, 49), melanoma cells (20), IMR-90
fibroblasts (28), and hematopoietic cells (50), demonstrating that PKC
can mediate cell cycle arrest at the G1/S boundary and/or
in G2/M phase. Thus, PKC isozyme-mediated inhibition of
cell cycle progression is not unique to intestinal epithelial cells,
but may reflect a widespread function of one or more PKC family
members. Moreover, our data implicating PKC , in particular, in
growth suppression in intestinal epithelial cells are in keeping with
emerging information from a number of systems linking this isozyme with
negative growth regulation. For example, specific activation of PKC
in K562 erythroleukemia cells results in cytostasis and megakaryocytic
differentiation (24), whereas PKC
II plays a requisite role in
proliferation of these cells (30, 51). Similarly, overexpression of PKC
in R6 rat embryo fibroblasts results in marked growth inhibition (25), while overexpression of PKC
I (25) or
(17) enhances the
growth of these cells. Overexpression of PKC
in F9 teratocarcinoma cells (52), B16 melanoma cells (19), CHO cells (53), or 3Y1 fibroblasts
(54) has also been shown to result in inhibition of cell growth/cell
cycle progression. Taken together with our findings indicating that
appropriately compartmentalized endogenous PKC
can negatively
modulate cell cycle progression in intestinal epithelial cells, these
data suggest that the biological role of PKC
is specifically
associated with negative growth-regulatory signaling.
Our studies in the IEC-18 model indicate that PKC isozyme(s), and PKC
in particular, act by initiating a cascade of events resulting in
increased levels of hypophosphorylated, transcriptionally repressive
Rb. It is now well established that Rb is a major regulator of cell
cycle progression; in its hypophosphorylated form, Rb represses
transcription of genes essential for entry into S phase and thus
induces cell cycle arrest. PKC agonists induced rapid (within 90 min)
hypophosphorylation/activation of Rb in IEC-18 cells. Importantly, this
change in Rb phosphorylation state occurred prior to cell growth
arrest, supporting a cause-and-effect relationship between these
events. Thus, PKC-mediated accumulation of the growth-suppressive form
of Rb could account for the observed G1 block in cell cycle progression. The involvement of Rb in PKC-mediated cell
cycle-inhibitory effects is further supported by studies in human
umbilical vein endothelial cells in which PDBu treatment was shown to
induce G1 arrest and Rb hypophosphorylation (14) and in NIH
3T3 cells, where ectopic expression of PKC
was shown to block
normal growth factor-induced phosphorylation of the Rb protein (31). In
addition, overexpression of PKC
in 3Y1 fibroblasts has been shown
to decrease E2F transcriptional activity, further linking PKC to
activation of the growth-suppressive function of Rb (54).
Insight was also obtained into the mechanism(s) by which PKC
isozyme(s), and PKC in particular, modulate the activation state of
the Rb protein. The phosphorylation state and activity of Rb are
controlled by both positive and negative regulators (37). Cyclin·cdk
complexes phosphorylate, and thus functionally inactivate, Rb during
the course of G1. The activity of cdks is regulated by
cyclin binding, by positive and negative phosphorylation events, and by
the accumulation or activation of cdk-inhibitory proteins such as
p21waf1/cip1 and p27kip1 (46). Our data demonstrate
that PKC isozyme(s), or PKC
alone, can mediate induction of Cip/Kip
family cdk inhibitors in IEC-18 cells. The time course of
p21waf1/cip1 induction closely paralleled the appearance of
hypophosphorylated Rb in response to PKC agonists, suggesting that PKC
isozyme-mediated G1 arrest occurs, at least in part, as a
result of increased expression of cdk inhibitory molecules and
subsequent inhibition of Rb phosphorylation. Further evidence for this
pathway comes from the observation that p21waf1/cip1 expression
is induced in response to PMA treatment in leukemic cells (55, 56),
that NIH 3T3 cells overexpressing PKC
show elevated levels of both
p21waf1/cip1 and p27kip1 (31), and that PMA treatment
of synchronized melanoma cells inhibits the down-regulation of
p21waf1/cip1 and p27kip1 seen at the G1/S
border in these cells (57). Our demonstration that the DAG analogue
DiC8 also induces the expression of p21waf1/cip1
confirms the involvement of PKC isozyme(s) in mediating this effect.
While the molecular mechanisms underlying PKC isozyme-mediated control
of cdk inhibitor levels remain to be determined, it is noteworthy in
regard to p21waf1/cip1 induction that the activity of the p53
transcription factor, a major inducer of this cdk inhibitory protein
(reviewed in Ref. 46), has been shown to be positively modulated by PKC
phosphorylation (58). Thus, treatment of IEC-18 cells with PKC agonists
may result in phosphorylation and activation of p53 and subsequent induction of p21waf1/cip1 gene expression. However, evidence
also exists for p53-independent induction of p21waf1/cip1 (59,
60); the involvement of these pathways in PKC-mediated p21waf1/cip1 induction in IEC-18 cells is currently under
investigation in our laboratory.
The physiological relevance of the findings presented in this report
regarding PKC isozyme control of cell growth and cell cycle progression
in the intestinal epithelium is supported by our previous demonstration
that marked changes in the expression and subcellular distribution of
four members of the PKC family, including PKC , coincide
precisely with cell growth arrest in small intestinal epithelial crypts
in situ (27). Similar regulation of PKC isozymes has
recently been reported in rat (61) and human (62) colonic epithelium,
where marked increases in expression of several PKC isozymes coincide
with cell growth arrest in the upper region of colonic crypts. It
should be noted that, although PKC agonists did not induce the
expression of differentiation markers in IEC-18 cells, our findings do
not exclude a possible role of PKC-mediated cell cycle control in
regulating downstream differentiation events in the intestinal
epithelium in situ. The data presented also offer a
perspective on the role of disturbances in the PKC enzyme system in
development of colonic neoplasms. Human and rat colonic tumors have
been shown to exhibit reduced levels of PKC activity (63-65) and PKC
isozyme expression (66-69) relative to paired adjacent normal colonic
mucosa. Decreased levels of PKC activity have also been observed in
colonic adenomas (63), indicating that changes in PKC isozyme
regulation occur early in the multistage process of colon
carcinogenesis. It is noteworthy in this regard that PKC
has been
found to be markedly down-regulated in both rat (66, 69) and human
colonic adenocarcinomas.2 Furthermore,
overexpression of PKC
I in HT-29 human adenocarcinoma cells was
demonstrated to result in marked inhibition of cell growth and
reduction of tumorigenicity in nude mice (70), findings which led
Weinstein and colleagues to suggest that, in some cell types, PKC acts
as a tumor suppressor gene. Thus, reduced levels of PKC isozyme
expression in colonocytes may impair normal growth-inhibitory signaling, leading to increased cell growth and contributing to the
development of colonic neoplasia.
Taken together, the data presented indicate that PKC isozyme
activation, and PKC activation in particular, mediates inhibition of cell cycle progression in IEC-18 cells, preceded by rapid
accumulation of cdk inhibitory proteins and appearance of the
growth-suppressive form of Rb. The relevance of these findings to
regulation of cell growth in the intestinal epithelium in
situ is supported by evidence that activation of PKC isozymes,
including PKC
, coincides precisely with cell growth arrest (27) and
with induction of p21waf1/cip1 expression (71, 72) in the
mid-crypt region. Levels of p21waf1/cip1 and p27kip1
are thought to play a critical role in the decision of a cell to
proliferate or to withdraw from the cell cycle in response to
environmental signals (73, 74); thus, activation of PKC
may play an
important role in maintenance of balanced growth in intestinal
epithelial cells in situ by integrating environmental anti-mitogenic signaling with the process of cell division via regulation of cell cycle inhibitory proteins. The importance of subsequent functional activation of the Rb protein to maintenance of
cell cycle arrest in this system is emphasized by studies in transgenic
mice, where sequestration of Rb by SV40 large T antigen in villus cells
was shown to result in re-entry of these post-mitotic cells into the
cell cycle (75, 76). Thus, in the intestinal epithelium, our data
suggest that PKC functions as a physiological regulator of epithelial
tissue homeostasis, acting to limit the expansion of actively
proliferating populations by participating in the following program:
activation of PKC
by growth-inhibitory factors initiates a
signaling cascade, involving p53-dependent or -independent
mechanisms, which leads to induction of cdk inhibitory molecules,
suppression of phosphorylation of Rb in G1 (and modulation of other key substrates in S and/or G2/M phases), and
subsequent cell cycle arrest.
We thank Sulochana Dave for expert technical assistance and Dr. Adrian Black for critical review of the manuscript and for many helpful discussions.