Am J Physiol Gastrointest Liver Physiol 285: G1–G11, 2003.
First published March 5, 2003; 10.1152/ajpgi.00419.2002.
ANG II stimulates PKC-dependent ERK activation, DNA
synthesis, and cell division in intestinal epithelial cells
Terence Chiu, Chintda Santiskulvong, and Enrique Rozengurt
Department of Medicine, School of Medicine, CURE: Digestive Diseases Research Center
and Molecular Biology Institute, University of California, Los Angeles, California 90095
Submitted 26 September 2002; accepted in final form 26 February 2003
THE SEQUENTIAL proliferation, lineage-specific differentiation, migration, and cell death of epithelial cells of the
intestinal mucosa is a tightly regulated process that is
modulated by a broad spectrum of regulatory peptides
(3, 15, 27). The nontransformed IEC-6 and IEC-18
cells, derived from rat small intestinal crypt (22), have
provided an in vitro model to examine intestinal epithelial cell migration, differentiation, and proliferation
(8, 12, 23, 27). Previous studies have demonstrated
that the proliferation and migration of these intestinal
epithelial cells is regulated by polypeptide growth factors, including EGF, IGF-I, and hepatocyte growth
factor, which act via single-pass transmembrane tyrosine kinase receptors (2, 8, 20). Neuropeptides and
vasoactive peptides that signal through seven-trans-
membrane G protein-coupled receptors (GPCRs) also
act as potent cellular growth factors for a variety of cell
types (25, 26). However, the role of GPCRs, their ligands, and signal transduction pathways in intestinal
epithelial cell proliferation remain poorly understood.
PKC, a major target for the tumor-promoting phorbol esters, has been implicated in the signal transduction pathways that mediate important functions in
intestinal epithelial cells, including proliferation (1,
11) and carcinogenesis (21). It is known that intestinal
epithelial cells express multiple isoforms of the PKC
family, including ␣, ␤, ␦, ⑀, and ␨. Transgenic overexpression in murine colonic epithelium (19) or overexpression of certain PKC(s) in intestinal epithelial cells
in culture has been shown to promote growth (18). In
contrast to these growth-stimulatory roles, certain
PKCs, such as PKC-␣ (10, 11) and PKC-␦ (4), have been
implicated in the mediation of growth-inhibitory signals in intestinal epithelial cells. Furthermore, biologically active phorbol esters, which directly activate
PKCs, have been reported to inhibit cell cycle progression in logarithmically growing IEC-18 cells (10, 11).
Despite these studies in intestinal epithelial cells that
are predominantly in the S/G2 phase of the cell cycle,
the role of PKC in the regulation of the exit from the
G0/G1 phase into DNA synthesis (S phase) in IEC-18
cells has not been previously investigated.
The central aim of the present study is to examine
the role of PKC in the regulation of the transition from
the G0/G1 phase in DNA synthesis in IEC-18 cells. To
achieve this aim, we used phorbol esters as a pharmacological tool to directly activate PKC and the octapeptide ANG II to examine receptor-mediated PKC activation in IEC-18 cells. We have previously reported
that ANG II induces a dramatic increase in PKCdependent PKD activation in IEC-18 cells via the AT1
receptor coupled to Gq (6) and also stimulates in these
cells a PKC-dependent increase in the tyrosine phosphorylation of Pyk2, a nonreceptor tyrosine kinase that
has been implicated as an upstream element in
ERK1/2 activation (32). We used this model system to
elucidate the role of PKC signaling in the reinitiation
of the cell cycle of intestinal epithelial cells. We demonstrate, for the first time in IEC-18 cells arrested in
Address for reprint requests and other correspondence: E. Rozengurt, 900 Veteran Ave., Warren Hall, Rm. 11–124, Dept. of Medicine,
David Geffen School of Medicine at UCLA, Los Angeles, CA 900951786 (E-mail: [email protected]).
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. Section 1734
solely to indicate this fact.
IEC-18 cells; mitogen-activated protein kinase; mitogen/extracellular signal-regulated kinase; phorbol ester; angiotensin II; protein kinase C; deoxyribonucleic acid
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0193-1857/03 $5.00 Copyright © 2003 the American Physiological Society
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Chiu, Terence, Chintda Santiskulvong, and Enrique
Rozengurt. ANG II stimulates PKC-dependent ERK activation, DNA synthesis, and cell division in intestinal epithelial
cells. Am J Physiol Gastrointest Liver Physiol 285: G1–G11,
2003. First published March 5, 2003; 10.1152/ajpgi.00419.
2002.—PKC, a major target for the tumor-promoting phorbol
esters, has been implicated in the signal transduction pathways that mediate important functions in intestinal epithelial cells, including proliferation and carcinogenesis. With the
use of IEC-18 cells arrested in G0/G1, addition of phorbol
esters resulted in a modest increase in [3H]thymidine incorporation and a slight shift toward the S and G2/M phases of
the cell cycle, whereas the combination of EGF and phorbol
12,13-dibutyrate (PDB) synergistically stimulated DNA synthesis. To investigate the effects of receptor-mediated PKC
activation on mitogenesis, we demonstrated that ANG II
induced ERK activation, a response completely blocked by
pretreatment with mitogen/extracellular signal-regulated kinase inhibitors or specific PKC inhibitors. Furthermore,
ANG II stimulated an over threefold increase in [3H]thymidine incorporation that was corroborated by flow cytometric
analysis of the cell cycle to levels comparable to that achieved
by the combination of EGF and PDB. Taken together, our
results indicate that receptor-mediated PKC activation, as
induced by ANG II, transduces mitogenic signals leading to
DNA synthesis and cell proliferation in IEC-18 cells.
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ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
G0/G1, that addition of phorbol 12,13-dibutyrate (PDB)
combined with EGF synergistically and dramatically
stimulates mitogenesis to levels comparable with that
induced by ANG II. Our results indicate that ANG II
induces cell cycle progression via the PKC-dependent
pathway and thus indicate that phorbol ester-sensitive
PKCs play a positive role in promoting exit from G0/G1
and entry into S phase in IEC-18 cells.
MATERIALS AND METHODS
RESULTS
PKC signaling stimulates exit from G0/G1 and entry
in S phase in IEC-18 cells. Previous studies have reported that treatment of asynchronously growing
IEC-18 cells with biologically active phorbol esters,
which directly activate PKCs, induced transient cell
cycle delay in G0/G1 (10, 11). From these results, the
authors suggested that PKC mediates growth-inhibitory signaling in cells that are predominantly in S/G2
phase of the cell cycle. However, the role of PKC in the
regulation of the exit from the G0/G1 phase into DNA
synthesis (S phase) in IEC-18 cells has not been determined previously.
Initially, we examined the effect of PDB on the initiation of DNA synthesis by confluent and serumstarved cultures of IEC-18 cells incubated in the presence or absence of EGF, a well-known growth factor for
intestinal epithelial cells. DNA synthesis was assessed
by measuring [3H]thymidine incorporation in acid-precipitable material. Addition of PDB to quiescent cultures of IEC-18 cells induced a small increase in
[3H]thymidine incorporation (Fig. 1A). In striking contrast, exposure of IEC-18 cells to PDB (100 nM) in the
presence of EGF (5 ng/ml) led to a synergistic increase
(3.5-fold) in [3H]thymidine incorporation (Fig. 1A).
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Cell culture. IEC-18 cells were purchased from the American Type Culture Collection. Stock cultures of these cells
were maintained in DMEM supplemented with 5% FBS in a
humidified atmosphere containing 10% CO2 and 90% air at
37°C. For experimental purposes, cells were plated in
100-mm dishes at 3 ⫻ 105 cells/dish in DMEM containing 5%
FBS and were allowed to grow to confluency (5–7 days) and
then changed to serum-free DMEM for 18–24 h before the
experiment.
Western blot analysis for ERK2/ERK1 activation and PKC
isoforms. Serum-starved cultures of IEC-18 cells grown on
100-mm dishes were washed two times with DMEM and then
treated as described in the individual experiments. The cells
were lysed in 2⫻ SDS-PAGE sample buffer. After SDSPAGE, proteins were transferred to Immobilon-P membranes (Millipore) and blocked by 3–6 h incubation with 5%
nonfat milk in PBS, pH 7.2. Membranes were then incubated
overnight with a specific anti-phospho-ERK1/ERK2 monoclonal antibody (MAb; New England Biolabs) that recognizes
the phosphorylated state of Thr202 and Tyr204 of ERK1/2. The
same membranes were stripped and probed in a similar
fashion with goat anti-ERK2 polyclonal antibody.
For detection of PKCs or PKD, membranes were incubated
overnight with an antiserum that specifically recognizes the
phosphorylated state of Ser916 of PKD at a dilution of 1:500
or with antibodies that specifically recognize the different
PKC isoforms at a dilution of 1 ␮g/ml, in PBS containing 5%
nonfat dried milk.
Bound primary antibodies to immunoreactive bands were
visualized by enhanced chemiluminescence detection with
horseradish peroxidase-conjugated anti-mouse, anti-rabbit,
or anti-goat antibodies. Autoradiograms were scanned using a
GS-710 scanner (Bio-Rad), and the labeled bands were quantified using the Quantity One software program (Bio-Rad).
Assay of DNA synthesis. Confluent and serum-starved cultures of IEC-18 cells were washed two times with DMEM and
incubated with DMEM/Waymouth’s medium (1:1, vol/vol)
and various additions as described in legends for Figs. 1–8.
After 15 h of incubation at 37°C, [3H]thymidine (0.2 ␮Ci/ml,
1 ␮M) was added, and the cultures were incubated for a
further 4 h at 37°C. Cultures were then washed two times
with PBS and incubated in 5% TCA at 4°C for 20 min to
remove acid-soluble radioactivity, washed with ethanol, and
solubilized in 1 ml of 2% Na2CO3 and 0.1 M NaOH. The
acid-insoluble radioactivity was determined by scintillation
counting in 6 ml of Beckman Readysafe.
Flow cytometric/cell cycle analysis. The proportion of cells
in the G0/G1, S, G2, and M phases of the cell cycle was
determined by flow cytometric analysis. Confluent and serum-starved cultures of IEC-18 cells were washed two times
with DMEM and incubated with DMEM/Waymouth’s medium (1:1, vol/vol) containing various additions as described
in legends for Figs. 1–8. After 19 h of incubation at 37°C,
cultures were washed two times with PBS. Cells were then
detached by treatment with trypsin (0.025%), suspended in
DMEM containing 10% FBS, and centrifuged at 1,000 g for 5
min. Cells (106) were then resuspended and stained by adding 1 ml of a hypotonic DNA staining buffer containing
propidium iodide (0.1 mg/ml), sodium citrate (1 mg/ml),
RNase A (20 ␮g/ml), and Triton X-100 (0.3%). Samples were
kept at 4°C, protected from light for 30 min, and analyzed on
a FACScan (Becton-Dickinson, Franklin Lakes, NJ) using
the software CELLQuest version 3.3 and Modfit 3.1 (Verity
Software House, Topsham, ME).
Measurement of cell number. For experimental purposes,
5 ⫻ 104 IEC-18 cells were subcultured in 35-mm Nunc petri
dishes with 2 ml of DMEM containing 1% FBS. At day 0 (24
h after plating), cultures were washed two times with DMEM
to remove residual serum and replaced with DMEM/Waymouth’s medium (1:1, vol/vol) with or without ANG II as
described in legends for Figs. 1–8. Cell number was determined by removing the cells from the dish with a trypsinEDTA solution (0.5% trypsin in a Ca2⫹- and Mg2⫹-free PBS
with EDTA) and counting a portion of the resulting cell
suspension in a Coulter Counter. Cell counts were obtained
at day 0 (24 h after plating), day 1 (48 h after plating), and
day 2 (72 h after plating).
Materials. [3H]thymidine was from Amersham Pharmacia
Biotech (Piscataway, NJ). Bisindolylmaleimide I (GF109203X), bisindolylmaleimide V, PD-98059, U-0126, and Ro
-31-8220 were purchased from Calbiochem. ANG II, PDB,
EGF, PD-123329, and trypsin-EDTA solution (1⫻) were obtained from Sigma (St. Louis, MO). Anti-phospho-ERK1/2
MAb and phosphoserine 916 PKD antibody were obtained
from Cell Signaling Technology (Beverly, MA). Antibodies
(PKD C-20, PKC-␨ C-20, PKC-⑀ C-15, PKC-␦, and PKC-␣
C-15; anti-ERK2 polyclonal antibody) used in Western blot
analysis were obtained from Santa Cruz Biotechnologies
(Santa Cruz, CA). Losartan was generously provided by
Merck (Rathway, NJ) as exclusive licensee of E. I. du Pont de
Nemours. Other items were from standard suppliers or as
indicated in the text.
ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
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Fig. 1. Phorbol 12,13-dibutyrate (PDB) and EGF synergistically
stimulate thymidine incorporation. A: confluent and serum-starved
cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml
DMEM/Waymouth’s medium containing either 5 ng/ml EGF, 100 nM
PDB, or 5 ng/ml EGF and 100 nM PDB. After 15 h, 1 ␮Ci/ml
[3H]thymidine was added to the cultures, and 4 h later DNA synthesis was assessed by measuring the [3H]thymidine incorporated in
acid-precipitable material. Inset, confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml
DMEM/Waymouth’s medium containing vehicle or EGF at various
concentrations as indicated. [3H]thymidine incorporation was determined as described. cpm, Counts/min. Results shown means ⫾ SE;
n ⫽ 3 experiments. B: confluent and serum-starved cultures of
IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/
Waymouth’s medium containing PDB at various concentrations
without (F) or with (E) 5 ng/ml EGF. [3H]thymidine incorporation
was determined as described. Results are means ⫾ SE; n ⫽ 3
experiments.
Dose-response studies with EGF demonstrated that
maximal [3H]thymidine incorporation induced by this
growth factor was achieved at 1–5 ng/ml (Fig. 1A,
inset).
To investigate the synergistic interaction of PDB and
EGF on the initiation of DNA synthesis by IEC-18 cells
in more detail, we examined the effect of increasing
concentrations of PDB (0.01–1,000 nM) on [3H]thymidine incorporation by these cells incubated in the presence or absence of EGF. As shown in Fig. 1B, [3H]thymidine incorporation was synergistically increased
when these cells were exposed to increasing concentrations of PDB in the presence of a fixed concentration of
EGF (5 ng/ml). These findings suggested that PKC
mediates mitogenic signaling in cells costimulated
with EGF.
To substantiate that the stimulatory effect induced
by PDB on [3H]thymidine incorporation reflects an
increase in DNA replication through S phase of the cell
Fig. 2. Effect of PDB and EGF on progression through the cell cycle
in IEC-18 cells. A: confluent and serum-starved cultures of IEC-18
cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s
medium containing PDB at various concentrations without (⫺) or
with (⫹) 5 ng/ml EGF. After 19 h of incubation at 37°C, cultures were
washed two times with PBS, and cells were detached by treatment
with trypsin (0.025%). Cells (106) were then resuspended and stained
by 1 ml of a hypotonic DNA staining buffer and analyzed on a
FACScan (Becton-Dickinson) as described. B: %S phase shown in A.
F, ⫺EGF; E, ⫹EGF. Results are representative of 3 independent
experiments.
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cycle rather than alterations in transport and/or phosphorylation of [3H]thymidine, we used flow cytometric
analysis to determine the proportion of cells in the
various phases (G0/G1, S, and G2/M) of the cell cycle.
Confluent and serum-starved cultures of IEC-18 cells
were subjected to identical conditions to those for the
[3H]thymidine incorporation experiments and exposed
to various concentrations of PDB (0.01–1,000 nM) with
or without EGF. As shown in Fig. 2, serum starvation
of IEC-18 cells resulted in 81.1 ⫾ 2.1% of cells in G0/G1,
comparable to previous published results (10, 11), confirming that serum starvation induced the accumulation of IEC-18 cells into quiescence. Incubation with
PDB alone induced a slight shift toward S and G2/M
phase of the cell cycle (Fig. 2). When PDB was added in
the presence of EGF, the proportion of cells that entered S and G2/M increased synergistically in a dosedependent manner. Addition of 100 nM PDB combined
with EGF induced a marked increase in the proportion
of cells that entered S phase (from 11 and 14% in cells
exposed to PDB or EGF to 35% in cells stimulated with
PDB and EGF). These results further support the no-
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ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
tent activation of the PKC/PKD pathway in IEC-18
cells via an AT1 receptor pathway (6). In the present
study, we examined whether ANG II not only induces
PKC/PKD pathway activation but also alters the level
of classic, novel, and atypical PKC isoforms in these
cells.
To explore the effects of prolonged exposure of
IEC-18 cells to ANG on PKC isoforms, serum-starved
IEC-18 cells were treated with 200 nM ANG II for
various times (0.5–24 h), and levels of PKC-␣, PKC-␦,
PKC-⑀, and PKC-␨ were measured by Western blotting
using specific antisera directed against each of these
isoforms. For comparison, parallel cultures of IEC-18
cells were treated with 100 nM PDB. As shown in Fig.
3A, levels of PKC-␣, PKC-␦, and PKC-⑀ diminished
dramatically after PDB exposure. In contrast, prolonged exposure of IEC-18 cells to ANG II did not
deplete PKC levels and led to a persistent PKD autophosphorylation at Ser916, indicative of persistent PKC
activation (Fig. 3B).
ANG induces DNA synthesis and cell proliferation.
Having demonstrated that ANG II induces persistent
PKC without detectable PKC downregulation (Fig. 3),
our next objective was to determine whether ANG II
can stimulate DNA synthesis and cell proliferation in
IEC-18 cells. To investigate the effects of ANG II on S
phase entry in IEC-18 cells, serum-starved cultures of
these cells were incubated with increasing concentrations of ANG II, and DNA synthesis was assessed by
measuring [3H]thymidine incorporation in acid-precipitable material. As shown in Fig. 4A, ANG II induced a
marked increase in [3H]thymidine incorporation in a
concentration-dependent fashion, achieving half-maximal and maximal stimulation at 0.3 and 10 nM, respectively.
To determine which ANG receptor subtype mediates
the mitogenic effect of ANG II in IEC-18 cells, cultures
of these cells were pretreated with either the selective
AT1 receptor antagonist losartan or the selective AT2
receptor antagonist PD-123329 before addition of ANG
II. As shown in Fig. 4B, pretreatment with losartan
Fig. 3. Effects of PDB and ANG II on PKC isoforms in
IEC-18 cells. Confluent and serum-starved cultures of
IEC-18 cells were treated for various times with 100
nM PDB (A) or 200 nM ANG II (B) as indicated. Cell
lysates were analyzed by SDS-PAGE and transferred
to Immobilon membranes. Western blot analysis was
carried out using isoform-specific polyclonal antisera
against the different PKCs, PKD, or phosphoserine
(pS)-916 of PKD.
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tion that PKC activation acts synergistically with EGF
to stimulate progression through the cell cycle.
PKC downregulation can be dissociated from PKCmediated mitogenesis in IEC-18 cells. It is well known
that PKCs can mediate positive (PKC-⑀) and/or negative (e.g., PKC-␦) effects on cell cycle progression. Our
results (presented in Figs. 1 and 2) provide compelling
evidence showing that treatment of IEC-18 cells with
PDB, in the presence of EGF, stimulates [3H]thymidine incorporation and cell cycle progression of these
cells. These effects could be explained by either the
positive or the inhibitory limb of the dual actions attributed to PKC signaling on cell cycle progression. For
example, PKC signaling in the first 2–6 h of PDB
exposure could be sufficient to act synergistically with
EGF to induce DNA synthesis. Alternatively, PDBinduced downregulation of classic and novel isoforms of
PKC, a well-described effect of chronic treatment with
biologically active phorbol esters in many cell types,
could remove an inhibitory influence and thereby facilitate the positive effects of a synergistic growth factor
(i.e., EGF). To distinguish between these alternative
possibilities, it is necessary to dissociate PKC downregulation from PKC-mediated mitogenesis. In this
context, it would be useful to identify a stimulus that
induces PKC-dependent mitogenesis but does not
downregulate any of the PKC isoforms.
ANG II is known to exert its biological effects
through binding to the following two receptor subtypes:
AT1 and AT2 receptors, which are members of the
GPCR superfamily. Agonist binding to the AT1 receptor induces PLC-mediated hydrolysis of membrane
phosphoinositides, leading to the generation of two
second messengers: inositol 1,4,5-trisphosphate, which
stimulates Ca2⫹ mobilization from intracellular stores,
and diacylglycerol, which activates the classic and
novel isoforms of the PKC family. PKD/PKC-␮, a
serine/threonine protein kinase with structural, enzymological, and regulatory properties different from the
PKC family members (14, 30), has been identified as a
downstream target of PKCs in a variety of cell types.
Recently, we demonstrated that ANG stimulates po-
ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
completely prevented ANG II-induced [3H]thymidine
incorporation.
Interestingly, ANG II was more effective than EGF
(at 5 ng/ml) in stimulating [3H]thymidine incorporation in IEC-18 cells (Fig. 4C). Addition of 100 nM PDB
and 5 ng/ml EGF induced an increase in [3H]thymidine
incorporation by nearly fourfold, comparable with that
induced by ANG II. Furthermore, exposure of these
cells to both ANG II and EGF induced additive stimulation of DNA synthesis that reached a level almost
comparable with that promoted by addition of medium
containing 5% FBS.
To corroborate that ANG II induces cell cycle activation, we used flow cytometry to determine the distribution of IEC-18 cells through the various phases of
the cell cycle, treated in the absence or presence of
ANG II, EGF, or both. As shown in Fig. 5A, IEC-18
cells accumulated in the G0/G1 phase of the cell cycle
after serum starvation and addition of 50 nM ANG II
induced a marked increase (from 8 to 21%) of cells in S
phase of the cell cycle. Consistent with [3H]thymidine
incorporation data, EGF induced a modest increase of
cells in S phase, whereas the combination of ANG II
and EGF stimulated a marked shift toward S and G2/M
phase of the cell cycle.
To determine whether ANG II can stimulate IEC-18
cell proliferation in the absence of any other exogenously added growth factor, sparse cultures of these
cells were transferred to serum-free medium and then
stimulated with or without 100 nM ANG II. As shown
in Fig. 5B, there was nearly a doubling in the cell
number of cultures treated with ANG II for 24, 48, and
72 h compared with the controls.
The results presented in Figs. 4 and 5 indicate that
ANG II acts as a potent growth factor for IEC-18 cells,
and those depicted in Fig. 3 clearly demonstrate that
this agonist induces persistent PKC/PKD pathway activation without concomitant PKC downregulation. To
determine whether PKC downregulation is dissociable
from PKC-mediated mitogenesis in ANG II-treated
cells, we next determined the contribution of PKC to
the mitogenic activity of ANG II.
To test whether ANG II-induced DNA synthesis is
PKC dependent, cultures of IEC-18 cells were preincubated for 1 h with the selective PKC inhibitor GF-I (at
3.5 ␮M). Control cells received either an equivalent
amount of solvent or GF-V (also at 3.5 ␮M), a biologically inactive analog of GF-I, before addition of ANG II.
As shown in Fig. 6A, GF-I markedly reduced [3H]thymidine incorporation in response to ANG II stimulation by ⬃50%. These results indicate that the activity
of the PKCs is required for ANG II-induced mitogenesis in IEC-18 cells. Collectively, the results presented
in Figs. 3–6 imply that PKCs play a positive role in
ANG II-induced stimulation of the cell cycle in IEC-18
cells.
ANG II-stimulated ERK1/2 activation occurs rapidly
via AT1 receptor and is mitogen/extracellular signalregulated kinase dependent. To identify the signaling
pathways that participate in ANG II-induced, PKCmediated mitogenesis in IEC-18 cells, we examined
whether this agonist induces ERK1 (p44mapk) and
ERK2 (p42mapk) activation in these intestinal epithelial cells. These are the best-characterized isoforms of
the MAPK family of highly conserved serine/threonine
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Fig. 4. ANG II induces DNA synthesis and cell proliferation in
IEC-18 cells. A: ANG II induces DNA synthesis in IEC-18 cells in a
concentration-dependent fashion. Confluent and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml
DMEM/Waymouth’s medium containing increasing concentrations
of ANG II. After 15 h, 1 ␮Ci/ml [3H]thymidine was added to the
cultures, and 4 h later DNA synthesis was assessed by measuring
the [3H]thymidine incorporated in acid-precipitable material. Results are %maximum ⫾ SE; n ⫽ 3 experiments. B: confluent and
serum-starved cultures of IEC-18 cells were washed and incubated
at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG
II with the selective AT1 receptor antagonist losartan (10 ␮M),
selective AT2 receptor antagonist PD-123329 (10 ␮M), or vehicle
(control). [3H]thymidine incorporation was determined as described.
Results are means ⫾ SE; n ⫽ 3 experiments. C: ANG II is a more
effective mitogen than EGF. Confluent and serum-starved cultures
of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/
Waymouth’s medium containing vehicle, 5 ng/ml EGF, 100 nM PDB,
100 nM PDB and 5 ng/ml EGF, 50 nM ANG II, 5 ng/ml EGF and 50
nM ANG II, or 5% FBS. [3H]thymidine incorporation was determined as described. Results are means ⫾ SE; n ⫽ 3 experiments.
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ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
Cultures of IEC-18 cells were treated with increasing concentrations of ANG II and lysed, and the active
forms of ERK1/2 were detected by Western blotting
using an antibody that recognizes the dually phosphorylated forms of these enzymes. ERK1/2 activation was
a rapid consequence of ANG II stimulation of IEC-18
cells, reaching a maximum within 2.5 min (Fig. 7A).
Furthermore, ANG II induced ERK1/2 activation in a
kinases that are directly activated by phosphorylation
on specific tyrosine and threonine residues by the dualspecificity ERK kinase [or mitogen/extracellular signal-regulated kinase (MEK); see Ref. 31].
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Fig. 5. ANG II stimulates DNA synthesis. A: confluent and serumstarved cultures of IEC-18 cells were washed and incubated at 37°C in
2 ml DMEM/Waymouth’s medium containing vehicle, 5 ng/ml EGF, 50
nM ANG II, or both EGF and ANG II. After 19 h of incubation at 37°C,
cultures were washed two times with PBS, and cells were detached by
treatment with trypsin (0.025%). Cells (106) were then resuspended and
stained by 1 ml of a hypotonic DNA staining buffer containing propidium iodide (0.1 mg/ml), sodium citrate (1 mg/ml), RNase A (20
␮g/ml), and Triton X-100 (0.3%). Samples were kept at 4°C, protected
from light for 30 min, and analyzed on a FACScan (Becton-Dickinson).
Results shown are representative of 3 independent experiments. B:
ANG II stimulates IEC-18 cell proliferation; 5 ⫻ 104 IEC-18 cells were
plated on 35-mm Nunc petri dishes with 2 ml DMEM containing 1%
FBS. At day 0 (24 h after plating), cultures were washed two times with
DMEM to remove residual serum and replaced with DMEM/Waymouth’s medium (1:1, vol/vol) with (open bars) or without (filled bars)
100 nM ANG II. Cell number was determined by counting trypsinized
cells using a Coulter counter. Cell counts were obtained at day 0 (24 h
after plating) and 24 (day 1), 48 (day 2), and 72 (day 3) h after ANG II
exposure. *P ⬍ 0.01 vs. DMEM control for the respective day.
Fig. 6. Effect of inhibitors of mitogen/extracellular signal-regulated
kinase (MEK) and PKC on ANG II-induced DNA synthesis. A: effects
of GF-109203X on DNA synthesis in response to ANG II. Confluent
and serum-starved cultures of IEC-18 cells were washed and incubated at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM
ANG II either in the absence or in the presence of GF-109203X or its
inactive analog GF-V. B: effect of the MEK inhibitors PD-98059 or
U-0126 on DNA synthesis in response to ANG II. Confluent and
serum-starved cultures of IEC-18 cells were washed and incubated
at 37°C in 2 ml DMEM/Waymouth’s medium containing 50 nM ANG
II either in the absence or in the presence of 10 ␮M PD-98059 or 2.5
␮M U-0126. After 15 h, 1 ␮Ci/ml [3H]thymidine was added to the
cultures, and 4 h later DNA synthesis was assessed by measuring
the [3H]thymidine incorporated in acid-precipitable material. Results are means ⫾ SE; n ⫽ 3 experiments. *P ⬍ 0.01 vs. 50 nM ANG
II. [3H]thymidine incorporation in acid-precipitable material was
measured as described.
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ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
G7
concentration-dependent fashion, achieving half-maximal and maximal activation at 1 and 10 nM, respectively (Fig. 7B).
As shown in Fig. 7C, ANG II-induced ERK1/2 activation in IEC-18 cells was completely prevented by pretreatment of these cells with the selective AT1 receptor
antagonist losartan, whereas preincubation with the selective AT2 receptor antagonist PD-123329 before addition of ANG II had no effect. We conclude that ANG II
induces PKC/PKD activation, ERK1/2 activation, and
DNA synthesis via AT1 receptors in IEC-18 cells.
To determine whether ANG II-induced activation of
the ERKs is mediated by MEK in IEC-18 cells, cultures
of these cells were preincubated for 1 h in the absence
or presence of the specific MEK inhibitors PD-98059 (2)
or U-0126 (9) and subsequently stimulated with ANG
II. The results shown in Fig. 7D demonstrate that
exposure to either PD-98059 or U-0126 completely
prevented ERK1/2 activation in response to ANG II.
To establish whether DNA synthesis in response to
ANG II is mediated by MEK-dependent ERK1/2 activation, serum-deprived cultures of IEC-18 cells were
preincubated for 1 h in the absence or presence of the
specific MEK inhibitors PD-98059 or U-0126 and subsequently stimulated with ANG II. As shown in Fig.
6B, treatment with either PD-98059 or U-0126 attenuated [3H]thymidine incorporation in response to ANG
II stimulation by ⬃50%.
ANG-induced ERK1/2 activation is PKC dependent.
Because we demonstrated that ANG II is a potent
growth factor for IEC-18 cells (Figs. 4 and 5) through a
PKC-dependent pathway (Fig. 6A) and ERK1/2 activity is required for ANG II-stimulated DNA synthesis
(Fig. 6B), we next determined whether PKC is also
required for ERK1/2 activation in response to ANG II.
Initially, we investigated whether direct PKC activation by PDB can stimulate ERK phosphorylation. Stimulation of IEC-18 cells with PDB (100 nM) for various
times indicates that PDB induces ERK1/2 activation
rapidly, reaching a maximum within 2.5 min (Fig. 8A).
Furthermore, PDB induced ERK1/2 activation in a
concentration-dependent fashion, achieving half-maximal and maximal activation at 10–30 and 100 nM,
respectively (Fig. 8B).
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Fig. 7. ANG II induces ERK1/2 activation rapidly via AT1 receptor and is MEK dependent in IEC-18 cells. A: ANG
II induces p42mapk (ERK2) and p44mapk (ERK1) phosphorylation in a time-dependent manner. Confluent and
serum-starved cultures of IEC-18 cells were treated for various times with 10 nM ANG II at 37°C as indicated.
Western blot analysis using a specific anti-phospho-ERK1/ERK2 monoclonal antibody (MAb) that recognizes only
the activated forms phosphorylated on Thr202 and Tyr204 was performed after lysis of the cells with 2⫻ sample
buffer as described in MATERIALS AND METHODS. B: ANG II induces p42mapk (ERK2) and p44mapk (ERK1) phosphorylation
in a dose-dependent manner. Confluent and serum-starved cultures of IEC-18 cells were treated with various
concentrations of ANG II for 90 s at 37°C as indicated. ERK1/2 activation was determined by Western blot analysis
using a specific anti-phospho-ERK1/ERK2 MAb as described. C: top, confluent and serum-starved IEC-18 cultures were
incubated for 1 h with the selective AT1 receptor antagonist losartan (10 ␮M), selective AT2 receptor antagonist
PD-123329 (10 ␮M), or vehicle (control). The cultures were subsequently left unstimulated (⫺) or stimulated (⫹) for 2.5
min with 10 nM ANG II at 37°C. ERK1/2 activation was determined by Western blot analysis using a specific
anti-phospho-ERK1/ERK2 MAb as described. Middle, Western blot was also probed for total ERK, showing equal
loading. D: MEK inhibitors abolish ANG II-induced ERK2 and ERK1 phosphorylation. Top, confluent and serumstarved IEC-18 cells were incubated for 1 h with 10 ␮M PD-98059 or 2.5 ␮M U-0126. Control cells (⫺) received
equivalent amount of solvent. The cultures were subsequently unstimulated (⫺) or stimulated (⫹) for 2.5 min with 10
nM ANG II at 37°C. ERK1/2 activation was determined by Western blot analysis using a specific anti-phospho-ERK1/
ERK2 MAb as described. Middle, Western blot was also probed for total ERK, showing equal loading. C and D, bottom:
results are means ⫾ SE (n ⫽ 3) of the levels of ERK2 and ERK1 phosphorylation obtained from scanning densitometry
expressed as a percentage of the maximum increase in phosphorylation obtained with 10 nM ANG II. All Western blots
are representative of at least 3 independent experiments.
G8
ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
To determine whether PDB-induced activation of the
ERKs is mediated by MEK in IEC-18 cells, cultures of
these cells were preincubated for 1 h in the absence or
presence of the specific MEK inhibitor PD-98059 or
U-0126 and subsequently stimulated with PDB. The
results shown in Fig. 8C demonstrate that exposure to
either MEK inhibitor completely prevented ERK1/2
activation in response to PDB.
GPCRs are known to induce ERK1/2 activation
through multiple signal transduction pathways, including PKC-dependent and PKC-independent pathways. Because the results shown in Fig. 7 indicate that
ANG II induces ERK activation and those shown in
Fig. 8A indicate that PKC is a potential signaling
pathway that can lead to ERK activation, we examined
the contribution of PKCs to ANG II-induced ERK1/2
activation in IEC-18 cells. As shown in Fig. 8D, pretreatment of these cells with the specific PKC inhibitors GF-I and Ro-31-8220 prevented ANG II-induced
ERK1/2 activation. In contrast, preincubation of cultures with GF-V, a biologically inactive analog of GF-I,
had no effect on ANG II-induced ERK1/2 activation.
These results suggest that ANG II-induced ERK1/2
activation is largely mediated by PKC in IEC-18 cells.
These results do not exclude the possibility of a PKCindependent pathway, which may play a minor role in
ANG II-induced ERK activation in IEC-18 cells.
Taken together, these results imply that PKC plays
an important role in mediating ERK activation and
reinitiation of DNA synthesis induced by either pharmacological (PDB) or physiological (ANG II-AT1 receptor) stimulation of intestinal epithelial IEC-18 cells.
Consistent with this, concomitant pretreatment with
PKC inhibitor GF-I (2.5 ␮M) and MEK inhibitor
U-0126 (2.5 ␮M) did not have an additive inhibitory
effect on [3H]thymidine incorporation in response to
ANG II beyond that achieved by either inhibitor alone
(results not shown).
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Fig. 8. PDB-induced ERK1/2 phosphorylation is MEK dependent. A: PDB induces p42mapk (ERK2) and p44mapk
(ERK1) phosphorylation in a time-dependent manner. Confluent and serum-starved cultures of IEC-18 cells were
treated for various times with 100 nM PDB at 37°C as indicated. ERK1/2 activation was determined by Western
blot analysis using a specific anti-phospho-ERK1/ERK2 MAb as described. B: PDB induces p42mapk (ERK2) and
p44mapk (ERK1) phosphorylation in a dose-dependent manner. Confluent and serum-starved cultures of IEC-18
cells were treated with various concentrations of PDB for 90 s at 37°C as indicated. C: MEK inhibitors abolish
PDB-induced ERK2 and ERK1 phosphorylation. Top, confluent and serum-starved IEC-18 cells were incubated for
1 h with 10 ␮M PD-98059 or 2.5 ␮M U-0126. Control cells (⫺) received an equivalent amount of solvent. The
cultures were subsequently unstimulated (⫺) or stimulated (⫹) for 2.5 min with 100 nM PDB at 37°C. ERK1/2
activation was determined by Western blot analysis using a specific anti-phospho-ERK1/ERK2 MAb as described.
Results are means ⫾ SE (n ⫽ 3) of the level of ERK2 and ERK1 phosphorylation obtained from scanning
densitometry expressed as a percentage of the maximum increase in phosphorylation obtained with 100 nM PDB.
Middle, Western blot was also probed for total ERK, showing equal loading. D: Ro-31-8220 and GF-109203X
inhibits ANG II-induced ERK1/2 activation. Top, confluent and serum-starved IEC-18 cells were incubated for 1 h
with 2.5 ␮M Ro-31-8220 or 2.5 ␮M GF-109203X. Control cells (⫺) received equivalent amount of solvent or GF-V,
a biologically inactive analog of GF-1. The cultures were subsequently unstimulated (⫺) or stimulated (⫹) for 2.5
min with 10 nM ANG II at 37°C. The results are means ⫾ SE (n ⫽ 3) of the level of ERK2 and ERK1
phosphorylation obtained from scanning densitometry expressed as a percentage of the maximum increase in
phosphorylation obtained with 10 nM ANG II. Middle, Western blot was also probed for total ERK, showing equal
loading. All Western blots are representative of at least 3 independent experiments.
ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
DISCUSSION
nonreceptor tyrosine kinase that has been implicated
as an upstream element in ERK1/2 activation (32). In
the present study, we demonstrate that ANG II is a
potent growth factor for IEC-18 cells, as shown by
[3H]thymidine incorporation in DNA, flow cytometric
analysis to determine the proportion of cells in the
various phases of the cell cycle, and assays of cell
proliferation. The level of DNA synthesis induced by
ANG II is substantially higher than that promoted by
EGF and is comparable to that induced by the combination of EGF and PDB. Our results demonstrate that
the mitogenic activity of ANG II is mediated by an AT1
receptor subtype. Recently, we demonstrated that the
peptide agonist vasopressin via GPCR V1A receptor
also induced DNA synthesis and cell proliferation in
IEC-18 cells (7), indicating that the regulation of intestinal cell proliferation by GPCR agonists could be more
common than previously suspected. Of special interest
in the context of this study, treatment with ANG II did
not induce any detectable downregulation of PKC isoforms but produced a persistent activation of PKD, a
well-established downstream target of PKCs. Thus our
results indicate that depletion of PKCs is clearly not
responsible for the growth-stimulatory effect of ANG II
in IEC-18 cells.
Subsequently, we determined the contribution of
PKC to ANG II-induced mitogenesis and attempted to
identify intermediate steps between PKC and cell cycle
activation. Our results demonstrate that treatment
with selective PKC inhibitors attenuated DNA synthesis in response to ANG II, implying that PKC downregulation can be dissociated from PKC-mediated mitogenesis. Our results indicate that PKC plays a stimulatory role in promoting exit of epithelial intestinal
cells from quiescence. Because previous studies suggested that PKC delays cell cycle progression in growing cells (i.e., cells distributed in various phases of the
cell cycle), it is conceivable that PKCs could play different roles in cell proliferation at different stages of
the cell cycle (17). In this context, it is of interest that
our laboratory has recently demonstrated that overexpression of PKD potentiated DNA synthesis and cell
proliferation in Swiss 3T3 fibroblasts in response to
GPCR agonists (33).
The MAPKs are a family of highly evolutionary conserved kinases connecting cell-surface receptors to critical regulatory targets within cells, and they regulate
important cellular processes, including gene expression, cell proliferation, and cell motility (5). These
serine/threonine kinases are activated by a range of
extracellular signals via protein phosphorylation cascades (24), which relay mitogenic signals to the nucleus
(16), thereby modulating the activity of transcription
factors (29). The two best-characterized isoforms,
p42mapk (ERK2) and p44mapk (ERK1), are directly activated by phosphorylation on specific tyrosine and
threonine residues by the dual-specificity ERK kinase
(or MEK; see Ref. 28).
Here, we show that addition of ANG II to IEC-18
cells stimulates MEK-dependent ERK1/2 activation.
Furthermore, we have established that ANG II-in-
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The proliferation of epithelial cells of the intestinal
mucosa is a highly precise process that is modulated by
a broad spectrum of peptides (3, 15, 27), but the signal
transduction pathways involved in intestinal cell proliferation remain poorly characterized. Nontransformed IEC-18 cells, derived from rat ileal crypts, have
served as an in vitro model for studying intestinal
epithelial cell migration, differentiation, and proliferation (8, 12, 23, 27).
In actively cycling IEC-18 cells (cells growing in 5%
serum), treatment with biologically active phorbol esters transiently delayed cell cycle progression, which
was noted 2–6 h after treatment (10, 11). This cellcycle delay was associated with a rapid decrease in the
level of cyclin D1, which subsequently rebounded and
in fact increased over the basal levels at later times of
phorbol ester treatment (10). From these results, the
authors suggested that PKC mediates growth-inhibitory signaling in cells that are predominantly in S/G2
phase of the cell cycle. However, the role of PKC in the
regulation of the exit from the G0/G1 phase in DNA
synthesis (S phase) in IEC-18 cells has not been determined previously. The central aim of the present study
was to examine the role of PKC in the regulation of the
transition from the G0/G1 phase in DNA synthesis in
IEC-18 cells.
A salient feature of the results presented here is that
stimulation of IEC-18 cells with the combination of
EGF and PDB synergistically stimulated DNA synthesis as judged by either [3H]thymidine incorporation in
DNA or flow cytometric analysis to determine the proportion of cells in the various phases (G0/G1, S, and
G2/M) of the cell cycle. Our results demonstrate, for the
first time, that direct activation of PKC, as induced by
a biologically active phorbol ester, strikingly potentiates the ability of EGF to stimulate cell cycle progression in IEC-18 intestinal epithelial cells.
Phorbol ester-sensitive isoforms of PKC have been
implicated in both positive and negative regulation of
the cell cycle, and phorbol esters are well known to
produce downregulation of PKC proteins after their
acute catalytic activation. Consequently, the mitogenic
effects of PDB could be explained by either the stimulatory or the inhibitory limb of the dual actions attributed to PKC signaling on cell cycle progression. Specifically, PKC signaling in the first 2–6 h of PDB exposure could be sufficient to act synergistically with EGF
to induce DNA synthesis. Alternatively, PDB-induced
downregulation of classic and novel isoforms of PKC
could remove an inhibitory influence and thereby facilitate the positive effects of EGF. To distinguish between these possibilities, we attempted to identify a
stimulus that induces PKC-dependent mitogenesis but
does not downregulate any of the PKC isoforms.
Recently, we demonstrated that ANG stimulates potent activation of the PKC/PKD pathway in IEC-18
cells via an AT1 receptor pathway (6). We also found
that ANG II stimulates in these cells a PKC-dependent
increase in the tyrosine phosphorylation of Pyk2, a
G9
G10
ANG II STIMULATES PKC-DEPENDENT MITOGENESIS
We thank Nena Hsieh for technical assistance.
T. Chiu is a recipient of an American Gastroenterological Association/AstraZeneca Fellowship/Faculty Transition Award. This work
was supported by National Institutes of Health (NIH) Grants DK55003, DK-56930, and DK-17294 to E. Rozengurt. Flow cytometry
was performed in the University of California Los Angeles (UCLA)
Jonsson Comprehensive Cancer Center and Center for Acquired
Immunodeficiency Syndrome Research Flow Cytometry Core Facility that is supported by NIH Grants CA-16042 and AI-28697, by the
Jonsson Cancer Center, the UCLA AIDS Institute, and the UCLA
School of Medicine.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
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