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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
IEC-18 cells; mitogen-activated protein kinase; mitogen/extracellular signal-regulated kinase; phorbol ester; angiotensin II; protein kinase C; deoxyribonucleic acid
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 PKC-dependent 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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2x SDS-PAGE sample buffer. After SDS-PAGE, proteins were transferred to Immobilon-P membranes (Millipore) and blocked by 36 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 µg/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, 2, 3, 4, 5, 6, 7, 8. After 15 h of incubation at 37°C, [3H]thymidine (0.2 µCi/ml, 1 µM) 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, 2, 3, 4, 5, 6, 7, 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 µg/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 x 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, 2, 3, 4, 5, 6, 7, 8. Cell number was determined by removing the cells from the dish with a trypsin-EDTA 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 (GF-109203X),
bisindolylmaleimide V, PD-98059, U-0126, and Ro-31-8220 were purchased from
Calbiochem. ANG II, PDB, EGF, PD-123329, and trypsin-EDTA solution (1x)
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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Initially, we examined the effect of PDB on the initiation of DNA synthesis by confluent and serum-starved 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). Dose-response studies with EGF demonstrated that maximal [3H]thymidine incorporation induced by this growth factor was achieved at 15 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 (0.011,000 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 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 (0.011,000 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 dose-dependent 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 notion that PKC activation acts synergistically with EGF to stimulate progression through the cell cycle.
PKC downregulation can be dissociated from PKC-mediated 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 26 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, 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-µ, 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 potent 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 (0.524 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 auto-phosphorylation 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 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 µM). Control cells received either an equivalent amount of solvent
or GF-V (also at 3.5 µM), 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,
4,
5,
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 signal-regulated kinase dependent. To identify the signaling pathways that participate in ANG II-induced, PKC-mediated 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 kinases that are directly activated by phosphorylation on specific tyrosine and threonine residues by the dual-specificity ERK kinase [or mitogen/extracellular signal-regulated kinase (MEK); see Ref. 31].
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 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 1030 and 100 nM, respectively (Fig. 8B).
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 PKC-independent 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 µM) and MEK inhibitor U-0126 (2.5 µM) 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).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 26 h after treatment (10, 11). This cell-cycle 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 26 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 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-induced ERK1/2 activation is PKC dependent, suggesting that PKC is a critical kinase that lies upstream of MEK/ERK in these cells. Using selective inhibitors of MEK, we found that ERK1/2 inhibition attenuated ANG II-induced DNA synthesis. These findings indicate that ANG II-induced stimulation of the AT1 receptor subtype, endogenously expressed by IEC-18 cells, leads to PKC/PKD activation and stimulation of the ERK pathway and induces progression though the cell cycle of these cells arrested in G0/G1.
ANG II, the principal active component of the renin-ANG system (RAS), is traditionally recognized for its role in the regulation of systemic blood pressure and body fluid homeostasis. Recently, all RAS components [angiotensinogen, renin, and ANG-converting enzyme (ACE)] mRNA transcripts were found in human colonic mucosa (13). Furthermore, immunohistochemical examination of these colonoscopic biopsies revealed renin and ACE to be localized in vessel walls, mesenchymal cells in the lamina propria, and parts of the surface epithelium (13). Therefore, it is likely that ANG II is normally produced in intestinal tissues and may act as a local mediator in the control of intestinal functions, suggesting that, in addition to its well-known role as an endocrine hormone found in blood, ANG II may also act as a paracrine signaling peptide in the intestine.
In summary, we demonstrate, for the first time, that addition of PDB combined with EGF synergistically stimulates reinitiation of DNA synthesis in IEC-18 cells arrested in G0/G1. Our results also show that ANG II is a potent growth factor for IEC-18 cells via the AT1 receptor subtype. ANG II induces cell cycle progression via a PKC-dependent pathway, supporting the notion that phorbol ester-sensitive PKCs play a positive role in promoting exit from G0/G1 and entry in S phase in IEC-18 cells. Furthermore, ANG II-induced DNA synthesis is partially ERK dependent. Taken together, our results imply that PKC plays an important role in mediating early ERK activation and subsequent reinitiation of DNA synthesis induced by either pharmacological (PDB) or physiological (ANG II-AT1 receptor) stimulation of quiescent intestinal epithelial IEC-18 cells.
![]() |
ACKNOWLEDGMENTS |
---|
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 DK-55003, 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.
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|