Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095
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ABSTRACT |
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Protein kinase C (PKC) is implicated in the regulation of multiple important functions in intestinal epithelial cells, but the downstream signaling targets of PKCs in these cells remain poorly characterized. Here we report that treatment of normal rat intestinal cell lines IEC-6 and IEC-18 with phorbol 12,13-dibutyrate (PDBu) led to a rapid and striking PKC-dependent activation of protein kinase D (PKD; also known as PKCµ). Unlike conventional and novel PKCs, PKD did not undergo downregulation in response to prolonged (24 h) exposure of IEC-6 or IEC-18 cells to PDBu. PKD was also rapidly activated in these cells by lysophosphatidic acid (LPA) or angiotensin in a concentration-dependent fashion via a PKC-dependent pathway. EC50 values were 0.1 µM and 2 nM for LPA and angiotensin II, respectively. LPA-induced PKD activation was prevented selectively by treatment with pertussis toxin. PKD activation was tightly associated with an increase in PKD autophosphorylation at serine 916. Our results identify PKD as a novel early point of convergence and integration of Gi and Gq signaling in intestinal epithelial cells.
IEC-18 cells; lysophosphatidic acid, protein kinase C downregulation; epithelial restitution; Gi and Gq
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INTRODUCTION |
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PROTEIN KINASE C
(PKC), a major target for the tumor-promoting phorbol esters, has been
implicated in the signal transduction pathways that mediate a wide
range of biological responses in response to growth factors, hormones,
neuropeptides, and cellular oncogenes (40, 43, 53).
Molecular cloning has demonstrated the presence of multiple related PKC
isoforms including conventional PKCs (,
1,
2, and
), novel PKCs (
,
,
, and
), and
atypical PKCs (
and
), all of which possess a highly conserved
catalytic domain (35, 41, 43). The different PKC isoforms
exhibit distinct patterns of expression in different cell types and
tissues as well as distinct subcellular distributions, leading to the view that they play distinct, rather than redundant, roles in signal
transduction (52).
The sequential proliferation, lineage-specific differentiation,
migration, and cell death of the epithelial cells of the intestinal mucosa are a tightly regulated process that is modulated by a broad
spectrum of regulatory peptides (8, 27, 55). The signaling
mechanisms involved, however, remain incompletely understood. The PKC
family has been implicated in the regulation of multiple important
functions in intestinal epithelial cells including differentiation (65), proliferation (1, 14, 68), migration
(4), apoptosis (68), and
carcinogenesis (9, 28, 46, 56). It is known that
intestinal epithelial cells express multiple isoforms of the PKC
family, including ,
,
,
, and
. These cells have emerged
as an important model system to elucidate isoform-specific PKC
signaling. Mice with transgenic overexpression of PKC
2
in the intestinal epithelium exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced preneoplastic lesions in the colon (39). PKC
has been shown to act as an oncogene when overexpressed in intestinal
epithelial cells (46). PKC also has been implicated in the
downregulation of the tumor suppressor gene Cdx-2 by oncogenic Ras in
vivo (30). Furthermore, loss of PKC
expression in
sporadic human and chemically induced colonic cancers may confer a
relative growth advantage during colonic malignant transformation
(56). Despite these findings indicating a critical role
for PKCs in intestinal epithelial cell proliferation and
carcinogenesis, little is known about the mechanisms of PKC signaling
in these cells. In particular, downstream signaling targets for PKCs in
epithelial cells remain poorly characterized.
Protein kinase D (PKD; also known as PKCµ) is a serine/threonine protein kinase with distinct structural features and unique enzymological properties (26, 61). In particular, the catalytic domain of PKD shows very low homology to the conserved kinase subdomain of the PKCs and displays a distinct substrate specificity (42, 61, 64). In contrast to all known PKCs, the NH2-terminal region of PKD contains a pleckstrin homology (PH) domain that regulates enzyme activity (22) and lacks a sequence with homology to a typical PKC autoinhibitory pseudosubstrate motif (61). However, the amino-terminal region of PKD contains a tandem repeat of cysteine-rich, zinc finger-like motifs that binds phorbol esters with high affinity (21, 61) and mediates PKD translocation to the cell membrane (32). PKD/PKCµ can be activated in vitro by diacylglycerol (DAG)/phorbol esters in the presence of phosphatidyl-L-serine (10, 64, 74), indicating that PKD/PKCµ is a phorbol ester/DAG-stimulated protein kinase (54).
More recently, a second mechanism of PKD activation has been identified
that involves PKD phosphorylation (23, 34, 66, 74).
Specifically, treatment of intact fibroblasts with biologically active
phorbol esters (74), bryostatin (33), growth
factors, and G protein-coupled receptor (GPCR) agonists (45, 63,
75) induces PKD activation that persists during cell disruption
and immunoprecipitation. Treatment with PKC-selective inhibitors
prevents PKD activation by all these factors (33, 54, 74,
75). Furthermore, cotransfection of PKD with constitutively
active mutants of PKC and PKC
dramatically increased the
catalytic activity of PKD (67, 74) and led to
complex formation between PKD and PKC
(67).
These findings revealed a link between PKCs and PKD and implied
that PKD lies downstream of PKCs in a novel signal transduction network
(54, 66).
Nontransformed IEC-6 and IEC-18 cells, derived from fetal rat intestinal crypt cells (48), have provided an in vitro model with which to examine intestinal epithelial cell migration, differentiation, and proliferation (11, 16, 49, 55). To further elucidate PKC-mediated signaling in epithelial cells, we examined whether PKD is endogenously expressed and regulated via PKC in IEC-6 and IEC-18 cells. Our results demonstrate that treatment of these cells with the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu) induces a striking increase in PKD activity but, unlike classic and novel PKCs, does not deplete PKD in these cells. Lysophosphatidic acid (LPA) and angiotensin that signal through heptahelical receptors coupled to heterotrimeric G proteins also promote a rapid PKC-dependent PKD activation in IEC-6 and IEC-18 cells. Although LPA and angiotensin receptors are thought to couple to Gq (37, 38, 51) and thereby to phospholipase C (PLC) (13) in fibroblasts, prior treatment of IEC-6 cells with pertussis toxin (PTx), which inactivates Gi, selectively attenuated PKD activation by LPA. Our results identify PKD activation as a novel early point of convergence and integration of Gi and Gq signaling in intestinal epithelial cells.
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MATERIALS AND METHODS |
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Cell culture. IEC-6 and IEC-18 cells were purchased from American Type Culture Collection. Stock cultures of these cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in a humidified atmosphere containing 10% CO2-90% air at 37°C. For experimental purposes, cells were plated in 100-mm dishes at 3 × 105 cells/dish in DMEM containing 5% FBS, grown to near confluency (5-7 days), and then changed to serum-free DMEM for 18-24 h before the experiment.
Immunoprecipitation. Cultures of IEC-6 cells, treated as described in the individual experiments, were washed and lysed in 50 mM Tris · HCl, pH 7.6, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol (DTT), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and 1% Triton X-100 (lysis buffer A). Cell lysates were clarified by centrifugation at 15,000 g for 10 min at 4°C. PKD was immunoprecipitated at 4°C for 2-4 h with the PA-1 antiserum (1:100), as previously described (74). The immune complexes were recovered with the use of protein A coupled to agarose.
Kinase assay of PKD.
PKD autophosphorylation was determined in an in vitro kinase assay by
mixing 20 µl of PKD immunocomplexes with 10 µl of a phosphorylation
mixture containing (final concentration) 100 µM [-32P]ATP (specific activity 400-600 cpm/pmol),
30 mM Tris · HCl, pH 7.4, 10 mM MgCl2, and 1 mM
DTT. After 10 min of incubation at 30°C, the reaction was stopped by
washing with 200 µl of kinase buffer and then adding an equal volume
of 2× SDS-PAGE sample buffer (200 mM Tris · HCl, pH 6.8, 2 mM
EDTA, 0.1 M Na3VO4, 6% SDS, 10% glycerol, and
4% 2-mercaptoethanol), followed by SDS-PAGE analysis (64,
74). The gels were dried, and the 110-kDa radioactive band
corresponding to autophosphorylated PKD was visualized by autoradiography. Autoradiographs were scanned in a GS-710 Calibrated Imaging Densitometer (Bio-Rad), and the labeled band was quantified with the Quantity One software program.
Western blot analysis. Quiescent cultures of either IEC-6 or IEC-18 cells grown on 100-mm dishes were washed twice with DMEM and then treated as described in the individual experiments. The cells were lysed in 2× SDS-PAGE sample buffer. After SDS-PAGE was carried out on 8% gels, proteins were transferred to Immobilon-P membranes (Millipore) and blocked by 3-6 h of incubation with 5% nonfat milk in phosphate-buffered saline, pH 7.2. Membranes were then incubated overnight with an antiserum that specifically recognizes the phosphorylated state of serine 916 of PKD (pS916) at a dilution of 1:500 or with antibodies that specifically recognize the different PKC isoforms at a dilution of 1 µg/ml in phosphate-buffered saline containing 5% nonfat dried milk. Horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5,000; Amersham) was then applied for 1 h at room temperature after three washes with PBS containing 0.05% Tween. Immunoreactive bands were detected by Western blotting with ECL (enhanced chemiluminescence) reagents (Amersham).
Measurement of intracellular Ca2+
concentration.
Intracellular Ca2+ concentration
([Ca2+]i) was measured with the fluorescent
indicator fura 2. Confluent and quiescent cultures of IEC-6 cells,
grown on 9 × 22-mm coverslips, were washed twice with DMEM and
then incubated for 30 min in DMEM containing 1 µM fura 2-AM at
37°C. The cultures were then washed twice with Hanks' buffered salt
solution (pH 7.2) supplemented with 35 mM NaHCO3, 1.3 mM
CaCl2, 0.5 mM MgCl2, 0.4 mM MgSO4,
and 0.1% bovine serum albumin (calcium buffer). The coverslips were
then incubated for a further 15 min in calcium buffer and then
transferred to a quartz cuvette containing 2 ml of the same buffer.
Fluorescence was monitored via a Hitachi F-2000 Fluorospectrophotometer
with dual-excitation wavelengths of 340 (1) and 380 nm
(
2) and an emission wavelength of 510 nm while the
incubation media were continually stirred at 37°C.
[Ca2+]i was determined from the formula
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Materials.
[-32P]ATP (370 MBq/ml) was from Amersham
International. Bisindolylmaleimide I (GF-I), Ro-31-8220, and PTx were
purchased from Calbiochem. Angiotensin II, LPA, PDBu, wortmannin,
rapamycin, PD-098059, and genistein were purchased from Sigma. Protein
A-agarose was from Boehringer Mannheim. PA-1 antiserum was raised
against the synthetic peptide EEREMKALSERVSIL that corresponds to the carboxy-terminal region of the predicted amino acid sequence of PKD, as
previously described (64, 74). Antibodies (PKD C-20, PKC
C-20, PKC
C-15, PKC
C-15, PKC
, and PKC
C-15) used in Western blot analysis were obtained from Santa Cruz Biotechnologies (Palo Alto, CA). An antiserum that specifically recognizes pS916 was
generously provided by Dr. Doreen Cantrell (Imperial Cancer Research
Institute, London, UK). Other items were from standard suppliers or as indicated in the text.
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RESULTS |
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Expression of PKD in rat intestinal epithelial cell lines.
As a first step toward defining the regulation of PKD in epithelial
cells, lysates from the rat intestinal epithelial cell line IEC-6 were
analyzed for the presence of PKD by Western blotting with the use of a
specific antibody that recognizes the COOH-terminal region of this
enzyme. A major immunoreactive band of 110 kDa, which corresponds to
the molecular mass of PKD (61), was detected in lysates of
this cell line (Fig. 1A,
left). In some experiments, we noticed that PKD isolated
from IEC-6 cells migrated in SDS-PAGE gels as a doublet. The detection
of this (doublet) band was completely blocked when the immunoblots were
incubated with the antibody in the presence of the synthetic peptide
EEREMKALSERVSIL, which corresponds to the COOH-terminal region of the
predicted amino acid sequence of PKD.
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PDBu induces PKD activation in IEC-6 cells.
To determine whether biologically active phorbol esters can induce PKD
activation in intact IEC-6 cells, we treated cultures of these cells
with PDBu for increasing times, lysed the cells, and immunoprecipitated
PKD with PA-1 antiserum. The resulting immunocomplexes were incubated
with [-32P]ATP, and the incorporation of
32P into PKD was analyzed by SDS-PAGE and autoradiography.
As shown in Fig. 1B, PKD isolated from unstimulated IEC-6
cells had low catalytic activity. Treatment of IEC-6 cells with PDBu
induced a rapid and striking increase in PKD kinase activity that was maintained during cell lysis and immunoprecipitation. PKD activation was detectable within 1 min and reached a maximum (~10-fold) after 10 min of PDBu stimulation. These results demonstrate that PKD activation
is one of the early events induced by phorbol esters in epithelial cells.
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Long-term exposure to PDBu downregulates PKCs but not PKD.
We next examined the effect of long-term treatment with PDBu on the
level and serine 916 phosphorylation of PKD from IEC-6 cells. As shown
in Fig. 3, Western blotting with a PKD
antibody revealed that exposure to PDBu induced a marked
electrophoretic retardation of PKD that was evident 0.5 and 2 h
after addition of PDBu. The apparent decrease of PKD at these times is
the result of decreased detectability of phosphorylated PKD at serine
916 (34, 50). In agreement with this explanation, the
decrease of both electrophoretic mobility and detectability coincided
temporally with a striking increase in serine 916 phosphorylation (Fig.
3).
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LPA induces PKD activation in IEC-6 cells. Next, we determined whether PKD is regulated in IEC-6 cells through receptor-mediated pathways. Recently, LPA, a major bioactive lipid of serum (12), was implicated in intestinal epithelial wound healing by modulation of intestinal epithelial cell migration and proliferation in the IEC-6 cell model (59). LPA activates several signaling pathways via heptahelical receptors linked to Gq, Gi, and G12, including PLC-mediated hydrolysis of membrane phosphoinositides leading to the generation of the second messengers inositol 1,4,5-trisphosphate, which stimulates Ca2+ mobilization from intracellular stores, and DAG, which activates classic and novel PKCs (15, 17, 37). Here we examined whether LPA can stimulate PKD activation in intact IEC-6 cells.
Treatment of IEC-6 cells with increasing concentrations of LPA induced a striking increase in PKD activity and serine 916 phosphorylation in a concentration-dependent manner (Fig. 4A). Half-maximal and maximal PKD activation was achieved at 0.1 and 1 µM LPA, respectively (Fig. 4B). Consistent with these results, syntide-2 phosphorylation assays also demonstrated that LPA induces a marked increase in PKD activity in IEC-6 cells (Fig. 4B, inset).
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LPA stimulates PKD activation via PKC.
To determine the role of PKCs in PKD activation induced by LPA, we
treated cultures of IEC-6 cells with various concentrations of
Ro-31-8220 (72) before LPA stimulation. As shown in Fig. 5A, treatment with Ro-31-8220
potently blocked PKD activation induced by subsequent addition of LPA
in a concentration-dependent fashion. In contrast, Ro-31-8220 added
directly to the in vitro kinase assay, even at concentrations
(0.25-2.5 µM) that abrogated LPA-induced PKD activation in
intact IEC-6 cells, did not inhibit PKD activity. Similar results were
obtained when the PKC inhibitor GF-I was used instead of Ro-31-8220
(results not shown). These results imply that Ro-31-8220 and GF-I do
not inhibit PKD activity directly but interfere with LPA-mediated PKD
activation in intact cells by blocking PKC. Furthermore, depletion of
PKCs by treatment with PDBu for 24 h abrogated LPA-induced PKD
activation. (Fig. 5B).
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LPA stimulates PKD activation via a PTx-sensitive pathway.
LPA binds to a seven-transmembrane domain receptor(s) that couples to
Gq, Gi, and G12 signaling
(17, 38). To clarify the G protein pathways leading to
LPA-induced PKD activation, we examined the effect of treatment with
PTx, which catalyzes ADP ribosylation and inactivation of G proteins of
the Gi/Go family. As shown in Fig.
6, exposure of IEC-6 cells to increasing
concentrations of PTx for 3 h completely blocked the increase in
PKD activity induced by subsequent LPA stimulation in a
concentration-dependent manner. Half-maximal and maximal inhibition of
LPA-induced PKD activation was achieved at ~1 and 10 ng/ml PTx,
respectively. In contrast, exposure of parallel cultures to 100 ng/ml
PTx for 3 h did not interfere with PKD activation induced by PDBu,
which directly activates PKC and bypasses receptor-mediated pathways
(results not shown). These results indicate that LPA-stimulated PKD
activation is mediated through Gi in IEC-6 cells.
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Angiotensin induces PKD activation in IEC-6 cells via PKC.
In fibroblasts, PKD is rapidly activated by peptide agonists that
signal through heptahelical receptors coupled to Gq
(73, 75). Recent results indicate that Gq
activation is sufficient to stimulate sustained PKD activation via PKC
and show that the endogenous G
q mediates PKD activation
in response to acute bombesin receptor stimulation (73).
Previous reports indicated that IEC-6 cells endogenously express the
AT1 subtype of angiotensin receptor, which signals via
Gq (57, 58). We verified that addition of angiotensin (5-50 nM) to IEC-6 cells induced a PTx-insensitive increase in cytosolic Ca2+ concentration (Fig. 6,
inset). Here, we examined whether angiotensin stimulates PKD
activation in these epithelial intestinal cells.
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PKD activation integrates Gi and Gq
signaling pathways in IEC-6 cells.
To determine the effect of PTx on PKD activation induced by angiotensin
in IEC-6 cells, we treated cultures of these cells with 100 ng/ml PTx
for 3 h and then challenged with either angiotensin or LPA for 10 min. PKD activity was determined by in vitro kinase assays after
immunoprecipitation, Western blotting with the pS916 antibody, and
syntide-2 phosphorylation assays (Fig.
9). In agreement with the results
presented in Fig. 6, treatment with PTx profoundly inhibited the
activation and the serine 916 phosphorylation of PKD induced by LPA. In
contrast, angiotensin-induced PKD activation was not affected by an
identical PTx treatment, as assessed by all the assays used. These
results imply that angiotensin signals PKD activation via
Gq and indicate that PKD integrates pathways mediated by
Gi- and Gq-linked receptors in epithelial
cells.
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PKD activation by PDBu, LPA, and angiotensin in IEC-18 cells.
To substantiate the findings obtained with IEC-6 cells, we also
examined PKD expression and regulation in IEC-18 cells, a different
crypt intestinal cell line derived from rat ileal epithelium (48). Initially, we confirmed that PKD is expressed in
IEC-18 cells, as judged by Western blot analysis of either cell lysates or PA-1 immunoprecipitates (Fig.
10A). Next, we examined the
effect of PDBu on PKD expression and regulation.
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DISCUSSION |
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The PKC family plays a central role in the signal transduction pathways that mediate important functions in intestinal epithelial cells including differentiation (65), proliferation (1, 14), migration (4), and carcinogenesis (46). The present challenge is to identify downstream targets for PKCs that transmit signals to the cell interior and participate in the regulation of processes that control intestinal epithelial cell function.
PKD is a novel serine/threonine protein kinase that can be
distinguished from PKC isoforms by a variety of criteria including catalytic domain structure, substrate specificity, the presence of a PH
domain, and absence of a pseudosubstrate autoinhibitory motif, which is
present in all known PKCs upstream of the first cysteine-rich domain
(54). PKD has been implicated in the regulation of
endothelial growth factor receptor signaling (5),
Na+/H+ antiport activity (19),
Golgi organization and function (24, 47), nuclear
factor-B-mediated gene expression (25), and cell
migration (7). As a first step to elucidate the
mechanism of activation of PKD in intestinal epithelial cells, we
examined the regulation of this enzyme in IEC-6 and IEC-18 cells,
derived from fetal rat intestinal crypt cells. These cells have
provided an in vitro model to examine intestinal epithelial cell
migration, restitution, and proliferation (11, 16, 49,
55).
Our results demonstrate that treatment of intact IEC-6 cells with the tumor-promoting phorbol ester PDBu induces rapid PKD activation. The conversion of PKD into this activated state occurs within minutes of phorbol ester stimulation of intact IEC-6 cells and, thus, is one of the early events induced by phorbol esters in these cells.
It is well established that long-term exposure to biologically active
phorbol esters induces downregulation of conventional and novel PKC
isoforms in mammalian cells. Binding of phorbol esters to these PKCs is
mediated by a tandem repeat of cysteine-rich motifs, the C1 region, and
is associated with their translocation to cellular membranes
(52). Interestingly, PKD also possesses a tandem repeat of
cysteine-rich motifs (21, 26, 61) and is translocated to
the plasma membrane in response to phorbol ester stimulation
(32). However, little is known about the regulation of PKD
level in response to long-term treatment with phorbol esters. The
results presented here indicate that, in contrast to classic and
conventional PKCs, prolonged exposure to PDBu did not induce downregulation of PKD protein under conditions that produced striking depletion of PKCs ,
, and
. These results, corroborated in IEC-6 and IEC-18 cells, identify another important functional difference between PKD and phorbol ester-sensitive isoforms of PKC.
To investigate the physiological significance of the PKC/PKD pathway in intestinal epithelial cells, we examined PKD regulation in these cells via receptor-mediated pathways. LPA, a major bioactive lipid of serum, has been shown to elicit a broad spectrum of biological responses in many cell types (15, 17, 37). LPA binds to seven-transmembrane domain receptor(s) and activates several heterotrimeric G proteins including Gq, Gi, and G12, which are responsible for transducing LPA signals into multiple biological responses (17, 38). In this study, we report that LPA stimulation promotes a rapid and concentration-dependent PKD activation in intact IEC-6 cells through a PKC-dependent pathway.
In fibroblasts, LPA-induced PKC activation is thought to be mediated by LPA receptor coupling to PTx-insensitive Gq (37). In contrast, our results with IEC-6 cells demonstrate that PKC-dependent PKD activation induced by LPA was attenuated markedly and selectively by prior treatment of these cells with low concentrations of PTx. These results identify a novel PTx-sensitive molecular response in the action of LPA and demonstrate, for the first time, the involvement of a Gi-dependent pathway leading to PKD activation in epithelial cells.
It is known that the -subunits, rather than
-subunits, of
heterotrimeric Gi proteins preferentially activate the
2 and
3 isoforms of PLC (13,
51). Unlike PLC-
2, which is expressed only in
certain cells of hematopoietic origin, PLC-
3 is widely distributed (13, 51). Recently, the LPA receptor subtype
EDG-2 has been shown to induce a rapid PLC-dependent increase in the cytoplasmic Ca2+ concentration via PTx-sensitive
Gi (3), whereas the other LPA receptor
subtypes cloned so far (e.g., EDG-4 and EDG-7) induce Ca2+
fluxes via PTx-insensitive Gq (3, 6). Here we
found that treatment of IEC-6 cells with PTx markedly attenuated
Ca2+ mobilization in response to LPA. Our results,
therefore, are consistent with a model in which LPA induces
PKC-dependent, PTx-sensitive PKD activation via a pathway involving
EDG-2 and Gi in IEC-6 cells.
Recent results with transfected COS-7 cells indicate that
Gq activation is sufficient to stimulate
sustained PKD activation via PKC and show that the endogenous
G
q mediates PKD activation in response to acute bombesin
receptor stimulation (73). Here we have shown that
addition of angiotensin to IEC-6 cells induced a very rapid activation
of endogenous PKD that was prevented by inhibitors of PKC. In contrast
to the results obtained with LPA, treatment of IEC-6 cells with PTx did
not affect PKD activation in response to angiotensin. These results
demonstrate that inhibition of LPA-induced PKD activation by PTx is
selective and suggest that angiotensin induces PKD activation via
Gq in IEC-6 cells. Thus PKD is activated in
intestinal epithelial cells by agonist stimulation of GPCRs that signal
via Gi and Gq.
PKD activation is accompanied by an increase in its phosphorylation at multiple sites (23, 34, 66). Previous work has used the site-specific phosphopeptide antibody approach to identify a phosphorylation site within the COOH-terminal region of PKD at serine residue 916 (34). Mutational analysis has indicated that serine 916 is an in vivo autophosphorylation site for active PKD. Using the pS916 antibody, we have shown here a striking increase in the phosphorylation of serine 916 of endogenous PKD in response to both pharmacological (phorbol ester) and physiological (LPA, angiotensin) stimulation of IEC-6 and IEC-18 cells.
Recently, LPA has been shown to stimulate intestinal epithelial wound healing by enhancing intestinal epithelial cell migration in the IEC-6 cell model as well as in vivo (59). LPA induced IEC-6 cell migration, like PKD activation, through a PTx-sensitive pathway. Other studies have indicated that PKCs play a role in intestinal epithelial cell motility (4), and PKD has been implicated in cell migration and metastasis in breast cancer cells via its molecular association with the cytoskeletal proteins paxillin and cortactin (7). Recently, we found that PKD extracted from either IEC-6 or IEC-18 cells coimmunoprecipitates with paxillin and that treatment of these cells with angiotensin or PDBu stimulated cell migration (unpublished observations). Thus an attractive possibility is that PKD contributes to intestinal epithelial cell migration and wound healing, a proposition that warrants further experimental work.
In conclusion, our findings indicate that PKD is activated in intact IEC-6 cells by biologically active phorbol esters, LPA, and angiotensin through a PKC-dependent signal transduction pathway. Our results, showing that PKD can function downstream of PKC in intestinal epithelial cells, raise the possibility that PKD mediates some of the biological responses elicited by PKC in these cells. Unlike conventional and novel PKCs, PKD does not undergo downregulation in response to prolonged (24 h) exposure to PDBu. LPA induced PKD activation through a PTx-sensitive pathway involving Gi, whereas angiotensin promoted PKD activation via PTx-insensitive Gq. The salient features of the results obtained with IEC-6 cells were corroborated with IEC-18 cells. We propose that PKD constitutes an early molecular point of convergence and integration of Gi and Gq signaling in intestinal epithelial cells.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-55003 and DK-56930. T. Chiu is the recipient of an Eisai-Janssen Research Fellowship.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Rozengurt, 900 Veteran Ave., Warren Hall, Rm. 11-124, Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1786 (E-mail: erozengurt{at}mednet.ucla.edu).
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.
Received 7 September 2000; accepted in final form 6 November 2000.
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