PKD in intestinal epithelial cells: rapid activation by phorbol esters, LPA, and angiotensin through PKC

Terence Chiu and Enrique Rozengurt

Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta 1, beta 2, and gamma ), novel PKCs (delta , epsilon , eta , and theta ), and atypical PKCs (zeta  and lambda ), 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 alpha , beta , delta , epsilon , and zeta . These cells have emerged as an important model system to elucidate isoform-specific PKC signaling. Mice with transgenic overexpression of PKCbeta 2 in the intestinal epithelium exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced preneoplastic lesions in the colon (39). PKCepsilon 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 PKCalpha 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 PKCepsilon and PKCeta dramatically increased the catalytic activity of PKD (67, 74) and led to complex formation between PKD and PKCeta (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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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.

Exogenous substrate phosphorylation by immunoprecipitated PKD was carried out by mixing 20 µl of the washed immunocomplexes with 20 µl of a phosphorylation mixture containing 2.5 mg/ml syntide-2 (PLARTLSVAGLPGKK), a peptide based on phosphorylation site two of glycogen synthase. After 10 min of incubation at 30°C, the reaction was stopped by adding 100 µl of 75 mM H3PO4 and spotting 75 µl of the supernatant on P-81 phosphocellulose paper. Free [gamma -32P]ATP was separated from the labeled substrate by washing the P-81 paper four times for 5 min in 75 mM H3PO4. The papers were dried, and the radioactivity incorporated into syntide-2 was determined by Cerenkov counting.

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 (lambda 1) and 380 nm (lambda 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
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB> <FR><NU>(R − R<SUB>min</SUB>)</NU><DE>(R<SUB>max</SUB> − R)</DE></FR> × <FR><NU>F<SUB>min</SUB> &lgr;<SUB>2</SUB></NU><DE>F<SUB>max</SUB> &lgr;<SUB>2</SUB></DE></FR>
where R, Rmin, and Rmax are the ratios of the emission at 510 nm following excitation at 340 and 380 nm, Fmax is the fluorescence after the addition of 40 µM digitonin, and Fmin is the fluorescence after the Ca2+ in the solution has been chelated with 10-20 mM EGTA. The value of the dissociation constant (Kd) used was 224 nM.

Materials. [gamma -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, PKCzeta C-20, PKCepsilon C-15, PKCeta C-15, PKCdelta , and PKCalpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Protein kinase D (PKD) expression and activation by phorbol 12,13-dibutyrate (PDBu) in IEC-6 cells. A: Western blot analysis of IEC-6 cell lysates and PKD immunoprecipitates (IP). Left: confluent and quiescent cultures of IEC-6 cells were solubilized with 2× sample buffer. The lysates were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out using the antibody against the COOH-terminal region of mouse PKD (C-20) in the absence (-) or presence (+) of the immunizing peptide (50 µg/ml) that corresponds to the COOH-terminal region of the predicted amino acid sequence of PKD. Right: confluent and quiescent cultures of IEC-6 cells were lysed with lysis buffer A. The lysates were immunoprecipitated with the PA-1 antiserum in the absence or presence of the immunizing peptide (20 µg/ml). The immunoprecipitates were analyzed by Western blotting using the PA-1 antiserum as described in MATERIALS AND METHODS. B: PKD activation in IEC-6 cells. Confluent and quiescent cultures of IEC-6 cells were treated for various times with 100 nM PDBu at 37°C as indicated. Cultures were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum, and PKD activity in the immunocomplexes was determined by an in vitro kinase (IVK) assay as described in MATERIALS AND METHODS, followed by SDS-PAGE and autoradiography. Identical experiments were done in parallel where Western blot analysis was performed on cells lysed with 2× sample buffer by using an antibody raised against the phosphorylated state of serine 916 (pS916). Inset: representative autoradiogram of IVK assay and pS916 Western blots. The level of PKD autophosphorylation in the IVK experiments was quantified by scanning densitometry. The results, expressed as percentages of the maximum increase in phosphorylation, are means ± SE (n = 3) plotted as a function of time.

A prominent PKD band was also obtained when lysates from the same intestinal epithelial cell lines were immunoprecipitated with the PA-1 antiserum (see MATERIALS AND METHODS), and the immunoprecipitates were analyzed by Western blotting with the use of a different antibody directed against PKD (Fig. 1A, right). Detection of the 110-kDa band was blocked by the inclusion of the immunizing peptide during the immunoprecipitation (Fig. 1A). These results demonstrate the expression of PKD in IEC-6 cells.

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 [gamma -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.

Recently, an antiserum specifically recognizing the phosphorylated form of a PKD COOH-terminal residue, serine 916, was developed and used to detect in vivo autophosphorylation at this site by active PKD (34). Thus the pS916 antiserum provides a novel approach for detecting conversion of PKD to an active form within cells. Here, lysates from IEC-6 cells stimulated with PDBu for various times were analyzed by SDS-PAGE, followed by Western blot analysis with the pS916 antiserum. PDBu stimulation induced a dramatic increase in the immunoreactivity of the PKD band indicative of phosphorylation at serine 916 (Fig. 1B, inset). An increase in PKD autophosphorylation was detectable within 1 min and reached a maximum (~10-fold) after 5-10 min of PDBu stimulation.

Stimulation of intact IEC-6 cells with increasing concentrations of PDBu for 10 min induced a striking dose-dependent increase in PKD activation, as judged by assays of in vitro PKD kinase activity after immunoprecipitation or by Western blotting with pS916 antibody to detect PKD autophosphorylation in intact cells (Fig. 2A). Half-maximal PKD activation by PDBu was achieved at 10-25 nM (Fig. 2B).


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Fig. 2.   PDBu activates PKD in a dose-dependent and protein kinase C (PKC)-dependent manner. A: PDBu activates PKD in a dose-dependent manner. Top: confluent and quiescent cultures of IEC-6 cells in IVK experiments were treated with various concentrations of PDBu for 10 min at 37°C as indicated. The cultures were lysed in lysis buffer A and immunoprecipitated with PA-1 antiserum, and PKD activity was determined by an IVK assay as described in MATERIAL AND METHODS. The autoradiogram shown is representative of 3 independent experiments. Bottom: identical experiments were carried out in parallel cultures where Western blot analysis with pS916 antiserum was performed following lysis of the cells with 2× sample buffer as described in MATERIALS AND METHODS. The Western blot shown is representative of 3 independent experiments. B: scanning densitometry. The results shown are means ± SE (n = 3) of the level of PKD activation from IVK obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained with 100 nM PDBu. Inset: confluent and quiescent cultures of IEC-6 cells were either kept in serum-free DMEM (-) or treated with 100 nM PDBu for 10 min at 37°C. After lysis with lysis buffer A and immunoprecipitation with PA-1 antibody, PKD activity in the immunocomplexes was then measured by syntide-2 phosphorylation, as described in MATERIALS AND METHODS. C: Ro-31-8220 (Ro) and bisindolylmaleimide I (GF-I) inhibit PKD activity. Confluent and quiescent IEC-6 cells were incubated for 1 h with different concentrations of the PKC inhibitors Ro or GF-I. The cultures were subsequently stimulated for 10 min with 100 nM PDBu at 37°C. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum, and PKD activity was determined by an IVK assay as described in MATERIALS AND METHODS.

As an independent measure of PKD activation induced by PDBu in IEC-6 cells, we also examined phosphorylation of an exogenous substrate using syntide-2 (29, 36), a peptide identified as an excellent model substrate for PKD (42, 61, 64, 67). Consistent with the results of autophosphorylation assays, syntide-2 phosphorylation assays also showed that IEC-6 cells stimulated with PDBu had dramatically increased PKD activity (Fig. 2B, inset). Together, the results presented in Figs. 1 and 2 provide multiple lines of evidence indicating that exposure of intact IEC-6 cells to PDBu induces rapid and striking PKD activation.

We also determined the role of PKCs in PKD activation induced by PDBu. Cultures of IEC-6 cells were treated with various concentrations of either Ro-31-8220 or GF-I, selective inhibitors of phorbol ester-sensitive isoforms of PKC (31, 60, 72) but not PKD (33, 74, 75), before PDBu stimulation. As shown in Fig. 2C, the increase in PKD activity induced by PDBu was prevented by treatment with either Ro-31-8220 or GF-I. These findings imply that PDBu activates PKD in IEC-6 cells through a PKC-dependent pathway.

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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Fig. 3.   Effect of PDBu on PKD and PKC levels in IEC-6 cells. A: Western blot analysis of PKCs and PKD following addition of PDBu. Confluent and quiescent cultures of IEC-6 cells were incubated for various times with 200 nM PDBu at 37°C. At the indicated times, the cultures were lysed by adding 2× sample buffer, and Western blot analysis was performed with the use of isoform-specific polyclonal antisera against the different PKCs or PKD, as described in MATERIALS AND METHODS. B: PKD activity after prolonged PDBu treatment. Confluent and quiescent cultures of IEC-6 cells were incubated for 24 h either without (-) or with (+) 200 nM PDBu at 37°C and washed with serum-free DMEM. After 30 min at 37°C in serum-free media, these cells were then incubated for a further 10 min either without or with 100 nM PDBu as indicated. The cells were then lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was determined by an IVK assay as described in MATERIALS AND METHODS, followed by SDS-PAGE and autoradiography.

Interestingly, continued exposure to PDBu did not lead to downregulation of PKD in IEC-6 cells. In contrast, serine 916 phosphorylation, indicative of PKD activity, declined gradually over the 24-h period. A plausible interpretation of this decline is that continuous exposure to PDBu induced the depletion of PKC isoforms that are required for PKD activation and autophosphorylation at serine 916. To test this possibility, we analyzed the lysates from IEC-6 cells incubated with PDBu for various times for the presence of PKCs alpha , delta , epsilon , and zeta . As shown in Fig. 3, treatment with PDBu induced a dramatic downregulation of PKCs alpha , delta , and epsilon  that was apparent by 6 h and extensive at 24 h. These findings indicate that, in contrast to classic and novel PKCs, PKD does not undergo downregulation upon chronic treatment with PDBu. The reversal in the phosphorylation of serine 916, indicative of PKD activity, correlated with the downregulation of the classic and novel PKCs and supports the hypothesis that one or more of these PKCs are required for PDBu-induced PKD activation.

To confirm the requirement of PKC in mediating PDBu-induced PKD activation, we treated IEC-6 cells with PDBu for 24 h to produce extensive depletion of classic and novel PKCs and then challenged with PDBu for 10 min. As shown in Fig. 3B, the increase in PKD activity induced by acute exposure to PDBu was abrogated by prior treatment with this PKC agonist for 24 h. The results shown in Fig. 3 provide additional evidence indicating that PKC(s) is required for PKD activation and demonstrate that prolonged exposure to PDBu induces differential depletion of PKCs but not PKD.

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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Fig. 4.   Lysophosphatidic acid (LPA) activates PKD in a time- and dose-dependent manner. A: LPA activates PKD in a dose-dependent manner. Top: confluent and quiescent cultures of IEC-6 cells in IVK experiments were incubated with various concentrations of LPA for 5 min at 37°C. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was determined by an IVK assay as described in MATERIALS AND METHODS, followed by SDS-PAGE and autoradiography. The autoradiogram shown is representative of 3 independent experiments. Bottom: an identical experiment was carried out in parallel where Western blot analysis using pS916 antiserum was performed following lysis of the cells with 2× sample buffer as described in MATERIALS AND METHODS. The Western blot shown is representative of at least 3 independent experiments. B: scanning densitometry. The results shown are means ± SE (n = 3) of the level of PKD activation by IVK obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained with 3 µM LPA. Inset: confluent and quiescent cultures of IEC-6 cells were either kept in serum-free DMEM (-) or treated with 5 µM LPA for 5 min at 37°C. PKD activity in the immunocomplexes was then measured by syntide-2 phosphorylation, as described in MATERIALS AND METHODS. C: LPA activates PKD in a time-dependent manner. Confluent and quiescent cultures of IEC-6 cells were treated for various times with 5 µM LPA as indicated. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was determined by an IVK assay, as described in MATERIALS AND METHODS, followed by SDS-PAGE and autoradiography (inset, IVK). Identical experiments were carried out in parallel cultures where Western blot analysis with pS916 antiserum was performed following lysis of the cells with 2× sample buffer, as described in MATERIALS AND METHODS (inset, pS916). The Western blot shown is representative of 3 independent experiments. The results shown are means ± SE (n = 3) of the level of PKD activation obtained from scanning densitometry of IVK and are expressed as percentages of the maximum increase in phosphorylation obtained from cells incubated with 5 µM LPA for 5 min at 37°C.

PKD activation was a rapid consequence of the addition of LPA to IEC-6 cells (Fig. 4C). A marked increase in PKD activity was evident at as early as 30 s of LPA stimulation. These results indicate that LPA induces rapid PKD activation at concentrations that are present physiologically in serum.

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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Fig. 5.   LPA induces PKD activation through a PKC-dependent pathway. A: Ro and GF-I inhibit PKD activity. Confluent and quiescent IEC-6 cells were incubated for 1 h with different concentrations of the PKC inhibitor Ro (top blot and black-down-triangle ). Control cells (-) received equivalent amounts of solvent. The cultures were subsequently stimulated for 5 min with 5 µM LPA at 37°C. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay, as described in MATERIALS AND METHODS. Parallel cultures were treated with 5 µM LPA for 5 min at 37°C (bottom blot and triangle ), lysed with lysis buffer A, and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay in the absence or presence of the indicated concentrations of Ro. Graphed data represent average values of the level of PKD activation obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained with 5 µM LPA for 5 min at 37°C. B: PKD activity after prolonged PDBu treatment. Confluent and quiescent cultures of IEC-6 cells were incubated for 24 h either without (-) or with (+) 200 nM PDBu. The cultures were washed with DMEM and incubated for 30 min at 37°C in DMEM. The cells were then incubated for a further 5 min without or with 5 µM LPA at 37°C. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay, as described in MATERIALS AND METHODS. The autoradiogram shown is representative of 3 independent experiments. C: genistein (Gen), LY-294002 (LY), PD-098059 (PD), rapamycin (Rap), and wortmannin (Wort) do not inhibit PKD activity. IEC-6 cells were incubated for 1 h with either 50 µM Gen, 20 µM LY, 20 µM PD, 20 nM Rap, 100 nM Wort, or an equivalent amount of solvent (-). Cells were subsequently challenged for 5 min with 5 µM LPA at 37°C. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay, as described in MATERIALS AND METHODS. The autoradiogram shown is representative of 3 independent experiments.

In contrast to the results obtained with PKC inhibitors, either treatment with the protein tyrosine kinase inhibitor genistein, which prevents LPA-mediated Ras activation (20, 62), the MEK inhibitor PD-098059 (2), which prevents extracellular signal-regulated kinase activation, and the phosphatidylinositol 3-kinase inhibitors LY-294002 and wortmannin (44, 71) or interference with activation of the phosphatidylinositol 3-kinase downstream target p70 ribosomal S6 kinase (p70S6K) with rapamycin (18, 69, 70) did not affect PKD activation in response to LPA in IEC-6 cells (Fig. 5C). The results presented in Fig. 5 indicate that LPA induces PKD activation through PKC in IEC-6 cells.

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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Fig. 6.   PKD activation induced by LPA in IEC-6 cells is blocked by pertussis toxin (PTx) in a dose-dependent manner. Top: confluent and quiescent IEC-6 cells were washed with DMEM and incubated with different concentrations of PTx, as indicated, for 3 h. Cells were then stimulated with (+) or without (-) 5 µM LPA for 5 min at 37°C. Cultures were lysed with lysis buffer A and then immunoprecipitated with PA-1 antiserum. Immunoprecipitates were subjected to IVK assay, SDS-PAGE, and autoradiography. The autoradiograph shown is representative of 3 independent experiments. Bottom: results are means ± SE (n = 3) of the level of PKD activation obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained with 5 µM LPA for 5 min at 37°C () or from cells incubated with PTx but without LPA (). Inset: increase in intracellular Ca2+ concentration ([Ca2+]i) induced by LPA is inhibited by PTx. Confluent IEC-6 cells grown on 9 × 22-mm coverslips were washed twice with DMEM and then incubated without or with 100 ng/ml PTx for 3 h in DMEM at 37°C. [Ca2+]i following the addition of 5 µM LPA or 100 nM angiotensin II was determined as described in MATERIALS AND METHODS. The bar graph shows the percentage of increase over basal [Ca2+]i ± SE (n = 6).

It is known that the beta gamma -subunits rather than alpha -subunits of heterotrimeric Gi proteins preferentially activate the beta -isoforms of PLC (13, 51). The LPA receptor subtype EDG-2 induces a rapid PLC-dependent increase in the cytoplasmic Ca2+ concentration via a PTx-sensitive Gi pathway (3), whereas the other LPA receptor subtypes (e.g., EDG-4 and EDG-7) induce Ca2+ fluxes via PTx-insensitive Gq (3, 6). The rapid and transient increase in the cytoplasmic Ca2+ concentration induced by LPA in IEC-6 cells was strikingly reduced (~80%) by prior treatment of the cells with PTx (Fig. 6, inset). These findings indicate that LPA induces Ca2+ mobilization, like PKD activation, via Gi in IEC-6 cells and suggest the involvement of the EDG-2 receptor in the action of LPA in these cells.

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 Galpha q activation is sufficient to stimulate sustained PKD activation via PKC and show that the endogenous Galpha 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.

Stimulation of intact IEC-6 cells with increasing concentrations of angiotensin for 90 s induced a striking, dose-dependent increase in PKD activation, as judged by assays of in vitro PKD kinase activity after immunoprecipitation with PA-1 antiserum or Western blotting of cell lysates with pS916 antibody to detect PKD autophosphorylation (at serine 916) in intact cells (Fig. 7A). Half-maximal and maximal PKD activation by angiotensin was achieved at 2 and 10 nM, respectively (Fig. 7B, left). In addition, syntide-2 phosphorylation assays also demonstrated that angiotensin induced a marked increase in PKD activity in IEC-6 cells (Fig. 7B, inset).


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Fig. 7.   Angiotensin II activates PKD in a time- and dose-dependent manner. A: angiotensin II (ANG II) dose-response curve. Top: confluent and quiescent cultures of IEC-6 cells were treated with various concentrations of ANG II for 90 s at 37°C as indicated. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay as described in MATERIALS AND METHODS. The autoradiogram shown is representative of at least 3 independent experiments. Bottom: identical experiments were carried out in parallel cultures where Western blot analysis with pS916 antiserum was performed following lysis of the cells with 2× sample buffer, as described in MATERIALS AND METHODS. The Western blot shown is representative of 3 independent experiments. B: scanning densitometry. The results shown are means ± SE (n = 3) of the level of PKD activation obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained from cells incubated with 100 nM ANG II for 90 s at 37°C. Inset: confluent and quiescent cultures of IEC-6 cells were either kept in serum-free DMEM (-) or treated with 100 nM ANG II for 90 s at 37°C. PKD activity in the immunocomplexes was then measured by syntide-2 phosphorylation, as described in MATERIALS AND METHODS. C: ANG II time course. Top: confluent and quiescent cultures of IEC-6 cells were treated for various times with 100 nM ANG II as indicated. Cells were lysed with lysis buffer and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay, as described in MATERIALS AND METHODS. The autoradiogram shown is representative of at least 3 independent experiments. Bottom: identical experiments were carried out in parallel cultures where Western blot analysis with pS916 antiserum was performed following lysis of the cells with 2× sample buffer, as described in MATERIALS AND METHODS. The Western blot shown is representative of 3 independent experiments. D: scanning densitometry. The results shown are means ± SE (n = 3) of the level of PKD activation obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained from cells incubated with 100 nM ANG II for 90 s at 37°C.

Treatment of IEC-6 cells with angiotensin induced a very rapid increase in PKD activation, which was detectable as early as 15 s after agonist stimulation and reached maximal stimulation within 1 min, as shown by in vitro kinase assays after immunoprecipitation or by Western blotting with the pS916 antibody (Fig. 7, A and B).

Treatment of IEC-6 cells with either GF-I or Ro-31-8220 potently blocked PKD activation induced by subsequent addition of angiotensin in a concentration-dependent fashion. In contrast, GF-I or Ro-31-8220 did not inhibit PKD activity when added directly to the in vitro kinase assay, even at concentrations (0.25-1 µM) that prevented angiotensin-mediated PKD activation in intact IEC-6 cells (Fig. 8, A and B). Exposure of IEC-6 cells to other kinase inhibitors including genistein, PD-098059, wortmannin, or rapamycin did not affect PKD activation in response to angiotensin (results not shown). In addition, depletion of PKCs by prolonged exposure to PDBu abrogated angiotensin-induced PKD activation (Fig. 8C). Thus angiotensin, like LPA, induces PKD activation in intact IEC-6 epithelial cells via PKC.


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Fig. 8.   ANG II induces PKD activation through a PKC-dependent pathway. A: Ro and GF-I inhibit PKD activity. Confluent IEC-6 cells were incubated for 1 h with different concentrations of the PKC inhibitor GF-I (top left) or Ro (top right). Control cells (-) received equivalent amounts of solvent. The cultures were subsequently stimulated for 90 s with 100 nM ANG II at 37°C. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay, as described in MATERIALS AND METHODS. Parallel cultures were treated with 100 nM ANG II for 90s at 37°C. Cells were lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was then determined by an IVK assay in absence or presence of the indicated concentrations of GF-I (bottom left) or Ro (bottom right) added to the incubation mixture. B: scanning densitometry. The results shown are means ± SE (n = 3) of the level of PKD activation obtained from scanning densitometry and are expressed as percentages of the maximum increase in phosphorylation obtained from cells incubated with 100 nM ANG II for 90 s at 37°C. , values corresponding to PKD activity from cells incubated with GF-I (left) or Ro (right); triangle , values corresponding to PKD activity determined by an IVK assay in the absence or presence of indicated concentrations of GF-I (left) or Ro (right) added to the incubation mixture. C: PKD activity after prolonged PDBu treatment. Confluent and quiescent cultures of IEC-6 cells were treated for 24 h either without (-) or with (+) 200 nM PDBu at 37°C and washed with serum-free DMEM. After 30 min at 37°C in serum-free media, these cells were then incubated for a further 90 s either without or with 100 nM ANG II as indicated. The cells were then lysed with lysis buffer A and immunoprecipitated with PA-1 antiserum. PKD activity was determined by an IVK assay, as described in MATERIALS AND METHODS, followed by SDS-PAGE and autoradiography.

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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Fig. 9.   Effect of PTx on PKD activation induced by ANG II and LPA in IEC-6 cells. Confluent and quiescent IEC-6 cells were washed with DMEM and incubated with 100 ng/ml PTx or an equivalent amount of solvent for 3 h. Cells were then stimulated with 100 nM ANG II for 90 s or 5 µM LPA for 5 min. PKD activity was measured by syntide-2 phosphorylation by using the immunocomplexes (B), IVK assay (A, top), and Western blot for pS916 (A, bottom). Autoradiograms shown are representative of at least 3 independent experiments.

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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Fig. 10.   PKD expression and the effect of PDBu on PKD and PKC levels in IEC-18 cells. A: Western blot analysis of IEC-6 cell lysates and PKD immunoprecipitates. Confluent and quiescent cultures of IEC-18 cells were solubilized with 2× sample buffer. The lysates were analyzed by SDS-PAGE and transferred to Immobilon membranes. Left: Western blot analysis was carried out by using the antibody against the COOH-terminal region of mouse PKD (C-20) in the absence (-) or presence (+) of the immunizing peptide that corresponds to the COOH-terminal region of the predicted amino acid sequence of PKD (50 µg/ml). Right: Western blot analysis of PKD immunoprecipitates. Lysates from confluent and quiescent cultures of IEC-6 cells were immunoprecipitated with the PA-1 antiserum in the absence (-) or presence (+) of the immunizing peptide (20 µg/ml). B: Western blot analysis of PKCs and PKD following addition of PDBu. Confluent and quiescent cultures of IEC-18 cells were treated for various times with 200 nM PDBu as indicated. Cell lysates were analyzed by SDS-PAGE and transferred to Immobilon membranes. Western blot analysis was carried out by using isoform-specific polyclonal antisera against the different PKCs or PKD.

Treatment of IEC-18 cells with PDBu induced a striking and transient increase in the phosphorylation of serine 916 and electrophoretic retardation of endogenously expressed PKD but did not reduce the level of PKD (Fig. 10B). An identical long-term treatment with PDBu depleted the conventional PKCalpha and novel PKCs delta  and epsilon  but not atypical PKCzeta , which is known to be unresponsive to phorbol esters. The results shown in Fig. 10 with IEC-18 cells are in very good agreement with those shown in Fig. 3 with IEC-6 cells and confirm that PKD is activated but not depleted by long-term treatment with PDBu.

We also determined the time course and dose response of PKD activation in response to PDBu, LPA, and angiotensin. Lysates of IEC-18 cells treated with 100 nM PDBu, 5 µM LPA, or 100 nM angiotensin for various times or with increasing concentrations of these agonists for a fixed time were analyzed by Western blotting with the pS916 antibody to detect PKD autophosphorylation at serine 916. Stimulation of IEC-18 cells with these agonists induced PKD phosphorylation in a dose- and time-dependent fashion (Fig. 11, A-C). In other experiments, we verified that exposure to PDBu, LPA, and angiotensin induced striking PKD activation using syntide-2 phosphorylation assays (results not shown). Treatment of IEC-18 cells with PTx (100 ng/ml for 3 h) attenuated PKD autophosphorylation at S916 induced by LPA but did not interfere with the stimulatory effect of either PDBu or angiotensin (Fig. 11D). These results with IEC-18 cells substantiate the notion that PKD activation is a novel early molecular event in the response of epithelial cells to activation of Gi- and Gq-coupled receptors.


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Fig. 11.   PKD activation in IEC-18 cells by PDBu, LPA, and ANG II. A: confluent cultures of IEC-18 cells were treated with various concentrations of PDBu for 10 min as indicated (left); confluent and quiescent cultures of IEC-18 cells were treated for various times with 100 nM PDBu as indicated (right). B: confluent cultures of IEC-18 cells were treated with various concentrations of LPA for 5 min as indicated (left); confluent cultures of IEC-18 cells were treated for various times with 5 µM LPA as indicated (right). C: confluent cultures of IEC-18 cells were treated with various concentrations of ANG II for 90s as indicated (left); confluent cultures of IEC-18 cells were treated for various times with 100 nM ANG II as indicated (right). D: confluent IEC-18 cells were washed with DMEM and incubated with 100 ng/ml PTx or an equivalent amount of solvent for 3 h. Cells were then stimulated with 100 nM ANG II for 90 s, 5 µM LPA for 5 min, or 100 nM PDBu for 10 min. In all cases, Western blot analysis with pS916 antiserum was performed following lysis of cells with 2× sample buffer, as described in MATERIALS AND METHODS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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 alpha , delta , and epsilon . 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 beta gamma -subunits, rather than alpha -subunits, of heterotrimeric Gi proteins preferentially activate the beta 2 and beta 3 isoforms of PLC (13, 51). Unlike PLC-beta 2, which is expressed only in certain cells of hematopoietic origin, PLC-beta 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 Galpha q activation is sufficient to stimulate sustained PKD activation via PKC and show that the endogenous Galpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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ABSTRACT
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RESULTS
DISCUSSION
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