Gastrin's trophic effect in the colon: Identification of a signaling pathway mediated by protein kinase C

Peptides,Vol. 14, pp. 1119-1124, 1993 Printedin the USA.

0196-9781/93$6.00 + .00 Copyright© 1993PergamonPressLtd.

Gastrin's Trophic Effect in the Colon: Identification of a Signaling Pathway Mediated by Protein Kinase C R I H A B R. YASSIN, l H A R R I S R. C L E A R F I E L D A N D K E V I N M. L I T T L E

Division o f Gastroenterology, H a h n e m a n n University, Philadelphia, PA 19102-1192 Received 28 April 1993 YASSIN, R. R., H. R. CLEARFIELD AND K. M. LITTLE. Gastrin's trophic effect in the colon: Identification of a signaling pathway mediated by protein kinase C. PEPTIDES 14(6) 1119-1124, 1993.--In previous studies we have reported that gastrin exerts atrophic effect on rat colonic epithelial cells in vitro. The effect of gastrin appeared to be mediated through a protein kinase C mechanism. In this study, we have characterized the role of protein kinase C in the gastrin-inducedstimulation. Gastrin, in a time- and dose-dependent manner, increased protein kinase C translocation from the cytosol to the membrane, an index of enzyme activation. Maximum translocation occurred in 1 to 2 min followingexposure to gastrin (10-s M), before declining back to baseline level within 5 min. Gastrin did not change total protein kinase C activity in the colonic cells. Staurosporine, an inhibitor of protein kinase C, totally abolished the basal as well as the gastrin-stimulatedactivity of protein kinase C. The tumor promoter phorbol 12-myristate 13-acetate also stimulated colonic epithelial protein kinase C. However, prolonged treatment of cells with phorbol inhibited their subsequent response to gastrin stimulation. The response to gastrin was also prevented by the gastrin receptor antagonist proglumide. These observationssuggestthat protein kinase C mediates the stimulatory effect ofgastrin on colonic epithelial cells, possiblythrough a receptor mechanism. Gastrin

Protein kinase C

Signaltransduction

Phorbolesters

THE hormone gastrin has been shown to exert a growth-promoting effect in the digestive tract and the pancreas (10,11,30). Chronic administration of pharmacological doses of gastrin to laboratory animals induces gastric mucosal hyperplasia (20). In short-term experiments, single or multiple injections of gastrin or pentagastrin stimulate protein, RNA, and DNA synthesis in the oxyntic mucosa of the stomach, duodenum, and ileum (l 012,30). A decrease in circulating gastrin due to antrectomy results in atrophy of the gastric and duodenal mucosa (9). Gastrin has also been shown to stimulate the growth responses of epithelial cultures derived from gastric and intestinal mucosa (5,25) and to enhance cancer growth in experimental animals as well as in culture (15,16,21). Intracellular gastrin has been detected in tumor cells, and human gastric and coiorectal cancer cells produce a gastrin-like substance that can react with antigastrin antibodies (19,26). The trophic effect of gastrin is independent of its secretagogue action, but dependent on gastrin binding to specific cell surface receptors (28). Gastrin receptors have been located on gastric and colonic mucosal membranes of the rat and on normal human colonic mucosa and adenocarcinoma of the colon (23,28). Though an autocrine regulatory mechanism can be ascribed to gastrin in gastric and coiorectal cancer (19), its physiological influence in

Trophic hormones

the colon and the mechanisms by which it activates colonic cells are still controversial. In previous investigations, we have reported that gastrin stimulates protein, RNA, and DNA synthesis in rat colonic epithelial cells in vitro (31). The stimulatory effect of gastrin appeared to be mediated through protein kinase C activation (32). In our effort to elucidate the molecular events in signal transduction mechanisms regulating colonic epithelial cell proliferation, we characterized the role of protein kinase C in the gastrininduced stimulation. The experiments described in this study support a significant role for protein kinase C in transducing gastrin signals in the colon. METHOD

Materials Gastrin (G2-17) was purchased from Research Plus (Bayonne, N J), [32p]ATP (3000 Ci/mmol, 2 mCi/ml) and protein kinase C assay kit from Amersham (Arlington Heights, IL), essential and nonessential amino acids from Gibco Laboratories (Grand Island, NY), 2-mercaptoethanol from Biorad Laboratories (Richmond, CA), NP-40 from Hoefer Scientific (San Francisco, CA), and all other chemicals and supplies from Sigma

t Requests for reprints should be addressed to Rihab R. Yassin, Ph.D., Division of Gastroenterology, Mail Stop 131, Hahnemann University, Broad and Vine, Philadelphia, PA 19102-1192.

1119

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FIG. 1. DEAE-cellulosepurification profile of soluble protein kinase C activity from colonic epithelial cells. Protein kinase C was extracted as described in the Method section, was applied to DEAE-cellulose columns, and then eluted sequentially with the indicated concentrations of NaCl in homogenizing buffer. The data are from two representative purifications.

Chemical Company (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA).

Isolation of Colonic Epithelial Cells Colonic epithelial cells were isolated as described previously by us (31). Briefly, fasted male Sprague-Dawley rats (approximately 200 g) were terminated with CO2 asphyxiation, and their colons from the colocecal junction to the anal verge were surgically excised. Colons were involuted and cut into several concentric strips. The strips were incubated with 50-75 U/ml of collagenase (type II, Worthington Biochemical Corp., Freehold, NJ) in a miUipore-flltered buffer containing (mM): NaC1 122, KC14.8, NaHCO3 25.2, KH2PO4 1.2, MgSO4.7H20 1.2, CaC12 1.3, glucose 11, glutamine 1.0, pH 7.2. The medium also ineluded 2% essential and 1% nonessential amino acids, and 0. !% bovine serum albumin. Incubation with collagenase was carried out for I h in a 37°C rotary shaker bath that was aerated with 95% O2 and 5% CO2. Following enzymatic digestion, cells were loosened from the colonic strips by shaking the strips in the eollagenase medium with forceps. The cell suspension was strained through a 65-t~M nylon mesh (Tetko, Elmsford, NY). Epithelial cells were pelleted by centrifugation at 75 X g for 5 min. The cells were washed three times, pelleted by differential centrifugation, and finally resuspended in fresh buffer and checked for viability and morphology as described previously (31). The cell suspension was balanced for 10-30 min in an oxygen-enriched shaker bath prior to any experimentation.

Extraction of Protein Kinase C From Colonic Cells Colonic epithelial cells were incubated with or without stimulating agents for the desired time intervals, and the reaction was terminated by the rapid addition of an ice-cold homogenizing buffer. The buffer contained (raM): Tris-HC120, pH 7.5, EGTA 0.5, EDTA 2, phenylmethylsulfonylfluoride 2, benzamidine 0.5, 2-mercaptoethanol 5, and leupeptin 10 mg/1 (29). The preparations were centrifuged at 200 X g for 5 min at 4°C. The su-

pernatants were discarded and the pellets were resuspended in chilled homogenizing buffer and were sonicated on ice for 5-10 s. The sonicates were ultracentrifuged at 100,000 × g for 60 min at 4°C. The resulting supernatants (cytosolic fraction) were removed and kept on ice. The pellets were resuspended in 300500 tA of chilled homogenizing buffer containing 0.1% NP-40, and were shaken for 1 to 2 h at 4°C. The suspensions were centrifuged at 13,000 X g for 20 min at 4°C, and resulting supernatant represented the membrane fractions (8). The cytosolic or membrane fractions were used directly or were further purified on DEAE-cellulose columns (Polyprep columns, 0.8 X 4 cm, Biorad Laboratories, Richmond, CA) that were prewashed with homogenizing buffer. In the preliminary studies, the columns were eluted with an increasing concentration of NaCI in homogenizing buffer ranging from 0.1 to 400 mM. As most of protein kinase activity was eluted with higher NaCI concentration, the loaded columns were washed with 5 vol. of buffer, followed by 10 vol. of buffer containing 30 m M NaC1, then 90 m M NaC1. Finally, protein kinase C was eluted in 1 ml of buffer containing 300 m M NaC1. Figure 1 illustrates the DEAE-purification profile of protein kinase C.

Assay of Protein Kinase C Protein kinase C was assayed, as described previously (32), by measuring the incorporation of 32p from [3,32P]ATP into a specific peptide substrate (Amersham). Briefly, the reaction mixture consisted of(75 #1 final volume): 40 m M Tris-HCl, pH 7.5, 1 m M calcium acetate, 2.5 m M dithiothreitol, 15 m M magnesium acetate, 50 # M [~,32p]ATP, 0.7 mol % L-phosphatidylL-serine plus 2 ug/ml phorbol 12-myristate 13-acetate, 75 ~m peptide substrate, and cytosolic or membrane preparations. The reaction was initiated by the addition of [32P]ATP, and the tubes were incubated for 15 rain at 25°C. The reaction was then terminated, and aliquots from each sample were spotted on specific binding papers. The papers were allowed to soak, and then were washed twice individually and processed for counting as described previously (32). Protein content of each sample was measured with the Bradford method, using bovine serum al-

GASTRIN AND PROTEIN KINASE C STIMULATION

1121

bumin as a standard (1). The assays were constantly assessed for linear range of enzyme activity with regard to protein concentration and all incubation conditions. Assays were performed in duplicate and the results were expressed as picomoles of 32p incorporated in the peptide substrate/minutes/~tg protein.

Effect of Staurosporine on Protein Kinase C Activity We examined the effect of the potent protein kinase C inhibitor staurosporine on protein kinase C activity. The microbial alkaloid staurosporine strongly inhibited the basal as well as the gastrin-stimulated activity of protein kinase C (Fig. 3). A higher inhibition was seen with larger concentrations of staurosporine.

Statistical Analysis A one-way analysis of variance (ANOVA) was used to test for statistical significance between paired or unpaired data and a Scheffe test was used for multiple comparisons. All values are mean + SEM and p values less than 0.05 were considered statistically significant. RESULTS

Effect of Gastrin on Protein Kinase C Distribution Under unstimulated conditions, 72% of colonic epithelial protein kinase C activity was associated with the cell cytosol and only 28% was associated with the cell membrane. On the other hand, when the cells were stimulated with gastrin (10 -s Air), a rapid activation of protein kinase C was induced, as measured by its translocation from the cytosol to the membrane (Fig. 2). Maximum activation was observed in the 1 min following exposure to gastrin, at which time 66% of protein kinase C activity was associated with the cell membrane. The activation was transient, and the membrane-associated activity declined to baseline value within 5 min, despite the continuous presence of gastrin. Gastrin did not change total protein kinase C activity (cytosol + membrane) in the colonic cells. The concentration of gastrin used in these studies was the one we reported to be most effective in inducing a growth-promoting response (31,32). Concentrations higher or lower than 10-s M were less effective in mobilizing protein kinase C (data not shown).

Responsiveness of Colonic Epithelium to Phorbol Esters The tumor promoter phorbol 12-myristate 13-acetate (PMA) is a strong activator of protein kinase C (3). We tested the effect of PMA on protein kinase C translocation in the colonic cells. Phorbol 12-myristate 13-acetate (1.0 ~tg/ml) induced a rapid and strong mobilization of protein kinase C from the cytosol to the membrane and also increased total protein kinase C activity (cytosol + membrane) (Fig. 4). However, this initial activation was followed by a time-dependent decrease in both cytosolic and membrane-associated activities. The effect of prolonged exposure of colonic cells to PMA (down-regulation) on protein kinase C activation elicited by subsequent stimulation with gastrin is also illustrated in Fig. 4; PMA inhibited the stimulated increase in membrane-associated protein kinase C activity observed with 10-8 M gastrin.

Effect of Proglumide on Protein Kinase C Activation To investigate whether protein kinase C activation is part of a receptor-mediated pathway, colonic cells were incubated with the gastrin receptor antagonist proglumide. Table 1 shows that proglumide (10 mM) totally abolished the increase in protein kinase C translocation to the membrane induced by 10-8 M gastrin. DISCUSSION In previous studies, we have shown that gastrin exerts a growth-promoting effect on colonic epithelial cells in vitro

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FIG. 2. Time course of gastrin-stimulated increase in membrane-associated protein kinase C activity. Colonic epithelial cells were incubated with gastrin (10-s M) for the indicated time intervals and the reaction was terminated by the addition of chilled homogenizing buffer. Protein kinase C was extracted and assayed as described in the Method section. The data are expressed as percentage of total protein kinase C activity. Each value represents the mean _+SEM. *p < 0.01, **p < 0.05 vs. control.

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Condition FIG. 3. Effect of staurosporine on protein kinase C activity. Colonic epithelial cells were incubated with staurosporine (20 or 40 ng/ml) or with staurosporine + gastrin (10 8 M) under optimum conditions. Protein kinase C was extracted and assayed as described in the Method section. Each value represents the mean _+SEM. For cytosolic activity: F(5, 31 ) = 22.14, **p < 0.01 vs. control, for 3-13 determinations. For membrane-associated activity: F(5, 32) = 8.49, *p < 0.05, **p < 0.01 vs. gastrin, for 3-16 determinations.

(31,32). The experiments described in this study define a signaling pathway of gastrin, mediated by protein kinase C activation. Incubation of colonic cells with gastrin induced a rapid activation of protein kinase C, as measured by its translocation from the

2.00

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Time (rain) FIG. 4. Time-dependent effect of PMA on protein kinase C activity. Colonic epithelial cells were treated with 1.0 ~tg/ml PMA for the indicated time lengths, and protein kinase C was extracted and assayed as described in the Method section. For the PMA effect on gastrin, cells were stimulated with l0 -a M gastrin (no longer than 2 min) following a 30-min incubation or longer (not shown) with PMA. Each value represents mean _+ SEM. For cytosolic activity: F(4, 10) = 47.27, *p < 0.01 vs. control, for 3 determinations. For membrane-associated activity: F(5, 1 l) = 19.69, *p < 0.01 vs. control, for 2-4 determinations.

GASTRIN AND PROTEIN KINASE C STIMULATION TABLE 1 EFFECT OF PROGLUMIDE ON PROTEIN KINASEC ACTIVATION Protein KinaseC (pmol/min/ugprotein) Condition

Cytosol

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Control Gastrin (10-s M) Proglumide (10 mM) + gastrin (10 aM)

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Values are mean + SEM. Colonic epithelial cells were stimulated with gastrin ( 10-s M) for 1 min and the reaction was terminated by the rapid addition of chilled homogenizing buffer. Protein kinase C was extracted and assayed as described in the Method section. Proglumide (10 mM) was added together with gastrin when indicated. * p < 0.01 vs. control, n = 2-16.

the susceptibility of the activated form of protein kinase C to modifications by proteolysis, thus generating a form with altered requirements for its physiological activators, Ca 2+, phosphatidylserine, and diacylglycerol (29). The concentration response for protein kinase C activation was equivalent to the concentration required to stimulate protein and DNA synthesis (31). Staurosporine, a compound with great potency for inhibiting protein kinase C (18,27), inhibited both the basal and the gastrinstimulated activities of protein kinase C. This finding is consistent with our previous observation that deactivation of protein kinase C suppresses the increase in epithelial protein synthesis elicited by gastrin (32). Our data also showed that phorbol 12-myristate 13-acetate rapidly and strongly activated protein kinase C and increased its induction. The tumor promotor phorbol esters and related compounds are strong activators of protein kinase C (3,13). However, in many cells, prolonged or repeated treatment with phorbol esters results in a complex time-dependent deactivation of protein kinase C and depletion of the enzyme mass (7,14). In our system, prolonged treatment with PMA inhibited the colonic cells' response to subsequent stimulation with gastrin. Based on the previously available knowledge, this timedependent inhibitory effect of PMA can be explained by two mechanisms: a) PMA triggers a negative feedback autoregulatory control mechanism, and b) PMA induces the downregulation of protein kinase C activity (2,7,14,17). With regard to the autoregulatory mechanism, it has been suggested that activation of protein kinase C may induce receptor uncoupling, dissociating receptor occupancy from the subsequent generation of second messengers (2). Alternatively, activation of protein

1123 kinase C may lead to modification in the binding kinetics of receptors coupled to the phospholipase C pathway (4,17). It has been shown that the phorbol ester 12-O-tetradecanoylphorbol13-acetate (TPA) and the synthetic diacylglycerol 1-oleoyl-2acetyl-sn-glycerol (OAG) decrease the number of muscarinic and gastrin receptors on gastric parietal cells, without altering receptor binding affinity (4). With regard to the concept of downregulation of protein kinase C activity by PMA, it has been shown that phorbol esters induce a persistent activation of protein kinase C, increasing enzyme translocation from the cytosol to the membrane, and leading to subsequent degradation of the membrane-associated activity (7,13). The catalytically active fragment of protein kinase C cannot always be recovered from the TPA-treated cells, possibly due to degradation by proteolysis (13). Therefore, and in agreement with many observations (2,4,17), it appears that protein kinase C plays a dual stimulatory and inhibitory role in controlling colonic cell activation. In this study, we have also demonstrated that proglumide, a gastrin receptor antagonist (12,22), prevented the increase in protein kinase C activation induced by gastrin. Furthermore, rechallenging colonic cells with the optimum dose of gastrin following their initial stimulation produced no effect on protein kinase C distribution (data not shown). These results indicate that gastrin stimulates protein kinase C through a specific receptor-mediated pathway. Gastrin has been shown to act as a cellular growth factor inside and outside the gastrointestinal system, but evidence of its trophic effect has been largely controversial (9-12,22). Our studies have indicated that gastrin promotes colonic epithelial protein, RNA, and DNA synthesis. This growth-promoting influence cannot be observed under all conditions, indicative of the involvement of other factors (22). Our studies have also demonstrated that gastrin's effect is mediated through the protein kinase C mechanism. The presence of protein kinase C in colonic cells and evidence of its involvement in the regulation of colonic cell proliferation induced by colon growth and tumor promoters have been recently reported (6,7,29). Therefore, it also appears that gastrin operates through such a signal transduction pathway. This, however, does not rule out the involvement of additional operating mechanisms. Although the exact course of the stimulated increase in protein kinase C activity cannot be fully deduced from the present data, our observations point strongly toward a receptor-activated pathway. ACKNOWLEDGEMENTS This work was supported by grant DK44190-02 from the National Institutes of Health. We wish to thank Mrs. Jeanne Myers for typing the manuscript.

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