Gen. Pharmac. Vol. 23, No. 4, pp. 715-718, 1992 Printed in Great Britain. All rights reserved

0306-3623/92 $5.00 + 0.00 Copyright .~ 1992Pergamon Press Ltd

ENHANCEMENT OF GASTRIC MUCOSAL EPIDERMAL GROWTH FACTOR A N D PLATELET-DERIVED GROWTH FACTOR RECEPTOR EXPRESSION BY SUCRALFATE B. L. SLOMIANY,J. LIU, J. P. KEOGH, J. PIOTROWSKI,and A. SLOMIANY Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, University Heights, 110 Bergen Street, Newark, NJ 07103-2400, U.S.A. [Tel. (201) 456-7052] (Received 29 October 1991)

Almtract--l. The effect of an anti-ulcer agent, sucralfate, on the expression of gastric mucosal epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors was investigated. 2. Gastric mucosal cell membranes, isolated from the stomach of groups of rats, one receiving twice daily for 3 consecutive days a dose of 100 mg/kg sucralfate, and the other only the vehicle, were used as source for EGF and PDGF receptors. 3. Binding assays revealed the presence of both types of receptors, activation of which led to the elevation of tyrosine kinase activity as evidenced by a marked increase in membrane protein tyrosine phosphorylation patterns. 4. The specific receptor binding in the control group was 2.46 fmol/mg protein for EGF and 1.46 fmol/mg protein for PDGF, whereas the respective binding values in the sucralfate treated group increased by 61 and 65%. 5. The results suggest that sucralfate is capable of enhancement of epithelial proliferative activities through the stimulation of gastric mucosal EGF and PDGF receptors.

MATERIALS AND METHODS

INTRODUCTION The maintenance of gastric mucosal protection under the adverse environment of luminal contents depends upon a delicate balance of a n u m b e r of factors which control the processes of mucosal repair and restitution. Primary among the factors which are of considerable interest are epidermal growth factor (EGF), and platelet-derived growth factor ( P D G F ) (Carpenter, 1987; Konturek, 1988; Decker and Harris, 1989). E G F and P D G F are potent mitogens widely recognized for their effect on a variety of functions associated with cellular proliferation (Carpenter, 1987). The biological effects of E G F and P D G F are mediated by receptors located on the target cell surfaces. In both cases, the ligands binding to the receptors result in activation of the intrinsic receptor tyrosine kinase activity and phosphorylation of the receptors on tyrosine residues (Carpenter, 1987; Kelly et al., 1991). This autophosphorylation is a central event towards mediating the proliferative effects of E G F , as well as P D G F . Hence, the agents capable of affecting the expression of receptor activity could influence the extent of cellular proliferation. A m o n g the agents in this category is an antiulcer drug, sucralfate, the effects of which in mediating the growth factors action in the stomach has been attributed to its ability to prolong the factors luminal availability (Nexo and Poulsen, 1987; Szabo et al., 1991). In the present study, using gastric mucosal cell membranes from animals subjected to prolonged sucralfate administration, we show that the drug enhances the epithelial proliferative activities through increase in E G F and P D G F receptors expression.

Materials

Mouse [t25I]EGF (specific activity 100/aCi//~g) and [I~I]PDGF (specific activity 23/aCi//~g) were purchased from Amersham Corp. (Arlington Heights, III.), and mouse EGF and human PDGF from Sigma Chemical Co. (St Louis, Mo.). Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophores (SDS-PAGE) were from Bio-Rad (Rockville Center, N.Y.), BCA protein assay kit from Pierce (Rockford, II1.), and 5-bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium from Oncogene Science Inc. (Manhasset, N.Y.). Goat anti-mouse alkaline phosphatase conjugated IgG and O-phospho-l-tyrosine were obtained from Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Anti-phosphotyrosine monoclonal lgG was from Upstate Biotechnology Inc. (Lake Placid, N.Y.). Rats were supplied by Taconic Farms Inc. (Germantown, N.Y.). All other chemicals were of reagent grade. Animal treatment The study /n vivo was conducted with two groups (each

consisting of 12 animals) of male Sprague--Dawley rats weighing 180-200 g, and maintained on a regular chow diet. Each animal in the first group was given intragastrically, twice daily for 3 consecutive days, a dose of I00 mg/kg body wt sucralfate in 1 ml of 5% gum arabic in saline, whereas animals in the control group were exposed to daily doses of vehicle consisting of 5% gum arabic in saline. Following the last dose, rats were fasted for 16hr in individual wirebottomed cages and then killed by decapitation before their stomachs were removed. Gastric membrane preparation

The dissected stomachs were opened along the greater curvature, rinsed with ice-cold saline in 0.05 M phosphate buffer pH 7.2, and the mucosal cells were collected by scraping the mucosa with a blunt spatula on an ice-cold 715

716

B.L. SLOMIANY et al.

glass plate (Slomiany et al., 1990b). Scrapings were placed in ice-cold buffer (2.5mM Tris-HCl, pH 7.0, 250mM sucrose, 2.5mM EDTA, l mM phenyl methyl sulfonyl fluoride (PMSF)) and homogenized for I min in a polytron tissuemizer. The homogenates were centrifuged at 400g for 15 min at 4¢C and NaCI/MgSO4 was added to the supernatants to form final concentrations of 0.I M and 0.2 mM, respectively. After centrifugation of the supernatants at 40,000g for I hr at 4°C, the pellets were resuspended in 0. I M sodium phosphate buffer (pH 7.2) and aliquots of such prepared gastric membrane samples were stored at - 7 0 C until use. Protein concentration of the resuspended pellets were estimated using the BCA protein assay kit. Receptor binding assay For EGF binding assays, membrane samples (300pg protein/assay) were incubated in triplicate with [t2~I]EGF (0.18nM, 44,000cpm) and 5raM Tris-HC1 (pH7.0), 125 mM sucrose, 75 mM NaCI, 0.5 mM CaCI 2, 0.5% bovine serum albumin (BSA) in a final volume of 200 #1. Unlabeled EGF was added to the incubations to form a final concentration of 0.25~M in the experiments where nonspecific binding was being estimated. Incubates were maintained for I hr at room temperature, and then stopped by addition of 1 mM ice-cold buffer containing 10 mM Tris-HC1 (pH 7.0) and 0.5% BSA. Membrane-bound [I:~I]EGF was separated from the unbound [~:Sl]EGF by centrifugation at 10,000g for 10min at 4~C. The pellet was washed with l ml of ice-cold buffer, centrifuged, and counted in a gamma counter. For PDGF binding assays, the conditions were identical except that [~ZSl]PDGF (0.016 nM, 14,000 cpm) was used in place of [t:SI]EGF, and PDGF (0.66 riM) was used in place of EGF. The incubation buffer was Ham's F-12 medium with 25raM HEPES (pH 7.0) and 0.25% BSA, and the stopping buffer was ice-cold 0.1 M phosphate (pH 7.4) with 0. 1% BSA. Specific binding for both PDGF and EGF was determined by subtracting nonspecific binding, measured in experiments incorporating excessive amounts of unlabeled growth factor, from total binding which was measured in experiments where no unlabeled growth factor was added. Cell membrane protein phosphoo'rosine Aliquots of gastric membranes were centrifuged for 20 min at 10,000g and 4"C, the pellet was solubilized in 2 ml of a buffer containing 0.1% Triton X-100, 10% glycerol, 1 mM PMSF and 1.25 mM EDTA. The mixture was mixed constantly at 4~C for 1 hr, after which it was centrifuged at 200,000g for 90min at 4~C. To a volume of mixture containing 300 t~g of solubilized protein, dithiothreitol and CaC12 were added to form 20 mM final concentration. The solution was incubated with or without EGF (I ~M) or PDGF (1.4 nM), or with O-phospho-l-tyrosine (0.I raM) for l hr. After incubation, 10~M ATP was added for 15 rain. The reaction was stopped by adding a buffer containing 170mM Tris-HCl, 10% SDS, 100mM dithiothreitol and heating to 100°C for 8 rain. Immunoblotting was performed according to the manufacturer's instructions (Upstate Biotechnology Inc.). Briefly, samples were run on Table 1. Effect of sucralfate on EGF binding to gastric mucosal cell membranes

Type of Total Specific Bound [*2~I]EGFbound samples (cpm) (cpm) (%) (fmol/mg protein) Control 1073 + 50 466 __ 10 1.06 2.46 __0.10 Sucralfate 1516_+202 749 ± 120 1.70 3.96 ± 0.79* Gastric mucosal cell membranes were prepared from the stomachs of groups of rats, one receivingtwice daily for 3 consecutive days a dose of sucralfate, and the other receiving only the vehicle. Each value represents the means _+SD of 5 experiments performed in triplicate. *P < 0.05.

7.5% SDS-PAGE after which proteins were electrophoretically transferred onto 0.2pM nitrocellulose membranes. 10% BSA was used to block the membranes, mouse antiphosphotyrosine monoclonal IgG (I0 pg/ml, 1 hr) as detecting antibody, and goat anti-mouse alkaline phosphatase conjugated IgG (0. I/a g/ml, 120 min) as secondary antibody. Visualization was achieved using 5-bromo-4-chloro-3-indole phosphate and nitro blue tetrazolium. Sucralfate The antiulcer drug, sucralfate, lot No. 31498, was donated by the Marion Laboratories (Kansas City, Mo.). The drug was stored at 4'C in the dark and was suspended in 5% gum arabic saline shortly before each administration. The drug or vehicle was given orally in a volume of I ml through a dull metal tubing attached to a 2 ml syringe. Statistical analysis All experiments were carried out in triplicate, and the results are expressed as means + SD. Student's t-test was used to determine significance, and P values of 0.05 or less were considered significant. RESULTS The results of binding assays c o n d u c t e d with mucosal cell m e m b r a n e p r e p a r a t i o n s using [125I]EGF a n d [J:sI]PDGF revealed the presence of b o t h types o f receptors in gastric mucosa. The E G F binding experiments gave a m e a n specific binding value o f 2 . 4 6 f m o l / m g m e m b r a n e protein for the control g r o u p and a value o f 3.96 fmol/mg m e m b r a n e protein for the sucralfate-treated g r o u p (Table 1). This represents a 6 1 % increase in E G F binding with sucralfate. Table 2 shows the data on the mucosal receptor binding for P D G F . The specific binding values for P D G F in the control g r o u p averaged !.46 f m o l / m g m e m b r a n e protein, and increased by 6 5 % in the m e m b r a n e p r e p a r a t i o n s from animals treated with sucralfate (Table 2). With b o t h types o f receptors, an increase in nonspecific binding with sucralfate was also observed. In the case o f E G F receptor, this value increased by 2 6 % (Table 1) a n d by 84% in the case of P D G F receptor (Table 2), thus indicating the ability of sucralfate to p r o m o t e the interaction of E G F a n d P D G F with b o t h specific non-specific binding sites in gastric mucosa. Figure 1 illustrates the effect o f E G F a n d P D G F o n gastric mucosal cell m e m b r a n e protein tyrosine phosphorylation. E x a m i n a t i o n o f the protein tyrosine p h o s p h o r y l a t i o n p a t t e r n s using a n t i p h o s p h o t y r o s i n e a n t i b o d y revealed that b o t h E G F a n d P D G F caused a m a r k e d increase in proteins p h o s p h o r y l a t i o n . Furthermore, the p h o s p h o p r o t e i n profile o b t a i n e d with P D G F were quite similar to t h a t o b t a i n e d with E G F , Table 2. Effect of sucralfate on PDGF binding to gastric mucosal cell membranes Type of Total Specific Bound [~:~I]PDGFbound samples (cpm) (cpm) (%) (fmol/mg protein) Control 3188 _+355 389 + 106 3.18 1.46 _+0.38 Sucralfate 5806+ 849 635 4- 144 5.19 2.40_+.0.53* Gastric mucosal cell membranes were prepared from the stomachs of groups of rats, one receiving twice daily for 3 consecutive days a dose of sucralfate, and the other receiving only the vehicle. Each value represents the means _+SD of 5 experiments per-

formed in triplicate. *P < 0.05.

Sucralfate and gastric mucosal EGF and PDGF receptors and contained a large number of proteins with wide range of molecular weights, which suggests that the two growth factors exerts their action through a common mechanism. In both cases, an increase in phosphorylation of the proteins in the region (170-180 kDa) corresponding to those of E G F and P D G F receptors has also occurred. DISCUSSION

Sucralfate, a basic aluminum salt of sucrose octasulfate, is a potent antiulcer agent, increasingly popular as the drug of choice in peptic ulcer therapy (Hunt, 1991; Asaka et al., 1991). Over the years, numerous studies demonstrated that the beneficial effect of sucralfate in ulcer healing occurs through the stimulation of endogenous prostaglandin generation, binding to the ulcer crater protein, inhibition of peptic erosion of mucus gel, and stimulation of mucus glycoprotein synthesis and secretion (Nagashima and

180 kDa-

116 k D a -

86 kDa~,~.~ . . ~ , ~

58 kDa-

4 8 kD,o,:

••

.. ,/

36, k D o -

..,.r

C I

EGF 2

PDGF 3

OPT 4

Fig. I. Effect of EGF and PDGF on gastric mucosal cell membrane proteins tyrosine phosphorylation. Membrane preparations were treated for l hr as follows: lane I, no growth factors (C); lane 2, I pM EGF; lane 3, 1.4nM PDGF; lane 4, 0.1 mM O-phospho-l-tyrosine (OPT). ATP substrate (10 ,aM) was then added and the reaction stopped after 15 min. Following membrane solubilization and SDSPAGE, the proteins were transferred onto nitrocellulose membranes and probed with mouse anti-phosphotyrosine monoclonal lgG. Visualization was achieved using alkaline phosphatase conjugated goat anti-mouse IgG and 5-bromo4-chloro-3-indole phosphate/nitro blue tetrazolium. Molecular weights correspond to the positions at which molecular weight markers separated when run parallel with the membrane samples. G P 23/~-J

717

Yoshida, 1979; Samloff, 1983; Hollander et al., 1984; Slomiany et al., 1985a, b, 1989, 1991; Szabo and Hollander, 1989). Furthermore, the evidence has been obtained that sucralfate evoked increase in mucin secretion is reflected in enhanced gastric mucosal phosphoinositide turnover (Slomiany et al., 1990a, 1991). This indicates that the drug is capable of initiation of the chain of events linking extracellular signals to intracellular responses leading to a cascade of regulatory protein phosphorylation, the process closely associated with cellular proliferative activities. Since sucralfate is also known to enhance efficacy of E G F and other mitogenic peptides in the stomach (Nexo and Paulsen, 1987; Konturek, 1988; Szabo et al., 1991), and since biological effects of these mitogens are mediated by receptors located on the target cell surfaces (Carpenter, 1987), the studies presented herein evaluated the effect of sucralfate administration on the mucosal expression of E G F and P D G F receptors. The results obtained revealed the presence of both types of receptors in gastric mucosa, demonstrated that their activation with E G F and P D G F results in an elevation of tyrosine kinase activity, and showed that the mucosal cell membranes from the animals subjected to prolonged sucralfate administration exhibit a marked increase in E G F and P D G F receptor expression. The specific binding values for gastric mucosal E G F (2.46 fmol/mg membrane protein) and P D G F (1.46 fmol/mg membrane protein) obtained here are in line with those reported by other studies (Konturek, 1988; Decker and Harris, 1989; Slomiany et al., 1990b; Wang et al., 1990). Upon ligand binding, both types of receptors caused an increase in membrane proteins tyrosine phosphorylation, including those in the region corresponding to E G F and P D G F receptors, and yielded similar phosphoprotein profiles, thus supporting the view that P D G F and EGF receptor systems are coordinated in some manner (Decker and Harris, 1989). Indeed, both E G F and P D G F stimulate their receptors dimerization, a prerequisite for ligandinduced activation of tyrosine kinase, and P D G F is known to participate in the regulation of E G F receptor function (Countaway et al., 1989; Li and Schlessinger, 1991). Our demonstration that the gastric mucosal cell membranes from the animals subjected to prolonged sucralfate administration show a significant (61-65%) increase in EGF and P D G F specific receptor binding attests to the effect of sucralfate on the proliferative activity of gastric mucosa. This effect of sucralfate, until now, has been attributed solely to the drug's ability to bind growth factors (e.g. EGF and b F G F ) and prolong their luminal availability (Nexo and Paulsen, 1987; Szabo et al., 1991). In the light of the findings presented here, it is apparent that the remarkable ability of sucralfate to repair, restore and maintain gastric mucosal integrity results, at least in part, from its effect on the mucosal expression of E G F and P D G F receptors. An increased availability of these receptors thus allow for efficient utilization of the mitogens, binding of which initiate a series of events that lead to enhanced cellular proliferation and thus to the restitution of gastric mucosal integrity.

718

B.L. SLOMI^SVet al.

Acknowledgements--This work was supported by Grant

#DK21684-15 from the National Institute of Diabetes and Digestive and Kidney Diseases, and Grant #AA05858-10 from the National Institute of Alcohol Abuse and Alcoholism, NIH, USPHS. REFERENCES Asaka M., Takeda H., Saito M., Murashima Y. and Miyazaki T. (1991) Clinical efficacy of sucralfate in the treatment of gastric ulcer. Am. J. Med. Suppl. 91, 71-73. Carpenter G. (1987) Receptors for epidermal growth factor and other polypeptide mitogens. A. Rez,. Biochem. 56, 881-914. Countaway J. L., Girones N. and David R. J. (1989) Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells. J. biol. Chem. 264, 13,642-- 13,647. Decker S. J. and Harris P. (1989) Effects of platelet-derived growth factor on phosphorylation of the epidermal growth factor receptor in human skin fibroblasts. J. biol. Chem. 264, 9204-9209. Hollander D., Tarnawski A., Gergely M. and Zipser R. D. (1984) Sucralfate protection of gastric mucosa against alcohol-induced necrosis: a prostaglandin mediated process. Scand. J. Gastroent. Suppl. 19, 97-102. Hunt R. H. (1991) The treatment of peptic ulcer disease with sucralfate: a review. Am. J. Med. Suppl. 91, 102-106. Kelly J. D., Haldeman B. A., Grant F. J., Murray M. J., Seifert R. A., Rowen-Pope D. F., Cooper J. A. and Kazlaukas A. (1991) Platelet-derived growth factor (PDGF) stimulates PDGF receptor subunit dimerization and trans-phosphorylation. J. b i o l . Chem. 266, 8987-8992. Konturek S. J. (1988) Role of epidermal growth factor in gastroprotection and ulcer healing. Scand. J. Gastroent. 23, 129-133. Li W. and Schlessinger J. (1991) Platelet-derived growth factor (PDGF)-induced disulfide-linked dimerization of PDGF receptor in living cells. Molec. cell. Biol. 11, 3756-3761. Nagashima R. and Yoshida N. (1979) Sucralfate, a basic aluminum salt of sucrose octasulfate. I. Behav-

iors in gastroduodenal pH. ArznemitteI-Forsch. 29, 1668-1676. Nexo E. and Poulsen S. S. (1987) Does epidermal growth factor play a role in the action of sucralfate? Scand. J. Gastroent. Suppl. 22, 45-49. Samloff M. I. (1983) Inhibition of peptic aggression by sucralfate. The view from the ulcer crater. Scand. J. Gastroent. Suppl. 18, 7-11. Slomiany B. L., Laszewicz W. and Slomiany A. (1985a) In t'itro inhibition of peptic degradation of porcine gastric mucus glycoprotein by sucralfate. Scand. J. Gastroent. 20, 857-860. Slomiany B. L., Piotrowski J., Tamura S. and Slomiany A. (1991) Enhancement of the protective qualities of gastric mucus by sucralfate: role of phosphoinositides. Am. J. Med. Suppl. 91, 30-36. Slomiany B. L., Laszewicz W., Murty V. L. N., Kosmala M. and Slomiany A. (1985b) Effect of sucralfate on the viscosity and retardation of hydrogen ion diffusion by gastric mucus glycoprotein. Comp. Biochem. Physiol. ~C,, 311-314. Slomiany A., Mizuta K., Piotrowski J., Nishikawa H. and Slomiany B. L. (1990a) Gastric mucosal protection by sucralfate involves phosphoinositides participation. Int. J. Biochem. 22, 1179-t 183. Slomiany B. L., Piotrowski J., Okazaki K., Grzelinska E. and Slomiany A. (I 989) Nature of the enhancement of the protective qualities of gastric mucus by sucralfate. Digestion 44, 222-231. Slomiany B. L., Liu J., Yao P., Wu-Wang C. Y., Keogh J. P., Wang S. L. and Slomiany A. (1990b) Characterization of the epidermal growth factor receptor in the gastric mucosa. Digestion 47, 181-190. Szabo S. and Hollander D. (1989) Pathways of gastrointestinal protection and repair: mechanism of action of sucralfate. Am. J. Med. 86, 23-31. Szabo S., Vattay P., Scarbrough E. and Folkman J. (1991) Role of vascular factors, including angiogenesis, in the mechanisms of action of sucralfate. Am. J. Med. Suppl. 91, 158-160. Wang S. L., Wu-Wang C. Y., Slomiany A. and Slomiany B. L. (1990) Characterization of epidermal growth factor receptor in rat buccal mucosal ceils. Int. J. Biochem. 22, 669~75.