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(The FASEB Journal. 2002;16:1515-1525.)
© 2002 FASEB

Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway

MICHELLE C. BURESI, ANDRÉ G. BURET, MORLEY D. HOLLENBERG and WALLACE K. MacNAUGHTON1

Mucosal Inflammation Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1

1Correspondence: Mucosal Inflammation Research Group, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, T2N 4N1, Canada. E-mail: wmacnaug{at}ucalgary.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase-activated receptor 1 (PAR-1) is activated by thrombin and induces chloride secretion by intestinal epithelial cells. To elucidate further the mechanisms whereby PAR-1 stimulates secretion, monolayers of SCBN intestinal epithelial cells were studied in modified Ussing chambers. Short circuit current responses were determined after basolateral application of thrombin and the PAR-1-activating peptide, Ala-parafluoro-Phe-Arg-cyclohexyl-Ala-Citrulline-Tyr (Cit-NH2) in the presence or absence of a variety of signal transduction and cyclo-oxygenase (COX) pathway inhibitors. Increased kinase activity was monitored by immunoprecipitation and Western blot analysis of target phosphoproteins. The PAR-1-induced chloride secretory response was significantly attenuated by inhibitors of the EGF receptor tyrosine kinase, Src-kinase, MEK1/2, as well as by inhibitors of cytosolic phospholipase (cPL) A2, COX-1 and COX-2. PAR-1-induced activation of cPLA2, as shown by Western blot of phosphoserine residues, was blocked in cells treated with the MEK inhibitor U0126, indicating that the MEK-ERK1/2 MAP kinase pathway mediated PAR-1-induced cPLA2 phosphorylation. Our data show that PAR-1-induced chloride secretion in SCBN cells involves Src, EGF receptor trans-activation, activation of a MAPK pathway, phosphorylation of cPLA2, COX activity, but not PGF2{alpha} or PGE2. These findings may be of clinical importance in inflammatory diseases of the intestine where secretory dysfunction is evident and thrombin levels are elevated.—Buresi, M. C., Buret, A. G., Hollenberg, M. D., MacNaughton, W. K. Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway.


Key Words: epidermal growth factor • PAR-1 • inflammatory bowel disease • PCR


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
PROTEINASE-ACTIVATED RECEPTORS (PARS) are a recently characterized class of G-protein-coupled receptors (GPCRs) that are activated by proteolytic cleavage of their NH2 termini by specific serine proteases (1) . The new NH2 terminus acts as a ‘tethered ligand’ that binds to other extracellular domains of the receptor to initiate G-protein-dependent signaling. PAR-1, the prototypical thrombin receptor, was the first of four PARs that have been cloned (2 , 3) . PAR-1 is found in diverse cell types including epithelial cells, platelets, endothelial cells, fibroblasts, monocytes, T cell lines, osteoblast-like cells, smooth muscle cells, neurons, and glial cells, and in certain tumor cell lines (1 , 4 , 5) . Given the wide tissue distribution of PAR-1, it is likely that this receptor plays an important role in a variety of cellular functions.

The involvement of thrombin in inflammation is well described (6) , and its participation via activation of PAR-1 has been demonstrated in platelet aggregation, vasodilatation and vasoconstriction, increased vascular permeability, and granulocyte chemotaxis (7) . Thrombin levels are increased during inflammatory bowel disease (IBD) (8 , 9) and this proteinase has been implicated in the pathogenesis of the disease (8) . For example, Crohn’s disease patients show various coagulation abnormalities and, indeed, intestinal vascular injury has been proposed as a major pathogenic factor (9) . In addition, ulcerative colitis is associated with a thrombotic tendency (10) . Although thrombin levels have not been directly measured in the intestinal lamina propria, the coagulation system has been shown to be significantly altered in the colonic mucosa of patients with IBD (11) . Given these findings and the increased microvascular permeability characteristic of inflamed tissues, it is likely that active thrombin would be in a position to affect epithelial function in the inflamed gut.

The intestinal epithelium represents the first line of defense against luminal bacteria, bacterial products, and food antigens. This role is carried out in part through the ability of crypt enterocytes to secrete chloride, which promotes the movement of water in a basolateral-to-apical direction. This serves to flush away potentially harmful luminal contents and thus slows or prevents the translocation of bacteria and antigens into the lamina propria of the intestine (12) . Hyperresponsiveness to secretagogues can lead to excess water and electrolyte loss. Thus, responsiveness of the epithelium to secretagogues must be tightly regulated. The loss of regulation of secretory function through either increased or decreased responsiveness to secretagogues or through the exposure of enterocytes to mediators that normally might not have contact with the epithelium may contribute to the etiology of, or symptoms associated with, inflammatory or other pathological conditions of the gut.

We have recently shown that activation of PAR-1 induces calcium-dependent chloride secretion in a nontransformed intestinal epithelial cell line (4) . However, the cellular mechanism whereby this response occurs is unknown. PAR-1 couples to several different G-proteins, resulting in activation of phospholipase C, phosphoinositide hydrolysis, and formation of inositol trisphosphate (IP3) and diacylglycerol (DAG), leading to Ca2+ mobilization and activation of protein kinase C (13) . It has been suggested that PAR-1 activation may also lead to the stimulation of mitogen-activated protein (MAP) kinase pathways, although the signaling mechanisms by which GPCRs activate these pathways are complex and not fully understood. The trans-activation by GPCRs of receptor tyrosine kinases such as the EGF receptor has been demonstrated in different cell systems (14) , including ion-transporting intestinal epithelial cells (15) . G-protein signaling can interact with a receptor tyrosine kinase pathway at several levels. It has been suggested that the ß{gamma} subunits of Gi protein can activate nonreceptor tyrosine kinases, such as those of the Src family (16) . In turn, Src kinases can phosphorylate the EGF receptor. Alternately, a GPCR-induced increase in calcium can lead to the activation of Pyk-2 and subsequently of Src family kinases (17) . In addition to upstream tyrosine kinases, reactive oxygen species and metalloproteinases (MMPs) have been implicated in the mechanisms of EGFr trans-activation by GPCRs in some systems (14 , 17 18 19) . MMP-dependent GPCR-induced EGFr trans-activation is thought to involve the MMP-induced shedding of the heparin binding EGF-like growth factor, transforming growth factor (TGF) {alpha} (18) . The released ligand could subsequently bind to and activate the EGFr leading to stimulation of MAP kinase pathways (18) .

PAR-1 may also stimulate cPLA2 to release arachidonic acid and, through cyclo-oxygenase activity, increase prostaglandin (PG) synthesis (20 , 21) . Prostaglandins, particularly those of the E series, are potent secretagogues and act as ‘final common mediators’ of the secretory effects of other compounds (22 , 23) . We have shown that PAR-1 activation stimulates PGE2 synthesis in the CCD-18Co colonic myofibroblast cell line (24) .

Given these observations, we sought to determine the signaling pathways responsible for PAR-1-induced chloride secretion in an intestinal epithelial cell line. We have focused our attention on EGFr-stimulated MAP kinase pathways and have also investigated the ability of activated PAR-1 to stimulate chloride secretion through cyclo-oxygenase-dependent pathways. We provide evidence for a unique link between MAP kinase activation and cyclo-oxygenase activity in regulating PAR-1-induced chloride secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell line
SCBN is a nontransformed intestinal epithelial cell line capable of vectorial chloride transport (4 , 25 , 26) . SCBNs from passage numbers 23–33 were grown as described previously (25 , 26) . Cells were grown to confluence (~5 days) in either 75 cm2 culture flasks, on Snapwell semipermeable supports, on 24 mm diameter Transwell semipermeable supports (Corning Inc., Corning, NY), or on Lab Tek II chamber slides (Nalge Nunk International, Naperville, IL) coated with 10 µg/cm2 collagen type IV (Sigma Chemicals, Mississauga, ON). Cells in Snapwells, Transwells, or chamber slides were fed every day with Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), L-glutamine, streptomycin, and tylosin. Cells in culture flasks were fed fresh media every 2–3 days.

Assessment of chloride secretion
To measure PAR-1-induced chloride secretion, SCBN monolayers were grown to confluence on Snapwell semipermeable supports. Confluence was determined by the increase in resistance across the monolayers as measured by an electrovoltohmmeter (EVOM, World Precision Instruments, Sarasota, FL). Only monolayers with a resistance of >1000 Ohms/cm2 were used. Snapwells were mounted in modified Ussing chambers and bathed on the apical side with Krebs buffer containing 10 mM mannitol and on the basolateral side with Krebs buffer containing 10 mM glucose. Krebs buffer contained 115 mM NaCl, 2.0 mM KH2P04, 2.4 mM MgCl2, 25 mM NaHCO3, 8 mM KCl, and 1.3 mM CaCl2. The transepithelial potential difference was clamped to zero volts by applying a short circuit current (Isc) with a voltage clamp apparatus (EVC-4000, World Precision Instruments). The changes in Isc ({Delta}Isc) were an indicator of changes in the net electrogenic electrolyte flux across the monolayer. Isc was recorded with a digital data acquisition system (MP100, BioPac, San Diego, CA) and analyzed with AcqKnowledge software (version 3.1.3, BioPac).

PAR-1 activation was accomplished with the basolateral addition of either thrombin or the PAR-1-activating peptide (PAR-1-AP) Ala-parafluoro–Phe-Arg-cyclohexyl-Ala-citrulline-Tyr-NH2 (Cit-NH2) (27) . When used, inhibitors and antagonists were added to the buffers bathing both the basolateral and apical sides of the monolayers. In studies of prostanoid receptor involvement in PAR-1-mediated secretion, EP and FP receptors were desensitized by basolateral application of 2.5 µM PGE2 or 5 µM PGF2{alpha}, respectively. Receptor desensitization was verified by a second challenge with prostaglandin.

Immunocytochemistry
COX expression in SCBN cells has not been described before and therefore was determined by immunocytochemistry. SCBN cells were grown to confluence on collagen-coated Lab-Tek II chamber slides as described above. All procedures were conducted at room temperature and cells were washed three times for 5 min in Tris-buffered saline (TBS) between each step unless otherwise stated. Cells were fixed using ice-cold methanol for 20 min and incubated for 30 min with 0.1% sodium azide and 0.3% hydrogen peroxide to inhibit endogenous peroxidases. Nonspecific binding was blocked by incubating cells for 30 min in blocking medium made up of eight parts DMEM, two parts FBS, and 10 mg/mL bovine serum albumin. Cells were then stained for 1 h with mouse monoclonal IgG2B (1:200) or IgG1 (1:100) directed against COX-1 and COX-2, respectively (Cayman Chemical, Ann Arbor, MI), or with the corresponding negative control IgG isotypes (DAKO Diagnostics Canada, Mississauga, ON) added directly to the blocking medium. Cells were then stained for 1 h with biotin-labeled secondary antibody (DAKO, rabbit-anti-mouse) diluted 1:400 in TBS before being incubated for 1 h with streptavidin-biotin-HRP complex (DAKO) diluted 1:200 in Tris-HCl (6 mL 0.2 M Tris, 9.5 mL 0.1 M HCl, 9.5 mL distilled water, pH 7.6). Cells were incubated with AEC solution (DAKO) for 15–20 min, then washed once with TBS and once with water. Cells were counterstained with Mayer’s hematoxylin (Fluka, Ronkonkoma, NY) for 2 min, washed with water for 5 min, and slides were mounted with Aquaperm (VWR, Edmonton, AB).

Immunoprecipitation and Western blot
SCBNs were grown to confluence in 75 cm2 culture flasks. Cells were rinsed with phosphate-buffered saline (PBS), then incubated for 2 h in serum-free medium. Cells were harvested using a cell scraper and transferred to 1.5 mL microcentrifuge tubes, where they were exposed for 3 min to the vehicle control, HEPES, the PAR-1 agonists thrombin (10 U/mL) and Cit-NH2 (20 µM), or EGF (125 ng/mL). The 3 min point was chosen as it correlates with the peak change in Isc observed after exposure of SCBN monolayers to PAR-1 activators in Ussing chamber experiments. This technique of harvesting cells from flasks was chosen since SCBN cells grown on polycarbonate filter supports (and treated basolaterally with PAR-1 agonists before reactions were stopped using ice-cold buffer) yielded results similar to those obtained from cells grown in flasks, but the amount of protein obtained was very low and changes in phosphorylation state as detected with our IP-Western blot protocol were difficult to assess. Cells to be used for cPLA2 immunoprecipitation were also treated with the MEK inhibitor U0126 (10 µM) or the vehicle control DMSO 20 min before PAR-1 activation. Tubes were then placed on ice and centrifuged at 10,600 g at 4°C for 10 min to pellet cells. Cells were rinsed once with cold PBS and resuspended in 500 µL lysis buffer [1% Triton X-100, 1.0 µg/mL leupeptin, 1.0 µg/mL antipain, 1.0 mM EDTA pH 9.0, 1.0 mM NaF, 1.0 mM Na vanadate, 0.1 mg/mL phenylmethylsulfonyl fluoride (PMSF) and 1% protease inhibitor mixture (Sigma)]. A further 0.1 mg/mL of PMSF was then added to each sample and these were left on ice for 45 min. Samples were then centrifuged at 15,300 g for 10 min at 4°C and supernatants were transferred to new tubes.

Immunoprecipitation was performed according to standard techniques using antibodies against cPLA2 (Santa Cruz Biotechnology, Santa Cruz, CA) or EGF receptor (Upstate Biotechnology, Lake Placid, NY) and protein A/G PLUS agarose beads (Santa Cruz Biotechnology). Cytoplasmic PLA2 or EGF receptors were identified by Western blot and probed with antibodies against phosphoserine residues (Sigma) or phosphotyrosine residues (Transduction Laboratories, Lexington, KY), respectively.

Measurement of prostaglandin synthesis
SCBN cells grown to confluence on 24 mm Transwells were incubated for 1 h in serum-free medium, then rinsed with PBS. The basolateral surfaces of the cells were exposed for 5 min to the vehicle control HEPES, thrombin (10 U/mL), Cit-NH2 (20 µM), or the positive control, arachidonic acid (10 µM). Supernatants were collected and placed into microcentrifuge tubes on ice. Transwell inserts were transferred to ice-cold PBS and cells harvested in serum-free DMEM. Cell suspensions were then transferred to microcentrifuge tubes and cycled three times between a 40°C water bath and liquid nitrogen to lyse cells. Lysates were centrifuged at 15,300 g for 5 min at 4°C and supernatants were transferred to new tubes. PGE2 and PGF2{alpha} levels were assessed using commercially available EIA kits (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.


   MATERIALS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS
RESULTS
 DISCUSSION
 REFERENCES
 
The PAR-1-activating peptide Cit-NH2 was synthesized in-house as a carboxamide by the University of Calgary Peptide Synthesis Facility (director, Dr. Dennis McMaster). Thrombin (human, specific activity 2800 NIH units/mg protein), PD153035, AG1296, PP1, and AACOCF3 were purchased from Calbiochem (San Diego, CA). Piroxicam, wortmannin, bisindolylmaleimide, U0126, SQ 29548, and SB203580 were purchased from Sigma-Aldrich (Oakville, ON). NS-398, PGF2{alpha}, and PGE2 were from Cayman Chemical (Ann Arbor, MI). Human recombinant EGF was from Austral Biological (San Ramon, CA). BB2116 and SC-560 were generous gifts from Drs. Chris Overall (University of British Columbia) and Frank Degner (Boehringer-Ingelheim), respectively.

Statistics
Data are presented as the mean ± standard error of the mean. Comparison of more than two groups was made using ANOVA with a post hoc Tukey test. Comparison of two groups was made using Student’s t test for unpaired data. An associated probability (P) value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS
 RESULTS
DISCUSSION
 REFERENCES
 
Chloride secretion by SCBN cells was measured as the change in short circuit current ({Delta}Isc) expressed as µA/cm2. In the presence of the vehicle control (DMSO), SCBN cells responded to basolateral application of PAR-1 agonists with a rapid, transient increase in Isc as previously observed (4) . This increase was significantly diminished when cells were preincubated with the Src kinase inhibitor PP1 (1 µM) (28) , the EGF receptor tyrosine kinase inhibitor PD153035 (1 µM) (29) or the MEK inhibitor U0126 (10 µM) (30) (Fig. 1 ). Conversely, the change in Isc elicited by PAR-1 activation was not blocked by the PDGF receptor kinase inhibitor AG1296 (2 µM) (31) , the PKC inhibitor bisindolylmaleimide (5 µM) (32) , the PI-3 kinase inhibitor wortmannin (100 nM) (30) , or the p38 MAP kinase inhibitor SB203580 (10 µM) (30) . The general MMP inhibitor BB2116 (200 nM) (33) or preincubation with EGF 60 ng/mL also did not inhibit the PAR-1-induced response and, in fact, potentiated it (Fig. 1) . The mechanisms underlying the potentiating effect of these agents were not investigated in these studies.



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  		Figure 1. The effect of inhibitors on the short circuit current ({Delta}Isc) response to serosal application of A) thrombin (5 U/mL) or B) Cit-NH2 (10 µM). The short circuit current responses to thrombin and Cit-NH2 were both blocked by the EGF receptor tyrosine kinase inhibitor PD153035 (1 µM), the Src inhibitor PP1 (1 µM), and the MEK 1/2 inhibitor U0126 (10 µM). The responses were not blocked by the PDGF receptor tyrosine kinase inhibitor AG1296 (AG; 2 µM), the protein kinase C (PKC) inhibitor bisindolylmaleimide (Bisindolyl; 5 µM), the IP3 kinase inhibitor wortmannin (100 nM), the p38 MAP kinase inhibitor SB203580 (10 µM), or the general inhibitor of matrix metalloproteinases, BB2116 (200 nM). Preincubation with 60 ng/mL EGF had no effect on Isc in itself, but potentiated the secretory response to PAR-1 activation. *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle control (DMSO). n = 4–8 per group.

To test whether the EGF receptor was being phosphorylated after PAR-1 activation, EGF receptor was immunoprecipitated from cells treated with the vehicle HEPES, the PAR-1 agonists thrombin (10 U/mL) or Cit-NH2 (20 µM), or EGF (125 ng/mL). The intensity of tyrosine phosphorylation of EGFr was assessed by Western blot with subsequent densitometry. EGFr showed significantly increased tyrosine phosphorylation in cells treated with the PAR-1 agonists or EGF compared to those treated with HEPES (Fig. 2 ).



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  		Figure 2. A) Western blot showing tyrosine phosphorylation of EGF receptor immunoprecipitated from SCBN cells treated with the vehicle (HEPES), thrombin (10 U/mL), Cit-NH2 (20 µM), or EGF (125 ng/mL). B) Densitometry of Western blots reveals that phosphorylation of tyrosine residues on the EGF receptor is significantly increased after treatment with thrombin, Cit-NH2, or EGF compared to treatment with the vehicle, HEPES. *P < 0.05; **P < 0.01. vs. vehicle control (HEPES). n = 7–8.

Because of the important role of prostanoids in mediating chloride secretory responses in intestinal epithelial cells, experiments were conducted to assess the involvement of various components of the prostaglandin synthetic pathway after PAR-1 activation. To examine whether the response to PAR-1 agonists involved MAP kinase activation with subsequent phosphorylation and activation of cPLA2, cPLA2 was immunoprecipitated from cells treated or not with the MEK inhibitor U0126 before exposure to the control, HEPES, the PAR-1 agonists thrombin (10 U/mL) and Cit-NH2 (20 µM), or EGF (125 ng/mL). The intensity of serine phosphorylation of cPLA2 was then assessed by Western blot. In cells treated with thrombin, Cit-NH2, or EGF, the intensity of serine phosphorylation of cPLA2 was significantly increased vs. those treated with the vehicle control as determined by densitometry. In cells treated with U0126 before PAR-1 agonists, the increase in phosphorylation was abolished (Fig. 3 ).



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  		Figure 3. A) Western blot showing serine phosphorylation of cytosolic PLA2 immunoprecipitated from SCBN cells treated with the vehicle (HEPES), thrombin (10 U/mL), and Cit-NH2 (20 µM) with or without pretreatment with the MEK inhibitor U0126 (10 µM) or EGF (125 ng/mL). B) Densitometry of Western blots shows that phosphorylation of serine residues on cytosolic PLA2 is increased after treatment with thrombin or Cit-NH2 when compared to treatment with the vehicle, HEPES. The increased phosphorylation of cPLA2 after PAR-1 activation is significantly diminished by preincubation of cells with the MEK inhibitor U0126 (10 µM). *P < 0.05; **P < 0.01. vs. vehicle control (HEPES). {Psi}P < 0.05 vs. corresponding agonist without U0126 pretreatment. n = 8–13.

Prostanoid synthesis from arachidonic acid proceeds primarily through the activities of COX-1 and COX-2. However, COX expression in SCBN cells has not been described. Therefore, we used immunocytochemistry to evaluate the presence of COX-1 and COX-2 in unstimulated SCBNs. Almost all cells exhibited COX-1, evident as diffuse positive staining compared to the antibody control (Fig. 4 ). COX-2 reactivity appeared as intense staining in ~30% of cells (Fig. 4) . We then determined the involvement of COX-1 and COX-2 in the chloride secretory response to PAR-1 activation. PAR-1-induced chloride secretion, as assessed in the Ussing chambers, was significantly diminished by inhibitors of COX pathway proteins (Fig. 5 ). Preincubation of cells with 30 µM AACOCF3, an inhibitor of cytosolic PLA2 (34) , caused the Isc response to PAR-1 agonists to be significantly reduced, thus implicating cPLA2 and the subsequent liberation of arachidonic acid. When cells were preincubated with the nonselective COX inhibitor piroxicam (10 µM) (35) , subsequent addition of PAR-1 agonists elicited a decrease rather than an increase in Isc and thus in baseline chloride secretion. This decrease was abolished in the presence of chloride-free buffer (Fig. 5) . The same observation was made after exposure of the cells to the COX-2 inhibitor NS-398 at concentrations selective for COX-2 (30 µM) (36 , 37) (Fig. 5 , lower trace). Preincubation with another selective COX-2 inhibitor, celecoxib (2.5 µM) (37) , also significantly reduced the secretory response to PAR-1 activation. In the presence of the selective COX-1 inhibitor SC-560 (300 nM) (38) , the Isc response to Cit-NH2 was abrogated and the response to thrombin was significantly decreased compared to that elicited in the presence of the vehicle (Fig. 5) .



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  		Figure 4. Micrographs of SCBN cells grown to confluence and stained for A) COX-1 and B) COX-2. A positive immunocytochemical reaction is indicated by red staining. Diffuse COX-1 immunoreactivity is evident in almost all cells, whereas COX-2 staining is more intense and is observed in ~30% of the cells. C, D) Controls for COX-1 and COX-2 staining, respectively, using an unrelated IgG.



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  		Figure 5. A) Representative traces of short circuit current responses to thrombin (5 U/mL) after preincubation with the vehicle DMSO (top trace) or the selective COX-2 inhibitor, NS-398 (30 µM, bottom trace). A reversal of current is observed after PAR-1 activation in the presence of the COX-2 inhibitor. Both tissues responded equally to the adenylate cyclase activator forskolin (FSK; 10 µM). Effect of cyclo-oxygenase inhibitors on the short circuit current responses to B) thrombin (5 U/mL) and C) Cit-NH2 (10 µM). The nonselective COX inhibitor piroxicam (10 µM) and the selective COX-2 inhibitor NS-398 (NS; 30 µM) reversed the current change elicited by PAR-1 activation. The decreased Isc elicited by PAR-1 activation in the presence of piroxicam was abolished in chloride-free buffer. The other selective COX-2 inhibitor, celecoxib (2.5 µM), and the cPLA2 inhibitor AACOCF3 (30 µM) significantly reduced the PAR-1-induced secretory response. Preincubation with the selective COX-1 inhibitor SC-560 (300 nM) abolished the current change elicited by Cit-NH2 and significantly reduced the Isc response to thrombin. *P < 0.05; **P < 0.01; ***P < 0.001vs. vehicle control (DMSO). {psi}P < 0.05 vs. piroxicam in the presence of chloride. n = 4–8 per group.

The involvement of cPLA2 and COX in PAR-1-induced chloride secretion suggested that a metabolite of arachidonic acid was involved. Since PGE2 and PGF2{alpha} have been shown to act as chloride secretagogues in various systems, we sought to determine whether PAR-1 activation stimulated production of these prostanoids. SCBN cells showed increased prostaglandin synthesis when treated with the COX substrate arachidonic acid (data not shown), indicating that the COX pathway was functional in these cells. As shown in Fig. 6 , activation of PAR-1 by either thrombin or Cit-NH2 caused a significant increase in both PGE2 and PGF2{alpha} levels in supernatants, indicating synthesis and release of the prostanoids from the cells. In cell lysates, PGE2 levels were significantly increased after stimulation with Cit-NH2 but not thrombin. PGF2{alpha} levels were significantly increased in cell lysates after stimulation with either PAR-1 agonist.



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  		Figure 6. Effect of PAR-1 activation on prostaglandin synthesis in SCBN cells. A) Basolateral application of the PAR-1 agonists thrombin (10 U/mL) and Cit-NH2 (20 µM) to SCBN monolayers elicited a significant increase in PGE2 and PGF2{alpha} release as measured in the media in which the cells were incubated (supernatants). B) Application of Cit-NH2 but not thrombin significantly increased PGE2 levels in cell lysates. PGF2{alpha} levels were significantly increased in cell lysates after stimulation with either PAR-1 agonist. *P < 0.05; ***P < 0.001 vs. vehicle control. n = 7–12 per group.

In SCBN monolayers grown on Snapwells and mounted in Ussing chambers, pretreatment with the thromboxane receptor antagonist SQ29548 (5 µM) (39) did not significantly affect the Isc response to thrombin or Cit-NH2. To determine the role of EP or FP receptors, SCBN monolayers on Snapwells were exposed to 2.5 µM PGE2 or 5 µM PGF2{alpha}, respectively, to desensitize the receptors. Receptor desensitization was confirmed by a lack of response to a second application of the PG (Fig. 7 ).Pretreatment with either PGE2 or PGF2{alpha} did not abrogate the PAR-1-induced increase in Isc, but rather significantly enhanced it (Fig. 7) .



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  		Figure 7. A) Representative traces of Isc responses to Cit-NH2 (10 µM) after pretreatment with the vehicle DMSO (top trace) or after desensitization of EP and FP receptors by repeated basolateral application of PGE2 (2.5 µM, middle trace) or PGF2{alpha} (5 µM, bottom trace), respectively. Receptor desensitization was verified by the lack of response to a second challenge with prostaglandin. Neither PGE2 nor PGF2{alpha} pretreatment reduced the increased Isc caused by exposure to Cit-NH2. Short circuit current responses elicited by B) thrombin (5 U/mL) or C) Cit-NH2 (10 µM) after pretreatment with the TXA2 receptor antagonist SQ29548 (5 µM) or after desensitization of EP and FP receptors by basolateral application of PGE2 (2.5 µM) or PGF2agr; (5 µM), respectively. SQ29548 had no effect on the PAR-1-induced {delta}Isc. The PAR-1-induced response was potentiated by pretreatment with PGE2 or PGF2{alpha}. **P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle control (DMSO).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous studies showed that PAR-1 activation caused chloride secretion in the SCBN intestinal epithelial cell line (4) , but the stimulus-secretion coupling mechanism remained unknown. We now show that this response occurs through stimulation of the EGF receptor tyrosine kinase via trans-activation and MAP kinase phosphorylation of cPLA2, with a subsequent COX-dependent activation of chloride secretion.

EGF receptor trans-activation, with subsequent activation of the ERK 1/2 MAP kinase pathway, is a recognized signal transduction pathway commonly associated with mediation of cell growth and differentiation (14) . EGFR trans-activation has been demonstrated after thrombin-induced activation in rat smooth muscle cells (40) , human astrocytoma cells, and rat fibroblast cells (14 , 18 , 41) . This signal transduction pathway has also been shown to regulate intestinal epithelial cell transport of ions in response to secretagogues, which increase intracellular levels of Ca2+ (15) . Activation of basolateral muscarinic cholinergic receptors on T84 colonic epithelial cells leads to EGF receptor trans-activation with a subsequent suppression of chloride secretion through IP-3 kinase-dependent and -independent pathways (42 , 43) . Here we showed that stimulation of EGF receptor trans-activation and subsequent stimulation of the ERK 1/2 MAP kinase pathway resulted in an increase in apically directed chloride secretion from SCBN cell monolayers. These conflicting findings may be explained by the cell lines from which the data were derived. The inhibitory effects of EGF receptor trans-activation on chloride secretion were demonstrated in the colonic carcinoma cell line T84 (15) , which does not express COX-2 (personal observations). We have demonstrated in this study that SCBN cells express both COX enzymes and that the secretory response to PAR-1 activation is both COX-1 and COX-2 dependent. Native intestinal epithelial cells express both COX isoforms, particularly in the inflammatory milieu (44) . We observed a decrease in Isc after PAR-1 activation in cells in which COX activity had been inhibited. This decrease was dependent on the presence of chloride in the buffer. This result suggests that two pathways—one COX dependent and stimulatory, and one COX independent and inhibitory—are activated after PAR-1 activation. Furthermore, we observed that blockade of COX-2 abolishes chloride secretion in SCBNs despite the finding that this enzyme is expressed in a minority of cells. It is possible that only those cells expressing the enzyme actually secrete chloride or that COX-2 products are acting in a paracrine fashion to activate chloride secretion in surrounding cells. More studies are required to determine the signaling pathway mediating PAR-1-induced inhibition of basal chloride secretion.

EGF receptor activation and subsequent activation of the ERK 1/2 MAP kinase pathway are commonly associated with activation of Src family kinases (45) . This appears to be the case in SCBN cells since the Src kinase inhibitor PP-1 significantly reduced the chloride secretory response to PAR-1 activation. The ability of PP-1 to inhibit the platelet-derived growth factor receptor (PDGF) kinase has been reported (28) , but our study ruled out a role for the PDGF receptor since AG1296, a specific inhibitor of PDGF kinase, did not affect PAR-1-induced chloride secretion in these cells. Others have shown that a Src-like kinase is involved in chloride conductance in CFTR-deficient lymphocytes (46) . We did not determine which Src kinase was involved in the signaling pathway mediating PAR-1-induced chloride secretion, but others have shown that EGF receptor trans-activation is associated with activation of c-Src (17) .

Transactivation of the EGF receptor can occur in a number of ways. The activation of a calcium-dependent tyrosine kinase such as Pyk-2 has been suggested and indeed has been demonstrated in stimulus-secretion coupling pathways regulating chloride transport from T84 cells (17) . We did not investigate Pyk-2 activation in our studies. A mechanism involving the release of reactive oxygen species has also been implicated (47) , and recent data have demonstrated the involvement of matrix MMPs in EGF receptor kinase trans-activation in some systems. This latter process involves the activation of transmembrane MMPs that cleave bound EGFr ligands, such as TGF-{alpha} or heparin binding EGF, to secondarily activate the EGF receptor (14 , 17 18 19) However, our data suggest that MMP-induced liberation of a surface-bound EGFr ligand is not involved in the secretory response to PAR-1 activation, since the MMP inhibitor BB2116 (48) did not affect PAR-1-induced chloride secretion.

Although we clearly showed through use of the selective inhibitor PD153035 and immunoblotting for phosphorylated EGFr that EGFr activation was involved in PAR-1-induced secretion, application of EGF itself, even at high concentrations (125 nM), did not elicit a secretory response in SCBN cells. However, application of EGF did potentiate secretory responses to subsequent PAR-1 activation by an unknown mechanism. Similarly, EGF had no direct effect on secretion in T84 cells in other studies (49) . This apparently paradoxical observation does not necessarily negate trans-activation of the EGF receptor by PAR-1 since receptor tyrosine kinases may display diverging or pluripotent signaling activity. Thus, the signaling pathways acting through the EGF receptor may differ depending on cellular compartmentalization of the receptor, the presence or absence of ligand, the type of ligand, and the receptor subunit isotypes involved in subsequent homo- or heterodimerization of the receptor (50) . Recently it has been shown that different signaling responses from EGFr can be elicited depending on the tyrosine residue phosphorylated through either trans-activation or ligand binding (51 , 52) . Our experiments showed that pretreatment with EFG, though not eliciting chloride secretion on its own, potentiated the secretory response to PAR-1 activation. This potentiation may be due to differences in phosphorylation pattern under these experimental conditions. Finally, parallel activation of other signaling proteins, such as Src, may be required to elicit the PAR-1 response, as may be the case in our studies.

It has recently been shown that thrombin-induced epidermal growth factor receptor trans-activation can mediate activation of the p38 MAP kinase pathway (53) . However, treatment of SCBN monolayers mounted in Ussing chambers with the p38 MAP kinase inhibitor SB203580 was without effect on the secretory responses to PAR-1 activation, suggesting that this pathway is not involved. The third MAP kinase pathway, c-Jun amino-terminal kinase, was not investigated in these studies.

In addition to the involvement of EGFr trans-activation in PAR-1-induced epithelial chloride secretion, we have also implicated COX-1- and COX-2-dependent production of prostaglandins. Our data suggest that pathways involved in liberation of arachidonic acid are activated after exposure of SCBN cells to PAR-1 activators. Several enzymes can liberate arachidonic acid from plasma membrane phospholipids, including diacylglycerol lipase and the phospholipases (54 , 55) . Involvement of DAG lipase was not explored in this study, but our data clearly implicate cPLA2 since we have shown that PAR-1 activation induced serine phosphorylation of cPLA2, as shown by Western immunoblot of phosphorylated cPLA2. The cPLA2 inhibitor AACOCF3 partially blocked the change in Isc evoked by PAR-1 activation of SCBN monolayers mounted in Ussing chambers. Residual changes in Isc in the presence of AACOCF3 may be due to DAG lipase activity or to incomplete enzyme inhibition.

The dependence of the secretory response to PAR-1 activation on both the ERK 1/2 MAP-kinase pathway and COX could have two explanations: 1) that these are parts of the same pathway, and act in series or 2) that these are separate pathways acting in parallel. Our data suggest that these two signaling pathways are acting in series since the cPLA2 phosphorylation induced by PAR-1 activation was blocked by the MEK inhibitor U0126, showing that cPLA2 activation was dependent on the ERK 1/2 MAP kinase pathway. Others have shown that the ERK 1/2 MAP kinase pathway can phosphorylate and activate cPLA2. For example, cPLA2 phosphorylation and activation after Fc{gamma}RII receptor binding in human neutrophils is dependent on the ERK 1/2 and p38 MAP kinase pathways (56) . Our study is the first to demonstrate this unique pathway in epithelial cells and to link it to activation of chloride secretion.

Activation of cPLA2, which liberates arachidonic acid, the substrate for COX-dependent PG synthesis, led us to investigate further the role of COX activity in PAR-1-stimulated chloride secretion. Constitutive expression of both COX-1 and COX-2 was observed by immunocytochemistry. Both isoforms were implicated in the secretory response to PAR-1 activation since the changes in Isc elicited by thrombin and Cit-NH2 were blocked not only by the nonselective COX inhibitor piroxicam, but also by the selective COX-1 inhibitor SC-560 and the selective COX-2 inhibitors NS398 and celecoxib. We have previously shown that both isoforms are involved in arachidonic acid-induced chloride secretion in mouse colon in vitro (57) . Others have also recently shown that COX-2 mediates chloride and water secretion in a rat model (58) .

Besides studying COX isoforms, we sought to determine the prostaglandin synthesized by SCBN epithelial cells after PAR-1 activation with thrombin or the PAR-1-AP, Cit-NH2. We and others have shown that PAR-1 stimulation elicits prostaglandin release from other cells. PAR-1 activation in human fibroblasts results in PGE2 synthesis (24 , 59) and in platelets results in thromboxane release (60) . We studied PG synthesis in two ways. First, we determined PG release by measuring PGE2 and PGF2{alpha} concentrations in the media in which cells were incubated during exposure to PAR-1 activators. Second, since it is possible that PGs can be acting as intracellular messengers to mediate PAR-1-induced chloride secretion, we measured PGE2 and PGF2{alpha} concentrations in lysates from washed cells. PAR-1 activation in SCBN cells resulted in significant increases in PGE2 and PGF2{alpha} release in response to thrombin and Cit-NH2 as measured in media. In cell lysates, PGE2 levels were significantly increased after stimulation with Cit-NH2 but not thrombin. PGF2{alpha} levels were significantly increased in cell lysates after stimulation with either PAR-1 agonist. More studies are required to unequivocally demonstrate whether the PGs involved in PAR-1-stimulated chloride secretion are acting extra- or intracellularly. The fact that the selective TXA2 receptor antagonist SQ29548 did not affect PAR-1-induced chloride secretion suggests that thromboxane is not involved.

In Ussing chambers, addition of 2.5 µM of either PGE2 or 5 µM PGF2{alpha} to SCBN cells leads to a chloride secretory response similar in magnitude to that induced by PAR-1 activation and prevents further PGE2 or PGF2{alpha} (respectively) induced secretion, presumably through receptor desensitization. We used PG receptor desensitization in the absence of selective PG receptor antagonists to determine whether either PGE2 or PGF2{alpha} were involved in PAR-1-induced changes in Isc. Pretreatment with either PG potentiated rather than reduced the subsequent response to PAR-1 activation, suggesting that these are not the primary PGs mediating the response. Although the mechanism by which this response occurs is unknown, others have shown that mediators that increase cAMP, as does PGE2, are capable of potentiating calcium-dependent secretory responses in the gut. Desensitization studies using pretreatment with prostaglandins may not be conclusive and may be complicated by the fact that prostaglandins such as PGE2 and PGF2{alpha} have numerous cellular effects. Further studies are thus required to determine which prostanoid is involved in PAR-1 induced chloride secretion.

In conclusion, we have delineated a complex MAP kinase- and cPLA2/COX-dependent signaling pathway that couples epithelial PAR-1 activation to chloride secretion. The findings of this study have broad implications for the function of chloride-transporting epithelia that may, in the case of ongoing inflammation, constitutively express both COX-1 and COX-2 and be exposed to active thrombin.


   ACKNOWLEDGMENTS
 
This work was supported primarily by grants from the Canadian Institutes for Health Research (W.K.M., M.D.H.), with ancillary support from the Natural Sciences and Engineering Research Council of Canada (A.G.B.). M.C.B. is supported by M.D.-Ph.D. studentships from the Alberta Heritage Foundation for Medical Research (AHFMR) and the Canadian Institutes for Health Research. W.K.M. is an AHFMR Scholar. The authors thank Dr. Michelle Seymour for assistance with the immunocytochemistry.

Received for publication February 21, 2002. Revision received May 21, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 

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