Raf-1 Kinase Inhibitory Protein (RKIP) Mediates Ethanol-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells*

Background: The molecular mechanism by which ethanol (EtOH) sensitizes pancreatic acinar cells to secretagogue stimulation is largely undefined. Results: Ethanol stimulates PKC-dependent RKIP phosphorylation, and RKIP ablation prevents EtOH-induced sensitization of secretagogue Ca2+ signaling and aberrant chymotrypsin activation. Conclusion: RKIP mediates the cytotoxic effects of EtOH on pancreatic acinar cells. Significance: Modulation of RKIP expression may have therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis. Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca2+ homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca2+ signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca2+ signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca2+ signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.

Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca 2؉ homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin-and carbachol-regulated Ca 2؉ signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca 2؉ signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signalregulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin-and carbachol-stimulated Ca 2؉ signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.
Excessive consumption of ethanol (EtOH) (Ն2-5 drinks/ day) is a major risk factor for pancreatitis (1,2), a necro-inflammatory disease of varying severity that in the United States results in ϳ200,000 hospitalizations annually at a cost of over $2 billion (3)(4)(5). Although EtOH and its metabolites exert numerous effects on all cell types of the pancreas, the sensitization of pancreatic acinar cells to secretagogue signaling, premature intracellular zymogen activation, and deregulation of intracellular Ca 2ϩ homeostasis have been identified as critical early events in the pathogenesis of the disease (6 -8).
Under normal physiological conditions, the cytoplasmic concentration of free Ca 2ϩ ([Ca 2ϩ ] cyto ) in pancreatic acinar cells is tightly regulated. The activation of G protein-coupled receptors (GPCRs) 2 by pancreatic secretagogues, such as cholecystokinin (CCK) and acetylcholine, leads to the opening of second-messenger-gated Ca 2ϩ channels located on the endoplasmic reticulum (ER) and various apical acidic vesicles (9 -14). The released Ca 2ϩ from these internal stores, in turn, stimulates the Ca 2ϩ -induced Ca 2ϩ influx through plasma membrane Ca 2ϩ channels (15)(16)(17)(18) resulting in a transient and localized rise in [Ca 2ϩ ] cyto , mainly in the apical region of the cell.
Limiting the secretagogue-induced increase in [Ca 2ϩ ] cyto are ATP-dependent Ca 2ϩ pumps that return the [Ca 2ϩ ] cyto to resting levels by both extruding Ca 2ϩ against its concentration gradient back into the extracellular space and by re-sequestering Ca 2ϩ into the intracellular stores. Together, the coordinated regulation of the various ligand-gated Ca 2ϩ channels, ATP-dependent pumps, and proteinaceous Ca 2ϩ buffers produce the transient, localized, oscillatory increases in [Ca 2ϩ ] cyto that are responsible for the normal meal-induced secretion of fluids and zymogens into pancreatic ducts and, eventually, into the small intestine where they are activated to facilitate nutrient digestion (19 -21).
Despite progress in understanding the critical role of aberrant Ca 2ϩ signaling in alcohol-induced acinar cell dysfunction, the underlying molecular mechanism(s) by which EtOH sensitizes acinar cells to secretagogue stimulation remains largely undefined. In this study we identify Raf-1 kinase inhibitory protein (RKIP) as an essential mediator of EtOH-induced sensitization of CCK-and carbachol-regulated Ca 2ϩ signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of RKIP expression may have a future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.
AR42J Cell Line-The rat AR42J cell line was selected for these studies because it exhibits a variety of features common to normal pancreatic acinar cells, including expression of secretagogue receptors (e.g. cholecystokinin 1 and muscarinic acetylcholine receptors), zymogen granules, and components of the Ca 2ϩ -dependent-regulated secretory pathway (39). AR42J cell cultures were maintained in Ham's F12K medium supplemented with L-glutamine (2 mM) and 20% fetal bovine serum (FBS) in a humidified atmosphere of 95% air and 5% CO 2 at 37°C.
Mouse Primary Acinar Cells-Mouse pancreatic acinar cells were isolated using a modification of the enzymatic dissociation method previously described (40). Briefly, the pancreata from 2-3 mice were removed, perfused with saline, washed, and minced with fine scissors in a cell isolation solution containing phosphate-buffered saline with Ca 2ϩ and Mg 2ϩ , pH 7.4, supplemented with 0.1% bovine serum albumin (BSA) and 0.01% soybean trypsin inhibitor. The minced tissue was digested for 15 min in 3 ml of 37°C isolation buffer containing 0.3 mg/ml collagenase type IV (Sigma). The enzyme digestion was facilitated mechanically by continuous pipetting the minced tissue suspension with a 10-ml pipette for 15 min. The digested tissue (i.e. clusters of acini) were washed with 6 ml of cold isolation buffer and collected by centrifugation (1000 rpm for 2 min). The washing step was repeated twice to remove small debris and blood cells. The smaller acinar cell clusters were further separated from the larger acini by filtering through a sterile 860-m stainless steel mesh. After centrifugation (1000 rpm for 2 min), the supernatant was removed, and the cell pellets were resuspended in 5 ml of oxygenated Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1% antibiotic/antimycotic, and 1% soybean trypsin inhibitor and then plated on either laminin-coated culture plates for biochemical experiments or coverslips for intracellular Ca 2ϩ imaging (see "Ratiometic Intracellular Ca 2ϩ Imaging Microscopy" below). All cells were incubated in a humidified atmosphere of 95% air and 5% CO 2 at 37°C and used for experimentation within 24 h of isolation. For quality control, acinar cell viability was assessed by trypan blue staining with Ͼ95% viability obtained for each experiment presented below.
Isolation of Phosphorylated Protein-Phosphorylated proteins where isolated using the phosphoprotein enrichment kit (Pierce Protein Research Products, catalog no. 90003), Thermo Fisher Scientific, Inc. Rockford, IL). The affinity columns in the Pierce Phosphoprotein Enrichment kit contain a proprietary metal that interacts with negative charges from phosphate groups. The optimized buffer conditions enable specific capture of phosphoproteins from complex biological samples. Briefly, cells were first washed twice with a non-phosphatebased HEPES (50 mM) buffer, pH 7.4, scraped from the culture plate in 1 ml of lysis solution supplemented with CHAPS detergent (0.25%), EDTA-free Halt Protease Inhibitors, and Halt Phosphatase Inhibitors (Thermo Fisher Scientific catalog nos. 78425 and 78420, respectively), incubated on ice for 45 min with periodic vortexing, and spun in a centrifuge at 10,000 ϫ g for 20 min (4°C) to pellet cellular debris. The protein concentration of the supernatants were determined using the Bio-Rad protein assay reagent, and 2.5 mg of total protein from each assay condition was applied to a phosphoprotein affinity column equilibrated with 5 ml of cell lysis solution containing 0.25% CHAPS. The protein samples were loaded onto the columns by centrifugation at 1000 ϫ g for 1 min at 4°C. Each column was then washed 3 times to remove unbound proteins by adding 5 ml of lysis solution with 0.25% CHAPS and spinning the column in a 50-ml conical tube at 1000 ϫ g for 1 min at 4°C. The bound phosphoproteins were eluted in a high salt elution buffer containing 75 mM sodium phosphate buffer, pH 7.5, 500 mM NaCl, and 0.25% CHAPS. 1 ml of elution buffer was added to each column, which was then incubated at room temperature with occasional agitation for 3 min. The eluate was collected by centrifugation at 1000 ϫ g for 1 min at 4°C. The elution procedure was repeated 4 times for a total of five elution fractions (5 ml total). The phospho-RKIP content of the pooled fractions was assessed by Western blotting.
Western Blotting-Cells were treated as indicated in the figure legends, and protein extracts were prepared using either the phosphoprotein enrichment kit or a cell lysis solution containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, 5 mM ␤-glycerophosphate, 1 mM NaF, 1 mM PMSF, 1 protease inhibitor mixture tablet (Roche Diagnostics) per 50 ml of solution. Insoluble cellular material was removed by centrifugation at 14,000 rpm for 15 min, and the concentration of proteins in the supernatant was determined using the Bio-Rad protein assay reagent and BSA as a standard. 20 -40 g of soluble protein extract was loaded per well of a Bis-Tris⅐HCl-buffered polyacrylamide gel (Invitrogen), resolved by electrophoresis, and transferred onto a PVDF membrane (Millipore Corp., Bedford, MA). For immunostaining, PVDF membranes were incubated in blocking solution containing 5% (w/v) nonfat milk dissolved in Tris-buffered saline plus 0.1% Tween 20 (TBST) for 1 h at room temperature. All primary antibodies were diluted in TBST containing 1% nonfat milk. All blots were incubated with primary antibody overnight at 4°C. After washing, the membranes were probed with a horseradish peroxidase-conjugated secondary antibody. Immunoreactive protein bands were detected by using ECL TM Western blotting detection reagent (Amersham Biosciences) and Kodak X-Omat film.
Ratiometic Intracellular Ca 2ϩ Imaging Microscopy-Isolated acinar cells were loaded with 2 M Fura-2-AM solution containing 0.025% Pluronic F-127 at 25°C for 35 min as previously described (41). The cells were washed and resuspended in Krebs-Ringers Henseleit solution consisting of NaCl (125 mM), KCl (5 mM), KH 2 PO 4 (1.2 mM), MgSO 4 (1.2 mM), CaCl 2 (2 mM), glucose (6 mM), and HEPES (25 mM), pH 7.4. Fura-2-loaded cells were then transferred to either laminin or BD Cell-TAK TM (BD Biosciences)-coated glass coverslips (25-mm diameter) for 1 h or 15 min, respectively, at 37°C. EtOH (100 mM) was added to the cells for 10 min at room temperature. Changes in the concentration of free cytosolic Ca 2ϩ ([Ca 2ϩ ] cyto ) were monitored with a Nikon Diaphot inverted microscope (Nikon Instrument Group, Garden City, NY) equipped with a Nikon 40ϫ (1.3 NA) Neofluor objective. The fluorescent light source was a PTI DeltaScan RD-1 ratio fluorescence spectrometer system (Photon Technology International, Inc., Birmingham, NJ) equipped with a light-path chopper and dual-excitation monochrometers. The Fura-2 was alternately excited at 340 and 380 nm, and fluorescence emission was monitored through a 510-nm bandpass filter with a Roper Coolsnap EZ digital camera. Data were obtained from single acinar cells and from single cells of doublets and triplet cell clusters. All experiments were performed at room temperature.
Suppression of RKIP Expression Using shRNA-RNA interference constructs containing four gene-specific shRNA sequences (Rat Pebp1 Gene ID 29542) were obtained from Ori-Gene Technologies (Rockville, MD) and analyzed for their effectiveness at suppressing RKIP protein levels in AR42J cells. Of the four shRNA expression constructs, infection of AR42J cells with a retrovirus containing the RKIP-specific sequence (5Ј-GCCGCTGTCATTACAGGAGGTGGATGAGC-3Ј) resulted in the greatest reduction in RKIP protein levels (Fig. 4A). To control for off-target effects, control cultures were infected with a virus expressing a non-targeting 29-mer scrambled shRNA sequence (OriGene, catalog no. TR30013). Retroviruses were made by simultaneously transfecting HEK293FT cells (Invitrogen) with the shRNA expression plasmid plus plasmids encoding MLV gag-pol and vesicular stomatitis virus envelope protein.
Transfection efficiency was Ͼ80% as determined by green fluorescent protein expression from the shRNA vector. Cell supernatants from the transfected HEK293FT cells were collected and filtered through a 0.45-m filter to remove cell debris and stored at Ϫ80°C. AR42J cells were plated in 12-well plates so that they were ϳ50% confluent at the time of infection (ϳ24 h post-plating). To infect the cells, culture medium was removed, and 1 ml of room temperature HEK293FT cell supernatant containing virus was added. Infected cells were maintained in a humidified atmosphere of 95% air and 5% CO 2 at 37°C under puromycin dihydrochloride (10 g/ml) selection.
Chymotrypsin Assay-Pancreatic acinar cells were isolated as described above ("Mouse Primary Acinar Cells"), and chymotrypsin activity was assayed using a modification of a previously described procedure (42). Briefly, acinar cells were pretreated with EtOH (100 mM) for 10 min and stimulated with different concentrations of CCK or vehicle over a time course. At the end of each incubation period, the cells and culture medium were transferred to an ice-cold 1.5-ml centrifuge tube, homogenized with a small pestle, and spun in a centrifuge at 1000 ϫ g at 4°C for 1 min. A portion of the supernatant proteins (20 l) from each sample was added to a 96-well plate containing 70 l of enzyme assay buffer (50 mM Tris, pH 8.1, 150 mM NaCl, I mM CaCl 2 , 0.01% BSA) per well. The chymotrypsin activity assay was initiated by adding 10 l of fluorogenic chymotrypsin substrate II (EMD Chemicals, Inc., Gibbstown, NJ) to each well (t ϭ 0). The cleavage of substrate was monitored at 440 nm over a time course using a Gemini XPS Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA). Data points were collected at 15-min intervals for up to 60 min. The enzyme activity in each sample was normalized to the concentration of total protein (using the Bio-Rad protein assay reagent) in an equivalent volume of supernatant from the same sample.
Statistical Analysis-Statistical analysis (Student's t test) was performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA). Statistical significance was assumed if p Յ 0.05.
To begin testing the hypothesis that RKIP is a downstream target of EtOH-induced PKC activity and regulator of CCK receptor sensitization, we first pretreated AR42J cells either with the PKC inhibitor, 2- which inhibits the classical PKC isozymes, PKC␣, -␤I, -␤II, and -␥ (52), or its solvent dimethyl sulfoxide (DMSO) for 1 h and then exposed the cells to EtOH (100 mM) or an equivalent volume of its diluent (H 2 O) for 10 min. After enriching for phosphorylated proteins by affinity chromatography ("Experimental Procedures"), the phosphoproteins in the column eluates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using a rabbit polyclonal antibody to RKIP. Fig. 1B shows representative immunoblots depicting the levels of phosphorylated RKIP in the column eluates and the levels of total RKIP protein in an equivalent volume of each protein extract before being applied to the phosphoprotein affinity resin (i.e. column input). Densitometric analyses from three independent experiments showed that the level of phosphorylated RKIP increased ϳ2-fold in cells treated with EtOH when compared with controls (i.e. cells treated with either DMSO or GF109203X and H 2 O) (Fig. 1C). Furthermore, the EtOH-induced increase in phosphorylated RKIP was significantly inhibited in cells pretreated with GF109203X (*, p Ͻ 0.03, EtOH alone versus EtOH plus GFX109203).
Having demonstrated that RKIP is a downstream target of EtOH-stimulated, GF109203X-sensitive PKC activity, we next determined the effects of EtOH treatment on ERK activation (i.e. ERK phosphorylation) and secretagogue-induced Ca 2ϩ signaling. If RKIP is mediating the effects of EtOH on CCKinduced signal transduction, the model depicted in Fig. 1A predicts that phosphorylation of RKIP should reduce RKIPmediated inhibition of the Raf/MEK/ERK pathway and enhance GPCR-mediated signaling by inhibiting GRK-dependent FIGURE 1. EtOH exposure induces RKIP phosphorylation and enhances both basal and CCK-stimulated signaling. A, shown is a pathway model illustrating the relationship between PKC-dependent RKIP phosphorylation and inhibition of the Raf/MEK/ERK signaling pathway and GRK. B, representative Western blots show levels of phosphorylated RKIP protein in column eluates after enrichment of AR42J protein extracts on phosphoprotein affinity columns (2.5 mg of protein extract was loaded on each column, and 20 g of protein from the eluate was load per lane). AR42J cells were pretreated with 2 M GF109203X or DMSO for 1 h and then exposed to 100 mM EtOH or H 2 O for 10 min. To ensure that equivalent amount of RKIP protein was loaded on to each column, 5 g of AR42J protein extract from each sample was resolved on a separate gel, transferred to PDVF membranes, and probed for RKIP levels (column input). C, a bar graph summarizes densitometry data from three independent experiments. The data represent the ratio of phosphorylated RKIP (column eluate) to the total amount of RKIP in the column input multiplied by 100. Error bars are S.E. Statistical analysis (Student's t test) was performed using GraphPad Prism 5 software. Densitometry was performed using ImageJ software (imagej.nih.gov). D, an immunoblot of AR42J cell protein extracts shows the effects of EtOH exposure on basal and CCK-8-stimulated phosphorylation of ERK1 and -2 (pERK1,2) and RKIP at Ser-153 (pS153-RKIP). Blots where reprobed with an antibody to total ERK1,2 to ensure the equal loading and transfer of proteins for each sample. E, an immunoblot shows the effects of GF109203X (2 M) pretreatment (1 h) on EtOH-induced phosphorylation of ERK1 and -2 using freshly isolated mouse pancreatic acinar cells. receptor desensitization. Consistent with this model, immunoblots of AR42J cell extracts revealed that EtOH pretreatment stimulated an increase in the basal levels of phosphorylated (activated) ERK1 and ERK2 (pERK1,2) (Fig. 1D) that was associated with a concomitant increase in the level of Ser-153-phosphorylated RKIP (pS153-RKIP) (Fig. 1D). Combined treatment with EtOH and CCK (0.1 nM) further increased the levels of pERK1,2 when compared with either agent alone (Fig. 1D). CCK treatment alone also increased the level of Ser(P)-153-RKIP (Fig. 1D), which is consistent with previous reports showing that CCK treatment activates PKC in rodent pancreatic acinar cells (26,51,53,54). However, the combined treatment with EtOH and CCK did not further increase Ser(P)-153-RKIP, suggesting that CCK can also activate ERK by mechanisms independent of the release of RKIP-mediated inhibition of Raf.
Similar to AR42J cells, exposing freshly isolated mouse pancreatic acinar cells to EtOH increased the levels of activated ERK1 and ERK2 (Fig. 1E). Both the basal and EtOH-stimulated levels of pERK1,2 were reduced in cells pretreated for 1 h with GF109203X (Fig. 1E). Finally, exposing AR42J cells to EtOH potentiated CCK-induced increases in the concentration of free cytosolic Ca 2ϩ ([Ca 2ϩ ] cyto ) when compared with control (H 2 O treated) cells (Fig. 1F). Collectively, these data support the hypothesis that RKIP mediates the EtOH-induced, PKC-dependent, sensitization of secretagogue (CCK) signaling in AR42J and freshly isolated pancreatic acinar cells.

PKC⑀ Mediates EtOH-induced RKIP Phosphorylation and Sensitization of CCK-stimulated Ca 2ϩ
Signaling-PKC⑀ has also been shown to be activated upon exposure of isolated rat acinar cells to EtOH (26), but it is not inhibited by GF109203X. Therefore, to specifically assess the function of PKC⑀ in EtOHinduced RKIP phosphorylation, we inhibited its activation with a PKC⑀ translocation inhibitor peptide (EAVSLKPT) as previously described (55,56). This peptide corresponds to the V1 region of PKC⑀ that is responsible for membrane translocation (activation) of the kinase. Functionally, the peptide blocks PKC⑀ activity by competitively inhibiting the binding of the enzyme to its endogenous membrane-anchoring protein (55,56). To ensure the effectiveness of the inhibitor peptide in blocking PKC⑀ activation, AR42J cells were pretreated either with the PKC⑀ translocation inhibitor peptide (10 M) or a negative-control peptide with the sequence (LSETKPAV) (10 M) for 30 min and then exposed to EtOH or an equivalent volume of H 2 O for 10 min. After treatment, the cellular membrane and cytoplasmic protein fractions were isolated by differential centrifugation as previously described (57). Immunoblot analyses to determine PKC⑀ levels in the two subcellular fractions showed that cells treated with the negative-control peptide exhibited relatively high levels of PKC⑀ in the cytoplasmic fraction and little enzyme associated with cell membranes (Fig. 2A). Pretreatment with the PKC⑀ inhibitor peptide caused a small increase in the level of cytoplasmic PKC⑀ and reduced membrane quantities to below the level of antibody detection ( Fig.  2A). Exposing the control peptide-treated cells to EtOH for 10 min decreased cytoplasmic PKC⑀ levels and increased the amount of enzyme in the membrane fraction ( Fig. 2A), indicating EtOH-induced enzyme activation. The stimulatory effect of EtOH on PKC⑀ membrane translocation was blocked in cells pretreated with the PKC⑀ inhibitor peptide (Fig. 2A). Together these data show that, similar to previous reports using primary rat acinar cells (26), EtOH treatment of AR42J cells induces PKC⑀ membrane translocation (i.e. activation). Furthermore, they demonstrate that the PKC⑀ inhibitor peptide effectively blocks enzyme activation.
Having established the effectiveness of the PKC⑀ inhibitor peptide in AR42J cells, we next determined the function of PKC⑀ in EtOH-induced RKIP phosphorylation and CCK-stimulated Ca 2ϩ signaling. After pretreatment either with the inhibitor or control peptides and exposure of the cells to EtOH or H 2 O, the phosphorylated proteins were enriched by affinity chromatography, resolved by SDS-PAGE, and transferred to PVDF membranes. Immunoblots of the column eluates revealed an increase in the level of phosphorylated RKIP in cells treated with control peptide and exposed to EtOH, whereas the stimulatory effect of EtOH on RKIP phosphorylation was completely blocked in cells treated with the PKC⑀ inhibitor peptide ( Fig. 2B). Analyses of the secretagogue-stimulated Ca 2ϩ responses showed that PKC⑀ inhibition prevented the EtOHinduced potentiation of CCK-induced increases in [Ca 2ϩ ] cyto (Fig. 2C). A summary analysis of data from three independent experiments demonstrated an ϳ3-fold increase in the 340/ 380-nm ratio of Fura-2 fluorescence in the initial CCK-stimulated Ca 2ϩ peak compared with controls when the cells were pretreated with negative-control peptide and exposed to EtOH (Fig. 2D). The EtOH-induced potentiation of the CCK-stimulated Ca 2ϩ response was completely blocked in cells treated with the PKC⑀ inhibitor peptide (Fig. 2D) (*, p Ͻ 0.01, control peptide ϩ EtOH versus PKC⑀ inhibitor ϩ EtOH).

Suppression of RKIP Expression Prevents EtOH-induced Sensitization of CCK Signaling in AR42J
Cells-Next, we evaluated whether suppression of RKIP expression mimicked the effects of PKC inhibition on secretagogue-stimulated increases in [Ca 2ϩ ] cyto . To suppress RKIP protein expression, AR42J cells were infected with a retrovirus containing a gene-specific shRNA sequence targeting RKIP. Control cultures were infected either with a virus containing a non-targeting (NT) control shRNA or the empty retroviral expression construct. An immunoblot of protein extracted revealed almost complete suppression of RKIP expression in cells transduced with RKIP shRNA when compared with either the NT-shRNA control or empty viral vector (Fig. 3A). Evaluation of CCK-induced Ca 2ϩ signaling showed that, compared with H 2 O-treated cells (Fig. 3, B and D), EtOH exposure potentiated CCK-stimulated increases in [Ca 2ϩ ] cyto in cells infected either with NT-shRNA (Fig. 3C) or the empty expression vector (data not shown) but not in cells infected with the retrovirus containing RKIP shRNA (Fig. 3E). A comparison of the effects of EtOH exposure on CCK-stimulated increases in [Ca 2ϩ ] cyto over a range of agonist concentrations revealed that NT-shRNA-infected cells exhibited an enhanced CCK-induced Ca 2ϩ responses over the range of agonist concentrations when compared with cells exposed to  an equivalent volume of H 2 O (Fig. 3F). In contrast, EtOH exposure failed to potentiate CCK-stimulated Ca 2ϩ signaling over the same range of CCK concentrations when RKIP expression was suppressed (Fig. 3G). Competition binding experiments with 125 I-labeled CCK confirmed that neither exposure to 100 mM EtOH nor suppression to RKIP expression affected the levels or relative affinity of the CCK receptors expressed by AR42J cells (Fig. 3H). Together these studies demonstrate that suppression of RKIP expression is functionally equivalent to PKC inhibition (Figs. 1F and 2C) when evaluating the effects of EtOH exposure on secretagogue-stimulated Ca 2ϩ signaling, suggesting the RKIP is a downstream mediator of PKC-dependent CCK receptor sensitization.
Acinar Cells from RKIP Null Mice Are Insensitive to EtOHinduced Sensitization-To confirm the function of RKIP in normal pancreatic acinar cells, we isolated cells from the pancreata of both RKIP null (RKIP Ϫ/Ϫ ) and wild-type (RKIP ϩ/ϩ ) control mice. Immunoblots confirmed the absence of RKIP protein expression in the pancreatic tissue (PT) and isolated pancreatic acinar cells (PACs) from RKIP Ϫ/Ϫ mice (Fig. 4A). Similar to AR42J cells, the acinar cells from RKIP ϩ/ϩ mice pretreated with EtOH (100 mM) for 10 min exhibited enhanced CCK-induced Ca 2ϩ responses when compared with cell exposed to an equivalent volume of H 2 O (Fig. 4B), and the EtOH-induced sensitization of the CCK-stimulated Ca 2ϩ response was observed over a range of agonist concentrations (Fig. 4C). In contrast to cells from wild-type animals, EtOH had no effect on the sensitivity of the CCK-stimulated Ca 2ϩ responses in acinar cells isolated from RKIP Ϫ/Ϫ mice (Fig. 4, D  and E). A dose-response study with the RKIP Ϫ/Ϫ acinar cells showed no differences in the CCK-induced Ca 2ϩ responses between the EtOH-and H 2 O-pretreated cells over a range of agonist concentrations (Fig. 4F). A similar RKIP-dependent sensitization, the carbachol-stimulate Ca 2ϩ response, was observed in acinar cells pretreated for 10 min with 50 mM EtOH (Fig. 5). Together these data establish that RKIP plays a critical role in EtOH-induced sensitization of secretagogue-stimulated Ca 2ϩ signaling in pancreatic acinar cells.
Loss of RKIP Expression Prevents the Sensitizing Effect of EtOH on CCK-induced Chymotrypsin Activation-A hallmark of the pathogenesis of pancreatitis is the aberrant activation of the digestive enzymes within the acinar cell (for review, see Halangk and Lerch (58)). EtOH exposure has been shown to enhance secretagogue-induced intra-acinar trypsin and chymotrypsin activation in isolated rodent acinar cells (24,28). To assess the role of RKIP in EtOH-induced chymotrypsin activation, acinar cells were isolated from RKIP Ϫ/Ϫ and wild-type mice, exposed to EtOH or H 2 O for 10 min, and then stimulated either with CCK or vehicle. To validate the functionality of the isolated acinar cells, we assessed the cleavage of the fluorogenic chymotrypsin substrate in response to a high concentration of CCK (10 nM). The super-physiological concentration of CCK induced chymotrypsin activity in cells from both wild-type and RKIP Ϫ/Ϫ mice (Fig. 6, A and B, CCK (10 nM) versus Control, t ϭ 60 min), confirming previous studies which show that in the absence of a sensitizer like EtOH, secretagogue hyperstimulation is sufficient to induce intracellular digestive enzyme activation (35,51,59). Furthermore, pretreatment of acinar cells either from RKIP ϩ/ϩ or RKIP Ϫ/Ϫ mice with H 2 O followed by stimulation with a physiologic concentration of CCK (0.1 nM) had no effect on basal chymotrypsin activity (Fig. 6, A and B, Control versus H 2 O ϩ CCK, t ϭ 60 min). In contrast, acinar cells from RKIP ϩ/ϩ mice exposed to EtOH (100 mM) showed an ϳ3.5-fold increase in chymotrypsin activity in response to 0. 1 nM CCK compared with controls (Fig. 6A, EtOH ϩ CCK versus H 2 O ϩ CCK or Control, t ϭ 60). EtOH pretreatment, however, failed to potentiate CCK-induced chymotrypsin activity in acinar cells from RKIP Ϫ/Ϫ mice (Fig. 6B, EtOH ϩ CCK versus H 2 O ϩ CCK or Control, t ϭ 60). A similar RKIP-dependent aberrant intracellular chymotrypsin activation was observed in acinar cells pretreated for 10 min with 50 mM EtOH and stimulated with a low concentration (1 M) of carbachol (Fig. 6, C and D). Together, these data demonstrate that RKIP expression is critical for EtOH-induced sensitization of secretagoguestimulated chymotrypsin activation in isolated pancreatic acinar cells.

DISCUSSION
Although alcohol abuse has been long recognized as a risk factor for acute and chronic pancreatitis, the molecular mechanism mediating the sensitizing affect of EtOH on secretagogue signaling in acinar cells has remained largely unresolved (26,27). In this study we show that RKIP is a substrate for EtOHinduced PKC-dependent phosphorylation and a critical mediator of EtOH-induced sensitization of secretagogue signaling. Collectively, the data presented are consistent with the model diagramed in Fig. 7. Acute exposure of pancreatic acinar cells to EtOH induces the PKC-dependent phosphorylation of RKIP (pRKIP) (Fig. 7, 1), decreasing both RKIP-mediated inhibition of the Raf/MEK/ERK pathway (Fig. 7, 2), and GRK-mediated secretagogue receptor desensitization (Fig. 7, 3), resulting in an increase in basal ERK activation as well as secretagogue-stimulated increases in [Ca 2ϩ ] cyto , ERK phosphorylation, and aberrant intracellular chymotrypsin activation (Fig. 7, 4), a hallmark of pancreatitis. Additionally, CCK stimulation alone increased cellular levels of phosphorylated RKIP, suggesting that RKIP is a physiological modulator of agonist-induced receptor desensitization (Fig. 7, 5).
To elucidate the molecular mechanism by which EtOH sensitizes secretagogue signaling, we have focused on the peptide hormone CCK, which binds with high affinity to the CCK1 receptor, a Class A or rhodopsin-like GPCR. Similar to other members of this class of GPCRs, agonist activation of CCK1 receptor is followed by its rapid phosphorylation and desensitization (60,61). Agonist-dependent GPCR desensitization, in general, is initiated by GRKs, a family of serine/threonine kinases (62) that phosphorylates specific amino acid residues within the third intracellular loop domain and/or C terminus of the activated receptor, creating binding sites for the adapter protein ␤-arrestin, which then blocks further receptor interactions with heterotrimeric G proteins and signaling (for review, Kelly et al. 2008 (63)).
RKIP has been identified as an antagonist of GRK-dependent GPCR desensitization (47) and thus functions to enhance or prolong agonist-induced signaling. For example, PKC-phosphorylated RKIP has been shown to inhibit GRK2-mediated phosphorylation of the isoproterenol (agonist)-activated ␤ 2adrenergic receptors with the result of potentiating agoniststimulated cAMP production and inhibiting receptor internalization (47). The potential for RKIP to enhance GPCR signaling led us to investigate its role in EtOH-induced sensitization of secretagogue signaling in pancreatic acinar cells. Herein, we show that RKIP mediates the toxic effects of EtOH on CCK signaling by enhancing both agonist-induced increases in ERK activation and [Ca 2ϩ ] cyto . The dysregulation of acinar cell Ca 2ϩ homeostasis is a critical event in acinar cell death and the early pathophysiology of pancreatitis (36,64). We show that EtOH stimulates the PKC-dependent phosphorylation of RKIP, which is associated with both enhanced ERK activation and CCK-induced increases in [Ca 2ϩ ] cyto . Furthermore, we showed either inhibition of PKC or loss of RKIP expression is sufficient to prevent the sensitizing effects of EtOH on both CCK-induced Ca 2ϩ signaling and, importantly, on intracellular chymotrypsin activation, a hallmark of pancreatitis.
Previous studies have shown that EtOH activates both the classical PKC isozymes PKC␣ and the novel PKC⑀ (26,50,51). Consistent with these observations, we show that EtOH-induced phosphorylation of RKIP is inhibited in cells treated with the classical PKC inhibitor, GF109203X, as well as the PKC⑀specific inhibitory peptide (Figs. 1C and 2B). Although the phosphorylation of RKIP by classical PKC isozymes is relatively well documented in the scientific literature, the role of the novel PKC isozymes is ambiguous. This is due in part to an earlier study by Corbit et al. (44) in which they evaluated the ability of various PKC isozymes to phosphorylate RKIP using purified recombinant proteins in a cell-free kinase assay. In that study they show that RKIP is robustly phosphorylated by the classical PKC isozymes -␣, -␤I, -␤II, -␥, and atypical PKC primarily at Ser-153, as the substitution of Ser-153 with alanine prevented most of RKIP phosphorylation. By comparison, the phosphorylation of RKIP by the purified novel PKC isozymes -␦, and -⑀ was very weak but, based on the data shown, specific for Ser-153. However, despite the evidence for weak Ser-153 phosphorylation by PKC␦ and -⑀, the authors concluded that RKIP is not a substrate for novel PKC isozymes (44). Our own in silico analyses of RKIP protein sequences from human, rat, and mouse using the Scansite 2.0 software (65) identified Ser-153 in humans and rats and Thr-153 in mice with high stringency for classical PKC consensus sites but low stringency PKC␦ sites. Neither Ser-153 nor Thr-153 was predicted to a PKC⑀ consensus site using the Scansite software. High stringency indicates that the sequence motif indentified in the query is within the top 0.2% of all matched sequences contained in the Swiss-Prot protein database. Low stringency motifs sequences are in the top 5%.
How can the PKC⑀-dependent phosphorylation of RKIP induced by EtOH that we observed be explained? A recent structural analysis of RKIP protein may provide one possible explanation (66). Mammalian RKIP, like other members of the evolutionarily conserved phosphatidylethanolamine-binding protein (PEBP) superfamily, is structurally characterized by a conserved ligand binding pocket that is capable of binding chemically distinct ligands, including but not limited to phosphatidylethanolamine and the protein Raf-1. Importantly, occupancy of the RKIP binding pocket appears to exert an allosteric effect on RKIP, modulating both its association with and phosphorylation by specific protein kinases involved in its regulation (66). Allosteric regulation of pancreatic acinar cell RKIP by EtOH or its metabolites may explain the enhanced phosphorylation by PKC⑀ observed in Fig. 2. Alternatively, it is also possible that the cell-free assay used by Corbit et al. (44) lacked critical cellular components, such as specific phospholipids or yet to be identified proteins present in pancreatic acinar cells that occupy the RKIP binding site, altering RKIP affinity for specific PKC isozymes.
Finally, our data suggest that in addition to mediating the toxic effects of EtOH, RKIP also regulates normal physiological secretagogue signaling in pancreatic acinar cells. Physiologic concentrations of secretagogues such as acetylcholine and CCK trigger zymogen exocytosis by repetitively releasing Ca 2ϩ from multiple intracellular stores (20). The demonstration that stimulation of AR42J cells with a physiologic concentration of CCK (0.01 nM) induced an increase in Ser(P)-153-RKIP (Fig. 1D) levels suggests that under physiologic conditions CCK can modulate its own receptor desensitization (homologous desensitization) by stimulating the phosphorylation of RKIP that in turn reduces the activity of GRK at the agonist-activated receptor. Although the physiological role of RKIP-mediated attenuation of CCK receptor desensitization was not specifically addressed in our study, we hypothesize that RKIP-dependent modulation of GRK may be part of the mechanism by which pancreatic secretagogues generate the oscillatory changes in [Ca 2ϩ ] cyto associated with normal meal-induced secretion. Future studies will examine the validity of this hypothesis.
Alcohol abuse is a leading risk factor for both recurrent acute and chronic pancreatitis. Understanding the molecular mechanisms by which EtOH and its metabolites exert their toxic effects on pancreatic acinar cells can help to inform the development of new therapeutic strategies for the prevention or treatment of alcohol-associated pancreatitis. Modulation of RKIP expression may have future utility in this endeavor.