Biochimica et Biophysica Acta 1813 (2011) 2145–2156

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Biochimica et Biophysica Acta

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PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerousimportant cellular signaling cascades

Veronica Sancho a, Marc J. Berna b, Michelle Thill c, R.T. Jensen a,⁎a Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1804, USAb Universitätsklinikum Eppendorf, Medizinische Klinik I, 20246 Hamburg, Germanyc Universitätsklinikum Eppendorf, Klinik und Poliklinik für Augenheilkunde, 20246 Hamburg, Germany

Abbreviations: CCK, COOH-terminal octapeptide otetradecanoylphorbol-13-acetate; GI, gastrointestinal;immunoprecipitation; Co-IP, co-immunoprecipitation;G protein-coupled receptor; PYK2, proline-rich tyrosinekinase; IKK, IκB kinase; AKT, protein kinase B; VIP, vasohepatocyte growth factor; EGF, epidermal growth factgrowth factor; PDGF, platelet-derived growth factor; TGbeta; IGF-1, insulin-like growth factor 1; bFGF, basicprotein kinase A; DAG, diacylglycerol; 8-Br-cAMP, 8-Brosphate; TCR, T-cell receptor; PLC, phospholipase C; HRP,ERK, mitogen-activated protein kinase; IRS-1, insulincaspase recruitment domain (CARD) carrying memberguanylate kinase (MAGUK) family proteins; MALT-1, muctranslocation gene 1; Cbl, Casitas B-lineage lymphomalymphoma/leukemia 10 protein; PI3K, phosphatidylinadhesion kinase β; NFκβ, nuclear factor-kappa B; PAR2,PAR4, protease-activated receptor 4⁎ Corresponding author at: NIH/NIDDK/DDB, Bldg. 10,

1804, Bethesda, MD 20892-1804, USA. Tel.: +1 301 496E-mail address: robertj@bdg10.niddk.nih.gov (R.T. Je

0167-4889/$ – see front matter. Published by Elsevierdoi:10.1016/j.bbamcr.2011.07.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 February 2011Received in revised form 12 July 2011Accepted 13 July 2011Available online 23 July 2011

Keywords:PKCθ activationPancreatic aciniCCKSignalingPancreatic growth factorsPKC

The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to havea restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis,T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance ininsulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions.However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmittersandgrowth factors. In thepresent studyweused rat pancreatic acinar cells to explore their ability to activate PKCθand the possible interactions with important cellular mediators of their actions. Particular attention was paid tocholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processesaffecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activationwas stimulated only by hormones/neurotransmitters activating phospholipaseC. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCKA-receptoractivation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125FAK and IKKα/β,but not basal/stimulatedenzymesecretion.AlsoCCK-andTPA-inducedPKCθ activationproducedan increment inPKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence inpancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cellsignaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokineexpression, and pathological responses like pancreatitis and cancer growth).

f cholecystokinin; TPA, 12-O-CCK-JMV, CCK-JMV-180; IP,

PKD, protein kinase D; GPCR,kinase 2; FAK, focal adhesionactive intestinal peptide; HGF,or; VEGF, vascular endothelialFβ, transforming growth factorfibroblast growth factor; PKA,mo-cyclic adenosinemonopho-horseradish peroxidase; MAPK/receptor substrate 1; CARMA,of the membrane associatedosa-associated lymphoid tissueproto-oncogene; Bcl-10, B-cellositol-3-; Kinase, CAKβ, cellprotease-activated receptor 2;

Rm. 9C-103, 10 Center Dr. MSC4201; fax: +1 301 402 0600.nsen).

B.V.

Published by Elsevier B.V.

1. Introduction

Protein kinase C θ (PKCθ) belongs to the threonine/serine kinasesuperfamily PKC [1]. In mammals this superfamily is comprised of 12isoformsdivided in3groupsdependingon their activation requirements:conventional PKC isoforms (α, βI, βII and γ), which activation dependsonDAGandCa2+; novel (δ, ε,η and θ),whose activationdependsonDAGbut not Ca2+ and atypical (λ/ι, μ and ζ), whose activation is independentfrom both DAG and Ca2+ [1]. Once activated by phosphorylation andcofactor binding [2], PKCs stimulate serine/threonine phosphorylation ofmany cellular proteins including other kinases, cytoskeletal proteins,structural proteins, enzymes, adapter proteins and receptors, and theiractivation hasmultiple effects in both normal and pathological processesincluding differentiation, proliferation, apoptosis, cell death, secretion,adhesion and cell migration [3].

The novel PKCθ isoform, which is the most recently described [4],was originally thought to have a restrictive distribution with highexpression in T-cells [4], brain [4] and skeletal muscle [4]. Its activationand effect on various cellular processes have been primarily studied in

2146 V. Sancho et al. / Biochimica et Biophysica Acta 1813 (2011) 2145–2156

these tissues, especially in T-cells [5]. Numerous subsequent studiesshow that PKCθ is more widely distributed than originally described [4]and it has been detected in a number of other tissues such as in testis [4],intestinal epithelial cells [6] and mast cells [7]; and in several humantumor and tumor cell lines [such as human gastrointestinal stromaltumor [8], human colorectal cancer [9] and gastric cancer cells KATO-III[10]].

Studies demonstrate that PKCθ activation plays an importantfunction in various tissues including T cell antigen receptor (TCR)activation, proliferation, apoptosis [5], insulin secretion [11], insulinsignaling in muscle [12], barrier permeability in intestinal epithelium[6], thrombus formation in platelets [13] and mast cell activation [7].However, there is little information on the activation of PKCθ bygastrointestinal (GI) hormones/neurotransmitters and growth factors.

Pancreatic acinar cells are an excellent model system to studykinase activation by GI hormones/neurotransmitters and growthfactors because many GI hormones/neurotransmitters and growthfactors can alter pancreatic acinar function and signaling cascadesincluding phospholipases (A, C, D), adenylate cyclase, tyrosine kinasesand other serine/threonine kinases [14–19]. In pancreatic acinar cellsfrom normals or animals with pancreatic disorders (pancreatitis,pancreatic cancer), PKC activation, including conventional and othernovel PKCs (PKCδ and PKCε), has been implicated in several processes.These include enzyme secretion, activation of proteases, inflammatoryresponses, growth and apoptotic pathways stimulated by variouspancreatic hormones/neurotransmitters or growth factors, as well asother stimulants [20–24]. At present, it is unclear whether PKCθ ispresent in pancreatic acinar cells, whether any pancreatic neurotrans-mitter/hormones or growth factors can activate it or whether itparticipates in any of signaling cascades mediating either thephysiological or pathological processes caused by pancreatic neuro-transmitter/hormones or growth factor stimulation of pancreatic acinarcells.

The purpose of the present study was to address these issues andto determine whether PKCθ is present in pancreatic acinar cells, ifgastrointestinal hormones/neurotransmitters can activate this novelprotein kinase, PKCθ, and if so, to provide insights into the possiblemechanisms of its interactions with various known important cellularmediators of the actions of these pancreatic stimulants. Particularattention was paid to the hormone/neurotransmitter, cholecystokinin(CCK), because it is not only a physiological regulator of pancreaticacinar cell function, it is also important in a number of importantpathological processes affecting acinar cell function, such as pancre-atitis [23,25,26].

2. Materials and methods

2.1. Materials

Male Sprague–Dawley rats (150–250 g) were obtained fromthe Small Animals Section, Veterinary Resources Branch, NationalInstitutes of Health (NIH), Bethesda, MD. Rabbit anti-phospho-proteinkinase C θ (PKCθ) pT538, rabbit anti-PKCθ, rabbit anti-phospho-Srcfamily (Y416), mouse monoclonal anti-phospho p44/42 mitogen-activated protein kinase (MAPK) (T202/Y204) (E10), rabbit anti-Akt,rabbit anti-RafA, rabbit monoclonal anti-RafB (55C6), rabbit anti-RafC,rabbit anti-protein kinaseD (PKD), rabbit anti-protein kinase δ (PKCδ),rabbit anti-14-3-3-γ, rabbit monoclonal anti-Bcl-10 (C78F1), rabbitanti-Mucosa-associated lymphoid tissue translocation gene 1 (MALT-1),rabbit anti-c-Cbl, rabbit anti-phospho Akt (T308), rabbit anti-phospho-IκB kinase (IKK) IKKα (Ser180)/IKKβ (Ser181), rabbit monoclonal anti-phospho Raf C (56A6), rabbit phospho-PKD (Ser744/748), rabbitphospho-FAK (Tyr397), rabbit phospho-Pyk2 (Tyr402), rabbit phospho-PKCδ (Tyr311), rabbit anti-α/β tubulin antibodies and nonfat drymilk were purchased from Cell Signaling Technology, Inc. (Beverly,MA). Mouse monoclonal anti-PKCθ (E-7) antibody, mouse monoclonal

anti-Lyn (H-7), mouse anti-pan Src, bovine anti-goat horseradishperoxidase (HRP)-conjugate and anti-rabbit-HRP-conjugate antibodieswere from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mousemonoclonal anti-PKCθ (clone 27) was purchased from BD Biosciences(San Jose, CA). Mouse monoclonal anti-cadherin (36/E) antibody wasfrom BD Transduction laboratories (Lexington, KY). Mouse monoclonalanti-calpain (15C10) antibody was from Biosource International,Inc. (Camarillo, CA). Rabbit anti-phosphatidylinositol-3-kinase p85(PI3K) was purchased from Upstate Biotechnology (Lake Placid, NY).Tris/HCl pH 8.0 and 7.5 were from Mediatech, Inc. (Herndon, VA).2-mercaptoethanol, protein assay solution, sodium lauryl sulfate (SDS)and Tris/Glycine/SDS (10×) were from Bio-Rad Laboratories (Hercules,CA). MgCl2, CaCl2, Tris/HCl 1 M pH 7.5 and Tris/Glycine buffer (10×)were fromQuality Biological, Inc. (Gaithersburg,MD).Minimal essentialmedia (MEM) vitamin solution, Dulbecco's Modified Eagle Medium(DMEM), Waymouth's medium, basal medium Eagle (BME) aminoacids 100×, Dulbecco's phosphate buffered saline (DPBS), glutamine(200 mM), Tris–Glycine gels, L-glutamine, fetal bovine serum (FBS),0.05% trypsine/EDTA solution, penicillin–streptomycin, Alexa594, Alexa488-conjugated anti-rabbit secondary antibodies and glycerol werefrom Invitrogen (Carlsbad, CA). COOH-terminal octapeptide of chole-cystokinin (CCK), hepatocyte growth factor (HGF), bombesin, insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF),vasoactive intestinal peptide (VIP), endothelin and secretin were fromBachem Bioscience Inc. (King of Prussia, PA). CCK-JMV-180 (CCK-JMV)was obtained from Research Plus Inc., Bayonne, NJ. Epidermal growthfactor (EGF), thapsigargin, platelet-derivedgrowth factor (PDGF), vascularendothelial growth factor (VEGF), deoxycholic acid, protein kinase Cisoenzyme sample kit andmyristolated PKCθ pseudosubstrate were fromCalbiochem (La Jolla, CA). Carbachol, insulin, transforming growth factorbeta (TGFβ), dimethyl sulfoxide (DMSO), 12-O-tetradecanoylphobol-13-acetate (TPA), 8-bromoadenosine 3′5′ cyclic monophosphate sodium(8-Bromo-cAMP), L-glutamic acid, glucose, fumaric acid, pyruvic acid,trypsin inhibitor, HEPES, TWEEN® 20, Triton X-100, phenylmetha-nesulfonylfluoride (PMSF), ethylenediaminetetraacetic acid (EDTA),ethylene glycol tetraacetic acid (EGTA), sucrose, sodium-orthovanadate,sodiumazide, Nonidet P40, sodiumpyrophosphate,β-glycerophosphate,sodium fluoride, dithiothreitol, AEBSF, MOPS (3-(N-morpholino)propa-nesulfonic acid), methanol and CeLytic™MCell Lysis Reagent were fromSigma-Aldrich, Inc. (St. Louis, MO). Albumin standard, Protein G agarosebeads and Super Signal West (Pico, Dura) chemiluminescent substratewere from Pierce (Rockford, IL). Protease inhibitor tablets, pepstatin,leupeptine and aprotine were from Roche (Basel, Switzerland). Purifiedcollagenase (type CLSPA) was from Worthington Biochemicals (Free-hold, NJ). Nitrocellulose membranes were from Schleicher and SchuellBioscience, Inc. (Keene, NH). Biocoat collagen I Cellware 60 mm disheswere from Becton Dickinsen Labware (Bedford, MA). Albumin bovinefraction V was from MP Biomedical (Solon, OH). NaCl, KCl, acetone,phosphoric acid and NaH2PO4 were from Mallinckrodt (Paris, KY). HEK293 cells were from ATCC (Manassas, VA). Dominant negative PKCθadenovirus, Quick Titer™ Adenovirus Quantification Kit and ViraBind™Adenovirus Purification Kit were from Cell Biolabs, Inc. (San Diego, CA).Ad-CMV-Null was from Vector Biolabs (Philadelphia, PA). RNA PCR Kit,DNA-polymerase (AmplitaqGold), 10×PCRbuffer anddeoxynucleotideswere from Applied Biosystems (Foster City, CA). L-364,718 (3S(−)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl-1H-indole-2-carboxamide) and L-365,260 (3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N′-(3-methylphenyl)urea) were from Merck, Sharp and Dohme (WestPoint, PA). YM022 ((R)-1-[2,3-dihydro-1-(2′-methyl-phenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3-(3-methylphenyl)urea) and SR27897 (1-[[2-(4-(2-chlorophenyl)-thiazol-2-yl)amino-carbonyl] indolyl]acetic acid were from Tocris Bioscience (Ellisville,MO). Phadebas reagent was fromMagle Life Science (Lund, Sweden).PKC Assay Kit and Histone H1 were from Millipore (Temecula, CA).[γ-32P]ATP (3000 Ci/mmol) was from Perkin Elmer (Waltham, MA).

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2.2. Methods

2.2.1. Tissue preparationPancreatic acini were obtained by collagenase digestion as previously

described [17]. Standard incubation solution contained 25.5 mM HEPES(pH 7.45), 98 mM NaCl, 6 mM KCl, 2.5 mM NaH2PO4, 5 mM sodiumpyruvate, 5 mM sodium glutamate, 5 mM sodium fumarate, 11.5 mMglucose, 0.5 mMCaCl2, 1 mMMgCl2, 1 mMglutamine, 1% (w/v) albumin,0.01% (w/v) trypsin inhibitor, 1% (v/v) vitamin mixture and 1% (v/v)amino acid mixture.

2.2.2. Acini stimulationAfter collagenase digestion, dispersed acini were pre-incubated in

standard incubation solution for 2 h at 37 °C with or without inhibitorsas described previously [17,27]. Protein concentration was measuredusing theBio-Radprotein assay reagent. Equal amounts of sampleswereanalyzed by SDS-PAGE and Western blotting.

2.2.3. cDNA preparationRandom hexamer-primed first strand cDNA was obtained with RT

(RNA PCR Kit) from mRNA from rat muscle, pancreas and brain(Stratagene, Clontech and Bio-chain, respectively).

2.2.3.1. PCR. Primers for PKCθ was selected through analysis of the ratPKCθ mRNA sequence (GenBank accession no AB020614.1). The senseand antisense sequences of the PKCθ primer were as follows: sense,5′-TAGAAAGGGAGGCCAAGGAT-3′ (nucleotides 236–256); and anti-sense, 5′-CTGAAGGGTGGGTCAATCTC-3′ (nucleotides 367–386) givinga PCR product size of 151 bp. The presence of the PKCθ mRNA wasdetermined in cDNA samples from ratmuscle, pancreas and brain, usinggenomic DNA as negative control. The PCR was conducted with40 cycles, which were within the linear amplification range. The PCRbegan with a cycle of 94 °C for 10 min, followed by 40 cycles of 94 °Cfor 40 s (denaturing), 58 °C for 40 s (annealing), and 72 °C for 40 s(elongation). A final extension period of 94 °C for 5 min concluded theamplification. PCR products were size-fractionated on agarose gels,stained with ethidium bromide and visualized under UV light.

2.2.4. Western blotting/immunoprecipitationWestern blotting and immunoprecipitation were performed as

described previously [19]. The intensity of the protein bands wasmeasured using Kodak ID Image Analysis, which were assessed in thelinear detection range.When re-probingwas necessarymembraneswereincubated in Stripping buffer (Pierce, Rockford, IL) for 30 min at roomtemperature, washed twice for 10 min in washing buffer, blocked for 1 hin blocking buffer at room temperature and re-probed as described above.

2.2.5. TranslocationTranslocation studies were performed as described previously [16].

Briefly, after stimulation, acini were resuspended in membrane lysisbuffer (50 mMTris/HCl pH 7.5, 150 mMNaCl, 0.1% sodium azide, 1 mMEGTA, 0.4 mM EDTA, 0.2 mM sodium orthovanadate, 1 mM PMSF, andone protease inhibitor tablet per 10 ml). After homogenization, lysateswere cleared by centrifugation. The supernatant (cytosol and mem-brane fraction) was centrifuged for 30 min at 4 °C and 60,000×g. Thepellet containing the membrane fraction was washed twice inmembrane lysis buffer, resuspended in lysis buffer (50 mM Tris/HClpH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodiumazide, 1 mMEGTA, 0.4 mMEDTA, 0.2 mMsodiumorthovanadate, 1 mMPMSF, and one protease inhibitor tablet per 10 ml), sonicated and thencentrifuged at 10,000×g for 15 min at 4 °C. Equal amounts of proteinwere subjected to SDS-PAGE and analyzed by Western blotting.

2.2.6. Immunocytochemistry and immunofluorescence imagingAfter treating isolated pancreatic acini with orwithout stimulants as

indicated, cells were fixed, transferred to glass slides and blocked as

previously described [28], then slides were incubated with a rabbitanti-pT538 PKCθ antibody and a mouse anti-cadherin antibody at adilution of 1:500 overnight at 4 °C. Reactivity was demonstrated byincubation with an Alexa 488-conjugated anti-rabbit or with an Alexa555-conjugated anti-mouse secondary antibody, respectively, at adilution of 1:500 for 2 h RT. Negative controls consisted of replacementof primary antibody with an isotype-matched control. Slides wereanalyzed as previously reported [28].

2.2.7. Amylase releaseAmylase release from isolated pancreatic acinar cells was measured

as previously described [29,30]. Amylase activity was determined withthe Phadebas reagent and expressed as % of total cellular amylasereleased into the extracellular medium during the incubation time.

2.2.8. Co-immunoprecipitation (Co-IP)Co-IP studies were performed as previously described [14]. Briefly,

cells were lysated with CelLytic Buffer (CelLytic™M Cell Lysis Reagent0.2 mM sodiumorthovanadate, 1 mMPMSF, and one protease inhibitortablet per 10 ml), and lysates (750 μg/ml) were incubated with 4 μg ofthe required antibody and with 25 μl of protein G-agarose at 4 °C,overnight. The immunoprecipitates were washed with phosphate-buffered saline and analyzed by SDS-PAGE and Western blotting.

2.2.9. Virus infection and culture aciniPancreatic acini were isolated as described above, infected with

either Ad-CMV-Null (empty adenovirus, as infection control) ordominant negative PKCθ adenovirus at 1×109 VP/ml concentration, asdescribed previously [18]. After 6 h, stimulants were added and cellslysed as described above).

2.2.10. PKCθ kinase activation assayPKCθ kinase activation was measured as previously described [14].

Briefly, after isolation and incubation with 10 nM CCK or 1 μM TPA, 1and 2.5 min, respectively, pancreatic acinar cellswere lysated, PKCθwasimmunoprecipitated with 4 μg BD PKCθ antibody and 25 μl Protein Gagarose beads overnight, 4 °C Kinase assays were performed on thePKCθ immunoprecipitates in two different ways: one using the PKCsubstrate peptide (QKRPSQRSKYL) provided by the PKC kinase assaykit fromMillipore (Temecula, CA), following the directions provided bythe manufacturer; and the other using Histone H1 (1 μg/sample). Thesubstrate peptide was spotted on to p81 filter paper and processedaccording to themanufacturer's instructions.When PKCθ kinase activitywas measure as increments in the 32P-phosphorylation of the proteinhistone H1, the reaction was terminated by the addition of 15 μl ofloading buffer and 5 min at 95 °C, samples were subjected to SDS-PAGEelectrophoresis, gels dried and analyzed in a phosphor imager(InstantImager, Packard Instruments Co.

2.2.11. Statistical analysisAll experiments were performed at least 3 times. Data are

presented as mean±SEM and were analyzed using the Student'st-test for paired data using the software StatView (SAS Institute,Casy, NC). p valuesb0.05 were considered significant. Curve fitting,EC50 and tmax were determined using the GraphPad 5.0 software.

3. Results

3.1. Presence of PKCθ mRNA and protein in pancreas

It is well established that four PKC isoforms (α, δ, ε, ζ) are expressedin rat pancreatic acini [14,24], but also there are contradictory dataregarding the presence in this tissue of the novel isoform PKCθ[24,29,31,32]. In order to clarify this point, both studies of the presencein rat pancreas of PKCθ mRNA and PKCθ protein were determined(Fig. 1). PCRusing specific primers for PKCθmRNAwith cDNAsobtained

Fig. 1. Presence of PKCθmRNA and protein in pancreas. Panel A: PCR results using PKCθspecific primers and cDNA from rat muscle and rat brain, known to contain PKCθ, andfrom rat pancreas. Panel B: Presence of the protein PKCθ in isolated pancreatic acini.Lysates from pancreatic acini were run in parallel with different human recombinantPKC isoforms (α, δ, ε, θ and ζ) and detected by Western blotting (WB) with a specificanti-PKCθ antibody (Cell signaling). Panel C: Specificity of PKCθ antibodies wasexamined by immunoprecipitation (IP) of the PKC isoforms (α, δ, ε, θ and ζ) frompancreatic acini lysate with a specific anti-PKCθ anti-mouse antibody (BD Biosciences)and detection with a specific anti-PKCθ anti-rabbit antibody (Cell Signaling). A singleband was obtained just in the case of PKCθ isoform, showing the specificity of theantibodies used.

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from brain, muscle (tissues known to express this enzyme) and ratpancreas was performed, and it resulted in a single band of 151 bp inall cases (Fig. 1, Panel A, Rows 1–3). To determine whether the PKCθprotein is present in pancreatic acini, a Western blot was performwith different human recombinant PKC isoforms running in parallelwith lysate obtained from rat pancreas acini (Fig. 1, Panel B).Immunodetected with a PKCθ total antibody (Cell Signaling),resulting in a single band of 80 kDa in the pancreatic acinar lysateand only with recombinant human PKCθ (Fig. 1, Panel B, Rows 1 and4), showing the presence of PKCθ protein in rat pancreatic acini. Thespecificity of the PKCθ antibody used was assessed by immunopre-cipitation of the known isoforms present in the lysates of the pancreasacini (α, δ, ε, ζ) with PKC specific antibodies as well as with a PKCθantibody (BD Bioscience), with subsequent analysis by Western blot,using a different total PKCθ antibody (Cell Signaling) for the immuno-detection. Only one band of 80 kDa was detected in the lanecorresponding to the immunoprecipitationwith the total PKCθ antibody(Fig. 1, Panel C, Row 5), demonstrating the specificity of the antibodiesfor PKCθ.

3.2. Ability of various pancreatic secretagogues and pancreatic growthfactors to stimulate PKCθ phosphorylation (pT538) in rat pancreatic acini

In order to establish whether PKCθ is not only expressed but alsoactivated by known pancreatic secretagogues or growth factors [15], ratpancreatic acini were incubated in the absence and presence of severalgastrointestinal hormones (CCK, carbachol, bombesin, secretin, VIP)known to activate pancreatic acinar cells and cause enzyme secretion[15]. The pancreatic secretagogues that activate phospholipase C (CCK,carbachol and bombesin) stimulated a significant increase in the PKCθphosphorylation in threonine 538 (pT538) (Fig. 2, Panel A, Rows 2–4).Neither of the gastrointestinal hormones (VIP and secretin) that causean increase in the cAMP,nor the cAMPanalog, 8-Bromo-cAMPwere ableto increase thephosphorylation of PKCθ T538 (Fig. 2, Panel A, Rows 5–8).As a measurement of PKCθ activity the phosphorylation of T538 ofPKCθwas assessed because it is known that an increase in the threonine538 phosphorylation of PKCθ is directly related to the activation of thisenzyme [33].

None of the known pancreatic growth factors (insulin, EGF, PDGF,VEGF, bFGF, IGF, TGFβ and HGF) [15] were able to activate PKCθ andstimulate threonine 538 phosphorylation of PKCθ (Fig. 2, Panel B,Rows 3–10). This lack of stimulationwas not due to the inability of thegrowth factors to activate acinar cells because each stimulated pS473Akt phosphorylation (data not shown).

In order to determine if any of the CCK stimulating effect on pT538PKCθ phosphorylation was caused by the presence/activation of CCKA

or CCKB receptors in the pancreatic acinar cell, isolated acinar cellswere first incubated with either CCK, gastrin, or a known CCKA

receptor agonist (A71378) [34] (Fig. 2, Panel C, Lanes 1–4). Gastrin didnot produce any increase in pT538 PKCθ phosphorylation, and that theCCK activation of PKCθ was mimicked by the incubation of the cellswith the CCKA receptor agonist. Moreover, when the acinar cells wereincubated with CCK and two different CCKA receptor antagonists[L364,718 or SR27897] [30,35] the increment in PKCθ phosphorylationobserved in the sole presence of CCK was completely inhibited, butnot in the presence of CCKB receptor antagonists [L365,260 or YM022][30,36] (Fig. 2, Panel C, Lanes 5–10). These results demonstrate thatthe observed effect of CCK in PKCθ phosphorylation is only due to theactivation of the CCKA receptors.

3.3. Dose–response effect of CCK and CCK-JMV on PKCθ T538phosphorylation in rat pancreatic acini

As CCK has an important role in both the physiology andpathophysiology of the pancreas [15,37], we focused our study in theactivation of PKCθ exerted by this hormone in rat pancreatic acini.Increasing concentrations of CCK produced a biphasic increase in T538phosphorylation of PKCθ with concentrations from 0.1 nM to 10 nMcausing increasing stimulation, and then concentrations from 100 to1000 nM CCK causing less stimulation (Fig. 3, Panel A). The maximalstimulation occurred with 10 nM CCK (239±25% of control=100±10% of maximal response) and CCK's half-maximal effect (EC50)occurred with 0.174±0.008 nM (Fig. 3, Panel A). The CCKA receptor inpancreatic acini can exist in two different activation states, a low and ahigh-affinity state, and the activation of the different states activatesdifferent cell signaling cascades [18,26,38–40]. Inorder to determine thecontribution of each activation receptor state to the activation of PKCθby CCK, pancreatic acini were incubated in the presence of increasingconcentrations of CCK-JMV, known to be an agonist of the CCKA highaffinity state and an antagonist of the low affinity CCKA receptor state inrat pancreatic acini [18,19]. CCK-JMV stimulated threonine 538phosphorylation of PKCθ in a monophasic manner with concentra-tions from 10 nM to 1000 nM (Fig. 3) with an EC50 of 1.262±0.033 nM (Fig. 3, Panel A), and therefore was 7-times less potent thanCCK. CCK-JMV caused 32% of the maximal stimulation of T538 PKCθphosphorylation caused by CCK (Fig. 3, Panel A). These results

Fig. 2. Ability of various pancreatic secretagogues and growth factors to stimulate PKCθphosphorylation (pT538) in rat pancreatic acini. Panel A: Isolated pancreatic acini wereincubated in the absence or presence of different pancreatic secretagogues for 5 min,and then lysed.Western blots were analyzed using anti-pT538 PKCθ and anti-total PKCθ(PKCθ), as loading control. Top: Results of a representative blot of 5 independentexperiments are shown. Bottom: Means±S.E. of 5 independent experiments. Results areexpressed as % of control. *pb0.05 compared to the control group. Panel B: Isolatedpancreatic acini were incubated in the absence or presence of different growth factorsfor 5 min and HGF for 10 min, and then lysed. Western blots were analyzed using anti-pT538 PKCθ and anti-total PKCθ (as loading control). Results of a representative blot of4 independent experiments are shown. *Significantlygreater thancontrol.Panel C: Isolatedpancreatic acini were incubated in the absence or presence of CCK, gastrin or A71378 for1 min, orpreincubated for 5 min in thepresenceof L364,718, YM022, L365,260or SR27897and then in the additional presence of CCK 10 nM for 1 min, and then lysed.Western blotswere analyzed using anti-pT538 PKCθ and anti-total PKCθ (as loading control).

Fig. 3. Dose–response effect of CCK and CCK-JMV on PKCθ phosphorylation in ratpancreas acini. Panel A: Isolated pancreatic acini were incubated in the absence orpresence of CCK and CCK-JMV for 1 min, lysed and subjected to Western blotting asdescribed in Fig. legend 2. Top: Results of a representative blot of 4–5 experiments isshown. Bottom: Means±S.E. of 4–5 independent experiments, with CCK or CCK-JMV,respectively. Results are expressed as % of maximal stimulation caused by CCK. *pb0.05compared to the control with no additions. Panel B: Isolated pancreatic acini wereincubated in the absence or presence of CCK or TPA for 2.5 min, and then lysed. PKCθwas immunoprecipitated from equal amount of protein (1 mg/ml) and subjected tokinase activity measurement, as outlined in the Materials and methods. This result is arepresentative experiment of 5 others.

Table 1Effect of CCK and TPA on PKCθ kinase activity.

Stimulant % Kinase Activity

Substrate peptide Histone H1

None 100±1 100±2CCK (10 nM) 139±4⁎⁎ 142±4⁎⁎

TPA (1 μM) 235±22⁎⁎ 173±30⁎

PKCθ kinase activity is expressed as the percentage of phosphorylation of either asubstrate peptide or Histone H1 protein, as outlined in Materials and methods. Resultsare expressed as means±S.E. of 5 independent experiments.⁎ pb0.0001.⁎⁎ pb0.005 vs control.

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support the conclusion that CCK stimulation of PKCθ activation ismediated 30% by the high affinity state CCKA receptor and 70% byactivation of the low affinity CCKA receptor state. As a CCKconcentration of 10 nM produces the maximal stimulation of PKCθphosphorylation, it was selected for the rest of the study.

To further demonstrate that CCKwas activating PKCθ, we studied itsability to stimulate and increase in PKCθ kinase activity (Table 1, Fig. 3,Panel B). Both CCK and TPA produced a significant increment in thephosphorylation of the Histone H1 protein (Fig. 3, Panel B) and PKCsubstrate peptide (Table 1) which was assessed after performing PKCθ

immunoprecipitation from pancreatic acinar cells, previously stimulat-ed with CCK or TPA (Fig. 3, Panel B).

3.4. Time course of CCK stimulation of PKCθ T538 phosphorylation in ratpancreatic acini

StimulationofpT538 threoninephosphorylationbyCCKwas rapid andthe time course was biphasic (Fig. 4). Specifically, CCK stimulated a rapidinitial increase reaching amaximumwithin 1 min (tmax: 0.78±0.08 min)(maximum: 197±19% of control). Subsequently, the magnitude of

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pT538 phosphorylation stimulated by CCK was significantly less thanthemaximal but was greater than the basal level for 30 min (145±11%of control) (Fig. 4).

Because phospholipase C (PLC) stimulating pancreatic hormones,which subsequently activate PKC, stimulated PKCθ T538 phosphoryla-tion, the ability of TPA, a known PKC activator, to stimulate PKCθ T538phosphorylation at different times, was also studied (Fig. 4). TPAproduced, as in the case of CCK, a rapid (1 min) and significant increase(188±13% of control) in PKCθ pT538 phosphorylation (Fig. 4). Thisincrease was maintained for 5 min, and then the stimulation slowlydecreased over a 30 min period but still remained significantly abovethe control level (30 min, 171±23%of control).WithTPA, the tmax value(1.60±0.06 min) was longer than that seen with CCK stimulation.

Fig. 5. Ability of CCK and TPA to stimulate translocation of PKCθ to the cell membranesin rat pancreatic acinar cells. Isolated acini were incubated with or without CCK or TPAfor 1 or 5 min, respectively. Samples were processed as described in Materials andmethods to obtain subcellular fractions, and then subjected to Western blotting. PanelA: Results from one experiment representative of 5 separate experiments. In the upperpanel, membranes were analyzed using anti-PKCθ antibody. Lower 2 panels: To assess

3.5. Ability of CCK and TPA to stimulate translocation of PKCθ to the cellmembranes in rat pancreatic acinar cells

Because PKCθ translocation to the plasma membranes as well as itspT538 phosphorylation are needed for activation [33,41] we examinedthe ability of CCK and TPA to cause translocation of total PKCθ. CCK orTPA produced a significant increment in the membrane associatedPKCθ after 1 min or 5 min incubation, 216±34 and 189±59%of controlpb0.05, respectively (Fig. 5, Panel A (Lanes 1–3) and Panel B). Theseincubation times were chosen because this is when both stimulantsexerted their maximum effects on PKCθ phosphorylation (Fig. 4). Toconfirm that the separation of the cytosol and membrane fractionswas adequate, equal protein amounts of each fraction were subjectedto Western blot and immunodetection was performed either withE-cadherin (membrane fraction) or calpain (cytosol marker) antibody(Fig. 5, Panel A). This immunodetection also showed equal loading ineach experiment condition and good separation of the cytosol andmembrane fractions (Fig. 5, Panel A).

We also studied the translocation of PKCθ to the membrane afterstimulation with CCK or TPA by immunofluorescense-cytochemistry,

Fig. 4. Time course of CCK and TPA stimulation of PKCθ T538 phosphorylation in ratpancreatic acini. Isolated pancreatic acini were incubated in the absence or presence ofCCK or TPA for the indicated times, lysed and subjected toWestern blotting as describedin Fig. legend 2. Top: Results of a representative blot of 5 independent experiments areshown. Bottom: Means±S.E. of 5 independent experiments. Results are expressed as %of control. *pb0.05 compared to the control value (i.e. 0 time).

the effectiveness of subcellular fractionation, the cytosol and membrane fractions wereanalyzed using anti-calpain antibody, a marker for the cytosol fraction, and anti-E-Cadherin Ab, a marker for the membrane fraction. Panel B shows the mean values±S.E.of 5 independent experiments. Results are expressed as % of control. *pb0.05 comparedto the control group.

and we observed that in the absence of CCK or TPA, PKCθ washomogenously distributed through the cytoplasm (Fig. 6, Panel A).After TPA or CCK treatment, the phosphoT538 PKCθ was increased inthe membrane in both cases (Panels B and C, Fig. 6, respectively), withthe increase caused by TPA more prominent. This results were similarto those obtain by the membrane fractionating approach (Fig. 5).

3.6. Ability of CCK and TPA to stimulate the association of PKCθ withvarious cellular proteins in rat pancreatic acinar cells

PKCθ is reported to interact with a number of other importantcellular signaling proteins in different cell types including Src [41], PKD[42], Raf [42], CARMA and IKK [43], Cbl [44], 14-3-3 [45], Blc-10 [46],MALT-1 [47] and Akt [12], kinases known to be particularly importantin regulating growth and apoptosis. In order to determine the possibleinteraction of PKCθ with these proteins in the pancreatic acini, westudied the ability of CCK or TPA to stimulate a direct interaction of PKCθwith these proteins by performing co-immunoprecipitation (Co-IP)studies (Fig. 7). We first tested 2 different PKCθ antibody's abilities toimmunoprecipitate PKCθ, one that recognized the N terminal sequenceof PKCθ (BD Bioscience) and another that recognizes the C terminalsequence of PKCθ (Santa Cruz: SC) (Fig. 7, Panel A). We observed thatboth PKCθ antibodieswere able to immunoprecipitate the PKCθ isoformfromhuman recombinant PKCθprotein solution and from rat pancreaticacini incubated in the absence or presence of CCK (10 nM) and TPA(1 μM), respectively. We next investigated the Co-IP of PKCθ and the

Fig. 6. PKCθ immunofluorescense-cytochemistry in rat pancreatic acini with andwithout TPA or CCK treatment. Pancreatic acini were incubated with no additions, TPA(1 μM) or CCK (10 nM) for 10 min. After stimulation cells were washed, fixed,permeabilized and transferred onto poly-l-lysine coated glass slides by cytocentrifuga-tion as described in Materials and methods. Cells were then labeled using polyclonalrabbit anti-pT538 PKCθ (Cell Signaling) and mouse anti E-cadherin primary antibodies.Specific binding was detected using an Alexa 488- and Alexa 555-conjugated secondaryantibodies, respectively, so that green staining represents staining for pT538 PKCθ andred staining represents E-cadherin (in inserts). Nuclei were counterstained using DAPI(blue). Fluorescent images were collected using a Leica CTR5000 microscope. Panel Ashows acini treated with incubation buffer only (control). Panels B–C show cells treatedfor 10 min with 1 μM TPA or 10 nM CCK. Shown are the results of a typical experimentrepresentative of 4 independent experiments. Cells shown are representative of N90%of total cells present.

Fig. 7. Ability of CCK and TPA to stimulate the association of PKCθ with Akt, RafA, RafCand Lyn, in rat pancreatic acinar cells. Panel A: Human recombinant PKCθ proteinsolution and pancreatic acinar lysates were immunoprecipitated with anti-C terminal(Santa Cruz: SC) or N terminal anti-PKCθ antibody (BD Bioscience: BD), or directlysubjected to Western blot (WB alone), and subsequent immunodetected with adifferent anti-PKCθ antibody (Cell Signaling: CS). Panel B: Isolated pancreatic acini wereincubated for 2.5 min in the absence and presence of CCK or TPA. Equal amounts ofproteins were immunoprecipitated with an anti-PKCθ (BD) or anti-Lyn antibody, andthen subjected to Western blotting, using as first antibody anti-Akt, anti-PKCθ (CellSignaling), anti-RafA or anti-RafC antibody. Results are representative of 3 independentexperiments. Panel C: Isolated pancreatic acini were incubated for 2.5 min in theabsence and presence of CCK or JMV. Equal amounts of proteins were immunopreci-pitated with an anti-PKCθ (BD) or anti-Lyn antibody, and then subjected to Westernblotting, using as first antibody anti-PKCθ (Cell Signaling), anti-RafA or anti-RafCantibody. Results are representative of 3 independent experiments.

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different proteins after incubation with CCK (10 nM) or TPA (1 μM),concentrations that caused a maximal PKCθ activation. CCK (10 nM)(Fig. 7, Panel B, Lane 2) and TPA (1 μM) (Fig. 7, Panel B, Lane 3) producedan increase in the association of PKCθ with Akt, RafA, RafC and the Srckinase, Lyn (Fig. 7, Panel B, Rows A–D), but not with MALT-1, Bcl-10,14-3-3γ or Cbl (data not shown). To ensure that the antibody used forimmunoprecipitation was not the cause of lack of association, werepeated the Co-IP experiments using the other PKCθ antibody for theimmunoprecipitation and MALT-1, Bcl-10, 14-3-3γ or Cbl antibody for

the immunodetection, and also in the reverse direction: immunopre-cipitating with MALT-1, Bcl-10, 14-3-3γ or Cbl antibody and immuno-detection with both PKCθ antibodies, and we did not obtain anystimulated association with PKCθ by after incubation with CCK or TPA.

In order to establish whether this direct association of PKCθ withother kinases was produced at low (physiological) or high (supraphy-siological) CCK concentrations, Co-IP experimentswere performedwithCCK concentrations that occupy the high or low affinity receptor states(CCK concentrations: 0.1–1–10 nM) and also with CCK-JMV, known tobe anagonist only of the CCKAhigh affinity state and anantagonist of thelow affinity CCKA receptor state in rat pancreatic acini [18,19]. And wehave found that the direct association of PKCθ with either Lyn, RafA orRafC was observed also at low CCK concentration and with CCK-JMV(Fig. 7, Panel C). CCK-JMV stimulated an association of 33±12% of that

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exerted by CCK10 nM, a value thatwas not different from that observedin the T538 PKCθ phosphorylation exerted by JMV in relation to CCK10 nM (Fig. 3, Panel A).

3.7. Effects of PKCθ inhibition by PKCθ pseudosubstrate on thephosphorylation of various cellular signaling molecules

In order to investigate further the role of this PKCθ activation instimulating various intracellular signaling cascades in pancreatic acini,we used a PKCθ inhibitor, a PKCθ pseudosubstrate. This is a cell-permeable myristolyated peptide, and a selective and competitiveinhibitor of this PKC isoenzyme that includes amino acids 113–125 ofthe pseudosubstrate sequence. The pseudosubstrate produced aninhibition of the pT538 phosphorylation of PKCθ stimulated by CCK(48±3% vs CCK alone pb0.003) or TPA (57±4% vs TPA alone pb0.003)(Fig. 8, RowA), but it did not affect thephosphorylation of another novelPKC isoform, PKCδ, known to be present in the pancreas and to beactivated by CCK or TPA [17,48] (Fig. 8, Row B). The possibility ofblocking the activation of PKCθ give us the opportunity to study theeffect of this inhibitor on the activation of kinases known to bestimulated in pancreatic acini by CCK, such as PKD, Src, RafC, RafA, PYK2,FAK, IKK, Akt, and p44/42 MAPKs and to assess whether they could beactivated by PKCθ. The inhibition of PKCθ by the PKCθ pseudosubstrateinhibitor decreased the phosphorylation of PKD stimulated by eitherCCK or TPA (Fig. 8, Row C, Lanes 2–3) (19.4±3.69 and 19.1±3.0 foldsover control, respectively, both pb0.05) to values still above the control,but significantly reduced (69±3% and 75±3%, respectively vsstimulant alone, both pb0.04; Fig. 8, Row C, Lanes 5–6). Similarly,CCK- and TPA-stimulated Src pY416 phosphorylation was inhibited(59±3% and 46±6% vs stimulant alone, pb0.02 and pb0.04, respec-tively; Fig. 8, Row D, Lanes 2–3 and 5–6), RafC (39±6% and 33±64 vsstimulant alone, pb0.02 and pb0.01, respectively; Fig. 8, Row E, Lanes2–3 and 5–6). The PKCθ pseudosubstrate partially inhibited CCK or TPAphosphorylation of pY402 of PYK2 (73±5% and 71±8% vs stimulantalone, both pb0.05, respectively; Fig. 8 Row F, Lanes 2–3 and 5–6),

Fig. 8. Effects of PKCθ inhibition by PKCθ pseudosubstrate in the phosphorylation of PKD,Src, RafC, PYK2, FAK and IKK. Isolated pancreas acini were preincubated with or without20 μM PKCθ pseudosubstrate for 3 h and then incubated in the absence or additionalpresence of CCK (10 nM) or TPA (1 μM) for 2.5 and 5 min, respectively, and then lysed.Western blots were analyzed using anti-pT538 PKCθ, anti-pY311 PKCδ, anti-pS744/748PKD, anti-pY416 Src, anti-pS338 RafC, anti-pY402 PYK2, anti-pY397 FAK, anti-pS180/181IKKα/β and, as loading control, anti-PKCθ and anti-α/β tubulin antibodies. Results arerepresentative of 10 independent experiments.

pY397 phosphorylation of p125FAK (73±5% and 71±8% vs stimulantalone, pb0.02 and pb0.01, respectively; Fig. 8, Row G, Lanes 2–3 and5–6)andpS180/181phosphorylation of IKKα/β (55±6%and56±5%vsstimulant alone, pb0.04 and pb0.03, respectively; Fig. 8, Row H, Lanes2–3 and 5–6). However, the increment/decrement in the phosphory-lation state of other kinases produce by CCK or TPA in pancreatic acinarcells, such as Akt and p44/42MAPKs, RafA, or p85 PI3Kwas not affectedby the presence of the PKCθ inhibitor, supporting the conclusion thatactivation of PKCθ in pancreatic acini is not implicated in the activationof these enzymes by CCK or TPA.

3.8. Effects of PKCθ inhibition by dominant negative PKCθ adenovirus onthe phosphorylation of various cellular signaling molecules

In order to confirm the results obtained with the PKCθ pseudosub-strate inhibitor, we used another approach, inhibition by a dominantnegativePKCθ adenovirus. This virus contains in its genomea copyof thePKCθ DNA human sequence with a K/R point mutation at the ATPbinding site which renders it inactive. The infection of pancreatic aciniwith this adenovirus produces over-expression of the mutated PKCθ,where it can function as a dominant negative. Isolated pancreatic aciniwere infected for 6 h in the absence or presence of 109pfu/mg [18]empty adenovirus (control of infection) or the dominant negative PKCθadenovirus, and then incubated in the absence or additional presenceof CCK and TPA. After the 6 hour incubation, an overexpression inPKCθ could be observed (Fig. 9, Panel A, RowA).Moreover, in no case did

Fig. 9. Effects of PKCθ inhibition by dominant negative PKCθ adenovirus in thephosphorylation of PKD, Src, RafC, PYK2 and FAK. Isolated pancreas acini were culturedas described in Materials and methods and infected without and with an emptyadenovirus or a dominant negative PKCθ adenovirus for 6 h, incubated in the absence oradditional presence of CCK (10 nM) or TPA (1 μM) for 2.5 and 5 min, respectively, andthen lysed. Western blots were analyzed using: Panel A: anti-PKCθ, anti-pT538 PKCθ,anti-pY311 PKCδ and, as loading control, anti-α/β tubulin antibodies; Panel B: anti-pS744/748 PKD, anti-pY416 Src, anti-pS338 RafC, anti-pY402 PYK2, anti-pY397 FAKand, as loading control, anti-α/β tubulin antibodies. Results are representative of 4independent experiments.

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the infection with the empty adenovirus resulted in unusual PKCθexpression or result in a change in the ability by CCK or TPA to stimulatepT538 phosphorylation of PKCθ itself (Fig. 9, Panels A and B, Rows A–F,Lanes 4–6). These results supported the conclusion that if any change inthe phosphorylation of the enzymewas observed it would be due to thedominant negative PKCθ, and not because of the adenovirus infection.Over-expression of the dominant negative PKCθ did not alter theincrement in the PKCδ phosphorylation induced by CCK or TPA (Fig. 9,Panel A, Row C), but it produced a completely inhibition of the pT538PKCθ phosphorylation (Fig. 9, Panel A, Row B), showing that theinhibition of the PKCθ dominant negative adenoviruses is specific and itis not affecting the activation of another novel PKC isoform, PKCδ, whichis known to be expressed and activated by CCK in the pancreatic acini[17,48].

The over-expression of the dominant negative PKCθ produced amarked decrease in the stimulation caused by CCK or TPA in thephosphorylation of PKD, Src, RafC, PYK2 and FAK (Fig. 9, Panel B, RowsA–E, Lanes 1–3 vs 7–9), similar to what was observed when cells werepreincubated in the presence of the PKCθ pseudosubstrate inhibitor(Fig. 8).

3.9. Effects of PKCθ inhibition in amylase release

In order to study the possible implication of PKCθ activation in thestimulation of amylase release in rat acinar cells, isolated acini werepreincubated in the presence of 20 μM PKCθ pseudosubstrateinhibitor and subsequently incubated with different stimulants(Table 2). The preincubation with the PKCθ inhibitor did not produceany modification in the stimulation in the amylase secretion by CCK,carbachol or VIP, all known stimulants of pancreatic acinar cellssecretion (Table 2).

4. Discussion

The purpose of the present study was to determine whethergastrointestinal hormones/neurotransmitters can activate the novelprotein kinase, PKCθ, and if so, to provide insights into the possiblemechanisms of its interactions with various known important cellularmediators of the actions of these GI stimulants. This was performedstudying pancreatic acinar cells after first determining whetherPKCθ is present in the cells. Particular attention was paid to thehormone/neurotransmitter, cholecystokinin (CCK), because it is notonly a physiological regulator of pancreatic acinar cell function, it isalso important in a number of important pathological processesaffecting acinar cell function, such as pancreatitis [23,25,26].

The ability of GI hormones/neurotransmitters and GI growth factorsto activate PKCθ was investigated using pancreatic acinar cells for anumber of reasons. First, pancreatic acini are known to be highlyresponsive and activated by numerous GI hormones/neurotransmitters

Table 2Effect of PKCθ inhibition on amylase secretion.

Secretagogue Amylase secretion (% total)

−PKCθ inhibitor +PKCθ inhibitor

None 1.7±0.2 1.7±0.2CCK (0.03 nM) 5.1±0.8 5.8±0.9

(0.1 nM) 7.1±0.6 7.4±0.6(100 nM) 4.5±0.4 4.5±0.3

Carbachol (1 μM) 6.8±0.7 7.0±0.5(1 mM) 6.2±0.7 6.3±0.6

VIP (10 nM) 3.4±0.6 3.6±0.4

Pancreatic acini were pre-incubated for 2 h with 20 μM PKCθ pseudosubstrate and thenfor 15 min with the indicated secretagogues. Amylase secretion is expressed as thepercentage of the total acinar cell amylase released into the medium during theincubation. Results are means±S.E. from 6 different experiments.

and growth factors, and this cell systemhas beenwidely used as amodelsystem to study the cellular signaling and effects of these stimulants[14–17]. Second, there is very little information on the ability ofgastrointestinal hormones/neurotransmitters or growth factors toactivate this novel PKC. Third, previous studies have demonstratedactivation of other novel PKCs (PKCδ, ε), aswell as the conventional PKC,PKCα, are important in mediating some of CCK's physiological andpathophysiological effects in the pancreas, aswell as the effects on thesecells by a number of other gastrointestinal hormones/neurotransmittersand growth factors [14,20,21,24,48,49].

A number of our results support the conclusion that PKCθ is presentin pancreatic acinar cells, even thoughprevious studies havepresentedconflicting results. In a recent review of PKCs in pancreatic acinar cells,as well as in four different studies [20,50–52], only four of the 12known PKCs isoformswere reported to be present in pancreatic acinarcells and PKCθ was not one of them (PKCα, δ, ε and ζ). However, inother studies the presence of PKCθ has been found in two pancreaticacinar tumor cell lines (TUC3 and BMRPA.430) [29,32], reported to bepresent weakly in one study using immunoblotting in isolated ratpancreatic acinar cell lysates [32] and to be present in acinar cells fromhuman pancreas in 15% of the cases studied [31], as well as to bepresent in guinea pig pancreatic duct cells [53] and pancreatic islet ratβ-cells [11]. We established that PKCθ was present in rat pancreaticacini using three different approaches. First, we established that PKCθwas present using immunoblotting and detection with antibodiesspecific for PKCθ, which under the experimental conditions used in thepresent study, did not cross-react with other PKCs, including novelPKCs (δ, ε) known to be present in acinar cells [20,50–52]. Thespecificity of the results for pancreatic acinar cells was established bypreparing dispersed isolated rat pancreatic acini and detecting PKCθ inthese cells and comparing the result with known PKC standardsassessed under identical assay conditions, which established thespecificity of the antibodies used for the detection of PKCθ. Second, wedetected the presence of PKCθmRNA by PCR using primers specific forPKCθ mRNA and demonstrated that the PCR product was similar withthat found in two other tissues known to contain the PKCθ isoform,muscle and brain. Third, we demonstrated by immunofluorescenceusing a specific PKCθ antibody, the presence of PKCθ in isolated ratpancreatic acini in the cytoplasm and its subsequent translocation tothe acinar cell membrane after the acini were exposed to a knownphysiological stimulant of pancreatic acinar function, CCK [25].

In this study we next assessed the ability of various GI hormones/neurotransmitters, as well as GI growth factors to activate PKCθ. Anumber of our results support the conclusion that pancreaticstimulants that interact with G-protein-coupled receptors in acinarcells and stimulate phospholipase C (PLC)-mediated pathways, but notother cascades, nor pancreatic growth factors, activate PKCθ. First, onlycarbachol, bombesin and CCK, all known to activate the PLC-cascade inpancreatic acinar cells [15], and not GI hormones/neurotransmitterswhich activate adenylate cyclase (VIP or secretin) or stimulatedirectly PKA (8-Br-cAMP) [15] stimulated T538 PKCθ phosphoryla-tion. Second, none of the known pancreatic growth factors (insulin,EGF, PDGF, VEGF, bFGF, IGF and HGF) stimulated T538 PKCθphosphorylation. This lack of activation by growth factors was notdue to their inability to stimulate pancreatic acinar cells becauseprevious studies show that specific receptors for these growthfactors exist on these cells and their activation stimulates a numberof cellular signaling cascades [15,19]. Moreover, we demonstrated thatthe increase activation of PKCθ by CCK is due to the activation ofCCKA receptor only, andnot due to thebindingof CCK to apossibleCCKB/gastrin receptor that could be present in the rat pancreatic acinar cell orother accompanying cells. Although in other species, CCKB receptors arepresent in pancreatic acinar cells and they are present in the ratpancreatic acinar cell line, AR42J cells, or results are consistent withother studies which demonstrate only CCKA receptors on rat pancreaticacini [15,40,54].

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To assess activation of PKCθ in the present study we used threedifferent approaches, first stimulation of T538 PKCθ phosphorylation,second stimulation of PKCθ translocation, and third measurement ofstimulation of PKCθ kinase activity. T538 PKCθ phosphorylation wasassessed because previous studies showed that PKCθ is phosphory-lated in 3 different sites (T538, S695 and S676), with its activation ofkinase catalytic activity [1,2]. The phosphorylation of the T538 siteplays an essential role in PKCθ activation, becausemutation of this siteleads to a PKCθ that does not undergo activation or translocation withknown stimulants [33]. Furthermore, there is a close correlationbetween the PKCθ T538 phosphorylation state and its kinase catalyticactivity [47]. For that reason, increments in phosphorylation of T538PKCθ have been widely used to assess PKCθ activation in a number oftissues including, not only in T cells or T cell lines [10,47], but also in anumber of other cells [13]. Thirdly, we also demonstrated PKCθ'sactivation by demonstrating its translocation to membranes whenstimulated by some of these agents. Once phosphorylated by itsactivators, PKC isoforms translocate to the membrane where they canphosphorylate various protein/substrates. In fact, after stimulation PKCθhas been shown to translocate tomembranes in T cells [55],muscle cells[56] andmast cells [7].We demonstrate that in rat pancreatic acinar cellwith stimulation by CCK there is an increase membrane-associatedPKCθ. These results were established through two different approaches,first using Western blotting and immunodetection of PKCθ withspecific PKCθ antibodies in fractionatedmembranes from rat pancreaticacinar cell and demonstrating an increase of PKCθ in membranes afterincubation with CCK or TPA (PKC activator); and second, usingimmunofluorescence in isolated rat pancreatic acinar cells with specificPKCθ antibodies, treatment with CCK or TPA resulted in the accumu-lation of phospho-T538 PKCθ in themembrane. These results have somesimilarities anddifferences fromprevious studies of the ability of variousagents to activate PKCθ in other tissues. They are similar in that a numberof other GI hormones/neurotransmitters in other systems can alsoactivate PKCθ. Specifically, muscarinic cholinergic agents activate PKCθin muscle [57] and bombesin-related peptides in human antral G cells[58]. There are no studies reporting the ability of agents that activateadenylate cyclase/PKA to stimulate PKCθ. However, our results withactivation of PKCθ differ from studies with other novel PKC isoforms inother tissues, which demonstrate both dBcAMP/8-Br-cAMP/CPT-cAMPor agents that activate adenylate cyclase, can stimulate their activation.Specifically, synthetic cAMP analogs activate PKCδ in hepatocytes [59]and VIP activates PKCδ in cortical astrocytes [60]. Our results withgrowth factors differ from studies reporting that insulin is able toincrease PKCθ activation in myocytes [56], EGF increases T538 PKCθphosphorylation in A431 cells [61] and that IGF-1 increases PKCθcatalytic activity in rhabdomyosarcoma cell line [62]. These resultsdemonstrate that a selected group of GI hormones/neurotransmittersthat activate the PLC cascade can activate PKCθ in pancreatic acinar cells.However, our resultswith growth factors differ from the results reportedin some other tissues suggesting that PKCθ responsiveness may betissue-specific. Furthermore, the lack of response to cAMP/PKA agents inpancreatic acinar cells, suggests PKCθ responsiveness to this signalingcascade, differs from that reported for theGI hormone/neurotransmitterVIP, as well as for other stimulants, for other novel isoform of PKCs inother tissues, suggesting that the PKCθ responsiveness to the agentsmayalso be tissue specific or novel PKC subtype specific.

Numerous studies support the conclusion that in pancreatic acinarcells, the CCKA receptor exists in two different states, a high affinityand low affinity state, each of which activates different signalingcascades [15,26,63]. In the present study CCK's stimulation of T538PKCθ phosphorylation was detectable at 0.1 nM, with a half-maximaleffect at 0.174 nM and a maximal effect at 10 nM in pancreatic acinarcells. These results have both similarities and differences from theability of CCK to stimulate other novel PKC kinases in pancreatic acinarcells. Specifically, CCK-stimulated maximal PKCmu (PKD1) activationat a similar concentration to that observed for PKCθ (10 nM), however

10-fold higher CCK concentrations were required to fully activatePKCδ (100 nM). These results demonstrate that different novel PKCsin pancreatic acinar cells have different dose–response curves for CCKactivation. Several results from this study support the conclusion thatfull activation of PKCθ by CCK in pancreatic acini requires activation ofboth, the high and low, CCKA affinity receptor states. Activation ofPKCθ in pancreatic acinar cells by CCK occurs over a 5-log-fold rangeof CCK concentrations, with PKCθ stimulation occurring with CCKconcentrations which has been shown in other studies to activateboth the high and low affinity CCKA receptor states [15,38]. Thisconclusion is supported by the results with the synthetic analog, CCK-JMV, which, in rat pancreatic acinar cells, functions as an agonist of thehigh affinity CCK receptor state and an antagonist of the low affinityreceptor state [15,38,39]. CCK-JMV produced 30% of themaximal PKCθactivation exerted by CCK. As CCK-JMV is an agonist of only the highaffinity CCKA receptor state [39], these results with CCK-JMV demon-strate that CCK causes 30% of its maximal PKCθ activation throughactivation of the high affinity CCKA receptor state and the remaining70%of maximal activation is due to activation of low affinity CCKA receptorstate. These results show similarities and differences from CCKA

receptor-mediated phosphorylation of other kinases or proteins inpancreatic acinar cells. PKCθ activation by CCK is similar to CCK-mediated activation of the Src kinase, Lyn [64], PYK2/CAKβ [17], p125FAK

[27], paxillin [16] and the novel PKC, PKCmu (PKD1) [18] in pancreaticacinar cells, in that in each case, maximal activation requires CCKARactivation of both the high and low states. However, these differentsignaling cascades differ in the relative importance of the high affinityand low affinity CCK receptor states. Activation of the high affinity CCKA

receptor state is responsible for only 20% of the maximal activation ofPKCmu (PKD1) [18] and PYK2/CAKβ [17], whereas it mediates 50% ofthe maximal stimulation of p125FAK and paxillin phosphorylation [65]and mediates 60% of Lyn activation [64]. These results with PKCθactivation differ from the results seen with CCK activation in pancreaticacinar cells of the novel PKC, PKCδ [14] or stimulation of phosphory-lation of the adaptor protein, CrKII, [66] where they are mediated onlythrough activation of the low affinity CCKA receptor state. In contrast,CCK-mediated activation of PI-3 kinase and phospholipase D inpancreatic acinar cells is mediated entirely by the activation of thehigh affinity receptor state [67]. These results demonstrate that not onlydo the CCKA receptor states differ in their coupling to pancreatic cellularkinase cascades, but evenwith different novel PKCs in the same cell, theCCKAR receptor activation states show differential coupling.

In other cells activated by various stimulants, PKCθ activation isknown to be important in the stimulation of a number of importantcellular signaling proteins [5], including Src, [41], PKD [42], Raf [42,68],CARMA and IKK/NFκβ [43], Cbl [44,47], 14-3-3 [45], Blc-10 [46],MALT-1[47], PI3K [19], ERK [13], IRS-1 [12], p125FAK [69] and Akt [12]. Inpancreatic acinar cells, stimulation by growth factors and GPCRs causesactivation of a number of these cellular signaling proteins, including Srckinases [64], PKD [18,49], Raf [70], IKKα/β/NFκβ [49], PI3K [19,71],p44/42 MAPKs [72], p125FAK, PYK2 and paxillin and Akt [19], whichhave been shown to be important in mediating cell activation, enzymesecretion [73,74], proliferation and apoptosis [19], cell survival [75] andprotein synthesis [76]. Furthermore, activation of a number of thesesignaling pathways such as NFκβ plays a critical role in pathologicalprocesses such as inflammatory and cell death responses of pancreaticacinar cells in acute pancreatitis [21,49,72]. A number of our resultssupport the conclusion that PKCθ activation by CCK or TPA plays animportant role in the activation of a number of these cellular signalingcascades inpancreatic acinar cells. First, usinga specific pseudosubstratePKCθ inhibitor [22] we observed a decrease in the CCK- and TPA-stimulated phosphorylation/activation of PKD1 (PKCmu), Src, RafC,PYK2, p125FAK and IKKα/β, but no effect on phosphorylation/activationof AKT or p44/42 MAPKs. Second, with specific inhibition of the PKCθactivation by the overexpression, in isolated pancreatic acinar cells, of aPKCθ dominant negative form by adenovirus infection, we found also a

2155V. Sancho et al. / Biochimica et Biophysica Acta 1813 (2011) 2145–2156

decrease in the CCK- and TPA-stimulated phosphorylation/activation ofPKD1 (PKCmu), Src, RafC, PYK2, FAK and IKKα/β, but no effect onAKTorp44/42 MAPKs phosphorylation/activation. That these results werespecific for PKCθ were supported by the findings that neither thepseudosubstrate PKCθ inhibitor nor the dominant negative PKCθ,altered the activity of PKCδ, another novel PKC expressed in acinarcells [14]. These results show similarities and some importantdifferences from results with PKCθ activation reported in other studiesin other tissues. They are similar to results with PKD activation in COS-7and Jurkat cells,whichwasdependent onPKCθ activation [42] and in thecase of activation of the IKKα/β-NFκβ pathway in T cells by CD3/CD28,which is also dependent on PKCθ activation [5]. They differ in that wefound no alteration by the inhibition of PKCθ activation in thephosphorylation/activation of AKT or p44/42 MAPK with pancreaticacinar cells stimulated by either CCK or TPA, which are well-establishedstimulants in pancreatic acini of both AKT [19] and p44/42 MAPK [77].Previous studies have provided evidence that other two novel PKCs(PKCδ, PKCε) found in pancreatic acinar cells, are activated by CCK andTPA [14,20,21,48], and their activation is important for a number ofphysiological and pathological responses of the acinar cell [49]. Ourresults support the conclusion that PKCθ is also involved in theactivation of many of these cellular signaling cascades involved inseveral of these effects in pancreatic acinar cells, and in future studies,which of the three novel kinases is most important in producing thesevarious cell effects will need to be investigated.

Our results not only provide evidence for the participation of PKCθ inthe activation of other kinases in pancreatic acinar cells, but also supportthe conclusion that it can affect various signaling cascades by a directassociation of this novel PKC isoform, observed not only at high but alsoat low doses, with different protein/kinases implicated in variouscellular signaling cascades. This conclusion is supported by the results ofour co-immunoprecipitation experiments,which showed that both CCKand TPA stimulated the association of PKCθ with the Src kinase, Lyn;AKT; RafA and RafC, but not an association with p85 PI3K, PKD1, RafB,Bcl-10, Cbl, MALT-1, tubulin or 14-3-3 protein. These results havesimilarities and differences with other studies in other tissues. They aresimilar in the case of the direct association of PKCθwith amember of theSrc kinase family, because in Jurkat, RBL-2H3 and T cells co-stimulationby CD3/CD28, produces an increase in the direct association of PKCθwith Src kinases (Src, Lyn, Lck and Fyn) [41,46,55]; and in the case of Rafproteins in Cos-1 transfected cells with PKCθ and Raf proteins, uponstimulation with TPA, a direct association of PKCθ with RafC and RafBoccurred [68]. Our results show also differences with other studies inother tissues,which reported a direct association of PKCθwith PI3K, Cbl,Bcl-10, MALT-1, tubulin and 14-3-3 protein [44,45,47]. They also differfrom other studies in that for the first timewe find that with pancreaticacinar cells activation of PKCθ stimulated a direct associate with AKT,which has been shown to be important in mediating pancreaticprocesses including enzyme secretion or apoptosis and in pathologicalprocesses, such as pancreatitis and pancreatic cancer growth [19,78].

The role of other PKC novel isoforms in amylase release in ratpancreatic acinar cells is controversial with one study [79] concludingthat PKCδ is not implicated in this secretory effect of CCK or carbachol inacinar cells from guinea pigs or PKCδ−/− rats. Another study [20],though, proposed that the PKCδ isoform, and not the PKCε, is importantin mediating amylase release. Our results support the conclusion that,PKCθ, is not implicated in the amylase secretion produced by CCK,carbachol or VIP, as its inhibition did not alter the amylase releaseevoked by secretagogues either activating phospholipase C or adenylatecyclase.

Acknowledgements

Thiswork is partially supported by the Intramural Research Programof the NIDDK, NIH.

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