nhibition of Bile Salt–Induced Apoptosis by Cyclic AMPnvolves Serine/Threonine Phosphorylation of CD95
OLAND REINEHR and DIETER HAUSSINGERlinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, Dusseldorf, Germany
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ackground & Aims: Cyclic AMP (cAMP) inhibits bilealt–induced hepatocyte apoptosis; the underlyingechanisms are unclear. Methods: The effects of cAMP
n taurolithocholate-3-sulfate-(TLCS)- or glycochenodes-xycholate (GCDC)-induced CD95 (Fas/APO-1) activa-ion and apoptosis were studied in 24-hour cultured ratepatocytes and in perfused rat liver. Results: TLCSnduced a rapid oxidative stress response, c-Jun-N-termi-al kinase (JNK) and epidermal growth factor (EGF)eceptor (EGF-R) activation, subsequent EGF-R/CD95 as-ociation and CD95 tyrosine phosphorylation, CD95embrane targeting, death-inducing signal complex
DISC) formation and hepatocyte apoptosis. None ofhese responses was triggered by cAMP; however, cAMPnduced H89-sensitive serine/threonine phosphorylationf CD95. Similar data were obtained with GCDC, anotherroapoptotic bile acid. cAMP did not prevent the TLCS-nduced oxidative stress response, JNK activation andGF-R/CD95 association, however abolished EGF-R ac-ivation and subsequent CD95 tyrosine phosphorylation,D95 membrane trafficking, and DISC formation in a89-sensitive way. Also in presence of TLCS, cAMP in-uced rapid Ser/Thr phosphorylation of CD95 within 10in. The effects of cAMP on the various steps of CD95
ctivation were also found in the intact perfused rativer. Evidence is given that a cAMP-induced Ser/hr phosphorylation favors internalization of CD95. Con-lusions: Inhibition of bile salt–induced apoptosis byAMP involves both PKA-dependent Ser/Thr phosphory-ation of the CD95 and inhibition of EGF-R activation,hich results in an inhibition of CD95 tyrosine phosphor-lation, CD95 membrane targeting, and DISC formation.D95 regulation involves complex phosphorylations withD95-tyrosine phosphorylation favoring CD95 mem-rane trafficking and DISC formation, whereas CD95er/Thr phosphorylation inhibits these processes.
ydrophobic bile acids can induce hepatocyte apop-tosis in vivo and in vitro,1–10 which plays an
mportant role in the pathogenesis of cholestatic liverisease. Different mechanisms are involved in bile acid–nduced apoptosis, including ligand-independent CD95CD95 receptor, also known as Fas/APO-1) activation,3,5
-Jun-N-terminal kinase (JNK),11,12 protein kinase C
PKC),2,13 cathepsin B,14 oxidative stress,15,16 and CD95embrane targeting.17,18 However, not all bile salts are
roapoptotic, e.g., tauroursodesoxycholic acid even protectsepatocytes against apoptosis, which is otherwise inducedy hydrophobic bile acids, such as taurolithocholateTLCS) and glycochenodeoxycholate (GCDC).6,8,19–23
cAMP was recently shown to inhibit bile acid–inducedpoptosis in isolated hepatocytes24–26 and to modulatehe apoptotic program in other cell types.27 However, theechanisms underlying the protective effect of cAMP on
ile acid–induced apoptosis are incompletely under-tood, although it was suggested that inhibition of he-atocyte apoptosis by cAMP is mediated by activationf protein kinase A (PKA) and phosphatidylinositol-3-inase (PI-3-kinase).25,26
Recent studies provided a more detailed insight intohe mechanisms that trigger CD95 membrane targetingnd activation in response to hydrophobic proapoptoticile acids,12,18 such as TLCS or GCDC. These bile acidsnduce an almost immediate oxidative stress responseith subsequent antioxidant-sensitive activation of the
pidermal growth factor receptor (EGF-R) and JNK.18
his JNK response is required for an association of theGF-R with CD95, with subsequent EGF-R–catalyzedyrosine phosphorylation of CD95, which provides theignal for CD95 membrane trafficking, recruitment ofas-associated death domain (FADD) and caspase 8death-inducing signal complex [DISC] formation), andnduction of apoptosis. Inhibition of this complex CD95argeting and activation process by antioxidants, JNK orKC inhibitors, inhibitors of EGF-R tyrosine kinase
Abbreviations used in this paper: carboxy-H2-DCFDA, carboxy-2,7-ichlorofluorescin diacetate; CD95, CD95 receptor APO-1/Fas; DB-AMP, dibutyryl-adenosine 3�,5�-cyclic monophosphate; DISC, death-nducing signal complex; EGF-R, epidermal growth factor receptor;ADD, Fas-associated death domain; GCDC, glycochenodeoxycholate;NK, c-Jun-N-terminal kinase; PI-3-kinase, phosphatidylinositol-3-ki-ase; PKA, protein kinase A; ROS, reactive oxygen species; TCDC,aurochenodeoxycholate; TLCS, taurolithocholate-3-sulfate.
250 REINEHR AND HAUSSINGER GASTROENTEROLOGY Vol. 126, No. 1
ctivity or inhibitors of EGF-R activation, abolishes thepoptotic action of hydrophobic bile acids. Interestingly,ot only proapoptotic bile acids18 but also CD95 li-and28 induces this complex interaction between CD95nd EGF-R, indicating its importance for apoptosis in-uction in hepatocytes in general. Because cAMP washown to prevent CD95 membrane trafficking andaspase 8 activation,12,24 we studied the effects of cAMPn CD95 phosphorylation and the different steps in-olved in TLCS-induced CD95 activation. The data showhat cAMP inhibits TLCS-induced EGF-R activation andrevents CD95 tyrosine phosphorylation but not theGF-R/CD95 association. This prevents CD95 tyrosinehosphorylation and, consequently, CD95 membranerafficking and DISC formation. Furthermore, cAMPriggers Ser/Thr phosphorylation of CD95 via activationf protein kinase A, which may reflect an internalizationignal for CD95.
Materials and MethodsMaterials
The materials used were purchased as follows: collag-nases from Boehringer (Mannheim, Germany); William’s Eedium and fetal calf serum from Gibco Life Technologies
Gaithersburg, MD); taurolithocholate-3-sulfate (TLCS) andlycochenodeoxycholate (GCDC) from Sigma-Aldrich (Dei-enhofen, Germany); penicillin/streptomycin from BiochromBerlin, Germany); carboxy-2,7-dichlorofluorescin diacetatecarboxy-H2-DCFDA)29 from Molecular Probes (Eugene, OR);89 from Tocris Cookson Ltd. (Bristol, United Kingdom);
nd LY294002,30 wortmannin31 (PI-3-kinase inhibitors) fromalbiochem (Bad Soden, Germany). The antibodies used wereurchased as follows: rabbit anti-CD95, rabbit anti-FADD,nd mouse anti-caspase 8 antibodies from Santa Cruz Biotech-ology (Santa Cruz, CA); goat anti-rabbit Cy3-conjugatedntibody from Dianova (Hamburg, Germany); rabbit anti-hospho-JNK-1/-2 and rabbit anti-phospho-EGF-R antibod-es from BioSource Inc. (Camarillo, CA); sheep anti-EGF-Rnd mouse anti-phospho-tyrosine antibodies from Upstateiotechnology (Lake Placid, NY); mouse anti-phospho-serine
clone 16B4) and mouse anti-phospho-threonine (clone 14B3)rom Biomol (Hamburg, Germany); horseradish peroxidase-onjugated anti-mouse IgG and anti-rabbit IgG from Bio-RadHercules, CA). All other chemicals were from Merck (Darm-tadt, Germany) at the highest quality available.
Cell Preparation and Culture
Hepatocytes were isolated from livers of male Wistarats, fed ad libitum with a standard diet by a collagenaseerfusion technique as described previously.32 Aliquots of.5 � 106 cells were plated on collagen-coated 6-well culturelates (Falcon, Heidelberg, Germany) and cultured as pub-ished recently33 in 5% CO -supplemented room atmosphere.
2
he viability of the hepatocytes was more than 95% as assessedy trypan blue exclusion.
Measurement of Reactive Oxygen Species
Hepatocytes were cultured for 24 hours on collagen-oated glass coverslips (diameter 30 mm) in 6-well culturelates (Falcon, Heidelberg, Germany). Reactive oxygen speciesROS) generation was measured at the single-cell level asescribed recently.18 Cells were loaded with 5 �mol/L car-oxy-H2-DCFDA for 10 minutes and then washed extensivelyn PBS before mounting in a perfusion chamber on an inverteduorescence microscope (Zeiss, Oberkochen, Germany) with.5 mL PBS. The temperature was kept at 37°C. Hepatocytesere excited at 488 nm, and the emission was measured at 515
o 565 nm with a photon counting tube (Hamamatsu H3460-4; Hamamatsu, Herrsching, Germany). Autofluorescence wasubtracted and was less than 1% of the total fluorescence ofye-loaded cells. Noise during recordings of carboxy-H2-CFDA fluorescence was less than 3% of the total photon
ount. When 10 �mol/L H2O2 was applied at the end of thexperiments, the photon count always markedly increased,howing that the sensitivity limit of carboxy-H2-DCFDA didot restrict the detection of increases in reactive oxygen inter-ediates.To rule out a possible interaction between the dye and the
ubstances used, i.e., TLCS, DB-cAMP, and H89, these com-ounds were added to the dye in a cell-free system. None of theatter compounds increased the measured fluorescence of theye (n � 5). H2O2 served as a positive control.
Western Blot Analysis
At the end of the incubation period, the medium wasemoved, and the cells were immediately lysed in Western blotample buffer containing 20 mmol/L Tris-HCl (pH 7.4), 140mol/L NaCl, 10 mmol/L NaF, 10 mmol/L sodium pyrophos-
nd protease inhibitor cocktail (Boehringer, Mannheim, Ger-any). Samples were then transferred to sodium dodecyl sul-
ate (SDS)/polyacrylamide gel electrophoresis (PAGE), andubsequent proteins were blotted to nitrocellulose membranessing a semidry transfer apparatus (Pharmacia Biotech,reiburg, Germany) as recently described.12,34 Blots werelocked for 2 hours in 5% (wt/vol) BSA containing 20 mmol/Lris, pH 7.5, 150 mmol/L NaCl, and 0.1% Tween 20 (TBS-T)nd then incubated at 4°C overnight with the first antibodyantibodies used: anti-phospho-JNK-1/-2 [1:1000]; anti-phos-ho-EGF-R, anti-phospho-serine, and anti-phospho-threonine1:2500]; anti-EGF-R [1:5000]; and anti-CD95, anti-FADD,nti-caspase 8, and anti-phospho-tyrosine [all 1:10,000]). Fol-owing washing with TBS-T and incubation with horseradisheroxidase-coupled anti-mouse, anti-sheep, or anti-rabbit IgGntibody (all diluted 1:10,000) at room temperature for 2ours respectively, the blots were washed extensively andeveloped using enhanced chemiluminescent detection (Am-rsham Pharmacia Biotech, Uppsala, Sweden). Blots were ex-
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osed to Kodak X-OMAT AR-5 film (Eastman Kodak Co.,ochester, NY).
CD95 Immunoprecipitation
Hepatocytes were cultured on collagen-coated culturelates (diameter 10 cm; Falcon, Heidelberg, Germany) at aensity of 8 � 106 cells/well. Cells were harvested in lysisuffer containing 136 mmol/L NaCl, 20 mmol/L Tris-HCl, 10
(vol/vol) glycerol, 2 mmol/L EDTA, 50 mmol/L �-glycer-phosphate, 20 mmol/L Na-pyrophosphate, 0.2 mmol/L pefa-lock, 5 �g/mL aprotinin, 5 �g/mL leupeptin, 4 mmol/Lenzamidin, 1 mmol/L Na-vanadate, and 1 % (vol/vol) Triton-100. The lysates were kept for 10 minutes on ice and then
entrifuged at 10,000g for 25 minutes at 4°C, and aliquots ofhe supernatants were taken for protein determination usinghe Bio-Rad protein assay (Bio-Rad Labs.). Equal proteinmounts (200 �g) of the respective samples were taken, addedo an identical volume of lysis buffer, and incubated for 2 hourst 4°C with a polyclonal rabbit anti-rat CD95 antibody (di-ution 1:100; Santa Cruz). In the following, 10 �L of protein- and 10 �L of protein-G-agarose (Santa Cruz) were added,
nd samples were incubated at 4°C overnight. Immunopre-ipitates were washed 3 times using a buffer containing 136mol/L NaCl, 20 mmol/L Tris-HCl, 10 % (vol/vol) glycerol,mmol/L EDTA, 50 mmol/L �-glycerophosphate, 20 mmol/La-pyrophosphate, 0.2 mmol/L pefablock, 5 �g/mL aprotinin,�g/mL leupeptin, 4 mmol/L benzamidin, 1 mmol/L Na-
anadate, and 0.1 % (vol/vol) Triton X-100 and then trans-erred to Western blot analysis as described above.
CD95 Trafficking
For determination of membrane surface trafficking ofD95, cells were cultured for 24 hours on collagen-coatedlass coverslips (diameter 30 mm) in 6-well culture platesFalcon, Heidelberg, Germany) and subsequently exposed tohe bile salts indicated. All inhibitors were preincubated for 30inutes before bile salts were instituted. Permeabilized and
onpermeabilized cells were stained as published recently33
sing a polyclonal rabbit anti-CD95 antibody (dilution 1:500n PBS) and a secondary anti-rabbit Cy3-conjugated antibody.ells were visualized using an Axioskop (Zeiss, Oberkochen,ermany) and images were taken with a 3CCD-Camera (Intas,ottingen, Germany). CD95 membrane trafficking was de-ned as the appearance of fluorescent spotting on the surface ofhe nonpermeabilized cells compared with the nonpermeabi-ized control cells. For each condition, at least 100 cells perndependent experiment from at least 3 different cell prepara-ions were scored for CD95 membrane trafficking.
Subfractionation Studies
CD95 phosphorylation was also studied in cytosolicnd membrane fractions. For this, cells were cultured for 24ours on collagen-coated 10-cm culture plates and subse-uently exposed as described before. Cells were lysed in auffer containing 10 mmol/L Tris, 30 mmol/L mannitol, and0 mmol/L CaCl (pH 7.5). After centrifugation of the samples
2
5 minutes, 1200g), the supernatants were subjected to ultra-entrifugation (35 minutes, 40,000g) to separate cytosolicrom membrane fractions, which underwent CD95 immuno-recipitation and Western blotting as described above forD95-tyrosine phosphorylation, total CD95-amount, GAPDH,nd annexin II. GAPDH and annexin II served as markers forhe cytosolic and the membrane fraction, respectively, andere taken as loading controls. GAPDH was not detectable in
he membrane fraction, and annexin II was not detectable inhe cytosolic fraction, indicating a high efficacy of separation.
Cyclic AMP Measurements
For determination of cellular cAMP ([cAMP]i) levels,epatocytes were cultured on collagen-coated culture platesdiameter 10 cm; Falcon) at a density of 8 � 106 cells/well.fter the incubation with the respective agonists, cells werearvested in 0.1 N HCl for up to 10 minutes and the lowH-cAMP ELISA from R&D Systems (Wiesbaden, Germany)as used according to the manufacturer’s recommendations.liquots from each sample were taken for protein determina-
ion using the Bio-Rad protein assay and total [cAMP]i/�grotein was determined.To confirm the data obtained by the enzyme-linked immu-
osorbent assay, an additional radioimmunosorbent assay pro-ided by Amersham (Biosciences’s cAMP assay system; Buck-nghamshire, United Kingdom) was performed according tohe manufacturer’s recommendations. Samples were extractedy homogenization in the recommended assay buffer contain-ng 4 mmol/L EDTA; aliquots from each sample were taken forrotein determination, and then samples were boiled for 5inutes in a water bath to coagulate proteins. Again, total
cAMP]i/�g protein was determined.
Protein Kinase A (PKA) Assay
For determination of PKA activity, hepatocytes wereultured on collagen-coated culture plates (diameter 10 cm;alcon) at a density of 8 � 106 cells/well. After the incubationith the respective agonist, the Protein-Kinase-Assay-Kit
rom Calbiochem (San Diego, CA) was used according to theanufacturer’s recommendations. Aliquots from each sampleere taken for protein determination using the Bio-Rad pro-
ein assay, and PKA activity per total protein amount perample was determined. Relative increase in PKA activity wasalculated in relation to the untreated control.
TUNEL Technique
The TUNEL method employs the terminal deoxynu-leotidyl transferase-mediated X-dUTP nick-end labelling ofITC-conjugated deoxyuridine triphosphate. Briefly, cellsere rinsed twice in PBS, fixed in paraformaldehyde (4 %ol/vol in PBS) for 45 minutes at room temperature, washed inBS, permeabilized in Triton X-100 (0.1 % vol/vol in PBS) for0 minutes at 4°C and then intensely washed again as de-cribed recently.33 The manufacturer’s protocol was then fol-owed (Roche Diagnostics, Mannheim, Germany). Using auorescence microscope (Zeiss, Oberkochen, Germany) the
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252 REINEHR AND HAUSSINGER GASTROENTEROLOGY Vol. 126, No. 1
umber of apoptotic cells was determined by counting theercentage of fluorescein-positive cells. At least 100 cells fromindependent experiments from 3 different cell preparationsere counted for each condition.
Liver Perfusion
Livers of male Wistar rats (160–180 g) were perfusedn situ in a nonrecirculating system with Krebs-Henseleituffer (KHB) at 37°C, equilibrated with 5%/95% (vol/vol)02/O2, and supplemented with lactate (2.1 mmol/L) andyruvate (0.3 mmol/L) at a perfusate flow of 3.5–4.5 mL/in/g liver as described previously.34 TLCS (10 �mol/L),B-cAMP (10 �mol/L), their combination, or dimethyl sul-
oxide (DMSO) as control were added to the influent after 30inutes of perfusion. A liver lobe was excised before TLCS,B-cAMP, or DMSO addition (t � 0 minutes), and then
dditional lobes were excised 30 and 60 minutes after the onsetf TLCS, DB-cAMP, or DMSO infusion in a way that keptortal pressure constant. For further determinations, samplesere then homogenized with an Ultraturrax (Janke & Kunkel,taufen, Germany) at 4°C in the appropriate lysis bufferndicated.
Statistics
Results from at least 3 independent experiments arexpressed as means � SEM. n Refers to the number of inde-endent experiments. Results were analyzed using the Studenttest: P � 0.05 was considered statistically significant.
ResultsEffects of cAMP and TLCS on CD95Phosphorylation Pattern
Because cAMP was shown to inhibit TLCS-in-uced CD95 membrane targeting, caspase activation,nd hepatocyte apoptosis,24 its effect on CD95 phosphor-lation was studied. In line with recent data18 and ashown in Figure 1A, TLCS (100 �mol/L) induced within0 minutes CD95 tyrosine phosphorylation, which per-isted for more than 12 hours. No Ser/Thr phosphoryla-ion of CD95 was observed during the first 3 hours ofLCS addition; however, thereafter, Ser/Thr phosphory-
ation of CD95 started to increase. On the other hand, noD95 Tyr phosphorylation in response to DB-cAMP wasbserved during 18 hours of exposure; however, DB-AMP triggered within 10 minutes a sustained Ser/Thrhosphorylation of CD95 (Figure 1B). When TLCS andB-cAMP were added simultaneously, Ser/Thr phos-horylation of CD95 occurred within 10 minutes andersisted for 18 hours, but no Tyr phosphorylation wasnduced (Figure 1C ). These data show that DB-cAMPrevents TLCS-induced CD95 Tyr phosphorylation andnduces rapid and persistent Ser/Thr phosphorylation ofD95. Furthermore, these data show that CD95 is sub-
ect to complex phosphorylation. As shown in Figure 1D,B-cAMP concentrations of 1 �mol/L were sufficient to
nduce CD95 Ser/Thr phosphorylation and to inhibitD95 Tyr phosphorylation in presence of TLCS. Similarndings were obtained with GCDC, another proapop-otic bile acid (Figure 1E ).
Effect of cAMP on TLCS-Induced JNK-,EGF-R-, and CD95 Activation and Apoptosis
As shown previously,18 TLCS induces a rapidxidative stress response in hepatocytes, which triggersntioxidant-sensitive JNK activation and EGF-R activa-ion as initial steps in the machinery leading to CD95embrane trafficking and DISC formation. EGF-R acti-
ation in response to TLCS was also sensitive toenistein, suggestive for the involvement of a yet un-nown tyrosine kinase activity.18,28 We therefore studiedhe effect of cAMP on TLCS-induced oxidative stress,GF-R, and JNK activation.As shown by carboxy-H2-DCFDA fluorescence, DB-
AMP had no effect on the TLCS-induced oxidativetress response: TLCS (100 �mol/L) increased within 2inutes ROS formation 2.15 � 0.16-fold (P � 0.05)
ompared with the control level (n � 3). If DB-cAMP100 �mol/L) and TLCS were added simultaneously,OS formation increased 1.92 � 0.03-fold (n � 3)
ompared with control in the absence and 2.06 � 0.10-old (n � 3) compared with control in the presence of89 (5 �mol/L).As shown in Figure 2, DB-cAMP (100 �mol/L) by
tself had no effect on EGF-R or JNK phosphorylation,hereas TLCS triggered EGF-R activation within 1inute and JNK activation within 15 minutes. Coad-inistration of DB-cAMP, however, largely prevented
he TLCS-induced EGF-R activation. This inhibitoryffect of cAMP on TLCS-induced EGF-R activation wasully abolished in presence of H89 (5 �mol/L) (FigureA ), indicating transmission of this effect by proteininase A. On the other hand, DB-cAMP had no effect onhe TLCS-induced JNK activation during the first hour;owever, thereafter, JNK phosphorylation declined (Fig-re 2B), in line with previous data.24
Interestingly, DB-cAMP had no effect on the TLCS-nduced association of the EGF-R with CD95 (Figure 3);owever, DB-cAMP fully prevented TLCS-inducedD95 Tyr phosphorylation (Figure 1C ) in a H89-sensi-
ive way (Figure 4). Similar findings were obtained whenCDC was used as proapoptotic bile acid (Figures 1D
nd 4). Likewise, the cAMP-induced Ser/Thr phosphor-lation of CD95, which occurred in absence and presencef TLCS (Figure 1), was sensitive to inhibition by H89,ndicating the involvement of PKA. These data suggest
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hat prevention of bile salt–induced CD95-Tyr phos-horylation by cAMP is not due to a failure of CD95 tossociate with the EGF-R but is due to an inhibition ofile salt–induced EGF-R-tyrosine kinase activation byAMP. The data further suggest the involvement of PKAn mediating cAMP-induced Ser/Thr phosphorylation ofD95 and prevention of bile salt–induced CD95-Tyrhosphorylation. In line with the recent demonstrationhat CD95-Tyr phosphorylation is a prerequisite forD95 membrane trafficking and recruitment of FADDnd caspase 8 to CD95,18,28 cAMP fully prevented CD95embrane trafficking and DISC formation (Figure 4).gain, the inhibitory effects of DB-cAMP on CD95embrane trafficking and DISC formation were abol-
shed by H89.The effects of DB-cAMP and H89 on TLCS-induced
D95 activation were also reflected at the level of TLCS-nduced apoptosis, as detected by TUNEL staining.ere, TLCS (100 �mol/L) induced within 12 hours
poptosis in 40.2% � 1.3% (n � 3) of the hepatocytes,ompared with 1.2% � 0.2% (n � 3) in control cellsnd in line with previous data.18 When DB-cAMP (100mol/L) was simultaneously added together with TLCS,LCS-induced apoptosis was largely prevented, and, after2 hours exposure to TLCS, only 3.3% � 0.3% (n � 3)f the hepatocytes were TUNEL-positive. Preincubationith H89 (5 �mol/L) abolished the antiapoptotic DB-
AMP effect and 37.6% � 2.2% (n � 3) of the cellshowed apoptosis when incubated for 12 hours withLCS, DB-cAMP, and H89 (Figure 5, Table 1). When,owever, cAMP was added 150 minutes after TLCS
igure 1. Bile salt–induced CD95 phosphorylations. Hepatocytesere cultured for 24 hours and then exposed to TLCS (100 �mol/L),CDC (100 �mol/L), and/or DB-cAMP (100 �mol/L) for the timeeriods indicated. Samples were collected, and CD95 was immuno-recipitated as described in the Materials and Methods section andhen CD95 was detected for phosphorylation on serine/threonine/yrosine residues by Western blotting. Total CD95 served as a loadingontrol. (A) TLCS induces CD95-Tyr phosphorylation within 30 min-tes, which was maximal at about 3 hours and slowly decreasedhereafter. After about 5 hours, a CD95-Ser/Thr phosphorylationecomes detectable. (B) DB-cAMP induces CD95-Ser/Thr phosphory-ation within 10 minutes but no CD95-Tyr phosphorylation. (C) Simul-aneous addition of TLCS and DB-cAMP prevents CD95-Tyr phosphor-lation and induces CD95-serine/threonine phosphorylation withinetics similar to that induced by DB-cAMP alone. (D) DB-cAMPnhibits TLCS-induced CD95-tyrosine phosphorylation in a dose-de-endent way when incubated together with TLCS for 60 minutes. Oneicromolar DB-cAMP was sufficient to induce CD95-Ser/Thr phos-horylation and to inhibit CD95-Tyr phosphorylation. (E) DB-cAMPnhibits GCDC-induced CD95-tyrosine phosphorylation in a dose-de-endent way when incubated together with GCDC for 60 minutes. Oneicromolar DB-cAMP was sufficient to induce CD95-Ser/Thr phos-horylation and to inhibit CD95-Tyr phosphorylation.
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254 REINEHR AND HAUSSINGER GASTROENTEROLOGY Vol. 126, No. 1
xposure, i.e., after a time period sufficient to induceLCS-induced CD95-Tyr phosphorylation, membrane
rafficking, and DISC formation,18 the antiapoptotic ef-ect of cAMP was largely abolished (Table 1).
igure 2. Effect of cAMP on bile salt–induced EGF-R- and JNK-activa-ion. Hepatocytes were cultured for 24 hours and then exposed toontrol medium, TLCS (100 �mol/L), and/or DB-cAMP (100 �mol/L)or the time periods indicated. The PKA-inhibitor H89 (5 �mol/L) wasreincubated 30 minutes prior to the bile salt addition. EGF-R phos-horylation and JNK phosphorylation were detected by immunoblot-ing. Representative blots from 3 independent experiments arehown. (A) TLCS induces within 1 minute a marked EGF-R phosphor-lation, which was abolished when incubated together with DB-cAMPn a H89-sensitive way, suggesting that cAMP prevents TLCS-inducedGF-R activation in a PKA-dependent manner. (B) TLCS, but not DB-AMP, induces JNK-1 protein phosphorylation within 15 minutes,hich lasts for up to 6 hours. cAMP has no effect on the TLCS-inducedNK-1 activation during the first hour, whereas JNK-1 phosphorylationfter 3 hours was inhibited. Similar data were obtained for JNK-2rotein phosphorylation (not shown).
Inhibition of PI-3-kinase by LY294002 (10 �mol/L)30
r wortmannin (100 nmol/L)31 did not significantly af-ect the inhibition by cAMP of the TLCS-induced acti-ation of the CD95 system (Figure 4), indicating thatI-3-kinase survival pathways,35 as described for TCDC-
nduced DISC formation,18 are not involved.
Effects of Adenosine and Forskolin onTLCS-Induced CD95 Activation
As shown in Figure 6 and in line with previousata,36,37 exposure of 24-hour cultured rat hepatocytes todenosine (100 �mol/L) or forskolin (100 �mol/L) led torapid increase of hepatocellular cAMP levels (Figure
A ) and PKA activity (Figure 6B). As expected, DB-AMP (100 �mol/L) also increased PKA activity (FigureB). PKA activation under these conditions occurredithin minutes, was maximal after about 30 minutes,
nd slowly declined thereafter (Figure 6B). As shownn Figure 7, stimulation of PKA by adenosine or forsko-in also induced Ser/Thr phosphorylation of CD95 and
igure 3. cAMP does not prevent TLCS-induced EGF-R/CD95-associ-tion. Hepatocytes were cultured for 24 hours and then exposed toLCS (100 �mol/L) and/or DB-cAMP (100 �mol/L) for the timeeriods indicated. CD95 was immunoprecipitated and EGF-R-associ-tion was detected by immunoblotting. Representative blots from 3ndependent experiments are shown. In line with previous data,18
LCS induced CD95/EGF-R association (A), whereas DB-cAMP had noffect (B), DB-cAMP did not alter the TLCS-induced CD95/EGF-R as-ociation (C), indicating that inhibition of TLCS-induced CD95-tyrosinehosphorylation by cAMP does not occur at the level of CD95/EGF-Rssociation.
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January 2004 cAMP AND CD95 PHOSPHORYLATION 255
imicked the effects of exogenously added DB-cAMP onLCS-induced EGF-R activation, CD95-tyrosine phos-horylation, and subsequent DISC formation in a H89-ensitive way (Figure 7).
igure 4. Effects of cAMP and H89 on TLCS- and GCDC-induced activa-ion of the CD95 system. Hepatocytes were cultured for 24 hours andhen exposed to control medium, TLCS (100 �mol/L), GCDC (100 �mol/), or DB-cAMP (100 �mol/L). If indicated, the PKA-specific inhibitor H895 �mol/L) or the PI-3-kinase-specific inhibitors LY294002 (10 �mol/L)r wortmannin (100 nmol/L) were preincubated for 30 minutes. Samplesere collected and either underwent Western blotting for EGF-R phos-horylation (1) or immunoprecipitation for CD95 with subsequent West-rn blotting for EGF-R (2), phospho-serine/threonine/tyrosine (3), orISC-formation as indicated by caspase 8 and FADD immunoblotting (4).otal CD95 served as loading control. Representative blots of 3 inde-endent experiments are shown. (1) Bile salt–induced EGF-R phosphor-lation was detected after 10 minutes of bile salt addition and wasnhibited by DB-cAMP in a H89-sensitive but LY294002- and wortmannin-nsensitive way. (2) EGF-R/CD95 association induced by TLCS or GCDCas measured after 60 minutes of bile salt addition and was not affectedy DB-cAMP. (3) CD95 phosphorylations were detected after 60-minutencubation of bile salts and/or DB-cAMP. Whereas TLCS and GCDCnduced CD95-Tyr phosphorylation, DB-cAMP induced phosphorylation ofD95 on Ser/Thr residues. When DB-cAMP was incubated together withLCS or GCDC, bile salt–induced CD95-Tyr phosphorylation was inhib-ted, whereas CD95-Ser/Thr phosphorylation still occurred in a PKA-nhibition-sensitive way. (4) DISC-formation was measured 3 hours afterile salt addition. TLCS and GCDC induced caspase 8 and FADD asso-iation with CD95, which was abolished in a H89-sensitive way whenLCS or GCDC were incubated together with DB-cAMP. Inhibition of PI-3inase with either wortmannin or LY294002 did not alter DB-cAMP–ediated effects on TLCS-induced activation of the CD95-system.
Late PKA Activation by TLCS
TLCS had no effect on cAMP levels and PKActivity during the first 5 hours of exposure (Figure 8).owever, thereafter, cAMP levels increased and activa-
ion of PKA became apparent (Figure 8), which wasccompanied by the appearance of Ser/Thr phosphory-ated CD95 (Figures 1A and 9A ) and a decline in CD95yr phosphorylation (Figure 1A ). Similar data with re-
pect to CD95 serine/threonine phosphorylation werebtained with GCDC (not shown).
Functional Significance of CD95 Ser/Thr-Phosphorylation
CD95-Tyr phosphorylation was recently identi-ed as a trigger for CD95 membrane trafficking andISC formation in response to proapoptotic stimuli such
s hydrophobic bile acids,18 CD95 ligand, or hyperos-olarity.28 Whereas under control conditions, unperme-
bilized hepatocytes exhibited almost no CD95 mem-rane staining (0.2% � 0.1% of the cells exhibit CD95embrane staining), TLCS (100 �mol/L) addition in-
uced CD95 membrane trafficking (10.8% � 0.7%),hich was inhibited by DB-cAMP (1.1% � 0.2%) in a89-sensitive way (10.2% � 1.4%).
igure 5. cAMP abolishes TLCS-induced apoptosis in a H89-sensitiveay. Hepatocytes were cultured for 24 hours and then exposed toontrol medium (F) or TLCS (100 �mol/L, E) for the given timeeriods. When indicated, H89 (5 �mol/L) was preincubated for 30inutes prior to the TLCS addition (Œ). DB-cAMP (100 �mol/L) wasither added together with TLCS (0�, �/�H89, {) or after 150inutes of TLCS incubation (150�, ■ /�H89, ‚). The percentage of
UNEL-positive hepatocytes was determined as described in the Ma-erials and Methods section. TLCS induced a time-dependent in-rease in apoptotic cells, which was enhanced by the inhibition of PKAnd inhibited by DB-cAMP (in a H89 sensitive way), especially if givenimultaneously with TLCS. The antiapoptotic effect of cAMP wastrongly blunted when cAMP was added 150 minutes after TLCS.
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256 REINEHR AND HAUSSINGER GASTROENTEROLOGY Vol. 126, No. 1
The effects of DB-cAMP and H89 on TLCS-inducedD95 membrane trafficking were also confirmed inD95 immunoblots from membrane and cytosolic frac-
ions of hepatocytes (Figure 9B). Within 3 hours ofxposure to TLCS, an enrichment of tyrosine-phospho-ylated CD95 in the membrane fraction became detect-ble, which was not observed when TLCS was addedogether with DB-cAMP (Figure 9A ). Under these con-itions, the cytosolic, but not the membrane fraction,ontained Ser/Thr-phosphorylated CD95. As furtherhown in Figure 9, H89 fully abolished the cAMP effects
igure 6. cAMP generation and PKA activation in response to adeno-in and forskolin in 24-hour cultured rat hepatocytes. (A) Hepatocytesere cultured for 24 hours and then exposed to control medium (E),denosine (100 �mol/L, ■ ), or forskolin (100 �mol/L, }) for the timeeriods indicated. Measurement of intracellular cAMP was performeds described in the Materials and Methods section. All agonists led tosignificant cAMP-generation within 30 seconds (P � 0.05; n � 3).asal cAMP level was 21 � 1 fmol cAMP/�g protein (n � 21). (B)fter 24 hours of culture, hepatocytes were exposed to control me-ium (E), adenosine (100 �mol/L, ■ ), forskolin (100 �mol/L, }), orB-cAMP (100 �mol/L, Œ) for up to 180 minutes. PKA activity waseasured as described in the Materials and Methods section andas elevated significantly for all agonists used (P � 0.05; n � 3).
n the TLCS-induced CD95 phosphorylation and distri-ution between the membrane and cytosolic fraction.hen, however, cAMP was added 150 minutes after
LCS exposure, Ser/Thr-phosphorylated CD95 wasound in the membrane and the cytosolic fraction, butimultaneous CD95 Tyr phosphorylation was only foundn the membrane fraction.
As shown previously,12,18 and in Figure 10A, TLCS-nduced CD95 membrane trafficking is maximal afterbout 3 hours of TLCS exposure and, thereafter, slowlyeclines. Because this decline roughly paralleled the ap-earance of Ser/Thr-phosphorylated CD95 (compare withigure 1A ), the possibility was addressed as to whetherD95-Ser/Thr phosphorylation might act as a signal forD95 internalization. Therefore, hepatocytes were incu-ated with TLCS together with H89 to inhibit the lateKA activation by TLCS (see Figure 8B). As shown inigure 10A and 10B, H89 not only abolished the lateLCS-induced CD95-Ser/Thr phosphorylation during
he 18-hour incubation period (compare Figure 10B andigure 1A ), but also prevented the decrease of CD95embrane staining (Figure 10A ). In another set of ex-
eriments, hepatocytes were exposed to TLCS (100
able 1. TLCS-Induced Apoptosis in Rat Hepatocytes
Condition TUNEL staining (%)
ontrol 1.2 � 0.2LCS 40.2 � 1.3a
� DB-cAMP 3.3 � 0.3b
� H89 � DB-cAMP 37.6 � 2.2c
� LY294002 � DB-cAMP 3.5 � 0.4b
� wortmannin � DB-cAMP 2.8 � 0.3b
� DB-cAMP (after 150 min) 33.2 � 1.8b
� H89 � DB-cAMP (after 150 min) 43.3 � 2.6c
�H89 43.9 � 1.5c
B-cAMP 0.9 � 0.2c
89 1.3 � 0.2c
Y294002 1.0 � 0.2c
ortmannin 1.2 � 0.3c
OTE. Hepatocytes were kept in culture for 24 hours and then ex-osed for 12 hours to TLCS (100 �mol/L), which increased theumber of hepatocytes with positive TUNEL staining to 40.2% � 1.3%P � 0.05a) when compared with 1.2% � 0.2% in control cells. Incu-ation of DB-cAMP (100�mol/L), H89 (5 �mol/L), LY294002 (10mol/L), or wortmannin (100 nmol/L) had no significant effect onepatocyte apoptosis when compared with control (P 0.05,c).B-cAMP largely prevented TLCS-induced apoptosis when incubated
ogether with TLCS. This effect was sensitive to inhibition of PKA by89, which was added 30 minutes prior to the bile salt exposure.Y294002 and wortmannin did not alter the protective effect of cAMPn TLCS-induced apoptosis. DB-cAMP, when given after a 150-minuteLCS-preincubation (see Figure 9), still inhibited TLCS-induced apo-tosis (33.2 � 1.8). H89, when added together with TLCS, had noffect on TLCS-induced apoptosis. Data are given as means � SEMnd are from 3 independent experiments for each condition.Statistically significant (P � 0.05) inhibition of the TLCS-inducedpoptosis.No significant inhibition.
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January 2004 cAMP AND CD95 PHOSPHORYLATION 257
mol/L) and, after 150 minutes preincubation, i.e., at aime point when CD95 Tyr phosphorylation and mem-rane staining were near-maximal (Figure 10A ), DB-AMP was added to induce CD95-Ser/Thr phosphoryla-ion (Figure 10C ). As shown in Figure 10A, under theseonditions, cAMP significantly accelerated CD95 disap-earance from the plasma membrane. In these experi-ents, DB-cAMP induced rapid Ser/Thr phosphoryla-
ion of CD95 but had no effect on the CD95 Tyrhosphorylation (Figure 10C, compare with Figure 1A ).These findings suggest that CD95 Ser/Thr phosphor-
lation may either act as a signal for internalization ofembrane-bound CD95 or as a signal preventing CD95embrane targeting. This view was further strengthened
n cell subfractionation studies using hepatocytes that
igure 8. Effect of TLCS on cAMP levels and PKA activity. Hepato-ytes were cultured for 24 hours and then exposed to control mediumE) or TLCS (100 �mol/L, F). cAMP-levels (A) and PKA activity (B)ere measured as described in the Materials and Methods section.LCS increased cAMP levels and PKA activity (*indicates P � 0.05;� 3) after 6–7 hours of TLCS exposure. This suggests that the late
LCS-induced CD95-Ser/Thr phosphorylation is caused by TLCS-in-uced cAMP generation and PKA activation.
igure 7. Inhibition of the TLCS-induced CD95 activation by aden-sine and forskolin. Hepatocytes were cultured for 24 hours andhen exposed to control medium, TLCS (100 �mol/L), adenosine100 �mol/L), or forskolin (100 �mol/L). When indicated, the PKAnhibitor H89 (5 �mol/L) was preincubated for 30 minutes. Sam-les were collected and either underwent Western blotting forGF-R phosphorylation (1) or immunoprecipitation for CD95 withubsequent Western blotting for EGF-R (2), phospho-serine/threo-ine/tyrosine (3), or DISC formation as indicated by caspase 8 andADD immunoblotting (4). Total CD95 served as loading control.epresentative blots of 3 independent experiments are shown. (1)LCS-induced EGF-R phosphorylation was detected after 10 min-tes of bile salt exposition and was sensitive to adenosine ororskolin addition in a H89-sensitive way. (2) EGF-R/CD95 associ-tion induced by TLCS was measured after 60 minutes of bile saltddition and was not affected by adenosine or forskolin. (3) CD95hosphorylations were detected after 60-minute incubation ofLCS, adenosine, or forskolin. Whereas TLCS induced CD95-Tyrhosphorylation, adenosine and forskolin induced phosphorylationf CD95 on Ser/Thr residues. When the latter compounds were
ncubated together with TLCS, TLCS-induced CD95-Tyr phosphory-ation was blunted, and CD95-Ser/Thr phosphorylation still oc-urred in a H89 sensitive way. (4) DISC formation was measured 3ours after bile salt addition. TLCS-induced caspase 8 and FADDssociation with CD95 was abolished by adenosine or forskolin inH89-sensitive manner.
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258 REINEHR AND HAUSSINGER GASTROENTEROLOGY Vol. 126, No. 1
ad been exposed to TLCS for 12 hours. At this timeoint of TLCS exposure, spontaneous CD95 Ser/Thrhosphorylation in addition to the rapid TLCS-inducedD95 Tyr phosphorylation has already occurred (FigureA ). As shown in Figure 9A, under these conditionsyr-phosphorylated CD95 was enriched in the plasmaembrane, and no increase of Ser/Thr-phosphorylatedD95 was found in this fraction. Conversely, Ser/Thr-hosphorylated CD95 was found in the cytosolic fraction,nd this amount was increased by cAMP in a H89-sensitiveay. These data again suggest that PKA-induced CD95-er/Thr phosphorylation may act to inhibit CD95 external-zation or to augment CD95 internalization.
In Vivo Relevance of the cAMP-MediatedInhibition of the Bile Salt–InducedActivation of the CD95 System
To examine whether the pathways of CD95 mem-rane trafficking can also be identified in the intact liver,at livers were perfused in situ with TLCS (10 �mol/L) inhe presence and absence of DB-cAMP (10 �mol/L).GF-R and CD95 phosphorylation were assessed in liverpecimens taken before and 30 and 60 minutes afterLCS addition to the perfusate. As shown in Figure 11,LCS induced within 30 minutes an activation of theGF-R and CD95-tyrosine phosphorylation as it wasbserved in isolated hepatocytes (see above). As is furtherhown in Figure 11, DISC formation occurred after 60inutes of TLCS perfusion, which was again sensitive toB-cAMP. Apart from the TLCS-induced CD95 activa-
ion, CD95 translocation from the cytosol to the mem-rane was investigated by subfractionation studies ofiver specimens. After 30 minutes of TLCS perfusion, annrichment of CD95 in the membrane fraction was ob-erved, suggestive for CD95 membrane trafficking. ThisD95 translocation was again sensitive to DB-cAMP
Figure 11). DB-cAMP itself did neither activate EGF-Ror induce CD95-Tyr phosphorylation, DISC formation,r CD95 membrane trafficking but induced within 30inutes CD95-Ser/Thr phosphorylation, in line with the
ndings obtained from isolated hepatocytes.
DiscussionMechanism of cAMP-Induced Inhibition ofCD95 Activation by Hydrophobic Bile Acids
Recent data indicated that CD95 activation inepatocytes is a complex process,12,18 which is not onlyriggered by hydrophobic bile acids but also by otherroapoptotic stimuli, such as CD95 ligand and hyperos-olarity.28 This process of CD95 activation involves an
lmost instantaneous oxidative stress response, which
igure 9. Enrichment of Tyr-phosphorylated CD95 in the membranend Ser/Thr-phosphorylated CD95 in the cytosolic fraction of ratepatocytes. Twenty-four–hour cultured hepatocytes were exposedo control medium, TLCS (100 �mol/L), and DB-cAMP (100mol/L) for the given time periods. When indicated, H89 (5mol/L) was added 30 minutes prior to the bile salt addition.B-cAMP was either added together with TLCS (cAMP 0�) or 150inutes after TLCS (cAMP 150�). Samples were collected, andembrane and cytosolic fractions were separated by ultracentrif-gation as described in the Materials and Methods section. CD95as immunoprecipitated first and then CD95 phosphorylationsere detected by immunoblotting. CD95 served as a loading con-
rol. A high efficacy of separation is shown by the exclusive detec-ion of the membrane marker annexin II in the membrane fractionnd the cytosolic enzyme GAPDH in the cytosolic fraction only.D95 membrane trafficking was detected using CD95 immunoblot-
ing in the latter fractions. Annexin II and GAPDH served as aoading control. (A ) TLCS induced within 3 hours an enrichment ofyr-phosphorylated CD95 in the membrane fraction, which wasbolished in the presence of DB-cAMP in a H89-sensitive manner.imultaneously, cAMP induced a CD95-Ser/Thr phosphorylation,hich was only detectable in the cytosolic fraction. Prolongedxposure (12 hours) to TLCS also induced CD95-Ser/Thr phosphor-lation, which was cytosolic. When cAMP was added 150 minutesfter TLCS, Ser/Thr phosphorylation of CD95 occurred in both theytosolic and membrane fraction. The findings suggest that CD95-er/Thr phosphorylation may provide a signal for its internalizationnd/or prevention of its externalization. (B) TLCS induced a CD95nrichment in the membrane fraction, indicating a TLCS-inducedD95 membrane trafficking. This CD95 membrane trafficking wasbolished by addition of DB-cAMP in a H89-sensitive way.
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riggers a tyrosine kinase-dependent EGF-R activationnd JNK activation. This JNK signal together with aKC signal is required for a CD95/EGF-R associationith subsequent CD95 tyrosine phosphorylation by theGF-R tyrosine kinase activity, which occurs after about
igure 10. CD95 internalization is induced by a PKA-mediated CD95-er/Thr phosphorylation. Twenty-four–hour cultured hepatocytes werexposed to TLCS (100 �mol/L). When indicated, H89 (5 �mol/L) wasdded 30 minutes prior to the TLCS addition, or DB-cAMP (100mol/L) was incubated after 150 minutes of TLCS exposure. CD95as then immunostained in nonpermeabilized hepatocytes to detectD95 membrane localization (A), or samples were taken for CD95
mmunoprecipitation and subsequent detection of CD95 phosphory-ation grade using immunoblotting technique (B and C). (A) TLCS (E)nduced a CD95 membrane trafficking, which was maximal at about 3ours and declined thereafter. This decline was prevented by H89reincubation (Œ). On the other hand, when DB-cAMP was added after50 minutes, i.e., a time point when TLCS-induced CD95 membranerafficking was maximal, CD95-internalization was accelerated (■ ).Indicates significant difference from TLCS-induced CD95 membranerafficking (P � 0.05). (B) When H89 was incubated together withLCS, “late” CD95-Ser/Thr phosphorylation was abolished (for TLCSlone see Figure 1A). (C) When DB-cAMP was added after a 150-inute TLCS-preincubation, CD95-Ser/Thr phosphorylation was in-uced on top of CD95 Tyr phosphorylation (see Figure 1). The datauggest that CD95-Ser/Thr phosphorylation may provide a signal forD95 internalization, whereas CD95-Tyr phosphorylation is a prereq-iste for CD95 membrane trafficking.
0 minutes of TLCS or GCDC addition, respectively.18
yr phosphorylation of CD95 then triggers CD95 mem-rane trafficking and DISC formation. As shown in theresent study, cAMP inhibits this process of bile salt–
igure 11. Inhibition of TLCS-induced activation of the CD95 system byAMP in perfused rat liver. Rat livers were perfused as described in theaterials and Methods section for 60 minutes with TLCS (10 �mol/L),B-cAMP (100 �mol/L), or DMSO as control. Liver samples were takent the time points indicated (t � 0 minutes immediately prior to TLCSnfusion). Representative blots from 3 independent experiments for eachondition are shown. (A) EGF-R-tyrosine phosphorylation (P-EGF-R), aarker for EGF-R activation, was detected by Western blotting. TotalGF-R served as loading control. CD95 was immunoprecipitated asescribed in the Materials and Methods section and assessed for Ser/hr/Tyr phosphorylation. Caspase 8 and FADD association with CD95DISC formation) was detected using immunoblotting technique. TotalD95 served as loading control. (B) CD95 membrane trafficking wasetected by Western blotting of total CD95 recovered in membrane andytosolic fractions (see Materials and Methods section). Annexin II andAPDH served as loading controls for the latter fractions.
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260 REINEHR AND HAUSSINGER GASTROENTEROLOGY Vol. 126, No. 1
nduced CD95 activation at different steps. cAMP has noffect on the oxidative stress response induced by TLCS,nd, in line with this, it did not affect TLCS-inducedNK activation during the first 60 minutes of TLCSddition. cAMP, however, fully prevented bile salt–in-uced EGF-R activation without affecting its associationith CD95. The latter observation is in line with therevious finding that EGF-R/CD95 association occurs ineponse to TLCS irrespective of the EGF-R tyrosinehosphorylation state but requires JNK activation.18 Be-ause the EGF-R is a known substrate for JNK-catalyzeder/Thr phosphorylation,38,39 one might speculate thatNK-induced EGF-R phosphorylation favors EGF-R/D95 association. The mechanisms underlying the inhi-ition of TLCS- or GCDC-induced EGF-R tyrosinehosphorylation by cAMP are unclear; however, PKA-ependent mechanisms play a crucial role because H89bolished the inhibitory cAMP effect on the EGF-Rctivation. TLCS-induced EGF-R activation is genisteinensitive, suggestive for the involvement of a not yetdentified tyrosine kinase activity.18,28 However, it is atresent unclear whether this tyrosine kinase activity is aotential target for inhibition by PKA. Inhibition of bilealt–induced EGF-R activation may well explain thenhibition of bile salt–induced CD95 Tyr phosphoryla-ion by cAMP and, as suggested by previous studies,18,28
revention of CD95 tyrosine phosphorylation is suffi-ient to block bile salt–induced CD95 membrane traf-cking, DISC formation, and apoptosis. Interestingly,owever, cAMP also induced a rapid Ser/Thr phosphor-lation of the CD95, indicating that CD95 is subject toomplex regulation by phosphorylation. cAMP-induceder/Thr phosphorylation of CD95 was H89-sensitive,gain suggestive for an involvement of PKA.
Functional Significance of CD95Phosphorylations
CD95 Tyr phosphorylation was recently shown toe essential for CD95 membrane trafficking in responseo hydrophobic bile acids, hyperosmolarity, or CD95igand addition.18,28 It is apparently also required forISC formation; however, this latter step is also con-
rolled by PI-3-kinase signals,18 which are activated byCDC but not by GCDC and TLCS.5 With regard to theewly described CD95 Ser/Thr phosphorylation in re-onse to cAMP, another site of apoptosis control at earlyteps of CD95 activation was found.
Interestingly, also prolonged exposure of hepatocyteso TLCS (5 hours) or GCDC induced PKA activation,hich was accompanied by the appearance of Ser/Thr-hosphorylated CD95 and a decrease of the number ofepatocytes showing CD95 membrane staining. These
rocesses may reflect a delayed counter regulatory, anti-poptotic response of the cells. As shown by subfraction-tion studies, Ser/Thr-phosphorylated CD95 was mainlyytosolic, whereas Tyr-phosphorylated CD95 was en-iched in the plasma membrane. These findings suggesthat Ser/Thr phosphorylation of CD95 may act either asn internalization signal and/or as a signal that preventsD95 membrane trafficking. In this respect, it is inter-sting to note that cAMP-induced Ser/Thr phosphoryla-ion of the endothelin A-(ETA)-receptor in hepatic stel-ate cells40 and of the adenosine A1 receptor inDT1MF-2 smooth muscle cells41 act as signals for
eceptor internalization. Interestingly, Ser/Thr phosphor-lation of CD95 can be induced in membrane-boundD95 on top of CD95 Tyr phosphorylation, i.e., whenAMP was added 150 minutes after TLCS addition.nder these conditions, the decline in CD95 membrane
taining was accelerated, in line with the view that CD95er/Thr phosphorylation may act as a CD95 internaliza-ion signal. Such a role of CD95 Ser/Thr phosphorylationould also explain why inhibition of PKA by H89
argely abolishes the spontaneous decrease of CD95embrane staining.
CD95 Phosphorylation Pattern andInhibition of Apoptosis
The finding that CD95 is subject to complexhosphorylations sheds a new light on the induction ofile salt–induced apoptosis and its prevention. PKActivation is apparently 1 major antiapoptotic mecha-ism through both induction of Ser/Thr phosphorylationnd inhibition of Tyr phosphorylation of the CD95. Inine with this, H89 fully blocked both the cAMP-in-uced changes in CD95 phosphorylation and the cAMP-nduced inhibition of TLCS-induced CD95 activationnd apoptosis. However, in neutrophils, also PKA-inde-endent pathways for the antiapoptotic action of cAMPave been suggested.42 These, however, apparently playittle or no role in the prevention of bile salt–inducedepatocyte apoptosis by cAMP.Hepatocytes are type II cells with respect to apoptosis
nduction, which require mitochondrial amplification ofhe CD95-induced death signal to sufficiently activateffector caspases and to induce cell death.43 Accordingly,nhibition of bile salt–induced CD95 activation by an-ioxidants, JNK-and PKC inhibitors, and cAMP willlso inhibit the downstream mitochondrial amplificationrocess. Thus, the reported cAMP and antioxidant-in-uced inhibition of bile acid-induced cytochrome c re-ease from mitochondria may be a downstream conse-uence of cAMP- and antioxidant-induced inhibition ofD95 activation.18,24 These considerations, however, do
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January 2004 cAMP AND CD95 PHOSPHORYLATION 261
ot rule out additional sites of action of cAMP andntioxidants in the apoptotic cascade. Figure 12 summa-izes our current view on the mechanisms involved inLCS-induced CD95 activation and its pharmacologicanipulation.
Svingen PA, Kaufmann SH, Gores GJ. Toxic bile salts inducerodent hepatocyte apoptosis via direct activation of Fas. J ClinInvest 1999;103:137–145.
2. Jones BA, Rao Yp, Stravitz RT, Gores GJ. Bile salt–induced apo-ptosis of hepatocytes involves activation of protein kinase C.Am J Physiol 1997;272:G1109–G1115.
3. Miyoshi H, Rust C, Roberts PJ, Burgart LJ, Gores GJ. Hepatocyteapoptosis after bile duct ligation in the mouse involves CD95.Gastroenterology 1999;117:669–677.
4. Yerushalmi B, Dahl R, Devereaux MW, Gumpricht E, Sokol RJ. Bileacid induced rat hepatocyte apoptosis is inhibited by antioxidants
igure 12. Regulation of the CD95 system by proapoptotic bile saltsnd cAMP. Proapoptotic bile acids such as TLCS, TCDC, or GCDCenerate oxidative stress in rat hepatocytes, which triggers EGF-R-yrosine phosphorylation and JNK-activation. This JNK signal mayonverge with a PKC-signal, to allow association of activated EGF-Rith CD95, which is a substrate for EGF-R-tyrosine kinase activity.ollowing EGF-R-mediated CD95-Tyr phosphorylation, CD95 mem-rane trafficking and DISC formation (FADD and caspase 8/CD95ssociation) and apoptosis are induced. *TCDC only induces DISCormation and apoptosis after inhibition of a PI-3-kinase survival path-ay.5 cAMP inhibits bile salt–induced apoptosis through a PKA-depen-ent (1) CD95-Ser/Thr phosphorylation, which may provide a signalor CD95-internalization, and (2) inhibition of CD95 Tyr phosphoryla-ion, which is a prerequisite for CD95 membrane trafficking and DISCormation. Inhibition of CD95 Tyr phosphorylation by cAMP is due to aKA-dependent inhibition of bile salt–induced EGF-R phosphorylation.late activation of PKA by hydrophobic bile acids may be involved in
he termination of their apoptotic action (dashed line).
and blockers of the mitochondrial permeability transition. Hepa-tology 2001;33:616–626.
5. Takikawa Y, Miyoshi H, Rust C, Roberts P, Siegel R, Mandal PK,Millikan RE, Gores GJ. The bile acid–activated phosphatidylinosi-tol 3-kinase pathway inhibits CD95 apoptosis upstream of bid inrodent hepatocytes. Gastroenterology 2001;120:1810–1817.
6. Benz C, Angermuller S, Otto G, Sauer P, Stremmel W, Stiehl A.Effect of tauroursodeoxycholic acid on bile acid-induced apopto-sis in primary human hepatocytes. Eur J Clin Invest 2000;30:203–209.
7. Benz C, Angermuller S, Kloters-Plachky P, Sauer P, Stremmel W,Stiehl A. Effect of S-adenosylmethionine versus tauroursodeoxy-cholic acid on bile acid–induced apoptosis and cytolysis in rathepatocytes. Eur J Clin Invest 1998;28:577–583.
8. Rodrigues CM, Fan G, Ma X, Kren BT, Steer CJ. A novel role forursodeoxycholic acid in inhibiting apoptosis by modulating mito-chondrial membrane pertubation. J Clin Invest 1998;101:2790–2799.
0. Faubion WA, Gores GJ. Death receptors in liver biology andpathobiology. Hepatology 1999;29:1–4.
1. Leppa S, Bohmann D. Diverse functions of JNK signaling andc-Jun in stress response and apoptosis. Oncogene 1999;18:6158–6162.
2. Graf D, Kurz AK, Fischer R, Reinehr R, Haussinger D. Taurolitho-cholic acid-3 sulfate induces CD95 trafficking and apoptosis in ac-Jun N-terminal kinase dependent manner. Gastroenterology2002;122:1411–1427.
3. Beuers U, Throckmorton DC, Anderson MS, Isales CM, ThaslerW, Kullak-Ublick GA, Sauter G, Koebe HG, Paumgartner G, BoyerJL. Tauroursodeoxycholic acid activates protein kinase C in iso-lated rat hepatocytes. Gastroenterology 1997;113:1306–1341.
4. Roberts LR, Kurosawa H, Bronk SF, Fesmier PJ, Agellon LB,Leung WY, Mao F, Gores GJ. Cathepsin B contributes to bilesalt–induced apoptosis of rat hepatocytes. Gastroenterology1997;113:1714–1726.
5. Sokol RJ, Winkelhofer-Roob BM, Devereaux MW, McKim JM.Generation of hydroperoxides in isolated rat hepatocytes andhepatic mitochondria exposed to hydrophobic bile acids. Gastro-enterology 1995;109:1249–1256.
6. Sokol RJ, McKim JM, Goff MC, Ruyle SZ, Devereaux MW, Han D,Packer L, Everson G. Vitamin E reduces oxidant injury to mito-chondria and the hepatotoxicity of taurochenodeoxycholic acid inthe rat. Gastroenterology 1998;164–174.
7. Sodeman T, Bronk SF, Roberts PJ, Miyoshi H, Gores GJ. Bile saltsmediate hepatocyte apoptosis by increasing cell surface traffick-ing of CD95. Am J Physiol Gastrointest Liver Physiol 2000;278:G992–G999.
8. Reinehr R, Graf D, Haussinger D. Bile salt–induced hepatocyteapoptosis involves epidermal growth factor-dependent CD95-ty-rosine phosphorylation. Gastroenterology 2003;125:839–853.
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Received July 23, 2003. Accepted September 25, 2003.Address requests for reprints to: D. Haussinger, Prof., Medizinische
niversitatsklinik, Moorenstrasse 5, D-40225 Dusseldorf, Germany.-mail: [email protected]; fax (49) 211 811 8838.Supported by Deutsche Forschungsgemeinschaft through Sonder-
orschungsbereich 575 “Experimentelle Hepatologie” (Dusseldorf).The authors thank C. Holneicher and N. Schumacher for expert help
n liver perfusion and S. Becker for excellent technical assistance.
