Deoxycholic Acid Activates the c-Jun N-terminal Kinase Pathway via FAS Receptor Activation in Primary Hepatocytes

ROLE OF ACIDIC SPHINGOMYELINASE-MEDIATED CERAMIDE GENERATION IN FAS RECEPTOR ACTIVATION*
      We have shown previously that bile acids can activate the JNK pathway and down-regulate cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis. In this study, the mechanism(s) by which deoxycholic acid (DCA) activates the JNK pathway were examined. FAS receptor (FAS-R) and acidic sphingomyelinase (ASM)-deficient hepatocytes were resistant to DCA-induced activation of the JNK pathway. Activation of the JNK pathway (2-3-fold) in response to tumor necrosis factor-α was similar in both wild-type and FAS-R-/- hepatocytes. In wild-type and FAS-R-/- hepatocytes, ceramide elevation was detected as early as 2 min and peaked at 10 min after DCA treatment. In contrast, ASM-/- hepatocytes were defective in DCA-induced ceramide generation. Treatment with DCA resulted in movement of FAS-R to the cell surface, which was blocked upon treatment with brefeldin A. However, brefeldin A failed to block DCA-mediated JNK activation in wild-type hepatocytes. DCA-induced JNK activation was independent of either the epidermal growth factor receptor activation or free radical generation. Addition of ASM to rat hepatocytes activated JNK and down-regulated CYP7A1 mRNA levels. In conclusion, these results show that DCA activates JNK and represses CYP7A1 mRNA levels in primary hepatocytes via an ASM/FAS-R-dependent mechanism that is independent of either the epidermal growth factor receptor or free radical generation.
      Bile acids are steroid molecules that are synthesized from cholesterol in the liver. They function as important regulators of cholesterol metabolism in the hepatocyte by modulating the expression of genes encoding cholesterol-degrading enzymes (bile acid biosynthetic enzymes) as well as cholesterol and phospholipid transport proteins (
      • Vlahcevic Z.R.
      • Heuman D.M.
      • Hylemon P.B.
      ,
      • Vlahcevic Z.R.
      • Pandak W.M.
      • Stravitz R.T.
      ). However, under certain pathophysiological conditions such as cholestasis, bile acids can promote cellular apoptosis and necrosis (
      • Greim H.
      • Trulzsch D.
      • Czygan P.
      • Rudick J.
      • Hutterer F.
      • Schaffner F.
      • Popper H.
      ,
      • Guicciardi M.F.
      • Gores G.J.
      ,
      • Higuchi H.
      • Gores G.J.
      ). Bile acid structure appears to play an important role in the regulation of their various physiological functions in the cell (
      • Vlahcevic Z.R.
      • Heuman D.M.
      • Hylemon P.B.
      ). Generally, hydrophobic bile acids such as lithocholic acid (
      • Allan R.N.
      • Thistle J.L.
      • Hoffman A.F.
      ,
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ), chenodeoxycholic acid (
      • Faubion W.A.
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.
      • Roberts P.J.
      • Svingen P.A.
      • Kaufmann S.H.
      • Gores G.J.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ), and deoxycholic acid (DCA)
      The abbreviations used are: DCA
      deoxycholic acid
      PKC
      protein kinase C
      ERK
      extracellular signal-regulated kinase
      JNK
      c-Jun N-terminal kinase
      CYP7A1
      cholesterol 7α-hydroxylase
      SHP
      small heterodimer partner
      LRH-1
      liver receptor homolog-1
      ASM
      acidic sphingomyelinase
      NSM
      neutral sphingomyelinase
      FAS-R
      FAS receptor
      TNF-α
      tumor necrosis factor-α
      EGF
      epidermal growth factor
      EGFR
      EGF receptor
      PBS
      phosphate-buffered saline
      GST
      glutathione S-transferase
      DAG
      diacylglycerol
      NAC
      N-acetylcysteine
      lpr
      lymphoproliferative.
      1The abbreviations used are: DCA
      deoxycholic acid
      PKC
      protein kinase C
      ERK
      extracellular signal-regulated kinase
      JNK
      c-Jun N-terminal kinase
      CYP7A1
      cholesterol 7α-hydroxylase
      SHP
      small heterodimer partner
      LRH-1
      liver receptor homolog-1
      ASM
      acidic sphingomyelinase
      NSM
      neutral sphingomyelinase
      FAS-R
      FAS receptor
      TNF-α
      tumor necrosis factor-α
      EGF
      epidermal growth factor
      EGFR
      EGF receptor
      PBS
      phosphate-buffered saline
      GST
      glutathione S-transferase
      DAG
      diacylglycerol
      NAC
      N-acetylcysteine
      lpr
      lymphoproliferative.
      (
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Qiao D.
      • Stratagouleas E.D.
      • Martinez J.D.
      ,
      • Qiao L.
      • Song I.H.
      • Youwen F.
      • Park J.S.
      • Gupta S.
      • Gilfor D.
      • Amorino G.
      • Valerie K.
      • Sealy L.
      • Engelhardt J.F.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ) are more potent regulators of gene expression and promoters of liver injury than the more hydrophilic bile acids, such as ursodeoxycholic acid (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Schliess F.
      • Kurz A.K.
      • von Dahl S.
      • Haussinger D.
      ).
      Recently, bile acids have been shown to activate multiple signaling pathways within the hepatocyte that can not only regulate gene expression but can alter cell survival and proliferation. Bile acids have been reported to activate various isoforms of protein kinase C (PKC) (
      • Stravitz R.T.
      • Rao Y.P.
      • Vlahcevic Z.R.
      • Gurley E.C.
      • Jarvis W.D.
      • Hylemon P.B.
      ), extracellular signal-regulated kinase (ERK) (
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Qiao D.
      • Stratagouleas E.D.
      • Martinez J.D.
      ,
      • Qiao L.
      • Song I.H.
      • Youwen F.
      • Park J.S.
      • Gupta S.
      • Gilfor D.
      • Amorino G.
      • Valerie K.
      • Sealy L.
      • Engelhardt J.F.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Qiao D.
      • Chen W.
      • Stratagouleas E.D.
      • Martinez J.D.
      ,
      • Rao Y.P.
      • Studer E.J.
      • Stravitz R.T.
      • Gupta S.
      • Qiao L.
      • Dent P.
      • Hylemon P.B.
      ), c-Jun N-terminal kinase (JNK) (
      • Gupta S.
      • Stravitz R.T.
      • Dent P.
      • Hylemon P.B.
      ,
      • De Fabiani E.
      • Mitro N.
      • Anzulovich A.C.
      • Pinelli A.
      • Galli G.
      • Crestani M.
      ,
      • Wang L.
      • Lee Y.K.
      • Bundman D.
      • Han Y.
      • Thevananther S.
      • Kim C.S.
      • Chua S.S.
      • Wei P.
      • Heyman R.A.
      • Karin M.
      • Moore D.D.
      ), and phosphatidylinositol 3-kinase (
      • Rust C.
      • Karnitz L.M.
      • Paya C.V.
      • Moscat J.
      • Simari R.D.
      • Gores G.J.
      ,
      • Takikawa Y.
      • Miyoshi H.
      • Rust C.
      • Roberts P.
      • Siegel R.
      • Mandal P.K.
      • Millikan R.E.
      • Gores G.J.
      ). Studies from our laboratories have demonstrated that activation of the JNK pathway by bile acids plays a pivotal role in regulating cholesterol 7α-hydroxylase (CYP7A1) gene expression, the rate-limiting enzyme in the neutral pathway of bile acid biosynthesis (
      • Gupta S.
      • Stravitz R.T.
      • Dent P.
      • Hylemon P.B.
      ). We have shown that activation of this pathway leads to induction of the repressor protein SHP (small heterodimer partner), which interacts with the transcription factor liver receptor homolog 1, a known positive regulator of CYP7A1, and represses its transcriptional activity. A similar network of nuclear receptor interactions for feedback repression of CYP7A1 has also been described for the bile acid receptor, farnesoid X receptor (
      • Lu T.T.
      • Makishima M.
      • Repa J.J.
      • Schoonjans K.
      • Kerr T.A.
      • Auwerx J.
      • Mangelsdorf D.J.
      ,
      • Goodwin B.
      • Jones S.A.
      • Price R.R.
      • Watson M.A.
      • McKee D.D.
      • Moore L.B.
      • Galardi C.
      • Wilson J.G.
      • Lewis M.C.
      • Roth M.E.
      • Maloney P.R.
      • Willson T.M.
      • Kliewer S.A.
      ). Recent studies show that cholic acid feeding in both wild-type and SHP knock-out mice increases the levels of the active, phosphorylated JNK in vivo (
      • Wang L.
      • Lee Y.K.
      • Bundman D.
      • Han Y.
      • Thevananther S.
      • Kim C.S.
      • Chua S.S.
      • Wei P.
      • Heyman R.A.
      • Karin M.
      • Moore D.D.
      ). Furthermore, it was shown that treatment of primary hepatocytes from wild-type and SHP-/- mice with the JNK inhibitor, SP600125, increases the basal expression levels of CYP7A1. However, additional studies in SHP knock-out mice also demonstrate the existence of other JNK-dependent but SHP-independent pathways regulating CYP7A1. It has been reported that bile acids can also feedback-regulate CYP7A1 transcription through the nuclear receptor hepatocyte nuclear factor-4α (
      • De Fabiani E.
      • Mitro N.
      • Anzulovich A.C.
      • Pinelli A.
      • Galli G.
      • Crestani M.
      ). Activation of the MEKK1/JNK pathway by bile acids has been shown to decrease the trans-activation potential of hepatocyte nuclear factor-4α, thereby down-regulating CYP7A1 transcription. Bile acids have also been shown to be able to activate liver macrophage cytokine production that can then initiate a cascade that activates JNK in the hepatocyte, a process that decreases the transcriptional activation of CYP7A1 (
      • Miyake J.H.
      • Wang S.L.
      • Davis R.A.
      ). More recently, the nuclear bile acid receptor farnesoid X receptor was shown to regulate directly the expression of fibroblast growth factor-19, a secreted growth factor that signals through the receptor tyrosine kinase, fibroblast growth factor receptor-4 (
      • Holt J.A.
      • Luo G.
      • Billin A.N.
      • Bisi J.
      • McNeill Y.Y.
      • Kozarsky K.F.
      • Donahee M.
      • Wang D.Y.
      • Mansfield T.A.
      • Kliewer S.A.
      • Goodwin B.
      • Jones S.A.
      ). In turn, activated fibroblast growth factor receptor-4 repressed CYP7A1 gene expression via a JNK-dependent pathway. Collectively, the above studies clearly underscore the importance of the JNK signaling pathway in regulation of CYP7A1 gene expression by bile acids. However, the molecular events by which bile acids activate this pathway in hepatocytes have not yet been fully elucidated.
      In other studies, we have shown that low concentrations of DCA cause activation of the Raf-1/MEK/ERK signaling cascade via activation of the epidermal growth factor receptor (EGFR) (
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Rao Y.P.
      • Studer E.J.
      • Stravitz R.T.
      • Gupta S.
      • Qiao L.
      • Dent P.
      • Hylemon P.B.
      ). Activation of this pathway was found to be a protective response of primary hepatocytes to DCA, as a blockade of DCA-induced ERK1/2 activation dramatically increased apoptosis ∼8-fold within 6 h of exposure. Apoptosis was dependent on bile acid-induced, ligand-independent activation of the FAS death receptor (FAS-R; CD95). Studies by other groups (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Faubion W.A.
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.
      • Roberts P.J.
      • Svingen P.A.
      • Kaufmann S.H.
      • Gores G.J.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ) indicate that toxic bile acids can activate FAS-R by directly increasing its cell surface density via induction of FAS-R trafficking to the plasma membrane. The increased density of cell surface FAS-R likely promotes its oligomerization, formation of a death-inducing signal complex, and subsequent induction of apoptosis. Both PKC and JNK have been implicated in FAS-R trafficking to the plasma membrane (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ). However, PKC and JNK may have multiple targets in the apoptotic cascade. More recent studies (
      • Brenner D.A.
      ,
      • Liedtke C.
      • Plumpe J.
      • Kubicka S.
      • Bradham C.A.
      • Manns M.P.
      • Brenner D.A.
      • Trautwein C.
      ,
      • Roulston A.
      • Reinhard C.
      • Amiri P.
      • Williams L.T.
      ) have suggested that JNK signaling may have both pro- and anti-apoptosis signaling effects in hepatocytes. Indeed, studies from our laboratory demonstrate that bile acid-induced activation of the FAS receptor can generate opposing signals, receptor-induced JNK1 activation, resulting in cell death, and receptor-induced JNK2 activation, resulting in protection and metabolism regulation (
      • Qiao L.
      • Song I.H.
      • Youwen F.
      • Park J.S.
      • Gupta S.
      • Gilfor D.
      • Amorino G.
      • Valerie K.
      • Sealy L.
      • Engelhardt J.F.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ). Hence, depending upon the concentration, structure, and conjugation state of the bile acid being presented to the hepatocyte, it can either initiate an apoptotic response or promote cell survival via an interaction of multiple signaling pathways and transcription factors.
      Clustering or aggregation of cell surface receptor molecules upon ligand binding has been reported for a variety of receptors, including tumor necrosis factor (
      • Natoli G.
      • Costanzo A.
      • Guido F.
      • Moretti F.
      • Levrero M.
      ), insulin (
      • Bourguignon L.Y.
      • Jy W.
      • Majercik M.H.
      • Bourguignon G.J.
      ), epidermal growth factor (
      • Bourguignon L.Y.
      • Bourguignon G.J.
      ), FAS (
      • Grassme H.
      • Jekle A.
      • Riehle A.
      • Schwarz H.
      • Berger J.
      • Sandhoff K.
      • Kolesnick R.
      • Gulbins E.
      ,
      • Cremesti A.
      • Paris F.
      • Grassme H.
      • Holler N.
      • Tschopp J.
      • Fuks Z.
      • Gulbins E.
      • Kolesnick R.
      ,
      • Grassme H.
      • Cremesti A.
      • Kolesnick R.
      • Gulbins E.
      ,
      • Fanzo J.C.
      • Lynch M.P.
      • Phee H.
      • Hyer M.
      • Cremesti A.
      • Grassme H.
      • Norris J.S.
      • Coggeshall K.M.
      • Rueda B.R.
      • Pernis A.B.
      • Kolesnick R.
      • Gulbins E.
      ), CD40 (
      • Grassme H.
      • Jendrossek V.
      • Bock J.
      • Riehle A.
      • Gulbins E.
      ), L-selectin (
      • Junge S.
      • Brenner B.
      • Lepple-Wienhues A.
      • Nilius B.
      • Lang F.
      • Linderkamp O.
      • Gulbins E.
      ), immunoglobulin (
      • Graziadei L.
      • Riabowol K.
      • Bar-Sagi D.
      ), and T-cell receptors (
      • Boniface J.J.
      • Rabinowitz J.D.
      • Wulfing C.
      • Hampl J.
      • Reich Z.
      • Altman J.D.
      • Kantor R.M.
      • Beeson C.
      • McConnell H.M.
      • Davis M.M.
      ,
      • Ratcliffe M.J.
      • Coggeshall K.M.
      • Newell M.K.
      • Julius M.H.
      ). These receptors are known to cluster in distinct cholesterol- and sphingolipid-rich domains of the cell membrane, termed rafts (
      • Smart E.J.
      • Graf G.A.
      • McNiven M.A.
      • Sessa W.C.
      • Engelman J.A.
      • Scherer P.E.
      • Okamoto T.
      • Lisanti M.P.
      ). Evidence suggests that membrane rafts are the specific sites for ceramide generation in response to various agonists and stress signals (
      • Cremesti A.E.
      • Goni F.M.
      • Kolesnick R.
      ). Cellular ceramide is generated either by hydrolysis of sphingomyelin or by de novo biosynthesis (
      • Kolesnick R.N.
      • Kronke M.
      ). Sphingomyelin hydrolysis can be catalyzed by at least three different sphingomyelinases: acid (ASM), neutral (NSM), and alkaline sphingomyelinases. The current model of FAS-R signaling suggests that engagement of FAS-R by its ligand or anti-Fas antibody results in translocation of ASM from an intracellular pool to the outer leaflet of the plasma membrane to hydrolyze sphingomyelin and release ceramide in membrane rafts (
      • Grassme H.
      • Jekle A.
      • Riehle A.
      • Schwarz H.
      • Berger J.
      • Sandhoff K.
      • Kolesnick R.
      • Gulbins E.
      ,
      • Grassme H.
      • Cremesti A.
      • Kolesnick R.
      • Gulbins E.
      ). Local release of ceramide in these rafts results in the reorganization of rafts into larger platforms within which FAS-R aggregates and clusters. Clustering of FAS-R appears to facilitate oligomerization of downstream FAS effectors, bringing it in close contact with other signaling molecules and/or excluding inhibitory signaling mechanisms, all of which promote FAS-mediated signaling. Modification of rafts by ceramide, generated via activation of ASM, has also been shown to play an important role in non-apoptotic signaling pathways that require clustering of specific receptors (
      • Gulbins E.
      • Kolesnick R.
      ). Furthermore, rafts altered by ASM activation may be involved in non-receptor-mediated signaling. Activation of the FAS and TNF receptor by stress stimuli (irradiation, heat shock, or UV light) has been observed to occur independently of the FAS ligand or TNF, respectively (
      • Gulbins E.
      • Kolesnick R.
      ,
      • Rehemtulla A.
      • Hamilton C.A.
      • Chinnaiyan A.M.
      • Dixit V.M.
      ,
      • Boldin M.P.
      • Mett I.L.
      • Varfolomeev E.E.
      • Chumakov I.
      • Shemer-Avni Y.
      • Camonis J.H.
      • Wallach D.
      ,
      • Gulbins E.
      ). It has been proposed that some stress stimuli might alter the conformation of FAS-R or change the composition of rafts permitting trapping of unliganded FAS-R molecules. This may result in low level receptor activation sufficient to initiate intracellular signaling.
      In the present studies, we show that expression of the FAS receptor in primary hepatocytes is critically involved in activation of the JNK pathway by bile acids. Moreover, ceramide generated by ASM seems to be a key player in JNK activation in hepatocytes. These data provide a mechanistic basis for the activation of the JNK pathway by physiologic concentrations of bile acids via activation of ASM and generation of ceramide.

      EXPERIMENTAL PROCEDURES

      Materials—DCA, Bacillus cereus sphingomyelinase (NSM), human placental sphingomyelinase (ASM), ATP, diethylenetriaminepenta acetic acid, bovine brain type III ceramide, brefeldin A, dexamethasone, and thyroxine were purchased from Sigma. Anti-JNK1 and rhodamine-conjugated goat anti-Armenian hamster IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Jo2 hamster anti-mouse FAS IgG was from BD Pharmingen (San Diego, CA). Genistein, AG1478, and DAG kinase were from Calbiochem. Cardiolipin (heart) was from Avanti Polar Lipids (Alabaster, AL). N-Octyl-β-d-glucopyranoside and [γ-32P]ATP were supplied by ICN Biomedicals Inc. (Costa Mesa, CA).
      Primary Hepatocyte Cultures—Primary rat hepatocyte monolayer cultures were prepared from male Harlan Sprague-Dawley rats (200-300 g) using the collagenase-perfusion technique of Bissell and Guzelian (
      • Bissell D.M.
      • Guzelian P.S.
      ). Hepatocytes were plated on collagen-coated culture dishes in serum-free William's E medium containing penicillin (100 units/ml), dexamethasone (0.1 μm), and thyroxine (1 μm). Before plating, cells were judged to be greater than 90% viable using trypan blue exclusion. Cells were routinely incubated for 22 h at 37 °C in humidified 5% CO2. Mouse hepatocytes were isolated from lymphoproliferative (lpr; FAS-/-) (a gift from Dr. Prakash Nagarkatti, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA) and wild-type (C57BL/6) male mice or from male ASM knock-out mice (a gift from Dr. Richard Kolesnick, Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York), maintained in an sv129 × C57BL/6 background, propagated using heterozygous breeding pairs, and genotyped as described previously (
      • Paris F.
      • Grassme H.
      • Cremesti A.
      • Zager J.
      • Fong Y.
      • Haimovitz-Friedman A.
      • Fuks Z.
      • Gulbins E.
      • Kolesnick R.
      ). Experimental mice were 8-12-weeks-old when sacrificed.
      JNK Activity Assay—After treatments, hepatocytes (8.5 × 105 cells/35-mm dish) were washed with ice-cold PBS followed by homogenization in cold lysis buffer (25 mm HEPES, pH 7.4, 5 mm EDTA, 5 mm EGTA, 50 mm NaCl, 1 mm Na3VO4, 1 mm sodium pyrophosphate, 0.05% SDS, 0.05% sodium deoxycholate, 1% Triton X-100, 5 mm NaF, 0.1% 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 1 μm microcystin-LR, and 40 μg/ml each of pepstatin A, aprotinin, and leupeptin). Cell supernatants (400 μg) were incubated with 1 μg of anti-JNK1 antibody at 4 °C for 2-3 h. The immune complexes were isolated by the addition of protein A-agarose beads. The immunoprecipitates were recovered by centrifugation and washed (10 min) sequentially with lysis buffer, PBS, and kinase assay buffer (25 mm HEPES, pH 7.4, 15 mm MgCl2, 0.1 mm Na3VO4, and 0.1% (v/v) 2-mercaptoethanol). JNK activity was determined by incubating the washed immunoprecipitates in a reaction mixture containing 40 μl of kinase assay buffer, 0.1 mm ATP, 1 μm microcystin-LR, 10 μCi of [γ-32P]ATP, and 10 μg of recombinant GST-c-Jun (amino acids 1-169) for 20 min at 37 °C. Reactions were terminated by adding 5× SDS-PAGE sample buffer and boiling for 5 min. Phosphorylated GST-c-Jun was resolved in 10% SDS-PAGE and the gels dried and autoradiographed, and the radioactivity incorporated in GST-c-Jun was determined by laser scanning the autoradiograms. Measurement of JNK activity upon addition of either DCA or TNF-α in primary cultures of rat or mouse hepatocytes revealed a species-specific difference in JNK response, with primary rat hepatocytes showing a more robust activation of JNK as compared with primary mouse hepatocytes (Table I).
      Table IJNK activity in primary rat and mouse hepatocytes
      Treatment
      a Primary hepatocytes were treated with DCA (50 μm; 60 min) or TNF-α (10 ng/ml; 20 min), and JNK activity was determined as described under “Experimental Procedures.”
      JNK activity
      % control
      b Values are mean ± S.E., n = 3-4. All values are statistically significant when compared with controls.
      Rat hepatocytes
      Control100
      DCA553.7 ± 110
      TNF-α378.4 ± 61
      Mouse hepatocytes
      c Mouse hepatocytes were isolated from sv129 × C57BL/6 background mice.
      Control100
      DCA183.0 ± 18.8
      TNF-α265 ± 62
      a Primary hepatocytes were treated with DCA (50 μm; 60 min) or TNF-α (10 ng/ml; 20 min), and JNK activity was determined as described under “Experimental Procedures.”
      b Values are mean ± S.E., n = 3-4. All values are statistically significant when compared with controls.
      c Mouse hepatocytes were isolated from sv129 × C57BL/6 background mice.
      Measurement of Endogenous Ceramide—Lipids were extracted, and mass amounts of ceramide in cellular extracts were measured by the DAG kinase enzymatic method (
      • Olivera A.
      • Spiegel S.
      ). Briefly, hepatocytes were washed with PBS and scraped into 1 ml of cold methanol containing 2.5 μl of concentrated HCl. Lipids were extracted by adding 2 ml of chloroform, 1 m NaCl (1:1, v/v), and phases were separated. An aliquot (100 μl) of the chloroform phase was dried under nitrogen gas. Bovine brain type III ceramide was used as a standard. The enzymatic reaction was started by the addition of 100 μl of 50 mm imidazole, 1 mm diethylenetriaminepenta acetic acid, 12.5 mm MgCl2, 50 mm NaCl, 1 mm EGTA, 10 mm dithiothreitol, 1 mm ATP, 1.5% N-octyl-β-d-glucopyranoside, 1 mm cardiolipin, DAG kinase (0.01 unit), and [γ-32P]ATP (1 μCi). After incubation at 37 °C for 35 min with 15 min of sonication at room temperature in between, lipids were extracted by the addition of 500 μl of chloroform:methanol:HCl (100:100:1) and 100 μl of 1 m NaCl. Labeled ceramide 1-phosphate and phosphatidic acid (50-μl organic layer) were resolved by TLC with chloroform:acetone:methanol:acetic acid:water (10:4:3:2:1). Bands corresponding to ceramide were quantified with a Bio-Rad PhosphorImager.
      Immunofluorescence—Hepatocytes were cultured at 10-20% confluence on collagen-coated glass coverslips in 4-well dishes for 24 h. After treatment, the cells were washed with PBS and fixed in cold methanol:acetone (1:1) for 10 min at -20 °C. After washing three times in PBS for 5 min each, the cells were blocked in 0.5% bovine serum albumin in PBS for 30 min at room temperature and incubated with the primary antibody, Jo2, diluted in PBS, 0.5% bovine serum albumin (1:500) overnight at 4 °C. For measurements of total cellular FAS, hepatocytes were fixed in 3.7% formaldehyde in PBS for 10 min at 4 °C and permeabilized with 0.1% Triton X-100 in PBS for 3 min at 4 °C prior to incubation with the primary antibody. Cells were washed again with PBS and then incubated with rhodamine-conjugated goat anti-Armenian hamster IgG at 1:100 dilution for 60 min at room temperature. After washing with PBS, the cells were stained with 5 μm 4′,6′-diamidino-2-phenylindole dihydrochloride in PBS for 5 min. The cells were washed again with PBS, followed by distilled water, and mounted in Crystal-Mount. Fluorescence staining was viewed with an Olympus model IX70 inverted phase microscope (Olympus America, Melville, NY). Fluorescence was quantified via Image-Pro® Plus analysis software and expressed as integrated optical density.
      Quantitation of CYP7A1 mRNA—Total RNA was prepared from cultured hepatocytes using guanidinium thiocyanate CsCl centrifugation. CYP7A1 mRNA levels were determined by RNase protection assay as described previously (
      • Stravitz R.T.
      • Rao Y.P.
      • Vlahcevic Z.R.
      • Gurley E.C.
      • Jarvis W.D.
      • Hylemon P.B.
      ). Rat cyclophilin mRNA was used as an internal control.
      Statistical Analyses—Data were analyzed by Student's t test. Level of significance was set at p < 0.05.

      RESULTS

      Previous studies from our laboratory have shown that bile acids at physiological concentrations (5-50 μm) significantly and rapidly increased JNK activity in primary rat hepatocytes (
      • Gupta S.
      • Stravitz R.T.
      • Dent P.
      • Hylemon P.B.
      ). Recent studies from our laboratory and others have suggested that toxic bile acids and bile acids at pathophysiological concentrations (>150 μm) activate the FAS receptor independently of its ligand, resulting in hepatocyte apoptosis (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Faubion W.A.
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.
      • Roberts P.J.
      • Svingen P.A.
      • Kaufmann S.H.
      • Gores G.J.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ,
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Takikawa Y.
      • Miyoshi H.
      • Rust C.
      • Roberts P.
      • Siegel R.
      • Mandal P.K.
      • Millikan R.E.
      • Gores G.J.
      ). In many cell types, activation of the FAS receptor has been reported to trigger activation of the JNK pathway (
      • Wallach D.
      • Varfolomeev E.E.
      • Malinin N.L.
      • Goltsev Y.V.
      • Kovalenko A.V.
      • Boldin M.P.
      ). We have recently shown that in JNK1-/- and JNK2-/- hepatocytes, treatment with DCA leads to JNK pathway activation, with DCA-induced JNK1 activation, resulting in cell death, and DCA-induced JNK2 activation, resulting in protection (
      • Qiao L.
      • Song I.H.
      • Youwen F.
      • Park J.S.
      • Gupta S.
      • Gilfor D.
      • Amorino G.
      • Valerie K.
      • Sealy L.
      • Engelhardt J.F.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ). To investigate whether the FAS receptor is involved in activation of the JNK pathway by bile acids, hepatocytes isolated from wild-type (C57BL/6) and FAS-R-/- (lpr) mice were treated with 50 μm DCA. As reported previously, JNK was activated in wild-type hepatocytes following DCA treatment (Fig. 1A). However, hepatocytes from FAS-R-/- mice failed to activate JNK in response to DCA. As a positive control, TNF-α activated JNK similarly (2-3-fold) in both wild-type and FAS-R-/- hepatocytes (Fig. 1B).
      Figure thumbnail gr1
      Fig. 1Effect of DCA and TNF-α on JNK activity in primary hepatocytes from wild-type and FAS knock-out mice.A, primary hepatocytes from wild-type (wt) and FAS receptor knock-out mice (FAS-/-) were treated with 50 μm DCA for 60 min. After treatments, hepatocytes were harvested and assayed for JNK activity as described under “Experimental Procedures.” Values are mean ± S.E.; n = 6. *, p < 0.005. Statistical significance was calculated between DCA-treated samples. B, autoradiogram depicting JNK activity in wild-type and FAS-/- hepatocytes in the absence (NA) and in the presence of TNF-α (10 ng/ml for 20 min).
      We have shown previously (
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Rao Y.P.
      • Studer E.J.
      • Stravitz R.T.
      • Gupta S.
      • Qiao L.
      • Dent P.
      • Hylemon P.B.
      ) that DCA can also activate the ERK pathway via activation of the EGF receptor. Activation of EGFR, as determined by increased receptor tyrosine phosphorylation, was found to be dependent upon reactive oxygen species-mediated inhibition of anti-EGFR protein-tyrosine phosphatase activity. A recent report (
      • Reinehr R.
      • Graf D.
      • Haussinger D.
      ) suggests that bile acid-induced EGFR activation can also activate the FAS-R via increased EGFR-mediated FAS-R tyrosine phosphorylation, which triggers FAS-R membrane targeting and death-inducing signal complex formation. To determine whether activation of EGFR by DCA also plays a role in activation of the JNK pathway, we individually pretreated primary rat hepatocytes with 5 μm AG1478, a specific tyrphostin inhibitor of EGFR, 100 μm genistein, a nonspecific tyrosine kinase inhibitor, or the antioxidant, N-acetylcysteine (NAC; 20 mm). Activation of the JNK pathway by DCA was not blocked in the presence of AG1478, genistein, or NAC, suggesting that DCA-mediated JNK activation is independent of the activation of the EGF receptor (Fig. 2, A and B). Moreover, activation of the ERK pathway by DCA (50 μm) was similar in both wild-type and FAS-R-/- mice (180 and 195% of untreated controls, respectively).
      Figure thumbnail gr2
      Fig. 2Effect of tyrosine kinase inhibitors and NAC on DCA-induced JNK activity in primary rat hepatocytes.A, hepatocytes were pretreated for 30 min with Me2SO (vehicle control), 100 μm genistein, or 5 μm AG1478. The cells were then subsequently treated with 50 μm DCA for 60 min (+) or left untreated (-). JNK activity was assayed in cell lysates as described under “Experimental Procedures.” B, hepatocytes were pretreated with either vehicle control or 20 mmN-acetylcysteine (NAC) for 30 min, followed by treatment with 50 μm DCA for 60 min (+). The cells were then harvested and assayed for JNK activity. Representative autoradiograms of two independent experiments, each done in duplicate, are shown.
      Because recent studies (
      • Grassme H.
      • Jekle A.
      • Riehle A.
      • Schwarz H.
      • Berger J.
      • Sandhoff K.
      • Kolesnick R.
      • Gulbins E.
      ,
      • Grassme H.
      • Cremesti A.
      • Kolesnick R.
      • Gulbins E.
      ) suggest that ASM-mediated ceramide generation is critically involved in FAS-R signaling by promoting FAS-R clustering in ceramide-enriched membrane rafts, we examined the ability of DCA to enhance ceramide levels in wild-type, FAS-R-/-, and ASM-/- hepatocytes. Cells were treated with 50 μm DCA over a period of 30 min, and ceramide levels were assayed by the DAG kinase enzymatic method. Ceramide levels increased to 163 ± 22% of control levels (p < 0.05) after 2 min in wild-type (sv129 × C57BL/6) hepatocytes and remained elevated for at least 20 min (Fig. 3). However, hepatocytes isolated from ASM-/- mice failed to generate ceramide in response to treatment with DCA. In contrast, hepatocytes from both wild-type (C57BL/6) and FAS-R-/- mice responded by rapid ceramide elevation to DCA treatment (Fig. 4). Addition of exogenous sphingomyelinase to generate ceramide and hence by-passing FAS-R in FAS-R-/- cells failed to activate JNK (Fig. 5A). These data suggest that the machinery for ceramide generation is intact in FAS-R-/- hepatocytes, and it is the lack of the FAS receptor per se that is responsible for the inability of these cells to activate JNK in response to bile acids or exogenous ceramide addition. Moreover, these studies support the notion that ceramide generated via activation of ASM by bile acids seems to play a key role in FAS-R-mediated JNK activation in mouse hepatocytes.
      Figure thumbnail gr3
      Fig. 3Time course of ceramide generation in primary hepatocytes from littermate wild-type and ASM knock-out mice. Primary hepatocytes from wild-type (wt) and ASM knock-out mice (ASM-/-) were treated with 50 μm DCA for up to 30 min. After treatments, hepatocytes were harvested and lipids extracted for ceramide measurement as described under “Experimental Procedures.” All values are means ± S.E., n = 3. *, p < 0.01. Statistical significance was calculated between ceramide values of wild-type and ASM-/- mice at 5 min.
      Figure thumbnail gr4
      Fig. 4Time course of ceramide generation in primary hepatocytes from wild-type and FAS knock-out mice. Primary hepatocytes from wild-type (wt) and FAS knock-out mice (FAS-/-) were treated with 50 μm DCA for up to 20 min. After treatments, hepatocytes were harvested and lipids extracted for ceramide measurement as described above. All values are means ± S.E., n = 4-6. *, p < 0.05; **, p < 0.001; #, p < 0.001. Statistical significance was calculated between control and treated samples in wild-type and FAS-/- mice.
      Figure thumbnail gr5
      Fig. 5Effect of DCA and/or NSM on JNK activity in primary hepatocytes from FAS or ASM knock-out mice.A, primary hepatocytes from wild-type (wt) and FAS knock-out mice (FAS-/-) were treated with 1 unit/ml of bacterial sphingomyelinase (NSM) for 60 min. After treatments, hepatocytes were harvested and assayed for JNK activity. Values are mean ± S.E., n = 6. *, p < 0.001. Statistical significance was calculated between NSM-treated samples. B, primary hepatocytes from littermate wild-type (wt) and ASM knock-out mice (ASM-/-) were treated with either 50 μm DCA or with 2 units/ml bacterial sphingomyelinase (NSM) for 60 min. After treatments, hepatocytes were harvested and assayed for JNK activity. Values are mean ± S.E., n = 4. *, p < 0.01. Statistical significance was calculated between DCA-treated samples.
      To test further this hypothesis, we determined activation of the JNK pathway by DCA in hepatocytes isolated from wild-type (sv129 × C57BL/6) and ASM-/- mice. We observed that genomic deletion of the alleles to express ASM prevented DCA-mediated JNK activation and that addition of bacterial sphingomyelinase (NSM) to generate ceramide or C2-ceramide addition rescued the phenotype (Fig. 5B and data not shown). Thus, in the presence of FAS-R, but the absence of ASM, exogenous ceramide activated JNK.
      Initiation of FAS signaling relies normally on Fas-L-induced activation of the receptor with ASM-mediated ceramide release playing a critical role in receptor-activated apoptotic and non-apoptotic pathways (
      • Grassme H.
      • Jekle A.
      • Riehle A.
      • Schwarz H.
      • Berger J.
      • Sandhoff K.
      • Kolesnick R.
      • Gulbins E.
      ,
      • Cremesti A.
      • Paris F.
      • Grassme H.
      • Holler N.
      • Tschopp J.
      • Fuks Z.
      • Gulbins E.
      • Kolesnick R.
      ,
      • Grassme H.
      • Cremesti A.
      • Kolesnick R.
      • Gulbins E.
      ,
      • Gulbins E.
      • Kolesnick R.
      ,
      • Paris F.
      • Grassme H.
      • Cremesti A.
      • Zager J.
      • Fong Y.
      • Haimovitz-Friedman A.
      • Fuks Z.
      • Gulbins E.
      • Kolesnick R.
      ,
      • Kirschnek S.
      • Paris F.
      • Weller M.
      • Grassme H.
      • Ferlinz A.
      • Riehle Z.
      • Fuks R.
      • Kolesnick R.
      • Gulbins E.
      ,
      • De Maria R.
      • Rippo M.R.
      • Schuchman E.H.
      • Testi R.
      ,
      • Wajant H.
      • Pfizenmaier K.
      • Scheurich P.
      ). However, recent studies provide evidence that some stress stimuli, like bile acids (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Faubion W.A.
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.
      • Roberts P.J.
      • Svingen P.A.
      • Kaufmann S.H.
      • Gores G.J.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ,
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Takikawa Y.
      • Miyoshi H.
      • Rust C.
      • Roberts P.
      • Siegel R.
      • Mandal P.K.
      • Millikan R.E.
      • Gores G.J.
      ), the anti-tumor reagent ET-18-OCH3 (
      • Gajate C.
      • Mollinedo F.
      ), ganciclovir (
      • Beltinger C.
      • Fulda S.
      • Kammertoens T.
      • Meyer E.
      • Uckert W.
      • Debatin K.M.
      ), UV (
      • Aragane Y.
      • Kulms D.
      • Metze D.
      • Wilkes G.
      • Poppelmann B.
      • Luger T.A.
      • Schwarz T.
      ), and cell shrinkage (
      • Fumarola C.
      • Zerbini A.
      • Guidotti G.G.
      ), induce Fas-L-independent aggregation of the FAS receptor. These stimuli were found to promote increased cell surface trafficking of FAS-R from the Golgi complex and its subsequent spontaneous “concentration”-driven aggregation. Some of the stress stimuli mentioned above were also found to activate ASM resulting in ceramide release and cell death. Because of our findings, we next examined the distribution of FAS-R on the cell surface after treatment of hepatocytes from both wild-type and ASM-/- with DCA. For these studies, mouse hepatocytes were stimulated with DCA for up to 45 min, and cell surface FAS-R was detected with a rhodamine-conjugated secondary antibody. Total cell surface fluorescence was quantitated and expressed as integrated optical density units. Treatment with DCA resulted in an ∼6-fold (p < 0.05) increase in cell surface fluorescence after 45 min in wild-type cells (Fig. 6A and Table II). ASM-/- hepatocytes failed to translocate FAS-R onto the cell surface after stimulation with DCA (Fig. 6B and Table II). Hepatocytes from both wild-type and ASM-/- mice displayed equal amounts of total cell FAS-R as measured in permeabilized cells (data not shown).
      Figure thumbnail gr6
      Fig. 6Effect of DCA on cell surface immunofluorescence of FAS in primary hepatocytes from wild-type and ASM knock-out mice.A and B, primary hepatocytes from littermate wild-type (wt) and ASM knock-out mice (ASM-/-) were treated with 100 μm DCA or diluent in the absence or presence of 10 μg/ml of brefeldin A for the indicated times. After treatments, the cells were fixed, and cell surface FAS was localized by fluorescence microscopy using Jo2 and rhodamine-conjugated goat anti-Armenian hamster IgG as the primary and secondary antibodies, respectively. Nuclei were imaged by 4′,6′-diamidino-2-phenylindole dihydrochloride staining. Representative fluorescence microscopy images are shown (n = 3).
      Table IIQuantitation of cell surface FAS immunofluorescence in ASM mice
      Treatment
      a Primary hepatocytes from wild-type and ASM-/- mice were treated with 100 μm DCA or diluent in the absence or presence of 10 μg/ml of brefeldin A for the indicated times. Cell surface FAS was localized by fluorescence microscopy as described under “Experimental Procedures.”
      Integrated optical density
      Wild-typeASM-/-
      %control
      b Fluorescence was quantified via Image-Pro® Plus analysis software and expressed as integrated optical density. Values are mean ± S.E, n = 3.
      − Brefeldin A
      Diluent100100
      DCA-30 min676.5 ± 219.178.2 ± 6.6
      DCA-45 min581.3 ± 155.6
      c p < 0.05.
      75.8 ± 12
      d Statistically not significant (compared with diluent control).
      + Brefeldin A
      Diluent230 ± 25.8118.8 ± 35.3
      DCA-30 min163.3 ± 68.276.1 ± 16.4
      DCA-45 min178 ± 77.9
      d Statistically not significant (compared with diluent control).
      70 ± 16.4
      a Primary hepatocytes from wild-type and ASM-/- mice were treated with 100 μm DCA or diluent in the absence or presence of 10 μg/ml of brefeldin A for the indicated times. Cell surface FAS was localized by fluorescence microscopy as described under “Experimental Procedures.”
      b Fluorescence was quantified via Image-Pro® Plus analysis software and expressed as integrated optical density. Values are mean ± S.E, n = 3.
      c p < 0.05.
      d Statistically not significant (compared with diluent control).
      During bile acid-induced hepatocyte apoptosis, a Golgi-associated and microtubule-dependent pathway has been implicated in FAS-R trafficking to the plasma membrane (
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ). Thus, we pre-incubated primary hepatocytes for 30 min with brefeldin A, a drug that blocks Golgi-dependent protein secretion (
      • Chardin P.
      • McCormick F.
      ). Brefeldin A prevented surface translocation of FAS-R upon stimulation with DCA in wild-type hepatocytes (Fig. 6A and Table II). However, no effect on DCA-induced JNK activation was observed when primary mouse hepatocytes were pre-treated with brefeldin A (Fig. 7, A and B). These studies argue that although bile acids can promote FAS-R trafficking to the plasma membrane, FAS-R movement to this location is not required to activate JNK in primary hepatocytes.
      Figure thumbnail gr7
      Fig. 7Effect of brefeldin A on DCA-induced JNK activity in primary hepatocytes.A, primary mouse hepatocytes (sv129 × C57BL/6 background) were pretreated with either ethanol (vehicle) or 10 μg/ml of brefeldin A for 30 min. The cells were then subsequently treated with 100 μm DCA for 60 min (+) or left untreated (-). JNK activity was assayed in cell lysates as described under “Experimental Procedures.” Values are mean ± S.E., n = 3. *, p < 0.001; **, statistically not significant; ***, p < 0.05. Statistical significance was calculated with respect to vehicle (-DCA)-treated sample. B, representative autoradiogram depicting JNK activity in mouse hepatocytes treated with brefeldin A prior to treatment with DCA.
      Bile acid-induced activation of the JNK pathway in primary rat hepatocytes down-regulates expression of the CYP7A1 gene (
      • Gupta S.
      • Stravitz R.T.
      • Dent P.
      • Hylemon P.B.
      ). Our data support a pivotal role of ASM/ceramide in FAS-R-mediated JNK activation. Hence, we determined whether addition of exogenous ASM (human placental sphingomyelinase) would also activate JNK and down-regulate CYP7A1. Because the CYP7A1 gene is turned off in mouse hepatocytes in culture,
      S. Gupta, R. Natarajan, S. G. Payne, E. J. Studer, S. Spiegel, P. Dent, and P. B. Hylemon, unpublished observations.
      we performed these studies in primary cultures of rat hepatocytes. When rat hepatocytes were treated with 1 unit/ml of ASM, JNK activity was strongly induced (Fig. 8). Activation of JNK by exogenous ASM was comparable with that for activation by DCA. As expected, addition of either ASM or DCA resulted in an ∼50% decrease in CYP7A1 mRNA levels within 6 h (Fig. 9).
      Figure thumbnail gr8
      Fig. 8Effect of DCA and exogenous ASM on JNK activity in primary rat hepatocytes. Primary rat hepatocytes were treated with either DCA (50 μm) or acidic sphingomyelinase (1 unit/ml) for 60 min. After treatments, hepatocytes were harvested and assayed for JNK activity. Values are mean ± S.E., n = 4. *, p < 0.01; **, p < 0.001. Statistical significance was calculated between control and treated samples.
      Figure thumbnail gr9
      Fig. 9Effect of DCA and exogenous ASM on CYP7A1 mRNA levels in primary rat hepatocytes. Primary rat hepatocytes were treated with either DCA (50 μm) or acidic sphingomyelinase (1 unit/ml) for 6 h. After treatments, hepatocytes were harvested for total RNA and CYP7A1 mRNA levels measured by RNase protection assay. Values are mean ± S.E., n = 4. *, p < 0.001; **, p < 0.05. Statistical significance was calculated between control and treated samples.

      DISCUSSION

      The present studies were designed to provide mechanistic information by which bile acids activate the JNK pathway in primary hepatocytes. The results demonstrate the following: (i) FAS-R is required for DCA-mediated JNK activation, which is independent of EGFR signaling and free radicals; (ii) FAS receptor trafficking to the plasma membrane does not play a role in JNK activation by DCA; and (iii) ceramide generation via ASM is an essential pre-requisite for FAS-R and JNK activation and down-regulation of CYP7A1 gene expression. These data suggest that DCA activates ASM in primary hepatocytes. The increase in ceramide levels then causes activation of the FAS receptor predominantly located internally, resulting in JNK activation and CYP7A1 mRNA down-regulation. Hence, the molecular ordering of events according to the present data is DCA → ASM activation → ceramide generation → FAS-R activation → JNK activation → CYP7A1 transcriptional repression.
      The role of FAS-R in programmed cell death (apoptosis) has been well documented, and hepatocyte apoptosis is a common pathologic feature of cholestatic liver diseases. Bile acid-induced apoptosis has been linked to the activation of the FAS receptor and, more recently, also to the TRAIL-R2/DR5 death receptors (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Faubion W.A.
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.
      • Roberts P.J.
      • Svingen P.A.
      • Kaufmann S.H.
      • Gores G.J.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ,
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Higuchi H.
      • Bronk S.F.
      • Takikawa Y.
      • Werneburg N.W.
      • Takimoto R.
      • El-Deiry W.
      • Gores G.J.
      ). Exposure of hepatocytes to TNF-α also can elicit an apoptotic response (
      • Liedtke C.
      • Plumpe J.
      • Kubicka S.
      • Bradham C.A.
      • Manns M.P.
      • Brenner D.A.
      • Trautwein C.
      ,
      • Roulston A.
      • Reinhard C.
      • Amiri P.
      • Williams L.T.
      ). Data from our laboratory and others have shown that apoptosis in hepatocytes, mediated by DCA and conjugated bile acids (glycochenodeoxycholic acid, taurochenodeoxycholic acid, and taurolithocholic acid), is dependent upon ligand-independent signaling from the FAS receptor (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Faubion W.A.
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.
      • Roberts P.J.
      • Svingen P.A.
      • Kaufmann S.H.
      • Gores G.J.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ,
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ). We have recently shown that DCA-induced JNK1 signaling was cytotoxic, whereas DCA-induced JNK2 signaling was cytoprotective in primary hepatocytes (
      • Qiao L.
      • Song I.H.
      • Youwen F.
      • Park J.S.
      • Gupta S.
      • Gilfor D.
      • Amorino G.
      • Valerie K.
      • Sealy L.
      • Engelhardt J.F.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ). In this paper, we show that DCA can activate the JNK pathway in wild-type but not FAS-R-/- hepatocytes. Because TNF-α activated JNK similarly in both wild-type and FAS-R-/- hepatocytes, this suggests that the mechanism of activation of the JNK pathway by bile acids is different from its activation by inflammatory cytokines in primary hepatocytes. Recently, it was shown that bile acid-mediated activation of the FAS receptor was dependent upon reactive oxygen species-mediated activation of the EGF receptor (
      • Reinehr R.
      • Graf D.
      • Haussinger D.
      ). However, our data show that neither genistein, AG1478, nor NAC blocks DCA-mediated JNK activation in primary rat hepatocytes, arguing that bile acid-mediated FAS-R activation does not require prior phosphorylation by the EGFR. Activation of JNK by DCA significantly repressed CYP7A1 mRNA levels. This is similar to our previous data where we found that bile acid-induced JNK activation down-regulated CYP7A1 gene expression in the absence of cytotoxicity (
      • Gupta S.
      • Stravitz R.T.
      • Dent P.
      • Hylemon P.B.
      ). Thus, the present data identify CYP7A1 as a new JNK-regulated downstream target of DCA-activated FAS-R in primary hepatocytes.
      Our notion of activation of JNK by FAS receptor is supported by previous studies showing that Jo2 or FAS ligand can stimulate JNK activation (
      • Wallach D.
      • Varfolomeev E.E.
      • Malinin N.L.
      • Goltsev Y.V.
      • Kovalenko A.V.
      • Boldin M.P.
      ). Although death receptor-mediated JNK activation is often associated with the induction of apoptosis, several studies have shown that inhibition of JNK has either no effect or a weak attenuating effect on death receptor-mediated apoptosis (
      • Wajant H.
      • Pfizenmaier K.
      • Scheurich P.
      ). Thus, JNK activation is not an obligatory step in FAS-R-induced cell death. FAS-R-mediated activation of JNK and AP-1 in the absence of apoptosis induction has also been described recently (
      • Wollert K.C.
      • Heineke J.
      • Westermann J.
      • Ludde M.
      • Fiedler B.
      • Zierhut W.
      • Laurent D.
      • Bauer M.K.
      • Schulze-Osthoff K.
      • Drexler H.
      ) in cultured cardiomyocytes and has been implicated in cardiac hypertrophy. In agreement with a role of FAS-R-mediated JNK activation in cardiac hypertrophy, this response and JNK activation were diminished in lpr (FAS-R-/-) mice (
      • Badorff C.
      • Ruetten H.
      • Mueller S.
      • Stahmer M.
      • Gehring D.
      • Jung F.
      • Ihling C.
      • Zeiher A.M.
      • Dimmeler S.
      ).
      How does FAS-R activate JNK? FAS-R has been shown to initiate JNK activation by caspase-dependent and caspase-independent pathways (
      • Wallach D.
      • Varfolomeev E.E.
      • Malinin N.L.
      • Goltsev Y.V.
      • Kovalenko A.V.
      • Boldin M.P.
      ,
      • Toyoshima F.
      • Moriguchi T.
      • Nishida E.
      ). Evidence from several groups suggests that FAS-R has the capability to induce JNK activation in the absence of apoptosis by a DAXX-ASK1-dependent but caspase-independent pathway (
      • Wajant H.
      • Pfizenmaier K.
      • Scheurich P.
      ,
      • Yang X.
      • Khosravi-Far R.
      • Chang H.Y.
      • Baltimore D.
      ,
      • Chang H.Y.
      • Yang X.
      • Baltimore D.
      ). Brenner et al. (
      • Brenner B.
      • Koppenhoefer U.
      • Weinstock C.
      • Linderkamp O.
      • Lang F.
      • Gulbins E.
      ) showed that FAS-R-induced apoptosis was mediated by a Ras- and Rac1-regulated activation of JNK. We found that the bile acid taurocholate induced JNK activity via a Ras-independent but cdc42-dependent manner in primary rat hepatocytes.
      S. Gupta, R. Natarajan, S. G. Payne, E. J. Studer, S. Spiegel, P. Dent, and P. B. Hylemon, unpublished observations.
      How FAS-R signals to JNK may be specific to a particular type of signal and/or cell and the intensity of the signal being presented to the cell. However, we cannot exclude additional mechanisms of bile acid-mediated FAS-R/JNK activation.
      Recent reports (
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ) show that hydrophobic and potentially toxic bile acids at high concentrations induce apoptosis by FAS receptor trafficking to the plasma membrane via a Golgi- and microtubule-dependent pathway. In addition, recent studies demonstrated that a JNK- and PKC-mediated association between EGFR and FAS-R was essential for bile acid-induced FAS movement to the membrane (
      • Reinehr R.
      • Graf D.
      • Haussinger D.
      ). In agreement with the above data, we show that DCA also increases FAS receptor concentration on the cell surface of primary mouse hepatocytes. Moreover, pre-treatment with a Golgi-dependent protein secretion inhibitor, brefeldin A, attenuates the increase in cell surface FAS-R. Surprisingly, however, pretreatment with either brefeldin A or AG1478 failed to block JNK activation by DCA. This finding suggests that under our conditions, a protein secretion pathway leading to shuttling of FAS-R to the cell surface is probably not required for bile acid-mediated JNK activation. However, our data do not rule out additional mechanisms of bile acid stimulation of FAS-R trafficking to the plasma membrane. Our findings demonstrated that there was an absence of FAS-R translocation to the cell surface in ASM-/- mice upon stimulation with DCA. At the Golgi, ceramide has been shown previously (
      • van Blitterswijk W.J.
      • van der Luit A.H.
      • Veldman R.J.
      • Verheij M.
      • Borst J.
      ) to drive lipid raft formation and vesicular transport toward the plasma membrane. Studies using radiolabeled and/or fluorescently labeled bile acids demonstrate bile acid association with the Golgi network (
      • Crawford J.M.
      • Barnes S.
      • Stearns R.C.
      • Hastings C.L.
      • Godleski J.J.
      ,
      • Kitamura T.
      • Gatmaitan Z.
      • Arias I.M.
      ,
      • Suchy F.J.
      • Balistreri W.F.
      • Hung J.
      • Miller P.
      • Garfield S.A.
      ). Hence, it is conceivable that bile acid targeting to the Golgi initiates ASM-mediated ceramide generation, resulting in vesicular trafficking of FAS to the plasma membrane.
      Our data indicate a central role of ASM in bile acid-mediated activation of the JNK pathway. The failure of ASM-/- cells to release ceramide and activate JNK upon DCA treatment is in agreement with findings that other stimuli, e.g. irradiation (
      • Gulbins E.
      ,
      • Morita Y.
      • Perez G.I.
      • Paris F.
      • Miranda S.R.
      • Ehleiter D.
      • Haimovitz-Friedman A.
      • Fuks Z.
      • Xie Z.
      • Reed J.C.
      • Schuchman E.H.
      • Kolesnick R.N.
      • Tilly J.L.
      ) and UVA-light exposure (
      • Huang C.
      • Wy M.
      • Ding M.
      • Bowden G.T.
      • Dong Z.
      ), also fail to generate ceramide and activate JNK in ASM-deficient cells. There are numerous reports in the literature (
      • Grassme H.
      • Jekle A.
      • Riehle A.
      • Schwarz H.
      • Berger J.
      • Sandhoff K.
      • Kolesnick R.
      • Gulbins E.
      ,
      • Cremesti A.
      • Paris F.
      • Grassme H.
      • Holler N.
      • Tschopp J.
      • Fuks Z.
      • Gulbins E.
      • Kolesnick R.
      ,
      • Grassme H.
      • Cremesti A.
      • Kolesnick R.
      • Gulbins E.
      ) of early ceramide elevation upon FAS-R ligation and activation. Ceramide generation has been shown to be essential for FAS-R clustering, an event required for optimal FAS-R signaling in some cells. Moreover, ceramide can also serve as a second messenger, leading to induction of the JNK pathway (
      • Kolesnick R.N.
      • Kronke M.
      ). In our system, the ability of DCA to increase ceramide levels in both wild-type and FAS-R-/- hepatocytes and the failure of exogenous ceramide to activate JNK in FAS-R-/- cells suggest a hierarchical organization of ceramide and the FAS pathway, with sphingomyelinase-mediated ceramide generation being upstream of FAS-R/JNK signaling. A recent report (
      • Grassme H.
      • Cremesti A.
      • Kolesnick R.
      • Gulbins E.
      ) has suggested that ligand-bound FAS-R and ceramide constitute the minimal requirements for FAS-R clustering. However, our data and the findings of others (
      • Graf D.
      • Kurz A.K.
      • Fischer R.
      • Reinehr R.
      • Haussinger D.
      ,
      • Faubion W.A.
      • Guicciardi M.E.
      • Miyoshi H.
      • Bronk S.
      • Roberts P.J.
      • Svingen P.A.
      • Kaufmann S.H.
      • Gores G.J.
      ,
      • Sodeman T.
      • Bronk S.F.
      • Roberts P.J.
      • Miyoshi H.
      • Gores G.J.
      ,
      • Qiao L.
      • Studer E.
      • Leach K.
      • McKinstry R.
      • Gupta S.
      • Decker R.
      • Kukreja R.
      • Valerie K.
      • Nagarkatti P.
      • El Deiry W.
      • Molkentin J.
      • Schmidt-Ullrich R.
      • Fisher P.B.
      • Grant S.
      • Hylemon P.B.
      • Dent P.
      ,
      • Takikawa Y.
      • Miyoshi H.
      • Rust C.
      • Roberts P.
      • Siegel R.
      • Mandal P.K.
      • Millikan R.E.
      • Gores G.J.
      ) show that bile acids can activate the FAS-R independent of the FAS ligand. It is possible that bile acids could potentially interact with FAS-R and alter its conformation and/or change the composition of lipid rafts via promoting ASM translocation to the cell surface, thereby permitting trapping of unliganded FAS-R molecules. This may then result in low level receptor activation sufficient to initiate intracellular signaling. However, additional studies will be needed to answer these questions.
      In summary, our data provide evidence for a novel function of a death receptor, i.e. regulation of a gene, CYP7A1, involved in cholesterol metabolism (bile acid biosynthesis). We show that DCA activates JNK and down-regulates CYP7A1 in primary hepatocytes via an ASM/FAS-R/JNK-dependent mechanism that is independent of either the EGFR or free radical generation.

      Acknowledgments

      We thank Pat Bohdan and Emily Gurley for their excellent technical help. We gratefully acknowledge Dr. P. Nagarkatti (Virginia Commonwealth University) for Fas receptor null mice and Dr. Richard Kolesnick (Memorial Sloan-Kettering Cancer Center, New York) for ASM null mice.

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