1. CD95 Ligand Is a Proliferative and Antiapoptotic Signal in Quiescent Hepatic Stellate Cells 
Roland Reinehr, Annika Sommerfeld, Dieter Häussinger 
Gastroenterology 
Volume 134, Issue 5, Pages e7 (May 2008)
DOI: /j.gastro
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2. Figure 1
(A) CD95 expression and (B) apoptosis induction in cultured HSCs. HSCs were isolated and cultured as described in Materials and Methods. CD95 expression increased during culture but is already present in quiescent HSCs (day 1–2) (A; n = 3). In the presence of CHX (0.5 μmol/L), CD95L induced apoptosis in HSCs cultured for 7 (open triangles) or 14 days (closed squares) but not in quiescent HSCs (d1; closed circles) (B; n = 3).
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3. Figure 2
Death receptor ligand-induced EGFR phosphorylation in quiescent HSCs. HSCs were cultured for up to 48 hours and then exposed to CD95L (100 ng/mL), TNF-α (10 ng/mL or 1 μg/mL), TRAIL (100 ng/mL), or EGF (50 ng/mL) for 30 minutes. When indicated, GM6001 (25 μmol/L), a broad-spectrum inhibitor of MMPs,25 a neutralizing anti-EGF antibody (10 μg/mL), or N-acetylcysteine (NAC, 30 mmol/L) were added 16 hours, 2 hours, or 30 minutes, respectively, before addition of CD95L. Phosphorylation of EGFR tyrosine residues Y845, Y1045, and Y1173 was analyzed by Western blot using phospho-specific antibodies (A; n = 3) or by EGFR immunoprecipitation and subsequent detection for EGFR tyrosine phosphorylation (B; n = 3). Total EGFR served as loading control. CD95L, TNF-α, and TRAIL induce EGFR tyrosine phosphorylation, which was sensitive to MMP inhibition and EGF-neutralizing antibodies. For longer time course, see Supplementary Figure 2.
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4. Figure 3
Characterization of death receptor ligand-induced EGFR phosphorylation in quiescent HSCs. HSCs were cultured for up to 48 hours and then exposed to CD95L (100 ng/mL), TNF-α (1 μg/mL), or TRAIL (100 ng/mL) for 30 minutes. When indicated, a mixed MMP2/9 inhibitor (10μmol/L) or a selective MMP2 inhibitor (10 μmol/L) was preincubated for 3 hours, whereas the Src kinase inhibitors PP-2 (10 μmol/L)29 or SU6656 (10 μmol/L)30 were preincubated for 30 minutes. EGFR phosphorylation was determined by EGFR immunoprecipitation and subsequent detection for EGFR tyrosine phosphorylation. Total EGFR served as loading control (A). Src activation was determined using a phospho-Src-Y418 specific antibody, while activation of Fyn and Yes was detected after Fyn or Yes immunoprecipitation, respectively, and subsequent determination of activating Y418 phosphorylation. Total Src, Fyn, and Yes served as loading controls (B). (A) CD95L-, TNF-α– and TRAIL-induced EGFR tyrosine phosphorylation is sensitive to combined inhibition of MMP2/MMP9, whereas selective MMP2 inhibition was ineffective. The inhibitory effects of PP-2 and SU6656 indicate that Src kinases are upstream elements of EGFR phosphorylation (n = 3). (B) CD95L, TNF-α, and TRAIL induced a phosphorylation of Src but had little effect on Fyn or Yes. In contrast, taurolithocholylsulfate led to a marked Yes phosphorylation (n = 3).
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5. Figure 4
CD95L-induced Erk phosphorylation and BrdU incorporation in HSCs. HSCs were isolated and cultured for up to 48 hours (A and B) and then exposed to either CD95L (100 ng/mL), TNF-α (10 ng/mL or 1 μg/mL), or TRAIL (100 ng/mL). When indicated, GM6001 (25 μmol/L) or EGF-neutralizing antibodies (10 μg/mL) were added 16 hours or 2 hours, respectively, before addition of CD95L. BrdU incorporation in control cells was arbitrarily set to 1. (A) CD95L-induced Erk phosphorylation in quiescent HSCs was detected by Western blot using a phospho-specific antibody and is sensitive to MMP inhibition or EGF-neutralizing antibodies (n = 3). (B) CD95L significantly increased BrdU incorporation (#P < .05; n = 6) compared with untreated control cells (set as 1) in a GM6001 and an EGF-neutralizing antibody-sensitive manner (*P < .05; n = 3). Also, TNF-α (n = 6), TRAIL (n = 5), EGF (50 ng/mL; n = 3), and PDGF (10 ng/mL; n = 3) increased BrdU incorporation in quiescent HSCs (#P < .05). (C) CD95L-induced increase of BrdU incorporation in HSCs cultured for 1 (n = 6), 7 (n = 3), or 14 days (n = 3).
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6. Figure 5
CD95L-induced CD95 tyrosine nitration in quiescent HSCs. HSCs were isolated as described in Materials and Methods and cultured for either 2, 7, or 14 days (A and B) or 24 hours (C). (A) Time course: HSCs were exposed to CD95L (100 ng/mL) for the time periods indicated. Thereafter, CD95 was immunoprecipitated and detected for CD95 tyrosine nitration. Total CD95 served as loading control. In quiescent HSCs (d2), but not in HSCs cultured for 7 or 14 days, CD95L induced within 15 minutes CD95 tyrosine nitration, which persists at least 3 hours (n = 5). (B) Dose dependence: HSCs were exposed to CD95L for 3 hours at the concentrations indicated (n = 5). (C) Total protein tyrosine nitration in quiescent HSCs: Quiescent HSCs (24 hours in culture) were exposed for 60 minutes to either CD95L (100 ng/mL), TNF-α (10 ng/mL or 1 μg/mL), TRAIL (100 ng/mL), or peroxynitrite (ONOO −, 500 μmol/L for 15 minutes). Total protein tyrosine nitration was detected by Western blot (left panel; n = 6). In another set of experiments, CD95 was immunoprecipitated and detected for nitrotyrosine 3 hours after addition of death receptor ligand (right panel; n = 6). Bar graph shows densitometric analysis of CD95 tyrosine nitration in comparison with control experiments (no death receptor ligand added; arbitrarily set to 1). CD95L, TNF-α, and TRAIL induced a significant increase in CD95 tyrosine nitration (#P < .05; n = 3–6).
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7. Figure 6
Characterization of CD95L-induced CD95 tyrosine nitration in quiescent HSCs. HSCs were isolated and cultured for up to 48 hours. CD95 was immunoprecipitated and detected for CD95 tyrosine nitration by Western blot. Total CD95 served as loading control. In the densitometric analysis, the extent of CD95 tyrosine nitration under control conditions (CD95L added, but no inhibitors) was arbitrarily set to 1. (A) Inhibitor profile: CD95L (50 ng/mL) was added for 3 hours. When indicated, uric acid (200 μmol/L), apocynin (300 μmol/L), l-NMMA (1 mmol/L), 2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA; 20 μmol/L), or catalase (8000 U/mL) was preincubated for 30 minutes before addition of CD95L. CD95L-induced CD95 tyrosine nitration was slightly but significantly (*P < .05) inhibited by catalase, whereas uric acid, apocynin, l-NMMA, and BAPTA were ineffective (n = 6). (B) CD95L-induced CD95 tyrosine nitration is sensitive toward DUOX inhibitors: CD95L (50 ng/mL) was added for 1 hour; thiouracil, methimazole, or aminotriazole (1 mmol/L each) were preincubated for 30 minutes (n = 4). Thiouracil, methimazole, or aminotriazole significantly decreased CD95L-induced CD95 tyrosine nitration (*P < .05).
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8. Figure 7
CD95L-induced activation of the CD95 system in quiescent and activated HSCs and liver parenchymal cells (PC). HSCs and PC were cultured for 2 days (quiescent HSCs), 14 days (activated HSCs), or 24 hours (PC) and then exposed to CD95L (100 ng/mL). When indicated, CHX (0.5 μmol/L) was coadministered. (A) CD95L-induced activation of the CD95 system: Yes, EGFR, and CD95 were immunoprecipitated as described in Materials and Methods and analyzed by Western blot. Activating Yes Y418 phosphorylation and EGFR tyrosine phosphorylation (EGFR-Y-P) were detected 1 minute after CD95L exposure. EGFR/CD95 association, CD95 tyrosine nitration (CD95-Y-NO2), and CD95 tyrosine phosphorylation (CD95-Y-P) were detected 60 minutes after addition of CD95L, whereas DISC formation (ie, caspase-8/CD95 and FADD/CD95 association) was determined 3 hours after CD95L exposure. Total Yes, EGFR, and CD95 served as respective loading controls. As described previously,34 CD95L induced Yes phosphorylation in PC but not in HSCs. EGFR phosphorylation was induced in both HSCs and PC, but EGFR/CD95 association in HSCs required addition of CHX. Only in quiescent HSCs did CD95L induce CD95 tyrosine nitration. In PCs, CD95L triggered CD95 tyrosine phosphorylation, which was also observed in activated HSCs, when CHX was coadministered, that is, a condition allowing for EGFR/CD95 association. Only when CD95 tyrosine phosphorylation occurred was DISC formation observed, indicating the pivotal role of CD95 tyrosine phosphorylation for activation of the CD95 system.21,39 (B) CD95L/CHX-induced JNK phosphorylation in HSCs: JNK1/2 phosphorylation was detected in quiescent and activated HSCs at the time points indicated by use of phospho-specific antibodies. Total JNK served as a loading control. CD95L/CHX but not CD95L alone induced JNK1/2 phosphorylation. TNF-α (10 ng/mL or 1 μg/mL; 30 minutes) served as a positive control (n = 3). For longer time course, see Supplementary Figure 7.
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9. Figure 8
CD95L-induced signaling in hepatocytes and quiescent and activated HSCs. Hepatocytes: CD95L induces ROS formation and Yes-dependent EGFR phosphorylation. Simultaneous JNK activation allows for EGFR/CD95 association and subsequent EGFR-catalyzed CD95 tyrosine phosphorylation as prerequisites for DISC formation and apoptosis induction. Quiescent HSCs: CD95L induces ligand-dependent EGFR activation through an Src- and MMP-dependent mechanism, which triggers mitogenic signaling. A simultaneously induced CD95 tyrosine nitration prevents proapoptotic CD95 signaling. Activated HSCs (myofibroblast): Like in quiescent HSCs, CD95L induces ligand-dependent EGFR activation but no inactivating CD95 tyrosine nitration. No proapoptotic signaling occurs, because the failure to produce a JNK signal prevents CD95/EGFR association and consequently CD95 tyrosine phosphorylation. The missing JNK signal is introduced by further addition of CHX and under these conditions, CD95/EGFR association occurs, which allows for EGFR-catalyzed CD95 tyrosine phosphorylation, subsequent DISC formation, and apoptosis induction.
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10. Supplementary Figure 1
CD95-expression in cultured HSCSs. HSCs were immunocytochemically stained for CD95 after 1 day (quiescent phenotype as shown by co-staining for GFAP) or 14 days in culture (activated phenotype as shown by co-staining for αSMA), respectively (bar length 10 μm; n=3).
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11. Supplementary Figure 2
CD95L-induced epidermal growth factor-receptor (EGFR)-phosphorylation in quiescent HSCs. HSCs were cultured for up to 48h and then exposed to CD95L (100ng/mL). When indicated, GM6001 (25μmol/L), a broad spectrum inhibitor of matrix metalloproteinases,25 or a neutralizing anti-EGF antibody (10μg/mL)44 were added 16h or 2h, respectively, prior to CD95L-addition. Phosphorylation of EGFR-tyrosine residues Y845, Y1045, and Y1173 was analyzed by Western blot using phospho-specific antibodies. Total EGFR served as loading control. CD95L induces EGFR-tyrosine-phosphorylation, which was sensitive to MMP-inhibition and EGF-neutralizing antibodies (n=3).
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12. Supplementary Figure 3
Expression of matrix metalloproteinases (MMPs) in rat hepatic stellate cells (HSCs). HSCs were isolated and cultured for either 1-2, 7, or 14 days as described in the Methods section. Expression of MMP2, 9 and 13 at the mRNA or protein level was detected by PCR or Western blot and immunocytochemistry (bar length 10μm), respectively (n=3).
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13. Supplementary Figure 4
CD95L-, EGF-, and PDGF-induced Erk-phosphorylation in quiescent HSCs. HSCs were isolated and cultured for up to 48h as described in the Methods section and then exposed to either CD95L (100ng/mL), EGF (50ng/mL), or PDGH (10ng/mL) for the time periods indicated. CD95L-induced Erk-1/-2-phosphorylation in quiescent HSCs was detected by Western blot using phospho-specific antibodies. Total Erk-1/-2 served as a loading control. CD95L, EGF, and PDGF led to an increase in Erk-1/-2-phosphorylation (n=3).
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14. Supplementary Figure 5
CD95L does not affect HSCs-transdifferentiation to the myofibroblast phenotype. HSCs were cultured for 12h and then exposed to either control medium or CD95L (50ng/mL) for the time periods indicated. Total amount of αSMA-expression was detected by Western blot. γ-tubulin served as a loading control. CD95L had no effect on HSCS-transdifferentiation to the myofibroblast like phenotype with respect to αSMA-expression during culture for up to 14 days (n=3).
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15. Supplementary Figure 6
Expression of NADPH-oxidase subunits in cultured hepatic stellate cells (HSCs). HSCs were isolated and culture for either 1-2, 7, or 14 days as described in the Methods section. Expression of DUOX1/2, gp91phox (Nox2), or p47phox was detected at the protein level by either Western blot or immunocytochemistry (bar length 10 μm). DUOX1/2 is preferentially expressed in quiescent (d1-2) HSCs, whereas gp91phox (Nox2) and p47phox protein expression is higher in activated HSCs (d14) (n=3).
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16. Supplementary Figure 7
CD95L induces the formation of reactive oxygen species (ROS) and p47phox-phosphorylation in liver parenchymal cells (PC), but not in the hepatic stellate cells (HSCs). HSCs were isolated and cultured for either 1-2 days (quiescent HSCs) or 14 days (activated HSCs) and compared to 24h-cultured hepatocytes (PC). ROS generation (A) and activating p47phox-serine-phosphorylation (B) were determined as described in the Methods section. (A) CD95L-induced ROS generation: In PC, CD95L (100ng/mL) led to the previously decribed ROS generation,21,34 as assessed by DCFDA fluorescence, whereas in quiescent and activated HSCs, CD95L failed to induce significant ROS response (n=3). DCFDA flourescence in control cells was arbitrarily set as 1. (B) CD95L-induced p47phox-phosphorylation: CD95L (100ng/mL) induced within 1 min a marked p47phox-serine-phosphorylation in PC, but not in quiescent HSCs, regardless of whether cycloheximide (CHX; 0.5μmol/L) was present or not. Only weak p47phox-phosphorylation was observed in transformed HSCs (14d) (n=3). Total p47phox served as loading control.
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17. Supplementary Figure 8
CD95L-induced JNK-phosphorylation in quiescent and activated HSCs. HSCs were cultured for 2 days (quiescent HSCs) or 14 days (activated HSCs), respectively, and then exposed to CD95L (100ng/mL) for up to 24 h. When indicated, CHX (0.5μmol/L) was coadministered. JNK-phosphorylation was detected in quiescent and activated HSCs at the time points indicated by use of phospho-specific antibodies. CD95L/CHX, but not CD95L alone induced JNK-phosphorylation (n=3).
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