Bile acid-induced epidermal growth factor receptor activation in quiescent rat hepatic stellate cells can trigger both proliferation and apoptosis

Abstract

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47(phox) and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Czeta, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H2O2), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.

FIGURE 1.

Bile acid-induced EGFR phosphorylation in quiescent HSC. Quiescent HSC were exposed to TLCS (100 μmol/liter) for the time periods indicated or for 30 min to either TCDC, TC, TUDC, or GCDC (100 μmol/liter), CD95L (100 ng/ml), or EGF (50 ng/ml). When indicated, SU6656 (10 μmol/liter), an inhibitor of Src kinases (Yes, Fyn, and c-Src) (36), or AG1478 (5 μmol/liter), an EGFR-tyrosine kinase inhibitor (32), were added 30 min before TLCS addition. Phosphorylation of EGFR-tyrosine residues Tyr⁸⁴⁵, Tyr¹⁰⁴⁵, and Tyr¹¹⁷³ were analyzed by Western blot using phospho-specific antibodies. Total EGFR served as loading control (A and B). Yes, c-Src, and Fyn were analyzed for activating phosphorylation at position Tyr⁴¹⁸ by either immunoprecipitation (Yes, Fyn) or by use of a phospho-specific antibody (c-Src) as given under “Experimental Procedures.” Total Yes, c-Src, and Fyn served as respective loading controls (C). EGFR was immunoprecipitated and subsequently detected for Yes, c-Src, or Fyn by Western blot. Total EGFR served as loading control (D). Representative experiments from a series of three independent experiments are shown. A, in line with earlier studies (13), CD95L and EGF induced EGFR-tyrosine phosphorylation at positions Tyr⁸⁴⁵, Tyr¹⁰⁴⁵, and Tyr¹¹⁷³. In contrast, TLCS induced EGFR phosphorylation at positions Tyr⁸⁴⁵ and Tyr¹¹⁷³ only. AG1478 prevented EGFR autophosphorylation at position Tyr¹¹⁷³ but did not prevent TLCS-induced phosphorylation at position Tyr⁸⁴⁵. This may suggest that TLCS leads to a Src family kinase-mediated EGFR activation at position Tyr⁸⁴⁵, which is followed by EGFR-mediated autophosphorylation at position Tyr¹¹⁷³. B, like TLCS, the pro-apoptotic bile acids TCDC and GCDC, but not TC and TUDC, also trigger EGFR phosphorylation. C, TLCS, TCDC, and GCDC induced phosphorylation of Yes but not of Fyn. Only a weak c-Src phosphorylation was detectable. TC was ineffective, and TUDC induced only a weak Yes and c-Src phosphorylation. For statistical analysis see supplemental Fig. 1. Representative experiments from a series of three independent experiments are shown. D, TLCS, TCDC, and GCDC, but not TC and TUDC, trigger an association of Yes (but not of Fyn or c-Src) with the EGFR. For time courses of TLCS-induced Yes/EGFR association, see supplemental Fig. 2.

FIGURE 2.

Pharmacological characterization of bile acid-induced p47^phox, Yes, and EGFR phosphorylation in quiescent HSC. Quiescent HSC were exposed to either TLCS, TCDC, TC, TUDC, or GCDC (100 μmol/liter, each) for 30 min. When indicated, AY9944 (5 μmol/liter), desipramine (5 μmol/liter), PKCζ inhibitor (100 μmol/liter), chelerythrine (20 μmol/liter), apocynin (300 μmol/liter), diphenyleneiodonium (DPI, 10 μmol/liter), SU6656 (10 μmol/liter), PP-2 (10 μmol/liter), or AG1478 (5μmol/liter) were preincubated for 30 min, whereas the broad-spectrum matrix metalloproteinase inhibitor GM6001 (25 μmol/liter) (8) was added 16 h before TLCS addition. p47^phox, Yes, and EGFR were immunoprecipitated and then analyzed for activating p47^phox -serine phosphorylation, Yes-Tyr⁴¹⁸ phosphorylation, and EGFR-tyrosine phosphorylation by phospho-specific antibodies as described under “Experimental Procedures.” Total p47^phox, Yes, and EGFR served as respective loading controls. As in hepatocytes (23), TLCS-induced activation of the NADPH oxidase subunit p47^phox as well as Yes and EGFR phosphorylation was sensitive to inhibitors of sphingomyelinases (AY9944, desipramine) or protein kinase Cζ (PKCζ inhibitory pseudosubstrate, chelerythrine). In addition, TLCS-induced Yes phosphorylation was sensitive to inhibition of NADPH oxidases (apocynin, diphenyleneiodonium chloride). In line with the literature (7), Yes phosphorylation was also sensitive to SU6656 but not to PP-2. All compounds which inhibited NADPH oxidases and subsequent Yes activation also blocked EGFR-tyrosine phosphorylation. The data suggest a NADPH oxidase- and Yes-dependent EGFR transactivation. GM6001, which has been reported to inhibit bile acid-induced EGFR activation in cholangiocytes (8) or CD95L-induced EGFR activation in quiescent HSC (13), did not affect TLCS-induced EGFR phosphorylation in quiescent HSC. Similar effects were obtained with TCDC and GCDC but not with TC or TUDC. Representative experiments from a series of three independent experiments are shown.

FIGURE 3.

Bile acid-induced activation of mitogen-activated protein kinases in quiescent HSC. Quiescent HSC were exposed to TLCS (100 μmol/liter) for the time periods indicated. When indicated, CHX (0.5 μmol/liter) was added simultaneously with TLCS. Phosphorylation of Erk1/2, p38^MAPK, and JNK1/2 was analyzed by Western blot using phospho-specific antibodies. Total Erk1/2, p38^MAPK, and JNK1/2 served as loading controls. TLCS induced within 30 min a phosphorylation of Erk1/2 but only a weak JNK signal, and no p38^MAPK phosphorylation became detectable. Coadministration of TLCS/CHX resulted in a substantial JNK1/2 and p38^MAPK phosphorylation, whereas the Erk signal was suppressed. For statistical analysis see supplemental Fig. 4. Representative experiments from a series of six independent experiments are shown.

FIGURE 4.

Bile acid-induced BrdUrd incorporation, proliferation, and apoptosis in primary rat HSC. Quiescent (A–D) or activated HSC (A and B) were exposed to either TLCS (100 μmol/liter), CHX (0.5 μmol/liter), or TLCS/CHX for the time periods indicated. Cells were then tested for BrdUrd incorporation (A and D) or proliferation (B) as described under “Experimental Procedures.” If indicated, apocynin (300 μmol/liter), SU6656 (10 μmol/liter), AG1478 (5 μmol/liter), PD098059 (5 μmol/liter), or JNK inhibitor (L-JNKI-1, 5 μmol/liter) were added 30 min before TLCS or TLCS/CHX addition, respectively. In another set of experiments HSC were exposed to either control medium or TLCS for the time periods indicated (C) and then tested for α-smooth muscle actin (αSMA) expression as a surrogate marker for HSC transdifferentiation to myofibroblast-like cells by Western blot. γ-Tubulin served as the loading control. Representative experiments from a series of three independent experiments are shown. A, BrdUrd incorporation. TLCS stimulated BrdUrd incorporation in both quiescent and activated HSC within 48 h. The asterisk denotes statistical significance compared with control (p < 0.05); B, proliferation. The cell number also increased significantly within 48 h of TLCS addition in quiescent HSC but did not reach statistical significance in activated HSC. In contrast, coadministration of TLCS and CHX significantly decreased total cell number. The asterisk (*) denotes statistical significance compared with control (p < 0.05). C, HSC transdifferentiation to myofibroblast. Culture-dependent αSMA-expression was not affected by TLCS. D, Inhibitor profile. TLCS-induced increase in BrdUrd incorporation was sensitive to inhibition of NADPH oxidases, Yes-, EGFR-tyrosine kinase activity and Erk but not to JNK-inhibition. In contrast, upon TLCS/CHX coadministration, the otherwise observed TLCS-induced increase in BrdUrd incorporation did not occur but was reinstituted upon simultaneous JNK-inhibition. # denotes statistical significance compared with control (p < 0.05); */$ denotes significant inhibition compared with TLCS or TLCS/CHX, respectively (p < 0.05); n.s., (not significant, p > 0.05).

FIGURE 5.

Bile acid-induced apoptosis in quiescent HSC. Quiescent (A and B) or activated HSC (A) were either exposed to TLCS (100 μmol/liter), CHX (0.5 μmol/liter), or both TLCS and CHX for 24 h. In another set of experiments CD95L (100 ng/ml) was administered together with TLCS/CHX for 24 h. If indicated, AY9944 (5 μmol/liter), PKCζ inhibitor (100 μmol/liter), apocynin (300 μmol/liter), SU6656 (10 μmol/liter), AG1478 (5 μmol/liter), PD098059 (5 μmol/liter), or JNK inhibitor (L-JNKI-1, 5 μmol/liter) were added 30 min before TLCS/CHX coadministration. The percentage of apoptotic cells was detected using TUNEL staining as described under “Experimental Procedures.” A, apoptosis. Coadministration of TLCS and CHX induced within 24 h a significant increase in apoptotic cells with respect to positive TUNEL staining in both quiescent and activated HSC, whereas treatment with either TLCS or CHX failed to induce apoptosis in those cells. Whereas CD95L inhibited TLCS/CHX-induced apoptotic cell death in quiescent HSC, the number of apoptotic cells even increased in activated HSC. This is explained by CD95L-induced CD95-tyrosine nitration, which induces apoptosis resistance in quiescent HSC, whereas in activated HSC apoptosis is induced by CD95L/CHX (see Fig. 7B) (13). # denotes statistical significance compared with control (p < 0.05); n.s. (not significant) compared with control (p > 0.05); * denotes statistical significance compared with TLCS/CHX (p < 0.05). B, inhibitor profile. TLCS/CHX-induced apoptosis was sensitive to inhibitors of sphingomyelinases, PKCζ, NADPH oxidases, Yes, EGFR-tyrosine kinase activity and JNK, whereas Erk inhibition was ineffective. # denotes statistical significance compared with control (p < 0.05); * denotes significant inhibition compared with TLCS/CHX (p < 0.05); n.s. (not significant) compared with TLCS/CHX (p > 0.05).

FIGURE 6.

Bile acid-induced activation of the CD95 system in quiescent HSC. Quiescent HSC were exposed to TLCS (100 μmol/liter), CHX (0.5 μmol/liter) or CD95L (100 ng/ml), or a combination of TLCS/CHX or CD95L/CHX. Activation of p47^phox, Yes, EGFR, JNK, and CD95 as well as CD95-tyrosine nitration and DISC formation (i.e. association of FADD and caspase 8 to CD95) were detected as described under “Experimental Procedures.” p47^phox-serine phosphorylation, Yes-tyrosine phosphorylation, Yes/EGFR association, EGFR-tyrosine phosphorylation, and JNK1/2 phosphorylation were all detected after 30 min, CD95/EGFR association, CD95-tyrosine phosphorylation (CD95-Y-P), and CD95-tyrosine nitration (CD95-Y-NO₂) were detected after 60 min, whereas DISC formation was determined after 3 h of the respective incubations. Total p47^phox, Yes, EGFR, CD95, and JNK1/2 served as loading controls. Representative experiments from a series of three independent experiments are shown. A, EGFR activation. TLCS induced a p47^phox-serine phosphorylation that was followed by Yes activation, Yes/EGFR association, and subsequent EGFR transactivation, whereas CD95L failed to induce p47^phox or Yes activation. Neither CD95L and TLCS induced a marked JNK activation, which only occurred if CD95L or TLCS, respectively, was coadministered together with CHX. B, CD95 activation. Again, both CD95L and TLCS failed to induce a CD95/EGFR association, which has been reported to depend on a pronounced JNK activation (13, 24). Whereas CD95L induced CD95-tyrosine nitration in line with earlier findings (13), no CD95-tyrosine phosphorylation and subsequent DISC formation occurred even when coadministered together with CHX (13). In contrast, as TLCS did not induce a protective CD95-tyrosine nitration (13, 25), upon TLCS/CHX coadministration CD95-tyrosine phosphorylation and subsequent DISC formation became detectable.

FIGURE 7.

JNK activation switches bile acid-induced EGFR activation toward CD95 activation and subsequent apoptosis in quiescent and activated HSC. Quiescent (A and B) or activated HSC (B), respectively, were exposed to TLCS (100 μmol/liter), CHX (0.5 μmol/liter), CD95L (100 ng/ml), or TLCS/CHX or TLCS/CHX/CD95L was coadministered. When indicated, N-acetylcysteine (NAC, 30mmol/liter), SU6656 (10 μmol/liter), JNK inhibitor (L-JNKI-1, 5 μmol/liter) or AG1478 (5 μmol/liter) were added 30 min before TLCS/CHX were coadministration. Immunoprecipitation and Western blot analysis was performed as given in the legend to Fig. 6. A, inhibitor profile. In quiescent HSC, TLCS and CD95L induced EGFR phosphorylation but failed to induce a pronounced JNK1/2-signal, which is thought to be a prerequisite for CD95/EGFR association, and therefore, no CD95-tyrosine phosphorylation occurs. In addition, CD95L also induced a CD95-tyrosine nitration (13), which has been shown to prevent CD95-tyrosine phosphorylation and subsequent activation of the CD95 system (25), whereas no CD95-tyrosine nitration became detectable after TLCS treatment. Upon TLCS/CHX coadministration, a substantial JNK1/2 phosphorylation occurred which allowed for CD95/EGFR association. All maneuvers that prevented EGFR phosphorylation (i.e. N-acetylcysteine or SU6656), CD95/EGFR association (L-JNKI-1), or EGFR-tyrosine kinase activity (AG1478) also prevented TLCS/CHX-induced CD95-tyrosine phosphorylation, suggestive for an EGFR-mediated CD95-tyrosine phosphorylation and subsequent DISC formation, as has been previously reported in primary rat hepatocytes (6, 7). B, CD95L prevents TLCS/CHX-induced apoptosis in quiescent, but not in activated HSC. CD95L induced CD95-tyrosine nitration (13) and prevented TLCS/CHX-induced CD95-tyrosine phosphorylation and subsequent DISC formation in quiescent HSC. In activated HSC, however, no CD95-tyrosine nitration was triggered by CD95L, CD95-tyrosine phosphorylation was preserved, and CD95L did not inhibit TCLS/CHX-induced apoptosis (see Fig. 5A).

FIGURE 8.

Hydrogen peroxide-induced JNK activation couples bile acid-induced EGFR activation to CD95-mediated apoptosis. Quiescent HSC were exposed to TLCS (100 μmol/liter) and hydrogen peroxide (H₂O₂, 100 μmol/liter), or TLCS/H₂O₂ were coadministered. Immunoprecipitation and Western blot analysis was performed as given in the legend to Fig. 6. TUNEL staining was performed as given under “Experimental Procedures.” A and B, H₂O₂-induced JNK activation led to CD95/EGFR association, which was followed by CD95-tyrosine phosphorylation, DISC formation (A) and subsequent apoptosis as detected by TUNEL staining (B). Therefore, JNK activation switches TLCS-induced EGFR activation from proliferation to apoptosis. * denotes statistical significance compared with control (p < 0.05); # denotes statistical significance compared with TLCS alone (p < 0.05); n.s. (not significant, p > 0.05). C, dose response curve. H₂O₂ induced JNK phosphorylation starting at a dose of 1–10 μm in quiescent HSC, which resulted in CD95/EGFR association, CD95-tyrosine phosphorylation, and subsequent DISC formation.

FIGURE 9.

Gadd45β expression in primary rat HSC and hepatocytes. Primary rat hepatocytes (parenchymal cells (PC)) were isolated and cultured for 24 h as published recently (12). Primary rat HSC were cultured for 2–14 days as indicated. Then HSC and parenchymal cells were detected for Gadd45β-mRNA expression by reverse transcription-PCR (A) and for Gadd45β-protein expression by Western blot (B), respectively, as described under “Experimental Procedures.” β-Actin served as a loading control.

FIGURE 10.

Bile acid- and CD95L-induced proliferation and apoptosis in quiescent HSC and hepatocytes. This figure summarizes our current view on pro-apoptotic bile acid-, i.e. TLCS, and CD95L-induced signaling in primary rat quiescent HSC and hepatocytes. In hepatocytes both, CD95L and the pro-apoptotic bile acid TLCS led to an instantaneous ROS generation which has two functional consequences, activation of the Src-family kinase Yes and the mitogen-activated protein kinase JNK (6, 7, 12). Whereas Yes then transactivates the EGFR, JNK activation induces CD95/EGFR association, which is crucial for EGFR-mediated CD95-tyrosine phosphorylation, DISC formation, and apoptosis (6, 7, 12). In quiescent HSC, CD95L induced a rapid CD95-tyrosine nitration (13) and thereby resistance toward CD95-mediated apoptosis (13, 25). In addition, CD95L mediates Src- and MMP9-dependent EGF-shedding and subsequent ligand-dependent EGFR activation and Erk-dependent proliferation (13). In contrast, TLCS induced ROS-dependent Yes activation and subsequent Yes-mediated EGFR transactivation, resulting in an Erk-dependent HSC proliferation. However, upon induction of a substantial JNK activation by either CHX or hydrogen peroxide, CD95/EGFR association occurred, resulting in an EGFR-mediated CD95-tyrosine phosphorylation, DISC formation, and apoptosis (this study).