Potential conflict of interest: Nothing to report.
Cyclooxygenase-2 (COX-2)–derived prostaglandins participate in a number of pathophysiological responses such as inflammation, carcinogenesis, and modulation of cell growth and survival. This study used complementary approaches of COX-2 transgenic (Tg) and knockout (KO) mouse models to evaluate the mechanism of COX-2 in Fas-induced hepatocyte apoptosis and liver failure in vivo. We generated Tg mice with targeted expression of COX-2 in the liver by using the albumin promoter-enhancer–driven vector. The COX-2 Tg, COX-2 KO, and wild-type mice were treated with the anti-Fas antibody Jo2 (0.5 μg/g of body weight) for 4 to 6 hours, and the extent of liver injury was assessed by histopathology, serum aminotransferases, TUNEL staining, and caspase activation. The COX-2 Tg mice showed resistance to Fas-induced liver injury in comparison with the wild-type mice; this was reflected by the lower alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, less liver damage, and less hepatocyte apoptosis (P < 0.01). In contrast, the COX-2 KO mice showed significantly higher serum ALT and AST levels, more prominent hepatocyte apoptosis, and higher levels of caspase-8, caspase-9, and caspase-3 activity than the wild-type mice (P < 0.01). The COX-2 Tg livers expressed higher levels of epidermal growth factor receptor (EGFR) than the wild-type controls; the COX-2 KO livers expressed the lowest levels of EGFR. Pretreatment with a COX-2 inhibitor (NS-398) or an EGFR inhibitor (AG1478) exacerbated Jo2-mediated liver injury and hepatocyte apoptosis. Conclusion: These findings demonstrate that COX-2 prevents Fas-induced hepatocyte apoptosis and liver failure at least in part through up-regulation of EGFR. (HEPATOLOGY 2009.)
Cyclooxygenase-2 (COX-2) is a rate-limiting key enzyme that catalyzes the synthesis of prostaglandins (PGs) from arachidonic acid in a variety of tissues, including the liver. COX-2–derived PGs participate in a number of pathophysiological responses such as inflammation, carcinogenesis, and modulation of cell growth and survival. In the liver, COX-2 and PGs are implicated in hepatocyte growth and hepatocarcinogenesis. For example, liver regeneration following partial hepatectomy in mice and rats is associated with increases in the levels of PGs and the expression of COX-2, and the regenerative response is suppressed by treatment with COX inhibitors.1–7 COX-2 overexpression in hepatocytes has been shown to prevent apoptosis.8 PGs also stimulate DNA synthesis in primary cultures of neonatal and adult rat hepatocytes9–13; these effects are exerted in synergism with epidermal growth factor (EGF) in a manner consistent with comitogenic stimulation.12–14 The evidence for COX-2 and PGs in hepatocarcinogenesis is also compelling.15 For instance, the expression of COX-2 is increased in human and animal hepatocellular carcinomas (HCCs).16–22 COX-2 overexpression or PGE2 treatment increases the growth and invasiveness of cultured HCC cells.21, 23 Nonsteroidal anti-inflammatory drugs (COX inhibitors) and selective COX-2 inhibitors prevent the growth of HCC cells in vitro and in vivo through both COX-dependent and COX-independent mechanisms.15 All these findings underscore the importance of COX-2 and PG signaling in hepatocyte growth and liver tumorigenesis. It is noteworthy that epidermal growth factor receptor (EGFR) has been shown to mediate COX-2 and PGE2 effects in cultured primary hepatocytes and HCC cells in vitro.23, 24
EGFR is a key receptor tyrosine kinase in the liver that plays an important role in liver regeneration and hepatocarcinogenesis.25–32 In human HCC cells, COX-2–derived PGE2 transactivates (phosphorylates) EGFR in human HCC cells, and this mechanism is important for HCC cell growth and invasion.23 In primary hepatocytes, PGE2 has also been found to enhance EGFR signaling through modulation of downstream mitogenic signaling pathways.24 The role of EGFR in hepatocyte growth is exemplified by the fact that EGFR ligands (transforming growth factor-α or EGF) enhance the growth of cultured hepatocytes in vitro26, 27 and conditional deletion of EGFR in mice impairs liver regeneration in vivo.28 However, to date, the role of EGFR in COX-2–mediated and PG-mediated hepatic actions in vivo has not been addressed.
This study was designed to examine the mechanism of COX-2 in Fas-induced liver injury in vivo. We generated transgenic (Tg) mice with targeted expression of COX-2 in the liver by using the albumin promoter-enhancer–driven vector. The produced COX-2 Tg mice, along with COX-2 knockout (KO) and wild-type mice, were injected with the anti-Fas antibody Jo2 to document the extent of liver injury. Our data show that the COX-2 Tg mice resisted Fas-induced liver failure; this was reflected by the lower serum aminotransferase levels, less liver damage, less apoptotic hepatocytes, less caspase activation, and less poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, the COX-2 KO mice showed more prominent liver injury than wild-type mice under the same experimental conditions. Furthermore, hepatic expression of EGFR was highest in the COX-2 Tg mice, intermediate in the wild-type mice, and lowest in the COX-2 KO mice. Pretreatment with a COX-2 inhibitor (NS-398) or an EGFR inhibitor (AG1478) exacerbated Jo2-mediated liver injury. These results demonstrate that COX-2 prevents Fas-induced liver failure at least in part through up-regulation of EGFR.
Tg mice with targeted expression of COX-2 in the liver were developed with the well-established albumin promoter-enhancer–driven vector, as described in a separate study.33 Briefly, a transgene containing full-length human COX-2 complementary DNA under the control of mouse albumin enhancer/promoter was micro-injected into mouse zygotes (B6SJL/F1 eggs). The COX-2 Tg mice used in this study were derived from a Tg line that was backcrossed to C57BL/6 wild-type mice for more than five consecutive generations. The COX-2−/− mice and wild-type C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, MA), and the colonies were maintained at the University of Pittsburgh Animal Facility. The COX-2−/− mice (obtained from the Jackson Laboratory)34 were initially developed in the genetic background of C57BL/6 × 129Sv and were backcrossed to C57BL/6 mice for more than 12 generations. The mice at the age of 8 to 10 weeks were used for experiments, with age/sex-matched wild-type C57BL/6 mice as controls. The animals were kept at 22°C under a 12-hour light/dark cycle and received food and water ad libitum. The handling of the mice and experimental procedures were conducted in accordance with experimental animal guidelines.
Male C57BL/6 wild-type mice, COX-2 Tg mice, and COX-2 KO mice were used for experiments at the age of 8 to 10 weeks. The mice were intraperitoneally administered Jo2 (0.5 μg/g of body weight) to induce acute fulminant hepatic failure (the reagents were dissolved in a sterile, nonpyrogenic saline solution). The animals were sacrificed at specific time points to obtain blood and liver tissues. The liver tissues were rapidly excised, and the specimens were immediately cut into small fragments and subjected to standard formalin fixation and paraffin embedding for histological evaluation and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate digoxigenin nick-end labeling (TUNEL) staining. The remaining liver samples were immediately frozen in liquid nitrogen and stored at −80°C for the future preparation of tissue homogenates. The blood samples were centrifuged at 3000 rpm for 15 minutes, and the sera were collected and stored at −80°C. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with an automatic analyzer at the Chemistry Department of the University of Pittsburgh Medical Center. All experimental animals used in this study were treated according to the protocol approved by the University of Pittsburgh Animal Care and Use Committee (protocol 0303501).
In another set of experiments, mice were pretreated intraperitoneally with vehicle (dimethyl sulfoxide), NS-398 (5 mg/kg), or AG1478 (25 mg/kg) for 30 minutes before Jo2 administration, and the animals were sacrificed 4 hours after Jo2 administration to obtain blood and liver tissues.
Hematoxylin and Eosin Staining.
For histological analysis, liver tissue was fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections 5 μm thick were affixed to slides, deparaffinized, and stained with hematoxylin and eosin to determine morphological changes.
The extent of hepatocyte apoptosis was detected by TUNEL. TUNEL-positive cells were counted by the random selection of high-power fields (400×) distributed over six independent sections. The numbers of TUNEL-positive and TUNEL-negative cells were compiled, and the percentages of TUNEL-positive cells were calculated.
Analysis of Caspase Activity.
After Jo2 antibody challenge, caspase-3, caspase-8, and caspase-9 activity was measured in liver extracts from mice pretreated with or without inhibitors. Briefly, 30 μg of liver protein was incubated with 20 μM fluorogenic substrates (Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC for caspase-3, caspase-8, caspase-9 activity, respectively). The fluorescence signals were monitored with a fluorometer (Tecan, Genios) at an excitation wavelength of 400 nm and an emission wavelength of 510 nm, and the background signals were corrected. The caspase activity was expressed as the fold changes over the control samples (from corresponding wild-type mice).
Hepatic Overexpression of COX-2 Protects Mice Against Fas-Induced Liver Failure.
To investigate the effect of hepatocyte COX-2 on Fas-induced hepatocyte apoptosis, the COX-2 Tg mice and age/sex-matched wild-type mice were injected intraperitoneally with a single dose of the purified hamster anti-mouse Fas monoclonal antibody Jo2 (0.5 μg/g of body weight). The animals were sacrificed at specific time points (0, 4, and 6 hours) after Jo2 administration to obtain blood samples and liver tissues for liver enzyme and tissue analyses. The livers of the wild-type mice turned dark red after the Jo2 injection because of massive hepatic hemorrhaging, which was observed at 4 hours and became much more prominent at 6 hours. In contrast, the livers of the COX-2 Tg mice were completely normal at 4 hours and became slightly red at 6 hours (Fig. 1A). Accordingly, the wild-type mice showed significantly higher serum ALT and AST levels than the COX-2 Tg mice at both 4 and 6 hours (P < 0.01; Fig. 1B). Histological examination of the liver tissues revealed more prominent hepatocyte apoptosis and liver damage in the wild-type mice than in the COX-2 Tg mice (Fig. 2). In the wild-type group, multifocal hepatocyte apoptosis was observed at 4 hours, and massive hepatocyte apoptosis with hemorrhaging was observed at 6 hours. In contrast, the livers from the COX-2 Tg mice showed no significant histological abnormalities at 4 hours, and only mild scattered apoptosis was observed in the COX-2 Tg mice at 6 hours (Fig. 2A,B). The number of TUNEL-positive hepatocytes in the wild-type mice was significantly higher than that in the COX-2 Tg mice at both 4 and 6 hours (P < 0.01; Fig. 2C). These results indicate that hepatic overexpression of COX-2 protects the liver from Fas-induced hepatocyte apoptosis and liver injury.
Disruption of the COX-2 Gene Enhances Fas-Induced Liver Injury.
We next used the mice with homozygous deletion of COX-2 to determine their response to Jo2. The COX-2 KO mice and age/sex-matched wild-type control mice were injected intraperitoneally with Jo2 (0.5 μg/g of body weight), and the animals were sacrificed as described previously. The livers from the COX-2 KO mice turned dark red in color and showed more injury than the wild-type mice 4 hours after the Jo2 injection (Fig. 3A). Accordingly, the COX-2 KO mice showed significantly higher serum ALT and AST levels than the wild-type mice (P < 0.01; Fig. 3B). Histological examination of the liver tissues revealed more prominent hepatocyte apoptosis and liver damage in the COX-2 KO mice compared to the wild-type mice (Fig. 4). The number of TUNEL-positive hepatocytes in the COX-2 KO mice was significantly higher than that in the wild-type mice after Jo2 challenge (P < 0.01; Fig. 4). These findings indicate that COX-2 inactivation augments Fas-induced hepatocyte apoptosis and liver injury, and this is consistent with the observation that the COX-2 inhibitor NS-398 enhances Fas-induced liver injury (discussed later).
COX-2 Prevents Fas-Induced Caspase Activation.
Fas, after the binding of its ligand (eg, Jo2), undergoes trimerization and forms the death-inducing signaling complex, which recruits the initiator caspase-8 via an adaptor protein [Fas-associated protein with death domain (FADD)] and then activates caspase-8. Activated caspase-8 stimulates the caspase cascade leading to the activation of caspase-9 and caspase-3. Given the involvement of caspase-3, caspase-8, and caspase-9 in Fas-induced apoptosis, we next measured their activity in the COX-2 Tg and KO livers. As shown in Fig. 5A, although Jo2 injection increased the levels of hepatic caspase-3, caspase-8, and caspase-9 activity in the COX-2 KO and wild-type mice (P < 0.01 versus the corresponding saline treatment groups), the same concentration of Jo2 did not alter caspase activity in the COX-2 Tg mice. Moreover, the hepatic caspase-3, caspase-8, and caspase-9 activity in Jo2-treated COX-2 KO mice was significantly higher than that in the Jo2-treated wild-type mice (P < 0.01). Because PARP is one of the main cleavage targets of caspase-3 in vivo, we next performed western blot analysis to determine the levels of cleaved PARP in the liver tissues. As shown in Fig. 5B, Jo2 treatment for 4 hours induced the cleavage of PARP in the COX-2 KO and wild-type mice but not in the COX-2 Tg mice. It is noteworthy that more prominent PARP cleavage was observed in the COX-2 KO mice than in the wild-type mice. These results demonstrate that COX-2 signaling prevents Fas-induced caspase activation in the liver.
Hepatic Overexpression of COX-2 Induces Antiapoptotic Molecules.
To address the mechanisms by which COX-2 protects against Fas-induced hepatocyte apoptosis in our system, we further performed western blot analyses to determine the levels of several molecules that have been implicated in hepatocyte survival. Given that EGFR has been shown to play a key role in COX-2–mediated growth of transformed hepatocytes,23 we first investigated the levels of EGFR in the COX-2 Tg and KO mice. As shown in Fig. 6 and Supporting Fig. 1, the level of EGFR was increased in the COX-2 Tg liver and decreased in the COX-2 KO liver. This pattern of alteration was observed in mice with or without Jo2 treatment. Because EGFR is known to phosphorylate and activate protein kinase B (Akt) and this process is facilitated by phosphatase and tensin homolog (PTEN) phosphorylation, we next examined the phosphorylation levels of Akt and PTEN in the COX-2 Tg and KO livers. As shown in Fig. 6, the phosphorylation of Akt and PTEN was increased in the livers of COX-2 Tg mice and decreased in the COX-2 KO mice. The levels of myeloid cell leukemia-1 and B-cell lymphoma extra large, two downstream targets of Akt, were slightly increased in the COX-2 Tg mice and slightly reduced in the COX-2 KO mice (Fig. 6). These findings suggest a potential role of EGFR and related signaling in COX-2–mediated hepatocyte survival in vivo.
Inhibition of COX-2 and EGFR Exacerbates Fas-Induced Hepatocyte Apoptosis and Liver Injury.
To further investigate the role of COX-2 and EGFR in Fas-induced hepatocyte apoptosis, we next employed pharmacological inhibitors of COX-2 and EGFR in our animal models. For the COX-2 inhibitor protocol, wild-type mice and COX-2 Tg mice received an intraperitoneal injection of the COX-2 inhibitor NS-398 (5 mg/kg of body weight) 30 minutes before the administration of Jo2 (0.5 mg/kg of body weight); the animals were sacrificed 4 hours after the Jo2 injection. As shown in Fig. 7, pretreatment with NS-398 resulted in more prominent hepatocyte apoptosis in response to Jo2 challenge; this phenomenon was observed in both wild-type and COX-2 Tg mice. The number of TUNEL-positive hepatocytes in the wild-type and COX-2 Tg mice pretreated with NS398 was significantly higher than that in the mice pretreated with the vehicle control (P < 0.01; Fig. 7). The pretreatment of wild-type and COX-2 Tg mice with NS-398 induced significantly higher serum ALT and AST levels in comparison with the pretreatment with the vehicle control (P < 0.01; Fig. 8A). Furthermore, pretreatment with NS-398 also induced more caspase-8, caspase-9, and caspase-3 activation than pretreatment with vehicle in both wild-type and COX-2 Tg mice (P < 0.01; Fig. 8B).
For the EGFR inhibitor protocol, the COX-2 Tg mice were pretreated with the EGFR inhibitor AG1478 (25 mg/kg of body weight) or vehicle 30 minutes before the Jo2 injection. Histological examination of the liver tissues harvested 4 hours after the Jo2 injection showed more prominent hepatocyte apoptosis in AG1478-pretreated mice (Fig. 7). The number of TUNEL-positive hepatocytes in the AG1478-pretreated group was significantly higher than that in the control group (P < 0.01; Fig. 7). The mice pretreated with AG1478 showed significantly higher serum ALT and AST levels (Fig. 8A) and higher caspase-8, caspase-9, and caspase-3 activity (Fig. 8B; P < 0.01) than the mice pretreated with vehicle. These findings further support the role of EGFR in COX-2–mediated protection against Fas-induced liver injury in vivo.
Activation of Nuclear Factor Kappa B (NF-κB) Is a Potential Mechanism for COX-2–Induced EGFR Expression in the Liver.
Given that the EGFR promoter contains NF-κB binding site that is necessary for the transcriptional activity of EGFR and that inhibition of NF-κB decreases EGFR promoter activity,35 we sought to document NF-κB activation and binding to the EGFR promoter in mice with altered expression of COX-2. As shown in Supporting Fig. 2, the COX-2 Tg livers had higher levels of p65 NF-κB as well as phosphorylated p65 NF-κB than the wild-type livers. Moreover, the liver tissues from the COX-2 Tg mice showed increased binding of NF-κB to its response element in the EGFR promoter (Supporting Fig. 3). These findings suggest that COX-2 up-regulates and phosphorylates NF-κB, leading to its nuclear translocation and binding to the EGFR promoter. Thus, NF-κB activation may represent a potential mechanism for COX-2–mediated EGFR expression in the liver.
In this study, we present evidence for the involvement of COX-2 in hepatocyte survival by complementary genetic and pharmacological approaches. In the COX-2 Tg model, targeted expression of COX-2 in the liver was found to protect against Fas-induced liver injury. This is highlighted by the observation that Jo2-treated COX-2 Tg mice had lower aminotransferase levels, less liver tissue damage, fewer apoptotic hepatocytes, lower caspase-8, caspase-9, and caspase-3 activity, and less PARP cleavage in comparison with Jo2-treated wild-type mice. These findings indicate that COX-2 in hepatocytes is an important survival molecule that prevents Fas-induced hepatocyte apoptosis and liver injury. This conclusion is corroborated by the data from the COX-2 KO mouse model showing that deletion of COX-2 exacerbates Fas-induced liver injury. The role of COX-2 in hepatocyte survival is further supported by the pharmacological approach showing that pretreatment with the COX-2 inhibitor NS-398 enhanced Jo2-induced liver injury. All these findings demonstrate a protective effect of COX-2 against Fas-induced liver injury in vivo. Furthermore, our western blot analyses revealed that the COX-2 Tg livers expressed a higher level of EGFR than the wild-type controls; the COX-2 KO livers expressed the lowest level of EGFR. The direct involvement of EGFR in COX-2 actions is reflected by the observation that pretreatment with the EGFR inhibitor AG1478 augmented Jo2-induced liver injury. These results suggest that up-regulation of EGFR may be an important mechanism for COX-2–mediated resistance to Fas-induced liver injury.
EGFR is a receptor tyrosine kinase that controls a wide variety of biological responses such as proliferation, migration, and modulation of apoptosis.36 Our previous studies have shown that COX-2–derived PGE2 transactivates EGFR in cultured human HCC cells23; this phenomenon has also been documented in other transformed cell lines, including cholangiocarcinoma cells37 and colon cancer cells.38–40 However, in cultured primary hepatocytes, PGE2 does not appear to induce EGFR phosphorylation; instead, PGE2 acts in synergism with EGF by modulating mitogenic mechanisms downstream of EGFR.24 It remains unclear why COX-2–derived PGE2 transactivates EGFR in HCC cells but not in cultured primary hepatocytes. This scenario appears to coincide with the notion that G protein coupled receptors (including PG receptors) more often elicit EGFR transactivation in transfected or malignant cells in comparison with normal cells.41, 42 In the current study, we show that COX-2 up-regulates hepatic expression of EGFR in vivo.
Casado et al.8 reported a protective effect of COX-2 against Fas-induced hepatocyte apoptosis using a mouse model of targeted expression of COX-2 in the liver under the control of apolipoprotein E promoter; this part of the study is confirmed in our Tg model with targeted expression of COX-2 under the control of the albumin promoter. In the current study, we further used the COX-2 KO mice as a complementary model to validate the effect of COX-2. More importantly, the present study provides a novel mechanistic insight into COX-2 actions in the liver. Our results show that EGFR up-regulation plays a key role in COX-2–mediated protection against Fas-induced liver injury. Furthermore, our data suggest the involvement of NF-κB in COX-2–mediated EGFR expression in the liver.
Given that EGFR is able to phosphorylate and activate Akt and that this process is facilitated by PTEN phosphorylation, we also examined the phosphorylation levels of Akt and PTEN in the COX-2 Tg and KO livers. Our data indicate that the levels of p-Akt and p-PTEN were increased in the COX-2 Tg livers but reduced in the COX-2 KO livers. Additionally, the levels of myeloid cell leukemia-1 and B-cell lymphoma extra large, two downstream targets of Akt, were slightly increased in the COX-2 Tg mice and slightly decreased in the COX-2 KO mice. These findings suggest a potential role of EGFR and related signaling in COX-2–mediated hepatocyte survival in vivo.
Reinehr and colleagues43, 44 showed that Fas activation induces the phosphorylation and activation of EGFR, which in turn triggers Fas-tyrosine phosphorylation in hepatocytes; this process is proposed to be a prerequisite for Fas oligomerization and microtubule-dependent translocation of Fas to the plasma membrane, leading to subsequent FADD and caspase-8 recruitment and apoptosis induction. Therefore, the role of EGFR in hepatocytes is complex and involves both cell survival and proapoptotic actions. However, the exact reason for such a paradoxical effect of EGFR in hepatocyte survival is not known. Reinehr and colleagues45 showed that phosphorylation of Fas by EGFR is mediated by phospho-EGFR-Tyr845 and phospho-EGFR-Tyr1173 but not by phospho-EGFR-Tyr1045. On the other hand, activation of EGFR by its ligand (EGF and transforming growth factor-α) induces autophosphorylation of EGFR at distinct and overlapping tyrosine residues (including Tyr992, Tyr1068, Tyr1086, Tyr1148, and Tyr1173)46, 47; these phosphorylated tyrosine residues serve as docking sites for a range of adaptor proteins, whose recruitment leads to activation of the downstream cell survival signaling cascade including the Akt pathway. Therefore, EGFR may influence cell survival or Fas-mediated apoptosis on the basis of its phosphorylation at specific tyrosine residues. Further studies are needed to define the role of specific forms of phospho-EGFR in hepatocyte growth control and their interaction with other key signaling molecules.
Our experimental findings suggest that COX-2 is not a general cytoprotective mediator in the liver. The data presented in the current study show that COX-2 protects the liver from Fas-induced apoptosis via up-regulation of EGFR and activation of its downstream Akt signaling pathway. A separate study from our laboratory has shown that after the injection of lipopolysaccharide/D-galactosamine, COX-2 Tg mice exhibit more prominent liver tissue damage than wild-type mice, and the effect involves activation of c-Jun N-terminal kinase 2.33 Our unpublished data indicate that CCl4-induced acute liver injury is not significantly different between the COX-2 Tg and wild-type groups. Thus, hepatic COX-2 may mediate different responses depending on the context of the liver injury. Consequently, when or where to inhibit or enhance these pathways therapeutically requires careful consideration of the underlying cause of the liver injury as well as the potential benefit and/or risk associated with the intervention.
In summary, this study discloses an important role of EGFR in COX-2–mediated protection against Fas-induced hepatocyte apoptosis and liver failure in vivo. Given the importance of COX-2–derived PGs in several key aspects of liver pathobiology, including hepatocyte survival, liver regeneration, chronic hepatitis, liver injury, and hepatocarcinogenesis, further investigations are warranted to detail the mechanisms of COX-2 and PG actions in different liver diseases and animal models.