Previous studies have demonstrated that mice disrupted with the cyclooxygenase-2 gene showed much more severe liver damage compared with wild-type mice after liver injury, and prostaglandins (PGs) such as PGE1/2 and PGI2 have decreased hepatic injury, but the mechanisms by which prostaglandins exhibit protective action on the liver have yet to be addressed. In the present study, we investigated the mechanism of the protective action of PGI2 using the synthetic IP receptor agonist ONO-1301. In primary cultures of hepatocytes and nonparenchymal liver cells, ONO-1301 did not show protective action directly on hepatocytes, whereas it stimulated expression of hepatocyte growth factor (HGF) in nonparenchymal liver cells. In mice, peroral administration of ONO-1301 increased hepatic gene expression and protein levels of HGF. Injections of CCl4 induced acute liver injury in mice, but the onset of acute liver injury was strongly suppressed by administration of ONO-1301. The increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by CCl4 were suppressed by 10 mg/kg ONO-1301 to 39.4 and 33.6%, respectively. When neutralizing antibody against HGF was administered with ONO-1301 and CCl4, the decreases by ONO-1301 in serum ALT and AST, apoptotic liver cells, and expansion of necrotic areas in liver tissue were strongly reversed by neutralization of endogenous HGF. These results indicate that ONO-1301 increases expression of HGF and that hepatoprotective action of ONO-1301 in CCl4-induced liver injury may be attributable to its activity to induce expression of HGF, at least in part. The potential for involvement of HGF-Met-mediated signaling in the hepatotrophic action of endogenous prostaglandins generated by injury-dependent cyclooxygenase-2 induction is considerable.
- growth factor
prostaglandins (PGs) play an important role in maintaining local tissue homeostasis and evoking inflammatory responses. Hepatic expression of cyclooxygenase-2 (COX-2), a key enzyme for the elevated production of prostaglandins in response to proinflammatory stimuli and tissue pathology, is upregulated after liver injuries (4, 31). Transgenic mice that overexpressed COX-2 in hepatocytes were resistant to liver injury caused by activation of the Fas antigen and lipopolysaccharide (5, 21). Mice disrupted with the COX-2 gene developed much more severe liver damage than wild-type mice in a hepatic injury model, whereas PGE1/2 and PGI2 analogs decreased hepatic injury (35, 37). Thus, prostaglandins, particularly PGE1/2 and PGI2, play a role in the protection, regeneration, and pathophysiology of the liver. However, the mechanisms by which these effects occur are yet to be fully understood.
We previously showed that PGE1/2 and PGI2 analogs induce gene expression of hepatocyte growth factor (HGF) (20). HGF, purified and cloned as a mitogenic protein for hepatocytes (24, 28, 29), regulates development, survival, and proliferation of hepatocytes through the Met receptor. Gene expression of HGF increased in response to acute liver injuries, whereas administration of HGF promoted liver regeneration and suppressed apoptosis and necrosis of hepatocytes in animal models (16, 30). The hepatotrophic role of HGF was further demonstrated using a conditional knockout of the Met gene in mice (3, 10, 13). These results suggest a potential role of PGs in linking injury-related inflammatory responses to subsequent protection and regeneration of tissues through the induction of HGF.
ONO-1301 was developed as a new type of PGI2/IP receptor agonist lacking the typical prostanoid structures, including a five-membered ring and allylic alcohol (Fig. 1A) (11). Prostacyclin and its analogs are not stable in vivo, whereas ONO-1301 is chemically and biologically more stable than prostacyclin and its analogs because of the absence of prostanoid structures. Furthermore, the presence of a 3-pyridine radical in ONO-1301 confers inhibitory activity for thromboxane synthase, by which ONO-1301 escapes the desensitization of the action in vivo.
The present study examined the therapeutic action of ONO-1301 on liver injury and possible involvement of HGF in its action. ONO-1301 increased the expression of HGF and exerted protective action on the liver, and neutralizing antibody for HGF significantly, although not entirely, suppressed the protective action of ONO-1301. The proposal here is that ONO-1301 exerts a therapeutic action, at least in part, through an increase in HGF levels. ONO-1301 has considerable therapeutic value for liver diseases.
MATERIALS AND METHODS
Animal studies and reagents.
Female ICR mice (SLC, Hamamatsu, Japan), 8–9 wk old, were used. For acute hepatic injury, CCl4 in olive oil was injected intraperitoneally at 50 mg/kg of weight. Animals were killed under anesthesia administered by intraperitoneal injection of pentobarbital sodium salt. All animal experiments were carried out according to the Guidelines for Experimental Animal Care issued by the Prime Minister's Office of Japan and approved by the Committee on Animal Experimentation of Kanazawa University. ONO-1301 was obtained from ONO Pharmaceutical (Osaka, Japan), and recombinant human HGF was obtained from Kringle Pharma (Osaka, Japan).
Interleukin-1β (IL-1β) and CAY-10449 were purchased from Sigma-Aldrich (Sigma, St. Louis, MO) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Forskolin was acquired from Nakalai Tesque (Kyoto, Japan). H-89, PD-98059, and LY-294002 were from Cayman Chemical (Ann Arbor, USA). Bisindolylmaleimide (BIM) and KN-62 were from Calbiochem (San Diego, CA). Anti-phospho-CREB (Ser133) (87G3) and anti-CREB (48H2) antibodies were from Cell Signaling.
Preparation of antibody against HGF.
Recombinant rat HGF was expressed in Chinese hamster ovary cells and purified from the culture supernatant, essentially as described elsewhere (28, 29). The purity of rat HGF exceeded 98% as determined by SDS-PAGE and protein staining with Coomassie brilliant blue. Female Japanese White rabbits (Japan SLC) weighing ∼2 kg were immunized by subcutaneous injection of recombinant rat HGF (50 μg/rabbit) in complete Freund's adjuvant and boosted once or twice at 2-wk intervals by injecting rat HGF in incomplete Freund's adjuvant. The antiserum titer was monitored using an enzyme-linked immunosorbent assay (ELISA). IgGs from anti-rat HGF serum were purified using Protein A-Sepharose Fast Flow (GE Healthcare).
Adult male Wistar strain rats weighing 180–200 g were purchased from SLC Japan. Hepatocytes and nonparenchymal liver cells were isolated from Wistar rats by in situ collagenase perfusion of the liver and cultured as described previously (28, 29). The isolated hepatocytes were placed on a culture plate coated with type I collagen at a density of 5 × 105 cells/ml and cultured for 3 h in Williams' medium E supplemented with 5% FBS, 1 nM insulin, and 1 nM dexamethasone. The culture medium was changed and cultured for 20 h. Hepatocytes were cultured in fresh medium in the absence or presence of 5 mM CCl4, varying concentrations of ONO-1301, and/or 10 ng/ml HGF for 24 h. After in situ collagenase perfusion of the liver, the total cell suspension was centrifuged at 50 g for 1 min. Nonparenchymal liver cells were collected from the supernatant by centrifugation at 100 g for 3 min and washed three times by repetitive centrifugation. Nonparenchymal liver cells were cultured in Williams' medium E supplemented with 5% FBS. Normal human dermal fibroblasts and Madin-Darby canine kidney (MDCK, clone 3B) renal epithelial cells, a gift from Dr. R. Montesano (University of Geneva Medical School), were cultured in Dulbecco's modified Eagle's medium containing 10% FCS.
Measurement of HGF and enzyme activities.
Livers were excised and instantly placed in liquid nitrogen. Preparation of tissue extracts and measurement of tissue HGF concentrations were done as described previously (36). Briefly, tissues were homogenized in 10 volumes of buffer composed of 20 mM Tris·HCl (pH 7.5), 2 M NaCl, 0.01% Tween 20, 1 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA. The homogenate was centrifuged at 15,000 rpm for 30 min, and the resultant supernatant was used as tissue extract. Concentration of HGF in the tissue extract and culture supernatant was measured using an ELISA kit for mouse HGF or human HGF (B-Bridge International), as described previously (36). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using a transaminase CII-test kit (Wako Pure Chemical). Cytochrome P-450-2E1 (CYP2E1) activity in the hepatic microsomal fraction was determined by measuring chlorzoxazone to 6-hydroxychlorzoxazone using LC-MS/MS, as described previously (17).
RNA preparation and quantitative RT-PCR.
Total RNA was extracted using Sepasol-RNA I Super (Nacalai Tesque). First-strand cDNAs were synthesized using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) with oligo(dT)12–18 primers. The primer sequences for mRNA quantification are listed in Fig. 1B. The PRISM 7000 real-time PCR system (Applied Biosystems, Foster City, CA) and Power SYBR Green PCR Master Mix (Applied Biosystems) were used for amplification and online detection. Experimental samples were matched to the standard curve generated by amplifying serially diluted products using the same PCR protocol.
Western blot and immunocytochemistry.
Cells were lysed in a 1:1 mixture of 4× SDS-PAGE sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue) and lysis buffer composed of 50 mM Tris·HCl (pH7.5), 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin and 2% Triton X-100. After brief homogenization and ultrasonication, the samples were subjected to freeze-thawing and centrifuged at 10,000 g for 15 min at 4°C. Samples were separated by electrophoresis on 10% SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membrane was incubated with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 for 1 h, blotted with anti-phospho-CREB and anti-CREB antibodies (1:500) at 4°C overnight, and subsequently labeled with horseradish peroxidase-conjugated antibody against rabbit IgG (1:1,000) for 1 h. The signals were detected with ECL Plus detection reagents (GE Healthcare). For immunocytochemistry, cells were fixed in 4% paraformaldehyde for 15 min. After permeabilization with 0.2% Triton X-100 in PBS, the cells were blocked with 3% normal goat serum and 1% BSA in PBS for 1 h. The cells were incubated with anti-phospho-CREB and anti-CREB (1:500) at 4°C overnight in 3% normal goat serum and 1% BSA in PBS, followed by incubation with fluorescein isothiocyanate-labeled anti-rabbit IgG for 1 h.
For histopathology, tissue sections were analyzed by hematoxylin-eosin staining. The necrotic area was determined in the microscopic field of a 3.56 mm2 area/section in six tissue sections obtained from different animals (n = 6) in each group, using morphometric analysis with ImageJ software on hematoxylin-eosin sections. Apoptotic cell death was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, using a kit (In situ Apoptosis Detection Kit; Takara Bio). The number of apoptotic cells was determined in the microscopic field of a 3.56 mm2 area/section in six tissue sections obtained from different animals (n = 6) in each group using ImageJ software in images of ×200 microscopic fields.
We used a Student's t-test to determine statistical significance. P < 0.05 was considered significant.
Suppression of liver injury.
To determine the efficacy of ONO-1301 for liver injury, acute liver injury was caused by CCl4, and the mice were untreated or treated with ONO-1301 (Fig. 2). Based on our preliminary experiments and change in serum ONO-1301 (unpublished observations), ONO-1301 was administered to mice at 1 h before CCl4 injection and at 6 and 12 h after CCl4 injection. Serum ALT and AST increased markedly 24 h after intraperitoneal injection of CCl4, indicating the onset of acute liver injury. Oral administration of ONO-1301 suppressed the CCl4-induced increase in ALT and AST in a dose-dependent manner. ALT and AST were suppressed to 39.4 and 33.6%, respectively, by 10 mg/kg ONO-1301 compared with the values in control mice injected with CCl4 alone. CAY-10449, a specific IP receptor antagonist, largely reversed the suppressive effect of ONO-1301. These results indicate that ONO-1301 suppresses acute liver injury, and this protective action of ONO-1301 is predominantly mediated through its function as an IP receptor agonist.
Biological activities in liver cells in primary culture.
To determine whether ONO-1301 has a direct cytoprotective effect on hepatocytes, rat primary hepatocytes were cultured in the absence or presence of 5 mM CCl4 and/or ONO-1301 for 24 h, and cellular damage was evaluated by ALT activity in the culture supernatant (Fig. 3A). In a control culture without CCl4, the ALT level in the culture supernatant was not significantly changed by varying concentrations of ONO-1301 and 10 ng/ml HGF. When hepatocytes were treated with CCl4, ALT increased to a level 1.8-fold higher, indicating CCl4-induced damage. The increase in ALT with CCl4 was suppressed by 73.4% in the presence of HGF, indicating direct cytoprotective activity of HGF on hepatocytes. ONO-1301 did not significantly change the increase in ALT in either the absence or presence of HGF. Thus, ONO-1301 has no cytoprotective effect on hepatocytes, at least directly.
To understand the possible involvement of the HGF-Met pathway in the hepatoprotective action of ONO-1301, the effect of ONO-1301 on HGF production by rat nonparenchymal liver cells and human dermal fibroblasts in culture was determined (Fig. 3, B and C). Nonparenchymal cells isolated from rat liver and dermal fibroblasts were cultured in the absence or presence of ONO-1301, and the HGF level in the culture media was measured. For nonparenchymal liver cells, 10−10 M ONO-1301 increased HGF production, and a 3.2-fold increase was seen at 10−6 M (Fig. 3B). Hepatocytes in primary culture did not produce detectable levels of HGF even in the presence of ONO-1301 and IL-1β. The enhancement of HGF production by 10−6 M ONO-1301 was comparable to the action of IL-1β. Similarly, ONO-1301 enhanced HGF production in normal human fibroblasts (Fig. 3C).
Previous study indicated that PGE1/2 and PGI2 analogs enhanced HGF gene expression. Because biological activities of PGE1/2 and PGI2 are, respectively, mediated via EP and IP receptors, and activation of adenylate cyclase seems to be a common pathway triggered by these prostaglandins, we suspected the involvement of a cAMP-dependent pathway in enhancement of HGF expression by ONO-1301. We therefore analyzed the phosphorylation of a cAMP response element binding protein (CREB) and the effects of specific inhibitors on signaling molecules. Immunocytochemical and Western blot analysis indicated that treatment with ONO-1301 induced phosphorylation of Ser133 of CREB (Fig. 3, D and E), a critical event for activation of CREB to induce the transcription of target genes (22). Treatment with H-89, an inhibitor for protein kinase A, mostly suppressed the stimulatory effect of ONO-1301 on HGF production (Fig. 3F). Thus, ONO-1301 may enhance HGF expression through Ser133 phosphorylation of CREB induced by protein kinase A. On the other hand, BIM, an inhibitor for protein kinase C, significantly inhibited HGF production, whereas PD-98059 (mitogen/extracellular signal-regulated kinase inhibitor), KN-62 (calcium/calmodulin kinase-II inhibitor), and LY-294002 (phosphatidylinositol 3-kinase inhibitor) had no significant effect on HGF production. Taken together, these results indicate the possibility that upregulation of HGF expression by ONO-1301 in nonparenchymal liver cells and subsequent activation of the HGF-Met pathway in hepatocytes may be involved in the hepatoprotective action of ONO-1301.
In vivo change in HGF expression.
We analyzed changes in the expression of HGF in the livers of mice treated with the vehicle, ONO-1301, or CCl4, using quantitative real-time RT-PCR (Fig. 4A). During CCl4-induced acute liver injury, HGF mRNA did not increase at 3 h, and then it increased to a 1.4-fold higher level than the control at 8 h. Single administration of ONO-1301 enhanced hepatic HGF mRNA expression to a level 1.9-fold higher than that in control vehicle-treated mice. The hepatic HGF protein level in mice given ONO-1301 significantly increased to a level 1.5-fold higher than that in vehicle-treated mice at 8 h (Fig. 4B). Significant change was not seen in mice given CCl4. Thus a single administration of ONO-1301 increased HGF expression in whole liver to a higher degree and more quickly than the upregulation of HGF expression during CCl4-induced acute liver injury.
Previous studies demonstrated that several chemokines and growth factors, such as stromal cell-derived factor-1 (SDF-1), transforming growth factor-α (TGF-α), epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), and vascular endothelial cell growth factor (VEGF), participate in the regeneration and/or protection of the liver (7, 9, 15, 23). We considered the possibility that ONO-1301 might influence the expression of these factors. Therefore, we analyzed changes in hepatic mRNA expression of SDF-1, TGF-α, EGF, HB-EGF, and VEGF (Fig. 4C). ONO-1301 administration stimulated the expression of SDF-1, EGF, and HB-EGF, whereas ONO-1301 did not increase TGF-α and VEGF mRNA levels. These results implicate the potential involvement of SDF-1, EGF, and HB-EGF in the therapeutic effect of ONO-1301 on the liver. Because ONO-1301 did not significantly change the mRNA expression (Fig. 4C) and enzymatic activity (data not shown) of cytochrome P-450-2E1, an enzyme involved in the metabolism of CCl4, ONO-1301, may not exert therapeutic action by influencing CCl4 metabolism.
Neutralizing antibody against HGF.
To examine the involvement of the HGF-Met pathway in the hepatoprotective action of ONO-1301, we used the approach of neutralizing HGF in experimental animals. Anti-HGF antibody was raised in rabbits by immunization with recombinant rat HGF, and the specificity of the antibody was checked in biological assay (Fig. 5A). Rat, mouse, and human HGF induced cell scattering in a culture of MDCK cells. Anti-HGF antibody inhibited the cell scattering action of rat and mouse HGF but not human HGF, thereby indicating highly specific action for rat/mouse HGF. To determine the dose and interval for administration of anti-HGF antibody in animal models, anti-HGF antibody was injected in mice with CCl4-induced acute liver injury. When anti-HGF antibody was administered at different doses 2 h before CCl4 injection, the CCl4-induced increase in serum ALT and AST was further enhanced at 8 and 16 mg/kg IgG (Fig. 5B). The suppressive effect of ONO-1301 on the CCl4-induced increase in serum ALT and AST was largely cancelled when 8 mg/kg anti-HGF IgG was administered 2 h, 2 days, or 7 days before CCl4 injection (Fig. 5C). These experiments defined the dose, timing, and interval for administration of anti-HGF antibody, not only in this study but also in other studies for different disease models.
Neutralization of HGF in the ONO-1301-dependent suppression of liver injury.
When mice were injected with CCl4, ALT and AST significantly increased from 12 h later and reached a peak at 24 h. Therefore, changes in serum ALT and AST and liver histopathology were analyzed at 24 h after CCl4 injection. Serum ALT and AST increased to 15,686 and 15,739 IU/l, respectively (Fig. 6A). ONO-1301 suppressed the increase in serum ALT and AST to 406 and 450 IU/l, respectively. However, the suppressive effect of ONO-1301 on serum ALT and AST was mostly cancelled by neutralization of HGF; there was no significant change in serum ALT and AST values between the CCl4 group and the CCl4 + ONO-1301 + anti-HGF antibody group.
In mice injected with CCl4, histological examination of the liver indicated cell swelling and necrotic hepatocytes with eosinophilic cytoplasm in the centrolobular and midzonal areas (Fig. 6B). In the livers of mice given CCl4 + anti-HGF antibody, the expansion of the necrotic area was remarkably similar to that in CCl4-treated mice. The liver tissue showed no changes after ONO-1301 administration. However, the expansion of centroloblar lesions accumulated with necrotic/apoptotic hepatocytes was markedly decreased by ONO-1301 treatment in CCl4-treated mice. The necrotic area in the livers of CCl4-injected mice reached 31.0%, but it was decreased to 11.5% by ONO-1301 treatment (Fig. 6C). The necrotic area in mice given CCl4 and ONO-1301 was increased to 34.8% by the neutralization of HGF. These results suggest that the suppression of liver injury by ONO-1301 is attributable, at least in part, to HGF. Because apoptosis in hepatocytes participates in CCl4-induced liver damage (1, 33), we analyzed the changes in apoptotic hepatocytes using TUNEL analysis (Fig. 6D). Apoptotic hepatocytes reached 10.3% in CCl4-injected mice, whereas apoptotic hepatocytes in CCl4-injected mice were strongly decreased to 0.4% by treatment with ONO-1301 (Fig. 6E). The suppressive action of ONO-1301 on hepatocyte apoptosis was mostly cancelled by neutralization of HGF.
The elucidation of endogenous protective mechanisms triggered by liver injury is important, not only for an understanding of the pathophysiology of the liver but also for the design of therapeutic interventions. COX-2 is induced by a variety of stimuli, including proinflammatory cytokines, cellular stresses, and tissue injuries. Although mature hepatocytes lack COX-2 expression, regardless of proinflammatory cytokine stimulation, induction of COX-2 was seen in the liver after partial hepatectomy and alcoholic liver injury (4, 31). In mice lacking the COX-2 gene, the proliferation of hepatocytes after partial hepatectomy was decreased compared with wild-type mice (4). Transgenic mice expressing COX-2 in hepatocytes were resistant to Fas-mediated liver injury and lipopolysaccharide-induced liver injury (5, 21). Thus, prostaglandins generated in the liver play a role in the survival and proliferation of hepatocytes while the mechanisms by which prostaglandins exert these actions remain to be addressed.
In the present study, we found that 1) synthetic IP receptor agonist ONO-1301 stimulated expression of HGF in nonparenchymal liver cells, but not hepatocytes, in primary culture; 2) administration of ONO-1301 stimulated HGF expression in the liver and strongly suppressed CCl4-induced liver injury, and this protective effect was largely abolished by neutralization of endogenous HGF. These results suggest that HGF upregulated by ONO-1301 inhibited the necrotic and apoptotic death of hepatocytes, thereby leading to suppression of CCl4-induced liver injury. Compared with control mice, mice lacking the Met/HGF receptor gene in hepatocytes showed much augmented apoptosis and/or necrosis of hepatocytes caused by Fas activation or CCl4, whereas administration of HGF suppressed hepatocyte death caused by Fas activation and CCl4 (13, 16, 30). Increase in anti-apoptotic Bcl-xL expression and prevention of phosphatidylinositol 3-kinase- and protein kinase B-dependent FLIP degradation were noted as mechanisms for hepatoprotective action of HGF (16, 25). Taking into consideration the notion that COX-2 is induced following hepatic injury, we propose that PGI2 generated via COX-2 induction exhibits its biological action to suppress cell death of hepatocytes during liver injury.
In previous studies using rat hepatocytes in primary culture, the mitogenic activity of prostaglandins for hepatocytes was controversial. Some reports showed that prostaglandins alone did not stimulate basal DNA synthesis, whereas prostaglandins augmented the mitogenic action of EGF (6). Others indicated that PGI2 enhanced DNA synthesis of hepatocytes, whereas it was abolished by neutralization of TGF-α (14). Perhaps the discrepancy may be related to differences in experimental conditions; however, these results indicate that prostaglandins have no mitogenic activity for hepatocytes, at least directly. Changes in gene expression of EGF receptor ligands such as EGF, TGF-α, and HB-EGF occur following liver injury, and activation of EGF receptor is associated with the promotion of proliferation and survival in hepatocytes (7, 9, 23). We observed here that ONO-1301 increased expression of SDF-1, EGF, and HB-EGF in the liver. Taken together, these results indicate it is likely that the preventive effect of ONO-1301 on liver failure may be partly mediated by these chemokine and EGF receptor ligands, and this seems to explain why the protective action of ONO-1301 was largely, but not fully, cancelled by neutralization of HGF.
Gene expression of HGF is regulated by growth factors, cytokines, and prostaglandins; however, signaling pathways leading to induction of HGF gene expression have not been well characterized. Among these factors, prostaglandin receptors are G protein-coupled receptors from which different effectors and signaling pathways are evoked. EP2 and EP4 receptors for PGE1/2 and IP receptor for PGI2 activate adenylate cyclase upon ligand binding, thereby increasing the cAMP level. In the present study, we found that ONO-1301 rapidly induced phosphorylation of CREB-Ser133, and biological activity of ONO-1301 to enhance HGF production was mostly cancelled by a selective inhibitor for protein kinase A. Because CREB-Ser133 phosphorylation plays a major regulatory role in the transcriptional activation of target genes and protein kinase A can phosphorylate CREB-Ser133 (22), the activation of protein kinase A and CREB-Ser133 phosphorylation induced by protein kinase A seemed to be a main mechanism responsible for the induction of HGF expression by ONO-1301. On the other hand, activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate increased HGF expression, which was synergistically enhanced by the combination with PGE1 (20). Enhancement of HGF expression by ONO-1301 was significantly, but not mostly, decreased by inhibition of protein kinase C. Basal activity of protein kinase C may possibly be a prerequisite for the induction of HGF expression.
HGF exerts therapeutic action in different models in different tissues, including acute and chronic renal failure, pulmonary fibrosis, and cardiac ischemia and fibrosis (30). Likewise, recent studies demonstrated that ONO-1301 exhibited therapeutic action in various models, including pulmonary fibrosis, cardiac ischemia and fibrosis, and renal pathology (12, 26, 27). Based on the results from these previous studies, combined with results from our present study, HGF induced by ONO-1301 seems to explain, at least in part, the therapeutic effects of ONO-1301 on tissue pathology in different tissues. In particular, because the antifibrotic activity is characteristic of HGF among various growth factors, the involvement of HGF in the suppressive effect of ONO-1301 on tissue fibrosis is considerable. The activation and inactivation of the HGF-Met pathway are associated with an improvement in tissue fibrosis and an increased susceptibility to tissue fibrosis, respectively (10, 19, 30).
In conclusion, synthetic IP receptor agonist ONO-1301 suppressed the cell death of hepatocytes after CCl4-induced acute liver injury, thereby strongly suppressing liver damage, and this action was mainly attributable to its activity in inducing the expression of HGF. We suggest that endogenously generated prostaglandins such as PGE2 and PGI2 may exert hepatotrophic effects on the liver following COX-2 induction, through an increase in HGF- and Met-mediated signaling. ONO-1301 lacks the typical prostanoid/PGI2 structures, and, because of this, ONO-1301 is chemically more stable than prostaglandins. The therapeutic benefit of treating liver pathology with ONO-1301 or another chemically stable IP receptor agonist is considerable.
This work was partly supported by the Ministry of Education, Culture, Science, Sports, and Technology of Japan (no. 20390077 to K. Matsumoto, 21790312 to T. Nakamura).
No conflicts of interest are declared by the authors.
We are grateful for assistance from Scientific Editorial Services (Harrison, AR).
Present address of M. Nakayama: Division of Biological Informatics and Experimental Therapeutics, Akita University School of Medicine, 1-1-1, Hondo, Akita, 010-8543 Japan.
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