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Endocrinology Vol. 142, No. 10 4428-4440
Copyright © 2001 by The Endocrine Society

ARTICLES

Prostaglandin E2 (EP1) Receptor Agonist-Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes: The Involvement of TGF-{alpha}

Mitsutoshi Kimura, Sachie Osumi and Masahiko Ogihara

Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Josai University, 1-1, Keyakidai, Sakado City 350-0295, Japan

Address all correspondence and requests for reprints to: Masahiko Ogihara, Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Josai University, 1-1, Keyakidai, Sakado City 350-0295, Japan. E-mail: ogiharam{at}josai.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the effects of prostaglandin (EP) receptor subtype agonists on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes to elucidate their mechanisms of action. Maintained in short-term cultures (i.e. 3.5 h) in a serum-free, defined medium, hepatocyte parenchymal cells underwent DNA synthesis and proliferation in the presence of sulprostone (10-6 M), PGE2 (10-6 M), and 17-phenyl-trinor-PGE2 (10-9 M) in a time- and dose-dependent manner. PGE2 was less potent than 17-phenyl-trinor-PGE2 in stimulating hepatocyte mitogenesis. Sulprostone (10-6 M) and 11-deoxy-PGE1 (10-6 M) showed weak and insignificant stimulation, respectively, for hepatocyte mitogenesis. These effects of PGE2, 17-phenyl-trinor-PGE2, and sulprostone were abolished by treatment with a specific EP1 receptor antagonist, SC-51322, or the PLC inhibitor U-73122. The effects of these EP1 receptor agonists were potentiated by ionomycin and blocked by verapamil. Hepatocyte mitogenesis was almost completely blocked by specific inhibitors of growth-related signal transducers, such as genistein, wortmannin, PD98059, and rapamycin. A monoclonal antibody against TGF-{alpha} dose-dependently inhibited PGE2- and 17-phenyl-trinor-PGE2-induced hepatocyte mitogenesis. Treatment with the EP1 receptor agonists significantly increased the secretion of TGF-{alpha}, reaching a maximum within 5 min. The increase in TGF-{alpha} secretion was blocked by SC-51322, U-73122, somatostatin, and verapamil and potentiated by ionomycin. These results indicate that the proliferative mechanisms of action of EP1 receptor agonists are mediated through an increase in the autocrine secretion of TGF-{alpha}, which is dependent on the EP1 receptor/G-protein involved in PLC regulation/PLC/Ca2+ system. The locally secreted TGF-{alpha}, in turn, acts as a complete mitogen that stimulates the tyrosine kinase/MAPK pathway in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WE REPORTED PREVIOUSLY that a variety of growth factors, such as epidermal growth factor (EGF), insulin, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and TGF-{alpha}, rapidly induce DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes (1, 2, 3, 4). In addition, the action of these growth factors is modulated differently by {alpha}- and ß-adrenergic agonists. According to the results of previous studies, two classes of mitogens can be defined: 1) complete (direct or primary) mitogens (e.g. EGF, HGF, PDGF, and TGF-{alpha}), which, by themselves, in serum-free conditions can stimulate DNA synthesis and proliferation in quiescent hepatocyte populations; and 2) incomplete (secondary) mitogens or comitogens (e.g. catecholamines and PGs), which, by themselves, cannot stimulate proliferation in serum-free primary hepatocyte cultures but appear to promote cell growth by acting permissively in combination with complete mitogens (5, 6, 7, 8, 9). The relative importance of each factor in vitro and in vivo remains to be elucidated, and interactions between them are little understood as yet.

Among the substances classed as incomplete mitogens, PGs have diverse biological functions as chemical mediators for the maintenance of local homeostasis in nearly all mammalian tissues, including regulatory functions linked to inflammation, platelet aggregation, and contraction of smooth muscles (10). PGs are also thought to be involved in liver regeneration, because PGE2 in rat liver increases after partial hepatectomy and also because indomethacin, which is responsible for inhibiting DNA synthesis in the regenerating liver, prevents increases in PGE2 (11, 12). PGE2 is reported to be the main prostanoid secreted by sinusoidal endothelial cells in culture. Effects of exogenous PGs on hepatocyte DNA synthesis have also been studied in primary cultures of adult rat hepatocytes (13, 14, 15, 16, 17). Although the in vitro system has been useful for clarifying their mechanism of action, early studies examined the growth-promoting effects of PGs only in the presence of complete mitogens such as EGF and insulin. These primary mitogenic growth factors, which strongly influence hepatocyte DNA synthesis and proliferation by themselves, may in fact modulate the action of various PGs, even at lower concentrations. Therefore, little is known about the detailed dose-response relationships in PG-induced stimulation of hepatocyte growth responses and the cellular physiological mechanisms of PGs in the absence of exogenously added complete mitogens.

PGs produce this broad range of biological actions in various tissues by binding to specific receptors on the plasma membrane. Recently, various types of receptors for PGE2, PGF2{alpha}, PGI2, PGD2, and thromboxane A2 have been characterized using the pharmacological approach (10). PG receptors are members of the superfamily of G protein-coupled receptors that appear to have a single subunit structure containing seven membrane-spanning regions. Among them, there is further subdivision of the prostaglandin E2 (EP) receptors into four subtypes, recently defined at the molecular level: EP1, EP2, EP3, and EP4 (18, 19, 20, 21, 22). These subtypes are reported to differ somewhat in their signal transduction mechanisms.

We have recently shown that PGs from different series alone can stimulate DNA synthesis and proliferation in primary cultures of adult rat hepatocytes (23). This was an unexpected finding, for PGs are regarded as comitogenic growth factors that enhance the effects of primary mitogens such as insulin and EGF (5, 6, 7, 8, 9). In addition, the EP1 receptor subtype is not reportedly involved in the proliferative actions of PGE2 in primary cultured adult rat hepatocytes (17). The main purpose of the present study, therefore, was to investigate early stages in the growth-promoting effects of several EP receptor agonists [naturally occurring PGE2, its analog 17-phenyl-trinor-PGE2 (17-pt-PGE2), sulprostone, and 11-deoxy-PGE1] on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes and to investigate the EP receptor subtype mediation of these prostanoids in relation to the mechanism of intracellular signal transduction. Our results showed that among the EP receptors, only the EP1 receptor subtype was responsible for the proliferative actions on primary cultured hepatocytes, which is in direct contrast to the findings of the previous report (17). As to the mechanism of intracellular signal transduction, the EP1 receptor/G-protein involved in PLC regulation (Gq)/PLC/Ca2+ pathway and the tyrosine kinase/MAPK signaling pathway are closely implicated in the effects of EP1 receptor agonists on hepatocyte DNA synthesis and proliferation. In addition, we demonstrate that by stimulating the EP1 receptor/Gq/PLC/Ca2+ pathway, the EP1 receptor agonists induce secretion of TGF-{alpha}, which in turn increases hepatocyte mitogenesis via the tyrosine kinase/MAPK pathway. These findings indicate a novel mechanism of autocrine action for the indirect mitogen EP1 receptor subtype agonists PGE2 and 17-pt-PGE2 and, to a lesser extent, sulprostone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Wistar rats weighing 200–220 g were obtained from Saitama Experimental Animal Co. (Saitama, Japan). Adaptation to a light-, humidity-, and temperature-controlled room occurred during a minimum 3-d period before the start of the experiment. Rats were fed a standard diet and given tap water ad libitum. The animals used in this study were handled in accordance with the NIH’s Guidelines for the Care and Use of Laboratory Animals.

Hepatocyte isolation and culture
Rats were anesthetized by ip injection of sodium pentobarbital (45 mg/kg). Hepatocytes were isolated from the normal liver by the two-step in situ collagenase perfusion technique devised by Seglen (24) to facilitate disaggregation of the adult rat liver. In brief, dispersed hepatocytes were washed three times by slow centrifugation (50 x g, 1 min) of the cell suspension to remove cell debris, damaged cells, and nonparenchymal cells. Viability as tested by Trypan blue exclusion was more than 97%. Unless otherwise indicated, freshly isolated hepatocytes were plated onto collagen-coated plastic culture dishes (Sumitomo Bakelite Co., Tokyo, Japan) at a density of 3.3 x 104 cells/cm2 (3.0 x 105 cells/35-mm dish) and allowed to attach for 3 h on collagen-coated dishes in Williams’ medium E containing 5% newborn calf serum, 0.1 nM dexamethasone, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.10 µg/ml aprotinin in 5% CO2 in air at 37 C. The medium was then replaced by aspiration, and the cells were cultured further in serum- and dexamethasone-free Williams’ medium E supplemented with PGs such as PGE2, 17-pt-PGE2, sulprostone, and 11-deoxy-PGE1. When appropriate, the following agents were added: PGs with or without SC-51322, U-73122, U-73343, sphingosine, metaproterenol, 8-bromo-cAMP, UK-14304, H-89, 2,4-dideoxyadenosine (DDA), ionomycin, A23187, verapamil, diltiazem, somatostatin, and inhibitors of growth-related signal-transducing elements (i.e. genistein, wortmannin, PD98059, and rapamycin).

Measurement of DNA synthesis
Hepatocyte DNA synthesis was assessed by measuring the incorporation of [3H]thymidine into acid-precipitable materials. Briefly, after an initial attachment period of 3 h, the hepatocytes were washed twice with serum-free Williams’ medium E and cultured in a medium containing several PGs with or without testing agents for an additional 4 h or 21 h. The cells were pulsed at 1, 2, 3, or 19 h after PG stimulation for 2 h with [3H]thymidine (1.0 µCi/well) followed by 10% trichloroacetic acid precipitation as described previously (25). [3H]Thymidine incorporation into DNA was counted using a scintillation counter as cpm and normalized for cellular protein. Aphidicolin (10 µg/ml) was added to some wells to establish the level of nonreplicative DNA synthesis. Hepatocyte protein content was measured by a modified Lowry procedure with BSA as a standard (26). Data are expressed as dpm/h/mg cellular protein.

Counting nuclei
The number of nuclei rather than the number of cells was counted using a modified version of the procedure previously described by Nakamura et al. (27). Briefly, the primary cultured hepatocytes were washed twice with 2 ml of Dulbecco’s PBS (pH 7.4). Then, isolated liver cell nuclei were prepared for quantitation by exposure of the hepatocyte cultures to 0.25 ml of citric acid (0.1 M) containing Triton X-100 (0.1%) for 30 min at 37 C. An equal volume of the nucleus suspension was mixed with Trypan blue (0.3%) in Dulbecco’s PBS (pH 7.4), and the number of nuclei were counted in a hemocytometer. This procedure was performed because the hepatocytes had firmly attached to the collagen-coated plates and were not dispersed sufficiently by EDTA (0.02%)-trypsin (0.05%) treatment.

Neutralization of endogenous growth factors
For experiments using the neutralizing antibodies, serum-free primary cultured hepatocytes were treated with varying concentrations of EP1 receptor agonists in the presence and absence of monoclonal antibodies against EGF, HGF, IGF-I, and TGF-{alpha} (12.5, 25, 50, 75, and 100 ng/ml).

ELISA for TGF-{alpha}
TGF-{alpha} secretion into a culture medium after the addition of EP receptor agonists was determined by ELISA kits for TGF-{alpha}. Because unknown ingredient(s) of Williams’ medium E or MEM interfere with the ELISA assay system, we measured TGF-{alpha} secretion in PBS (pH 7.4) that contained 1.0 mM CaCl2 and 0.10 µg/ml aprotinin. In brief, hepatocytes were cultured in serum-containing Williams’ medium E at a density of 6.0 x 104 cells/cm2. After attachment for 3 h, the cultures were washed three times with PBS (pH 7.4). The culture medium was replaced with 1.0 ml of PBS containing 1.0 mM CaCl2 and 0.10 µg/ml aprotinin. Hepatocytes were preincubated in the conditioned medium for 5 min in humidified 5% CO2-95% air at 37 C and then incubated with EP1 receptor agonists and/or some pharmacological agents for various time periods. At each time point, the conditioned medium (50 µl) was collected and the medium samples were tested with an ELISA for TGF-{alpha} according to the manufacturer’s instructions (Calbiochem, Cambridge, MA). Absorbances were determined at a wavelength of 490 nm using a microplate reader (Bio-Rad Laboratories, Inc., Tokyo, Japan). A standard curve was obtained in a linear range from 25–800 pg/ml at a minimum detectable limit of about 12.5 pg/ml.

Measurement of MAPK activity
Phosphorylated MAPK isoforms (ERK 1 and ERK 2) were identified by Western blot analysis using anti-phospho-MAPK monoclonal antibody. In brief, cultured hepatocytes were washed once with ice-cold PBS (pH 7.4) and 0.2 ml of lysis buffer was added, then the hepatocytes were harvested. After centrifugation at 16,300 x g for 30 min at 4 C, the cell lysates were denatured in boiling water for 5 min. Samples of the supernatant (50 µg of protein) were subjected to SDS-PAGE using a 10% acrylamide resolving gel by the method of Laemmli. After electrophoresis, proteins were transferred to immobilon-P membranes (28).

For the detection of phosphorylated ERK1 [44 kDa (p44)] and ERK2 [42 kDa (p42)], the sheets were immersed in Tris-buffered saline containing 1% BSA. The sheets were then incubated with an antibody (1:2000 dilution) against phospho-MAPK and washed as described. Antibody binding was detected by enhanced chemiluminescence (ECL kit, Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) with donkey antirabbit IgG conjugated to horseradish peroxidase (1:3000 dilution). Densitometric analysis was performed using the NIH Image program version 1.60 for Macintosh. The data were calculated in arbitrary units and expressed as mean ± SEM.

Materials
The following reagents were obtained from Sigma (St. Louis, MO): aphidicolin, ionomycin, A23187, UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline), metaproterenol hemisulfate, 8-bromo-cAMP (8-bromoadenosine-3'-5'-cyclophosphate sodium), dexamethasone, somatostatin, verapamil hydrochloride, diltiazem hydrochloride and aprotinin. 17-pt-PGE2, PGE2, and SC-51322 (2-[3-[(2-furanylmethyl)-thiol]-1-oxopropyl]hydrazide) were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). 11-Deoxy-PGE1 and sulprostone were from Cayman Chemical Co. (Ann Arbor, MI). U-73122 (1-[6-[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]hexyl]-1H-pyrol-2,5-dione), U-73343 (1-[6-[17ß-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino]hexyl]-2,5-pyrrolidine-dione), sphingosine, DDA, and H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) were obtained from BIOMOL Research Laboratories, Inc., as were genistein, wortmannin, and rapamycin. PD98059 (2'-amino-3'-methoxyflavone) was obtained from Calbiochem-Behring (La Jolla, CA). Monoclonal antibodies against EGF, HGF, IGF-I, and TGF-{alpha} were obtained from BIOMOL Research Laboratories, Inc. Recombinant human TGF-{alpha} was obtained from PeproTech, Inc. (Rocky Hill, NJ). Williams’ medium E and newborn calf serum were purchased from Flow Laboratories (Irvine, Scotland). Collagenase (type II) was obtained from Worthington Biochemical Corp. Co. (Freehold, NJ). [methyl-3H]Thymidine (20 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA). The ELISA kit for TGF-{alpha} was obtained from Calbiochem. Anti-phospho-p44/42 MAPK monoclonal antibody was obtained from New England Biolabs, Inc. (Beverly, MA). All other reagents were of analytical grade.

Statistical analysis
Data are expressed as means ± SEM. Group comparisons were made by the ANOVA for unpaired data followed by post hoc analysis using Dunnett’s multiple comparison test. Differences at P < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of induced stimulation of hepatocyte DNA synthesis and proliferation by EP receptor agonists
We first examined the effects of several EP receptor agonists on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes in the absence of exogenously added peptide growth factors when freshly isolated hepatocytes were plated at low cell density (3.3 x 104 cells/cm2). The EP receptor agonists were added 3 h after plating when the change to serum-free culture was made, as described in Materials and Methods. While maintained in short-term culture in a defined medium, the hepatic parenchymal cells underwent time-dependent DNA synthesis and proliferation (i.e. an increase in the number of nuclei) in the presence of the EP1 receptor agonists PGE2 (10-6 M) and 17-pt-PGE2 (10-9 M). The onset of DNA synthesis was first observed about 2.5 and 2.0 h after the addition of 10-6 M PGE2 and 10-9 M 17-pt-PGE2, respectively (Fig. 1AGo). The reason why cultured hepatocytes initiate DNA synthesis in such a short period (2–2.5 h) is that the cells have already been primed during the 3-h attachment period (i.e. culture conditions consisting of a low cell density in 5% serum and a low dose of dexamethasone-containing medium). The mitotic activity of the hepatocytes in PGE2- and 17-pt-PGE2-treated cultures peaked at 4.0 and 3.5 h, respectively (Fig. 1BGo). Maximal stimulation for hepatocyte DNA synthesis and proliferation seen with these agents was approximately 6.0- and 1.3-fold, respectively. On the other hand, the DNA synthetic activity of the EP1/EP3 receptor subtype agonist sulprostone (10-6 M) reached a plateau at about 4 h and was sustained for 21 h. The number of nuclei induced by sulprostone increased significantly at about 4.0 h after the addition (Fig. 1Go). In contrast, the EP2/EP4 receptor subtype agonist 11-deoxy-PGE1 (10-6 M) produced no significant stimulation of hepatocyte DNA synthesis and proliferation during the early (4 h) and late phases (21 h) of culture (Fig. 1Go).



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  		Figure 1. Time course of the induced stimulation of hepatocyte DNA synthesis and proliferation by EP receptor agonists. Freshly isolated hepatocytes were cultured in Williams’ medium E containing 5% newborn bovine serum, 0.1 nM dexamethasone, 0.10 µg/ml aprotinin, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at a cell density of 3.3 x 104 cell/cm2. After an attachment period of 3 h (time zero), the medium was rapidly replaced with serum- and dexamethasone-free Williams’ medium E with or without 10-6 M PGE2, 10-9 M 17-pt-PGE2, 10-6 M sulprostone, or 10-6 M 11-deoxy-PGE1 and cultured for various lengths of time. Hepatocyte DNA synthesis and proliferation were determined as described in Materials and Methods. Rate of hepatocyte DNA synthesis is expressed as dpm/mg protein/h (A). Hepatocyte proliferation is expressed as the percent increase in total number of nuclei compared with that of the control culture (B). The results are expressed as means ± SEM of three experiments. *, P < 0.05; **, P < 0.01 compared with respective controls.

 
Dose-response effects of EP receptor agonists on hepatocyte DNA synthesis and proliferation
We next examined dose-response relationships between the various EP receptor agonists and DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. PGE2-induced DNA synthesis was dose dependent and reached a plateau at 5 x 10-7 M, with an ED50 of 2.5 x 10-8 M (Fig. 2AGo). DNA synthesis stimulated by 17-pt-PGE2 was also dose dependent and reached a plateau at 10-9 M, with an ED50 of 1.8 x 10-10 M (Fig. 2AGo). 17-pt-PGE2 is about 2 orders of magnitude more potent than PGE2 in stimulating hepatocyte DNA synthesis. Although PGE2 was less potent than 17-pt-PGE2, the maximal response induced by PGE2 was nearly the same. The relative magnitude of the proliferating effects of PGE2 and 17-pt-PGE2 roughly corresponded to their activities as stimulators of DNA synthesis, with ED50 values of 2.5 x 10-8 M and 1.7 x 10-10 M, respectively (Fig. 2B). Sulprostone induced dose-dependent stimulation of hepatocyte DNA synthesis, with an ED50 of 2.6 x 10-8 M (Fig. 2A). The maximal induction by sulprostone was seen at a concentration of 2.8 x 10-7 M. The maximal responses induced by sulprostone were significantly lower than those induced by PGE2 and 17-pt-PGE2. The relative magnitude of the proliferating effects of sulprostone roughly corresponded to the stimulatory activity for DNA synthesis, with an ED50 of 2.8 x 10-8 M (Fig. 2BGo). On the other hand, the DNA synthetic and proliferative effects of 11-deoxy-PGE1 on cultured hepatocytes were negligible, with concentrations ranging between 10-10 and 10-6 M (Fig. 2Go). Therefore, among the PGs we tested, 17-pt-PGE2 had the most potent DNA synthetic and proliferative effects, followed by PGE2, sulprostone, and 11-deoxy-PGE1 in that order.



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  		Figure 2. Dose-response effects of EP receptor agonists on hepatocyte DNA synthesis and proliferation. Freshly isolated hepatocytes were plated at a density of 3.3 x 104 cells/cm2 and cultured as described in the legend of Fig. 1Go. After medium change, hepatocytes were cultured with various concentrations of PGE2, 17-pt-PGE2, sulprostone, and 11-deoxy-PGE1 for a further 4 h. Rate of hepatocyte DNA synthesis is expressed as dpm/mg protein/h (A). Hepatocyte proliferation is expressed as the percent increase in total number of nuclei compared with that of the control culture (B). The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with respective controls (medium alone).

 
Effects of the specific EP1 receptor antagonist SC-51322 on hepatocyte DNA synthesis and proliferation induced by EP receptor agonists
To confirm EP1 receptor mediation of EP receptor agonist action, we investigated the effects of an EP1 subtype-specific antagonist, SC-51322, on the EP receptor agonist-induced hepatocyte DNA synthesis and proliferation at 4 h of culture. The DNA synthetic and proliferative effects of PGE2 (10-6 M) and 17-pt-PGE2 (10-9 M) on the primary cultured hepatocytes were inhibited by SC-51322 (10-9 to 10-6 M) in a dose-dependent manner (Fig. 3Go). SC-51322, when added alone, did not affect hepatocyte DNA synthesis or proliferation. The results confirmed that the EP1 subtype receptor is closely involved in the proliferative actions of PGE2 and 17-pt-PGE2. Because the effects of the EP1/EP3 receptor subtype agonist sulprostone (10-6 M) on hepatocyte DNA synthesis and proliferation were also blocked completely by SC-51322 (10-7 to 10-6 M), the results suggest that the EP3 subtype of EP receptors contributed less than the EP1 subtype to the proliferative action of sulprostone on these cells. The very weak effects of the EP2/EP4 receptor subtype agonist 11-deoxy-PGE1 (10-6 M) on hepatocyte DNA synthesis and proliferation were not influenced by SC-51322 (10-8 to 10-6 M). These results are in complete agreement with the effects being mediated via EP1 receptor subtypes.



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  		Figure 3. Effects of SC-51322 on hepatocyte DNA synthesis and proliferation induced by EP receptor agonists. Hepatocytes were plated at a density of 3.3 x 104 cells/cm2 and cultured as described in the legend of Fig. 1Go. After medium change, hepatocytes were cultured with 10-6 M PGE2, 10-9 M 17-pt-PGE2, 10-6 M sulprostone, or 10-6 M 11-deoxy-PGE1 in the presence or absence of SC-51322 (10-9 to 10-6 M) for 4 h. Rate of hepatocyte DNA synthesis is expressed as dpm/mg protein/h (A). Hepatocyte proliferation is expressed as the percent increase in total number of nuclei compared with that of the control culture (B). The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with respective controls.

 
Effects of an {alpha}2- and ß2-adrenergic agonist, 8-bromo-cAMP, DDA, and H-89 on the hepatocyte DNA synthesis and proliferation induced by PGE2 or 17-pt-PGE2
To determine how EP1 receptor agonists induce hepatocyte DNA synthesis and proliferation, we pharmacologically investigated the intracellular signal transduction events associated with EP1 receptor agonists in primary cultured hepatocytes. To clarify whether EP1 receptor agonists stimulate hepatocyte DNA synthesis and proliferation via an adenylate cyclase/PKA pathway, we investigated whether a direct inhibitor of adenylate cyclase, DDA, and an inhibitor of PLA, H-89, had inhibitory effects on the hepatocyte mitogenesis induced by PGE2 (10-6 M) or 17-pt-PGE2 (10-9 M). DDA (10-6 M) and H-89 (10-7 M) did not affect the EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation (Fig. 4Go), suggesting that adenylate cyclase and PKA may not contribute to hepatocyte mitogenesis induced by the EP1 receptor agonists. We also examined the effects of the {alpha}2- and ß2-adrenergic agonists on EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation during 4 h of culture. As shown in Fig. 4Go, hepatocyte DNA synthesis and proliferation induced by 10-6 M PGE2 or 10-9 M 17-pt-PGE2 was potentiated by the {alpha}2-adrenergic agonist UK-14304 (10-7 M), whereas it was inhibited by the ß2-adrenergic agonist metaproterenol (10-7 M) or a direct stimulator of PKA, 8-bromo-cAMP (10-7 M). The {alpha}2-adrenergic agonist, ß2-adrenergic agonist, and 8-bromo-cAMP by themselves did not significantly influence hepatocyte DNA synthesis or proliferation during 4 h of culture (data not shown).



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  		Figure 4. Effects of UK-14304, metaproterenol, 8-bromo-cAMP, DDA, and H-89 on hepatocyte DNA synthesis and proliferation induced by PGE2 or 17-pt-PGE2. Hepatocytes were plated at a density of 3.3 x 104 cells/cm2 and cultured as described in the legend of Fig. 1Go. Specific agonists or inhibitors were added with 10-6 M PGE2 or 10-9 M 17-pt-PGE2 immediately after medium change, and cells were cultured for a further 4 h. Concentrations were as follows: DDA, 10-6 M; H-89, 10-7 M; UK-14304, 10-7 M; metaproterenol, 10-7 M; 8-bromo-cAMP, 10-7 M. Rate of hepatocyte DNA synthesis is expressed as dpm/mg protein/h (A). Hepatocyte proliferation is expressed as the percent increase in total number of nuclei compared with that of the control culture (B). The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with respective controls.

 
Effects of U-73122, sphingosine, calcium channel blockers, calcium ionophores, and somatostatin on the hepatocyte DNA synthesis and proliferation induced by PGE2 or 17-pt-PGE2
To characterize possible involvement of the PLC/Ca2+/PKC pathway in the EP1 receptor-mediated stimulation of hepatocyte DNA synthesis and proliferation induced by PGE2 or 17-pt-PGE2, we investigated the effects of the specific PLC inhibitor U-73122 and a PKC inhibitor, sphingosine, on these responses. U-73122 (10-6 M) markedly attenuated PGE2 (10-6 M) or 17-pt-PGE2 (10-9 M) stimulation of hepatocyte DNA synthesis and proliferation during 4 h of culture (Fig. 5Go). Neither U-73343 (10-6 M), a close structural analog of U-73122 that has no such inhibitory action on PLC, nor sphingosine (10-6 M), a PKC inhibitor, significantly affected PGE2- or 17-pt-PGE2-induced hepatocyte DNA synthesis and proliferation during 4 h of culture. These inhibitors on their own did not show any significant effects on hepatocyte DNA synthesis and proliferation during 4 h of culture (data not shown).



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  		Figure 5. Effects of U-73122, sphingosine, verapamil, somatostatin, and ionomycin on hepatocyte DNA synthesis and proliferation induced by PGE2 or 17-pt-PGE2. Hepatocytes were plated at a density of 3.3 x 104 cells/cm2 and cultured as described in the legend of Fig. 1Go. Specific inhibitors and an agonist were added with PGE2 (10-6 M) or 17-pt-PGE2 (10-9 M) immediately after medium change, and the hepatocytes were cultured for a further 4 h. Concentrations were as follows: U-73122, 10-6 M; U-73343, 10-6 M; sphingosine, 10-6 M; verapamil, 10-6 M; somatostatin, 10-7 M; ionomycin, 10-7 M. Rate of hepatocyte DNA synthesis is expressed as dpm/mg protein/h (A). Hepatocyte proliferation is expressed as the percent increase in total number of nuclei compared with that of the control culture (B). The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with respective controls.

 
Similarly, to determine the possible involvement of Ca2+ mobilization in PGE1- or 17-pt-PGE2-stimulated hepatocyte DNA synthesis and proliferation, the cells were treated with the Ca2+ ionophore ionomycin or A23187 during 4 h of culture. Significant potentiation of both PGE2-induced hepatocyte DNA synthesis and proliferation was observed with ionomycin (10-7 M) or A23187 (10-6 M; data not shown) during 4 h of culture. These findings suggest that PLC-independent agents that increase intracellular Ca2+ concentration enhance PGE2-induced hepatocyte mitogenesis. Conversely, the ability of PGE2 to stimulate hepatocyte DNA synthesis and proliferation was almost completely blocked by Ca2+ channel blockers such as verapamil (10-6 M) and diltiazem (10-6 M; data not shown). Pharmacological profiles of the calcium ionophores and the Ca2+ channel blockers on the 17-pt-PGE2-stimulated hepatocyte DNA synthesis and proliferation were very similar to those of PG2 (Fig. 5Go). In addition, somatostatin (10-7 M), which inhibits the release of certain gastrointestinal and pancreatic hormones, presumably by affecting decreasing cytosolic Ca2+, strongly inhibited hepatocyte DNA synthesis and proliferation induced by these EP1 receptor subtype agonists. These inhibitors and stimulators on their own did not influence hepatocyte DNA synthesis and proliferation during 4 h of culture (data not shown). Our results suggest that EP1 agonists induce hepatocyte DNA synthesis and proliferation through the receptor-mediated activation of PLC and an extracellular Ca2+-dependent mechanism sensitive to calcium channel blockers but that this process is independent of the activation of PKC.

Effects of specific inhibitors of growth-related signal-transducing elements on hepatocyte DNA synthesis and proliferation induced by PGE2 or 17-pt-PGE2
We next investigated whether the mitogenic responses of primary cultured hepatocytes to the EP1 receptor agonists were mediated by signal transducers, such as receptor tyrosine kinase, PI3K, MAPK kinase, and ribosomal protein S6 kinase (p70 S6K), by using corresponding specific inhibitors of the signal transducers, namely those of genistein, wortmannin, PD98059, and rapamycin. As shown in Fig. 6Go, hepatocyte DNA synthesis and proliferation induced by PGE2 (10-6 M) and 17-pt-PGE2 (10-9 M) was almost completely blocked by genistein (5 x 10-6 M), wortmannin (10-7 M), PD98059 (10-6 M), and rapamycin (10 ng/ml). These inhibitors on their own did not affect the hepatocyte DNA synthesis and proliferation during 4 h of culture (data not shown). In terms of the intracellular signal transduction mechanisms of action of the EP1 receptor agonists, these pharmacological characterizations suggest that receptor tyrosine kinase, PI3K, MAPK kinase, and p70 S6K are closely involved in hepatocyte DNA synthesis and proliferation.



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  		Figure 6. Effects of specific inhibitors of growth-related signal transducers on hepatocyte DNA synthesis (A) and proliferation (B) induced by PGE2 or 17-pt-PGE2. Hepatocytes were plated at a density of 3.3 x 104 cells/cm2 and cultured as described in the legend of Fig. 1Go. Specific inhibitors of signal-transducing elements were added with 10-6 M PGE2 or 10-9 M 17-pt-PGE2 immediately after medium change, and cells were cultured for a further 4 h. The concentrations were as follows: genistein, 5 x 10-6 M; wortmannin, 10-7 M; PD98059, 10-6 M; rapamycin, 10 ng/ml. Rate of hepatocyte DNA synthesis is expressed as dpm/mg protein/h (A). Hepatocyte proliferation is expressed as the percent increase in total number of nuclei compared with that of the control culture (B). The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with respective controls.

 
Effects of monoclonal antibodies against TGF-{alpha} or IGF-I on hepatocyte DNA synthesis and proliferation induced by PGE2 or17-pt-PGE2
Figures 5Go and 6Go show that EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation are mediated through both the EP1 receptor/Gq/PLC/Ca2+ pathway and the receptor tyrosine kinase/MAPK cascade. However, how these associate with each other remains to be elucidated. Because PGs on their own have been reported to act as comitogens in vivo and in vitro, we hypothesized that the EP1 receptor agonists would selectively induce the secretion of primary mitogen(s) in an autocrine manner to stimulate hepatocyte mitogenesis. TGF-{alpha} or IGF-I are potential candidates for the primary growth factor, because hepatocytes express messages for TGF-{alpha} and IGF-I and the cells can synthesize and store these primary growth factors. To examine the possibility that TGF-{alpha} or IGF-I mediates EP1 agonist-induced hepatocyte DNA synthesis and proliferation in the primary culture system, we examined the effects of neutralizing monoclonal antibodies against TGF-{alpha} and IGF-I and compared the results with those of EGF and HGF. Fig. 7Go shows that the addition of a neutralizing monoclonal antibody against TGF-{alpha} dose-dependently inhibited the growth-promoting effect of PGE2 (10-6 M) and 17-pt-PGE2 (10-9 M) on hepatocyte DNA synthesis and proliferation. The median inhibitory concentration values for such synthesis and proliferation at 4 h of culture were 28 and 36 ng/ml, respectively. In contrast, the DNA synthetic and proliferative effects of EP1 agonists were not affected significantly by the treatment of hepatocytes with various concentrations of monoclonal antibody against IGF-I (1–100 ng/ml), EGF (1–100 ng/ml; data not shown), and HGF (1–100 ng/ml; data not shown). These monoclonal antibodies on their own did not significantly influence hepatocyte DNA synthesis and proliferation during 4 h of culture (data not shown). The results indicate that this elimination of the effects of PGE2 (10-6 M) and 17-pt-PGE2 (10-9 M) was specific for antibody against TGF-{alpha}. Accordingly, a novel model can be proposed in which the mitogenicity of the EP1 receptor agonists is mediated by the autocrine secretion of TGF-{alpha} and the subsequent stimulation of DNA synthesis and proliferation in primary cultures of adult rat hepatocytes.



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  		Figure 7. Effects of monoclonal antibodies against TGF-{alpha} or IGF-I on EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation. Hepatocytes were plated at a density of 3.3 x 104 cells/cm2 and cultured as described in the legend of Fig. 1Go. After medium change, hepatocytes were treated with 10-6 M PGE2 or 10-9 M 17-pt-PGE2 for 4 h in the presence or absence of TGF-{alpha}-neutralizing antibody or IGF-I-neutralizing antibody (1–100 ng/ml). Rate of hepatocyte DNA synthesis is expressed as dpm/mg protein/h (A). Hepatocyte proliferation is expressed as the percent increase in total number of nuclei compared with that of the control culture (B). The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with respective controls.

 
Effects of EP1 receptor agonists on TGF-{alpha} secretion by primary cultured hepatocytes: time course study and dose-response relationship
To verify the model proposed above, it is important to demonstrate that treatment of primary cultured hepatocytes with EP receptor agonists does in fact lead to increased secretion of TGF-{alpha}. After PGE2 or 17-pt-PGE2 treatment, there was a rapid and marked increase in medium TGF-{alpha} levels compared with the control (Fig. 8AGo). A time course study showed that a detectable increase began within 3 min of treatment with PGE2 (10-6 M) or 17-pt-PGE2 (10-9 M). The levels remained significantly increased for the duration of the experiment. TGF-{alpha} secretion stimulated by 17-pt-PGE2 (10-9 M) increased more rapidly and was significantly higher than that induced by PGE2 (10-6 M) during a 5-min period (Fig. 8A). The medium TGF-{alpha} levels after stimulation with sulprostone (10-6 M) were significantly lower than those induced by PGE2 (10-6 M) or 17-pt-PGE2 (10-9 M) within a 30-min period. 11-Deoxy-PGE1 had no significant effects on TGF-{alpha} secretion. As shown in Fig. 8BGo, PGE2 and 17-pt-PGE2 induced a significant increase in the medium TGF-{alpha} levels in a dose-dependent manner, with ED50 values of 2 x 10-8 M and 5 x 10-10 M, respectively. The treatment of hepatocytes with 17-pt-PGE2 (10-11 to 10-7 M) resulted in a more potent increase in medium TGF-{alpha} levels than that of PGE2 (10-9 to 10-6 M). The addition of sulprostone (10-9 to 10-6 M) to the culture mildly increased the medium levels of TGF-{alpha}, whereas the addition of 11-deoxy-PGE1 (10-9 to 10-6 M) had no significant effect. These findings provide direct evidence for the DNA synthetic and proliferative role of EP1 receptor agonists in primary cultured hepatocytes.



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  		Figure 8. Time course of medium TGF-{alpha} levels stimulated by EP receptor agonists (A), and the dose-dependent effects of EP receptor agonists on medium TGF-{alpha} levels (B). Freshly isolated hepatocytes were plated at a cell density of 6.0 x 104 cells/cm2 and cultured for 3 h as described in the legend of Fig. 1Go. After an attachment period of 3 h (time zero), the medium was washed three times with PBS (pH 7.4) and replaced with PBS containing 1.0 mM CaCl2 and 0.10 µg/ml aprotinin. The cells were preincubated for 5 min, PGE2 (10-6 M), 17-pt-PGE2 (10-9 M), sulprostone (10-6 M), and 11-deoxy-PGE1 (10-6 M) were added to the cultures immediately, and hepatocytes were cultured further. At each time point (1, 3, 5, 10, 20, and 30 min), medium levels of TGF-{alpha} were determined with an ELISA kit for TGF-{alpha} (A). Dose-dependent effects of each EP receptor agonist on the medium levels of TGF-{alpha} were determined at 10 min of incubation (B). The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with the respective controls.

 
EP1 receptor agonist-induced secretion of TGF-{alpha} by primary cultured hepatocytes: effects of inhibitors or stimulators of the adrenergic receptor/adenylate cyclase pathway and the EP1 receptor/PLC pathway
To investigate the signal-transducing mechanisms that mediate the EP1 receptor agonist-induced increase in medium TGF-{alpha} levels, we examined the effects of {alpha}2- and ß2-adrenergic agonists on EP1 receptor agonist-induced increase in medium TGF-{alpha} levels. As shown in Fig. 9AGo, the increase in medium levels of TGF-{alpha} induced by 10-6 M PGE2 or 10-9 M 17-pt-PGE2 was not affected significantly by the {alpha}2-adrenergic agonist UK-14304 (10-7 M), the ß2-adrenergic agonist metaproterenol (10-7 M), or 8-bromo-cAMP (10-7 M). To test the possibility that EP1 receptor agonists stimulate the increase in medium levels of TGF-{alpha} via an adenylate cyclase/PKA pathway, we also investigated whether a direct inhibitor of adenylate cyclase, DDA, and an inhibitor of protein kinase A, H-89, have inhibitory effects on the medium levels of TGF-{alpha} induced by PGE2 (10-6 M) or 17-pt-PGE2 (10-9 M). DDA (10-6 M) and H-89 (10-7 M) did not affect EP1 receptor agonist-induced increase in the medium levels of TGF-{alpha} (Fig. 9A); 10-6 M PGE2- or 10-9 M 17-pt-PGE2-induced secretion was not affected by the PKC inhibitor sphingosine (10-6 M). Neither DDA, H-89, sphingosine, {alpha}2-adrenergic agonist, ß2-adrenergic agonist, nor 8-bromo-cAMP alone significantly influenced the medium levels of TGF-{alpha} at 30 min of culture (data not shown).



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  		Figure 9. EP1 receptor agonist-induced secretion of TGF-{alpha} by primary cultured hepatocytes. Effects of inhibitors or stimulators of the adrenergic receptor/adenylate cyclase pathway (A) and the EP1 receptor/PLC pathway (B). Hepatocytes were plated at the cell density of 6.0 x 104 cells/cm2 and cultured as described in the legend of Fig. 8Go. Specific inhibitors or stimulators were added with or without PGE2 (10-6 M) or 17-pt-PGE2 (10-9 M) immediately after 5 min of preincubation, and hepatocytes were cultured further for 10 min. The concentrations were as follows: UK-14304, 10-6 M; metaproterenol, 10-6 M; 8-bromo-cAMP, 10-7 M; DDA, 10-6 M; H-89, 10-7 M; sphingosine, 10-6 M; SC-51322, 10-7 M; U-73122, 10-6 M; U-73343, 10-6 M; ionomycin, 10-7 M; verapamil, 10-6 M; somatostatin, 10-7 M. Medium levels of TGF-{alpha} were determined using an ELISA kit for TGF-{alpha}. The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with the respective controls.

 
We also examined the effects of SC-51322, U-73122, somatostatin, calcium channel blockers, and calcium ionophores on this process. As shown in Fig. 9BGo, PGE2 (10-6 M)-induced increase in the medium levels of TGF-{alpha} was completely blocked by the EP1 receptor antagonist SC-51322 (10-7 M). This indicates that TGF-{alpha} is released from primary cultured hepatocytes as a result of EP1 receptor stimulation. U-73122 (10-6 M) completely inhibited the PGE2 (10-6 M)-induced increase in medium TGF-{alpha} levels, suggesting that PLC is closely involved in TGF-{alpha} secretion by hepatocytes. U-73343 (10-6 M), a close structural analog of U-73122, which has no such inhibitory action on PLC, did not significantly affect PGE2 (10-6 M)-induced increase in medium TGF-{alpha} levels.

Next, we evaluated the effects of somatostatin, which inhibits the release of certain gastrointestinal and pancreatic hormones, presumably by affecting decreasing cytosolic Ca2+ levels and attenuating cAMP levels, on PGE2 (10-6 M)-induced increase in the medium levels of TGF-{alpha} and examined the signal transduction mechanism involved. Treatment of cultured hepatocytes for 10 min with somatostatin (10-7 M) caused a marked inhibition of PGE2-induced TGF-{alpha} secretion. In addition, PGE2-induced TGF-{alpha} secretion was also blocked by verapamil (10-6 M) or diltiazem (10-6 M; data not shown). On the other hand, this increased TGF-{alpha} level was potentiated by ionomycin (10-7 M) as well as A23187 (10-6 M; data not shown). These inhibitors or Ca2+ ionophores did not affect the basal TGF-{alpha} levels independently (data not shown). The pharmacological profiles of the effects of SC-51322, U-73122, somatostatin, Ca2+ channel blockers, and calcium ionophores on 17-pt-PGE2 (10-9 M)-stimulated medium levels of TGF-{alpha} were very similar to those of PGE2 (Fig. 9BGo). The results suggest that activation of the EP1 receptor/Gq/IP3 pathway and mobilization of intracellular Ca2+ led to a rapid secretion of TGF-{alpha} from primary cultured hepatocytes into the conditioned medium. These observations also indicate the need for influx of extracellular calcium in the process of EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation, which is mediated through TGF-{alpha} secretion.

EP1 receptor agonist-stimulated TGF-{alpha} secretion by primary cultured hepatocytes: effects of specific inhibitors on growth-related signal transducers
To investigate the signal-transducing mechanisms that mediate EP1 agonist-induced TGF-{alpha} secretion, we investigated whether or not this process is mediated by growth-related signal transducers such as receptor tyrosine kinase, PI3K, MAPK kinase, and p70 S6K by using corresponding specific inhibitors of the signal transducers, namely genistein, wortmannin, PD98059, and rapamycin. PGE2 (10-6 M) and genistein (5 x 10-6 M), when added in combination, caused a rapid increase in the medium levels of TGF-{alpha} (Fig. 10). PGE2 (10-6 M)-induced TGF-{alpha} secretion was unaffected by wortmannin (10-7 M), PD98059 (10-6 M), and rapamycin (10 ng/ml). These inhibitors did not affect the basal TGF-{alpha} levels when added alone. The pharmacological profiles of these inhibitors for 17-pt-PGE2 (10-9 M)-induced TGF-{alpha} secretion were very similar to those of PGE2 (Fig. 10Go), demonstrating that these inhibitors of the signal transducers inhibited PGE2- or 17-pt-PGE2-induced hepatocyte DNA synthesis and proliferation without affecting TGF-{alpha} secretion.



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  		Figure 10. EP1 receptor agonist-induced secretion of TGF-{alpha} by primary cultured hepatocytes. Effects of inhibitors of growth-related signal transducing elements. Hepatocytes were plated at the cell density of 6.0 x 104 cells/cm2, and cultured as described in the legend of Fig. 8. Specific inhibitors of signal-transducing elements were added with or without PGE2 or 17-pt-PGE2 immediately after 5 min of preincubation, and the hepatocytes were cultured further for 10 min. The concentrations were as follows: genistein, 5 x 10-6 M; wortmannin, 10-7 M; PD98059, 10-6 M; rapamycin, 10 ng/ml. Medium levels of TGF-{alpha} were assayed using an ELISA kit for TGF-{alpha}. The results are expressed as means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01 compared with the respective controls.

 
Activation of p42 MAPK by EP1 receptor agonists and blockade by PD98059
To confirm the notion that EP1 receptor agonists induce hepatocyte DNA synthesis and proliferation through MAPK activation, we investigated whether or not TGF-{alpha} and EP1 receptor agonists can stimulate MAPK activity. In addition, we examined the effects of the MAPK kinase inhibitor PD98059 on MAPK activity induced by EP1 receptor agonists or TGF-{alpha}. Fig. 11AGo shows that exogenously added TGF-{alpha} (human recombinant) caused an initial rapid increase in the phosphorylation of MAPK isoform p42 MAPK, peaking about 3.0-fold at 5 min after the addition, followed by a sharp decrease. PGE2 (10-6 M) and 17-pt-PGE2 (10-9 M) stimulated the phosphorylation of p42 MAPK, peaking about 3.0-fold at about 25 and 20 min, respectively, followed by slow decreases. Incubation of hepatocytes with sulprostone (10-6 M) caused MAPK activity to increase slowly to a plateau that was 1.6-fold greater than the control. In contrast, both TGF-{alpha} and the EP1 receptor agonists did not significantly affect MAPK isoform p44 MAPK activity (data not shown). Fig. 11BGo shows that PD98059 (10-6 M) completely abolished the TGF-{alpha} or EP1 receptor agonist-induced increase in p42 MAPK activity. In addition, p42 MAPK activation induced by EP1 receptor agonists was abolished by genistein (5 x 10-6 M) or wortmannin (10-7 M) treatment (data not shown). These results demonstrate that TGF-{alpha} and EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation is actually mediated through the activation of p42 MAPK. Furthermore, the results suggest that stimulation of hepatocyte DNA synthesis and proliferation induced by the EP1 receptor agonists was mediated through the activation of the tyrosine kinase/PI3K/MAPK pathway.



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  		Figure 11. Fig. 11. Activation of p42 MAPK by EP1 receptor agonists or TGF-{alpha} and blockade by PD98059. Freshly isolated hepatocytes were plated at a cell density of 3.3 x 104 cells/cm2 and cultured for 3 h as described in the legend of Fig. 1. After an attachment period of 3 h (time zero), the medium was rapidly replaced with serum- and dexamethasone-free Williams’ medium E. Hepatocyte MAPK activity stimulated by EP1 receptor agonists or TGF-{alpha} was determined at various time points in the absence (A) or presence (B) of PD98059 (10-6 M) as described in Materials and Methods. Arrows indicate additions to the incubation medium. The results are expressed as means ± SEM of three independent experiments. *, P < 0.05, **, P < 0.01 compared with the respective controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Primary growth factors, also known as complete mitogens, reportedly have growth-stimulating effects on primary cultured hepatocytes through intracellular signal transduction, which brings about changes in the plasma membrane receptor to nuclear DNA synthesis and proliferation (5, 6, 7, 8, 9). Growth modulators or comitogens, such as PGs and catecholamines, have been shown only to modulate the effects of the primary growth factors. However, as shown in Figs. 1Go and 2Go, we demonstrated that PGE2 and a synthetic PGE2 analog, 17-pt-PGE2 (a specific EP1 receptor subtype agonist), significantly induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes in the absence of exogenously added primary growth factors. Moreover, we demonstrated that sulprostone (an EP1/EP3 subtype agonist) was weakly mitogenic for primary cultured hepatocytes. 11-Deoxy-PGE1 (an EP2/EP4 subtype agonist) had no such effects. SC-51322, an EP1 receptor subtype-specific antagonist (29), almost completely blocked this EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation (Fig. 3Go), confirming that the EP1 receptor agonists apparently act as complete mitogens in primary cultures of adult rat hepatocytes. These results are in direct contrast to those of another report, which showed EP3 receptor mediation, but not EP1 receptor mediation, of hepatocyte DNA synthesis and proliferation (17). That report also stated that PGs act as comitogenic growth factors through the EP3 subtype coupled with the Gi protein in primary cultured adult rat hepatocytes. A possible reason for this discrepancy may be related to differences in culture conditions (e.g. hormonal condition and initial plating density). In addition, we observed that the cell density-independent nature of the EP1 receptor agonists for hepatocyte mitogenesis at high plating densities (1.0 x 105 cells/cm2) are similar to those of insulin (2), PDGF (3), IGF-II (30), and TGF-{alpha} (31) (data not shown). However, postreceptor mechanisms responsible for the proliferative action of the EP1 receptor agonists as well as intracellular signal transduction mechanisms remain to be clarified.

It has been reported that signal transduction mechanisms appearing to serve as mediators for prostanoid receptors include 1) stimulation of adenylate cyclase via the Gs protein, 2) inhibition of adenylate cyclase via the Gi protein, 3) stimulation of phosphatidylinositol-PLC via Gq, and, possibly 4) increased intracellular Ca2+ through a phosphatidylinositol-dependent process (10). Accordingly, we investigated pharmacologically which signal transduction pathways are mediated by EP1 subtype receptor stimulation. EP1 receptor agonist-stimulated hepatocyte mitogenesis was inhibited by the PLC inhibitor U-73122 (32) but not by the inactive analog of U-73122 (i.e. U-73343) (Fig. 5Go). Therefore, the functional EP1 receptors contained in hepatocytes in primary culture appear to stimulate phosphatidylinositol-PLC and increase Ca2+ mobilization by PGE2 or 17-pt-PGE2 as a primary mechanism in EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation. If this is true, then these responses could also be stimulated by the Ca2+ ionophore ionomycin or A23187, which increase Ca2+ influx into cultured hepatocytes. Conversely, the Ca2+ channel blockers verapamil and diltiazem could inhibit such synthesis and proliferation. The results shown in Fig. 5Go are consistent with this notion. On the other hand, it may be unlikely that Gq/PLC/PKC or adenylate cyclase/PKA contributes to EP1 receptor agonist-induced mitogenesis, because an inhibitor of PKC, sphingosine (33), a direct inhibitor of adenylate cyclase, DDA (34), and an inhibitor of PKA, H-89 (35), did not affects such DNA synthesis and proliferation (Fig. 4Go). However, there seems to be cross-talk between the EP1 subtype pathway and an adenylate cyclase/cAMP pathway, because EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation was potentiated by UK-14304 (31) and inhibited by metaproterenol and 8-bromo-cAMP.

Specific inhibitors of growth-related signal-transducing elements such as genistein (36), wortmannin (37), PD98059 (38), and rapamycin (39) attenuated EP1 receptor agonist-stimulated hepatocyte DNA synthesis and proliferation (Fig. 6). This demonstrates that mitogenic signaling through the EP1 receptor agonist pathway also requires the activation of tyrosine kinase, PI3K, MAPK kinase, and p70 S6K. Other studies have reported that growth factors such as PDGF, EGF, and insulin all evoke autophosphorylation of tyrosine residue on their respective receptors, which leads to the activation of tyrosine kinase toward other cellular components, including PI3K, MAPK, and p70 S6K, which further results in DNA synthesis and subsequent cell proliferation (40, 41, 42). However, the complete cascade of sequential phosphorylation events remains to be definitively established.

Although both the EP1 receptor/Gq/PLC/Ca2+ signaling pathway and the tyrosine kinase signaling cascade are critically involved in EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation, the links between the EP1 receptor/Gq/PLC/Ca2+ pathway and the tyrosine kinase signaling cascade have not been characterized clearly. We reported previously that an {alpha}1-adrenergic receptor- or vasopressin receptor-mediated pathway, which uses the PLC/Ca2+ pathway, has no significant effects on hepatocyte DNA synthesis and proliferation but only modulates the growth-promoting effects of primary growth factors such as EGF and HGF (1, 4). Moreover, there is little evidence as yet that EP1 receptor/Gq/PLC/Ca2+ pathways directly stimulate (or phosphorylate) elements of the tyrosine kinase-signaling pathway to induce cell proliferation (10). Therefore, any such association between the EP1 receptor/Gq/PLC/Ca2+ pathway and the receptor tyrosine kinase system is speculative at this time. To this end, therefore, we hypothesized that the EP1 receptor/Gq/PLC/Ca2+ pathway strongly stimulates secretion of a certain mitogen by cultured hepatocytes in an autocrine manner, which, in turn, induces the hepatocyte DNA synthesis and proliferation through stimulation of the downstream tyrosine kinase pathway. Two potential mitogens that could fulfill this requirement are TGF-{alpha} and IGF-I. TGF-{alpha} and IGF-I are reported to be cytokines that are synthesized and stored in parenchymal hepatocytes, and they are among the most active growth factors stimulating hepatocyte DNA synthesis and proliferation (5, 6, 7, 8, 9). To verify this hypothesis, we examined the effects of a monoclonal antibody against the putative growth factors TGF-{alpha} and IGF-I on EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation (Fig. 7Go) and compared the effects with those of monoclonal antibodies against HGF or EGF (data not shown). PGE2- or 17-pt-PGE2-induced hepatocyte DNA synthesis and proliferation was almost completely inhibited by a monoclonal antibody against TGF-{alpha} but not by those against IGF-I, HGF (data not shown), or EGF (data not shown) (Fig. 7Go). Therefore, the present results suggest that cytokine TGF-{alpha} is stored within the parenchymal hepatocytes and is triggered to secrete to the extracellular side via EP1 receptor stimulation. The TGF-{alpha} signaling system, which we have been studying extensively in primary cultures of adult rat hepatocytes (31), is mediated mainly through the tyrosine kinase/PI3K/MAPK/p70 S6K pathway. In addition, if the proliferative effects of EP1 receptor agonists are mediated through the secretion of TGF-{alpha}, the cell density-independent nature of the EP1 receptor agonists (data not shown) is consistent with that of TGF-{alpha} (31).

To confirm this hypothesis, it is important to demonstrate that EP1 receptor agonist treatments actually lead to TGF-{alpha} secretion into culture medium. As shown in Fig. 8Go, we have demonstrated for the first time that PGE2 and 17-pt-PGE2 rapidly stimulated TGF-{alpha} secretion into a conditioned medium; the maximal TGF-{alpha} level was about 0.025 ng/ml. In contrast, we have previously shown that exogenously added TGF-{alpha} (human recombinant) can stimulate DNA synthesis and proliferation in primary cultures of adult rat hepatocytes: 0.5 ng/ml TGF-{alpha} in the culture medium sufficiently stimulated DNA synthesis and proliferation (31). Therefore, the levels of TGF-{alpha} in the culture medium are about 1 order of magnitude lower than those of exogenously added TGF-{alpha} to stimulate hepatocyte DNA synthesis and proliferation. However, it is reasonable to consider the possibility that local TGF-{alpha} concentrations around the hepatocyte plasma membrane may become transiently much higher than in other extracellular spaces in the culture medium during times of autocrine secretion. Furthermore, the results in Fig. 8BGo demonstrate that EP receptor agonists in the order 17-pt-PGE2 > PGE2 > sulprostone >> 11-deoxy-PGE1 secrete TGF-{alpha} into the conditioned medium. This may be one of the main reasons why 17-pt-PGE2, with considerably less EP1/EP3 receptor agonist sulprostone or EP2/EP4 receptor agonist 11-deoxy-PGE1, was more potent than PGE2 in stimulating hepatocyte DNA synthesis and proliferation (Fig. 2Go). The TGF-{alpha}-releasing action of the EP1 receptor agonists, which is not a direct proliferating action, sufficiently explains the phenomenon of increased DNA synthesis and proliferation in primary culture of adult rat hepatocytes.

To confirm links between EP1 receptor agonist-induced TGF-{alpha} secretion and hepatocyte mitogenesis, we investigated the regulatory mechanisms associated with the rapid TGF-{alpha} secretion by EP1 receptor agonists. TGF-{alpha} secretion by primary cultured hepatocytes may be regulated by the EP1 receptor/Gq/PLC/Ca2+ pathway (Fig. 9BGo), because the stimulatory effects of these EP1 receptor agonists on medium levels of TGF-{alpha} were also antagonized by SC-51322, inhibited by U-73122, verapamil, and diltiazem, and potentiated by ionomycin. Blockade of EP1 receptor agonist-induced TGF-{alpha} secretion confirmed this hypothesis. In addition, somatostatin strongly inhibited EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation by inhibiting TGF-{alpha} secretion (Fig. 9BGo). Together, these observations show that Ca2+-mediated selective TGF-{alpha} secretion is an essential step in the stimulation of EP1 receptor agonist-induced hepatocyte DNA synthesis and proliferation. Within 5 min of treatment, hepatocytes started to release TGF-{alpha}, suggesting that TGF-{alpha} increase in the conditioned medium is not caused by de novo synthesis of TGF-{alpha} protein by hepatocytes. These results support the notion that PGE2, 17-pt-PGE2, and, to a lesser extent, sulprostone selectively act as TGF-{alpha} releasers to affect the hepatocyte’s acute phase response and that TGF-{alpha}, in turn, plays an essential role in the stimulation of hepatocyte DNA synthesis and proliferation.

Conversely, inhibitors of growth-related signal-transducing elements, such as genistein, wortmannin, PD98059, and rapamycin, had no significant effects on EP1 receptor agonist-induced increase in the medium levels of TGF-{alpha} (Fig. 10Go). Consequently, the suppression of EP1 receptor subtype signaling by these specific inhibitors of growth-related signal-transducing elements may occur downstream of TGF-{alpha} secretion. These inhibitors only blocked EP1 receptor subtype signaling by inhibiting corresponding signal transducer activities [e.g. tyrosine kinase and MAPK activity (Fig. 11Go)], thereby blocking hepatocyte DNA synthesis and proliferation (Fig. 6Go). Together, our findings confirm that the ability of the EP1 receptor agonists to promote hepatocyte mitogenesis is attributable to an indirect autocrine effect of the secreted TGF-{alpha}. EP1 receptor agonist-induced TGF-{alpha} secretion is regulated by the EP1 receptor/Gq/PLC/Ca2+ pathway. This is a novel mechanism of action compared with that of classic growth factors, including insulin, EGF, and HGF, and a similar indirect mechanism on cell growth has been reported in the human prostate cancer cell line TSU-Pr1, in which the proliferative effects of TGF-ß are mediated through the autocrine secretion of PDGF (43).

In conclusion, we provide here evidence that the EP1 receptor/Gq/PLC/Ca2+-dependent autocrine secretion of TGF-{alpha} occurring in response to EP1 receptor activation is an essential step in the stimulation of DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. The EP2/EP3/EP4 receptor subtypes play only a minor role in this process. In addition, our findings indicate the usefulness of culture conditions to the study of the autocrine loop in the proliferation of adult rat hepatocytes. Because PGE2 is the main prostanoid secreted by sinusoidal endothelial cells (11, 12), we have proposed that the hepatocyte proliferation regulation involves locally produced PGE2, generated in response to hepatic injury, acting as an inducer of hepatic regeneration in vivo. TGF-{alpha} is subsequently secreted by hepatocytes under the influence of EP1 receptor stimulation to play a fundamental role in the initiation of mitogenic responses. Further research on these mechanisms involving PGs may shed light not only on growth regulation of the liver but also on the therapeutic application of prostanoids for hepatic injury.


   Footnotes
 
Abbreviations: DDA, 2,4-Dideoxyadenosine; EGF, epidermal growth factor; EP, receptor for PGE2; Gq, G-protein involved in PLC regulation; HGF, hepatocyte growth factor; PDGF, platelet-derived growth factor; 17-pt-PGE2, 17-phenyl-trinor-PGE2; p44, phosphorylated ERK1 (