Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/Akt pathways in a murine hepatocyte cell line

Yuri Y. Sautin, James M. Crawford, and Stanislav I. Svetlov

Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida 32610


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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First published September 5, 2001; 10.1152/ajpcell.00077.2001.---Protective mechanisms for lysophosphatidic acid (LPA) against cell death caused by Clostridium difficile toxin, or tumor necrosis factor-alpha (TNF-alpha ) plus D-galactosamine, were investigated in a murine hepatocyte cell line AML12 expressing Edg2 LPA receptor. In these models of hepatocellular injury, LPA prevented hepatocyte damage, suppressed apoptosis, and enhanced cell survival in a dose-dependent fashion. The protective effects of LPA were abolished by wortmannin and LY-294002, specific inhibitors of phosphatidylinositol 3-phosphate kinase (PI 3-kinase), and by PD-98059 and U-0126, inhibitors of MEK1/MEK2. In nontreated hepatocytes, LPA elicited a gradual and sustained increase in phosphorylation of Erk1/Erk2 over 180 min of stimulation and downstream phosphorylation of p90RSK and transcription factor Elk-1. In C. difficile toxin-treated cells, LPA-induced phosphorylation of Erk1/Erk2 was rapid but transient, while p90RSK and Elk-1 phosphorylation did not change significantly. LPA stimulated phosphorylation of Akt in a time-dependent manner in both intact and toxin-treated AML12 hepatocytes. Wortmannin and LY-294002 abolished phosphorylation of Akt, further supporting activation of PI 3-kinase/Akt as a signaling pathway, which mediates hepatocyte protection by LPA. Taken together, these results demonstrate that LPA prevents cell apoptosis induced by C. difficile toxin and TNF-alpha /D-galactosamine in the AML12 murine hepatocyte cell line. Cell protection by LPA involves activation of the mitogen-activated protein kinase Erk1/Erk2 cascade and PI 3-kinase-dependent phosphorylation of Akt.

lysophosphatidic acid; phosphatidylinositol-3-phosphate kinase; Clostridium difficile; mitogen-activated protein kinase


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THE ROLE OF PEPTIDE GROWTH FACTORS, such as transforming growth factor-alpha (TGF-alpha ) acting through tyrosine kinase receptor, in hepatocyte survival and proliferation has been studied extensively (see Ref. 14 for review). The potential significance of newly recognized lysolipid phosphate growth factors, which signal via G protein-coupled receptors, is largely unknown.

Lysophosphatidic acid (LPA) is a key member of the lysolipid phosphate family, which serves an important role as secreted extracellular mediator. LPA is produced in membrane microvesicles from stimulated platelets, leukocytes, or tumor cells, and then is secreted outside on physiological activation, tissue injury, inflammation, and neoplasia (17, 29, 33). The principal effects of LPA are growth related, including induction of cellular proliferation, alterations in differentiation and survival, and suppression of apoptosis (see Ref. 18 for review). LPA also evokes cellular effector functions, which are dependent on cytoskeletal responses such as contraction, secretion, adhesion, and chemotaxis. The cellular effects of LPA are mediated predominantly via G protein-coupled receptors, encoded by the endothelial differentiation genes Edg2, Edg4, and Edg7 (1, 21, 25). As it binds to Edg receptor, LPA recruits Gi proteins, which may lead to a downstream activation of Erk1/Erk2 mitogen-activated protein (MAP) kinases and, via several intermediate effectors, induction of the serum response element (SRE)-driven cell proliferation (30, 39). Among the downstream effectors, which may be phosphorylated and activated by Erk1/Erk2, are p90RSK, also known as a ribosomal S6 kinase, and Elk-1, a transcription activator (38). However, the potential involvement of LPA in the stimulation of these pathways has not been investigated. The activation of G12/13 proteins stimulates the Rho-GTP pathways, which mediate cytoskeleton-dependent functions and activation of phosphatidylinositol 3-phosphate kinase (PI 3-kinase) and may also contribute to SRE-mediated transcription (16). Protein kinase B or Akt is known as an important downstream target for PI-3-kinase, which can be activated by a variety of growth factors and cytokines via phosphorylation on serine and threonine residues (10). Downstream effectors for Akt include transcription factors Forkhead, other protein kinases, and possibly Bcl-2 family members (see Ref. 27 for review). LPA was shown to inhibit fibroblast apoptosis, induced by serum deprivation, via activation of both MAP kinase and PI 3-kinase (13).

Little is known regarding the biological activities of LPA and underlying mechanisms in the liver. It has been shown recently that the LPA counterpart sphingosine-1-phosphate (S1P) induced proliferation of hepatic stellate cells (23, 48). Other authors reported growth inhibition by S1P in hepatic myofibroblasts (11) and in primary rat hepatocytes stimulated with hepatocyte growth factor (24). On the other hand, LPA was shown to increase motility and migration of a human hepatoma cell line (37). The ability of LPA to modulate the survival of hepatic cells in response to apoptotic agents, such as toxins or tumor necrosis factor-alpha (TNF-alpha )/D-galactosamine, has not been investigated. It has been reported that toxins from Clostridium difficile inhibit the small GTPases Rho, Rac, and cdc42 (6), which may lead in hepatocytes to depolymerization of the cytoskeleton and cell death (19, 35). On the other hand, in hepatocytes primed with transcriptional inhibitor D-galactosamine, TNF-alpha induced apoptosis, which was mediated via TNF-alpha receptor 1 and activation of caspase 3 (4). Cell apoptosis induced by the Rho-directed C. difficile toxin was prevented by activation of estrogen-inducible Raf-1 and Erk1/Erk2 MAP kinase in a highly growth factor-dependent line of lung fibroblasts (31). These findings highlight the ability of Erk1/Erk2 MAP kinase to generate survival signals that counteract cell death induced by the loss of matrix contact and cytoskeletal integrity.

In this study, we investigated the roles for LPA in survival of a murine hepatocyte cell line injured by C. difficile toxin or TNF-alpha /D-galactosamine, and we explored the cell protective mechanisms, which may be recruited by LPA. We employed hepatocytes obtained from mice transgenic for human TGF-alpha (hTGF-alpha ) (26, 46). hTGF-alpha -transformed hepatocytes (designated AML12 hepatocytes) are well differentiated and can be passaged for many times without loss of differentiation (46). Evidence has been accumulating that AML12 hepatocytes represent an adequate and reproducible model for long-term studies of hepatocyte proliferation, survival, and neoplastic transformation (5, 40). The present results reveal nearly complete protection of a murine hepatocyte cell line AML12 by LPA against the proapoptotic effects of toxin or TNF-alpha /D-galactosamine. In addition, the expression of Edg2 subtype LPA receptors in AML12 hepatocytes was detected. LPA stimulated PI 3-kinase-dependent phosphorylation of Akt, activation of MAP kinase Erk1/Erk2, and downstream phosphorylation of p90RSK and Elk-1, which may represent mechanisms responsible for the prosurvival effects of LPA in the AML12 hepatocyte cell line.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Materials. Oleoyl (C18:1) and palmitoyl (C16:0) LPA was obtained from Avanti Polar Lipids (Alabaster, AL). Cell culture media and reagents, Trizol and SuperScript One-Step RT-PCR kit were purchased from GIBCO BRL (Rockville, MD). Human TGF-alpha neutralizing antibody was from Chemicon (Temecula, CA). All other antibodies, U-0126 and PD-98059, were from Cell Signaling Technology (Beverly, MA). Radiolabeled [3H]thymidine (60 Ci/mmol) was purchased from Amersham Pharmacia (Piscataway, NJ). Mouse TNF-alpha was obtained from PeproTech (Rocky Hill, NJ). Other chemicals were products of Sigma (St. Louis, MO). C. difficile crude extract, containing exotoxins A and B (data not shown), was a gift from Dr. John Valentine (University of Florida College of Medicine, Gainesville, FL).

Cell cultures. AML12 hepatocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in cultures in DMEM/F-12 media (1:1) containing 10% fetal bovine serum, 5 µg/ml insulin, 3.35 ng/ml sodium selenite, 2.75 µg/ml transferrin, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphothericin B, and 40 ng/ml dexamethasone. Hepatocytes were grown at 37°C in humidified 7.5% CO2-92.5% air and passaged approximately every 7-10 days.

Primary mouse hepatocytes were isolated from male 129SeVe mice by the portal perfusion techniques developed by Seglen (43). Hepatocytes were suspended in DMEM medium containing 15% FCS and insulin (0.5 µg/ml), plated at a density of 2 × 105 cells/ml and cultured for 24 h before harvesting.

Assays of cell apoptosis, survival, and proliferation. Hepatocytes were plated on 24-well plates at a density of 4 × 104 cells/ml and cultured for 3-4 days to ~75-80% confluency. For cell proliferation assay, the hepatocytes were incubated in serum-free medium with various concentrations of LPA (0.1-50 µM). For apoptosis and survival studies, hepatocytes were treated with C. difficile toxin (10 ng/ml) or preincubated with D-galactosamine (1 mM, 30 min) followed by challenge with TNF-alpha (100 ng/ml) for 18 h in the presence and absence of LPA (0.1-25 µM). In separate experiments, AML12 hepatocytes were treated in the presence of PI 3-kinase antagonists, wortmannin (250 nM) and LY-294002 (25 µM), and MEK1/2 antagonists PD-98059 (50 µM) and U-0126 (10 µM), or hTGF-alpha neutralizing antibody (12 µg/ml). At the end of the incubations, cells were photographed using Hoffman modulation optics and processed for analysis of cell apoptosis, proliferation, and survival.

Hepatocyte apoptosis was assessed by measuring nuclear DNA fragmentation and release of DNA fragments into the cytoplasm using an apoptosis detection ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN). The cells were prelabeled with bromodeoxyuridine (BrdU) for 24 h. After the treatment, genomic DNA was precipitated, and the labeled fragments in the cytoplasm were determined by binding to an anti-DNA antibody-coated plate, followed by color development with anti-BrdU-POD complex using the same apoptosis detection kit.

Cell proliferation was determined by measuring DNA synthesis using [3H]thymidine incorporation. At the end of experimental incubations, [3H]thymidine (1 µCi/well) was added, and cells were cultured for an additional 6 h. Medium was aspirated, and cells were rinsed with PBS and then thoroughly washed twice with ice-cold 10% TCA and twice with ice-cold 90% ethanol. Cells were lysed with 1% Triton X-100, and radioactivity was counted in a liquid scintillation counter.

For cell survival studies, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test was employed, which directly reflects mitochondrial respiratory chain integrity. The MTT assay was performed as described in detail elsewhere (8).

RT-PCR analyses. RT-PCR was performed using a SuperScript One-Step RT-PCR kit. Template RNA (1 µg for each sample) was used with the following pairs of primers designed from the sequences: for LPA receptor (deposited in GenBank under accession no. U70622), forward primer 351-5'-GCCACAGAATGGAACACAG-3' and reverse primer 833-5'-GTAGAGGGGTGCCATGTTG-3'; for glyceraldehyde-3-phosphate dehydrogenase, forward primer 5'-GCCAGCCTCGTCTCATAGAC-3' and reverse primer: 5'-ATGTAGGCCATGAGGTCCAC-3'.

The conditions of RT-PCR were as follows: 1) RT step -50°C for 30 min, 2) 94°C for 2 min, 3) 94°C for 30 s, 4) 58°C for 30 s, 5) 72°C for 30 s, 6) repeat steps 3-5 for 35 cycles, and 7) 72°C for 1 min. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel.

Phosphorylation of MAP kinases p44/42 (Erk1/Erk2), p90RSK, Elk-1, and Akt. AML12 hepatocytes were incubated in the presence or absence of C. difficile toxin (10 ng/ml) for 18 h and were then stimulated with LPA (25 µM) for various times. To block the MEK1/2 and PI 3-kinase signaling pathways, cells were pretreated with 10 µM U-0126, 50 µM PD-98059, 500 nM wortmannin, 25 µM LY-294002, or vehicle for 60 min before stimulation with LPA. After stimulation, cells were washed with ice-cold PBS and scraped in the ice-cold Western blot buffer. Proteins (50 µg) were resolved by SDS-PAGE electroblotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked and incubated overnight at 4°C with primary rabbit antibodies to phosphorylated p42/44 MAP kinase (Thr-202/Tyr-204), Elk-1 (Ser-383), p90RSK(Ser-381), and Akt (Ser-473), and with the antibodies to corresponding nonphosphorylated kinases (1:1,000 each). Proteins were visualized using a goat anti-rabbit antibody conjugated to horseradish peroxidase and a chemiluminescence Phototope Western detection system. The optical density of the bands was quantified using the image acquisition and analysis system NIH-Scion.

Statistics. Nonpaired Student's t-test and one-way ANOVA followed by Fisher's test were used for statistical analysis. P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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LPA increases DNA synthesis in AML12 hepatocytes. In the absence of serum, LPA evoked a dose-dependent increase in DNA synthesis in nonconfluent AML12 hepatocytes, as indicated by [3H]thymidine incorporation (Fig. 1). An increase in [3H]thymidine incorporation was significant at the initial LPA concentration of 500 nM (Fig. 1). Twofold increases of [3H]thymidine incorporation were found at LPA concentrations between 5 and 50 µM. Oleic (C18:1) and palmitic (C16:0) residue-containing LPAs were nearly equally active (Fig. 1). The concentration range of LPA tested in this study falls into the higher limit of LPA levels of 0.56-6.3 µM reported for normal human plasma (42, 47).


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Fig. 1.   Concentration-dependent increase in DNA synthesis by lysophosphatidic acid (LPA) in AML12 hepatocytes. Subconfluent AML12 hepatocytes were incubated with various concentrations of oleoyl-LPA (, solid line) or palmitoyl-LPA (, dashed line) in serum-free media for 18 h. [3H]thymidine (1 µCi/well) was added for an additional 6 h, and [3H]thymidine incorporation was measured as described in MATERIALS AND METHODS in detail. Data shown are means ± SE of 3 experiments measured in quadruplicate.

LPA protects cell integrity, prevents apoptosis, and improves survival of AML12 hepatocytes treated with C. difficile toxin and TNF-alpha /D-galactosamine. Treatment of AML12 hepatocytes with C. difficile toxin induced the appearance of stress fibers and substantial loss of intercellular contacts (Fig. 2b) compared with untreated cells (Fig. 2a). In hepatocytes preincubated with D-galactosamine, TNF-alpha evoked cell blebbing and vacuolization, a typical apoptotic cell morphology (Fig. 2e). D-Galactosamine alone did not induce any morphological damage (Fig. 2d). Addition of LPA prior to or simultaneously with the toxin (Fig. 2c) and D-galactosamine (Fig. 2f) prevented the hepatocyte damage and protected cell integrity and cytoarchitechture. Both the toxin and TNF-alpha /D-galactosamine induced a remarkable DNA fragmentation and the release of DNA fragments into the cytoplasm, important molecular characteristics of apoptosis (Fig. 3). The levels of DNA fragments in cytoplasm were increased 10-fold by the toxin and 6-fold by TNF-alpha ; LPA significantly reduced the content of cytoplasmic DNA fragments (Fig. 3). The preventive effect of LPA on apoptosis induced by toxin was abolished by wortmannin and PD-98059, inhibitors PI 3-kinase and MEK1, respectively (Fig. 3A). Wortmannin attenuated the protective effect of LPA against TNF-alpha /D-galactosamine, while PD-98059 not only abolished the protection but even potentiated DNA fragmentation (Fig. 3B). The cellular damage caused by the toxin was accompanied by a profound decrease in hepatocyte viability. As shown in Fig. 4, overnight serum deprivation reduced AML12 hepatocyte viability by 30-35%. Treatment with C. difficile toxin in the absence of serum further decreased cell viability following 24 h of treatment (Fig. 4). Preincubation with LPA completely prevented the loss of hepatocyte viability in a dose-dependent manner (P < 0.01, ANOVA). Moreover, high concentrations of LPA restored the loss of viability by serum deprivation (Fig. 4). An increase in hepatocyte survival by LPA was abrogated by wortmannin and LY-294002, two different antagonists of PI 3-kinase, and by MAP kinase inhibitor PD-98059 (Fig. 5, B-D). Moreover, wortmannin, LY-294002, and PD-98059 potentiated the injurious effect of the toxin in both the absence and presence of LPA. Taken together, these data suggest that antiapoptotic mechanisms of LPA in AML12 hepatocytes challenged with C. difficile toxin and TNF-alpha /D-galactosamine involve both PI 3-kinase and MEK1/2 signaling cascades.


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Fig. 2.   Protective effect of LPA on Clostridium difficile toxin-induced cell damage in AML12 hepatocytes. Confluent AML12 hepatocytes were incubated in serum-free medium as follows: a: saline; b: toxin (10 ng/ml); c: toxin + LPA (25 µM); d: D-galactosamine alone (1 mM); e: D-galactosamine for 30 min followed by tumor necrosis factor-alpha (TNF-alpha ; 100 ng/ml); and f: TNF-alpha /D-galactosamine + LPA (25 µM). LPA was added 10 min before addition of toxin (c) or TNF-alpha (f). After 24 h of incubation, cells were photographed using Hoffman modulation contrast. Arrows indicate the formation of stress fibers in response to the toxin. Original magnification, ×300.



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Fig. 3.   Decrease by LPA of apoptosis induced by C. difficile toxin and TNF-alpha /D-galactosamine is abolished by phosphatidylinositol-3-phosphate kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase antagonists. Cultured AML12 hepatocytes were prelabeled with bromodeoxyuridine. Cells were preincubated for 10 min with PD-98059 (50 µM), LY-294002 (25 µM), wortmannin (250 nM), or vehicle. In TNF-alpha experiments, D-galactosamine (1 mM) was included in control samples. Cells were then treated with toxin (10 ng/ml) or D-galactosamine (1 mM) followed by TNF-alpha (100 ng/ml) for 24 h in the presence or absence of 25 µM LPA. After the treatment, genomic DNA was precipitated, and the levels of labeled DNA fragments in cytoplasm were measured by ELISA according to the manufacturer's instructions. Means of 3 experiments performed in duplicate are shown.



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Fig. 4.   Dose-dependent protection by LPA of cell viability in response to C. difficile toxin. AML12 hepatocytes were preincubated for 10 min with various concentrations of LPA. Cells were treated with the toxin (10 ng/ml) for 18 h in serum-free medium. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added for an additional 3 h, and cell viability was determined as described in MATERIALS AND METHODS. Data shown are means ± SE of 3 independent experiments. *P < 0.05 vs. toxin alone according to nonpaired Student's t-test. Overall effect of LPA was significant according to one-way ANOVA (P < 0.01).



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Fig. 5.   Protection by LPA of cell viability in C. difficile toxin-treated AML12 hepatocytes is abolished by PD-98059, LY-294002, and wortmannin. Hepatocytes were challenged with 25 µM LPA alone, 10 ng/ml toxin, and LPA plus toxin (A); or preincubated for 10 min with PD-98059 (50 µM), LY-294002 (25 µM), wortmannin (250 nM), or vehicle followed by treatment with C. difficile toxin for 18 h in the absence or in the presence of 25 µM LPA (B-D). MTT was added for an additional 3 h, and cell viability was determined as described in MATERIALS AND METHODS. Data depict means ± SE of 3 experiments conducted in triplicate. *P < 0.05 vs. toxin alone; NS, not significant vs. toxin alone.

To distinguish the role of stimulation by endogenously secreted hTGF-alpha in LPA effects, AML12 hepatocytes were preincubated with anti-hTGF-alpha neutralizing antibody. As shown in Fig. 6, preincubation with neutralizing antibody neither changed cell viability in intact AML12 hepatocytes nor affected the increase in cell survival by LPA in toxin-treated cells.


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Fig. 6.   The protective effect of LPA is not affected by human transforming growth factor-alpha (hTGF-alpha ) neutralizing antibody in AML12 hepatocytes treated with toxin. Hepatocytes were preincubated for 30 min with anti-hTGF-alpha antibody (12 µg/ml; +Ab) or vehicle (-Ab), and then challenged for 18 h with the toxin in the absence and presence of LPA. Cell viability was assessed using the MTT test as described in MATERIALS AND METHODS. Data of 3 experiments are shown as means ± SE. *P < 0.05, **P < 0.01 vs. toxin alone.

AML12 hepatocytes express Edg2 LPA receptor mRNA. The primers designed from a published mouse sequence were estimated to amplify a 483-bp fragment (351-833 nt) of the open reading frame. The 483-bp PCR product, corresponding to Edg2 receptor fragment, was readily detectable in AML12 hepatocytes (Fig. 7). PCR product of 483 bp was purified from the gel, and its 100% identity to the corresponding Edg2 fragment was confirmed by a sequence analysis in a DNA sequencer (Perkin Elmer, Applied Biosystems).


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Fig. 7.   Edg2 LPA receptor mRNA expression in the AML12 hepatocyte cell line. Total RNA was reverse transcribed and amplified by RT-PCR as described in MATERIALS AND METHODS in detail. RT-PCR products were separated on a 1.5% agarose gel containing ethidium bromide. The PCR product is a 483-bp fragment predicted from the designed pair of primers. A representative experiment from 5 performed with identical results is shown. Lack of Edg2 mRNA expression in the RALA255-10G hepatocyte cell line is shown as a negative control. The expression of mouse brain Edg2 LPA receptor represents a positive control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

LPA induces phosphorylation of Erk1/Erk2, p90RSK, and Elk-1 in AML12 hepatocytes. We observed a high level of phosphorylation of extracellular activated protein kinases Erk1/Erk2 in intact nonstimulated AML12 hepatocytes after 18 h of serum starvation (Fig. 8A). Interestingly, incubation with the toxin did not alter significantly the basal levels of phosphorylated Erk1/Erk2 (phospho-Erk1/Erk2), suggesting that the inhibition of small GTPases Rho and Rac by toxin does not affect basal MEK1/2 MAP kinase activity. LPA elicited a time-dependent, sustained increase in Erk1/Erk2 phosphorylation over 180 min of incubation (Fig. 8A). The values of phospho-Erk1/Erk2 were nearly doubled by LPA at 180 min of incubation in nontreated, serum-starved AML12 hepatocytes (Fig. 8B). In contrast, an increase in phospho-Erk1/Erk2 was transient in the toxin-challenged cells with a twofold increase at 2.5 min and a decline thereafter to baseline at 45 min (Fig. 8B). LPA evoked a remarkable phosphorylation of p90RSK at Ser-381, a phosphorylation site for Erk1/Erk2 (Fig. 9). Maximum phosphorylation was attained at 5 min, and phospho-p90RSK was still detectable at 45 and 180 min (Fig. 9). Toxin treatment slightly attenuated the levels of phospho-p90RSK, while both PD-98059 and U-0126 completely inhibited the phosphorylation of p90RSK in response to LPA (Fig. 9). The phosphorylation of Elk-1 at Ser-383 was also increased by LPA with maximum activity at 45 min (Fig. 9). Toxin decreased the levels of phospho-Elk1 modestly, while preincubation with PD-98059, but not U-0126, blocked the phosphorylation (Fig. 9).


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Fig. 8.   Stimulation by LPA of MAP kinase (MAPK) Erk1/Erk2 phosphorylation on Thr-202/Tyr-204 residues in AML12 hepatocytes. Confluent AML12 hepatocytes were treated, stimulated, and processed as described in detail in MATERIALS AND METHODS. Phosphorylated MAPK were detected by immunoblotting with the antibody specific for Thr-202/Tyr-204-phosphorylated MAPK (Erk1 and Erk2) (A). Aliquots of the cell lysates were analyzed simultaneously with antibody for total MAPK. The optical densities of phosphorylated MAPKs were normalized to the corresponding values for total MAPKs. Phosphorylation of MAPK is expressed as fold increases relative to the untreated, nonstimulated control (B). Western blot and densitometric analysis of a representative experiment from 4 performed are shown.



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Fig. 9.   LPA induced phosphorylation of p90RSK and Elk1 in AML12 hepatocytes. AML12 hepatocytes were treated with toxin or vehicle as described in detail in MATERIALS AND METHODS. After treatment, the cells were preincubated with PD-98059 (50 µM) or U-0126 (10 µM) before stimulation with LPA. Phosphorylated p90RSK and Elk1 were detected by immunobloting with the specific antibody to phospho-p90RSK (Ser-381) and phospho-Elk1 (Ser-383). A representative blot is shown.

LPA stimulates PI 3-kinase-mediated phosphorylation of Akt. Protein kinase B/Akt can be activated by phosphorylation on Ser-473 via the PI 3-kinase-dependent pathway (10). Levels of phospho-Akt were readily detectable in nonstimulated AML12 hepatocytes in the absence of C. difficile toxin (Fig. 10A). LPA induced a rapid increase in Akt phosphorylation with a maximum at 2.5 min followed by a decline at 45 min of stimulation (Fig. 10A). Significantly decreased basal levels of phospho-Akt were observed in the toxin-treated hepatocytes compared with resting cells (Fig. 10A). Nevertheless, stimulation with LPA was able to overcome this suppression and to substantially enhance the phosphorylation of Akt. The pattern of LPA stimulation was similar to that in nontreated AML hepatocytes, with a maximum of Akt phosphorylation observed at 2.5 min (Fig. 10A). Wortmannin (500 nM) and LY-294002 (25 µM) abolished Akt phosphorylation in both LPA-stimulated and resting AML12 hepatocytes (Fig. 10B), supporting a crucial role for PI 3-kinase in the upstream mediation of the Akt-dependent survival pathway in these cells.


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Fig. 10.   Phosphorylation of Akt on Ser-473 by LPA in AML12 hepatocytes via PI 3-kinase-dependent mechanism (A). Cells were serum deprived for 16-18 h with or without C. difficile toxin (10 ng/ml). Hepatocytes were then stimulated with LPA (25 µM) for various time intervals. B: serum-starved hepatocytes were pretreated with LY-294002 (25 µM) or wortmannin (500 nM) for 1 h before stimulation with 25 µM of LPA. Proteins were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies specific for Ser-473-phosphorylated Akt or total Akt. The results of a representative experiment from 3 performed are shown.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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There are several important aspects to this study. First, we have shown that, in hTGF-alpha -transformed murine hepatocytes, LPA prevents cell apoptosis caused by two different proapoptotic agents, C. difficile toxin and TNF-alpha in combination with a mild inhibitor of transcription, D-galactosamine. Second, cell protection by LPA involves activation of the MAP kinase Erk1/Erk2 cascade and PI 3-kinase-dependent signaling, possibly via phosphorylation of Akt. Third, we revealed using RT-PCR that Edg2 LPA receptor mRNA is present in this particular hepatocyte cell line.

LPA by itself was found to increase proliferation of AML12 hepatocytes in a dose-dependent manner (Fig. 1). The detectable mitogenic effect of LPA was observed at a concentration of 500 nM, which is merely 1.5 times higher than that detectable in normal mouse plasma (42) and was nearly twofold at 25-50 µM (Fig. 1). LPA has been shown to be mitogenic in a variety of cell types, including fibroblasts, smooth muscle cells, endothelial cells, astrocytes, and renal epithelial cells (36). Recently, the protective, prosurvival effects of LPA in several cells, including Schwann cells and fibroblasts, were reported (13, 45), predominantly involving MAP kinase and PI 3-kinase activation.

In this study, we examined whether LPA can modulate cell survival in AML12 hepatocytes stressed by two different proapoptotic stimuli, C. difficile toxin and TNF-alpha /D-galactosamine. AML12 hepatocytes challenged with the toxin developed a typical morphology of actin-dependent damage of the cytoskeleton. TNF-alpha /D-galactosamine induced cell blebbing and vacuolization, characteristics of apoptosis (Fig. 2, b and f). Addition of LPA before the agents prevented the hepatocyte damage and improved cytoarchitechture (Fig. 2c). Although C. difficile toxin has been shown to inhibit Rho, Rac, and cdc42 small GTPases and to induce cell death in different cell types (6, 31, 35), the detailed mechanisms, particularly in hepatocytes, are not clear. We found that the toxin evoked DNA fragmentation and release of DNA fragments into the cytoplasm, prominent features of apoptosis (Fig. 3A). These data are in accordance with several other observations, that toxins can induce apoptosis in various cell types, including renal tubular epithelial cells, via Rho-dependent disrupting of the cytoskeleton (3, 31). Cellular DNA fragmentation and release of the fragments into the cytoplasm, induced by the toxin, was strongly suppressed by LPA (Fig. 3A). Similarly, LPA significantly decreased DNA fragmentation in a different model of apoptosis evoked by TNF-alpha /D-galactosamine (Fig. 3B). In both models, the protective effect of LPA was reversed by wortmannin and PD-98059, suggesting that MAP kinase and PI 3-kinase cascades are involved as common mechanisms, which mediate the antiapoptotic effects of LPA. (Fig. 3, A and B). Hepatocellular injury and cell apoptosis elicited by the toxin was manifested by a decrease in hepatocyte survival (Fig. 4). LPA completely restored not only decrease of cell viability elicited by the toxin in serum-deprived hepatocytes, but, at higher doses, abolished the effect of serum starvation (Fig. 4). Prosurvival effects of LPA were started at a concentration of 100 nM and exhibited a clear dose dependency (Fig. 4), which suggests that the protective mechanisms recruited by LPA may be, at least in part, LPA receptor mediated. Both wortmannin and LY-294002, as well as PD-98059, abrogated the protective effect of LPA (Figs. 3 and 5). Complete inhibition of the protective effect of LPA by wortmannin, LY-294002, and PD-98059 suggests that both PI 3-kinase and MEK1/MEK2 downstream signaling play a role in prosurvival activity of LPA in AML12 hepatocytes. It has been well established that peptide growth factors such as epidermal growth factor (EGF) and TGF-alpha promote cell survival via both the MAP kinase cascade and PI 3-kinase activation (12, 41). Because overexpression of TGF-alpha is the intrinsic feature of AML12 hepatocytes, we examined the possibility that generation by LPA of survival signals may be a result of LPA potentiation of TGF-alpha effects or sensitization/priming of the cells to TGF-alpha , rather than the effects of LPA per se. As seen in Fig. 6, hTGF-alpha neutralizing antibody neither changed cell viability in intact AML12 hepatocytes nor affected the increase of these parameters by LPA in C. difficile toxin-treated AML12 hepatocytes (Fig. 6). Therefore, it appears that the prosurvival activities of LPA against the toxin-induced damage are attributable to the specific LPA-dependent pathways in AML12 hepatocytes. However, we cannot rule out that the authentic overexpression of hTGF-alpha in AML12 hepatocytes already triggered cell stimulation at the intracellular level, where it is inaccessible for neutralizing antibody.

It has been accepted that hepatocytes and the liver lack LPA receptors (1, 21, 25). Particularly, no Edg2 mRNA was detected in liver tissue during Northern blotting (1, 15, 21). Our data clearly indicate the presence of a 483-bp fragment corresponding to the Edg2 receptor in RT-PCR analyses of AML12 hepatocytes (Fig. 7). The 483-bp fragment, which encompasses nearly 25% of the entire open reading frame, was sequenced and identified as having 100% identity with the mouse Edg2 sequence (not shown). Less-sensitive Northern blot analyses used by other groups, compared with RT-PCR employed in the present studies, may account for this discrepancy. It has been shown that expression of Edg2 in HEK-293 and Chinese hamster ovary cells resulted in an increase in [3H]LPA binding, LPA-evoked stimulation of MAP kinase, and SRE-driven transcriptional activation (2). Therefore, the beneficial effects of LPA in AML12 hepatocytes may, at least at low concentrations, be attributed to Edg2 receptor-mediated signaling. Recently, Hooks and co-workers (22) provided evidence that the mitogenic effects of LPA in HEK-293 cells was Edg receptor independent and was regulated by a lipid phosphate phosphatase (22). Thus a contribution of nonreceptor-mediated mechanisms to LPA-dependent protective effects, particularly at high LPA concentrations, cannot be excluded in our models.

LPA induced a sustained increase in phosphorylation of Erk1/Erk2 over 180 min of stimulation in intact, serum-deprived AML12 hepatocytes (Fig. 8, A and B). This finding is consistent with data obtained using other cell types. For example, LPA stimulated phosphorylation of MAP kinase Erk1/Erk2 in endothelial cells, facilitating their proliferation and wound healing (30). LPA was able to stimulate phosphorylation of Erk1/Erk2 following incubation with the toxin; however, it was rapid but transient (Fig. 8A). This result may be due to an inhibition by toxin of Rac, which can stimulate the [extracellular signal-regulated kinase (Erk)] cascade either by promoting the phosphorylation of c-Raf or by direct facilitating of MEK1/MEK2 activation (9). It has been demonstrated that only sustained Erk1/Erk2 activation is associated with induction of cell proliferation, while transient activation can stimulate cellular metabolism, induce early gene transcription, and probably maintain cell viability (7, 28). The potential mechanisms of Erk1/Erk2 activation by LPA involve tyrosine phosphorylation of adaptor proteins and assembly of Ras activation complexes. As for many other G protein-coupled receptors, the scaffolds for LPA receptor-mediated Ras activation complexes may include LPA receptor itself, transactivated receptor tyrosine kinases, and integrin-based focal adhesions (20, 34). It has been reported very recently that LPA stimulates the shedding of pro-heparin-binding EGF-like growth factor (pro-HB-EGF), which comprises LPA receptor-mediated transactivation of the EGF receptor. Toxin B from C. difficile, which inhibits Rac, Rho, and cdc42, prevented LPA-induced shedding of pro-HB-EGF (44). In our study the toxin inhibited sustained, but not transient activation of Erk1/Erk2 and subsequent phosphorylation of p90RSK (Fig. 9). Therefore, it is plausible that both EGF receptor transactivation and direct LPA receptor-mediated activation of Ras complexes are involved in Erk1/Erk2 stimulation in AML12 hepatocytes.

Erk1/Erk2, activated in response to LPA, can further propagate the signal by phosphorylating numerous substrates, such as protein kinases, transcription factors, and membrane proteins. We have shown that, in AML12 cells, LPA induced phosphorylation of protein kinase p90RSK and transcription factor Elk-1 at Erk1/Erk2-specific sites, and these effects were attenuated by specific MEK1/MEK2 inhibitors (Fig. 9). These findings further elucidate the mechanism of LPA effects via the MAPK cascade. Once phosphorylated, Elk-1 can stimulate transcription from SRE, which is activated by a variety of growth factors. Substrates of p90RSK include BAD and CREB, important regulators of cell survival and gene expression (38).

Next, we suggested that the PI 3-kinase-dependent phosphorylation and activation of Akt plays a role in AML12 hepatocyte protection by LPA. LPA evoked a rapid phosphorylation of Akt on Ser-473 in both intact and toxin-challenged AML12 hepatocytes subjected to serum deprivation (Fig. 10A). Although basal levels of phospho-Akt were slightly lower in toxin-treated cells compared with nontreated cells, the patterns of Akt phosphorylation by LPA were similar, with a maximum at 2.5 min and decline thereafter (Fig. 10A). Thus, even in the presence of toxin, the phosphorylation and activation of Akt can be effectively increased by LPA, which may lead to an increase in cell survival. Preincubation with wortmannin or LY-294002, specific PI 3-kinase antagonists, abrogated the phosphorylation of Akt, both basal and LPA stimulated (Fig. 10B), further supporting the discovery that Akt phosphorylation in response to LPA in AML12 hepatocytes occurs downstream from PI 3-kinase activation. These results are in agreement with the data from other investigators (32), who demonstrated that LPA protection of renal proximal tubular cells and murine macrophages is associated with PI 3-kinase pathway activation.

In conclusion, these studies present evidence that LPA protects hTGF-alpha -transformed murine hepatocytes against cell death evoked by toxin and TNF-alpha , potent inducers of apoptosis with different mechanisms of action. We also demonstrate the presence of Edg2 LPA receptors at the mRNA level in this hepatocyte cell line. Intracellular signaling mechanisms of this protection, recruited by LPA, involve activation of the MAP kinase Erk1/Erk2 cascade and PI 3-kinase-dependent phosphorylation of Akt. Whether these LPA mechanisms are operative in nontransformed, primary hepatocytes, remains to be investigated.


    ACKNOWLEDGEMENTS

We thank Dr. John Valentine of the Department of Medicine, University of Florida College of Medicine, Gainesville, FL, for providing the C. difficile toxin. We are also indebted to Olga Tchigrinova for excellent technical assistance.


    FOOTNOTES

This work was sponsored in part by the Dr. Charles Trey Memorial Liver Scholar Award from the American Liver Foundation to S. I. Svetlov and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53512 to J. M. Crawford.

Address for reprint requests and other correspondence: S. Svetlov, Dept. of Pathology, Immunology, and Laboratory Medicine, 1600 SW Archer Rd., PO Box 100275, Gainesville, FL 32610 (E-mail: svetlov{at}pathology.ufl.edu).

First published September 5, 2001;10.1152/ajpcell.00077.2001

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 February 2001; accepted in final form 27 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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