Impaired Ras membrane association and activation in PPARalpha knockout mice after partial hepatectomy

Michael D. Wheeler1,2, Olivia M. Smutney2, Jennifer F. Check1, Ivan Rusyn3, R. Schulte-Hermann4, and Ronald G. Thurman1,2,dagger

1 Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, 2 Curriculum in Toxicology, School of Medicine, and 3 School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27599; and 4 Institut fur Krebsforschung, A-1090 Vienna, Austria


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-alpha (PPARalpha ) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPARalpha in cell proliferation after PH was tested. After PH, an increase in PPARalpha DNA binding was observed in wild-type mice, correlating with an increase in the PPARalpha -regulated enzyme acyl-CoA oxidase. In addition, the PPARalpha -regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPARalpha knockout (PPARalpha -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by ~60% in PPARalpha -/- mice, despite increases in TNF-alpha and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPARalpha -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPARalpha -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPARalpha -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPARalpha blunted the activation of cyclin-dependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPARalpha is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA.

hepatocyte proliferation; cell cycle regulation; RhoA; peroxisome proliferation-activated receptor-alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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THE ROLE OF LIPID AND LIPID metabolism in cell cycle control is not clearly understood (15, 29, 30). Peroxisome proliferator-activated receptor-alpha (PPARalpha ) acts as a regulator of lipid metabolism and homeostasis through transcriptional regulation of key enzymes, such as acyl-CoA oxidase, the initial rate-limiting enzyme in the conversion of long-chain fatty acids to acyl-CoA thioesters, and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, a rate-limiting enzyme in the biosynthesis of isoprenoids for protein isoprenylation (30). This suggests that PPARalpha may play a role in the regulation of prenylation of small GTPases necessary for cell cycle progression. Importantly, inhibition of the HMG-CoA reductase, an enzyme immediately downstream of HMG-CoA synthase, by lovastatin completely prevents mesangial cell proliferation in culture (14). Moreover, treatment of vascular smooth muscle cells with the HMG-CoA reductase inhibitor simvastatin nearly completely inhibited PDGF-induced DNA synthesis, retinoblastoma phosphorylation, and cdk activation (24). PPARalpha also regulates the expression of farnesyl pyrophosphate synthase (FPPS), the terminal enzyme in FPPS (44). Collectively, these observations support the hypothesis that isoprenoid synthesis via PPARalpha -dependent mechanisms is an important factor in regulation of the cell cycle.

Several hypolipidemic drugs, such as WY 14643 and clofibrate, have gained considerable interest as hepatic peroxisome proliferators and as potent carcinogens in rodent liver. Moreover, PPARalpha has recently been demonstrated to be necessary for hepatocyte proliferation due to peroxisome proliferators (37). The mechanisms underlying liver carcinogenesis due to peroxisome proliferators remain uncertain; however, recent evidence suggests mitogenic cytokines, such as TNF-alpha , play key roles (6, 42). Interestingly, TNF-alpha -induced hepatocyte proliferation has been shown to involve critical small GTPases Ras and RhoA (2). PPARalpha is a nuclear transcription factor responsible for the upregulation of a number of genes involved in peroxisomal beta -oxidation, cholesterol and isoprenoid synthesis (26, 48), yet, it is unclear how PPARalpha plays a role in hepatocyte proliferation.

The two-thirds partial hepatectomy (PH)/liver regeneration model is an in vivo model in which progression of hepatocyte proliferation occurs in a relatively synchronous manner and is regulated by a number of proinflammatory/mitogenic cytokines and growth factors. There is compelling evidence that TNF-alpha is necessary for liver regeneration in this model (1, 46). Since PPARalpha likely plays a role in upregulation of isoprenoid synthesis and TNF-alpha -induced cell proliferation involves Ras and RhoA, it was hypothesized that PPARalpha mediates hepatocyte proliferation in vivo after PH.

Recently, it was reported that the rate of DNA synthesis in PPARalpha -deficient mice was not different from controls 72 h after PH (40). Indeed, data presented here demonstrate that no difference in the rate of proliferation is observed at 72 h. However, since other studies (1, 47) report a peak DNA synthesis at 36-44 h in this model, it is possible that differences in DNA synthesis were overlooked. Thus our studies address the role of PPARalpha in the early stages of DNA synthesis (i.e., 0-48 h after PH) and, in contrast, clearly demonstrate an essential role for PPARalpha in mechanisms of maximal Ras and RhoA activation and cell cycle progression.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Antibodies used in these studies included the following: cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), ckd2 (sc-163; Santa Cruz Biotechnology), cdk4 (sc-260; Santa Cruz Biotechnology), phospho-RbT181 (BioSource, Camarillo, CA), p21Cip1 and p27Kip1 (BioSource), RhoA (kind gift of Dr. Channing Der, University of North Carolina), and pan-Ras (OP-40; Chemicon, Temecula, CA). The plasmid including glutathione-S-transferase Ras binding domain of Raf-1 (GST-RBD; binding substrate for activated GTP-bound Ras) was a kind gift of Dr. Adrienne Cox, University of North Carolina. Glutathione Sepharose 4B beads were obtained from Pharmacia (Piscataway, NJ), and protein A/G beads were purchased from Santa Cruz Biotechnology.

Animals. Mice deficient in the peroxisome proliferator-activated receptor-alpha (PPARalpha -/-) and the appropriate SV129 wild-type mice originally characterized and described by Lee et al. (25) have since been back-crossed more than five generations to C57Bl6 mice. Mice were anesthetized with pentobarbital sodium (60 mg/kg body wt). The left and median lobes of the liver, constituting ~70% of total liver mass, were removed by the method of Higgins and Anderson (19) through a midabdominal incision. Experimental animals were allowed to recover for 2-4 h on a 37°C warm plate. All surgeries were performed between 1,000 and 1,400 h to control for diurnal variation.

Histochemical analysis and 5-bromo-2-deoxyuridine incorporation. Animals were injected with 100 mg/kg ip 5-bromo-2-deoxyuridine (BrdU; Sigma, St. Louis, MO) 1 h before death. Livers were weighed, fixed in formalin, embedded in paraffin, and sectioned at 6 µm. A section of small intestine, a rapidly proliferating tissue, was collected as a positive control. Unstained slides were deparaffinized in xylene and hydrated in graded concentrations of alcohol and hydrolyzed with 4 N HCL. Sections were further digested with pepsin before incubation with anti-BrdU antibody (DAKO, Carpenteria, CA) diluted 1:200 in 1.0% BSA/PBS. Immunostaining was detected using secondary reagent and diaminobenzidine as recommended by the manufacturer (DAKO). Tissues were counterstained in hematoxylin, followed by dehydration and mounting. Parenchymal cells undergoing DNA synthesis were quantitated by determining the percentage of BrdU-positive nuclei in 10 random high-power fields per slide. BrdU-positive nonparechymal cells were excluded for calculations.

Acyl-CoA oxidase activity. Activity of the peroxisomal enzyme acyl-CoA oxidase, an accepted indicator of peroxisome induction (21), was measured from the amount of formaldehyde formed by the peroxidation of methanol by hydrogen peroxide, a product of peroxisomal beta -oxidation. Liver samples (~100 µg) were homogenized in 10 volumes of 0.25 M sucrose buffer. A reaction mixture containing (in mg) 13.9 palmitate 3.54 CoA, 68.9 ATP, 20.4 MgCl2, 66.7 NAD+, 45 fatty acid free BSA, 36.8 semicarbazide, 370 Tris base, 307 Tris · HCl, and 201 niacinamide, and 200 µl methanol, and 5 µl Triton X-100 (per 50 ml of buffer; pH 8.3) was warmed to 37°C and 1.4 ml was mixed with 200 µl of liver homogenate. The reaction was terminated after 10 min with 40% TCA. The solution was centrifuged to pellet protein, and 1 ml of supernatant was added to 400 µl of Nash reagent to measure formaldehyde (34). After the reaction was incubated for 60 min at 37°C, the absorbance was read at 405 nm (epsilon  = 6.58). Protein concentration was determined by the method of Bradford (8).

Electrophoretic mobility shift assay. For studies in whole liver, nuclear extracts were isolated as described by Dignam et al. (12) with minor modifications (43). The consensus DNA binding oligo for PPARalpha (Santa Cruz Biotechnology) was end labeled with [32P-gamma ]ATP using T4 kinase. Binding conditions for PPARalpha were characterized, and EMSA was performed as described elsewhere (4, 49). Briefly, nuclear extracts (20 µg) from liver tissue were preincubated with 1 µg poly(dI-dC), 20 µg BSA (Pharmacia Biotech, Piscataway, NJ) and 2 µl of a 32P-labeled DNA probe (10,000 counts · min-1 · µl-1, Cerenkov) containing 1 ng of double-stranded oligonucleotide in a total volume of 20 µl. Mixtures were incubated 20 min on ice and resolved on 5% polyacrylamide (29:1 cross-linking) and 0.4× Tris · HCl/boric acid/EDTA gels. After electrophoresis, gels were dried and exposed to X-OMAT LS Kodak film. For supershift and competition assay, anti-PPARalpha antibody (4 µg) or unlabeled oligonucleotide was added to the nuclear extract 15 min before incubation with the labeled probe.

Transcription factor array. Biotin-labeled DNA binding oligonucleotides (TranSignal probe mix; Panomics) were incubated with 10 µg of nuclear extract for 1 h to allow the formation of protein/DNA (or transcription factor/DNA) complexes. The protein/DNA complexes were then separated from the free probes by 2% agorase gel electrophoresis. Probes were then extracted from the gel, and ethanol was precipitated, resuspended, and hybridized to the TranSignal Array membrane overnight at 42°C. Membranes were incubated with horseradish peroxidase-labeled streptavidin (DAKO). Detection of signals was obtained using enhanced chemiluminescence imaging system.

RNase protection assay. Total RNA was isolated from liver tissue using RNA STAT 60 (Tel-Test, Friendswood, TX). RNase protection assays were performed using the RiboQuant multiprobe assay system (Becton Dickenson Pharmingen). Briefly, 32P-labeled RNA probes were transcribed with T7 polymerase using the multiprobe template set mCK-3b. RNA (20 µg) was hybridized with 4 × 105 counts/min of probe overnight at 56°C. Samples were then digested with RNase followed by proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation. Samples were resolved on a 5% acrylamide-bisacrylamide (19:1) urea gel. After drying, the gel was visualized by autoradiography.

Cytokine ELISA. Whole liver from wild-type and PPARalpha -/- mice was homogenized in buffer containing 25 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.1% NP-40 and a cocktail of protease and phosphatase inhibitors. Extracts were used for ELISA as described by the manufacturer's recommendations.

RT-PCR. cDNA was synthesized from 1.0 µg of total RNA from each sample in a 20-µl final volume of reaction buffer containing (in mM) 25 Tris · HCl (pH 8.3), 37.5 KCl, 10 dithiothereitol, 1.5 MgCl2, 10 of each 2-deoxynucleotide 5'-triphosphate (Perkin Elmer Cetus, Norwalk, CT), and 0.5 mg random hexamer primer (GIBCO-BRL). Samples were incubated for 45 min at 42°C, and the reaction was terminated by denaturing the enzyme at 95°C. The reaction mixture was diluted with distilled water to a final volume of 50 µl. Aliquots (5 µl) of synthesized cDNA were added to 45 µl of PCR mix containing 5 µl of 10 × PCR buffer, 1 µl of each deoxynucleotide (1 mM each), 0.5 µl of sense and antisense primers (0.15 mM), and 0.25 µl (1.25 U) of DNA polymerase (Boehringer-Mannheim). Primers used in these experiments contained the following sequences: FPPS: 5'-AAAATTGGCACTGACATCCAGG-3' (sense), 5'-GGGTGCTGCGTACTGTTCAATG-3' (antisense); HMG-CoA synthase: 5'-TGCCCTGGTAGTTGCAG-3' (sense), 5'-GCCTCTTTCTGCCACT-3' (antisense); glyceraldehyde-3-phosphate dehydrogenase (G3PDH): 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (sense), 5'-CATGTGGGCCATGAGGTCCACCAC-3' (antisense). The size of amplified PCR products was 236 bp for FPPS, 291 bp for HMG-CoA synthase, and 983 bp for G3PDH.

The reaction mixture was covered with mineral oil, and amplification of G3PDH was initiated by 1 min of denaturation at 94°C for 1 cycle, followed by multiple cycles (20-35 cycles) at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min using a GeneAmp PCR system 9800 DNA Thermal Cycler (Perkin Elmer Cetus). After the last cycle of amplification, samples were incubated for 7 min at 72°C. Conditions for the amplification of FPPS and HMG-CoA synthase were identical except 60°C was used for the annealing temperature for FPPS and 55°C for HMG-CoA synthase. The amplified PCR products were subjected to electrophoresis at 75 volts through 2% agarose/ethidium bromide gel for 1 h.

Preparation of mouse liver cytosolic and membrane extracts. Livers obtained from mice were homogenized in a buffer containing 10 mM Tris · HCl (pH 7.4), 1 mM EDTA, and 0.25 M sucrose and centrifuged at 10,000 g for 20 min at 4°C. The supernatant was then centrifuged at 100,000 g for 1 h at 4°C to prepare cleared cytosol. The resulting pellet was solubilized with 2% (vol/vol) Triton X-100 for 1 h at 4°C and then centrifuged at 100,000 g for 1 h at 4°C and was used as the membrane extract.

Western blot analysis. Nuclear extracts were separated by SDS-PAGE and transferred to immobilon-P membranes. For cdk2/4 expression, membranes were incubated overnight with anti-cdk2 (1:1,000 dilution, Santa Cruz Biotechnology) or anti-cdk4 (1:1,000 dilution; Santa Cruz Biotechnology) antibodies, followed by 2-h incubation with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:5,000 dilution; Amersham Pharmacia Biotech). Immunoblots were visualized by enhanced chemiluminescence autoradiography. For retinoblastoma (Rb) phosphorylation as a marker for cdk4/cyclin D1 activity, nuclear extracts were separated on a 10% SDS-PAGE gel. Immunoblots were performed using anti-phospho-specific Rb (RbT181; 1:500; BioSource, Camarillo, CA).

For Ras and RhoA localization, membrane, and cytosolic fractions prepared as described above were separated by 16% SDS-PAGE, transferred to immobilon-P membranes, and immunoblotted using antibodies against Ras (1:1,000 dilution) or RhoA (1:1,000 dilution). Gels were subsequently stained with Coomassie blue to control for equal loading.

Immunoprecipitation and in vitro kinase reaction. Samples (300 µg protein) in a final volume of 550 µl were incubated in the presence of 1% (vol/vol) Triton X-100 and 0.5% (wt/vol) sodium deoxycholate at 4°C for 1 h. After centrifugation at 10,000 g for 2 min, the supernatant was treated for 30 min with 1 µg of nonimmune IgG. Twenty microliters of a 50% slurry of protein A/G-agarose beads were added, and after 30 min of incubation, the solution was centrifuged at 10,000 g for 2 min. Supernatants were incubated for 2 h with 20 µl of anti-cyclin E antibody at 4°C, after which 50 µl of a 50% slurry of protein A/G-agarose beads were added, and the solution was incubated for a further 1 h. After centrifugation at 10,000 g for 2 min, the pellet was washed three times with 1 ml of wash buffer (50 mM HEPES, pH 7.4, containing 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 100 mM NaF, and 2 mM sodium orthovanadate).

Kinases from immunoprecipitated-associated complexes were then assayed by the transfer of phosphate from [-32P]ATP to the substrates histone H1 in a reaction buffer consisting of (in mM) 50 Tris (pH 7.4), 10 MgCl2, and 1 DTT, and 144 µM ATP (40 µCi of [-32P]ATP). Reactions were performed at 37°C for 30 min and stopped by the addition of Laemlli sample buffer. Samples were boiled for 5 min at 95°C, and the histone H1 proteins were separated on a 12% SDS-PAGE gel.

Expression of GST fusion proteins and pull-down assay. pGEX plasmids, including GST-RBD (binding substrate for activated GTP-bound Ras), were a kind gift from Dr. Adrienne Cox, University of North Carolina. The expression of the GST-RBD (Ras binding domain of Raf-1) fusion protein was performed as described previously (39). Briefly, 10 ml of an overnight culture of bacteria transformed with expression plasmid was inoculated into 100 ml of LB medium containing 50 µg of ampicillin/ml. The culture was placed on a shaking incubator at 37°C until an absorbance of 1 at 600 nm was reached. Production of recombinant protein was induced with 1 mM (final concentration) isopropylthiol-d-galactoside (GIBCO-BRL), and the culture was incubated for a further 3-4 h at room temperature. Cells were harvested by centrifugation and resuspended in 5 ml of 1% Triton X-100-PBS and 1 mM PMSF. The suspension was incubated on ice for 30 min before cells were lysed by sonication. Bacterial lysate was centrifuged at 12,000 g for 20 min at 4°C to remove the insoluble fraction. The GST-RBD was purified using glutathione-beads (Amersham Pharmacia Biotech). Purified GST-RBD-glutathione complex was incubated overnight with 500 µg of whole liver extract prepared as described above. The resulting precipitate was washed and resuspended in 2× Laemlli sample buffer and separated by 16% SDS-PAGE. Immunoblot was performed using anti-pan Ras antibody (1:1,000; Chemicon).


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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PPARalpha is involved in liver regeneration after PH. Rodent hepatocyte proliferation induced by peroxisome proliferators has been shown to require both TNF-alpha and PPARalpha (6, 7). The PH model is an established in vivo model of synchronized hepatocyte proliferation that requires TNF-alpha and other mitogenic cytokines (1, 46). Thus it was hypothesized that liver regeneration and hepatocyte proliferation after PH would also require PPARalpha . To test this hypothesis, two-thirds PH was performed on wild-type and PPARalpha -/- mice. DNA synthesis was determined in livers from wild-type and PPARalpha -/- mice by measuring BrdU incorporation at several time points between 0 and 72 h after resection (Fig. 1). Since it is likely that hepatocytes and nonparenchymal cells, such as bile duct epithelia, are undergoing mitosis after PH, only hepatocytes were included in the determination of cell proliferation. Wild-type mice had a significant increase in BrdU incorporation within 24 h after PH, which peaked around 42 h (Fig. 1A), consistent with other reports (47). In contrast, DNA synthesis was significantly blunted by 60-65% in PPARalpha -/- mice at 42 and 48 h after PH (P < 0.05, repeated measures ANOVA) (Fig. 1B). However, 72 h after PH, the rate of DNA synthesis was not significantly different between wild-type and PPARalpha -/- mice, confirming a previous report (40). To test whether PPARalpha was involved in production of TNF-alpha and other mitogenic cytokines after PH, RNase protection assays were performed on mRNA isolated from wild-type and PPARalpha -/- mice from 0 to 72 h after resection (Fig. 2). TNF-alpha and IL-6 mRNA levels peaked between 12 and 24 h after PH and were not different between wild-type and PPARalpha -/- mice.


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Fig. 1.   Hepatocyte proliferation after partial hepatectomy (PH). A: representative immunohistochemical staining of 5-bromo-2-deoxyuridine (BrdU) incorporation 42 h after PH in control [wild type, peroxisome proliferator-activated receptor-alpha (PPARalpha ) +/+] and PPARalpha knockout mice (PPARalpha -/-) (magnification, ×40). B: wild-type and PPARalpha -/- mice underwent PH and were killed at 0, 12, 24, 42, 48, and 72 h after surgery. BrdU incorporation was measured as described in the MATERIALS AND METHODS. Data are represented as means ± SE of three individual experiments. (*P < 0.05 by repeated measures ANOVA).



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Fig. 2.   Mitogenic cytokines after PH. Wild-type and PPARalpha -/- mice underwent PH and were killed at 0, 12, 24, 42, 48, and 72 h after surgery. RNA was isolated from liver and expression of mitogenic cytokines was evaluated by RNase protection assay as described in MATERIALS AND METHODS. Representative data of three individual experiments are shown.

ELISA for TNF-alpha was performed using liver homogenates from both wild-type and PPARalpha -/- mice. At 24 h after PH, TNF-alpha levels peaked at 4,573 ± 214 pg/mg protein, compared with 111 ± 12 pg/ml in untreated mice. In PPARalpha -/- mice, TNF-alpha levels rose to 3,528 ± 441 pg/ml, a slightly lower value than in wild-type mice but not significantly different. These data support the hypothesis that inhibition of liver regeneration in PPARalpha -/- mice was not due to an effect on mitogen production.

To verify whether deletion of PPARalpha influenced the acute stress response after PH, the activation multiple transcription factors were evaluated by transcription factor array, which allows simultaneous determinations of multiple transcription factors within the same sample. Nuclear extracts from untreated mice were used as a control, and no significant transcription factor binding was observed (data not shown) with the exception of GATA, which is highly active in fetal liver during erythogenesis, and sterol responsive element. In nuclear extract from wild-type mice 24 h after PH, a significant increase in stress response transcription factor binding was observed (Fig. 3). Most notably, activator protein-1, NFkappa B, cyclic AMP response element-binding protein-alpha , (CREBalpha ), CCAAT/enhancer binding protein, PPARgamma , and others were identified. Importantly, deletion of PPARalpha did not have an observable effect on the panel of transcription factors activated after PH. It is also important to note that quantitative assessment of transcription factor activation cannot be made by this approach. However, these data are consistent with the observation that cytokine production is not different between wild-type and PPARalpha -/- mice.


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Fig. 3.   Transcription factor activation after PH. A: nuclear extracts were isolated from wild-type and PPARalpha -/- mice 24 h after PH was used to evaluate the activation of transcription factors involved in acute stress response using transcription factor arrays as described in the MATERIALS AND METHODS. Control nuclear extracts from untreated mice are not shown. B: schematic diagram of the transcription factor array. The genes are spotted in duplicate in two rows (top, undiluted; bottom, 1:10 dilution). Columns along the side and bottom of the membrane are biotinylated DNA for normalization. Shaded genes indicate change after PH.

PPARalpha is necessary for induction of key enzymes involved in isoprenoid synthesis. PPARalpha is a transcription factor activated by a number of endogenous ligands including fatty acids leading to upregulation of enzymes involved in fat metabolism as well as sterol and isoprenoid synthesis (16, 22, 23). To first test the hypothesis that PPARalpha is activated after PH, an electrophoretic mobility shift assay was performed to evaluate PPARalpha DNA binding activity after PH. With the use of nuclear extracts from wild-type mice, a significant increase in PPARalpha DNA binding activity was observed 24 and 48 h after PH (Fig. 4A). As expected, this increase was not observed in nuclear extracts from PPARalpha -/- mice. To validate the specificity of PPARalpha DNA binding, nuclear extracts from livers after PH were compared with nuclear extracts isolated from livers of mice treated with WY 14643, classical agonist of PPARalpha (Fig. 4B). Twenty-four hours after 100 mg/kg WY 14643 treatment, a significant increase in PPARalpha DNA binding was observed compared with vehicle-treated mice. The PPARalpha DNA binding from animals after PH, although slightly less intense than after WY 14643 exposure, was observed. Competition assay using excess unlabeled DNA oligo blunted PPARalpha DNA binding and incubation with anti-PPARalpha antibody caused a shift in the mobility, confirming the translocation and activation of PPARalpha after PH in mice. Importantly, a time-dependent increase in the activity of the classical PPARalpha -regulated gene acyl-CoA oxidase was observed after PH; acyl-CoA oxidase activity was significantly blunted in PPARalpha -/- mice (Fig. 5A). These data are consistent with the activation of PPARalpha after PH and support a potential role for PPARalpha in liver regeneration.


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Fig. 4.   Activation PPARalpha after PH. A: wild-type and PPARalpha -/- mice underwent PH and nuclear extracts were isolated from whole liver. Samples were taken at 0, 24 and 48 h after surgery. PPARalpha activity was determined by electrophoretic mobility shift assay using a 32P-labeled specific DNA binding consensus sequence oligonucleotide as described in MATERIALS AND METHODS. B: nuclear extracts from untreated wild-type mice (control), mice treated with PPARalpha agonist WY 14643 (WY), mice that underwent sham operation (sham) or mice 48 h after PH were evaluated by EMSA using 32P-labeled specific DNA binding consensus sequence oligonucleotide as described in MATERIALS AND METHODS. For unlabeled probe competition and anti-PPARalpha antibody (Ab) supershift assays, nuclear extract from mice 48 h after PH were used. Data are representative of three or more experiments.



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Fig. 5.   Induction of PPARalpha -regulated genes. A: activity of acyl-CoA oxidase (ACO), the classical PPARalpha -regulated gene, was measured biochemically in wild-type and PPARalpha -/- mice at 0, 24, 48, and 72 h after PH. Data are means ± SE of three or more individual experiments. Wild-type and PPARalpha -/- mice underwent PH, and mRNA was isolated from liver at 0, 24, 48, and 72 h after surgery. Expression of farnesyl pyrophosphate synthase (FPPS) (B), 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (C), and the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (D) was evaluated by RT-PCR as described in MATERIALS AND METHODS. Representative data are shown.

To test the hypothesis that PPARalpha -responsive genes may be involved in cell proliferation after PH, mRNA for FPPS and HMG-CoA synthase were evaluated by RT-PCR (Fig. 5B). Both FPPS and HMG-CoA synthase were not significantly expressed in either wild-type or PPARalpha -/- mice before PH. However, after PH, a sustained increase in FPPS at 24-72 h was observed in wild-type animals, whereas only a modest increase at 24 h alone was observed in PPARalpha -/- mice. Similarly, HMG-CoA synthase was significantly increased at 24 and 48 h after PH in wild-type animals but not in PPARalpha -/- mice under these conditions.

PPARalpha is necessary for activation and membrane localization of Ras after PH. Initiation of G1/S phase transition after PH is regulated in part by small GTPases Ras and RhoA through their activation of cdk (32). The hypothesis is that PPARalpha is involved in the regulation of isoprenoid synthesis and therefore may influence prenylation and function of small GTPases, such as Ras and RhoA. Specifically, it is hypothesized that Ras and RhoA are concentrated in the cytosol in livers from PPARalpha -/- mice rather than in the membranes in which they are normally localized. To test this hypothesis, membrane and cytosolic extracts from livers of both wild-type and PPARalpha -/- mice were immunoblotted for Ras and RhoA at several time points after PH (Fig. 6A). Prior to PH, the ratio of membrane-bound to cytosolic Ras was nearly equal in wild-type mice, whereas Ras was largely concentrated in the cytosol in PPARalpha -/- mice. After PH, the relative levels of Ras in the membrane vs. cytosol were not significantly different in wild-type mice for <= 48 h. However, the amount of Ras in membrane extracts of PPARalpha -/- livers was decreased significantly compared with the amount of Ras in the cytosol after PH. Relative changes of RhoA in the membrane compared with cytosol were similar to Ras. The amount of membrane-bound RhoA was higher than the levels of cytosolic RhoA before PH and was not significantly different for <= 48 h after PH in wild-type animals. In contrast, in PPARalpha -/- mice, membrane-bound RhoA was significantly lower than RhoA in the cytosol initially and was barely detectable at any time point studied after PH.


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Fig. 6.   Activation and subcellular localization of Ras and RhoA. A: membrane and cytosolic fractions were isolated by differential centrifugation and localization of Ras and RhoA was evaluated by Western blot as described in MATERIALS AND METHODS at 0, 12, 24, and 48 h after PH. B: Ras activity was evaluated by glutathione-S-transferase Ras binding domain of Raf-1 (GST-RBD) precipitation of GTP-bound Ras in whole liver lysates from wild-type and PPARalpha -/- mice at 0, 12, 24, and 48 h after PH. Data are representative of three or more experiments.

Experiments were then performed to evaluate the levels of activated (i.e., GTP-bound) Ras after PH (Fig. 6B). With the use of a specific substrate for active GTP-bound Ras, GST-RBD fusion protein-activated Ras was precipitated from whole liver extracts from both wild-type and PPARalpha -/- mice 0-72 h after PH. In wild-type mice, the levels of GTP-bound Ras were significantly elevated nearly 10- to 12-fold at 12-24 h after PH. In contrast, GTP-bound Ras was elevated only three- to fourfold at 12 h in PPARalpha -/- mice. These data are indeed consistent with the findings that small GTPases Ras and RhoA are improperly localized in PPARalpha -/- mice, supporting the hypothesis that PPARalpha is necessary for lipid modification and subsequent activation of Ras and RhoA after PH.

PPARalpha plays an essential role in cell cycle regulation. The hypothesis that PPARalpha is necessary for regulation of the cell cycle was tested by evaluating critical parameters of early G1/S phase transition of the cell cycle. Cdk2 and -4 complex with cyclin E and cyclin D1, respectively, to trigger G1 to S phase transition of the cell cycle (31). The expression of both cdk2 and cdk4 was evaluated by Western blot in nuclear extracts from wild-type and PPARalpha -/- animals 0-42 h after PH. Expression of cdk2 was minimal; however, it was increased dramatically 24 and 42 h after PH in wild-type mice (Fig. 7A). Importantly, this increase in cdk2 expression did not occur in livers from PPARalpha -/- mice at any time after PH. Similarly, an increase in cdk4 was also observed, which peaked 24 h after PH in wild-type mice; increases in cdk4 were not detectable in livers from PPARalpha -/- mice. Expression of cyclin E and D1 were also measured by Western blot and were not changed under these conditions (data not shown). These findings are consistent with a recent report that overexpression of constitutively active Ras enhances liver regeneration after PH without a significant increase in cyclin D1 expression (28).


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Fig. 7.   Cell cycle regulation after PH. Wild-type and PPARalpha -/- (KO) mice underwent PH. A: expression of cyclin-dependent kinase (cdk2) and ckd4 was evaluated by Western blot as described in MATERIALS AND METHODS at 0, 12, 24, and 42 h after PH. Representative Coomassie-stained gel is shown to demonstrate equal protein loading. B: activity of cdk2/cyclin E (histone H1 phosphorylation) was evaluated by kinase assay and activity of cdk4/cyclin D1 complexes (RbT181 phosphorylation) was evaluated as described in MATERIALS AND METHODS at 0, 12, 24, and 42 h after PH. C: levels of p21Cip and p27kip1 were evaluated by Western blot as described in MATERIALS AND METHODS at 0, 12, 24, and 42 h after surgery. D: image densitometry of p21 and p27 expression was performed and is expressed as percentage of control. Data are representative of three individual experiments.

Activity of cdk/cyclin complexes responsible for cell cycle progression through G1/S transition was determined (Fig. 7B). Cdk2/cyclin E activity was evaluated by an in vitro kinase assay using histone H1 as a substrate. Activity was increased significantly 24 h after PH in wild-type animals. In contrast, cdk2 activity was not increased under identical conditions in livers from PPARalpha -/- animals. Similarly, cdk4 associates with cyclin D to phosphorylate Rb protein. Thus cell extracts were immunoblotted with an antibody specific for cdk4-specific hyperphosphorylated Rb (T181). In wild-type animals, T181 levels were undetectable under basal conditions but were increased significantly 24 and 48 h after PH. In contrast, phosphorylation of Rb was barely detectable in PPARalpha -/- mice under similar conditions. These data are consistent with impaired activation of cdk2 and cdk4 after PH in PPARalpha -/- mice. Moreover, these data support the hypothesis that PPARalpha is involved in the regulation of early G1/S transition after PH. The expression data, together with cdk/cyclin complex activity, strongly support the hypothesis that deletion of PPARalpha limits the capacity of hepatic regeneration, most likely through blunting Ras-dependent activation of the cell cycle.

Cdk/cyclin complexes responsible for cell cycle progression through G1/S transition are inhibited by the cell cycle inhibitors p21waf and p27kip1, which are highly expressed in nondividing cells. Transient degradation of these cell-cycle inhibitors is dependent on activation of Ras and RhoA. Therefore, it was hypothesized that p21 and p27 expression would be downregulated in wild-type but not in PPARalpha -/- animals. In extracts in both wild-type and PPARalpha -/- mice before PH, p21 levels are relatively high (Fig. 7C). However, p21 decreased rapidly from basal levels in wild-type mice as early as 12 h and remained suppressed for <= 48 h after PH. In contrast, p21 levels in PPARalpha -/- mice remained elevated at all time points studied, consistent with the findings that DNA synthesis was repressed in these mice. Similar to p21 expression, p27 levels were high under normal conditions in both wild-type and PPARalpha -/- mice. After PH in wild-type mice, p27 was rapidly degraded at 12 h but returned to control levels at 24 and 48 h. However, in PPARalpha -/- mice, the transient decrease in p27 levels at 12 h was not observed. Image densitometric analysis clearly demonstrates these changes in p21 and p27 expression (Fig. 7D). The expression of p21 was rapidly decreased to nearly 70% of control levels for <= 48 h after PH in wild-type mice yet remained unchanged in PPARalpha -/- mice. Similarly, p27 expression was transiently decreased by ~75% in wild-type mice at 12 h after PH but unchanged in PPARalpha -/- mice at all time points studied.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of PPARalpha in isoprenoid synthesis. It is well established that numerous genes encoding peroxisomal, mitochondrial, and microsomal enzymes contain functional PPARalpha -responsive elements in their promoter regions (22). PPARalpha -responsive genes are largely involved in lipid metabolism and beta -oxidation of fatty acids but also include enzymes involved in isoprenoid synthesis, such as HMG-CoA synthase and the terminal enzyme FPPS (5, 44). Although it is clear the PPARalpha participates in regulation, these genes are also controlled by a variety of other transcription factors as well. For example, there are PPAR, retinoid X receptor, Sp1, and CREB cis-element binding sites within HMG-CoA synthase promoter, which may control basal and inducible gene expression (18). Indeed, it is demonstrated in Fig. 5 that PPARalpha activity required for maximal upregulation of both FPPS and HMG-CoA synthase in vivo after PH. Basal expression and the modest increase in gene expression in the PPARalpha -/- mice after PH may be due to activation of other regulatory elements. It is also well documented that inhibition of HMG-CoA reductase with statins blunts G1/S phase progression in response to a number of growth stimuli in cultured cells (17). Since HMG-CoA reductase and synthase are in a linear pathway for sterol and isoprenoid synthesis, these observations are consistent with the hypothesis that these synthetic pathways are central to normal cell cycle and proliferative function. As mentioned above, inhibition of HMG-CoA reductase blunts cell proliferation in vitro and depletes lipid-modified Ras and RhoA from membranes (14, 24). Ras and RhoA have each been shown to play a pivotal role in early progression of the cell cycle (27, 35). Furthermore, inhibition of cell growth and proliferation by farnesyl transferase inhibitors is dependent on the inhibition of prenylation and membrane-association of small GTPases (11). Thus it was hypothesized that activation of PPARalpha plays a key role in the synthesis of farnesyl and geranylgeranyl pyrophosphate, which are required for the association of Ras and RhoA to membranes. Auer et al. (2) reported that mitogen-induced cell proliferation in hepatocytes required activation of Ras. It was shown here that membrane association of both Ras and RhoA is diminished in PPARalpha -/- mice compared with wild-type mice under normal conditions as well as at several time points after PH (Fig. 6). Importantly, the impairment in Ras membrane association is reflected as a decrease in Ras activity after PH (Fig. 1B). These data indeed support the hypothesis that PPARalpha is required for lipid-modification and membrane association of Ras and RhoA. These data are consistent with other reports (24) showing that inhibition of HMG-CoA reductase by mevinolin reduces the level of membrane-bound Ras and Ras-mediated signal transduction in cell culture. However, this is the first report linking the PPARalpha to small G protein function in vivo.

One critical question still unresolved is the mechanism involved in PPARalpha activation after PH. These experiments were done in the absence of any classical exogenous activator of PPARalpha , such as clofibrate or WY 14643. These data do, however, suggest that an endogenous ligand for PPARalpha exists and it may play an important yet undetermined role in the regulation of isoprenoid synthesis.

Regulation of cell proliferation by small GTPases. Mitogens, through activation of Ras and RhoA, stimulate the progression of hepatocytes from G1 into S phase by activating of both cdk4/cyclin D and cdk2/cyclin E activities (3). Recently, it was demonstrated that activation of RhoA or overexpression of geranylgeranylated RhoA facilitates cell cycle progression by stimulating p27Kip1 degradation (20, 35, 36). Cyclin/cdk complexes are inhibited by specific inhibitor proteins p21Cip and p27Kip1, which are highly expressed in most cells and serve as negative regulators of mitogen-induced, cell-cycle progression (9, 41). Previously, it was shown that activation of the Ras/Rho but not the Ras/Erk pathway is required for p27Kip degradation and G1 progression (45). Loss of p21 and p27 is difficult to accurately determine in vivo since their expression rapidly changes within 24 h of PH. It is demonstrated here that a very transient decline in p21 and p27 levels occurs after PH in wild-type mice but not in PPARalpha -/- mice (Fig. 7). Additionally, the increase in cdk2/cyclin E and cdk4/cyclin D activities in wild-type mice (Fig. 7) are correlated with the loss of p21 and p27. Ras and RhoA are required for full activation of cdk2/cyclin E and cdk4/cyclin D and degradation of p21 and p27. Very recently it was demonstrated that the overexpression of dominant-negative Ras using adenovirus blunted cell proliferation after PH in rats (28), clearly supporting a role for Ras in regulation hepatic regeneration. Since PPARalpha -/- mice have significantly less membrane-associated Ras and RhoA as well as lower levels of activated Ras after PH (Fig. 6), it is concluded that PPARalpha is necessary for maximal Ras and RhoA-mediated cell cycle progression.

Role of PPARalpha in cell proliferation. PPARalpha was recently shown to be required for hepatocyte proliferation and tumorigenesis due to a variety of lipophilic peroxisome proliferators, such as di(2-ethylhexyl) phthalate, and WY 14643 (33). It was also shown that peroxisome proliferator-induced cell proliferation was dependent on activation of Kupffer cells and production of mitogenic cytokines (42). Interestingly, PPARalpha does not seem to play a direct role in cytokine production (Figs. 2 and 3), consistent with the finding that Kupffer cells, which are the major source of TNF-alpha in the liver (10), do not express PPARalpha (38). Therefore, it is possible that there is a requirement for both Kupffer cell mitogen production and PPARalpha for peroxisome proliferator-induced hepatocyte proliferation. The two-thirds PH/liver regeneration model is an in vivo model in which progression of hepatocyte proliferation occurs in a relatively synchronous manner and is regulated by a number of proinflammatory, mitogenic cytokines and growth factors (13). Indeed, there is compelling evidence that TNF-alpha is necessary for liver regeneration (1). It has been shown that livers from mice deficient in TNF-alpha receptors do not regenerate after PH compared with wild-type mice (47). The data are consistent with other reports that TNF-alpha and other mitogenic cytokines are rapidly induced after PH (Figs. 2 and 3). Despite increases in the mitogenic signals, liver regeneration after PH was blunted in mice deficient in PPARalpha (Fig. 1). Loss of PPARalpha does not seem to impact liver size or hepatocyte cell number under normal conditions based on liver morphology in these studies and other reports (25).

It was recently reported (40) that the rate of DNA synthesis in PPARalpha -/- mice was not different from controls 72 h after PH and that DNA synthesis was actually higher in PPARalpha -/- mice than wild types at 96 h after PH. This is an interesting finding, suggesting that PPARalpha is not required for liver regeneration and cell proliferation but that deletion of PPARalpha delays the rate of liver regeneration. These data are indeed consistent with our findings, which show similar rates of cell proliferation in wild-type and PPARalpha -/- mice 72 h after PH. Many reports (1, 46) have demonstrated that DNA synthesis peaks at 42 h and the full hepatic restoration occurs within 72-96 h after PH in mice. This is the reason that earlier time points were the focus of this present study. By integrating our findings with previously published data, it can be reasonably concluded that deletion of PPARalpha delays the rate of liver regeneration after PH. In wild-type mice, the transient peak in cell proliferation (~40-50% of the cells) occurs within 42 h, but in PPARalpha -/- mice, the proliferative response is slower and less transient. This is also supported by long-term studies suggesting that PPARalpha -/- livers fully recover to their original mass after PH, most likely at a delayed rate (40). Studies here do not refute previous findings; rather, they address the hypothesis that PPARalpha is involved in the early burst of DNA synthesis and cell cycle progression (i.e., 0-48 h after PH). It is also hypothesized that this early phase is most dependent on the availability of small GTPases, such as Ras and RhoA, to maintain a robust proliferative response. Alternatively, the necessity of PPARalpha may be less obvious when the rate of cell proliferation is at or near basal levels.

The role of lipid metabolism and isoprenoid synthesis in liver regeneration is still poorly understood and has not been addressed clearly in vivo. More importantly, the association between PPARalpha -regulated lipid metabolism and the function of small GTPases has not been previously addressed. Activation of Ras and RhoA are necessary for cell cycle progression and maximal DNA synthesis, and prenylation is essential for their membrane-association and function. Importantly, these data are the first to demonstrate the importance of PPARalpha activation and induction of isoprenoid synthetic pathways (i.e., HMG-CoA synthase and FPPS) after PH, leading to the membrane-association and activation of Ras and RhoA. Thus these data support this novel hypothesis in vivo and provide mechanistic evidence for the role of PPARalpha -mediated Ras and RhoA membrane association in early DNA synthesis and cell proliferation in liver regeneration after PH.


    FOOTNOTES

dagger Deceased 14 July 2001.

Address for reprint requests and other correspondence: M. D. Wheeler, Univ. of North Carolina at Chapel Hill, CB# 7365, 3013 Thurston-Bowles Bldg, Chapel Hill, NC 27599 (E-mail: wheelmi{at}med.unc.edu).

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.

First published October 16, 2002;10.1152/ajpgi.00175.2002

Received 14 May 2002; accepted in final form 7 October 2002.


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