Miguel Á Fernández-Moreno

Francesco Bruni

Cristina Adán

Rosana Hernández Sierra

Paola Loguercio Polosa

Palmiro Cantatore

Rafael Garesse

Marina Roberti
email: marina.roberti@biologia.uniba.it

Prof. M Á Fernández-Moreno

Prof M Roberti

The Drosophila nuclear factor DREF positively regulates the expression of the mitochondrial transcription termination factor DmTTF

Keywords: DREF, DmTTF, mtRNApol, RNA interference, ChIP, embryogenesis Short title: DmTTF transcription regulation

The DNA Replication-related Element-binding Factor (DREF), which regulates transcription of a group of cell proliferation-related genes in Drosophila, controls also the expression of three genes involved in mitochondrial DNA (mtDNA) replication and maintenance. In this study, by in silico analysis, we have identified DNA Replication-related Elements (DRE) in the promoter region of a gene participating in mtDNA transcription, the Drosophila mitochondrial Transcription Termination Factor (DmTTF). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DRE elements control DmTTF transcription; moreover, gel-shift and ChIP assays demonstrated that the analysed DRE sites interact with DREF either in vitro and in vivo. Accordingly, DREF knockdown in S2 cells by RNA interference induced a considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the contrary, mitochondrial RNA polymerase (mtRNApol) lacks DRE elements in its promoter and is not regulated in vivo by DREF. In situ RNA hybridization studies showed that DmTTF was transcribed almost ubiquitously throughout all stages of Drosophila embryogenesis, whereas mtRNApol was efficiently transcribed from stages 11 th -12 th . Territories where transcription occurred mostly were gut and Malpighi tubes for DmTTF, and gut, mesoderm, pharyngeal muscle and Malpighi tubes for mtRNApol. The partial overlapping in the temporal and spatial mRNA expression patterns confirms that transcription of the two genes is differentially regulated during embryogenesis and suggests that DmTTF might play multiples roles in mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required.

INTRODUCTION

Mitochondria are semi-autonomous cytoplasmic organelles whose biogenesis requires the coordinate expression of nuclear and mitochondrial genomes. The coding capacity of animal mitochondrial DNA (mtDNA) is limited to 13 polypeptides of the oxidative phosphorylation system (OXPHOS), 22 tRNAs and 2 rRNAs; thus, the majority of mitochondrial proteins including respiratory complex polypeptides, metabolic enzymes and proteins involved in mtDNA replication and expression are nuclear encoded [START_REF] Garesse | Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes[END_REF][START_REF] Falkenberg | DNA replication and transcription in mammalian mitochondria[END_REF]. Many efforts have been made in order to clarify the mechanisms that regulate the expression of nuclear genes encoding mitochondrial proteins. In mammals two nuclear transcription factors, namely NRF-1 and NRF-2 (Nuclear Respiratory Factor 1 and 2), have been shown to regulate, in cooperation with co-activators of the PGC-1 family, the expression of many nuclear genes encoding proteins involved in mitochondrial biogenesis and function [START_REF] Scarpulla | Transcriptional paradigms in mammalian mitochondrial biogenesis and function[END_REF]. In Drosophila, erect wing, the NRF-1 orthologue that regulates myogenesis and neurogenesis, has never been demonstrated to coordinate mitochondrial protein gene expression [START_REF] Desimone | The Drosophila erect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein[END_REF][START_REF] Echevarría | Intergenomic nuclear-mitochondrial cross-talk in Drosophila melanogaster[END_REF]. Recently, in silico analysis of D. melanogaster genome has led to the identification of a single, ten nucleotide long sequence, named NRG (Nuclear Respiratory Gene) element, in more than 50% of genes with mitochondrial function including electron transport chain, oxidative metabolism and mitochondrial biogenesis [START_REF] Sardiello | Energy biogenesis: one key for coordinating two genomes[END_REF]. The DNA Replication-related Element Factor (DREF) is a Drosophila transcription regulatory protein consisting of an 80 kDa polypeptide forming an homodimer that specifically binds DRE sequences [START_REF] Hirose | Isolation and characterization of cDNA for DREF, a promoter-activating factor for Drosophila DNA replication-related genes[END_REF]. They constitute an eight nucleotide long palindromic consensus sequence that was initially shown to be required for the expression of genes involved in nuclear DNA replication and cell cycle control [START_REF] Hirose | Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase alpha and proliferating cell nuclear antigen[END_REF][START_REF] Yamaguchi | A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNA replication-related genes[END_REF][START_REF] Hyun | DREF is required for efficient growth and cell cycle progression in Drosophila imaginal discs[END_REF][START_REF] Matsukage | The DRE/DREF transcription regulatory system: a master key for cell proliferation[END_REF]. DREF may either function as a bona fide transcription factor, being its binding sites located up to more than 1 kb upstream of the transcription start point of the controlled genes [START_REF] Choi | Transcriptional regulation of the Drosophila caudal homeobox gene by DRE/DREF[END_REF][START_REF] Okudaira | Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway[END_REF][START_REF] Tsuchiya | Transcriptional regulation of the Drosophila rcf1 gene by the DRE-DREF pathway[END_REF], or may be part of the basal transcription machinery involved in recognizing core promoters [START_REF] Hochheimer | TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila[END_REF]. Interestingly, it has been demonstrated that DREF also controls the expression of genes, such as the mitochondrial single-stranded DNA-binding protein (mtSSB), the β subunit of the mitochondrial DNA polymerase (pol γ-β) and the mitochondrial transcription factor A (TFAM), whose products are required for mtDNA replication and maintenance [START_REF] Ruiz De Mena | Regulation of mitochondrial single-stranded DNA-binding protein gene expression links nuclear and mitochondrial DNA replication in Drosophila[END_REF][START_REF] Lefai | Differential regulation of the catalytic and accessory subunit genes of Drosophila mitochondrial DNA polymerase[END_REF][START_REF] Takata | Drosophila mitochondrial transcription factor A: characterization of its cDNA and expression pattern during development[END_REF], thus providing evidence for a coordinated regulation of nuclear and mitochondrial DNA replication. No information concerning the control by DRE/DREF system on proteins that are specifically involved in mtDNA transcription are available yet; therefore we wished to investigate on the possible dependence on DREF of the genes for the single subunit mitochondrial RNA polymerase (mtRNApol) and for the accessory factor DmTTF. DmTTF is a nuclear encoded 43 kDa protein, member of the mTERF protein family [START_REF] Roberti | DmTTF, a novel mitochondrial transcription termination factor that recognises two sequences of Drosophila melanogaster mitochondrial DNA[END_REF][START_REF] Linder | A family of putative transcription termination factors shared amongst metazoans and plants[END_REF]. It acts as a transcription termination factor in Drosophila mitochondria by arresting the progression of mtRNApol at its two DNA-binding sites, both of which are located at the 3' end of clusters of genes transcribed in convergent direction [START_REF] Roberti | In vitro transcription termination activity of the Drosophila mitochondrial DNA-binding protein DmTTF[END_REF][START_REF] Roberti | The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription[END_REF]. Moreover, it appears that DmTTF has a more broad effect on mitochondrial transcription since its depletion alters the level of those mitochondrial RNAs that map upstream of its binding sites, thus pointing towards a possible function also in transcription initiation [START_REF] Roberti | The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription[END_REF]. In this paper, by a combination of in vitro and in vivo experiments, we demonstrate that DREF binds two DRE sequences located about 50 nt upstream of the DmTTF transcription initiation site and that positively regulates the expression of DmTTF. On the contrary, no control by the DRE/DREF system was observed for the mtRNApol gene. In addition, by analyzing the temporal and spatial accumulation of both DmTTF and mtRNApol mRNAs during Drosophila embryogenesis, we found that the two mRNAs show common patterns of significant expression in gut and Malpighi tubes and distinct profiles in other regions.

MATERIALS and METHODS

Promoter constructs

Construction of p2000DDD and mutant derivatives. A 2076 bp DNA fragment upstream of the D. melanogaster DmTTF coding region (from -1964 to +112 considering +1 the transcription start point and being +113 the first nucleotide of the start codon) was obtained by PCR using total DNA from D. melanogaster derivative S2 cell line as template, and the forward primer dir(1339) (5´CCGGATCCCTTCAATTGTCACTTGTCATT3´) and the reverse primer rev(818) (5´GGCTCGAGCCCGAATTTGCTTAATTTACA3´). Primers include the recognition sequence (in italic) for BamHI and XhoI, respectively. The PCR product was digested with these enzymes and cloned into the corresponding sites of pXp2 vector, upstream of the luciferase reporter gene. The resulting construct (p2000DDD) was used to generate of a series of deletion and mutant clones. Mutagenesis of DRE element at position -42 (5´TAACGATA3´) on p2000DDD was achieved by three PCR steps and sub-cloning of the amplified fragment. In the first PCR p2000DDD (template) and primers dir(1339) and rev(820) (5´TCAAACAATTActaTTATCGGTTTTGAAAATAATGA3´) were used. In the second PCR the same template and primers dir(1839) (5´TCATTATTTCAAAACCGATAAtagTAATTGTTTGA 3´) and rev(818) were used. Primers rev(820) and dir(1839) are complementary and contain the DRE element at -42 (underlined) mutated (changes are in lower case). A third PCR was carried out using as template an equimolar mixture of the two obtained products and primers dir(1339) and rev(818); it yielded a 2076 bp fragment that is identical to the original contained in p2000DDD except for the mutated DRE element at position -42. The final PCR product was cloned into BamHI and XhoI sites of pXp2 to generate p2000DDd. Mutagenesis of DRE element at position -48 (5'AACCGATA3') was carried out as for DRE at -42, using the mutation containing primers rev(821) (5´TCAAACAATTATCGTTActaGTTTTGAAAATAATGA3´) for the first PCR and dir(1840) (5´TCATTATTTTCAAAACtagTAACGATA3´) for the second. Primer sequences show DRE at -48 underlined and mutation in lower case. The product was finally cloned to generate p2000DdD. The double mutant DRE element (-42/-48) containing construct was obtained in the same manner, using primers rev(822) (5´TCAAACAATTActaTTActaGTTTTGAAAATAATGA 3´) for the first PCR and dir(1841) (5´TCATTATTTTCAAAACtagTAAtagTAATTGTTTGA 3´) for the second. Primer sequences show DRE at -42 in italic and that at -48 underlined and mutations on DRE elements in lower case. The product was finally cloned in pXp2 to generate p2000Ddd. Construction of p1300DDD, p400DDD and mutant derivatives. Construct p1300DDD was made by PCR using p2000DDD as template and primers dir(1299) (5´CCAAGCTTAACTAGTTAACGCAATTAAGCAGC 3´; HindIII site in italic) and rev(818). The PCR product was digested with HindIII and XhoI and cloned into the corresponding sites of pXp2 vector. Construct p400DDD was made in the same manner, using as forward primer dir(1298) (5´CCAAGCTTTGTTCAAAATCCTACAAGTC3´; HindIII site in italic). Mutation of proximal DRE elements (-42/-48) on p400DDD was carried out using p2000Ddd as template, and primers dir (1299) and rev(818) yielding p400Ddd. To construct p400dDD (containing only the mutated form of DRE at -361) a PCR reaction was carried out using p2000DDD as template and primers dir(2875) (5´CCAAGCTTTGTTCAAAATCCTACAAGTCTTTGTTTAGtagTAATGTTCCG3´; HindIII site in italic; DRE site underlined; mutation in DRE in lower case) and rev(818). Finally, p400ddd (containing mutated DRE at -361, -48 and -42) was made by PCR using p2000Ddd as template and primers dir(2875) and rev(818). The product was digested with HindIII and XhoI and cloned into the corresponding sites of pXp2 vector. All constructs were confirmed by sequencing.

Transient transfection assay

Transient transfection assays in Drosophila S2 cells were performed as described in (17) using 5 µg of the luciferase gene containing plasmids. To measure luciferase activity, cells were harvested by centrifugation and washed with PBS. Cell lysis and luciferase measurements were carried out using the Luciferase Assay System (Promega) following manufacturer's recommendations. Promoter activities were calculated normalizing luciferase activities by the β-galactosidase activity from plasmid pAc5.1/V5-His/LacZ (Invitrogen), 1 µg of which was co-transfected. β-galactosidase activity was measured using the Beta-Glo Assay System (Promega) following manufacturer's instructions.

Gel mobility-shift assay

For the mobility-shift assay nuclear protein extract from S2 cells (prepared as reported in 17) was incubated with a double-stranded 36-mer oligonucleotide (from positions -61 to -26). The probe was 5' end-labelled with γ-32 P-ATP and T4 polynucleotide kinase and then purified using G-50 nick columns (Pharmacia). Binding reactions were carried out combining 10 µg of nuclear protein extract, 50000 cpm (corresponding to 30 fmoles) of DNA probe and 1 µg poly(dI-dC)•poly(dI-dC) (Pharmacia) in binding buffer (20% glycerol, 1 mM dithiothreitol, 20 mM HEPES pH 9.0, 5 mM MgCl 2 , 0.2 mM EDTA, 200 mM KCl). After an incubation of 30 min at 4 °C, the reaction products were electrophoresed onto a 6% polyacrylamide gel in 0.5X TBE. A 200-fold molar excess of nonradioactive wild type or DRE mutated oligonucleotides was used as homologous competitor. DRE mutations in cold competitors corresponded to the same nucleotides changed in constructs for luciferase assays. In order to produce a super-shifted band, specific anti-DREF serum (from L. Kaguni, Michigan State University) was added (1:200 dilution in PBS) after 15 min of binding reaction. Pre-immune serum was used at the same dilution as a negative control.

ChIP assays

Chromatin immunoprecipitation assays were carried out with DNA obtained from 0.45 g of D.melanogaster overnight embryos as previously described [START_REF] Nègre | Mapping the distribution of chromatin proteins by ChIP on chip[END_REF], with some minor modifications. Homogenized embryos were sonicated 5 times (10 seconds continuous pulses at 7 amplitude microns power) in a MSE Soniprep 150 sonifier with a microtip probe at 4°C, with 30 s cooling on ice between pulses, yielding DNA fragments mostly between 200 and 700 bp. Immunoprecipitation was made on chromatin extract without filtering through a centricon column. For immunoprecipitation, polyclonal antiserum anti-DREF from rabbit and anti-Eyegone from guinea pig and their respective preimmune serum were used. Chromatin extracts were immunoprecipitated twice, initially with the corresponding preimmune serum for 6 h (IgG control to remove non specific interactions) and then overnight at 4°C with polyclonal antiserum. Immunoprecipitated DNA was used for PCR amplification of proximal DRE sites using the following primers: Fw-42 (5´ TTGTGTATGAACCAGCACAC 3´) and Rev-42 (5´ AAACAGCTGATATGTTGCGG 3´) as forward and reverse primers, respectively, for amplifying a 225 bp fragment containing the -42/-48 DRE site; Fw-361 (5´ ATGTGGAGTAGTGCTTGTGA 3´) and Rev-361 (5´ CATTCTTAGACAGAGAGTCC 3´) as those for amplifying a 175 bp fragment including the -361 DRE site. The PCR products (after 30 cycles of 94ºC/30 seconds; 58ºC/30 seconds; 72ºC/30 seconds) were resolved by gel electrophoresis and visualized by ethidium bromide staining.

RNA interference and western blotting assay

Templates for the production of dsRNAs were PCR-derived fragments carrying at both ends the T7 promoter sequence. Each primer contained a 5' end sequence corresponding to the T7 polymerase promoter (5´TAATACGACTCACTATAGGGA3'), followed by a gene-specific sequence as indicated: DREF (accession number NM_078805) forward-primer nt 720-736, reverse-primer nt 1321-1306; LacZ (pUC18 vector accession number L09136) forward-primer nt 2512-2528, reverseprimer nt 362-348. The PCR products were phenol extracted, ethanol precipitated and used as templates for the MEGAscript ® RNAi kit (Ambion) to produce dsRNAs according to the manufacturer's instructions. S2 cell were maintained in Schneider's Drosophila Medium (Gibco-Invitrogen) supplemented with 10% FBS (Gibco-Invitrogen), 50 units/ml penicillin and 50 mg/ml streptomycin, at 25° C. For dsRNA treatment, cells were diluted to a final concentration of 1.0x10 6 cells/ml in 10 ml of Schneider's Drosophila Medium (no supplemented) in a 75-cm 2 flask. dsRNAs (15 µg per 10 6 cells) were added directly to the medium and cells were incubated at 25° C for 1 hour. Then, 10 ml of complete medium were added to obtain a cell density of 0.5x10 6 cells/ml followed by an additional incubation at 25° C for 72 hours. dsRNA treated and untreated S2 cells were then harvested, centrifuged at 1000 g for 4 min at 4°C and washed twice in PBS. Pellets were resuspended in 1X Laemmli buffer and proteins were fractionated on 12% SDS polyacrylamide gel and electroblotted onto PVDF membrane (Immobilon-P, Millipore). Membranes were incubated with affinity-purified anti-DREF antibodies (from M. Yamaguchi, Kyoto Institute of Technology) and with anti-actin antibodies (Sigma), it followed an incubation with anti-rabbit IgG (Santa Cruz Biotechnology). Protein bands were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare).

Real-Time RT-PCR assay

Total cellular RNA was extracted from dsRNA treated and untreated S2 cells by the RNeasy Midi Kit (Qiagen); 500 ng were reverse-transcribed in a final volume of 25 µl using the Enhanced AMV Reverse Transcriptase kit (Sigma), according to the manufacturer's instructions. Real-Time PCR was performed using the Power SYBR ® Green PCR Master Mix (Applied Biosystems) and the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Primers were designed using Primer Express 2.0 software (Applied Biosystem) resulting sequence positions as follows: nt 583-602 (forward), nt 683-663 (reverse) for DmTTF (accession number AY196479); nt 2352-2371 (forward), nt 2452-2434 (reverse) for mtRNA pol (accession number NM_134721); nt 1407-1429 (forward), nt 1480-1462 (reverse) for cytoplasmic 28S rRNA (accession number M21017), which was used as endogenous control. Each reaction was run in triplicate and contained 1 µl of reverse transcription reaction (1 µl of a 1:200 dilution was used for 28S rRNA) along with 200 nM primers in a final reaction volume of 30 µl. Amplification conditions were: 95°C for 10 min, then 40 cycles of 95°C for 15 sec and 60°C for 1 min. To ensure that only a single product was amplified, the melting curve analysis was performed using the Dissociation Curves software (Applied Biosystem). All PCR products were run on a 2.5% agarose gel to confirm specificity. Analysis of data was performed as previously described [START_REF] Roberti | The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription[END_REF]. In situ analysis of mtRNApol and DmTTF expression on Drosophila embryos was performed incubating previously treated embryos with antisense riboprobes, using the corresponding sense riboprobes as negative controls. To prepare the mtRNApol riboprobe, a fragment of its cDNA (positions 2067-4277, according to FlyBase annotation symbol CG4644) was previously PCR amplified and cloned into the EcoRV site of pGEMT-easy vector (Promega). In the same manner, to prepare the DmTTF riboprobe, a fragment of its cDNA (positions 113-1346 according to FlyBase annotation symbol CG18124) was cloned into the EcoRV site of pBluescript-KS + vector. Preparation of embryos, riboprobes and in situ experiments were carried out as in [START_REF] Fernández-Moreno | Drosophila melanogaster as model system to study mitochondrial biology. In Mitochondrial genomics and proteomics protocols[END_REF] with minor modifications: in point 2. With the aim to characterize the involvement of DREF in controlling transcription of DmTTF and mtRNApol genes in D.melanogaster, we searched for DRE consensus sequence TATCGATA in the 2 kb DNA region upstream and around DmTTF and mtRNApol transcription initiation site that, according to FlyBase (http://www.flybase.org/), are located 112 and 168 nt upstream of the translation start codon, respectively. In the case of mtRNApol promoter, no DREF binding motifs were identified. On the contrary, the analysis of DmTTF promoter allowed the identification of ten putative DRE elements scattered between position -42 and -1899, according to the transcription start point, which keep at least six out of the eight nucleotides of the DRE consensus sequence (see Fig. 2). Three of these elements are located within the proximal 400 bp region, specifically at -42, -48 and -361, and matches 7, 6 and 6 nucleotides out of the 8 forming DRE consensus, respectively (see Fig. 1A). The element in position -42 overlaps by two nucleotides with that in position -48. Comparison of the promoter proximal sequence in five Drosophila species (D. simulans, D. sechellia, D. melanogaster, D. yakuba and D. erecta) showed 100% conservation in the sequence of all the three DRE elements (Fig. 1A) as well as in the relative position of the two most proximal, with the position of the third DRE sequence being very similar (Fig. 1B).

In situ hybridization

Role of DRE sites in DmTTF promoter activity

Transient transfection experiments in S2 cells were carried out in order to analyze the role of DRE sequences in DmTTF promoter function. For this purpose, different deleted and/or point mutated versions of DmTTF upstream sequences were linked to a luciferase reporter gene in the pXp2 vector. As shown in Fig. 2, the luciferase activity promoted by a fragment of 2076 bp (p2000DDD), spanning positions -1964 to +112 according to the transcription start point, was fixed as 100%. Deletion mutant analysis showed that the removal up to 1577 bp from the distal end of the promoter sequence did not alter significantly transcription efficiency, since p1300DDD and p400DDD mutants show approximately the same luciferase activity than p2000DDD. The 400 bp promoter proximal region, that retains almost 90% of the total promoter activity, contains the three conserved DRE sequences located at positions -42, -48 and -361. Different combinations of these proximal DRE sites containing changes in the tri-nucleotide core of the consensus (positions 4-6) were generated in p2000DDD and p400DDD and used in transient transfection assays. Comparison of luciferase activity obtained with the wild-type and mutated versions of p2000 (Fig. 2) indicates that mutation of DRE at either -42 (p2000DDd) or -48 (p2000DdD) site causes a significant reduction of the promoter activity (about 30%); the reduction reaches about 50% when both sites are mutated (p2000Ddd). The relevance of the proximal double -42/-48 DRE sequences is confirmed by the double mutation in p400Ddd which causes an even higher reduction, approximately 60%, of the reporter gene expression than that in p2000Ddd. Finally, mutation of the distal -361 DRE sequence (p400dDD) causes 40% of reduction in promoter efficiency, showing that this third DRE site also participates in DmTTF transcription in S2 cells. In conclusion, this analysis indicates that (i) DRE elements are involved in DmTTF transcription, (ii) there are three functional DRE elements in DmTTF promoter and (iii) mutations on proximal DRE sites abolish 50%-60% of the promoter activity.

Binding of DREF to DmTTF promoter

To demonstrate that DRE sequences in DmTTF promoter are recognized by DREF, gel mobilityshift and ChIP assays were carried out. Gel mobility-shift experiments were performed using S2 cell nuclear extracts and a 32 P-labelled 36mer double stranded oligonucleotide containing the -42 and -48 DRE sites. The electrophoretic analysis (Fig. 3) shows the formation of two protein-DNA complexes (lane 2) that were completely abolished by an excess of homologous cold competitor (lane 3). Moreover, we used as cold competitor oligonucleotides containing a mutated version of each or both DRE sites. As shown in Fig. 3, the oligonucleotide containing mutation at -42 site still keeps a high competition capacity (lane 5), whereas the oligonucleotide mutated at -48 site shows a reduced capacity to compete (lane 4). As expected, the double mutant oligonucleotide is almost completely unable to compete (lane 6). These results suggest that the protein contacting the DNA probe is DREF; moreover, they indicate that the -48 element has a higher affinity for the protein than that at -42. To produce a clear evidence of the involvement of DREF in binding to DmTTF promoter, we performed the binding reaction in the presence of anti-DREF serum: as shown in Fig. 3 (lanes 7-9) we observed a clear super-shift of the retarded bands; super-shift was observed neither in the presence of an excess of homologous cold competitor nor in the sample containing the pre-immune serum. The obtained results clearly demonstrate that the two DNA-protein complexes contain DREF. To analyse the in vivo binding of DREF to DmTTF promoter we performed ChIP experiments on D.melanogaster whole embryos. Chromatin was immunoprecipitated with anti-DREF antibodies and the bound DNA was identified by PCR amplification with primers specific for either the -42/-48 and -361 DRE elements (Fig. 4). As a negative control, PCR amplifications were also made on chromatin immunoprecipitated with anti-Eyegone serum [START_REF] Yao | Eyg and Ey Pax proteins act by distinct transcriptional mechanisms in Drosophila development[END_REF]. As shown in Fig. 4, PCR amplified a clear, single and intense band for -42/-48 and -361 DRE elements when template was the chromatin immunoprecipitated by anti-DREF, but no amplification, or a very faint band for -42/-48 region, was observed when the template was the chromatin immunoprecipitated by preimmune and anti-Eyegone serum. These results demonstrated that DREF binds indeed in vivo to proximal DRE elements on DmTTF promoter region. ChIP experiments on SL2 cells also produced similar results to those made on embryos (data not shown).

Effect of DREF knock-down on DmTTF and mtRNApol expression in S2 cultured cells.

We finally investigated the effect of DREF knock-down on DmTTF endogenous gene transcription in Drosophila cultured cells. Moreover, since DREF is known to be involved in a complex network of regulatory pathways [START_REF] Matsukage | The DRE/DREF transcription regulatory system: a master key for cell proliferation[END_REF], and it could not be excluded that also mtRNApol expression could be indirectly regulated by the DRE/DREF system, we tested the effect of DREF depletion also on mtRNApol mRNA. To this purpose, we used RNAi procedure to obtain DREF depletion in S2 cells. After treating cells for 72 hours with a dsRNA encompassing 601 nt of DREF coding sequence, the level of DREF polypeptide was monitored by western blotting assay. As reported in Fig. 5A, a decrease of more than 90% of DREF level was obtained; the effect of DREF RNAi was specific as no effect was observed in cells treated with dsRNA containing the sequence of LacZ gene (mock control). Then, we measured the steady-state level of DmTTF and mtRNApol mRNAs using Real-Time RT-PCR. As shown in Fig. 5B, results obtained from five independent RNAi experiments indicate that in DREF-depleted S2 cells the level of DmTTF mRNA is about 55% of that in non-depleted cells. On the contrary, no effect of DREF depletion was observed on mtRNApol mRNA level. The decrease of DmTTF mRNA is consistent with the data obtained from the transient tranfection assay, and definitively ensures that DREF is a transcription factor controlling DmTTF gene expression. To study the spatial and temporal expression of DmTTF and mtRNApol during embryogenesis, we used whole-mount in situ RNA hybridization. Fig. 6A shows that DmTTF mRNA has a significant maternal deposit; until stage 12 th , the signal appears to be quite uniform except for ectoderm and polar cells, where there is no expression (only stage 6 th is shown; polar cells data are not shown). On stage 12 th , DmTTF mRNA level is increased in midgut (digestive tube) over a general background. From stage 13 th , gut and Malpighi tubes show a higher content of DmTTF transcript than the rest of the embryo. Fig. 6B shows that mtRNApol mRNA also has a significant maternal deposit that disappears before stage 4 th . Zigotic transcription starts approximately at stage 11 th , when hybridization signal appears exclusively in gut primordia. Approximately from stage 12 th a strong signal is present in gut, pharyngeal muscles and mesoderm and seems to be absent or undetectable in the rest of the embryo. Finally, from stage 13 th mtRNApol transcript is relatively highly expressed in gut, pharyngeal muscles, Malpighi tubes and somatic mesoderm.

DISCUSSION

DREF is a Drosophila key transcription factor which regulates the expression of a set of genes involved in nuclear DNA replication and cell cycle control [START_REF] Hirose | Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase alpha and proliferating cell nuclear antigen[END_REF][START_REF] Yamaguchi | A nucleotide sequence essential for the function of DRE, a common promoter element for Drosophila DNA replication-related genes[END_REF][START_REF] Hyun | DREF is required for efficient growth and cell cycle progression in Drosophila imaginal discs[END_REF][START_REF] Matsukage | The DRE/DREF transcription regulatory system: a master key for cell proliferation[END_REF]. Moreover, transcription regulation by DREF was demonstrated for the expression of protein factors directly involved in mtDNA replication and maintenance [START_REF] Ruiz De Mena | Regulation of mitochondrial single-stranded DNA-binding protein gene expression links nuclear and mitochondrial DNA replication in Drosophila[END_REF][START_REF] Lefai | Differential regulation of the catalytic and accessory subunit genes of Drosophila mitochondrial DNA polymerase[END_REF][START_REF] Takata | Drosophila mitochondrial transcription factor A: characterization of its cDNA and expression pattern during development[END_REF]. With the aim to provide a contribution to the study of mitochondrial biogenesis in D. melanogaster, we focused our attention on the putative regulation by DREF of two nuclear genes coding for mitochondrial proteins belonging to the mtDNA transcription apparatus: mtRNApol and the transcription termination factor DmTTF. We first analysed in silico the 5' flanking region of the DmTTF and mtRNApol genes searching for DRE elements. This allowed the identification of ten DRE sequences in the promoter region of DmTTF gene, the three most proximal of which are 100% conserved in the Drosophila species belonging to the melanogaster subgroup. No significant DRE elements were found in mtRNApol promoter. Afterwards, by both in vivo and in vitro experiments we clearly demonstrate that DRE elements are required for DmTTF promoter activity and that DREF positively regulates the expression of the mitochondrial transcription termination factor. Our study thus provides the first experimental evidence of a control exerted by DREF on a factor involved in mtDNA transcription. On the contrary, our data rule out the possibility that DREF may control, although indirectly, mtRNApol gene at least in S2 cells. Some of us found that DREF also stimulates the expression of another transcription factor, namely TFB2M, that is involved in transcription initiation (manuscript in preparation). Therefore our findings on mtDNA transcription machinery parallel what observed for mtDNA replication apparatus, that is, the accessory factors are controlled by DREF whereas the catalytic polypeptides, mtRNApol and pol γ-α [START_REF] Lefai | Differential regulation of the catalytic and accessory subunit genes of Drosophila mitochondrial DNA polymerase[END_REF], are not. The transcription termination factor DmTTF belongs to the mTERF protein family that includes three more members from D. melanogaster [START_REF] Linder | A family of putative transcription termination factors shared amongst metazoans and plants[END_REF][START_REF] Roberti | MTERF3, the most conserved member of the mTERF-family, is a modular factor involved in mitochondrial protein synthesis[END_REF]; as reported in FlyBase, they are indicated as CG7175, CG15390 and CG5047. The last polypeptide was named D-MTERF3 and was shown to be involved in mitochondrial protein synthesis [START_REF] Roberti | MTERF3, the most conserved member of the mTERF-family, is a modular factor involved in mitochondrial protein synthesis[END_REF]. We found that multiple DRE sequences are also present in the 5' flanking sequences of the genes for all these three proteins (unpublished observations), thus suggesting the existence of a control by DREF on all the genes belonging to the mTERF family and pointing towards a more general role of DREF in the control of mitochondrial biogenesis in Drosophila. This hypothesis is further supported by the recent finding that the A c c e p t e d M a n u s c r i p t DRE/DREF system is also a crucial regulator of the adenine nucleotide translocase (ANT) gene expression [START_REF] Kim | Transcriptional regulation of the Drosophila ANT gene by the DRE/DREF system[END_REF]. It was reported that hDREF, the human orthologue of the Drosophila factor, regulates cell proliferation and expression of ribosomal protein genes [START_REF] Ohshima | Identification of a human homologue of the DREF transcription factor with a potential role in regulation of the histone H1 gene[END_REF][START_REF] Yamashita | hDREF regulates cell proliferation and expression of ribosomal protein genes[END_REF]. Interestingly, we found that the gene for the human mitochondrial transcription termination factor mTERF contains a putative recognition site for hDREF in the 5´UTR region. A similar observation has been reported also for the human ANT gene [START_REF] Kim | Transcriptional regulation of the Drosophila ANT gene by the DRE/DREF system[END_REF]. These findings suggest that hDREF might have a role in controlling the expression of these genes and that some regulatory patterns of mitochondrial biogenesis could be shared by human and Drosophila. In situ RNA hybridization studies on Drosophila embryos here reported show that DmTTF mRNA is transcribed almost ubiquitously and constitutively. MtRNApol expression seems to be more efficient and restricted to specific embryonic territories. However, the late stages of embryogenesis DmTTF transcription becomes, over a general background, more efficient in Malpighi tubes and gut (digestive tube), regions in which mtRNApol is also transcribed. Thus, it is interesting that the mRNAs for DmTTF and mtRNApol, two proteins involved in the same process, share some expression regions but do not show identical temporal and spatial expression patterns during embryogenesis; this confirms that transcription of the two genes is differentially regulated. Moreover, the obtained data imply that higher levels of DmTTF might be required in territories such as Malpighi tubes and gut. A similar situation can be inferred from the data for DmTTF and mtRNApol gene transcription in Drosophila larvae and adult tissues reported in FlyAtlas database (http://www.flyatlas.org/) [START_REF] Chintapalli | Using FlyAtlas to identify better Drosophila melanogaster models of human disease[END_REF]. In particular, the enrichment of mtRNApol in some adult tissues with respect to the whole fly is slightly different from that of DmTTF (1.1 vs 0.8 in brain, 0.8 vs 1.2 in crop and 0.6 vs 1.0 in tubule). This imbalance is higher in larval tissues such as larval tubule (0.2 vs 1.0) or larval fat body (0.2 vs 1.1). Our findings and those from FlyAtlas suggest the possibility that DmTTF could play multiple roles in mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required. This possibility is also supported by DmTTF RNAi data [START_REF] Roberti | The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription[END_REF] which indicate a possible function of the termination factor also in transcription initiation. Therefore, we suggest that the variation of DmTTF relative amount might control, for example, the balance of different groups of mitochondrial transcripts during embryo development and, possibly, in larvae. Further experimental work is necessary for gaining evidences towards this direction.

Fig. 6 -DmTTF and mtRNApol expression during Drosophila

The temporal spatial distribution of Drosophila DmTTF (A) and mtRNApol (B) was examined by wholemount in situ hybridization with digoxigenin-labelled antisense RNA as described in Materials and Methods. The staining pattern of embryos is shown at the indicated stages. Abbreviations are the following: mg, midgut; amg, anterior midgut; pmg, posterior midgut; pm, pharyngeal muscles; ms, mesoderm; ec, ectoderm; mt, Malpighi tubes. All stages are shown as lateral view with dorsal at the top, except 14 th and 15 th that are shown as dorsal view. Both mRNAs display a significant maternal deposit. DmTTF mRNA is present in all stages and almost ubiquitously; after 12 th stage is transcribed more efficiently in gut and after 13 th stage it is done in gut and Malpighi tubes (the latter are out of focus in the dorsal view). MtRNApol mRNA maternal deposit disappears before 4 th stage until 11 th approximately when it is efficiently transcribed in gut. In late stages of the process, mtRNApol is efficiently and specifically transcribed in gut, mesoderm, Malpighi tubes and pharyngeal muscles (the latter are out of focus in the dorsal view). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t 4

Biochemical

  Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t 7

  3.3., PBT solution was replaced by PBS containing 0.1% (v/v) Tween-20 and the developing solution by 100 mM NaCl, 50 mM MgCl 2 , 100 mM Tris-HCl, pH 9.0, 0.1% (v/v) Tween-20. Biochemical Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t 8 RESULTS DRE elements are present in DmTTF promoter and are conserved in different Drosophila species.

Biochemical

  Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t Spatial and temporal expression of DmTTF and mtRNApol genes during D. melanogaster development.

Biochemical

  Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174

BiochemicalFig. 1 -Fig. 2 -

 12 FIGURE LEGENDS

Fig. 3 -

 3 Fig. 3 -Binding of DREF to the DRE sites in DmTTF promoter region. DREF binding was evaluated by gel-shift analysis using a nuclear extract (10 µg of proteins) from S2 cells and a radiolabelled 36-mer double-stranded oligonucleotide (positions -61 to -26, relative to DmTTF transcription initiation site) containing the -42 and -48 DRE sites. Homologous cold competitors, added to the assay in a 200-fold molar excess, were different versions of the same 36-mer oligonucleotide used as probe. With respect to the wild-type (DD), the versions mutated in the -48 site (dD), in the -42 site (Dd) or in both sites (dd) contain the same three nucleotide changes used for the luciferase assay constructs. The black arrows indicate the protein-DNA complexes, the open arrow indicates the super-shifted band produced by adding of anti-DREF serum to the binding reaction.

Fig. 4 -Fig. 5 -

 45 Fig. 4 -DREF binds to proximal DRE elements in DmTTF promoter in vivo. Overnight D. melanogaster embryos were fixed to cross-link proteins to DNA. Then, they were homogenized, cells were lysed and their DNA fragmented by sonication. Cell lysates were immunoprecipitated with control IgG (preimmune serum) or polyclonal antiserum raised against DREF or Eyegone. Immunoprecipitated DNAs were amplified by PCR with specific primers for -42/-48 DRE elements (panel A) and for -361 DRE element (panel B). DNA templates, immunoprecipitated in the different conditions, are indicated above each lane. Input lanes show the

BiochemicalBiochemicalBiochemical

  Fig.1A

  Fig.3

  Biochemical Journal Immediate Publication. Published on 25 Nov 2008 as manuscript BJ20081174 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081174 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

  Fig.4

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  Fig.6

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

© 2008 The Authors Journal compilation © 2008 Portland Press Limited

Acknowledgements

This work was supported by grants from: M.I.U.R., Italy, COFIN-PRIN 2005; Università di Bari, Progetto di Ricerca di Ateneo; Ministerio de Educación y Ciencia, Spain, Grant BFU2004-04591 (to RG); Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Grant PI070167 (to RG) and by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII. Madrid, Spain. Authors are grateful to Caterina Manzari (Dept. of Biochemistry and Mol. Biol., Univ. of Bari), Crescenzio Francesco Minervini (Dept. Genetics and Microbiol., Univ. of Bari), Mar Ruiz, Lara Salvany, Manolo Calleja (Centro de Biología Molecular Severo Ochoa, C/ Nicolás Cabrera, 1. UAM. 28049-Madrid, Spain) and Diego Villar (Instituto de Investigaciones Biomedicas "Alberto Sols" CSIC-UAM ) for their precious help in some experiments. Authors thank Masamitsu Yamaguchi (Kyoto Institute of Technology) and Laurie Kaguni (Dept. of Biochemistry, Michigan State University) for the kind gift of anti-DREF antibodies and Natalia Azpiazu (Centro de Biología Molecular Severo Ochoa) for the kind gift of anti-Eyegone antibodies.


Rafael Mayoral

Belen Mollá

Juana Maria Flores

Lisardo Boscá
email: lbosca@iib.uam.es

Marta Casado

Paloma Martín Sanz

& Martín-Sanz

Paloma Martín-Sanz

Constitutive expression of cyclooxygenase 2 transgene in hepatocytes protects against liver injury

Keywords: prostaglandins, lipopolysaccharide, concanavalin A, inflammation, apoptosis prostaglandins, lipopolysaccharide, concanavalin A, inflammation, apoptosis

INTRODUCTION

The acute or chronic liver injury, caused by viral infection, autoimmune reactions, alcohol ingestion, or the use of certain drugs, results in severe pathologies including fulminant hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Chronic hepatitis is a prolonged inflammatory process, with inflammatory cells infiltrating the liver parenchyma and triggering hepatocyte death. Persistent hepatocyte cell death is followed by a regenerative response in which viable hepatocytes are stimulated to proliferate to restore the loss of hepatic parenchyma [START_REF] Avila | New therapies for hepatocellular carcinoma[END_REF].

Cyclooxygenase-1 (COX-1) and -2 catalyze the first step in prostanoid biosynthesis [START_REF] Simmons | Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition[END_REF]. COX-1 is constitutively expressed in many tissues and seems to be involved in the housekeeping function of prostaglandins (PGs) [START_REF] Pilbeam | Differential regulation of inducible and constitutive prostaglandin endoperoxide synthase in osteoblastic MC3T3-E1 cells[END_REF], whereas COX-2 is induced by a variety of stimuli such as growth factors, pro-inflammatory stimuli, hormones and other cellular stresses [START_REF] Feng | Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide[END_REF]. Adult hepatocytes fail to induce COX-2 expression regardless of the pro-inflammatory factors used; however, Kupffer, stellate, immortalized mouse liver cells and fetal hepatocytes retain the ability to express COX-2 upon stimulation with LPS and pro-inflammatory cytokines [START_REF] Martin-Sanz | Expression of cyclooxygenase-2 in foetal rat hepatocytes stimulated with lipopolysaccharide and pro-inflammatory cytokines[END_REF][START_REF] Ledwith | Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol 12,13-myristate-type tumor promoters in immortalized mouse liver cells[END_REF]. In addition to this, we reported that partial hepatectomy (PH) induced COX-2 in hepatocytes and this contributed to the progression of cell cycle after PH [START_REF] Casado | Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy[END_REF][START_REF] Fernandez-Martinez | Thioacetamide-induced liver regeneration involves the expression of cyclooxygenase 2 and nitric oxide synthase 2 in hepatocytes[END_REF]. Expression of COX-2 has been also detected in human and animal models of cirrhosis [START_REF] Yamamoto | JTE-522, a cyclooxygenase-2 inhibitor, is an effective chemopreventive agent against rat experimental liver fibrosis1[END_REF], after hepatitis B and C virus infection [START_REF] Lara-Pezzi | The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression[END_REF][START_REF] Nunez | Increased intrahepatic cyclooxygenase 2, matrix metalloproteinase 2, and matrix metalloproteinase 9 expression is associated with progressive liver disease in chronic hepatitis C virus infection: role of viral core and NS5A proteins[END_REF], in hepatoma cell lines [START_REF] Kern | Proapoptotic and antiproliferative potential of selective cyclooxygenase-2 inhibitors in human liver tumor cells[END_REF] and in well-differentiated HCC [START_REF] Morinaga | Cyclooxygenase-2 mRNA is up-regulated in cirrhotic or chronic hepatitis liver adjacent to hepatocellular carcinoma[END_REF].

Despite these observations, the question of whether COX-2 expression is sufficient to induce tumorigenesis and the contribution of COX-2-dependent PGs to the inflammatory injury in liver has not been properly addressed [START_REF] Avila | New therapies for hepatocellular carcinoma[END_REF][START_REF] Canbay | Apoptosis: the nexus of liver injury and fibrosis[END_REF]. To gain insight into the role of PGs in acute and chronic liver diseases, we have developed a transgenic mouse that expresses human COX-2 constitutively in hepatocytes under the control of the human ApoE promoter. These mice exhibited a normal phenotype but they were resistant to Fas-mediated apoptosis [START_REF] Casado | Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes[END_REF]. Although it is widely accepted that PGs contribute to the pathology of various inflammatory diseases, which provide the rational for the development of COX selective inhibitors [START_REF] Gilroy | New insights into the role of COX 2 in inflammation[END_REF], different reports suggest that PGE 2 plays a protective role in various animal models of liver injury [START_REF] Kuzumoto | Significance and therapeutic potential of prostaglandin E2 receptor in hepatic ischemia/reperfusion injury in mice[END_REF]. Based on these data, we sought to examine the response of the COX-2-Tg mice in two well established models of inflammatory liver injury: LPS-injury in D-GalN preconditioned mice, an experimental model for fulminant hepatitis in sepsis, and the hepatitis induced by intravenous injection of ConA, a model relevant to autoimmune hepatitis. Our data clearly show that PGE 2 produced by COX-2 in hepatocytes enhances the levels of anti-apoptotic proteins, decreases the susceptibility to apoptosis after LPS/D-GalN challenge; attenuates the T-cell dependent hepatitis in response to ConA administration, and accelerates the expression of proteins that are involved in the proliferation of hepatocytes in regenerating liver after PH.

MATERIAL AND METHODS

Chemicals

Liver injury and regeneration models

To generate mice expressing a human COX-2-Tg in the liver, we used a pLiv-Le6 vector that contains the constitutive human ApoE gene promoter and its hepatic control region [START_REF] Casado | Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes[END_REF]. 12 weeks-old Tg animals (C57BL/6JxDBA) and its corresponding wild type (Wt) littermates were injected intraperitoneally (i.p.) with 1g/kg D-GalN plus 35 µg/kg LPS. ConA was intravenous (i.v.) injected through the tail vein at a dose of 20 mg/kg. Untreated animals received 0.5 ml of NaCl 0.9%. For PH, mice were anesthetized with a 92:7 mg/kg mix of ketamine/xylacine and subjected to midventral laparotomy with 70% liver resection (left lower and upper and right upper lobes) as described [START_REF] Casado | Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy[END_REF]. In some experiments, mice were treated daily for four days with DFU (1 mg/kg) to inhibit COX-2 [START_REF] Riendeau | Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor[END_REF]. Plasma was obtained by puncture of the retro-orbital vein plexus. Animals were sacrificed at six or eight h after LPS/D-GalN or ConA treatment, respectively. Livers were rapidly removed and freeze-clamped in liquid N 2 or collected in a solution containing 30% sucrose in PBS. Animals were treated according to the Institutional Care Instructions.

Cytokine analysis

The plasma levels of IFN-, TNF-, IL-1 and IL-6 were measured by fluorimetry using Bio-Plex Suspension Array System (Cytokine Assay) according to the manufacturer's instructions (Bio-Rad).

Determination of metabolites

PGE 2 was determined in liver tissue by specific immunoassay (Amersham). Briefly, liver samples were homogenized in 0.1 M phosphate buffer pH 7.5, containing 0.9% BSA and 0.5% Kathon (assay buffer). After centrifugation, the PGs were analyzed according to manufacturer's instructions. Amino transferases, ALT and ASP, alkaline phosphatase, ALP, and lactate dehydrogenase, LDH, were assayed spectrophotometrically in plasma in the School of Clinic Biochemistry, Faculty of Pharmacy, Complutense University, Madrid, Spain. Protein levels were determined with the Bradford reagent (Bio-Rad).

Histochemistry and immunofluorescence

Samples of liver were fixed in 10% buffered formalin, embedded in paraffin wax, sectioned at 3 µm and stained with hematoxylin and eosin (H&E). Histopathology analysis was carried out by the Department of Medicine and Animal Surgery from Faculty of Veterinary, Madrid, Spain. For immunofluorescence analysis, liver samples from 30% sucrose solution in PBS, were snap-frozen in 2-methylbutane immersed in liquid N 2 , and serial 8 m-thick sections were cut onto gelatinized glass with a Microm HM550 sledge cryostat [START_REF] Casado | Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes[END_REF]. The preparations were fixed with 4% paraformaldehyde pH 7 for 30 min at room temperature (RT), washed with PBS and permeabilized with methanol for 15 min at RT. For the detection and quantification of apoptosis, the TUNEL commercial kit for cell death detection (Roche) was used following the instructions of the manufacturer. TO-PRO-3 (Molecular Probes) was used for DNA staining.

Western blot analysis

Tissue samples (100 mg) were homogenized in a lysis buffer as described [START_REF] Casado | Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes[END_REF]. For Western blot analysis, extracts were boiled for 5 min in Laemmli sample buffer, and equal amounts of protein (50-70 g) were separated by 10-15% SDS-PAGE. The relative amounts of each protein were determined in total extracts with the following polyclonal or monoclonal antibodies: COX-2, COX-1 and PGES (cytosolic and membrane) (Cayman and Santa Cruz), inhibitors of apoptosis (xIAP) (B & D Systems), Bcl-2 family proteins (Santa Cruz and Abcam), TNFR pathway (Santa Cruz and Cell Signaling), cell cycle proteins, cyclin E, D1, PCNA (Santa Cruz), Akt, and p-Akt (Ser 473), AMPK and p-AMPK (Thr172) (Cell Signaling). After incubation with the corresponding anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibody, blots were developed by the ECL protocol (Amersham). Target protein band densities were normalized with p85. The blots were revealed, and different exposition times were performed for each blot with a charged coupling device camera in a luminescent image analyzer (Gel-Doc, Bio-Rad).

Caspase assays.

Livers were homogenized in 20 mM HEPES pH 7.5; 2 mM DTT and 10 % glycerol.

The extracts were centrifuged at 8000g for 15 min at 4ºC and 0.5 mg/ml of protein supernatant was used to measure caspase activities by cleavage of specific fluorogenic substrates in accordance with the supplier's instructions (BD PharMingen). Substrates were N-acetyl-DEVD-7-amino-4-trifluoromethylcoumarin for caspase 3 and N-acetyl-IETD-7-amino-4-trifluoromethylcoumarin for caspase 8. The linearity of caspase assays was determined over a 30 min reaction period.

Data analysis

Data are expressed as means ± standard deviation (SD). Statistical significance of differences between the control and transgenic groups was evaluated by the U of Mann-Whitney test. All tests have been calculated two-tail, and the significance has been considered at P 0.05.

RESULTS

Expression of h-COX-2 and PGE 2 levels in Tg mice after LPS/D-GalN treatment.

To achieve selective expression of COX-2 in hepatocytes, we generated transgenic (Tg) mice carrying the human isoform of COX-2 (h-COX-2) under the control of the human ApoE promoter and its endogenous hepatic control region [START_REF] Casado | Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes[END_REF].

The levels of h-COX-2, COX-1 and microsomal PGE synthase, which catalyzes the isomerization of PGH 2 to PGE 2 , were analyzed in liver extracts from Wt and Tg mice after LPS/D-GalN administration. As shown in Fig. 1A, h-COX-2 was significantly expressed in Tg animals but was not detectable in the Wt. Total COX-2 (human and endogenous) was also increased in Tg mice. Low levels of mPGES-1, which preferently couples with COX-2 activity to increase the production of PGE 2 , and COX-1 were present in Wt and Tg mice and did not change after treatment with LPS/D-GalN. The protein levels of cPGES were higher than those of mPGES-1 and did not change after the injury (Fig. 1A); mPGES-2 levels were negligible (not shown). Treatment of the animals with the COX-2 selective inhibitor DFU reduced ( 75%) the PGE 2 synthesis (Fig. 1B).

Inhibition of pro-inflammatory cytokines and liver injury in COX-2-Tg mice after LPS/D-GalN treatment.

LPS/D-GalN administration represents an experimental model of fulminant hepatitis in sepsis. LPS is one of the most potent TNF-inducers, but normally LPS or TNF-do not cause liver apoptosis unless coadministered with D-GalN which blocks transcription in the liver. To analyze the effect of PG synthesis on LPS/D-GalNdependent injury, the levels of the pro-inflammatory cytokines TNF-, IL-1 and IL-6 were determined. As Fig. 2A shows, a significant increase of circulating cytokine levels was observed in Wt mice by the effect of the injury, but this was much lower, about 50%, in COX-2-Tg mice. When COX-2-Tg mice were treated with DFU, cytokine levels resembled those of the Wt animals. The activities of hepatic injury markers AST, ALT, ALP and LDH were analyzed after 6 h of LPS/D-GalN administration. After hepatic injury, the increase in plasma levels of aminotransferases observed in COX-2-Tg mice was much lower than in the Wt ( 80%). When COX-2 was inhibited by treatment with DFU, the protection in the Tg animals was lost (Table I). A representative histological analysis of liver sections of Wt and Tg mice treated with LPS/D-GalN is shown in Fig. 2B. After 6 h following LPS/D-GalN administration, the liver from Wt mice exhibited an important focal necrosis of hepatocytes. In contrast, no evident lesions were observed in the livers of Tg mice. When Tg animals were treated with DFU, necrosis and a focal mild hepatitis were observed resembling the Wt liver.

To determine the contribution of apoptosis to liver injury, TUNEL of histological liver sections and measurement of the caspase 3 and 8 activities were carried out. As shown in Fig. 2C, a significant decrease in TUNEL staining was observed in Tg animals compared with Wt. Moreover, when Tg animals were treated with DFU, again TUNEL increased after LPS/D-GalN administration. The activity and protein levels of the active form of caspase 3 were markedly lower in Tg liver after LPS/D-GalN treatment than in Wt counterparts (40% and 30% for caspase 3 and 8, respectively; Fig. 4A-B).

COX-2 dependent PGs prevent liver injury induced by ConA.

ConA induced liver injury is mediated by the activation of the innate and adaptive immune cells including natural killer cells (NK), Kupffer cells and CD4 + T cells and their production of inflammatory cytokines, such as IL-6, IL-2, TNF-and IFN- [START_REF] Trautwein | Concanavalin A-induced liver cell damage: activation of intracellular pathways triggered by tumor necrosis factor in mice[END_REF]. Tg animals expressing COX-2 in liver exhibited a significant decrease of ConA-induced injury, mainly in IFN-, IL-1 and IL-6 levels (Fig. 3A), as well as on liver injury markers (Table II). Histological analysis of liver sections of Wt and Tg mice treated with ConA is shown in Fig. 3B. Livers from Wt mice exhibited focal mild areas of necrosis in hepatocytes whereas no remarkable lesions were observed in Tg mice.

Caspase 3-like activity was minimally induced in both Wt and Tg mice by the effect of ConA (note the difference of magnitude vs. the LPS/D-GalN model), and a minimal processing of procaspase 3 was observed in the Wt mice (Fig. 4C-D).

Altered expression of apoptotic related proteins after liver injury in h-COX-2 Tg mice.

To address the anti-apoptotic mechanism of COX-2 acting upon LPS/D-GalN or ConA mediated liver injury, we investigated the expression of apoptosis-related proteins. As Fig. 5A shows, the protein levels of TNFR1, TRADD and xIAP were equivalent in Wt and Tg mice and no differences were found after treatments. Bax was increased in Wt mice after injury and no changes were observed in Tg mice. Bcl-2 and

Mcl-1 levels were increased in Tg liver under basal conditions and were maintained after LPS/D-GalN treatment. TNFR2 and TRAF2 (TNF receptor-associated factor 2) decreased by the effect of the treatment in Wt but not in COX-2 expressing liver, indicating an increased sensitivity to TNF-induced apoptosis in Wt mice. Moreover, an increase in the Bcl-2/Bax (170%) ratio was observed in Tg mice after LPS/D-GalN treatment (Fig. 5C). Similar results were obtained when all these parameters were measured in Wt and Tg mice treated with ConA (Fig. 5B-D).

The PI3K/Akt and AMPK pathways play a central role in integrating diverse survival signals. An increase in the p-Akt/Akt (>3 fold) and more moderate in the p-AMPK /AMPK (1.5 fold) ratio were observed in Tg mice after LPS/D-GalN (Fig. 6A) or ConA injury (Fig. 6B). To confirm the contribution of PGs synthesis on Akt phosphorylation, we performed experiments in liver cell lines with stable expression of COX-2 as described previously [START_REF] Fernandez-Martinez | Cyclo-oxygenase 2 expression impairs serum-withdrawal-induced apoptosis in liver cells[END_REF]. A significant inhibition of the apoptosis induced by the effect of LPS/D-GalN in CHL-C cells (cells transfected with COX-2) comparing with the CHL-V cells (cells transfected with empty vector) was observed. Moreover, when cells were treated with the specific Akt inhibitor II, the number of apoptotic CHL-C cells was doubled indicating that Akt activation plays an important role in the inhibition of apoptosis promoted by PGs (Fig. 6C). To know the contribution of PGs to AMPK activation, CHL-C and CHL-V cells were treated with LPS/D-GalN and, as shown in Fig. 6D, AMPK was phosphorylated under basal conditions in agreement with the in vivo results (Fig. 6A). When CHL-V cells were treated with LPS/D-GalN, AMPK activation decreased; however, CHL-C or CHL-V cells treated with PGE 2 , maintained the phosphorylation state of AMPK. DFU treatment reversed theses effects.

To gain further insight into this differential response to liver injury in Wt and COX-2-Tg mice, we used a specific mouse apoptosis micro array to compare the expression profile after treatment with LPS/D-GalN. Fig. 7A summarizes both the magnitude of changes and the corresponding statistical significance of the 84 apoptosisrelated genes analyzed. We sorted the candidate genes whose expression threshold after LPS/D-GalN treatment was altered more than 2-fold (Fig. 7B). As we expected from the previous results, the majority of the up-regulated genes in Wt treated mice were proapoptotic (Apaf1, Casp1, Casp14, Casp4, Fasl, Tnf, Tnfsf10, Trp63, Trp73) and the down-regulated genes were anti-apoptotic (Api5, Bag3, Birc5, Dapk1, Mcl1, Sphk2, Traf2, Zc3Hc1). However, Tg mice exhibited opposite and attenuated changes of these genes (Api, Bag1, Bax, Plob, Traf2).

COX-2 Tg mice exhibit an accelerated hepatocyte proliferation after PH.

Because liver regeneration after PH implies a negligible apoptosis under normal conditions, we decided to evaluate the effect of the COX-2 Tg after PH. As previously described [START_REF] Casado | Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy[END_REF], COX-2 protein levels increased after PH and the levels of COX-1 and mPGES did not change (Fig. 8C). Fig. 8A shows similar survival rates for Wt and Tg mice after PH and no significant differences were found in the liver regeneration index at 80 h after PH (Fig. 8B). When cell cycle proteins were analyzed, significant differences were found in the levels of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E between Tg and the corresponding Wt mice. As shown in Fig. 8D-E, cyclin D1 was present in Tg mice at 0 h, increased at 12 h and remained elevated at 24 h after PH; cyclin E increased at 12 h and the raise of PCNA after PH was evidenced at 48 and 96 h.

DISCUSSION

We have studied the effect of constitutive expression of COX-2 in the hepatocyte on the liver response to three distinct insults: LPS/D-GalN, ConA and PH.

These Tg animals exhibited higher levels of some anti-apoptotic genes of the Bcl-2 family and were significantly resistant to LPS/D-GalN-induced injury, mainly through an inhibition of the apoptotic response characteristic of this model. It has been reported that D-GalN co-administered with LPS, represents an experimental model for fulminant hepatitis in sepsis due to massive hepatocyte apoptosis, mainly caused by an abrupt TNF-rise in the absence of transcription [START_REF] Nowak | LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNFp55 receptor[END_REF]. Under these conditions, COX-2 Tg animals exhibited a much lesser pro-inflammatory profile than the corresponding Wt animals. TNF-signals through two distinct membrane receptors: TNFR1 and TNFR2.

TNFR1, but not TNFR2 can directly activate the caspase 8 apoptotic pathway through its cytoplasmic death domain (DD) [START_REF] Hsu | TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways[END_REF]. Through TRADD, TNFR1 engagement also leads to IKK and JNK activation. While IKK activation has a clear antiapoptotic function, different results have been reported for the role of JNK activation in TNF--induced apoptosis [START_REF] Maeda | IKKbeta is required for prevention of apoptosis mediated by cell-bound but not by circulating TNFalpha[END_REF]. Our data show a decrease in TNFR2 by the effect of LPS/D-GalN treatment in Wt mice and this may be due to a down-regulation of the receptor described during endotoxemia [START_REF] Van Der Poll | Down-regulation of surface receptors for TNF and IL-1 on circulating monocytes and granulocytes during human endotoxemia: effect of neutralization of endotoxin-induced TNF activity by infusion of a recombinant dimeric TNF receptor[END_REF]. Although TNFR2 lacks a death domain, it has also been associated with the apoptotic response to TNF-. It is known that TNFR2 triggering induces degradation and depletion of TRAF2 and IAP proteins and accelerates TNFR1-dependent activation of caspase 8 [START_REF] Li | TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2[END_REF]. TRAF2 protein levels were depleted by the effect of the injury in Wt mice but not in the COX-2-Tg liver supporting the hypothesis that TRAF2 provides anti-apoptotic signals following TNF-stimulation.

Intravenous administration of ConA is an excellent model resembling immunemediated fulminant hepatic failure in humans. As reported [START_REF] Tiegs | A T cell-dependent experimental liver injury in mice inducible by concanavalin A[END_REF], ConA rapidly induced (6-24 h) clinical and histological evidenced of hepatitis, including elevation of transaminases, T cell infiltration and necrosis. ConA-induced liver injury is mediated by the activation of the innate and adaptive immune cells including NKs, Kupffer cells and CD4 + T cells and their production of inflammatory cytokines, such as TNF-and IFN-. IFN-plays a critical role in T cell-dependent liver injury initiated by ConA since IFN-KO mice are protected from hepatic injury [START_REF] Hong | Opposing roles of STAT1 and STAT3 in T cell-mediated hepatitis: regulation by SOCS[END_REF]. In agreement with this mechanism, treatment with ConA produced a rise in IFN-levels, an effect that was markedly reduced in COX-2 Tg mice, which suggests that PGs are interfering on immune cell activation, in particular lymphocytes. In this regard, it should be mentioned that previous work described an inhibitory effect by PGs on T cell activation [START_REF] Kim | Tumor-driven evolution of immunosuppressive networks during malignant progression[END_REF]. The activation of caspase 3 observed in ConA-treated mice was less than 80% of that observed after LPS/D-GalN challenge and, indeed, a negligible procaspase 3 processing to caspase 3 was observed in ConA treated animals in our conditions, suggesting that ConA is mainly promoting a cytolytic/necrotic response in agreement with the rapid rise in plasma levels of transaminases. Although in both liver injury models the expression of the COX-2 Tg exerted a very important protection, it is noteworthy mention that the cellular and molecular mechanisms underlying this effect appear to be different: protection against apoptosis in the LPS/D-GalN model, and attenuation of the immunecell dependent hepatocyte necrosis in the case of ConA administration.

The pro-inflammatory and cytoprotective functions of COX-2 in liver remain unclear, mainly because hepatocytes, but not other hepatic cells, fail to express COX-2 upon pro-inflammatory challenge [START_REF] Callejas | Regulation of cyclooxygenase 2 expression in hepatocytes by CCAAT/enhancerbinding proteins[END_REF]. It has been reported that expression of a COX-2 Tg causes hepatitis in senescent animals [START_REF] Yu | Expression of a cyclo-oxygenase-2 transgene in murine liver causes hepatitis[END_REF]; however, different reports demonstrated that PGE 2 administration protects against liver injury after E. coli infection by suppressing circulating TNF-and IL-12 levels and increasing IL-10 production [START_REF] Takano | Prostaglandin E2 protects against liver injury after Escherichia coli infection but hampers the resolution of the infection in mice[END_REF].

The PGE 2 -EP4 signalling pathway effectively protects against hepatic ischemia/reperfusion injury because EP4 agonists downregulated the expression of several pro-inflammatory cytokines, chemokines and adhesion molecules [START_REF] Kuzumoto | Significance and therapeutic potential of prostaglandin E2 receptor in hepatic ischemia/reperfusion injury in mice[END_REF]. PGE 2 downregulates Kupffer cell production of IL-1 and IL-6 during hepatic regeneration and LPS liver injury [START_REF] Treffkorn | PGE2 exerts its effect on the LPS-induced release of TNF-alpha, ET-1, IL-1alpha, IL-6 and IL-10 via the EP2 and EP4 receptor in rat liver macrophages[END_REF] and attenuates the acute phase response through a decrease in IL-6-induced -2 macroglobulin formation via a cAMP-dependent signalling [START_REF] Fennekohl | Induction by interleukin 6 of G(s)-coupled prostaglandin E(2) receptors in rat hepatocytes mediating a prostaglandin E(2)-dependent inhibition of the hepatocyte's acute phase response[END_REF]. Our results agree with these data and show that COX-2-dependent PGs cause an important decrease in the plasma levels of pro-inflammatory cytokines and a significant improvement in liver function after LPS/D-GalN or ConA challenge compared with the Wt counterparts. In this regard, Yin et al. [START_REF] Yin | Prostaglandin I(2) and E(2) mediate the protective effects of cyclooxygenase-2 in a mouse model of immunemediated liver injury[END_REF] by using COX-2 -/-mice demonstrated an increased susceptibility of COX-2 -/-mice to ConA-induced hepatitis.

Studies with different Tg mouse models of COX-2 also demonstrated the important anti-apoptotic role for PGs. Tg mice expressing COX-2 in mammary gland developed tumours, and these animals had reduced levels of the proapoptotic proteins Bax and Bcl-x S and elevated levels of the antiapoptotic protein Bcl-2 [START_REF] Liu | Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice[END_REF]. growth [START_REF] Oshima | Hyperplastic gastric tumors induced by activated macrophages in COX-2/mPGES-1 transgenic mice[END_REF]. Our previous results obtained with stable expression of COX-2 in liver cell lines and with mouse hydrodynamically transfected with a COX-2-GFP expression vector, supported the view that PGs produced by COX-2 protected the liver against Fas-mediated apoptosis [START_REF] Fernandez-Martinez | Cyclo-oxygenase 2 expression impairs serum-withdrawal-induced apoptosis in liver cells[END_REF]. In this work we show that PGs produced by COX-2 in liver, increased the levels of Bcl-2, Mcl-1, TNFR2 and TRAF2, all these hallmarks of apoptotic/anti-apoptotic pathway, and were confirmed by a PCR array analysis specific for apoptosis profile. Several signalling pathways have been proposed as mediators of COX-2-dependent inhibition of apoptosis; however, few reports describe the direct effect of COX-2 expression on apoptosis in liver or HCC cells. Leng et al. [START_REF] Leng | Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth through Akt activation: evidence for Akt inhibition in celecoxib-induced apoptosis[END_REF] reported an enhanced phosphorylation of Akt in Hep3B cells transiently or permanently transfected with a COX-2 expression vector; overexpression of COX-2 in rat intestinal epithelial cells increased their resistance to undergo apoptosis and resulted in increased Bcl-2 protein levels [START_REF] Tsujii | Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2[END_REF] and COX-2 induces a Mcl-1-dependent survival mechanism in human lung adenocarcinoma cells [START_REF] Lin | Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung adenocarcinoma CL1.0 cells. Involvement of phosphatidylinositol 3-kinase/Akt pathway[END_REF]. Targets of PGs through their EP receptors are the PI3K and the Akt/PKB pathways. The PI3K/Akt is important in modulating the mammalian target of rapamycin, which acts as a central sensor for nutrient/energy availability, thereby regulating cell growth. Activation of Akt and G subunit of EP receptors can also result in the accumulation of -catenin of the canonical Wnt pathway in the nucleus, which also leads to transcriptional activation [START_REF] Buchanan | Connecting COX-2 and Wnt in cancer[END_REF]. Akt improves cellular energy stores by accelerating cell proliferation and size, while induces antiapoptotic effects by phosphorylation and inactivation of the proapoptotic protein Bad, and by the upregulation of Bcl-2 and Mcl-1 [START_REF] Schulze-Bergkamen | Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95mediated apoptosis via Akt[END_REF]. Our results show that in the liver of COX-2-Tg mice, both Akt and AMP-kinase are phosphorylated indicating a reinforcement of survival pathways and a higher efficiency in energetic metabolism, preventing ATP depletion [START_REF] Havel | Update on adipocyte hormones: regulation of energy balance and carbohydrate/lipid metabolism[END_REF]. These circumstances concur in favouring liver protection after acute injury.

It has been described [START_REF] Casado | Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy[END_REF][START_REF] Rudnick | Prostaglandins are required for CREB activation and cellular proliferation during liver regeneration[END_REF] that PGs produced by COX-2 are important for the early steps of liver regeneration after PH. Because liver regeneration after PH is characterized by a negligible concurrence of apoptosis, we investigated whether the COX-2 Tg might influence the hepatocyte proliferation. Our results show that PGs anticipated the initial cell cycle progression without noticeable changes in the rate of mass recovery at the end (5-days) of the regeneration process. As potential mechanisms responsible for this rapid cell commitment for proliferation, it is known that PGs increase cyclin D1 levels through -catenin pathway [START_REF] Shao | Prostaglandin E2 Stimulates the beta-catenin/T cell factor-dependent transcription in colon cancer[END_REF], and that both AMPK and Akt A c c e p t e d M a n u s c r i p t

  Antibodies were from Santa Cruz Laboratories (Santa Cruz, CA), Becton & Dickinson (New Jersey, NY) and Cell Signaling (Boston, MA). 5, 5-Dimethyl-3(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone (DFU) was from Merck (Rahway, NJ). Reagents were from Roche Diagnostics (Mannheim, Germany) or Sigma Chemical Co. (St. Louis, MO). LPS (from Salmonella Thyphimurium), D-GalN and ConA were obtained from Sigma. Fluorescent probes were from Molecular Probes (Eugene, OR, USA) and Calbiochem (Merck KGaA, Darmstadt, Germany). Akt inhibitor II was from Merck (UK). Reagents for electrophoresis were obtained from Bio-Rad (Hercules, CA).

  A different study demonstrated hyperplasic gastric tumours in Tg mice expressing COX-2 and mPGES. This study relates PGE 2 with the recruitment, infiltration and activation of mucosal macrophages establishing a relationship between inflammation and tumour cell 11 Biochemical Journal Immediate Publication. Published on 01 Aug 2008 as manuscript BJ20081224 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081224A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .

 1 Figure 1. Expression of human COX-2 in Tg mice after LPS/D-GalN treatment. (A) Human and total COX-2, mPGES-1, cPGES and COX-1 protein expression in liver homogenates from Wt and COX-2-Tg animals with or without LPS/D-GalN treatment (6 hours) detected by Western blot and normalized with p85. Abs were from Cayman and Santa Cruz. (B) Intrahepatic PGE 2 concentrations were determined by ELISA in liver homogenates in the presence or absence of 1 mg/kg of DFU. Data are the means + SD of six animals per condition. *P<0.05 vs. the corresponding Wt animals treated under identical conditions. # P 0.05 vs. the corresponding Tg animals treated without DFU.

Figure 2 .

 2 Figure 2. Inhibition of pro-inflammatory cytokines and liver injury in COX-2-Tg mice after LPS/D-GalN treatment. (A) TNF-, IL-1 and IL-6 were measured after 2 h of treatment in plasma by fluorimetric analysis using Bio-Plex Suspension Array System (Bio-Rad). (B) Hematoxylin and eosin (H&E) analysis of liver sections of a 12weeks-old Wt and Tg mice after treatment. Figures were taken with a CVD Camera at 40X. Scale bar, 50µm. (a) Focal necrosis of hepatocytes in LPS/D-GalN treated Wt mice. (b) No lesions were observed in the liver of TG mice under the same conditions. (c-d) Diffuse moderate acute and focal mild hepatitis is observed in Wt and Tg mice treated with DFU respectively. (C) Apoptosis in liver sections taken 6 hours after injection with LPS/D-GalN. Confocal images were obtained with a Leica TCS SP2 Spectral microscope and a 63x/1.40 NA oil objective (Leica Microsystems, Wetzlar,

Figure 3 .

 3 Figure 3. COX-2 dependent PGs prevent liver injury induced by ConA. ConA was i.v. at 20 mg/kg to Wt and Tg mice. (A) Plasma levels of cytokines after 2 h of ConA treatment were measured by fluorimetric analysis using Bio-Plex (Bio-Rad). (B) Histological analysis of liver sections of a 12-weeks-old Wt littermate and Tg mice after ConA treatment and stained with H&E (40X; scale bar, 50 µm). Mild focal necrosis of hepatocytes in ConA-treated Wt liver whereas no remarkable lesions were observed in the liver of Tg mice. Results are mean ± SD of five animals per condition. *, ** denotes P<0.05 and P<0.001 vs. the corresponding Wt mice treated under identical conditions.

Figure 4 .

 4 Figure 4. Expression of COX-2 inhibits caspase like activities. Caspase 3 and 8 were measured as DEVDase and IETDase activities in Wt and Tg liver homogenates. Caspase activities were measured by fluorimetric assay with specific fluorogenic substrates (see Materials and Methods) after LPS/D-Gal N (A-B) or ConA (C-D) treatment. Note the different scale in the A-C panel. Liver extracts were also used to determine the amount of pro-caspase 3 by Western blot. Blots were normalized for protein content with p85. Data are the means + SD of five animals per condition. *P 0.05 vs. the Wt mice treated under identical conditions.

Figure 5 .

 5 Figure 5. Altered expression of apoptotic related proteins in COX-2-expressing liver. Representative Western blots showing the expression of Bcl-x L , Bcl-2, Bax, xIAP, Mcl-1, TNFR1, TNFR2, TRAF-2 and TRADD in Wt and Tg animals. Densitometric analysis of the expression of Bcl-2/Bax ratio after LPS/D-GalN (6 h) (A-C) or ConA (8 h) (B-D) treatment. The expression of target proteins was normalized to that of p85 and the ratios are expressed as percentage of the Wt value without treatment. Data are the means + SD of six animals per condition. * denotes P<0.05 vs. the Wt mice treated under identical conditions.

Figure 6 .

 6 Figure 6. Akt and AMPK activation in COX-2-Tg mice and in liver cell lines. Representative Western blots showing the expression of p-Akt/Akt and p-AMPK /AMPK in Wt and Tg animals. Densitometric analysis of the expression of p-Akt/Akt after LPS/D-GalN (6 h) (A) or ConA (8 h) (B) treatment. Ab for Akt, and p-Akt (Ser 473), AMPK and p-AMPK (Thr172) were from Cell Signaling. (C) CHL-C (stable transfected with pcDNA3hCOX-2) and CHL-V (transfected with pcDNA3) cells (50x10 3 ) were treated with 5 mM D-GalN plus 100 ng/ml LPS or 5 µM Akt inhibitor II for 12 h. After that, cells resuspended in PBS were PI-stained and cell viability was determined in a BD FACS Canto I cell cytometry. (D) CHL-C and CHL-V cells (1x10 6 ) were preincubated with 5 M PGE 2 or 5 M DFU for 1 h and then treated with 5 mM D-GalN plus 100 ng/ml LPS for 12 h. Protein levels of p-AMPK /AMPK were measured by Western blot and the densitometric analysis is shown. The expression of target proteins was normalized to that of p85 and the ratios are expressed as percentage of the Wt value without treatment. Data are the means + SD of six animals per condition or three independent experiments. *, ** denotes P<0.05 and P<0.001 vs. the Wt mice or the CHL-V cells treated under identical conditions.

Figure 7 .

 7 Figure 7. Transcriptional profiling of COX-2 expressing liver after LPS/D-GalN treatment. Total RNA was isolated from liver with TRIzol Reagent (Invitrogen, Carlsbad, California, USA) and further cleaned up using the Qiagen RNeasy MiniKit (Hilden, Germany) with one step of DNase I digestion following the manufacturer's instructions. 2 g of RNA were used for cDNA synthesis with RT 2 First Standard kit (SuperArray Bioscience, Frederick, MD). The mouse apoptosis PCR array was performed according to the manufacturer's protocol, using the Profiler PCR Array System and the SYBR Green/Fluorescein qPCR master mix (SuperArray Bioscience) on a MyiQ Real-Time PCR System (Bio-Rad). Gene expression was compared with the web-based software package for the PCR Array System (http://www.superarray.com/pcr/arrayanalysis.php); this software automatically performs all C t based fold-change calculations from the specific uploaded raw threshold cycle data. (A) Volcano plot shows the log2 fold change of apoptosis-related genes versus their p-values. Each point represents a plot containing log2 of the fold change in gene expression threshold for two conditions (saline vs. LPS/D-GalN) on the x axis and the p value on the y axis. Points over the horizontal blue line indicated the p-

Figure 8 .

 8 Figure 8. COX-2 expressing hepatocytes exhibit an accelerated proliferation after PH. For PH, Wt and Tg mice were subjected to midventral laparotomy with 70% liver resection and total extracts were prepared. (A) Survival rates of Wt and COX-2-Tg mice at 120 h after PH. (B) Liver regeneration index at the indicated times after PH. (C) COX-2, mPGES and COX-1 protein expression in liver homogenates from Wt and COX-2-Tg animals after PH detected by Western blot and normalized with p85. (D-E) Representative Western blots and densitometric analysis of PCNA, cyclin D1 and E in Wt and COX-2 Tg mice after PH. Results are the means + SD of the five animals. *, ** denotes P<0.05 and P<0.001 vs. the corresponding Wt condition treated under identical conditions, respectively.

TABLE I . Plasma Levels of Liver Injury Markers.

 I

	Mice	AST (UI/l)	ALT (UI/l)	ALP (UI/l)	LDH (UI/l)
	Wt Saline	84 ± 16	39 ± 7	355 ± 66	47 ± 9
	LPS/D-GalN	3750 ± 694	537 ± 99	400 ± 74	6381 ± 1180
	LPS/D-GalN + DFU	3697 ± 684	ND	529 ± 98	ND
	Tg Saline LPS/D-GalN LPS/D-GalN + DFU	71 ± 12 624 ± 108 1531 ± 265 # *	22 ± 4 66 ± 11 ND *	279 ± 48 219 ± 38 238 ± 41	33 ± 6 ND 734 ± 127 *

Table I . Plasma levels of liver injury markers after LPS/D-GalN treatment.

 I Plasma levels of aminotransferases (AST, ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) were assayed spectrophotometrically after 6 h of treatment from the different groups of animals. ND, not determined. Results are mean ± SD of five animals per condition. *P<0.05 vs. the corresponding Wt animals treated under identical conditions. # P 0.05 vs. the corresponding Tg animals treated without DFU. Journal Immediate Publication. Published on 01 Aug 2008 as manuscript BJ20081224 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081224

	Biochemical

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

This work was supported by grants SAF2007-60551, SAF2005-1758 and S-BIO-0283/2006. B. M. was supported by grant of FIS-RECAVA RD06/0014/0025. Ciberehd is funded by the Instituto de Salud Carlos III.