Marc Vermeir
email: mvermeir@its.jnj.com

Alex Hemeryck

Filip Cuyckens

Andres Francesch

Marc Bockx

Jos Van Houdt

Kathleen Steemans

Geert Mannens

Pablo Avilés

Roland De Coster

studies on the metabolism of trabectedin (YONDELIS) in monkey and man, including human CYP reaction phenotyping

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Graphical abstract

The in vitro metabolism of 14 C-trabectedin is qualitatively similar in monkey and man. Biotransformation occurs predominantly at its A subunit. At clinically relevant levels, CYP3A4 is the main CYP involved.

Introduction

Trabectedin (YONDELIS ® , previously known as ecteinascidin-743 or ET-743) is a marine alkaloid originally isolated from the Caribbean tunicate Ecteinascidia turbinata. Its chemical structure is characterized by three fused tetrahydroisoquinoline subunits (Fig. 1). Subunits A and B are fused into a monobridged pentacyclic skeleton. Subunit C is linked to the A-B core via a ten membered lactone bridge through a benzylic sulfide linkage. Trabectedin binds covalently to guanine residues in the minor groove of the DNA double helix, triggering a cascade of events which include interference with several transcription factors, DNA binding proteins and repair pathways, and disruption of the cell cycle.

This results in potent antitumor activity against a number of cell lines in vitro, and important antiproliferative effects in vivo against a variety of experimental tumors [START_REF] D'incalci | Preclinical and clinical results with the natural marine product ET-743[END_REF].

In clinical trials, single agent trabectedin has shown activity in soft tissue sarcoma, breast, prostate and ovarian carcinomas [START_REF] D'incalci | Preclinical and clinical results with the natural marine product ET-743[END_REF][START_REF] Zelek | A phase II study of Yondelis (trabectedin, ET-743) as a 24-h continuous intravenous infusion in pretreated advanced breast cancer[END_REF][START_REF] Fayette | ET-743: a novel agent with activity in soft-tissue sarcomas[END_REF]. Recently, intravenous trabectedin has been approved in Europe for use in patients with advanced soft tissue sarcoma after failure of antracyclines or ifosfamide, or who are unsuited to receive these agents [START_REF] Carter | Trabectedin. A review of its use in the management of soft tissue sarcoma and ovarian carcinoma[END_REF].

Several clinical trials are currently ongoing to further substantiate its clinical activity either as a single agent or in combination with other anticancer agents [START_REF] Carter | Trabectedin. A review of its use in the management of soft tissue sarcoma and ovarian carcinoma[END_REF] (see also:

http://www.clinicaltrials.gov).

Despite the long standing clinical use of YONDELIS ® , the metabolism of trabectedin remains largely unknown. Attempts to elucidate the compound's metabolism in in vitro systems [START_REF] Sparidans | Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man[END_REF][START_REF] Reid | Rat and human liver cytochrome P-450 Isoform Metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans[END_REF] were only partially successful. Sparidans et al. [START_REF] Sparidans | Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man[END_REF] identified a few A c c e p t e d M a n u s c r i p t 6 metabolites and degradation products in human liver microsomes. Reid et al. [START_REF] Reid | Rat and human liver cytochrome P-450 Isoform Metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans[END_REF] identified three oxidative metabolites in cDNA-expressed CYP systems. The metabolic fate of trabectedin in vivo has neither been well characterized [START_REF] D'incalci | Preclinical and clinical results with the natural marine product ET-743[END_REF][START_REF] Beumer | Hepatotoxicity and metabolism of trabectedin: a literature review[END_REF][START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF].

Beumer et al. [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF] reported on the metabolite profiling in urine and faeces samples from cancer patients after intravenous administration of [ 14 C]trabectedin. A large number of metabolites was detected, however, only a few putative metabolites were tentatively identified [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF]. The incomplete knowledge on trabectedin's in vivo metabolism may in part be explained by the fact that in vivo investigations are seriously hampered by several compound related factors, including the low dose, the complexity of metabolism and the extremely low metabolite concentrations [START_REF] Sparidans | Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man[END_REF][START_REF] Beumer | Hepatotoxicity and metabolism of trabectedin: a literature review[END_REF][START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF].

In vitro metabolism studies using [ 14 C]labelled trabectedin and hepatic subcellular fractions of various species have shown that the in vitro metabolic profile of man and monkey were qualitatively similar, whilst that of other species including rat and dog was quite different [START_REF] Vermeir | An interspecies comparison of the metabolism of the anticancer agent Yondelis TM (Trabectedin, ET-743)[END_REF]. Although these investigations provided a preliminary comparative metabolic profile across species, additional studies were needed to further substantiate the similarity between human and Cynomolgus monkey hepatic metabolism including characterization of biotransformation pathways and the identity of trabectedin metabolites. Species-differences in trabectedin metabolism is of particular relevance to the preclinical safety evaluation of trabectedin-induced hepatotoxicity, whose nature has been shown to be species-dependent [START_REF] Beumer | Hepatotoxicity and metabolism of trabectedin: a literature review[END_REF].

During preclinical risk assessment, results from in vitro and/or in vivo metabolism studies can assist in the selection of appropriate animal species for toxicological assessments [START_REF]Food and Drug Administration Draft Guidance for Industry: Safety Testing of Drug Metabolites[END_REF][START_REF] Atrakchi | A regulatory perspective on issues and approaches in characterizing human metabolites[END_REF]. In addition to the species selection for preclinical risk A c c e p t e d M a n u s c r i p t 7 assessment, the prediction of metabolism-based drug-drug interactions is another important aspect during drug development [START_REF] Bjornsson | The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) Perspective[END_REF][START_REF]Food and Drug Administration Draft Guidance for Industry: Drug Interaction Studies -Study Design, Data Analysis, and Implications for Dosing and Labeling[END_REF]. This is particularly relevant to anti-cancer agents as the target populations for these drugs are at considerable risk of drug-drug interactions [START_REF] Beijnen | Drug interactions in oncology[END_REF][START_REF] Blower | Drug-drug interactions in oncology: why are they important and can they be minimized?[END_REF][START_REF] Riechelmann | Potential for drug interactions in hospitalized cancer patients[END_REF]. The characterization of a compound's elimination pathways including the enzyme(s) responsible for its metabolism typically forms the basis to assess whether a drug may be the precipitant of a metabolism-based drug interaction [START_REF] Bjornsson | The conduct of in vitro and in vivo drug-drug interaction studies: a Pharmaceutical Research and Manufacturers of America (PhRMA) Perspective[END_REF][START_REF]Food and Drug Administration Draft Guidance for Industry: Drug Interaction Studies -Study Design, Data Analysis, and Implications for Dosing and Labeling[END_REF]. In vitro investigations using human hepatic subcellular fractions and heterologously expressed human CYP (cytochrome P-450) forms have indicated that virtually all major CYPs are able to metabolize trabectedin [START_REF] Sparidans | Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man[END_REF][START_REF] Reid | Rat and human liver cytochrome P-450 Isoform Metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans[END_REF], but that CYP3A4 is likely the major isoform [START_REF] Brandon | In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis ® , Trabectedin), a novel marine anti-cancer drug[END_REF].

The aims of the present in vitro studies were therefore twofold: first, to elucidate the metabolism of trabectedin in hepatic subcellular fractions of the Cynomolgus monkey and man, second, to delineate the involvement of CYP in trabectedin's human metabolism.

Materials and Methods

Chemicals

Radiolabelled [ 14 C]trabectedin (Fig. 1) with a specific activity of 2.02 MBq/mg and a radiochemical purity of 92.7% was synthesized at PharmaMar (Colmenar Viejo, Madrid, Spain). Unlabelled trabectedin of natural source (chemical purity of 98.8%), tri-deuterated trabectedin and unlabelled authentic reference compounds (Fig. 1) used for HPLC co-chromatography were kindly provided by PharmaMar. The were of the highest quality commercially available.

Biological reagents

For the in vitro metabolism experiments, pooled batches of human liver subcellular fractions were prepared from liver tissue obtained from male and female human kidney transplant donors (n=4). For the incubations with liver subcellular fractions of male Cynomolgus monkey, two pools (n=3 and n=4) purchased from Biopredic International (Rennes, France), were combined.

The pooled human liver microsomes (n=38) used for the CYP phenotyping experiments were obtained from BD Biosciences (San Jose, CA, USA). For the preparation of cDNA-expressed human P450 enzyme systems, constructs in E. coli cells [START_REF] Gillam | Expression of Modified Human Cytochrome P450 3A4 in Escherichia coli and Purification and Reconstitution of the Enzyme[END_REF][START_REF] Blake | Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli[END_REF] Human liver subcellular fractions were prepared following standard procedures [START_REF] Amar-Costesec | Analytical study of microsomes and isolated subcellular membranes from rat liver. II. Preparation and composition of the microsomal fraction[END_REF][START_REF] Wang | Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver[END_REF]. Protein and cytochrome P450 concentrations were determined according to the methods of Lowry [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF] and Omura and Sato [START_REF] Omura | The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature[END_REF], respectively.

Trabectedin metabolism in liver subcellular fractions

For the incubations with hepatic subcellular fractions, 1-mM stock solutions of Final solvent concentrations were 0.5% (v/v). To examine phase I metabolism, incubations of [ All enzymatic reactions in the different CYP phenotyping experiments were terminated by snapfreezing the samples in dry ice, after which they were stored at or below -18 °C until analysis.

Analytical methods

Radio-HPLC

Incubates of [ 14 C]trabectedin with liver subcellular fractions and with recombinant systems were analyzed by radio-HPLC. The samples were thawed in the presence of DMSO at a sample/DMSO ratio of 1:1 (v/v). The samples were sonicated and centrifuged for 10 min at 13,800 x g prior to HPLC analysis. For radioactivity determination, appropriate aliquots (5-50 µl) of the samples were mixed with 10 ml of Ultima Gold™ (Packard) scintillation cocktail. Liquid scintillation counting was performed using a Packard 2100 TR microprocessor controlled multi-user liquid scintillation system.

To limit chemical degradation, the radio-HPLC analysis was performed immediately The amounts of parent drug and of its major metabolites were calculated from the radioactivity peaks following reversed-phase radio-HPLC analysis. Data processing was performed using Millennium 32 Chromatography Software.

Metabolite identification

A In addition to metabolite identification by LC-MS/MS, metabolite characterization was also performed by comparison of the radio-HPLC chromatograms with HPLC chromatograms of unlabelled authentic compounds. For this purpose, a mixture of the parent drug and of various authentic substances was co-chromatographed with the radioactive samples on the reversed-phase column. The authentic substances were detected by UV-monitoring at 280 nm, the metabolites by radioactivity monitoring.

Quantification of trabectedin (CYP phenotyping experiments with chemical inhibitors and inhibitory antibodies)

The concentration of trabectedin in the incubates of human liver microsomes with CYP-specific chemical inhibitors and inhibitory antibodies was determined by LC-MS/MS. Hundred-microliter aliquots of the incubates were spiked with 10 ng of the stable isotope labelled internal standard (tri-deuterated trabectedin) and 250 µl of methanol was added. After vortex mixing and centrifugation, the supernatants were transferred to an injection vial and mixed with 400 µl of water. The supernatants were analyzed using column switching, coupled to tandem mass spectrometry for detection. Fifty-microliter aliquots of the supernatants were injected on a trapping column (10 x 2.1 mm I.D., packed with 3.5 µm Symmetry Shield RP). The loading mobile phase was a water/methanol mixture (95/5;v/v) pumped at 0.5 ml/min. After loading the samples, the column was washed by increasing the methanol content to

Data analysis

For metabolite profiling, the relative amounts of unchanged trabectedin and of its major metabolites were expressed as the percentage of radioactivity in the samples accounted for by the individual radiolabelled compounds. Qualitative as well as quantitative comparisons between man and monkey were made. In the experiments with recombinant systems for CYP phenotyping, only the disappearance of parent drug and concurrent appearance of additional radioactive peaks in the radiochromatograms were considered. In the experiments with diagnostic inhibitors and with CYP inhibitory antibodies, the concentration of trabectedin in the incubation mixtures with and without inhibitors as a function of incubation time was calculated.

A c c e p t e d M a n u s c r i p t 16

Results

Metabolite Profiles

[ 14 C]trabectedin (5 µM) was incubated for 120 minutes with 12,000 x g liver supernatant fractions of male Cynomolgus monkeys and man. The metabolite profiles of trabectedin in the incubates of both species were defined by radio-HPLC (Fig. 2). Metabolite designation was based on their retention time in the radio-HPLC chromatograms. As several radioactivity peaks were composed of two or more coeluting metabolites under the HPLC conditions used, a suffix (letter) was added to identify individual metabolites. The radio-HPLC chromatograms indicate that liver subcellular fractions of the Cynomolgus monkey and of human produced a similar pattern of radiolabelled metabolites, although some quantitative differences were noticeable. Four major chromatographic peaks were observed with retention times of about 28, 32, 37 and 42 min. Unchanged trabectedin accounted for 14.9% and 9.2% of the injected sample radioactivity in liver subcellular fractions of human and Cynomolgus monkey, respectively, indicating that the metabolism of trabectedin proceeded faster in monkeys than in humans. Due to incomplete radio-HPLC separation, the abundance of individual metabolites in the major peaks could not be determined. Relative amounts of co-eluting metabolites are given in Table 1. Visual inspection of the metabolite profiles in Fig. 2 revealed a higher abundance of the 4a/4b and 8b/8c metabolites in monkey fractions than in human fractions, suggesting a more active biotransformation of trabectedin via N-demethylation (metabolite 8b)

and/or via demethylation with additional mono-oxidation (metabolite 4b), di-oxidation (metabolite 4a) or tri-oxidation (metabolite 8c) in monkeys than in humans.

Structure Elucidation of Metabolites.

Trabectedin metabolites were identified by LC-MS/MS and HPLC co-chromatography using unlabelled trabectedin and authentic reference compounds. The MS spectrum of trabectedin showed a protonated molecular ion at m/z 762. Fragmentation of the parent ion produced characteristic fragment ions, useful for metabolite identification (Fig. 3). MS 2 spectra of the various metabolites are provided in Figs. 4 and5.

Because of the complexity of the molecule and the multiple potential sites for metabolism, it was generally not possible to identify the exact site of metabolic conversion based on the available LC-MS/MS data. Nonetheless, the data allowed for partial structure elucidation of the metabolites and identification of the molecular substructures undergoing metabolism.

The molecular ion fragments of the metabolites are listed in Table 2. By using a QTOF mass spectrometer, it was possible to obtain accurate masses for the pseudomolecular ions of the metabolites in MS and their product ions generated in MS 2 . The accurate masses obtained were processed using the elemental composition calculator of the Masslynx software, and possible molecular formulae were calculated. All measured accurate masses were within a 5 ppm error relative to the corresponding calculated masses (Table 3). Based on the QTOF measurements, the following metabolites were identified:

Metabolites 4b and 6a

The protonated molecular ions of metabolites 4b and 6a and several corresponding MS/MS product ions were shifted 2 amu (m/z 764) as compared to trabectedin (Table 2). This suggests that both metabolites are the result of a combined demethylation and oxidation reaction at the A domain of the molecule.

Metabolite 6b

Metabolite 6b showed the same protonated molecular ion (m/z 762) and corresponding MS/MS product ions at m/z 206, 224, 260 and 475 (Table 2) as compared to trabectedin. However, in comparison to the unchanged drug a different accurate mass was obtained (Table 3), indicating a different elemental composition.

The minor water loss from the molecular ion observed in full MS and the presence of an abundant product ion at m/z 206 suggested that the hydroxyl function at the carbinolamine moiety was still intact. Taken together, these data indicate that metabolite 6b is likely the result from a demethylation and a mono-oxidation combined with a loss of two hydrogen atoms at the A domain of trabectedin.

Metabolite 8b

The protonated molecular ion of metabolite 8b had lost 14 amu (m/z 748) relative to trabectedin. The same shift was also seen with selected product ions (Table 2) indicating a demethylation of these fragments. The fragmentation behaviour and retention time of metabolite 8b proved to be identical to that of the authentic reference compound ET-729 (N-demethylated trabectedin).

Metabolite 8c

The protonated molecular ion of metabolite 8c had a 16 amu shift (m/z 778) relative to trabectedin. The product ions at m/z 224 and 260 were unchanged, whereas the product ions at m/z 491, 509 and 527 were shifted 16 amu.

The product ion at m/z 224 can be explained as 206 + 18 since the fragment at m/z 252 had also shifted 18 amu relative to m/z 234 of the parent drug. Although a shift of 16 amu may suggest a simple oxidation, this was not in accordance with the accurate mass measurements (Table 3) that indicated an elemental composition of inferred from the accurate mass measurements (Table 3), the proposed metabolite is thought to be the result of an aliphatic ring opening at the methylene dioxybridge at the B subunit of trabectedin.

Metabolite 11a

The protonated molecular ion of metabolite 11a (m/z 778) and several product ions at m/z 222, 250, 491, 509 and 511 were shifted 16 amu as compared to unchanged drug (Table 2), while the fragment ions at m/z 224 and 260 were unchanged. These data indicate that metabolite 11a results from a monooxidation at the A subunit of trabectedin.

Metabolite 16b

The protonated molecular ion of metabolite 16b (m/z 794) and several product ions at m/z 238, 266, 507, 525 and 543 were shifted 32 amu as compared to trabectedin (Table 2), while the fragment ions at m/z 224 and 260 were unchanged. Collectively, these data indicate that metabolite 16b is the result of a double oxidation at the A domain of trabectedin.

Metabolite 16c

The protonated molecular ion of metabolite 16c had lost 14 amu (m/z 748) relative to trabectedin. The same 14 amu shift was observed for the small fragment peak (Fig. 5) at m/z 210. All other product ions were identical to those detected for the unchanged drug (Table 2). Metabolite 16c is therefore considered to be the result of a O-demethylation reaction at the C domain of trabectedin. and 511 (Table 2) as compared to trabectedin. However, in comparison with unchanged drug, a different accurate mass was observed (Table 3), indicating a different elemental composition. The abundant water loss from the molecular ion, seen in full MS and MS/MS, and the presence of a small product ion at m/z 206, suggested that the hydroxyl function at the carbinolamine moiety was still intact.

Metabolite 16d is therefore thought to be the result of a demethylation and a monooxidation in combination with a loss of two hydrogen atoms at the trabectedin A domain.

Metabolite 16e

The protonated molecular ion of metabolite 16e (m/z 790) and several product ions at m/z 503, 521, 523 and 539 (Table 2) were shifted 28 amu as compared to trabectedin. A loss of 46 amu (-HCOOH) from the product ions at m/z 521 and 539, giving product ions at m/z 475 and 493, respectively, and the absence of a water loss from the molecular ion suggesting the absence of the hydroxyl function at the carbinolamine moiety. This indicated that metabolite 16e results from carboxylic acid formation in combination with a mono-oxidation at the A subunit of trabectedin.

A proposed metabolic scheme of trabectedin based on the in vitro metabolic data for monkey and man is presented in Fig. were present in non-boiled samples of both human and monkey liver microsomes, constituting 4% of the injected radioactivity at the most (data not shown). These results indicate that trabectedin is not substantially metabolized via glucuronidation in vitro. However, the incubations with liver microsomes in the presence of UDPGA did not allow one to assess possible glucuronidation of phase I metabolites of trabectedin. Conversely, the omission of UDPGA in incubates with 12,000 x g supernatants in the presence of a NADPH-generating system likely prevented oxidative metabolites from undergoing subsequent glucuronidation. To further delineate the involvement of specific CYP isoforms in the metabolism of trabectedin, separate inhibition experiments were performed at a clinically relevant trabectedin drug level. To that aim, the effect of chemical and immunoinhibition on the disappearance of trabectedin was investigated in pooled human liver microsomes at a trabectedin concentration of 10 ng/mL (13 nM). In Fig. 7, the disappearance of as well as by selective inhibitory antibodies directed against CYP3A4.

CYP phenotyping

Discussion

Available data indicate that trabectedin is extensively metabolized in humans and that its oxidative metabolism is very complex, resulting in a large number of metabolites [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF]. Despite the longstanding clinical use of the compound, its metabolism has not been well defined. In vivo investigations have been especially difficult because of several compound related factors including the low dose, the extent and complexity of metabolism, the extremely low metabolite concentrations, low faecal extraction recoveries, and stability issues with trabectedin and some of its metabolites [START_REF] Sparidans | Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man[END_REF][START_REF] Beumer | Hepatotoxicity and metabolism of trabectedin: a literature review[END_REF][START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF]. Owing to these complicating factors, literature data on the identification of trabectedin metabolites are scarce. Sparidans et al. [START_REF] Sparidans | Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man[END_REF] identified a few metabolites and degradation products in in vitro incubates with human liver microsomes and plasma, but attempts to detect these metabolites in patient samples were unsuccessful. Reid and co-workers [START_REF] Reid | Rat and human liver cytochrome P-450 Isoform Metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans[END_REF] Radio-HPLC analysis of urine and faeces samples indicated the formation of a large number of trabectedin metabolites. However, the structure of only a few putative trabectedin metabolites (ET-745, ET-759A, ETM-259, ETM-217, and ET-204) was tentatively identified [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF].

Vermeir et al. [START_REF] Vermeir | An interspecies comparison of the metabolism of the anticancer agent Yondelis TM (Trabectedin, ET-743)[END_REF] investigated the in vitro metabolism of trabectedin in hepatic subcellular fractions of various species including humans. Data from that study suggested similarity in trabectedin's hepatic metabolism between Cynomolgus monkeys and humans, while that of other species including rat and dog were quite different. However, the data from that study were considered suboptimal and inconclusive in a sense that the identification of some metabolites was mainly based on co-chromatography with authentic reference compounds, leaving the majority of the biotransformation products unidentified.

To facilitate the identification of the major metabolites, several parameters were optimized in the current in vitro study, including the selection of the substrate concentration (5 µM instead of 1.6 µM) and the use of a highly sensitive QTOF Ultima mass spectrometer. In addition, the risk for degradation of trabectedin and/or its metabolites was minimized by immediate injection of the samples after thawing.

In the present work, the structure of major human and monkey in vitro metabolites was elucidated. Identified metabolites mainly resulted from biotransformations at the trabectedin A domain and included mono-oxidation and di-oxidation, carboxylic acid O-demethylation at the C subunit. Individual LC-MS/MS analyses of human and monkey samples showed that all metabolites identified in human liver fractions were also present in those of the Cynomolgus monkey. Radio-HPLC analysis showed that trabectedin's metabolism is faster in monkeys than in humans. In addition, trabectedin proved to be more actively metabolized via N-demethylation and/or via demethylation with additional mono-, di-or tri-oxidation in monkeys than in humans.

With the exception of ET-729, none of the proposed in vivo trabectedin metabolites identified by Beumer et al. [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF] were detected in the current in vitro study. This may be explained by several limitations of the in vivo metabolite profiling study. In the latter study, only metabolite fragments from available reference compounds were monitored in the in vivo samples by LC-MS/MS, which precluded the detection of trabectedin's oxidized metabolites, as identified in the current in vitro study. In addition, the low extraction efficiency (about 30%) from the faeces, i.e. the major route of excretion, and the instability of metabolites during sample workup may also have contributed to the apparent discrepancy between the current in vitro and previously published in vivo metabolite profiling data.

In patient samples, ET-729 was detected as a potential metabolite in faeces extracts, but ET-729 was also identified as chemical degradant [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF]. LC-MS analysis of in vivo samples from trabectedin-treated patients did not result in the detection of ET-729

A c c e p t e d M a n u s c r i p t 27 [START_REF] Sparidans | Search for metabolites of ecteinascidin 743, a novel, marine-derived, anti-cancer agent, in man[END_REF]. The present in vitro work showed that formation of this metabolite through N-demethylation is a genuine metabolic conversion.

In the in vivo metabolite profiling work of Beumer et al. [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF], several ETM-coded compounds resulting from breaking up of the trabectedin molecule to its individual tetrahydroisoquinoline subunits have been proposed as possible trabectedin metabolites. These compounds were not detected in the present in vitro work.

Formation of this kind of compounds via metabolic conversion is not very likely and it has not been described for any structurally-related compound. It is conceivable that they are in fact artefacts resulting from fragmentation of trabectedin and/or its metabolites in the source of the mass spectrometer. Both the dehydroxylated metabolite ET-745 and its corresponding N-dealkylated metabolite ET-731 were detected in the faeces of patients, but not in the current in vitro study. Beumer et al. [START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF] argued that the unusual dehydroxylation reaction probably occurs through the activity of intestinal bacterial flora.

Possible glucuronidation of trabectedin was studied in a separate set of experiments.

Because of the experimental design, possible glucuronidation of phase I metabolites of trabectedin could not be evaluated. The results suggested that trabectedin may be glucuronidated, albeit to a limited extent. Brandon and co-workers [START_REF] Brandon | In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis ® , Trabectedin), a novel marine anti-cancer drug[END_REF] have published similar findings, and additionally demonstrated conjugation by glutathione-S-transferase, but not by sulfotransferase. Although the kinetics of trabectedin UGTmediated glucuronidation have not been studied, the in vivo relevance of this pathway is questionable given the generally low affinity of UGT substrates towards A c c e p t e d M a n u s c r i p t 28 these enzymes [START_REF] Williams | Drugdrug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios[END_REF][START_REF] Kiang | UDP glucuronosyltransferases and clinical drug-drug interactions[END_REF]. This may also hold true for glutathione-S-transferase mediated conjugation as the amount of substrate consumed was limited [START_REF] Brandon | In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis ® , Trabectedin), a novel marine anti-cancer drug[END_REF].

From both a drug development and a regulatory perspective, metabolism data are important as they can be used to support the selection of animal species for toxicity testing and to support/explain clinical findings [START_REF] Lin | Role of pharmacokinetics and metabolism in drug discovery and development[END_REF]. Such data are considered particularly relevant to trabectedin, which has been shown to increase various markers of hepatotoxicity in all species, but with marked interspecies differences [START_REF] Beumer | Hepatotoxicity and metabolism of trabectedin: a literature review[END_REF].

Previous in vitro metabolism data [START_REF] Vermeir | An interspecies comparison of the metabolism of the anticancer agent Yondelis TM (Trabectedin, ET-743)[END_REF] suggested similarity in trabectedin's hepatic metabolism between Cynomolgus monkeys and humans. The in vitro metabolism data in the present study, together with the comparable in vivo toxicological profiles [START_REF] Verbeeck | Hepatotoxicological differences with ET-743 between Sprague Dawley rats and Cynomolgus monkeys[END_REF][START_REF] Vynckier | Hepatotoxicological differences with ET-743 in Sprague-Dawley Rats and Cynomolgus Monkeys[END_REF], provided further supporting evidence in favour of the use of the Cynomolgus monkey as relevant and predictive animal species to evaluate human trabectedin toxicity.

The involvement of CYP in the human hepatic metabolism of trabectedin has previously been studied by several investigators [START_REF] Reid | Rat and human liver cytochrome P-450 Isoform Metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans[END_REF][START_REF] Brandon | In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis ® , Trabectedin), a novel marine anti-cancer drug[END_REF]. Reid and co-workers [START_REF] Reid | Rat and human liver cytochrome P-450 Isoform Metabolism of Ecteinascidin 743 does not predict gender-dependent toxicity in humans[END_REF] incubated trabectedin (50 µM or 38. To determine the CYP isoform(s) involved at clinically relevant concentrations of trabectedin (ng/ml range), a second set of experiments was performed. In these experiments, a substrate depletion approach was adopted since it was technically not feasible to monitor the formation of the various trabectedin metabolites at low ng/ml substrate concentrations. A sensitive LC-MS/MS method was used to monitor the antibodies directed against CYP3A4. This suggests that CYP3A4 is the predominant isoform implicated in the hepatic metabolism of trabectedin at therapeutically achievable concentrations.

Brandon et al. [START_REF] Brandon | In-vitro cytotoxicity of ET-743 (Trabectedin, Yondelis), a marine anti-cancer drug, in the Hep G2 cell line: influence of cytochrome P450 and phase II inhibition, and cytochrome P450 induction[END_REF] suggested that in addition to CYP3A4, trabectedin concentrations in patients may also be increased through inhibition of CYP2C9, 2C19, 2D6, and 2E1. The authors based their conclusions on in vitro cytotoxicity measurements of trabectedin in HepG2 cells in the presence and in the absence of metabolic inhibitors and inducers. However, the use of HepG2 cells to study in vitro metabolism-based drug-drug interactions is inappropriate since this cell line is known to express extremely low levels of drug metabolizing enzymes, making it unsuitable for such investigations [START_REF] Brandon | An update on in vitro test methods in human hepatic drug biotransformation research: pros and cons[END_REF][START_REF] Vermeir | Cell-based models to study hepatic drug metabolism and enzyme induction in humans[END_REF]. In addition, the choice and concentration of the employed chemical inhibitors or inducers as diagnostic tools, was not optimal in most cases [START_REF]Food and Drug Administration Draft Guidance for Industry: Drug Interaction Studies -Study Design, Data Analysis, and Implications for Dosing and Labeling[END_REF].

In conclusion, data from the current studies confirmed that the metabolism of In vitro metabolic profiling in hepatic 12,000 x g supernatant fractions

Metabolite

Human Cynomolgus monkey 4a

√ (2) √ 4b √ √ Relative amount of 4a+4b ++ ++ 6a √ √ 6b √ √ Relative amount of 6a+6b + + 8a √ √ 8b √ √ 8c √ √ 9 √ √ Relative amount of 8a+8b+8c+9 +++ +++ 11a √ √ Relative amount of 11a+11b (1) + + 16b √ √ 16c √ √ 16d √ √ 16e √ √ Relative amount of 16b+16c+16d+16e ++ ++ UD √ √
Relative amount of unchanged drug (trabectedin) ++ ++ (1) 11b is a chemical degradant (2) A c c e p t e d M a n u s c r i p t Fig. 1 M a n u s c r i p t

N N R 1 O O H 3 C R 3 O R 2 OCH 3 CH 3 HO S O NH X HO O H H 3 CO R 4 A B C R 1 R 2 R 3 R 4 X

u s c r i p t 3 *

 3 ** Current affiliation: Ablynx N.V., Technologiepark 4, B-9052 Zwijnaarde, Belgium

A c c e p t e d M a n u s c r i p t 8 chemical

 8 inhibitors used in the CYP phenotyping experiments were obtained from the following sources: furafylline (Ultrafine Chemicals, Manchester, UK); 8-methoxypsoralen, quinidine and ketoconazole (Janssen Chimica, Geel, Belgium); trimethoprim and sulfaphenazole (Sigma-Aldrich, St.-Louis, MO, USA); diethyldithiocarbamate (Merieux, Lyon, France); troleandomycin (Pfizer, New York, NY, USA) and (±)-3-benzyl-phenobarbital (Synthesis BA/DMPK department of J&JPRD). Glucose-6-phosphate dehydrogenase, glucose-6-phosphate and NADP were obtained from Roche Diagnostics (Mannheim, Germany). All other chemicals

[ 14 C

 14 ]trabectedin in DMSO (radioactivity concentration of 1.08 MBq/ml) were prepared. The stock solutions of [ 14 C]trabectedin were prepared immediately before the incubations, in order to minimize chemical degradation. The incubations were carried out in a total volume of 2 ml at a final [ 14 C]trabectedin concentration of 5 µM (5.4 kBq/ml) for 120 min at 37 °C under continuous shaking at 100 oscillations/min.

A c c e p t e d M a n u s c r i p t 12 the

 12 samples were incubated for 0 and 20 min under conditions identical to those described for the chemical inhibitors. Control incubates with pre-immune IgG from rabbit were run in parallel.

  after thawing the samples. The HPLC apparatus consisted of a Waters 2695 Alliance Separation Module. The samples were chromatographed on a stainless steel column (30 cm x 4.6 mm i.d.) packed with Kromasil C-18 (5 µm, Akzo Nobel).

A c c e p t e d M a n u s c r i p t 13 On

 13 -line radioactivity detection of HPLC eluates was carried out with a Berthold Radioactivity Monitor LB 509 system. The eluates were mixed with Ultima-Flo™ (Packard) as a scintillation cocktail delivered by a Berthold LB 5035-3 pump at a flow rate of 4.0 ml/min. UV-detection was performed at 280 nm using a Waters 996-diode array detector. Elution started with a linear gradient at a flow rate of 1 ml/min from 100% solvent system A (0.1 M ammonium acetate pH 6.25) to 100% solvent system B (water/acetonitrile, 10:90, by vol.) over 60 min followed by continued elution in the latter solvent mixture for 5 min. Injected amounts of radioactivity in the samples ranged from about 18,000 to about 61,000 dpm.

M a n u s c r i p t 14 1

 14 QTOF (quadrupole time-of-flight) Ultima mass spectrometer (Waters, Millford, MA, USA) was used for metabolite identification. The mass spectrometer was equipped with a dual electrospray ionization probe and was operated in the positive ion mode at a resolution of 8000 (FWHM at m/z 409). The source temperature was 100 °C, desolvation temperature 250 °C and the cone voltage was set at 40 V. The second LockSpray TM ESI probe provided an independent source of the lock mass calibrant H 3 PO 4 .NH 4 + . The cluster ion at m/z 409.94184 was used as the calibrant in full MS, whereas the ion at m/z 392.91534 (daughter ion of m/z 490.9) was used as the lock mass in MS/MS mode. Data were acquired in the centroid mode with a scantime of A c c e p t e d s and processed using Masslynx v. 4.0 software. The same chromatographic conditions were used as for the radio-HPLC analyses.

A c c e p t e d M a n u s c r i p t 15 50%.

 15 After 3 minutes, the trapping column was back-flushed with a water/methanol mixture (5/95; v/v) at a flow rate of 0.5 ml/min and the analyte and its internal standard eluted onto the analytical column (30 x 2.1 mm ID; packed with 3 µm Atlantis Hilic silica) connected to the mass spectrometer (triple quadrupole API 4000). Ionisation was by turbo ion spray, operated in the positive mode. Trabectedin and its internal standard were monitored at mass transitions m/z 744.3 → 495 and 747.3 → 496, respectively.

  A c c e p t e d M a n u s c r i p t 17 LC-MS/MS analysis (see below) revealed that the trabectedin metabolites identified in hepatic subcellular fractions of Cynomolgus monkey were similar to those of human.

A c c e p t e d M a n u s c r i p t 18 Metabolite 4a

 184a The mass spectrum of metabolite 4a revealed a protonated molecular ion at m/z 780, corresponding to a 18 amu shift relative to trabectedin (m/z 762). A similar 18 amu shift was noticed in the MS/MS fragmentation of specific protonated molecular ion fragments (Table2) while the fragments at m/z 224 and 260 were unchanged. The product ions at m/z 234 and 493 are the result of water loss of fragments m/z 252 and 511, respectively. The LC-MS/MS data indicate that metabolite 4a is the result of a demethylation in combination with a double oxidation at the A domain of trabectedin.

A c c e p t e d M a n u s c r i p t 19 Metabolite 8a

 198a The protonated molecular ion of metabolite 8a had a 30 amu shift (m/z 792) relative to trabectedin. Several product ions at m/z 236, 264, 505, 523, 525 and 541 displayed a 30 amu shift as well. The loss of 46 amu from fragment ions at m/z 523 and 541, giving product ions at m/z 477 and 495, respectively, suggested carboxylic acid formation at the A domain of the molecule.

CA c c e p t e d M a n u s c r i p t 20 Metabolite 9

 209 38 H 40 N 3 O 13 S. The accurate mass measurements therefore suggested that metabolite 8c is likely the result of demethylation in combination with a triple oxidation, including an oxidation of the carbinolamine moiety to an amide, at the trabectedin A domain. The protonated molecular ion was observed at m/z 750, or 12 amu less than the drug substance (m/z 762). This 12 amu shift was also reflected in several product ions (

A c c e p t e d M a n u s c r i p t 21 Metabolite 16d

 2116d Metabolite 16d showed the same protonated molecular ion (m/z 762) and corresponding MS/MS product ions at m/z 206, 224, 260, 475, 493, 495

6 .

 6 Trabectedin was metabolized via different biotransformation pathways including N-demethylation, O-demethylation, carboxylic acid formation (alone or in combination with oxidation), mono-and di-oxidation, aliphatic ring opening, and demethylation in combination with mono-, di-or tri-A c c e p t e d M a n u s c r i p t 22 oxidation. Most of the metabolic conversions occurred at the trabectedin A domain including mono-oxidation (metabolite 11a) and di-oxidation (metabolite 16b), carboxylic acid formation with and without additional oxidation (metabolites 16e and 8a, respectively), and demethylation either without (metabolite 8b, ET-729) or with additional mono-oxidation (metabolites 4b and 6a), di-oxidation (metabolites 4a, 16d and 6b) or tri-oxidation (metabolite 8c). Only two metabolic conversions were detected at the B and C domains of trabectedin, viz. aliphatic ring opening of the methylene dioxybridge at the B domain (metabolite 9) and O-demethylation at the C subunit (metabolite 16c). Overall, demethylation and oxidation played a major role in phase I metabolism of the drug. All biotransformation pathways were observed in liver subcellular fractions of both the Cynomolgus monkey and human. Besides these metabolic products, LC-MS/MS analysis revealed the presence of traces of four cyano compounds in incubations with both monkey and human 12,000 x g liver supernatants. The occurrence of these compounds (coded 11b, 20, 21 and 22) in the incubates can hardly be explained by metabolism. They are considered to be degradation products rather than genuine metabolites. Their presence is probably due to cyano impurities in the [ 14 C]trabectedin batch. Compound 20, the cyano derivative of unchanged trabectedin, was observed in incubates with boiled liver subcellular fractions. The other compounds might be formed chemically as cyano derivatives of biotransformation products after demethylation and/or aliphatic ring opening. Upon incubation of [ 14 C]trabectedin with monkey and human liver microsomes in the presence of UDPGA, two peaks corresponding to unchanged drug substance and A c c e p t e d M a n u s c r i p t 23 the degradation compound 20, were predominantly present in radio-HPLC chromatograms of both boiled and non-boiled samples. Two additional minor peaks

[ 14 C

 14 ]trabectedin (5 µM) was incubated for 120 minutes with E. coli membranes, which stably express single human CYP isozymes together with NADPH-P450 reductase. Incubation with heterologously expressed CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5 resulted in disappearance of the parent compound and concurrent formation of radiolabelled metabolites. In contrast, CYP1B1, 2C19, and 4A11 did not metabolize the drug to a notable extent (data not shown).

A c c e p t e d M a n u s c r i p t 24 trabectedin

 24 from the incubation mixture as a function of incubation time is presented in the absence and in the presence of CYP-isoform selective chemical inhibitors and inhibitory antibodies. The results from these experiments showed that the disappearance of trabectedin from the incubation mixture was virtually completely inhibited by selective chemical CYP3A4 inhibitors (ketoconazole and troleandomycin)

A c c e p t e d M a n u s c r i p t 25 Recently,

 25 Beumer and co-workers[START_REF] Beumer | Metabolism of trabectedin (ET-743, Yondelis TM ) in patients with advanced cancer[END_REF] reported on in vivo metabolite profiling work obtained from a clinical mass balance study with [ 14 C]trabectedin in cancer patients.

A c c e p t e d M a n u s c r i p t 26 formation

 26 with and without additional oxidation, and demethylation either without (N-demethylation to ET-729) or with additional mono-, di-or tri-oxidation. Only two metabolic conversions were detected at the B and C domains of trabectedin, viz. aliphatic ring opening of the methylene dioxybridge at the B domain and

A c c e p t e d M a n u s c r i p t 29 in

 29 pooled human liver microsomes and CYP supersomes. Trabectedin was significantly metabolized in CYP2C9, 2C19, 2D6, 2E1 and 3A4 supersomes[START_REF] Brandon | In vitro characterization of the human biotransformation and CYP reaction phenotype of ET-743 (Yondelis ® , Trabectedin), a novel marine anti-cancer drug[END_REF]. In addition, they determined enzyme kinetic parameters for the overall metabolism of trabectedin in pooled human liver microsomes and CYP supersomes, and estimated the relative contributions of individual CYPs from these data. However, with exception of CYP3A4 and CYP2C9 supersomes, the calculated K m -values for the depletion of trabectedin were not comprised by the tested concentration range, impacting the reliability of the determined enzyme kinetic parameters, and the estimation of the contribution of individual CYPs.The present studies were aimed at further defining the involvement of CYP in the hepatic metabolism of trabectedin. In a first set of experiments, in which [ 14 C]trabectedin (5 µM or 3.81 µg/ml) was incubated with heterologously expressed human CYPs, depletion of the parent compound and concomitant formation of radiolabelled metabolites was observed after incubation with CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, 3A4 and 3A5. Virtually no metabolite formation was seen with CYP1B1, 2C19, and 4A11. In the latter experiments a supra-therapeutic concentration of trabectedin was used to allow the detection of newly formed radiolabelled metabolites.

A c c e p t e d M a n u s c r i p t 30 temporal

 30 depletion of a therapeutically relevant trabectedin concentration (10 ng/ml) from the incubation mixture. Selective chemical inhibitors and inhibitory antibodies were used as diagnostic tools. Results from these experiments showed that trabectedin metabolism was completely abrogated by selective chemical CYP3A4 inhibitors (ketoconazole and troleandomycin) as well as selective inhibitory

[ 14 C

 14 ]trabectedin is qualitatively similar in liver subcellular fractions of the Cynomolgus monkey and human. Notably, all human hepatic trabectedin metabolites are also produced by the Cynomolgus monkey, supporting the use of the Cynomolgus monkey as relevant animal model for the safety assessment of trabectedin in cancerA c c e p t e d M a n u s c r i p t 31 patients. Most of the metabolites result from biotransformations at the trabectedin A subunit, and demethylation and oxidation prove to be major metabolic conversions.

Fig. 1 :Fig. 2 :

 12 Fig. 1: Chemical structure of trabectedin and authentic reference compounds used

Fig. 3 :

 3 Fig. 3: Mass spectrometric fragmentation of trabectedin.

Fig. 4 :Fig. 5 :

 45 Fig. 4: MS 2 spectra of (a) metabolite 4a, (b) metabolite 4b, (c) metabolite 6a,

Fig. 6 :Fig. 7 :

 67 Fig. 6: Proposed in vitro metabolic pathways of trabectedin in hepatic 12,000 x g

  Fig. 2

Fig. 3

 3 Fig.3

Fig. 4

 4 Fig.4

Fig. 5

 5 Fig.5

Fig. 6

 6 Fig. 6

  14

Table 2 )

 2 . Based on the elemental composition (C 38 H 44 N 3 O 11 S)

Table 1 :

 1 Comparative metabolism of [ 14 C]trabectedin in hepatic 12,000 x g supernatant fractions of humans and Cynomolgus monkeys. Relative amounts are expressed as + (1-5%), ++ (>5-15%), +++ (>15%).

	Data from CYP phenotyping experiments demonstrated that virtually all major CYPs
	are able to metabolize trabectedin, but that CYP3A4 is the main human CYP isoform
	at clinically achievable levels. In the absence of clinical drug-drug interaction data,
	caution needs to be exerted when potent CYP3A4 inhibitors or inducers are added to
	an existing trabectedin regimen.

Table 3 :

 3 Chemical formulas and accurate mass measurements of trabectedin and metabolites in incubates of [ 14 C]trabectedin with hepatic 12,000 x g supernatant fractions of human and Cynomolgus monkey.

	detected

Acknowledgements

The authors are grateful to Dr. Cor Janssen, Dr. Tom Verhaeghe, Bjorn Verreet and Luc Diels, BA/DMPK department of J&JPRD, for technical assistance, and to Mieke Roels for administrative help.


Jana Reinemund

Kerstin Seidel

Ulrike M Steckelings

Daniela Zaade

Sabrina Klare

Franziska Rompe

Marlen Katerbaum

Jens Schacherl

Yaosi Li

Mario Menk

Jan H Schefe

Petra Goldin-Lang

Csaba Szabo

Gabor Olah

Thomas Unger

Heiko Funke-Kaiser
email: heiko.funke-kaiser@charite.de

Poly(ADP-ribose) polymerase-1 (PARP-1) transcriptionally regulates angiotensin AT2 receptor (AT2R) and AT2R binding protein (ATBP) genes

Keywords:

come

A c c e p t e d M a n u s c r i p t

ABSTRACT

The renin-angiotensin system (RAS) plays a crucial role in cardiovascular and neuronal (patho-)physiology. The angiotensin AT2 receptor (AT2R) seems to counteract the proinflammatory, prohypertrophic and profibrotic actions of the AT1 receptor. Recently, we identified a novel protein, termed "AT2R binding protein" (ATBP/ ATIP) which seems essential for AT2R mediated growth inhibition.

Poly(ADP-ribose) polymerase-1 (PARP-1) can act as a nuclear integrator of angiotensin II-mediated cell signalling, and has been implicated in the pathogenesis of cardiovascular and neuronal disease.

In this study, promoters of human AT2R and ATIP1 were cloned and two transcriptional start sites in the ATIP1 promoter were identified whereas only one was detected in the AT2R promoter. Promoter assays indicated that the exon 1intron 1 region of AT2R is necessary and sufficient for AT2R promoter activity.

Inverse cloning experiments indicated that this regulatory region is a promoter but not an enhancer element implicating (a) further start site(s) in this region.

Consistently, the exon 1-intron 1 region of AT2R was shown to tether the basal transcriptional machinery. Overexpression, pharmacological inhibition and ablation of PARP demonstrated that PARP-1 activates the ATIP1 gene but represses the AT2R on promoter and mRNA levels in vitro, and in brain tissue in vivo. Additional experiments indicated that AT2R activation does not modulate PARP-1 transcript levels but increases AT2R promoter activity, thereby creating a positive feedback mechanism.

Our results demonstrate that PARP-1 acts as novel node within the RAS network based on its ability to regulate downstream targets such as AT2R and its adapter protein ATBP. Abbreviations: RAS, renin-angiotensin system; AT1R, angiotensin AT1 receptor; AT2R, angiotensin AT2 receptor; PARP-1, poly(ADP-ribose) polymerase-1; SHP-1, SH2 domain-containing phosphatase 1; PLZF, promyelocytic zinc finger protein; ATBP, AT2R binding protein; ATIP, AT2 receptor interacting protein; MTSG, mitochondrial tumor suppressor gene 1; PAR, poly(ADP-ribose); MEFs, mouse embryonic fibroblasts; RLA , relative luciferase activity; ChIP, chromatinimmunoprecipitation; TBP, TATA box-binding protein

Introduction

The renin-angiotensin system (RAS) constitutes one of the best characterized systems within mammalian physiology and pathophysiology. It is causally involved in a broad spectrum of diseases ranging from arterial hypertension, cardiac hypertrophy, diabetic nephropathy and stroke to cancer and inflammation [START_REF] De Gasparo | International union of pharmacology. XXIII. The angiotensin II receptors[END_REF][START_REF] Kaschina | Angiotensin AT1/AT2 receptors: regulation, signalling and function[END_REF][START_REF] Unger | The role of the renin-angiotensin system in the development of cardiovascular disease[END_REF][START_REF] Lindberg | Angiotensin converting enzyme inhibitors for cancer treatment?[END_REF].

The effects of the RAS are mainly mediated by two receptors, the angiotensin AT1 receptor (AT1R) and the angiotensin AT2 receptor (AT2R) [START_REF] De Gasparo | International union of pharmacology. XXIII. The angiotensin II receptors[END_REF]. Several groups have demonstrated that AT2R activation can counteract the vasoconstrictory, prohypertrophic and pro-proliferative effects of AT1R activation [START_REF] De Gasparo | International union of pharmacology. XXIII. The angiotensin II receptors[END_REF][START_REF] Stoll | The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells[END_REF][START_REF] Volpe | Angiotensin II AT2 receptor subtype: an uprising frontier in cardiovascular disease?[END_REF], in addition to its ability to mediate neuroprotective effects [START_REF] Reinecke | Angiotensin II accelerates functional recovery in the rat sciatic nerve in vivo: A c c e p t e d M a n u s c r i p t 26 role of the AT2 receptor and the transcription factor NF-kappaB[END_REF][START_REF] Li | Angiotensin AT2 receptor protects against cerebral ischemia-induced neuronal injury[END_REF]. Nevertheless, the (patho)physiological role of AT2R is still controversial [START_REF] Widdop | Angiotensin AT2 receptors: cardiovascular hope or hype?[END_REF][START_REF] Levy | Can angiotensin II type 2 receptors have deleterious effects in cardiovascular disease? Implications for therapeutic blockade of the reninangiotensin system[END_REF]. We recently postulated that the type of adapter protein recruited to the AT2R determines its cellular effects [START_REF] Su | Stimulation of AT2 receptors: role in the effect of angiotensin II receptor antagonists[END_REF]. The AT2R can couple to certain G proteins [START_REF] Zhang | The AT2 receptor selectively associates with Gialpha2 and Gialpha3 in the rat fetus[END_REF][START_REF] Hansen | Functional reconstitution of the angiotensin II type 2 receptor and G(i) activation[END_REF] and the SH2 domain-containing phosphatase 1 (SHP-1) [START_REF] Feng | Gbeta gamma -independent constitutive association of Galpha s with SHP-1 and angiotensin II receptor AT2 is essential in AT2-mediated ITIM-independent activation of SHP-1[END_REF]. The physical interaction with the latter is probably related to the antiproliferative effects of the AT2R [START_REF] Widdop | Angiotensin AT2 receptors: cardiovascular hope or hype?[END_REF][START_REF] Cui | Pivotal role of tyrosine phosphatase SHP-1 in AT2 receptor-mediated apoptosis in rat fetal vascular smooth muscle cell[END_REF]. In contrast, recruitment of the promyelocytic zinc finger protein (PLZF) to the AT2R was shown to be associated with an increase in protein synthesis of cardiomyocytes and cardiac hypertrophy [START_REF] Senbonmatsu | A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy[END_REF].

Our group was recently able to identify ATBP (AT2R binding protein) as a novel protein with the ability to bind to the cytoplasmic C-terminus of the AT2R [START_REF] Wruck | Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein[END_REF].

ATBP is expressed in at least three different isoforms termed ATBP50, ATBP60 and ATBP135 [START_REF] Wruck | Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein[END_REF]. ATBP50 plays a crucial role concerning the transport of the AT2R from the Golgi compartment to the cell membrane, and mediates the inhibitory effects on MAP kinases as well as the antiproliferative effects of the AT2R in neuroblastoma cells [START_REF] Wruck | Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein[END_REF]. The ATBP gene was simultaneously cloned by A c c e p t e d M a n u s c r i p t 5 two other groups who termed this gene ATIP (AT2 receptor interacting protein) [START_REF] Nouet | Transinactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP[END_REF][START_REF] Benedetto | Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptorinteracting proteins[END_REF] and MTSG (mitochondrial tumor suppressor gene 1) [START_REF] Seibold | Identification of a new tumor suppressor gene located at chromosome 8p21.3-22[END_REF], respectively, and who also demonstrated the inhibitory effects of this protein on cell proliferation and MAP kinases. ATIP1 (= ATBP50) is the major isoform since it was initially identified by the yeast two-hybrid screenings [START_REF] Wruck | Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein[END_REF][START_REF] Nouet | Transinactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP[END_REF] , is expressed ubiquitously [START_REF] Wruck | Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein[END_REF][START_REF] Benedetto | Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptorinteracting proteins[END_REF] and constitutes the most abundant isoform e.g. in brain, heart, breast and ovary [START_REF] Benedetto | Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptorinteracting proteins[END_REF]. ATBP can also function as a tumor suppressor gene [START_REF] Seibold | Identification of a new tumor suppressor gene located at chromosome 8p21.3-22[END_REF] and very recently it was shown that a genetic copy number variant (CNV) affecting the ATBP gene locus as a deletion polymorphism is significantly associated with a decreased risk of breast cancer [START_REF] Frank | Copy number variant in the candidate tumor suppressor gene MTUS1 and familial breast cancer risk[END_REF].

PARP-1 is an ubiquitous nuclear protein catalysing the attachment of poly(ADPribose) (PAR) polymers to several different acceptor proteins such as histones and RNA polymerase II [START_REF] Kraus | PARP goes transcription[END_REF][START_REF] D'amours | Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions[END_REF]. PARP-1 is involved in pathophysiologies such as hypertension, stroke, neuronal injury, diabetes and inflammation [START_REF] Jagtap | Poly(ADP-ribose) polymerase and the therapeutic effects of its inhibitors[END_REF] thereby contributing to similar disease states as the RAS. Consistently, animal experiments indicated that cardiac hypertrophy and also endothelial dysfunction induced by angiotensin II are absent in homozygous PARP-1 deficient mice [START_REF] Pillai | Poly(ADP-ribose) polymerase-1-deficient mice are protected from angiotensin II-induced cardiac hypertrophy[END_REF][START_REF] Szabo | Angiotensin II-mediated endothelial dysfunction: role of poly(ADP-ribose) polymerase activation[END_REF]. Nevertheless, the regulatory mechanisms of this RAS-PARP interplay are largely unknown. Therefore, we analysed basal and PARP-mediated regulation of the AT2R and its interaction partner ATBP/ATIP on chromatin, promoter and mRNA levels. The human neuroblastoma cell line KELLY was a kind gift of the Physiology Department (Campus Mitte) of the Charité, Berlin, Germany. KELLY cells were cultured in RPMI (Invitrogen) supplemented with 10% FCS, 100 U/ml penicillin and 100 microg/ml streptomycin. For transfection experiments KELLY cells were cultured on plates coated with poly-L-lysine (0.005 mg/ml; Biochrom, Bremen, Germany). The human breast adenocarcinoma cell line MCF-7, a kind gift of G. Schönfelder (Toxicology Department, Charité), was cultured in high glucose DMEM supplemented with with 10% FCS and 1% penicillin and streptomycin (100 IU/ml). Mouse embryonic fibroblasts (MEFs) were obtained from Z.-Q. Wang (Leibniz Institute for Age Research, Jena, Germany) and were grown in high glucose DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 microg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 0.2% beta-mercaptoethanol (Invitrogen).

All cell lines were grown in a humidified atmosphere of 5% CO 2 in air and at 37°C. Cellular total RNA was isolated using the RNA Isolation kit from Macherey-Nagel (Düren, Germany).

For stimulation experiments the following small molecules were used: the nonselctive PARP inhibitor 3-aminobenzamide (3-AB; Sigma, Hamburg, Germany; 10 mM (EA.hy926) or 30 mM (KELLY)), the AT2R agonist Compound 21 (1 µM in A c c e p t e d M a n u s c r i p t 7 water or PBS; a kind gift of Vicore Pharma, Gothenburg, Sweden), the AT1R blocker irbesartan (10 µM in DMSO/PBS; applied 30 min before angiotensin II treatment; Sigma) and angiotensin II (0.1 µM in water; Sigma). Stimulation was performed 48 hours after transfection and 24 hours after starving. Harvest for RNA extraction or luciferase assay was performed 7 hours after stimulation.

Tissue samples

Tissue samples from homozygous PARP-1 knockout mice (PARP-1 -/-) derived from the knockout line which was described previously [START_REF] Wang | Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease[END_REF]. Total RNA of these samples was isolated with Trizol (Invitrogen, Karlsruhe, Germany) method. mRNA quantification by real-time PCR was performed based on 6 wild type and 6 knockout animals, each in technical triplicate. In brain tissue of these 6 + 6 mice, mRNA expression was evaluated based on three independent reverse transcriptions due to larger standard deviations.

The AT2R knockout mice were a kind gift of Michael Bader (Max Delbrück Center for Molecular Medicine (MDC), Berlin, Germany); real-time PCR data represent 2 animals in each group and technical triplicates.

Constructs

Serial deletion mutants of the human AT2R promoter were subcloned into the luciferase reporter vector pGL3-basic (Promega, Mannheim, Germany) using a common antisense primer located directly upstream of exon The "-1100"-construct" of human AT2R promoter was subcloned using an antisense primer located directly upstream of exon 1 (5´-aatagcaccagatctctggtttcct-3´) and the sense primer used for the "-1316"-construct. The "-216"-construct and "-148"-construct based on the antisense primer located directly upstream of exon 2 (see above) and the sense primers 5´-acgtcccagcgtctgagagaacgag-3´ and 5´taagtatgaacaatttatatataat-3´, respectively. The "-216-inverse"-construct was subcloned as the "-216"-construct but exchanging the restriction sites within the anchor primers.

Serial deletion mutants of the human ATIP1 promoter were subcloned into the luciferase reporter vector pGL3-basic (Promega) using a common antisense primer located directly upstream of the translational start site (5´- A PCR product (5´-gggaggatggcggagtcttcggata-3´ (sense primer) and 5´ttaccacagggaggtcttaaaattg-3´ (antisense primer)) comprising the complete open reading frame of human PARP-1 was subcloned into the expression vector pcDNA3.1-V5-His-TOPO (Invitrogen, Karlsruhe, Germany).

All constructs were confirmed by sequencing.

Quantitative RT-PCR

First-strand cDNA was synthesized by MLV-RT (Promega) using random hexamer primers (Promega). cDNA was quantified by real-time PCR using Power Mix on the pGL3-basic reporter vector we used following primers: 5´ctcgatatgtgcatctgtaaaagca-3´ and 5´-cttcatagccttatgcagttgctc-3´ (nested).

Promoter luciferase assays

Promoter reporter assays were performed as described previously [START_REF] Funke-Kaiser | Endothelin-converting enzyme-1 (ECE-1) is a downstream target of the homeobox transcription factor Nkx2-5[END_REF][START_REF] Schefe | A novel signal transduction cascade involving direct physical interaction of[END_REF] Sonification itself was performed using the Sonoplus HD 2070/ UW 2070 sonifier with the tip MS 72 (Bandelin Electronic, Berlin, Germany; output control (power %)= 100; time= 20 sec (once); constant duty cycle).

Quantitative PCR was performed as described above applying the following primers:

Human ATIP1 promoter around the transcriptional start site: 5´aggatctgtgacattattggtt-3´ (sense), 5´-aaaatgatgctcttcctctgaa-3´ (antisense) and human beta-actin promoter: 5΄-aatgctgcactgtgcggcga-3´ (sense), 5´ggcggatcggcaaaggcga-3´ (antisense).

Input (total) and genomic DNA served as positive controls. For combined ChIP and siRNA experiments, KELLY cells were transfected with siRNA against PARP-1 (#J-006656-05, Dharmacon, Chicago, USA) or a negative control siRNA (sc-37007, Santa Cruz) using Dharmafect (Dharmacon). 27 hours after transfection cells were fixed using 1% formaldehyde for 3 min at 37°C. did not exhibit significant luciferase activity whereas the 216 bp region itself displayed promoter activity (Fig. 2A). A similar effect was observed in human endothelial EA.hy926 cells (not shown). Furthermore, upstream promoter elements (> 316 bp relative to exon 2) do not contribute to human AT2R promoter activity to a major degree (Fig. 2A). To examine whether the 216 bp exon 1-intron 1 region constitutes an independent promoter or an enhancer element -with the latter defined by an orientation independence (31) -we subcloned this region in an inverse orientation into the reporter vector quasi abolishing promoter activity (Fig. 2B). This promoter property of the exon 1-intron 1 region of the AT2R gene implies an additional transcriptional start site. We therefore performed a further 5´-RACE experiment after transfection of the [-216] promoter construct using antisense primers complementary to the luciferase gene of the reporter plasmid in order to A c c e p t e d M a n u s c r i p t 16 increase the sensitivity. Sequencing of the RACE PCR product revealed the expected splicing of intron 1 (Fig. 1C). In addition, we observed a transcriptional start site about 60 bp upstream of the 5´ end of exon 1 (Fig. 1C). This indicates that the 216 bp region consisting of exon 1 and intron 1 of the AT2R gene is capable of tethering the basal transcriptional machinery.

Concerning the serial deletion mutants of the human ATIP1 promoter, the construct comprising 300 bp directly upstream of the translational start site (i.e., the [-300] construct) showed significant promoter activity (Fig. 2C). Extension to 500 bp further increased luciferase activity (Fig. 2C). These observations are consistent with the location of the two transcriptional start sites that reside in the [-300] and [-500] construct, respectively. Maximum promoter activity was measured using the [-700] construct of human ATIP1 promoter in epithelial MCF7 (Fig. 2C) as well as in endothelial EA.hy926 cells (Fig. 4).

PARP-1 represses the AT2R gene

To examine whether PARP-1 regulates the AT2R gene as a downstream target, human neuronal KELLY cells were incubated with the PARP inhibitor 3-AB. These cells were chosen because of their basal AT2R mRNA expression in contrast to 9 different human endothelial (EA.hy926), epithelial (HeLa-S3, HEK293, MCF7), glial (T98G, U-87MG, U-373MG) and neuronal (SK-N-AS, SH-SY5Y) cell lines (not shown). Pharmacological inhibition of PARP caused an increase in AT2R mRNA to over 150% compared to the vehicle control (Fig. 3A). To validate these findings, AT2R mRNA expression was quantified in embryonic fibroblasts derived from homozygous PARP-1 knockout mice. In wild type control cells AT2R mRNA was To analyse corresponding regulatory mechanisms on promoter level, serial deletion mutants of the human AT2R promoter were transfected in the presence or absence of the PARP inhibitor 3-AB. PARP inhibition caused a significant derepression of all constructs containing intron 1 but not of the [-1100] construct without this region (Fig. 3C). Consistently, an increase in luciferase reporter activity of all promoter constructs containing the intron 1 region was also observed in PARP-1 knockout cells (Fig. 3D).

Finally, the complete coding sequence of human PARP-1 was subcloned into an expression vector and cotransfected into neuronal KELLY cells (Fig. 3E). PARP-1 overexpression decreased the promoter activities of three different constructs of the AT2R regulatory region containing intron 1 but not of the [-1100] reporter plasmid without this sequence. The 216 bp exon 1-intron 1 region alone was repressed by PARP-1 cotransfection to a similar degree (i.e., to about 45%) compared to the full length construct (Fig. 3E). The exon 1-intron 1 region cloned in an inverse orientation was not modulated by PARP-1 cotransfection (data not shown).

To analyse the regulation of AT2R by PARP on the protein level, KELLY cells were incubated with 3-AB and AT2R expression was visualised by immunocytology (Fig. 3F). PARP inhibition caused a clear increase of the AT2R in the plasma membrane region. Consistently, PARP-1 ablation caused a general upregulation of the AT2R protein expression in MEF cells (Fig. 3F).

The direct involvement of PARP-1, i.e., the binding of PARP-1 to the upstream and downstream promoter region of the AT2R genes, was demonstrated in neuronal A c c e p t e d M a n u s c r i p t 18 cells by chromatin-immunoprecipitation (ChIP) (Fig. 3G). The specificity of these findings was confirmed by siRNA directed against PARP-1 and by a negative antibody control (Fig. 3G).

PARP-1 activates the ATIP1/ ATBP50 gene

To study putative effects of PARP-1 on ATIP1 gene expression, endothelial cells were incubated with the PARP inhibitor 3-AB causing a downregulation of ATIP1 mRNA to 24.3% compared to vehicle control (Fig. 4A). This finding was further confirmed in cells derived from PARP-1 knockout mice (Fig. 4B). PARP deficiency decreased ATBP50 mRNA to about 73.6% compared to wild type control cells.

Additionally, we quantified ATIP1 mRNA expression after PARP-1 knockdown by siRNA in human endothelial cells and observed a strongly reduced ATIP1 expression (about 20% compared to the control transfection) (Fig. 4F).

To further analyse whether this regulation occurs on the transcriptional level, the serial deletion mutants of human ATIP1 promoter were transfected into EA.hy926 cells in the presence or absence of the PARP inhibitor 3-AB. Consistently with the real-time PCR data, PARP inhibition caused a repression of all promoter luciferase reporter constructs tested (Fig. 4C). Moreover, these serial deletion mutants were transfected in cells derived from PARP-1 knockout mice and respective controls and, similar to 3-AB, a suppression of ATIP1 promoter activities by PARP-1 ablation could be observed (Fig. 4D). Vice versa, recombinant overexpression of human PARP-1 caused an increase in promoter activities of these deletion mutants (Fig. 4E).

Finally, we performed a quantitative ChIP to analyse the promoter regulation within the endogenous chromatin context. Consistent with the data on mRNA level,

Downstream effects of an AT2 receptor activation

To address whether AT2 receptor activation itself affects PARP-1 expression, i.e.

whether there is a reciprocal interaction between AT2R and PARP-1, we performed stimulation experiments using angiotensin II and the novel AT2R agonist compound 21 [START_REF] Wan | Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist[END_REF]. Real-time PCR analyses indicated that PARP-1 mRNA remains unchanged after AT2R activation (Fig. 5A). Consistently, quantification of PARP-1 mRNA in cardiac tissue and brain of AT2R knockout mice and respective controls by real-time PCR yielded similar expression levels, indicating that PARP-1 itself is not a downstream target of the AT2R (Fig. 5B).

In the context of downstream effects of the AT2R it was also analysed whether there is a feedback of the AT2 receptor on its own promoter. Since AT2R mRNA was almost undetectable in MEF cells derived from wild type mice (not shown and Fig. 3B) the stimulation experiments were only evaluated in MEF cells of homozygous PARP-1 knockout mice. AT2R activation caused a modest, but significant increase of both AT2R mRNA expression (Fig. 5C) and promoter activity of the 216 bp regulatory exon 1-intron 1 region (Fig. 5D).

ATBP50 mRNA was not modulated by AT2 receptor activation in PARP -/-and PARP +/+ MEF cells (data not shown).

The regulation of AT2R and ATBP50 genes by PARP-1 in vivo

A c c e p t e d M a n u s c r i p t 20 In order to analyse the downstream regulatory effects of PARP-1 on AT2R and ATBP50 gene in vivo, we measured transcript levels of these genes in homozygous PARP-1 knockout mice and respective wild type controls. PARP-1 deficiency decreased AT2R mRNA to 34.1% and 41.7% in cardiac and renal tissues, respectively (Fig. 6A,B). In contrast, in the brain, PARP-1 ablation caused a derepression of AT2R mRNA (Fig. 6C) consistent with the regulation in neuronal cells in vitro (Fig. 3). Basal PARP-1 mRNA expression was demonstrated by realtime PCR in heart, kidney and brain of wild type, but -as expected -not of PARP-1 -/-mice assuring that PARP ablation is responsible for the observed expression differences of the AT2R (data not shown) .

Regarding ATBP50 expression we did not observe expression differences between PARP-1 knockout and wild type mice in heart, kidney and brain by realtime PCR (data not shown).

A c c e p t e d M a n u s c r i p t 21

Discussion

The AT2 receptor and PARP are major players within human cardiovascular pathophysiology. In this work we deciphered a novel regulatory network in which PARP-1 constitutes a nodal point based on the findings that this nuclear factor acts as an upstream regulator of AT2R and ATIP1 genes (Fig. 7). Furthermore, AT2R activation causes a PARP-independent induction of AT2R promoter activity thereby creating a positive feedback loop (Fig. 7, left dotted line), in line with the literature [START_REF] Volpe | Angiotensin II AT2 receptor subtype: an uprising frontier in cardiovascular disease?[END_REF].

Additionally, a regulatory interplay between the transcriptional and posttranslational levels is present since PARP-1 regulates the transcription of the AT2R and also the level of ATIP/ ATBP, the direct protein-protein interaction partner of the AT2R [START_REF] Wruck | Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein[END_REF][START_REF] Nouet | Transinactivation of receptor tyrosine kinases by novel angiotensin II AT2 receptor-interacting protein, ATIP[END_REF] (Fig. 7, right dotted line).

The reciprocal regulation of AT2R and ATIP1 genes by PARP-1 in vitro -i.e., activation in the case of ATIP1 and repression in the case of AT2R -was shown for the promoter and mRNA levels as well as for the chromatin context regarding the ATIP gene. In this context it is important to note that the repressive effects of PARP-1 on the AT2R gene observed in neuronal cells in vitro are consistent with the regulation in brain tissue in vivo. On the protein level, an upregulation of the AT2R by pharmacological PARP inhibition or genetic PARP-1 ablation was observed by immunocytology (Fig. 3F). Nevertheless, tissue-specific mechanisms and/ or differences between the in vitro and in vitro gene regulation appear likely because PARP-1 ablation causes a decreased AT2R mRNA expression in heart and renal tissue in vivo.

Our experiments, consistent with a previous publication [START_REF] Warnecke | Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements[END_REF], also demonstrated the important regulatory function of the exon 1-intron 1 region of the AT2R gene is necessary and sufficient for AT2R promoter activity, whereas upstream promoter elements (> 316 bp relative to exon 2) do not contribute to promoter activity to a major degree (Fig. 2&3). Inverse cloning experiments (Fig. 2) revealed that this 216 bp region of the AT2R is a promoter but not an enhancer element implicating (a) further start site(s) in this region. This hypothesis was further confirmed by 5´-RACE (Fig. 1) which indicated that the exon 1-intron 1 region is capable of tethering the basal transcriptional machinery. The binding of PARP-1 to the AT2R promoter was demonstrated by ChIP (Fig. 3G), indicating that PARP-1 is directly involved in the regulation of the AT2R gene.

The AT2R is technically "hazardous" because of its very low expression levels in adult tissues [START_REF] De Gasparo | International union of pharmacology. XXIII. The angiotensin II receptors[END_REF][START_REF] Matsubara | Pathophysiological role of angiotensin II type 2 receptor in cardiovascular and renal diseases[END_REF]. This observation is also reflected on the promoter level based on low relative luciferase activities compared to other genes [START_REF] Schefe | A novel signal transduction cascade involving direct physical interaction of[END_REF][START_REF] Funke-Kaiser | Regulation of the major isoform of human endothelin-A c c e p t e d M a n u s c r i p t 31 converting enzyme-1 by a strong housekeeping promoter modulated by polymorphic microsatellites[END_REF] and also extends to the analysis of transcriptional start sites since a substantial number of 5´-RACE clones had to be sequenced (data not shown). In this context, our result of a single transcriptional start site within the TATA box positive AT2R promoter is consistent with the promoter architecture of a tissue-specific gene [START_REF] Carninci | Genome-wide analysis of mammalian promoter architecture and evolution[END_REF] in contrast to strong housekeeping genes associated with multiple start sites and the absence of a TATA motif [START_REF] Schefe | A novel signal transduction cascade involving direct physical interaction of[END_REF][START_REF] Funke-Kaiser | Regulation of the major isoform of human endothelin-A c c e p t e d M a n u s c r i p t 31 converting enzyme-1 by a strong housekeeping promoter modulated by polymorphic microsatellites[END_REF]. It is noteworthy that two transcriptional start sites upstream the TATA box were identified in human uterus RNA by primer extension analysis [START_REF] Warnecke | Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements[END_REF] (Fig. 1A) which might reflect tissue specific differences and could affect mRNA stability. Furthermore, processed, expressed pseudogenes of the AT2R seem to exist competing with the canonical transcript for detection (unpublished data). Consistently, it is important to note that publications analysing the AT2R may be biased by overexpression experiments to circumvent the low expression [START_REF] Reudelhuber | The continuing saga of the AT2 receptor: a case of the good, the bad, and the innocuous[END_REF]. Since the AT2R expression is high in fetal tissues [START_REF] Lavoie | Minireview: overview of the renin-angiotensin system--an endocrine and paracrine system[END_REF], a

A c c e p t e d M a n u s c r i p t 23 repressor of the AT2R gene has to be postulated. Thus, our present finding that the AT2R can be strongly repressed by PARP-1 is of physiological and putative therapeutic relevance. As discussed above, the role of the AT2R still remains controversial, but assuming that AT2R activation is beneficial to the nervous [START_REF] Reinecke | Angiotensin II accelerates functional recovery in the rat sciatic nerve in vivo: A c c e p t e d M a n u s c r i p t 26 role of the AT2 receptor and the transcription factor NF-kappaB[END_REF][START_REF] Li | Angiotensin AT2 receptor protects against cerebral ischemia-induced neuronal injury[END_REF][START_REF] Lucius | The angiotensin II type 2 (AT2) receptor promotes axonal regeneration in the optic nerve of adult rats[END_REF] and cardiovascular [START_REF] Metcalfe | Prevention of cardiac hypertrophy by angiotensin II type-2 receptor gene transfer[END_REF] system, an activation of this receptor could be a worthwhile therapeutic task. This can be achieved by direct receptor stimulation with small molecule agonists such as the novel compound 21 on the protein level [START_REF] Wan | Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist[END_REF]. Nevertheless, our data indicate that indirect activation of the AT2R protein by intervention on the transcriptional level using PARP inhibitors is a further exciting approach, at least in the nervous system, whereas the qualitative and quantitative contribution of AT2Rs to the beneficial cardiac effects of PARP inhibitors remains elusive. The constitutive receptor activation of the AT2R [START_REF] Berk | Angiotensin type 2 receptor (AT2R): a challenging twin[END_REF] also favours the latter. In this context it is of interest that pharmacological PARP inhibitors have successfully passed preclinical development regarding e.g. stroke, neuronal injury, myocardial infarction and heart failure, and have already entered human clinical trials for the indications myocardial infarction and different neoplasms [START_REF] Jagtap | Poly(ADP-ribose) polymerase and the therapeutic effects of its inhibitors[END_REF]42].

Two parameters are crucial to qualitatively and quantitatively assess the contribution of AT2Rs to the effects of PARP inhibitors. First, it must be known -as discussed above -if the AT2R per se exerts beneficial or detrimental functions in the respective biological context. Second, the number of target genes downstream of PARP-1 and their respective functions are important. PARP-1 affects transcription in the absence of DNA damage due to several different mechanisms ranging from direct sequence-specific DNA binding, via recruitment by transcription factors, to poly(ADP-ribosyl)ation of other transcription factors and histones [START_REF] Kraus | PARP goes transcription[END_REF][START_REF] D'amours | Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions[END_REF]. Besides AT2R and ATBP50 identified in this study, it was shown that the gene encoding alpha-synuclein can be directly regulated by PARP-1 [START_REF] Chiba-Falek | Regulation of alpha-synuclein expression by poly (ADP ribose) polymerase-1 (PARP-1) binding to the NACP-Rep1 polymorphic site upstream of the SNCA gene[END_REF].

A c c e p t e d M a n u s c r i p t 24 A microarray analysis indicated the direct or indirect effect of PARP-1 on the expression of about 90 genes in fibroblasts [START_REF] Simbulan-Rosenthal | Misregulation of gene expression in primary fibroblasts lacking poly(ADP-ribose) polymerase[END_REF]. PARP´s enzymatic activity is required for its transcriptional regulatory functions in some contexts but not in others [START_REF] Kraus | PARP goes transcription[END_REF][START_REF] Chiba-Falek | Regulation of alpha-synuclein expression by poly (ADP ribose) polymerase-1 (PARP-1) binding to the NACP-Rep1 polymorphic site upstream of the SNCA gene[END_REF][START_REF] Meisterernst | Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro[END_REF]. Our data illustrate that inhibition of the enzymatic activity of PARP using 3-AB and PARP-1 ablation/ siRNA exert similar effects, indicating that PARP´s enzymatic activity is involved in the regulation of AT2R and ATBP50 genes.

In conclusion, our data reveal that PARP-1 is an upstream transcriptional regulator of the AT2 receptor in vitro and in vivo. Pharmacological interference within this AT2R-PARP-AT2R network by approved (e.g. angiotensin AT1 receptor blockers), experimental (e.g. PARP inhibitors and AT2R agonists) or future (e.g. decoy oligonucleotides) drugs -and their respective combinations -represents a current or putative therapeutic approach in cardiovascular and neuronal disease. (E) KELLY cells were cotransfected with luciferase reporter constructs comprising serial deletion mutants of human AT2R promoter and a PARP-1 expression vector (dark grey columns) or an insertless control vector (white columns), respectively.

(F) KELLY cells were incubated with 3-AB or vehicle control for 10 hours; MEF cells dervied from PARP-1 knockout mice and wild type controls were cultured without 3-AB. AT2R expression was analysed by immunofluorescence staining.

The "overlay" panel represents the combination of the AT2R staining ("anti-AT2R")

and the nuclear staining with DAPI. Incubation without primary antibody ("secondary antibody only") served as negative control. Exposition was identical between intervention (i.e., 3-AB or PARP -/-) and respective contol. Numbers ("20x", "63x") indicate the magnification.

(G) KELLY cells were transfected with siRNA against PARP-1 (grey columns) or a negative control siRNA (white columns). ChIP was performed with antibodies against PARP-1. Immunoprecipation using IgG antibodies served as negative control. Transcription factor recruitment was quantified by real-time PCR using two different primer pairs located around the transcriptional start site ("TSS") or in the upstream region ("upstream") of the human AT2R promoter. The ordinate (E) EA.hy926 cells were cotransfected with luciferase reporter constructs comprising serial deletion mutants of the human ATIP1 promoter and a PARP-1 expression vector (dark grey columns) or an insertless control vector (white columns), respectively. Harvest was performed 24 h after cotransfection.

(F) EA.hy926 cells were treated with siRNA against human PARP-1, followed by RNA isolation after 24 hours. ATIP1 mRNA was quantified by real-time PCR.

  culture Human endothelial EA.hy926 cells were kindly provided by C.-J. Edgell (Chapel Hill, NC, USA) and cultured in high glucose DMEM (Invitrogen, Karlsruhe, Germany) supplemented with 10% FBS, 100 U/ml penicillin and 100 microg/ml streptomycin, 1 mM sodium pyruvate, 1% non-essential amino acids and 2% HAT (all from Invitrogen).

A c c e p t e d M a n u s c r i p t 8 3´

 8 [-516-construct] and 5´-ctggcaagggttcataagtcagccc-3´ [-316-construct].

  [-500-construct], 5´-agcatgcacagtgtgatgtggt-3´ [-300construct] and 5´-gtatttcttggccttgaagagtac-3´ [-100-construct].

A c c e p t e d M a n u s c r i p t 9 (

 9 Applied Biosystems, Darmstadt, Germany). Data analysis was performed according to the ∆∆C T method. Following primer pairs were used for real-time PCR: AT2R (human, 107 bp): 5'-aacattaccagcggtcttcac-3' (sense), 5'-ggaattgcatctaaatgcttatctgatggt-3' (antisense); AT2R (rat, 80 bp): 5´-aatccctggcaagcatcttatgt-3' (sense), 5´-cggaaataaaatgttggcaatg-3' (antisense); AT2R (mouse, 100 bp): 5´-agggagctcggaactgaaag-3' (sense), 5´-ttgccagtgtttaaagcagttatc-3' (antisense); PARP-1 (mouse, 151 bp): 5´-gcagcgagagtattcccaag-3´(sense), 5´-aagccatccacctcaacatc-3´ (antisense); ATIP1 (human, 86 bp): 5´-tgcttcgaaaccttcgactt-3' (sense), 5´-gaggattcttttgcctgctc-3' (antisense); ATBP50 (rat, 115 bp): 5´-tgggctcaggaaaaacactg-3' (sense), 5´-cctttcggtggacagcacat-3' (antisense); ATBP50 (mouse, 107 bp): 5´-ggctcaggaaaaacactgtca-3' (sense), 5´-ggaggacagcacatctggag-3' (antisense); 18s rRNA (human, rat, mouse, 108 bp): 5´-ccgcagctaggaataatggaata-3' (sense), 5´-tctagcggcgcaatacgaat-3' (antisense); beta-actin (mouse, 144 bp): 5´-gacaggatgcagaaggagattactg-3´ (sense), 5´-gctgatccacatctgctggaa-3´ (antisense).The identities of the AT2R-specific transcripts were confirmed by sequencing in KELLY cells, MEFs of PARP-1 knockout mice as well as cardiac tissue of PARP-1 +/+ and PARP-1 -/-animals.2.5 Small interfering RNA (siRNA)A c c e p t e d M a n u s c r i p t 10 siRNA directed against human PARP-1 (5'-gauuucaucuggugugauauu-3' (sense), 5'-uaucacaccagaugaaaucuu-3' (antisense); Dharmacon/ Thermo Fisher Scientific, Lafayette, Colorado, USA) and a siRNA negative control (sc-37007, Santa Cruz Biotechnology, Heidelberg, Germany) were transfected using HiPerFect (Qiagen, Hilden, Germany). Efficiency of transcript suppression (to 1.5% of control) was evaluated by real-time PCR.2.6 RNA ligase-mediated (RLM)-5´-RACETranscriptional start sites of human AT2R and ATIP1 genes were determined using the GeneRacer kit (Invitrogen, Karlsruhe, Germany) which only amplifies capped mRNA. Cardiac mRNA (left ventricle) was a generous gift from H. Witt (CCR, Berlin). Reverse transcriptions were performed with random hexamer primers followed by nested PCRs using indicated primers: 5´-(AT2R; antisense; second round PCR), 5´gagctgcaggataaatccac-3´ (ATIP1; antisense; first round PCR) and 5´acagtgcttctcctaaaccc-3´ (ATIP1; antisense; second round PCR). Reaction products were subcloned and sequenced. To determine transcriptional start sites

  using the Dual-Luciferase Reporter Assay System (Promega). Cotransfection of the phRL-null plasmid (encoding humanized renilla luciferase; Promega) served for standardisation. Relative luciferase activity (RLA) is defined as the mean value ofA c c e p t e d M a n u s c r i p t 11 the firefly-luciferase/ renilla-luciferase ratios of each construct related to the insertless reporter plasmid pGL3-basic [28]. RLA data represent the mean ± standard deviation of at least four single, parallel transfection experiments. 2.8 Chromatin-immunoprecipitation (ChIP)EA.hy 926 cells were treated with 10 mM 3-AB and respective control for 24 hours without starving. Fixation at a confluence of about 90% was performed using 1% formaldehyde (Merck, Darmstadt, Germany) in 1x PBS (Invitrogen) for 7 min at 37°C. Cells were then rinsed twice with ice-cold 1x PBS, scraped of in 1x PBS and centrifuged for 5 min at 440 x g. Each pellet was resuspended in 2.5 ml lysis buffer (1% SDS, 50 mM Tris-HCl, 1x Complete protease inhibitor cocktail (Roche, Mannheim, Germany), 5 mM EDTA (final concentration); pH 8.1), followed by a 20 min incubation on ice. Sonification, immunoprecipitation and reversal of crosslink were performed according to Bryant and Ptashne [30] using 3 µg antibody directed against the following antigens: TBP (sc-204, Santa Cruz Biotechnology) and RNA polymerase II (sc-5943, Santa Cruz Biotechnology).

A c c e p t e d M a n u s c r i p t 12 Calculation

 12 of transcription factor (TF) recruitment X (e.g. to human ATIP promoter) standardised to the recruitment of TBP to the TATA box region of the human beta-actin promoter was calculated according to X= 2 -[C T [IP(TF), PCR(ATIP)] -C T [input, PCR(ATIP)]] 2 -[C T [IP(TBP), PCR(β-actin)] -C T [input, PCR(β-actin)]] X was calculated separately for the incubation condition (i.e., 3-AB) [X(i)] and vehicle control [X(c)]. X(i) divided by X(c) served as a measure for relative PARP-1 recruitment. Indicated standard deviation is based on three on three real-time PCR measurements on each template.

A c c e p t e d M a n u s c r i p t 15 3. Results 3 . 1

 1531 Basal characterisation of human AT2R and ATIP promotersTo analyse the mechanisms of AT2R and ATIP gene expression we initially determined the respective transcriptional start sites by a modified 5´-RACE protocol which only amplifies capped mRNA. Two transcriptional start sites were identified in the human ATIP1 promoter whereas only one single start site -in appropriate distance to a TATA box -was detected in the human AT2R promoter (Fig.1A&B). To gain insight into activating and repressing promoter regions we subcloned serial deletion mutants of human AT2R and human ATIP1 promoters into a reporter vector. Transient transfections indicated that the 216 bp region consisting of exon 1 and intron 1, termed [-216], of human AT2R is necessary and sufficient for AT2R promoter activity since the construct [-1100] without this region

A c c e p t e d M a n u s c r i p t 17 almost

 17 undetectable (as assayed by a high C T value and faint bands in the corresponding agarose gel (not shown)). In contrast, AT2R expression was increased about 3-fold in PARP-1 deficient cells (Fig.3B).

A c c e p t e d M a n u s c r i p t 19 pharmacological

 19 PARP inhibition decreased the recruitment of TBP and especially of RNA polymerase II (Fig.4G).

A c c e p t e d M a n u s c r i p t 22 consisting

 22 of 216 bp. In human MCF7 and KELLY cells, this exon1-intron 1 region

Fig. 1 .

 1 Figure legendsFig. 1. Analysis of the transcriptional start sites of human AT2R and human ATIP1

Fig. 2 .Fig. 3 .

 23 Fig. 2. Promoter activities of human AT2R and human ATIP1 genes under basal

A c c e p t e d M a n u s c r i p t 34 (

 34 C) Serial deletion mutants of human AT2R promoter were transfected into KELLY cells cultured in 3-AB (30mM; grey columns) or vehicle (white columns) for 8 hours. Promoter activities of these luciferase reporter constructs are expressed as RLA as described above. (D) Serial deletion mutants of human AT2R promoter were transfected into MEF cells derived from homozygous PARP-1 knockout mice (grey columns) and wild type MEF control cells (white columns).

A c c e p t e d M a n u s c r i p t 35 representsFig. 4 .

 354 Fig. 4. PARP-1 activates the ATIP1 gene. (A) Serum-cultured endothelial

A c c e p t e d M a n u s c r i p t 36 (

 36 G) EA.hy926 cells were incubated with 3-AB (grey columns) or vehicle control (white columns) followed by ChIP using antibodies against TATA box-binding protein (TBP) or RNA polymerase II (pol II). Recruitment of these factorsstandardised to the recruitment of TBP to the beta-actin promoter -was quantified by genomic real-time PCR using primers located in the core promoter region of human ATIP1.

Fig. 5 .

 5 Fig. 5. Downstream effects of an AT2 receptor activation. (A) MEF cells derived

A c c e p t e d M a n u s c r i p t 37 Fig. 6 .

 376 Fig. 6. Regulation of the AT2R gene in vivo. AT2R-specific mRNA from heart (A),

Fig. 7 .

 7 Fig. 7. Transcriptional regulation of AT2R and ATIP1 genes. PARP-1 acts as a

  figure[START_REF] De Gasparo | International union of pharmacology. XXIII. The angiotensin II receptors[END_REF]

ATIP1

Figure

  Figure

Acknowledgements

This work was supported by grants from the European Union (Network of Excellence "InGenious HyperCare", no. 037093, JRP B1 -P15) and the German Research Foundation DFG (GK-754-II/ III, TP 7).