Estradiol receptor plays a key role in breast cancer and specific hormonal therapies blocking the receptor functions have been developed. Unfortunately, many patients become resistant to this treatment and develop metastatic breast tumors. The causes of breast tumor progression and hormonal therapy resistance are still debated. Many proteins are mislocalized in human cancers, and increasing evidence indicates that nuclear exclusion of estradiol receptor is involved in tumorigenesis of breast cancer cells and hormonal therapy resistance. Therefore, analysis of intracellular localization of estradiol receptor together with screening for specific compounds that redirect the mislocalized receptor to the correct subcellular compartment is a very promising approach to the discovery of novel anticancer compounds. We recently dissected estradiol receptor nuclear export in breast cancer cells and clarified the role of estradiol-activated PI3-K in this process. These findings and their impact on cell cycle progression in breast cancer cells will now be presented. The strategy of trapping estradiol receptor in nuclear compartment of target cells using new peptides mimicking the nuclear export signal of estradiol receptor will also be discussed.

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

ESTRADIOL ACTION IN TARGET CELLS

Estradiol influences many processes in mammals, including cell growth, cardiovascular health, bone integrity, immunity, cognition and behavior. It is well known that transcriptional effects of estradiol usually occur either through ligand-dependent binding of estradiol receptor(s), alpha or beta, to DNA sequences, or receptor association with proteins that in turn bind discrete nucleotide sequences, or the dismissal of co-repressors and recruitment of co-activators [START_REF] Lonard | Gene transcription: two worlds merged[END_REF]. The resulting fluctuations in mRNAs and proteins occur within hours or days following hormonal exposure. Many findings, however, show that regulation of these effects is also mediated by a complex and bidirectional interface between control of gene expression and modulation of signaling cascades activated by estradiol in target cells. Increasing evidence indeed shows that estradiol triggers signaling pathways usually engaged by growth factors, cytokines and extracellular matrix. This so-called 'non-genomic' action is independent of estradiol receptor (ER) transcriptional activity and occurs within seconds or minutes. In addition to estradiol, almost all members of the steroid hormone family, from corticosteroids (glucocorticoids and mineral corticoids) to other sex steroid hormones (progestin and androgens), exert rapid, non-genomic actions. These range from activation of Src, MAPKs, adenylyl cyclase and PI3-K to rises in intracellular-calcium concentrations. Depending on the cell context, these signals are related to different effects of steroid hormones, such as proliferation, survival, vasorelaxation, migration and differentiation (Cheskis et al., 2007 and refs therein).

Our lab has in the past made a major contribution to the understanding of how the estradiolstimulated signaling machinery works, with a specific focus on the interface between intracellular signaling and cell cycle progression. Particularly, we have identified and characterized ER/Src interaction, which plays an important role in the signaling network activated by estradiol, and intersects PI3-K signaling in breast cancer cells [START_REF] Migliaccio | Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells[END_REF]2000;[START_REF] Castoria | PI3-kinase in concert with Src promotes the S-phase entry of estradiol-stimulated MCF-7 cells[END_REF]. This network controls cell cycle progression in cultured MCF-7 cells [START_REF] Castoria | Non-transcriptional action of estrogen and progestin triggers DNA synthesis[END_REF][START_REF] Castoria | PI3-kinase in concert with Src promotes the S-phase entry of estradiol-stimulated MCF-7 cells[END_REF][START_REF] Castoria | Role of atypical PKC in estradiol-triggered G1/S progression of MCF-7 cells[END_REF] and cell growth in mouse models of tumorigenesis [START_REF] Varricchio | Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor {alpha} Tyrosine-Phosphorylated Peptide[END_REF]. Subsequent studies from our and other labs extended these findings to androgens and progestin in other cell types and to a rather complex signaling network (Cato et al., 2002 and refs therein).

This model of steroid hormone action underlines the proliferative role of the rapid hormonal action and the associated mechanisms depending on extra-nuclear localization of the classical steroid receptors. There is therefore a strong interest in defining links between signal transducing pathways and subcellular steroid receptor localization.

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

ESTRADIOL RECEPTOR TRAFFICKING

Although ER is principally localized in nuclei of target cells [START_REF] Stenoien | FRAP reveals that mobility of oestrogen receptor-alpha is ligand-and proteasome-dependent[END_REF], it has been reported that ER continuously shuttles between nuclei and cytoplasm, and that the antiestrogens ICI 182780 disrupts this process [START_REF] Dauvois | The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling[END_REF]. In addition, cellular conditions, ligand binding and protein-protein interactions can significantly influence the nucleocytoplasmic shuttling of ER (Pemberton & Paschal, 2005 and refs therein).

Ligand stimulation of different cell types recruits different signaling effectors to ERalpha or ERbeta, which in turn leads to signal transduction pathway activation (Song & Santen, 2006 and refs therein). In addition, a transcriptional incompetent ERalpha mutant permanently resides in cytoplasm and mediates S-phase entry triggered by estradiol activation of the Src/Ras/ERK pathway [START_REF] Castoria | Non-transcriptional action of estrogen and progestin triggers DNA synthesis[END_REF][START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF]. These findings indicate a proliferative function for this extra-nuclear receptor, which is independent of its nuclear localization and transcriptional activity.

Many events regulate ER localization and their derangement causes ER mislocalization and tumor progression. PRMT1-mediated ERalpha methylation is required for recruitment of Src, PI3-K and FAK to ERalpha, and a subset of human breast cancer shows high levels of methylated ERalpha that is exclusively localized in the cytoplasm [START_REF] Le Romancer | Regulation of estrogen rapid signaling through arginine methylation by PRMT1[END_REF]. The recently discovered cytoplasmic cross talk between ER and EGF signaling in breast cancer MCF-7 cells [START_REF] Migliaccio | Steroid receptor regulation of EGF signaling through Src in breast and prostate cancer cells: steroid antagonist action[END_REF] has therefore emerged as a critical factor in endocrine resistance through the control of subcellular localization of ER signaling components [START_REF] Gururaj | Novel mechanisms of resistance to endocrine therapy: genomic and nongenomic considerations[END_REF]. In addition to mediating Src recruitment to ER, association of the receptor with the adaptor protein MNAR [START_REF] Wong | Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade[END_REF] also plays a role in ER localization. MNAR sequesters ER in cytoplasm/membrane, thus enhancing non-genomic effects of ER [START_REF] Vadlamudi | Functional implications of altered subcellular localization of PELP1 in breast cancer cells[END_REF]. Tumorigenesis and anti-hormone resistance of breast cancer cells then follow [START_REF] Vadlamudi | Functional implications of altered subcellular localization of PELP1 in breast cancer cells[END_REF][START_REF] Gururaj | Novel mechanisms of resistance to endocrine therapy: genomic and nongenomic considerations[END_REF]. Furthermore, expression of a shortened form of the metastatic tumor antigen 1 (MTA1s) sequesters ER in the cytoplasm and leads to malignant phenotypes by enhancing ER cytoplasmic functions in hormone-dependent breast cancer cells [START_REF] Kumar | A naturally occurring MTA1 variant sequesters oestrogen receptor-alpha in the cytoplasm[END_REF].

These findings imply that cytoplasmic localization of ER provides a mechanism to control signal transduction-dependent functions, such as DNA synthesis and anchorage-dependent growth of target cells. They also suggest that ER localization has functional implications in breast cancer progression and endocrine resistance. However, new information on the molecular basis of ERalpha nuclear export is emerging, and the combined import-export cycle of ER can be viewed as a potential mechanism for transcription integration with signaling events initiated in the cytoplasm.

A c c e p t e d M a n u s c r i p t 5

ESTRADIOL RECEPTOR NUCLEAR EXPORT: THE ROLE OF CRM1

Whereas it is largely accepted that steroid receptors undergo ligand-dependent nuclear translocation, the mechanism of their nuclear export is as yet unclear. The best-characterized nuclear export pathway uses CRM1 as a receptor for proteins with leucine-rich nuclear export signals (NESs;[START_REF] Fornerod | CRM1 is an export receptor for leucinerich nuclear export signals[END_REF][START_REF] Fukuda | CRM1 is responsible for intracellular transport mediated by the nuclear export signal[END_REF]. Leptomycin B (LMB), through covalent binding to CRM1, inhibits CRM1-dependent nuclear export [START_REF] Kudo | Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1[END_REF].

Conflicting data have been reported on inhibition of steroid receptor nuclear export by LMB treatment. Steroid receptors lack classical hydrophobic leucine-rich NESs, but have sequences with limited homology to NESs (De Franco, 2001). In addition, they can interact with other proteins, such as 14-3-3 family members and p160 co-activators that contain the nuclear export signals and function as adapters for CRM1-dependent export of steroid receptors. Under some conditions, LMB treatment inhibits the nuclear export of these receptors (Pemberton & Paschal 2005, and refs therein). We recently dissected the nucleo-cytoplasmic shuttling of chimeric ERalpha in MCF-7 cells, focusing on its export from the nucleus. Estradiol treatment of quiescent cells induces nuclear translocation of ERalpha after 30 min, followed by a decrease in the nuclear receptor to the basal level within 1h. Using LMB as well as in vitro interaction assay, we discovered that hormone-bound ERalpha is exported from nuclei through the CRM1/exportin pathway. LMB treatment of MCF-7 cells prevents ERalpha nuclear export in 1h estradiol-treated cells, and the hormone induces a strong interaction between recombinant ERalpha and in vitro translated CRM1 [START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF].

Thus, we showed by a nuclear export assay that the leucine-rich 444-456 sequence of ERalpha contains a functional NES, which is sensitive to LMB treatment [START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF]. A Tat-conjugated peptide mimicking this amino acid sequence specifically retains ERalpha in the nuclear compartment of 1h estradiol-treated MCF-7 cells (Fig. 1A) and displaces the interaction between ERalpha and CRM1 in vitro [START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF]. These findings support the conclusion that the 444-456 sequence contains a putative NES. In addition, mutations in the core of this sequence impair the estradiol-induced nuclear export of full-length ERalpha in MCF-7 cells (Fig. 1B). The 444-456 sequence we mapped in the hormone-binding domain of ERalpha is conserved among the members of a steroid receptor family, such as ERbeta, progesterone receptor (PgR), androgen receptor (AR), glucocorticoid receptor (GR) and mineral corticoid receptor (MR). This homology suggests that such a sequence is responsible for CRM1dependent nuclear export of most steroid receptors [START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF].

By combining different approaches, we linked ERalpha nuclear export to DNA synthesis triggered by estradiol in target cells. The Tat-conjugated peptide we used to trap ERalpha in the nuclear compartment of MCF-7 cells (Fig. 1A) specifically reduces estradiol-induced S-phase entry in MCF-7 cells (Fig. 1C). Furthermore, the NES-ERalpha mutant ectopically expressed in A c c e p t e d M a n u s c r i p t 6 NIH3T3 fibroblasts fails to induce S-phase entry (Fig. 1D), whereas it activates gene transcription upon estradiol treatment of cells [START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF].

Altogether, these findings point to the role of ERalpha nuclear export in estradiol-regulated DNA synthesis in breast cancer cells.

ESTRADIOL RECEPTOR NUCLEAR EXPORT: THE ROLE OF PI3-K/AKT PATHWAY

There is substantial interest in defining how signal transduction pathways contribute to nuclear receptor localization. Nuclear import of GR, for instance, can be triggered by shear stress activation of MAPK and PI3-K [START_REF] Ji | Shear stress causes nuclear localization of endothelial glucocorticoids receptor and expression from the GRE promoter[END_REF], and MAPKs have also been implicated in the regulation of PgR and GR nuclear export [START_REF] Shank | Nuclear transport of steroid hormone receptors[END_REF]. Furthermore, EGFstimulated MAPK phosphorylates PgR, thus inducing nuclear exclusion and cytoplasmic degradation of PgR [START_REF] Qiu | Mitogen-activated protein kinase regulates nuclear association of human progesterone receptor[END_REF].

We recently observed a new link between estradiol activation of PI3-K/AKT pathway and the nuclear export of ERalpha. Fig. 2 (A andB) shows that over-expression of dominant-negative versions of p85alpha, the regulatory subunit of PI3-K, and AKT inhibits 1h estradiol-induced ERalpha nuclear export in MCF-7 cells, thus linking a rapid action of estradiol controls with ERalpha intracellular traffic.

Previous findings reported that FKHR nuclear export depends on its phosphorylation by AKT [START_REF] Biggs | Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1[END_REF], and data from our lab showed that estradiol activation of the PI3-K/AKT pathway is required to drive MCF-7 cells into S phase [START_REF] Castoria | PI3-kinase in concert with Src promotes the S-phase entry of estradiol-stimulated MCF-7 cells[END_REF]. In addition, an estradiol-dependent interaction between ERalpha and FKHR forkhead () has been described [START_REF] Schuur | Liganddependent interaction of estrogen receptor-alpha with members of the forkhead transcription factor family[END_REF]. Altogether, these findings lead us to hypothesize a role for FKHR in both estradiol-regulated ERalpha nuclear export and cell cycle arrest mediated by NES-ERalpha mutant.

We have recently shown that estradiol-activated PI3-K/AKT pathway leads to phosphorylation of the gatekeeper Ser 256 of FKHR in MCF-7 cells. In addition, it has been observed that expression of the FKHR mutant (FKHR-AAA), which cannot be phosphorylated by AKT, localizes in nucleus thereby inducing G1 arrest in cells [START_REF] Nakamura | Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN[END_REF]. Expression of this unphosphorytable mutant inhibits the estradiol-induced S-phase entry in MCF-7 cells [START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF]. Interestingly, over-expression of this mutant sequesters ERalpha in the nuclear compartment of 1h estradiol-treated MCF-7 cells (Fig. 2C). In turn, over-expression of NES-ERalpha mutant retains wild type FKHR in the nuclei at that time (Fig. 2D). Thus, estradiol simultaneously regulates ER and FKHR nuclear export. Since ERalpha knockdown by siRNA traps wild type FKHR in the nuclear compartment of 1h estradiol-treated MCF-7 cells, we concluded that in ERalpha-positive breast cancer cells FKHR moves from nucleus to cytoplasm via receptor-dependent mechanism [START_REF] Lombardi | Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells[END_REF]. These findings are corroborated by immunoprecipitation experiments showing that 30-min estradiol stimulation increases the association of wild type FKHR with ERalpha in MCF-7 cells. In the same experimental setting, A c c e p t e d M a n u s c r i p t 7 the mutant FKHR-AAA fails to do so (Fig. 2E). Thus, FKHR phosphorylation by estradiol is required for its association with receptor, the export of the FKHR/ER complex and the consequent release of FKHR-mediated cell cycle inhibition.

FKHR belongs to the FoxO subfamily of forkhead transcription factors that are negatively regulated by PI3-K/AKT pathway, and AKT exerts its proliferative effect both by induction of cyclin D1 and down-regulation of p27 expression. FoxO factors downregulate cyclin D1 and upregulate p27 expression [START_REF] Medema | AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27 kip1[END_REF][START_REF] Schmidt | Cell cycle inhibition by FoxO forkhead transcription factors involves downregulation of cyclin D1[END_REF][START_REF] Ramaswamy | A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR[END_REF]. In both cases, a transcriptional regulation mechanism seems to be involved so that cell cycle is stopped. Such inhibition is released upon FoxO phosphorylation by AKT (Birkenkamp & Coffer, 2003 and refs therein). Our findings, together with previous data on induction of cyclin D1 transcription by estradiol-activated PI3-K/AKT pathway in MCF-7 cells [START_REF] Castoria | PI3-kinase in concert with Src promotes the S-phase entry of estradiol-stimulated MCF-7 cells[END_REF], led us to hypothesize the model depicted in Fig. 3. According to this model, estradiol stimulation of the PI3-K/AKT pathway triggers FKHR phosphorylation and leads to the functionally associated export of FKHR-ERalpha complex. In this way, the inhibitory action of FKHR on cyclin D1 transcription is removed and breast cancer cells enter S phase. Impairment of ER nuclear export by site-directed mutagenesis of NES-ER or interference in the ER/CRM1 interaction by synthetic peptides inhibits FKHR export and blocks cell cycle progression.

In summary, our data reveal a novel link between non-genomic estradiol action and ER export, and identify a new role for ER in DNA synthesis. It is plausible that alterations in nuclear trafficking of steroid receptors influence proliferation and contribute to human proliferative diseases. Therefore, a detailed analysis of nuclear transport pathways and regulation of this process is expected to provide new insights into how subcellular compartmentalization contributes to cell regulation in both normal and patho-physiological states. Lastly, these findings support the notion that nuclear retention of ERalpha essentially antagonizes the proliferative action of the PI3-K/AKT pathway. This pathway is frequently deregulated in human cancers as a consequence of inactivation of the tumor suppressor PTEN (Di Cristofano & Pandolfi, 2000), or constitutive activation of AKT [START_REF] Testa | AKT plays a central role in tumorigenesis[END_REF]. In this respect, the strategy of trapping ER in the nuclear compartment may make an important contribution to the development of novel therapeutic strategies for human breast cancers that result from constitutive activation of the PI3-K/AKT pathway (Dillon et al., 2007 and refs therein)

  .

  ABBREVIATIONS LISTAR, androgen receptor; CRM1, chromosome region maintenance 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ER, estradiol receptor; FKHR, forkhead in rhabdomyosarcoma; GFP, green fluorescent protein; GR, glucocorticoid receptor; LMB, leptomycin B; MAPK, mitogen activated protein kinase; MNAR, modulator of non-genomic action of steroid receptors; MR, mineralcorticoid receptor; MTA1, metastatic tumor antigen; NES, nuclear export signal; PgR, progesterone receptor; PI3-K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PTEN, phosphatase and tensin homologue deleted on chromosome.

Fig. 2 .

 2 Fig. 2. Role of estradiol-activated PI3-K/AKT pathway in nuclear export of ER and FKHR.Panel A shows that expression of dominant negative forms of PI3-K (∆p85α) and AKT (AKT K -; dnAKT) retains ERalpha in nuclei of 60-min estradiol-treated MCF-7 cells. Images of one experiment with dn AKT are presented in B. In cells over-expressing dnAKT (green), the ERalpha (red) is localized in nuclei. Merged images are also presented. In C, over-expression of the mutant GFP-FKHR AAA traps ERalpha in the nuclei of 60-min hormone-treated cells. In D, the Myc tagged-NES-ERalpha mutant retains FKHR in nuclei of 60 min estradiol -treated cells. Panel E shows that 30 min estradiol treatment increases FKHR wt association with ERalpha in MCF-7 cells. Under the same conditions, the mutant FKHR AAA does not associate with ERalpha.

Fig. 3 .

 3 Fig. 3. Model of ERalpha nuclear export.The model of ERalpha nuclear export in MCF-7 cells is depicted. Estradiol-stimulated PI3K/AKT pathway leads to FKHR phosphorylation, thus triggering the associated export of FKHR/ER. Such an export removes the transcriptional repressor activity of FKHR on cyclin D1 promoter and fosters the DNA synthesis. This model is fully compatible with our previous findings on regulation of cyclin D1 transcription by estradiol-activated PI3-K/AKT pathway in MCF-7 cells.

  Fig.2

  Fig.3

ACKNOWLEDGEMENTS

This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (National and Regional Grants), Ministero dell'Università e della Ricerca Scientifica (PRIN 2006) and Regione Campania (Legge 5).

The authors declare that they do not have competing financial interests.


J S Bielfeld

Kruessel

A P Hess
email: hess@unikid.de

D M Baston-Buest

A Schanz

J Hirchenhain

P Bielfeld

J S Kruessel

Interleukin-1 system in the human fallopian tube -no spatial but a temporal regulation of mRNA and protein expression

Keywords: Interleukin-1 (IL-1) system, human fallopian tube, Interleukin-1 receptor

Interleukin-1 system in the human fallopian tube -no spatial but a temporal regulation of mRNA and protein expression

A c c e p t e d M a n u s c r i p t Interleukin-1 system in human fallopian tube page 3 type I (IL-1R tI), Interleukin-1 receptor antagonist (IL-1ra), implantation, spatial and temporal expression pattern

Introduction

The factors involved in the complex embryonic maturation and in the embryomaternal dialogue resulting in eutopic embryonic implantation are poorly understood.

The human fallopian tube is the place of fertilization and also provides the natural environment for the first five days of embryonic development (Hess et al., 2007b). It is, as well, the most common place for ectopic implantation. The preimplantation embryo produces several factors during its development to signal its presence to the maternal organism. Appropriate interaction between the preimplantation embryo and the maternal system is at least partly controlled by paracrine cytokines (Hess et al., 2007a;[START_REF] Popovici | Gene expression profiling of human endometrial-trophoblast interaction in a coculture model[END_REF]. Knowledge of expression patterns of these cytokines could possibly help to gain a better insight of the physiological course of implantation. Although multiple studies have examined the existence and influence of various cytokines at the embryonic-maternal interface (Hess et al. 2007a;[START_REF] Popovici | Gene expression profiling of human endometrial-trophoblast interaction in a coculture model[END_REF], as yet, little is known about cytokine production by the human fallopian tube [START_REF] Brabec | Cytokines/growth factors in the human fallopian tube[END_REF].

The Interleukin-1-system is composed of two agonists, Interleukin-1 (IL-1)

and Interleukin-1 (IL-1), one antagonist, the Interleukin-1 receptor antagonist (IL-1ra) and two membrane-bound receptors, Interleukin-1 receptor type I (IL-1R tI) and II (IL-1R tII) [START_REF] Dinarello | The interleukin-1 family: 10 years of discovery[END_REF][START_REF] Dimitriadis | Cytokines, chemokines and growth factors in endometrium related to implantation[END_REF]. All components of the IL-1 family in humans are located on chromosome 2 and the DNA-, RNA-and protein-structures are all well characterized for many species. Both agonists are initially synthesized as precursor proteins of 31 kDa. The mature proteins have a molecular weight of 17 kDa and although the amino acid sequences have a similarity

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

Interleukin-1 system in human fallopian tube page 5 of only ~22 %, they induce the same biological responses [START_REF] Dower | The cell surface receptors for interleukin-1α and interleukin-1β are identical[END_REF]. There is also a high similarity between the cDNA-sequences of IL-1 and - in mice and human [START_REF] March | Cloning, sequence and expression of two distinct human interleukin-1 complementary DNAs[END_REF]. Interleukin-1 receptors type I and II both possess a transmembraneous domain and their extracellular portions are homologous with similar binding affinities for the agonists and antagonist. There is also a soluble form of the IL-1R tII. The IL-1 receptor type I wich is found in low numbers on almost all cell surfaces whereas IL-1R tII is found primarily on white blood cells. Only the binding of either IL-1 or - to the IL-1 receptor type I results in signal transduction [START_REF] Sims | Interleukin-1 signaling occurs exclusively via the type I receptor[END_REF] with receptor type II and the soluble IL-1 receptor acting as competitors of the receptor type I [START_REF] Colotta | Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4[END_REF]. The IL-1 receptor antagonist binds with a high affinity to both receptors and prevents signal transduction by IL-1 and - [START_REF] Hannum | Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor[END_REF].

The IL-1 system is intimately involved in embryonic implantation. In humans, the IL-1R tI has been detected in total human endometrium (Simon et al., 1993b) and, more specifically, in endometrial epithelial cells with a maximum mRNA-and proteinexpression during the luteal phase (Simon et al., 1993a) -the time of embryonic attachment and implantation. IL-1-mRNA was detected in secretory human endometrium beginning on day 23 of the menstrual cycle [START_REF] Kauma | Interleukin-1 (IL-1), human leukocyte antigen HLA-DR, and transforming growth factor- (TGF-) expression in endometrium, placenta and placental membranes[END_REF]. In the last years, all major components of the IL-1-system, namely IL-1, IL-1ra and IL-1R tI were detected immunohistochemically in single preimplantation embryos [START_REF] De Los Santos | Role of Endometrial Factors in Regulating Secretion of Components of the Immunoreactive Human Embryonic Interleukin-1 System during Embryonic Development[END_REF]. In vitro fertilized, cultured human embryos have been shown to produce both IL-1 and IL-1 High concentrations [>60 pg/ml and >80 pg/ml] of these cytokines in culture media have been correlated with successful implantation after intrauterine transfer of these embryos [START_REF] Sheth | Prediction of successful embryo implantatiom by measuring interleukin-1-alpha and immunosuppressive factor(s) in preimplantation embryo cultute fluid[END_REF], although other authors could not detect IL-1 or - in culture fluids of human embryos [START_REF] Seifer | Absence of immunoreactive cytokines in supernatants of individual preimplantation human embryos[END_REF].

In mice, IL-1 and IL-1 have been detected and localized at mRNA-and proteinlevel in endometrial endothelial cells [START_REF] Tackacs | Detection of IL-1 and IL-1 gene expression by in situ hybridization. Tissue localization of IL-1 mRNA in the normal C57BL/6 mouse[END_REF] in increasing levels from day 3 of pregnancy peaking between days 4 and 5 [START_REF] De | Expression of interleukin 1, interleukin 6 and tumor necrosis factor  in mouse uterus during the pre-implantation period of pregnancy[END_REF]. Blastocyst implantation is known to occur late on day 4. It was shown that systemically administered recombinant human IL-1ra given intraperitoneally from day 3 to day 6 of pregnancy inhibited embryonic implantation in mice (Simon et al., 1994a). It was also demonstrated that in vitro cultured single mouse embryos at various stages of preimplantation development express different patterns of mRNA for IL-1, IL-1ra

and IL-1R tI (Simon et al., 1994b).

In summary, there is an increasing body of evidence that the IL-1 system is involved in early embryonic development and implantation. The aim of our study was to detect a potentially spatial and temporal IL-1-, IL-1ra-and IL-1R tI-expression pattern on mRNA-and protein-level in human fallopian tubes and to describe possible hormonally regulated changes since these changes might influence the time and place of embryonic implantation.

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

Interleukin-1 system in human fallopian tube page 7

Material and Methods

Patients

Patients who underwent hysterectomy or tubal surgery for reasons other than cancer (except one patient with intrauterine pregnancy diagnosed of cervical cancer)

were asked to participate in this study. Each participating patient signed an informed consent that was prior approved by the human investigations committee of the Heinrich-Heine University medical school. Menstrual phase was determined by the patient's history and dating was verified by histological examination of the endometrium according to [START_REF] Noyes | Dating the endometrial biopsy[END_REF] and serum-levels of estradiol, progesterone, LH and FSH were measured. The indications for surgery were as follows: hysterectomy for benign reasons: 19 patients; ectopic tubal pregnancies: 3 patients and one patient received a radical hysterectomy in the 9 th week of gestation due to cervical cancer. The mean age of the patients was 39 ± 7.9 yr, range 26 -51 yr.

Tissue samples from the fimbrial, ampullary and isthmic parts of the fallopian tubes were collected from both sides. Tissue samples were than split in half: one half was fixed in 4 % paraformaldehyde and later processed for immunohistochemistry, the other half was snap-frozen in liquid nitrogen and stored at -80 °C until RNAextraction.

RNA isolation

The isolation of RNA from the tissue samples was done as described previously [START_REF] Chomczynski | Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF] AG, Ebersberg, Germany). To ensure that the product detected resulted from amplification of cDNA rather than contaminating genomic DNA, primers were designed to cross intron/exon boundaries. The human -actin primers that were used to amplify an internal standard were obtained from Clontech Laboratories Inc., Palo Alto, CA, USA. The primer-sequences, locations on the cDNA and the sizes of the amplified fragments are listed in Table 1.

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

Interleukin-1 system in human fallopian tube page 9

Reverse transcription (RT)

For each mRNA to be detected, 20 µl RT-MasterMix were prepared (5 mM The RT-reaction was carried out in the DNA Thermal Cycler 480 by using a program with the following parameters: 42 ºC, 15 min; 99 ºC, 5 min; 4 ºC, . After the reaction was complete, samples were stored at -20 ºC until the PCR.

Polymerase Chain Reaction (PCR)

2.5 µl RT-products were mixed with 77.5 µl PCR-Mastermix containing 1.25 mM MgCl 2 , 1 × PCR-Buffer, 0.125 µM 3' and 5' primer-mix for either IL-1, IL-1ra, IL-1R tI or -actin, 1 mM dNTPs (each), 1.5 U/µl AmpliTaq ® DNA Polymerase, DEPC-treated dH 2 O ad 80 µl (Applied Biosystems) and the reaction-mix was covered with 50 µl light white mineral oil, put in the DNA Thermal Cycler 480 and heated to 95 ºC for 5 min to activate the enzyme. After completion of 30 cycles of 94 ºC for 45 sec, 54 ºC for 45 sec and 72 ºC for 60 sec, the reaction was terminated at 72 ºC for 7 min and cooled down to 4 ºC. PCR-products were stored at -20 ºC until 2 % agarose-gel electrophoresis was carried out in the presence of ethidiumbromide. After

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

Interleukin-1 system in human fallopian tube page 10 completion of electrophoresis, the agarose-gel was analyzed on the GelDoc 1000 system (Bio-Rad Laboratories, Hercules, CA, USA). DNA-size calculation was carried out by using the Molecular Analyst Software (Bio-Rad Laboratories).

Immunohistochemical staining procedure

The immunostaining procedure was performed on fallopian tubes sections by the avidin-biotin-peroxidase (ABC) method. Serial sections from each sample were mounted on slides, fixed in formalin and embedded in paraffin. The first section was processed for hematoxylin-eosin staining.

Sections were deparaffinized in xylene, rehydrated through graded alcohols, and washed in phosphate-buffered saline (PBS; Sigma). Endogenous peroxidases were blocked with 1% H 2 O 2 in 96% methanol, and non-specific binding was blocked with non-fat milk 5% in PBS at room temperature. After washing with 0.05% Tween ® 20 in PBS (PBS-T; Sigma), the slides were incubated at room temperature for 30min

with the primary antibodies: monoclonal mouse anti-human IL-1 antibody, monoclonal mouse anti-human IL-1R tI antibody, and polyclonal rabbit anti-human IL-1ra (all from Genzyme Corp., Cambridge, MA, USA) at 20 µg/ml, 50 µg/ml, and 20 µg/ml respectively. Control incubations included deletion of the primary antibody.

After rinsing with PBS-T, sections were incubated with a secondary antibody at

Results

RT-PCR of spatial expression pattern throughout the menstrual cycle

All 19 collected oviduct samples from the proliferative (n=8) and secretory (n=9) phase of the cycle and postmenopausal (n=2) women were divided into fimbrial, ampullary and isthmic parts and investigated separately by RT-PCR. From the total of 19 samples all were positive for β-actin mRNA expression and therefore considered for further investigation. The following investigation of IL-1β, Il-1ra and Il-1R tI expression showed that the presence or absence of the respective gene was constant regarding the spatial distribution. This shows that the gene expression of IL-1β, Il-1ra and Il-1R tI within the length of the fallopian tube has no spatial difference (Fig. 1).

RT-PCR of temporal expression pattern throughout the menstrual cycle

All 23 collected oviduct samples from the proliferative (n=8) and secretory This shows that the gene expression of IL-1β, Il-1ra and Il-1R tI is temporally regulated within the human cycle (Fig. 1 and2). Additionally IL-1β is expressed not only in all different hormonal situations investigated but also in oviducts from intraand extrauterine pregnancies.

Immunohistochemistry

The protein expression of IL-1β, Il-1ra and Il-1R tI of proliferative phase versus secretory phase samples was evaluated using immunohistochemistry. Figure 3 A-H shows representative samples from fallopian tubes of the secretory phase whereas Fig. 3 Interleukin-1 system in human fallopian tube page 14 1ra in the luminal epithelium and the small vessel endothelium of secretory phase oviducts whereas a staining for IL-1ra in proliferative oviducts was completely absent revealing a strict temporal expression of IL-1ra.

No staining was observed in the absence of either primary or secondary antibodies.

These protein data support the previous mRNA data showing an expression of 260

Il-1β in fallopian tubes of both cycle phases with an increase in the secretory phase as well as a pronounced expression of IL-1R tI in secretory phase oviducts. However, IL-1ra expression was seen in secretory phase samples only.

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

Interleukin-1 system in human fallopian tube page 15

Discussion

Factors of the embryo-maternal communication that have an influence on maturation and transport of an embryo as it transverses the oviducts are crucial for a successful implantation (Hess et al., 2007a). The fallopian tube on the maternal side plays an important role in providing the place for fertilization as well as an active component for transport and early development of the embryo. Within the oviduct, remarkable changes occur in dependence to the sex steroids estradiol in the proliferative phase and progesterone in the post-ovulatory secretory phase indicating a hormonal cyclicity of the oviduct comparable to the endometrium (Gardner et al., 1996). A growing body of evidence suggests that chemokines of the IL-1 family play an important role at the feto-maternal interface [START_REF] Krussel | Regulation of embryonic implantation[END_REF]. Therefore, the knowledge of normal expression patterns of the IL-1 family members could help in understanding the physiological processes leading to proper maturation and transport of the blastocyst within the fallopian tube and subsequently to a successful intrauterine implantation. Since we have shown before that the human embryo produces IL-1β when cultured in vitro [START_REF] Krussel | Expression of interleukin-1 system mRNA in single blastomeres from human preimplantation embryos[END_REF], it is reasonable that it also produces IL-1 β during its passage through the fallopian tube. Consequently, it is very likely that the fallopian tube as the early communication partner for the developing embryo itself expresses components of the IL-1 family. Therefore, our objective was to first investigate the spatial expression pattern of IL-1β, Il-1ra and Il-1R tI in the human fallopian tube and second to elucidate a possible hormonal influence. Furthermore we wanted to explore whether we could find a hint for an All collected oviduct samples from the proliferative and secretory phase of the cycle as well as the postmenopausal samples were divided into fimbrial, ampullary and isthmic parts before investigation and prepared separately. This led us to the observation that regardless of the original spatial belonging of the oviduct part, the mRNA expression of IL-1β, Il-1ra and Il-1R tI showed no differences at all. These findings on the mRNA level were also proven on the protein level by immunohistochemistry (data not shown). The data support the notion that although the oviduct is compromised of a changing architecture within its full length, the molecular base of gene expression remains the same in all parts of the fallopian tube.

Therefore, possible differences in gene expression regarding to the biopsy technique are erased which makes studies involving different parts of the oviduct comparable. endometrium cells throughout the cycle (Simon et al., 1993a). In unison with our immunohistochemistry results they detected an increase of IL-1β immunostaining from proliferative to secretory endometrium suggesting an additional role in supporting the implantation within the endometrium during the window of implantation. On the contrary, another study primarily focused on the expression of Il-1α by endometrial stromal cells reports a hormonally controlled expression of IL-1α but not for IL-1β or Il-1ra [START_REF] Pretto | Production of interleukin-1alpha by human endometrial stromal cells is triggered during menses and dysfunctional bleeding and is induced in culture by epithelial interleukin-1alpha released upon ovarian steroids withdrawal[END_REF]. Furthermore, it was shown that incubation of endometrial stromal cells with IL-1β upregulates the expression of IL-1β and IL-1ra [START_REF] Huang | Interleukin (IL)-1beta regulation of IL-1beta and IL-1 receptor antagonist expression in cultured human endometrial stromal cells[END_REF] suggesting that besides a hormonal regulation chemokines seem to influence the expression of Il-1 family members in endometrial stromal cells which could be likewise for the tube. In addition an immunohistochemical approach revealed IL-1β staining in the villous cytotrophoblast, syncytiotrophoblast and maternal decidual cells, suggesting an autocrine/paracrine role during the human implantation (Simon et al., 1994b). In contrast to the ligand, we detected mRNA expression of its receptor IL-1R tI in all samples investigated. The immunostaining showed a less intense staining in proliferative phase samples compared to secretory phase oviducts suggesting at least a moderate influence of the sex hormones. Furthermore it provides new evidence that the preimplantation embryo is able to communicate with the maternal side already during its transit through the fallopian tube by its own IL-1β secretion in consent to a study by [START_REF] Strakova | In Vivo Infusion of 520 Interleukin-1β and Chorionic Gonadotropin Induces Endometrial Changes that Mimic Early Pregnancy Events in the Baboon[END_REF] simulating endometrial changes of an early pregnancy in baboons by in vivo infusion of hCG and IL-1β.

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

Interleukin-1 system in human fallopian tube page 18

In contrast, IL-1ra mRNA was only detected in progesterone dominated secretory phase samples and in the intrauterine pregnancy sample suggesting a hormonal influenced regulation and further a possible protection mechanism to avoid a false implantation of the embryo within the fallopian tube. The IL-1ra expression seems to be a rather systemic effect independent of the embryos presence since it was equally expressed in both sides of the fallopian tubes of each patient in the secretory phase.

IL-1ra's possible protective effect becomes even more likely in the context that IL-1ra was not only present in all secretory phase samples and the one investigated sample with an intrauterine pregnancy but did not occur in anyone of the 3 tested fallopian tubes with an extrauterine pregnancy. This hypothesis is furthermore supported by findings of other groups describing a decrease in IL-1ra expression in the human endometrium at the time of implantation to possibly facilitate the embryos invasion into the maternal endometrium [START_REF] Simon | Immunohistochemical localization, identification and regulation of the interleukin-1 receptor antagonist in the human endometrium[END_REF]. Another study showed increased levels of IL-1ra in endometrial stromal cells of the secretory phase [START_REF] Fukuda | Interleukin-1 receptor antagonist expression in epithelial cells of human endometrium[END_REF]. Those differences though might be explained by the fact that the first study investigated the mRNA and protein expression for secreted IL-1ra and intracellular IL-1ra whereas the second study did not differentiate between those two forms. The findings of the first study positively correlate with a study in mice where systemically administered IL-1ra prevented embryonic implantation (Simon, 1994a).

However, this distinct inhibition of implantation might have been a mouse strain specific effect as a study by [START_REF] Abbondanzo | Reproduction in mice lacking a functional Type 1 IL-1 receptor[END_REF] Based on those findings in human as well as in the animal studies the IL-1β system appears to be involved in the establishment of pregnancy although the exact magnitude is still questionable due to the conflicting data in the literature.

Furthermore, rigorous species specific differences might attribute to an inconsistency in the importance of the IL-1 system in implantation as well.

To our knowledge this is the first study to report that the gene expression profile is From the total of 23 samples, all were positive for β-actin mRNA expression (M -DNA size marker, p -proliferative, s -secretory, m -menopausal, iup -intrauterine pregnancy, eup -extrauterine pregnancy).

  MgCl 2 , 1 × RT Buffer, 1 mM dNTPs (each), 2.5 µM Oligo d(T) 16 , 1 U/µl RNase Inhibitor, 2.5 U/µl MuLV Reverse Transcriptase [all Applied Biosystems, Foster City, CA, USA], and 1 µg total RNA, DEPC-treated H 2 O ad 20 µl) and filled into a 0.5 ml thin wall PCR-tube (Applied Scientific, South San Francisco, CA, USA). RT-MasterMix in PCR-tubes was covered with 50 µl of light white mineral oil (Sigma, St. Louis, MO, USA) and kept on ice until the RT.

  A c c e p t e d M a n u s c r i p t Interleukin-1 system in human fallopian tube page 11 Immunoreaction products were visualized by incubating sections with the substrate 200 solution in 0.1 M Tris-HCl pH 8.2 buffer blocking alkaline phosphatase activity with levamisole. Slides were counterstained with 25 % of hematoxylin, then cleared, cover slips applied and finally examined by light microscopy. Positive staining by the primary antibody was indicated by a pink precipitate. The relative intensity of the immunostaining was evaluated by two of the authors in a double-blind manner as 205 absent (0), weakly positive (+), moderate (++) or intense (+++) for biopsy specimens from each portion and each phase of the menstrual cycle.

  A c c e p t e d M a n u s c r i p t Interleukin-1 system in human fallopian tube page 12

(

  n=9) phase of the cycle and from postmenopausal (n=2) women and extra-(n=3) and intrauterine (n=1) pregnancies were separately investigated by RT-PCR for temporal expression patterns of IL-1β, IL-1ra and IL-1R tI. From the total of 23 samples, all were positive for β-actin mRNA expression and therefore considered for further investigation. The following investigation of IL-1β, Il-1ra and Il-1R tI expression showed that there was a temporal and therefore most likely hormonal regulation of two of the target mRNAs. IL-1R tI was expressed in all samples regardless the hormonal situation. IL-1ra mRNA could be detected in samples of the secretory phase and in intrauterine pregnancy, whereas IL-1β could be detected in proliferative andA c c e p t e d M a n u s c r i p t Interleukin-1 system in human fallopian tube page 13 secretory phase fallopian tubes and in samples of intra-and extra-uterine pregnancy.

  I-P shows representative samples from proliferative phase fallopian tubes. Panel A-G and I-O are magnified 200×, panel B-H and J-P 400×. Panels A and B represent the control of secretory phase oviducts, panel I and J represent the negative control (omission of first antibody) of proliferative phase oviducts. We couldshow a strong expression of IL-1β foremost in the luminal epithelium and also in the small vessels endothelium of secretory phase oviducts (Fig.3 C, D), whereas the staining for IL-1β in the luminal epithelium of proliferative phase oviducts was less intense and only a weak staining was localized in the small vessels endothelium (Fig.3 K, L). The IL-1R tI was shown to be expressed in the luminal epithelium, small vessels endothelium and glandular epithelium of secretory phase samples (Fig.3 E, F). In comparison the proliferative sample showed only a sporadic staining for the IL-1R tI in the luminal epithelium and no staining either in the vessel endothelium or in the glandular epithelium (Fig.3 M, N). Fig.3G and H show an intense staining of IL-

  or an imbalance of gene expression contributing to a risk of an extrauterine tubal pregnancy.

Furthermore, our aim

  was to elucidate whether the sex steroids estrogen and progesterone have an influence on the temporal expression of IL-1β, Il-1ra and Il-1R tI during the reproductive phase. Therefore, fallopian tubes from the proliferative and secretory phase of the menstrual cycle as well as oviducts from postmenopausal women were investigated. This showed that IL-1β was only present in samples from the proliferative and secretory phase but in none of the postmenopausal oviducts suggesting a regulation by sex steroids and a role in the process of human reproduction. These data highlight that the oviduct and the uterus underlie partially common most likely hormonally driven regulatory expression phenotypes since a study has shown that IL-1β is expressed as well by epithelial and stromal A c c e p t e d M a n u s c r i p t Interleukin-1 system in human fallopian tube page 17

  IL-1R tI knockout mice (1998). On the other hand a recent knock out study with male IL-1ra-/-mice showed a reduced capacity to fertilize oozytes of wild type females in vitro and a reduced litter size when mated with wild type females(Genaiem et al., 2009). The discrepancy of those studies might have been evolved due to the fact that Abbondanzo et al. e.g. have used an outbread strain with a mutated gene which are known to possibly have an influenced phenotype. Otherwise it might be conceivable that subsequent events in the signaling cascade after blocking a receptor with the receptor antagonist compared to a knock out of the receptor itself may vary and possibly lead to inconsistent results as shown above when data of IL-1R tI knock out studies are compared with IL-1ra knock out studies. With regards to the event of extrauterine tubal pregnancy in human it could be possible that not only the absence of IL-1ra but furthermore an inappropriate ratio of IL-1β and IL-1ra favoring IL-1β may serve as one underlying molecular mechanism. So far most investigations regarding the molecular base of extrauterine pregnancies were conducted in the mouse or baboon model. Therefore little is known in the human system. Huang et al. (2005) investigated fallopian tubes from extrauterine pregnancies versus normal healthy control tubes from tubal ligations. The PCR approach showed partially varying results from our data with an upregulation of IL-1R tI and IL-1ra mRNA simultaneous to an IL-1β downregulation in tubes with ectopic pregnancies. The study by Huang et al. applied a quantitative PCR approach using a competitor compared to the qualitative method which was used in this study showing no upregulation of IL-1RtI and a downregulation of IL-1β but rather the presence of both in fallopian tubes of extrauterine pregnancies. Presumptions about a A c c e p t e d M a n u s c r i p t Interleukin-1 system in human fallopian tube page 20 regulation can not be drawn therefore. However the upregulation of IL-1ra in extrauterine tubes is opposed to the absence of IL-1ra in extrauterine and healthy proliferative phase tubes as occurred in the present study. Both studies have investigated a relatively small number of fallopian tube samples most likely due to low availability which might explain the partly varying results.

  not altered within the different anatomical parts of the fallopian tube showing a constant gene expression pattern of all IL-1β system factors throughout the complete length of the oviduct investigated. Additionally, the restricted IL-1ra expression in secretory phase fallopian tubes and absence in extrauterine pregnancy tube samples might be an important mechanism on the molecular base to prevent ectopic pregnancies within the oviduct which needs further investigation. The finding that IL-1R tI is expressed in both cycle phases independent of a pregnancy in addition to the embryos own IL-1β expression supports the concept of an early embryo-maternal dialogue facilitating a proper implantation process.

Fig. 1

 1 Fig.1Representative RT-PCR-gelelectrophoresis for IL-1β (549 bp),

Figure 2 Fig. 2

 22 Figure 2 Fig. 2 Representative RT-PCR gelelectrophoresis for temporal expression patterns of

  Figure 3.

Fig. 3

 3 Fig. 3 Immunohistochemistry staining of representative samples from fallopian tubes

  Figures

  disproved the latter finding in IL-1R tI female knockout mice showing no deficiency in fertility besides a slightly smaller litter size as well as Cohen and Pollard could not show noticeable fertility

Acknowledgements

We thank all women who participated in this study. Also we would like to thank Ursula Löhrmann for her help with preparing the sections for immunohistochemical 400 staining. This work was funded by the German Research Society (Deutsche Forschungsgemeinschaft, DFG) to AP Hess (HE 3544/2-1).

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


Anca Azoitei

Dr Margarethe Spindler-Barth
email: margarethe.spindler-barth@uni-ulm.de

DNA Affects Ligand Binding of the Ecdysone Receptor of Drosophila melanogaster

Keywords: dimerization, hormone response element, insect, moulting hormone, nuclear receptor, retinoic acid receptor, ultraspiracle Ecdysone receptor, EcRE: ecdysone response element, Eos-FP: red fluorescent protein, Usp: Ultraspiracle, Vp16: herpes simplex transcriptional activator protein, RXR: Retinoic acid X-receptor, YFP: Yellow fluorescent protein

The heterodimers of all three ecdysone receptor (EcR) isoforms with Ultraspiracle (Usp), the invertebrate orthologue of RXR, bind ponasterone A with the same affinity in the absence of DNA. Ligand binding is stimulated by ecdysone response elements (EcREs) to different degrees depending on the receptor isoform, the heterodimerization partner, and the type of EcRE. Ligand binding to heterodimers with wtUsp is enhanced 5 fold with hsp27, Pal-1 and DR-1. In the presence of DNA substantial differences in ligand binding were observed, when the AB domain of wtUsp is replaced by the N-terminus of VP16, which is routinely used for determination of transcriptional activity to overcome the inhibitory action of the ABdomain of Usp. Enhanced dimerization in the presence of hormone response elements increases mainly the number of binding sites resulting in improved ligand binding, which is observed even if the C-domain of Usp is deleted. RXR, which can partially replace Usp function, confers high affinity ligand binding only in the presence of an EcRE.

Introduction

Three isoforms of EcR with different AB-domains are described for Drosophila (Bender et al., 1997). "In vivo" these isoforms are not functionally equivalent and cannot substitute each other during Drosophila development. One possibility for finetuning of the hormone response in order to achieve isoform specific effects is variation of the expression level of receptor isoforms dependent upon the target tissue and developmental stage [START_REF] Kamimura | Tissue-specific and stagespecific expression of two silkworm ecdysone receptor isoforms -ecdysteroiddependent transcription in cultured anterior silk glands[END_REF][START_REF] Jindra | Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wings of the tobacco hornworm, Manduca sexta[END_REF][START_REF] Verras | Developmental profiles and ecdysone regulation of the mRNAs for two ecdysone receptor isoforms in the Mediterranean fruit fly Ceratitis capitata[END_REF]. In addition the transcriptional capability of receptor isoforms varies particularly hormone induced transactivation. This has been shown for receptors expressed both in vertebrate (Beatty et al., 2006;[START_REF] Mouillet | Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor[END_REF] and insect cells [START_REF] Hu | Transcription activation by the ecdysone receptor (EcR/USP): identification of activation functions[END_REF][START_REF] Schubiger | Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor[END_REF]. Therefore the question arose, whether the various hormonal responses are due to differences in ligand binding affinity.

The ecdysteroid ponasterone A binds to the heterodimer EcR /Usp with high affinity [START_REF] Yao | Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation[END_REF]. Since the affinity of separately expressed ligand binding domains are in the same range, when fused to Gal4-AD and Gal4-DBD respectively and the addition of other tags like GST, His 6, or C-myc did not alter the affinity significantly [START_REF] Grebe | Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor[END_REF], it was concluded that the ligand binding domain is autonomous and that ligand binding is not affected by interaction with other receptor domains. On the other hand it is known from vertebrate nuclear receptors that interaction between the AF-1 region in the AB-domain and the ligand binding domain at the C-terminus occurs either indirectly via comodulators [START_REF] Tian | The N-Terminal A/B domain of the thyroid hormone receptor-beta2 isoform influences ligand-dependent recruitment of coactivators to the ligand-binding domain[END_REF][START_REF] Ikonen | Interaction between the amino-and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators[END_REF] or directly [START_REF] Tetel | Hormone-dependent interaction between the amino-and carboxyl-terminal domains of progesterone receptor in vitro and in vivo[END_REF]. Since the AB-domain is shaped into its active conformation by DNA (Lavery and McEwan, 2005) the interaction of AF-1 and AF-2 may require DNA binding of nuclear receptors as reported e.g. for the progesterone A c c e p t e d M a n u s c r i p t 4 receptor by [START_REF] Heneghan | Coactivator assembly at the promoter: efficient recruitment of SRC2 is coupled to cooperative DNA binding by the progesterone receptor[END_REF]. An influence of the ligand binding domains of EcR/Usp on the C-domain is reported e.g. DNA-binding of the receptor complex is enhanced severalfold in the presence of ligand (Antoniewski et al., 1993;[START_REF] Elke | DNA-binding properties of the ecdysteroid receptor-complex (EcR/USP) of the epithelial cell line from Chironomus tentans[END_REF]Braun and Spindler-Barth, unpublished results). We therefore tested, whether the C-domain also affects ligand binding.

In most insects tested thus far, ligand binding to EcR is only detectable after heterodimerization. In Drosophila weak ligand binding is observed with EcR alone, but is reinforced about 90 fold by Usp [START_REF] Grebe | Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster[END_REF][START_REF] Grebe | Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor[END_REF]. Usp increases the half life of the holo-receptor complex by reducing the dissociation rate of the ligand [START_REF] Grebe | Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor[END_REF]. Mutational analysis revealed that Usp partially rescues the ligand binding capabilities of mutated EcRs [START_REF] Grebe | Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster[END_REF]. We therefore examined, whether the influence of Usp is restricted to the E-domain, necessary for ligand induced dimerization, or whether other domains of Usp are also involved.

Several publications report that Usp can be replaced by the vertebrate orthologue RXR [START_REF] Panguluri | Functional characterization of ecdysone receptor gene switches in mammalian cells[END_REF][START_REF] Palli | The influence of heterodimer partner ultraspiracle/retinoid X receptor on the function of ecdysone receptor[END_REF][START_REF] Henrich | Juvenile hormone potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system[END_REF][START_REF] Thomas | Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle[END_REF], albeit with lower efficiency [START_REF] Nieva | Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)[END_REF]. We therefore examined the influence of RXR on ligand binding of the receptor heterodimer.

A c c e p t e d M a n u s c r i p t 5

Material and Methods

Cell culture and transfection

The Chinese hamster ovary (CHO-K1) cells were maintained in DMEM/F12 medium (with L-Glutamine) (Invitrogen, Carlsbad, Ca, USA) supplemented with 5% FCS.

Cells were cultivated in tissue culture dishes at 37°C in a humidified atmosphere with 5 % CO 2 . For transfection 1.5 -2x10 6 cells were seeded in 10 cm cell culture dishes.

After 24 h (90% confluency) the transfection was performed with 7.0 µg of DNA (3.5 µg DNA for each expression plasmid (EcR and Usp) using Lipofectamine 2000 (Invitrogen, Carlsbad, Germany) according to the prescription of the manufacturer.

The cells were incubated for 24 hours before harvesting.

The following expression plasmids coding for nuclear receptors were used: EcR isoforms: pcDNA3-dEcRA, pcDNA3-dEcRB1and pcDNA3-dEcRB2 was kindly provided by Dr.V.C. Henrich (University of Greensboro, NC, USA). The open reading frames were cloned between BamHI -XbaI sites in pcDNA3 vector (Invitrogen, Carlsbad, USA). The strategy of plasmid construction is described by [START_REF] Mouillet | Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor[END_REF]. Greensboro, USA) [START_REF] Henrich | Juvenile hormone potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system[END_REF].

Preparation of cell extracts

24 hours after transfection cells were gently detached from the surface with a scraper. The cells were rinsed with 5ml ice cold 1x PBS (138 mM NaCl, 2.7 mm KCl, 1.8 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , pH = 7.2) and transferred into 15 ml tubes. After centrifugation (260g, 4 min) the supernatant was discarded and the cell pellet was resuspended in 3 packed volumes of ice cold extraction buffer (20 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH = 0 7.9) supplemented with 2 mM DTT and protease inhibitors (aprotinin, leupeptin, pepstatin, 1µg/ml each). After three freeze/thaw cycles the lysate was centrifuged (18 000g, 30 min, 4°C). The clear supernatant was used for ligand binding assays. Protein content was determined according to Bradford (1949). Since the nuclear receptors especially Usp were not epressed at comparable levels (Braun et al., personal communication) separately expressed Usp was added to ensure that all EcR present was transferred into the heterodimer.

Ligand binding

Cell extracts prepared as described above were diluted with ligand binding dilution buffer (20 MM HEPES, 15 %(v/v) glycerol, 2 mM EDTA, 4 mM DTT, pH = 7.9)

A c c e p t e d M a n u s c r i p t 7 supplemented freshly with protease inhibitors (aprotinin, leupeptin, pepstatin, 1µg/ml each). Ligand-binding was determined with 3 [H]-Ponasterone A (specific activity 7.9 TBq / mmol) using a filter assay, as described previously [START_REF] Turberg | Properties of nuclear and cytosolic ecdysteroid receptors from an epithelial cell line from Chironomus tentans[END_REF]. Briefly cell extracts were incubated routinely with 7.5 nM 3 [H]-Ponasterone for 5 hrs at 4°C. For determination of K D -values various concentrations of 3 [H]-Ponasterone A were used. Free ligand was removed by a filter assay [START_REF] Turberg | Properties of nuclear and cytosolic ecdysteroid receptors from an epithelial cell line from Chironomus tentans[END_REF] using NC45 nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) washed with 3 x 4ml ice-cold washing buffer (20mM HEPES, 20mM NaCl, 10%glycerol (v/v), 1 mM EDTA, 1 mM ß-mercaptoethanol, pH = 7.9).

Bound radioactivity was measured with a liquid scintillation counter (TRicarb 1500, Perkin Elmer, Rodgau-Jügesheim, Germany) and the non specific binding determined by competition with 1µM 20-OH-Ecdysone subtracted. As additional control extracts from non transfected cells were used (<100cpm). The purity of 3 [H]-Ponasterone A was routinely checked by HPLC analysis before use. For calculation of K D -values the results were evaluated according to [START_REF] Scatchard | The attraction of proteins for small molecules and ion[END_REF] or calculated with Kaleida Graph, version 4.0 (Synergy Software, Reading, PA, USA). Comparable results were obtained with both methods; each experiment was repeated 3-6 fold.

The influence of different hormone response elements (280 ng / 100µl) was studied using the same receptor concentration for each set of experiments. The amount of receptor was determined by Scatchard analysis/Kaleidagraph. The following double stranded oligonucleotides were used. Hsp 27 was purchased from Thermo (Ulm, Germany) and Pal-1 and DR-1 were obtained from Biomers (Ulm, Germany). Controls with the same amount of nonspecific DNA of comparable size were performed and subtracted (< 150 cpm).

Statistical significance (P values less than 0.01) of differences among groups was determined on the basis of a two-sided, matched paired Student's t test.

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

Results

1. Ligand binding to EcR isoforms in the absence of Usp is reinforced by DNA.

Ligand binding affinity of EcR is about 90 fold lower in the absence of a heterodimerization partner as determined by kinetic measurements of receptor domains expressed in yeast cells [START_REF] Grebe | Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor[END_REF]. Control experiments revealed that during the time period needed for the experiments (5 hours) the receptor proteins remained functional concerning ligand binding even in the absence of hormone and /or dimerization partner. After ten hours 93.5+3.5 % (n= 6) of the ligand binding capacity of the receptor protein of each isoform is still present. We therefore conclude that no degradation occurred during the incubation time in the extracts. In the presence of the ecdysone response element (EcRE) DR-1 ligand binding is improved particulary if oligomeric EcREs are used (Fig. 1). The stimulatory effect varies depending on the EcR isoform and is significant only for EcR-A and EcR-B1, but not for EcR-B2 and indicates a modulatory effect of the AB domain of EcR. We can not discriminate, whether ligand binding is improved by an increase in homodimerization or whether there is a direct influence of the DBD complexed with the hormone response element on ligand binding. Determination of ligand binding revealed a K D = 3.3 x 10 -8 M / L for EcR-A in the presence of DR-1 (Fig 2). Our efforts to determine K D -values in the absence of DNA were not successful under our experimental conditions, although at a concentration of 10 nM/L 3 H-ponasterone A specific ligand binding above background level (100-150cpm) could be detected (Fig. 1).

All three isoforms of Drosophila EcR bind ligand with the same affinity, if complexed with Usp.

A c c e p t e d M a n u s c r i p t 10 The affinity of Ponasterone A (Table 1, Fig. 3) is the same for heterodimers of all three isoforms of EcR and wild type Usp in the absence of DNA. Since the ABdomain of Usp is routinely replaced by the N-terminal activation domain of the Vp16 transcription factor in order to overcome the inhibitory influence of the original Usp AB domain on transcriptional activity [START_REF] Henrich | The ecdysteroid receptor[END_REF], we additionally determined ligand affinity of the heterodimers with Vp16 AD -Usp CDE fusion proteins (UspI and II). No significant influence of the AB-domain of Usp on ligand binding was observed (Table 1). Deletion of the DNA binding domain of Usp, which also harbours a strong dimerization interface, does not affect ligand binding (Table 1). Fusion of EcR with a fluorescent protein such as YFP at the N-terminal end also does not impair ligand binding and the K D -values are only slightly increased (10.7% + 3.2%, n=6). The number of binding sites varies about 6 fold (table 1), which is not due to degradation processes in the extract, since about 96 % of the ligand binding capacity is still present 5 hours after the experiments were performed. [START_REF] Henrich | The ecdysteroid receptor[END_REF]. Therefore hormone response element oligomers were also tested (Fig. 4). No difference between EcRE monomers and oligomers was found with all heterodimers consisting of all EcR isoforms and wild type Usp.

Ligand binding of ecdysone receptor heterodimers with

In the presence of DNA the AB domain of Usp modifies ligand binding to EcR.

In transcriptional assays the AB-domain of Usp is replaced routinely by the Nterminus of Vp16 [START_REF] Henrich | The ecdysteroid receptor[END_REF] as mentioned above. In this case pronounced differences in ligand binding depending on the EcR isoform and the type of EcRE present are obtained (Fig. 5). In contrast to 1x hsp 27, Pal-1 and DR-1 monomers can enhance ligand binding in some circumstances e.g. for EcR-B1/UspI. The stimulatory effect of EcRE pentamers is less pronounced in case of EcR-A. A similar result is obtained with UspII, which lacks the hexapeptide of the original wtUsp adjacent to the N-terminus of the C-domain. (Fig. 6).

To investigate, whether the increase in ligand binding is due to altered ligand affinity or increased number of binding sites due to enhanced heterodimerization in the presence of DNA we determined K D -values and receptor concentrations for some selected receptor complexes. As shown for EcR-B1/UspII in the presence of 5xDR-1 (Fig. 7A andB), the number of binding sites increases in the presence of this EcRE from 0.19+ 0.024 nM/L in the absence of DNA to 1.14+0.28 nM/l in the presence of 5xDR1, but simultaneously the affinity of the ligand is slightly impaired (about 2fold in the presence of 5xDR1 as shown in Fig. 7. Comparable results were obtained also with heterodimers with other Usp variants and EcR-B1 (data not shown). As outlined previously in the absence of DNA ligand binding of cell extracts is not changed, if the C-domain of Usp is deleted and no influence of the EcR isoform is detectable (Table 1). However, in the presence of EcREs, deletion of the DNA binding domain of Usp selectively stimulates ligand binding. This effect is considerably higher in heterodimers with EcR-A compared to EcR-B1 and EcR-B2 both in the presence of DR-1 and PAL-1 monomers and pentamers (Fig. 8, for comparison see Fig. 5 and6).

Enhanced ligand binding in the presence of DNA is mainly due to improved dimerization.

To investigate, whether the increase in ligand binding is due to altered ligand affinity or increased number of binding sites due to enhanced heterodimerization in the presence of DNA we determined K D -values and receptor concentrations for some selected receptor complexes. As shown for EcR-B1/UspII in the presence of 5xDR-1 (Fig. 7A andB), the number of binding sites increases in the presence of the EcRE, but simultaneously the affinity of the ligand is slightly impaired. Comparable results were obtained also with heterodimers with other Usp variants and EcR-B1 (data not shown). Even in the absence of the DNA-binding domain of Usp (heterodimers with UspIII) the number of binding sites is increased in the presence of the EcRE. (Fig. 9) Since the same cell extracts were used for determination of ligand binding with and without DNA, it is reasonable to assume that the number of heterodimeric receptor Unpartnered EcR from Drosophila melanogaster like that from Leptinotarsa decemlineata [START_REF] Ogura | Molecular cloning, expression analysis and functional confirmation of ecdysone receptor and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata[END_REF] binds ponasterone A specifically, although with lower ligand affinity than the heterodimeric ecdysone receptor [START_REF] Grebe | Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster[END_REF].

Since ligand affinity to cell extracts containing only EcR is about 90 fold lower compared to heterodimers with Usp, the biological significance of hormone binding of unpartnered EcR was often considered to be neglectable, even though ligand induced changes in receptor function in the absence of a heterodimerization partner had been demonstrated repeatedly e.g. enhanced interaction with DNA (Braun, personal communication) and chromatin [START_REF] Cronauer | Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle[END_REF]. Recently Constantino et al. (2008) showed that ecdysone signalling is mediated in a certain developmental stage without participation of Usp or any other RXR like molecule. et al., 2006;Tremmel and Ruff, unpublished observations). This is not due to interaction with endogenous RXR, since the subclone of CHO-K1 used in our lab expresses only neglectable amounts of RXR. Even after cotransfection with plasmids coding for RXR only hormone induced transcriptional activity but not ligand binding in cell extracts in the absence of DNA is enhanced [START_REF] Nieva | Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)[END_REF]. It is important to determine the RXR content of the cells used for these experiments, because the RXR content may vary depending on the subclone of CHO-K1 cells. For example constitutively expressed RXR in CHO-cells is reported by [START_REF] Hannan | Cloning and characterization of LcEcR: a functional ecdysone receptor from the sheep blowfly Lucilia cuprina[END_REF].

Transcriptional activity is also stimulated in the presence of Muristerone A in

The increase in ligand binding in the presence of an EcRE (Fig. 1) shows that not only dimerization via the ligand binding domains, but also via C-domains in the presence of DNA positively affects the ligand binding domain of EcR presumably by stimulating homodimerization mediated by the strong dimerization interface in the DNA binding domain. Comparison with data reported by [START_REF] Grebe | Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor[END_REF] revealed that ligand binding is enhanced about 3-4-fold in the presence of DNA, but is still about 20-25 fold lower compared to ligand affinity of the heterodimer.

The AB -domain of EcR does not affect ligand binding of EcR/Usp in the absence, but in the presence of DNA.

Hormone binding of unpartnered EcR varies in the absence or presence of DNA and depending on the AB domains of the receptor isoforms (Fig. 1). In contrast ligand affinity of heterodimers with Usp is comparable for all EcR isoforms in the absence of DNA. Similar results were also reported by [START_REF] Perera | Studies on two ecdysone receptor isoforms of the spruce budworm, Choristoneura fumiferana[END_REF] for the ecdysone A c c e p t e d M a n u s c r i p t 16 receptor of Choristoneura fumiferana. The affinity of EcR/Usp is in accordance with the data reported previously for heterodimers with the EcR-B1 isoform [START_REF] Yao | Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation[END_REF] as well as with heterodimers encompassing only the ligand binding domains of EcR and Usp fused to Gal4-AD and Gal4-DBD respectively [START_REF] Grebe | Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster[END_REF].

Therefore, it was concluded that ligand binding of EcR/Usp is not influenced by other domains of the ecdysone receptor molecule. This is in agreement with experiments, which show that various tags at the N-terminal end do not affect ligand binding [START_REF] Grebe | Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster[END_REF]this paper) and hormonal stimulation of transcriptional activity is only slightly impaired [START_REF] Nieva | Ultraspiracle promotes the nuclear localization of ecdysteroid receptor in mammalian cells[END_REF].

However in the presence of DNA the AB-domain of EcR isoforms affects ligand binding of heterodimers (Figures 45678). Since binding of nuclear receptors to DNA results in allosteric interaction between DBD and N-terminal regions it was expected that the AB domain of EcR affects dimerization mediated by the C-domains, thus modifiying the number of heterodimers, but ligand affinity is also slightly impaired indicating a more complex interaction of the receptor domains. An influence of the AB-domain of nuclear receptors on ligand induced transcriptional activity has been reported a number of times for different isoforms of vertebrate nuclear receptors [START_REF] Tian | The N-Terminal A/B domain of the thyroid hormone receptor-beta2 isoform influences ligand-dependent recruitment of coactivators to the ligand-binding domain[END_REF][START_REF] Matthews | Estrogen signaling: a subtle balance between ER alpha and ER beta[END_REF]Bevan et al., 1999) due to interaction of the N-terminus with the ligand binding domain [START_REF] Takimoto | Functional properties of the Nterminal region of progesterone receptors and their mechanistic relationship to structure[END_REF].

Isoform specific interaction of N-and C-terminus of EcR was shown by FRET analysis (Tremmel and Schaefer, unpublished results). At present it is not clear, whether this interaction may also affect ligand binding.

Influence of the AB domain of the heterodimerization partner on ligand binding in the presence of DNA.

A c c e p t e d M a n u s c r i p t 17 Although ligand binding is stimulated in the presence of DNA there was no observed influence of the type of EcRE or the AB-domain of EcR on ligand binding to EcR/Usp heterodimers. However, this is true only if the original AB domain of wild type Usp is maintained (Fig. 4). Because transcriptional activity of heterodimers with wild type Usp and in particular to EcR-B1 is rather low even in the presence of hormone, the AB-domain is routinely replaced by the N-terminus of VP16 [START_REF] Henrich | The ecdysteroid receptor[END_REF].

Surprisingly this exchange has considerable consequences for ligand binding to the heterodimer in the presence of DNA (Fig. 56). These results show that Usp is not simply a passive partner as has been previously proposed [START_REF] Hu | Transcription activation by the ecdysone receptor (EcR/USP): identification of activation functions[END_REF], but plays an active role in receptor mediated hormone action. The results presented here further demonstrate that replacement of the original AB-domain of wild type Usp alters transcriptional activity of the heterodimer not only qualitatively but also quantitatively. In contrast to the heterodimers of all EcR isoforms and wild type Usp, receptor complexes with Vp16 -Usp fusion proteins modify ligand binding of the heterodimers in a complex way, dependent upon the EcR isoform and the type of EcRE. This means that the physiological consequences of isoform specific hormone effects obtained in the presence of VP16-Usp fusion proteins should be interpreted cautiously and do not necessarily reflect the physiological situation.

Deletion of the DNA binding domain of Usp selectively modifies ligand binding in the presence of DNA

According to [START_REF] Niedziela-Majka | Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter[END_REF], the C-domain of Usp has a profound influence on DNA binding of the receptor complex directing EcR to the 5´end of the hormone response element. Nevertheless heterodimerization via interaction of the ligand binding domains is still observed according to gel mobility shift assays (Beatty Although dimerization is considerably weaker in the absence of the C-domain of Usp (Azoitei et al., unpublished observation), ligand binding in the absence of DNA is not changed when compared to heterodimers with full length Usp´s, as mentioned above (Table 1). Since the stimulation of ligand binding by addition of EcREs is even more pronounced in the absence of the dimerization interface of the C-domain of Usp (compare Figures 456with Fig. 8 ), the type of dimerization, which can be mediated only by the E-domain of both nuclear receptors obviously allows a more efficient ligand binding.

RXR confers ligand binding to EcR only in the presence of DNA.

In the presence of a strong heterodimerization partner like Usp, dimerization via the ligand binding domains seems to be sufficient to confer high affinity ligand binding to EcR even in the absence of DNA, since ligand affinity is not impaired if the C-domain of Usp is deleted. If only weak dimerization mediated by the ligand binding domains is possible as is the case with EcR homodimers or heterodimers with RXR, dimerization via the C-domains, which does not seem possible in solution [START_REF] Rastinejad | Structural determinants of nuclear receptor assembly on DNA direct repeats[END_REF] but is reinforced in the presence of a suited EcRE, allows still high affinity ligand binding (Fig. 10).

Our results explain why RXR can replace Usp in transcriptional assays [START_REF] Panguluri | Functional characterization of ecdysone receptor gene switches in mammalian cells[END_REF][START_REF] Palli | The influence of heterodimer partner ultraspiracle/retinoid X receptor on the function of ecdysone receptor[END_REF][START_REF] Henrich | Juvenile hormone potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system[END_REF][START_REF] Thomas | Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle[END_REF] but not in other A c c e p t e d M a n u s c r i p t 19 functional tests, which do not require interaction with DNA such as nuclear import [START_REF] Nieva | Impact of heterodimerization on intracellular localization of the ecdysteroid receptor (EcR)[END_REF].

Physiological consequences of modulation of ligand binding by DNA and dimerization partners

The intense intra-and intermolecular interactions described here, which allow modulation of ligand binding by the AB-and C-domains of the receptor molecule, the type and number of EcREs and dimerization partners, provide further examples of the high flexibility of the nuclear receptor complexes such as the ecdysone receptor. This in turn offers multiple possibilities for fine-tuning of the hormonal response. As outlined previously, receptor domains like the ligand binding domain (Billas et al., 2003) or the C-domain [START_REF] Orlowski | Plasticity of the ecdysone receptor DNA binding domain[END_REF] (spec. activity: 7.9 TBq / mmol)) was used. Non specific binding was subtracted as described in material and methods. Fig. 2: Ligand binding of EcR-B1 (transfected in CHO-K1-cells) in the presence of the hormone response element 5x DR-1. 3 H-Ponasterone (specific activity: 7.9 TBq / mmol) was used as ligand (n=4, one representative experiment is shown). Data are analysed according to [START_REF] Scatchard | The attraction of proteins for small molecules and ion[END_REF] and Kaleida Graph (inset). K D = 33.9 nM/L receptor concentration = 0.11nM/L (calculated with Kaleidagraph), Non specific binding was subtracted as described in material and methods.

  Usp variants: Wild type Usp inserted into pEYFP-N1 (Clontech, Palo Alto, CA, USA) using EcoRI and SalI restriction sites was provided by Prof. Ozyhar (University of Wrozlav, Polen) and modified further by Dr. S. Braun (University of Ulm, Germany) to allow expression of a fusion protein encompassing aa 1-507 of Usp. DNA constructs coding for Usp variants (UspI, UspII and UspIII) were obtained from Dr. V.C. Henrich (University of North Carolina, Greensboro, USA). All Usp variants were cloned between EcoRI and HindIII into a pVP16 expression vector (Clontech, Heidelberg, the A/B domain of Drosophila wild type Usp by VP16-AD (Beatty et al., 2006). The VP16-dUspI variant includes six amino acids of B domain of wild type Usp adjacent to the N-terminus of the C-domain of Usp and the remaining carboxy-terminal part of the open reading frame (aa 98-507). The VP16-dUspII variant is identical to UspI, with the exception that the six amino acids are missing. A third variant, VP16-dUspIII, coding for amino acids 170-507, lacks the DNA binding domain. mRXR  was obtained from Dr. V.C. Henrich (University of North Carolina,

  Usp is increased in the presence of DNA To investigate whether interaction with DNA modulates ligand binding to EcR/Usp, we performed hormone-binding experiments in the presence of hormone response elements. Receptor concentrations in the absence of DNA were determined for each cell extract by Scatchard plot/Kaleidagraph to ensure that the same amount of ligand binding sites were used in each experiment. Monomers of the ecdysone response element hsp27, which are routinely used for DNA binding studies such as gel mobility shift assays, increase ligand binding to all EcR isoforms in the presence of wild type Usp about 4-6 fold (Fig 4A). For determination of transcriptional activity tandemly

  A c c e p t e d M a n u s c r i p t 11 arranged EcREs oligomers were often used to enhance the hormonal response

  A c c e p t e d M a n u s c r i p t 12 3.5. Deletion of the DNA binding domain of Usp improves ligand binding to EcR in the presence of DNA in an isoform specific manner.

  the presence of a suited EcRE due to increased dimerization of the receptor molecules.3.7. RXR can partially replace Usp in ligand binding assays only in the presence ofDNAIf Usp is replaced by RXR, no high affinity hormone binding is observed (data not shown). However, in the presence of a hormone response element, RXR also confers ligand binding to the heterodimer (Fig.10). Ligand affinity is in a comparable range compared to heterodimers with Usp, but the number of binding sites is reduced (concentration of EcR-B1/RXR = 0.07 nM/L compared to EcR/Usp concentration = 1.05 nM/L (see fig.7) in the presence of 5xDR1) indicating that heterodimerization of EcR with RXR is less efficient compared to formation of EcR/Usp complexes.

  The influence of hormone on nuclear receptor function is studied mainly by transcriptional assays. However ligand induced changes in receptor function are the result of a variety of different receptor properties e.g. dimerization and DNA binding of the receptor protein. Hormone and/or heterodimerization also enhance the concentration of the expressed receptor proteins (Ruff, unpublished results) in an isoform specific manner. Direct measurements of ligand binding allow evaluation of the impact of different receptor domains, DNA and dimerization partner on hormoneinduced effects. 4.1 Interaction of EcR with DNA improves ligand binding in the absence of a heterodimerization partner.

  with EcR to different degrees depending on the isoform used (Beatty

  , 2006). Therefore we expected that deletion of the DNA binding domain of Usp would also modify the impact of DNA binding on ligand affinity to the receptor complexes.

  are rather flexible, which certainly facilitates modification of receptor activity.

Fig. 1 :

 1 Fig.1: Influence of the hormone response element 5x DR1 on ligand binding to EcR

Fig. 3 :

 3 Fig. 3: Ligand binding of heterodimers of EcR isoforms and wild type Usp

Fig. 4 :

 4 Fig. 4: Influence of hormone response elements on ligand binding (Ponasterone A)

Fig. 5 :

 5 Fig. 5: Influence of hormone response elements on ligand binding (Ponasterone A)

Fig. 6 :

 6 Fig. 6: Influence of hormone response elements on ligand binding (Ponasterone A) to

  bars indicated by *, ** and + are significant (p<0.01, n= 3).

Fig. 7 :

 7 Fig. 7: Ligand binding to EcR-B1/UspII cotransfected in CHO cells in the presence of 5x DR1. 3 H-Ponasterone (specific activity 7.9 Bq/ mmol) was used as ligand. A) In

Fig. 8 :

 8 Fig. 8: Influence of hormone response elements on ligand binding (Ponasterone A) to

Fig. 9 :

 9 Fig. 9: Ligand binding to EcR-A/UspIII cotransfected in CHO cells. 3 H-Ponasterone

Fig. 10 :

 10 Fig. 10: Ligand binding of heterodimers of EcR -B1 and RXR cotransfected in CHO-1K cells in the presence of 5x DR1. 3 H-Ponasterone (specific activity: 7.9 TBq /

  ., Laval, M.,Lepesant, J.A., 1993. Structural features critical to the activity of an ecdysone receptor binding site. Insect Biochem.Mol. Biol. 23,105-114. Beatty, J., Fauth, Th., Callendar, J.L., Spindler-Barth, M., Henrich, V.C., 2006.Functional analysis of ecdysteroid receptor isoforms inDrosophila melanogaster in a cell culture system. Insect Mol. Biol. 15, 785-795. Bender, M., Imam, F.B., Talbot, W.S., Ganetzky, B., Hogness, D.S., 1997. Drosophila ecdysone receptor mutations reveal functional differences among receptor isoforms. Cell 91, 777-788. Bevan, C.L., Hoare, S., Claessens, F., Heery, D.M., Parker, M.G., 1999. The AF1 and AF2 domains of the androgen receptor interact with distinct regions of SRC1.Mol. Cell. 19, 8383-8392. Billas, I.M., Iwema, T., Garnier, J.M., Mitschler, A., Rochel, N., Moras, D., 2003. Structural adaptability in the ligand-binding pocket of the ecdysone hormone receptor.Nature 426,[91][92][93][94][95][96]

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Acknowledgements 3 H-Ponasterone A was a kind gift of Dr. H. Kayser (Syngenta, Basel, Switzerland). A plasmid coding for wild type Usp was kindly provided by Dr. S. Braun, who modified a precursor plasmid obtained by Dr. Ozyhar (Technical University of Wroclav, Poland) to allow expression of wild type Usp. Expression plasmids for EcR-A, EcR-B1, EcR-B2 and Usp I, II and III and RXR, were kindly provided by Dr. V.C. Henrich (University of North Carolina, Greensboro, NC, USA). Antibody AB11 was a gift of Dr. Kafatos (EMBL, Heidelberg, Germany). The monoclonal antibody Ag10.2 developed by Thummel and Hogness was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 55242 USA. Ponasterone A was purified by M. Burret (University of Ulm). We kindly acknowledge also his help by preparing the figures. The work was supported by a grant from the DFG to MSB (Spi 350/5-1 and 2) and a PhD-scholarship to AA (Graduate college 1041). The technical assistance of N. Möbius, the help of K. Belisle, who corrected and improved the English version of the manuscript is gratefully acknowledged.