Mária Szekeres
email: szekeres@eok.sote.hu

Gábor Turu

Anna Orient

Bence Szalai

Katinka Süpeki

Miklós Cserző

Péter Várnai

László Hunyady

Mechanisms of angiotensin II-mediated activation of aldosterone synthase in H295R human adrenocortical and rat adrenal glomerulosa cells

Keywords: adrenal cortex, aldosterone synthase, angiotensin II, calmodulin kinase, gene expression, mediated gene induction in adrenocortical cells

In adrenal zona glomerulosa cells angiotensin II (Ang II) is a key regulator of steroidogenesis. Our purpose was to compare the mechanisms of Ang II-induced changes in the expression level of early transcription factors NR4A1 (NGFIB) and NR4A2 (Nurr1) genes, and the CYP11B2 gene encoding aldosterone synthase in H295R human adrenocortical tumor cells and in primary rat adrenal glomerulosa cells. Real-time PCR studies have demonstrated that Ang II increased the expression levels of NR4A1 and NR4A2 in H295R cells within one hour after stimulation, which persisted up to 6 hrs; whereas in rat adrenal glomerulosa cells the kinetics of the expression of these genes were more rapid and transient. Ang II also induced prolonged nuclear translocation of Nurr1 and NGFIB proteins in both cell types. Studies using MEK inhibitor (PD98059, 20μM), protein kinase C inhibitor (BIM1, 3μM) and calmodulin kinase (CAMK) inhibitor (KN93, 10μM) revealed that in rat adrenal glomerulosa cells CAMK-mediated mechanisms play a predominant role in the regulation of CYP11B2. In accordance with earlier findings, in H295R cells MEK inhibition increased the expression of NR4A1, NR4A2 and CYP11B2 genes, however, it decreased the Ang II-induced gene expression levels, suggesting that ERK activation has a role in control of expression of these genes. No such mechanism was detected in rat glomerulosa cells. Sar 1 -Ile 4 -Ile 8 -AngII, which can cause G protein-independent ERK activation, also stimulated the expression of CYP11B2 in H295R cells. These data suggest that the previously reported CAMKmediated stimulation of early transcription factors NGFIB and Nurr1 has a predominant role in Ang II-induced CYP11B2 activation in rat adrenal glomerulosa cells, whereas in H295R cells ERK activation and G protein-independent mechanisms also contribute to this process.

Introduction

Angiotensin II (Ang II) is the major effector molecule of the renin-angiotensin system, which has major physiological and pathophysiological roles in different cell types and organs, such as regulation of vasoconstriction, aldosterone secretion, cell growth, proliferation, differentiation and tissue remodeling, inflammation, atherosclerosis, diabetic nephropathy and cancer. Ang II exerts its actions via AT 1 and AT 2 angiotensin receptors. Ang II-induced AT 1 receptor activation stimulates G proteindependent and -independent mechanisms, as well [START_REF] De Gasparo | International union of pharmacology. XXIII. The angiotensin II receptors[END_REF][START_REF] Hunyady | Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II[END_REF][START_REF] Oliveira | The angiotensin II AT1 receptor structure-activity correlations in the light of rhodopsin structure[END_REF]. AT 1 receptor activation via G q/11 protein stimulates phospholipases, IP 3 /DAG signaling, protein kinase C (PKC), phosphoinositide 3-kinase, receptor and non-receptor kinases, mitogen activated protein kinases (MAPK), etc. [START_REF] De Gasparo | International union of pharmacology. XXIII. The angiotensin II receptors[END_REF][START_REF] Hunyady | Structural determinants of agonist-induced signaling and regulation of the angiotensin AT1 receptor[END_REF][START_REF] Oliveira | The angiotensin II AT1 receptor structure-activity correlations in the light of rhodopsin structure[END_REF]. However, Ang II also induces G protein-independent signaling pathways, such as β-arrestin-mediated activation of MAP-kinases (McDonald et al., 2000;[START_REF] Wei | Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2[END_REF][START_REF] Szidonya | AT1 receptor blocker-insensitive mutant AT1A angiotensin receptors reveal the presence of G protein-independent signaling in C9 cells[END_REF]. In A c c e p t e d M a n u s c r i p t 3 adrenal glomerulosa cells Ang II induces G q/11 -mediated activation of Ca 2+ signal generation and PKC activation, which is followed by activation of diverse signaling pathways, including stimulation of MAP kinases, tyrosine kinases, lipoxygenase production, phospholipase D and phospholipase A 2 (Hajnoczky et al., 1992;[START_REF] Gu | 12-lipoxygenase pathway increases aldosterone production, 3',5'-cyclic adenosine monophosphate response element-binding protein phosphorylation, and p38 mitogen-activated protein kinase activation in H295R human adrenocortical cells[END_REF][START_REF] Spät | Control of aldosterone secretion: a model for convergence in cellular signaling pathways[END_REF][START_REF] Shah | Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells[END_REF][START_REF] Bollag | Characterization and phospholipase D mediation of the angiotensin II priming response in adrenal glomerulosa cells[END_REF]. Ang II can also activate G protein-independent signaling mechanisms of type 1 angiotensin receptor (AT1R), which can be exclusively activated by Sar 1 -Ile 4 -Ile 8 -AngII [START_REF] Wei | Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2[END_REF][START_REF] Daniels | Divergent behavioral roles of angiotensin receptor intracellular signaling cascades[END_REF].

In adrenal zona glomerulosa cells Ang II regulates aldosterone secretion by stimulating its synthesis. The initial phase of Ang II-induced stimulation of aldosterone production is mediated by increased availability of the precursor molecules, including cholesterol and NADPH, whereas long term activation of aldosterone synthesis requires increased expression of steroid-transporting and steroidogenic proteins, such as StAR protein and CYP11B2 or aldosterone synthase (Bassett and White et al., 2004;[START_REF] Spät | Control of aldosterone secretion: a model for convergence in cellular signaling pathways[END_REF][START_REF] Capponi | The control by angiotensin II of cholesterol supply for aldosterone biosynthesis[END_REF]. Mechanisms of the Ang II-induced expression of CYP11B2 have been investigated in H295R human adrenocarcinoma cells and primary glomerulosa cells [START_REF] Spät | Stimulus-secretion coupling in angiotensin-stimulated adrenal glomerulosa cells[END_REF][START_REF] Clyne | Calcium regulates human CYP11B2 transcription[END_REF][START_REF] Natarajan | Key role of P38 mitogenactivated protein kinase and the lipoxygenase pathway in angiotensin II actions in H295R adrenocortical cells[END_REF][START_REF] Spät | Control of aldosterone secretion: a model for convergence in cellular signaling pathways[END_REF][START_REF] Rainey | Adrenocortical cell lines[END_REF]Bassett and White et al., 2004). It has been suggested that paracrine delivery of steroid precursors to CYP11B2 may also contribute to aldosterone synthesis, which underlines the importance of this enzyme in aldosterone biosynthesis [START_REF] Vinson | Glomerulosa function and aldosterone synthesis in the rat[END_REF]. In H295R cells Ang II-induced activation of human CYP11B2 gene involves Ca 2+ signal-mediated activation of calmodulin kinases (CAMK), which regulates the transcription and/or activity of early transcription factors, such as members of the AP-1 complex (including FOS and JUN), the early growth response (EGR) factor family, CREB, Nur-related factor 1 (Nurr1 or NR4A2) and nerve growth factor-induced clone B (NGFIB or NR4A1) (Bassett and White et al., 2004;[START_REF] Bassett | The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production[END_REF][START_REF] Spät | Control of aldosterone secretion: a model for convergence in cellular signaling pathways[END_REF]. Early transcription factors NGFIB (NR4A1) and Nurr1 (NR4A2), which are important regulators of CYP11B2 expression, belong to the class 4 receptors of nuclear receptor (NR) family [START_REF] Li | NR4A1, 2, 3--an orphan nuclear hormone receptor family involved in cell apoptosis and carcinogenesis[END_REF][START_REF] Lonard | Nuclear receptor coregulators and human disease[END_REF].

Gene targets of Ang II in glomerulosa cells were defined in H295R cells (Romero et al., 2004, Bassett andWhite et al., 2004). Comparative microarray studies by [START_REF] Nogueira | Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells[END_REF] in human, rat and bovine cells revealed a similar pattern of Ang II-induced genes in these cells. Members of the NR family of transcription factors were among the most induced genes in all three cell types, which is consistent with the role of these proteins in the Ang II-induced regulation of steroidogenesis. Both in H295R cells and in rat glomerulosa cells NR4A2 and NR4A1 were the most induced NR genes [START_REF] Nogueira | Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells[END_REF]. In the present study we aimed to investigate the quantitative contribution of G protein-mediated signaling mechanisms, including PKC and CAMK pathways, and the G protein-independent mechanisms to the effect of Ang II on the expression of CYP11B2 and transcription factors Nurr1 (NR4A2) and NGFIB (NR4A1) in H295R human adrenocortical cells and in primary rat glomerulosa cells to evaluate the general relevance of these mechanisms in the regulation of aldosterone synthesis. were cultured in DMEM/-F12 (1:1) medium (with l-Glutamine, 15 mM HEPES, purchased from Invitrogen) supplemented with 2% Ultroser FS serum (Biosepra, Villeneuve-la-Garenne, France), 1% ITS-1 serum (Sigma-Aldrich) and 1% Pen/Strep antibiotics (Sigma-Aldrich). Cell monolayers were subcultured into culture flasks and transferred onto 6-well plates before used. Cells were incubated in 5% CO 2 at 37 °C. Cells were passaged 3-13 times.

Materials and Methods

Animals

Male Wistar rats (170-200 g, Charles River Laboratories-Semmelweis University, Budapest) kept on a standard semisynthetic diet were used with the approval (No. 109/003/2007) of the Animal Health and Food Control Office of Budapest. All procedures followed legal and institutional guidelines of animal care.

Preparation of rat glomerulosa cells

Rat glomerulosa cells were prepared from the adrenal capsular tissue with collagenase digestion, as described previously [START_REF] Spät | Angiotensin II and potassium activate different calcium entry mechanisms in rat adrenal glomerulosa cells[END_REF]. Isolated cells were plated on polylysine-coated 6-well plates and incubated in 5% CO 2 at 37 °C in a medium consisting of modified Krebs-Ringer-bicarbonate solution and M199 culture medium (final concentrations: 145 mM Na + , 3.6 mM K + , 0.5 mM Mg 2+ , 1.2 mM Ca 2+ ), [START_REF] Rohács | Capacitative Ca2+ influx in adrenal glomerulosa cells: possible role in angiotensin II response[END_REF] overnight. Collagenase type one was purchased from Worthington Biochemicals (Lakewood, NJ). Chemicals were purchased from Sigma-Aldrich.

Treatment protocols

H295R cells were kept serum-starved overnight prior to stimulation. Rat glomerulosa cells were maintained in a serum-free medium. In Protocol 1 dose response curves for Ang II (1 µM, Sigma-Aldrich) from 100 pM to 1 μM were obtained in both cell types. Cells were stimulated with Ang II for 1 and 6 hours separately. In Protocol 2 cells were stimulated with Ang II for 1-2-4-6 hours. In Protocol 3 MAPK kinase (MEK) inhibitor (PD98059, 20 μM, Sigma-Aldrich), PKC inhibitor (bisindolylmaleimide-1, BIM1, 3 μM and 10 μM, Merck Chemicals Ltd., Darmstadt, Germany), calmodulin kinase inhibitor (KN93, 10 μM, Calbiochem) and epidermal growth factor receptor (EGFR) inhibitor (AG1478, 1 μM, Calbiochem) were added half hour prior to 1-hour Ang II (100 nM) stimulation. In Protocol 4 cells were treated with PD98059, BIM1, KN93 and AG1478 prior to 6-hour Ang II-stimulation (1 μM). In Protocol 5 H295R cells were treated with 1 μM Ang II or 30 μM Sar 1 -Ile 4 -Ile 8 -AngII (Bachem, Torrance, CA) for 6 hours in order to investigate Ang II-induced G protein-independent effects.

Protocol for RNA extraction and real-time PCR

A c c e p t e d M a n u s c r i p t 5 Cells were washed twice with sterile PBS (with composition in mM: NaCl 137, KCl 2.7, Na 2 HPO 4 10.1, KH 2 PO 4 1.8, pH 7.4), and removed with trypsin-EDTA (1:4 dilution into PBS), total RNA was extracted (RNeasy mini kit, Qiagen) and reverse transcription was carried out according to manufacturer's instructions (Fermentas) was made.

Real-time PCR assays were performed on capillary-based LightCycler 1.5 System (Roche Applied Science) with SYBR Green method. Reactions were performed in a LightCycler 1.5 real-time PCR equipment (Roche) using Taq polimerase. Primers were designed (Table 1) and synthesized by Sigma-Genosys (Germany) or purchased from SuperArray Biosciences (Frederick, MD). Cycling conditions were: 10 min preincubation at 95 C, 45-50 cycles of 95 C 10 sec, 62 C 5 sec and 72 C 15 sec. Fluorescence data including melting curves were obtained. For normalization glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was used. Efficiency for each primer pair was determined by using serial dilutions of the PCR product. Fold ratios of gene expressions were calculated as follows:

Ratio=E ΔCt target gene / E ΔCt GAPDH Ct was calculated by the second derivative method using LightCycler 3.5 Software. ΔCt is the difference in Ct values obtained from control sample and pharmacologically treated sample.

Messenger RNA levels of aldosterone-synthesizing enzyme (CYP11B2) and early transcription factors nuclear receptor subfamily 4, group A, member 1 (NR4A1 or NGFIB) and nuclear receptor subfamily 4, group A, member 2 (NR4A2 or Nurr1) were determined in human and rat adrenocortical cells.

Immunostaining and confocal microscopy

Rat glomerulosa cells were kept in serum-free medium, whereas H295R cells were serum-starved overnight prior to stimulation. In Protocol 6 cells were stimulated with 1 µM Ang II for 24 hours, and the cells were restimulated with 1 µM Ang II after 12 hours. Cells were fixed with 4% paraformaldehide (diluted in PBS) on glass coverslips, permeabilized with 0.1 % Triton, incubated with primary and secondary antibodies. Nurr1 (sc-990) and Nur77 (NGFIB, sc-5569) antibodies were purchased from Santa Cruz Biotechnology, and visualized rhodamine-labeled anti-rabbit antibody (Jackson ImmunoResearch-Izinta Trading Co., Hungary). Rhodamine fluorescence was detected with confocal microscopy and images were analyzed with Zeiss LSM Image Browser (Carl Zeiss GmbH) and ImageJ softwares.

Statistical analysis

Data are presented as Means±SEM. For paired data Student's t-test was applied; for multiple comparisons one-way or two-way ANOVA with repeated measures followed by Tuckey's post hoc test were applied. Fold changes of gene expression levels are plotted against control. P<0.05 was considered significant.

Dose-response curves of Ang II-induced expression of early transcription factors NGFIB (NR4A1) and Nurr1 (NR4A2) in H295R and rat adrenal glomerulosa cells (Protocol 1)

1 hour Ang II-stimulation from 100 pM to 1 μM caused a dose-dependent increase in the expression of early transcription factor genes, NR4A1 and NR4A2 both in H295R cells and in rat glomerulosa cells (Fig. 1, n=3-6) that reached maximum at 100 nM or 1 μM Ang II concentration after 1 hour or 6 hours stimulation, respectively. The maximum expression level for NR4A1 and NR4A2 was 15.9±2.0-and 23.1±6.2-fold in H295R cells, and 20.5±3.8-and 22.7±5.5-fold in rat glomerulosa cells, respectively. In case of rat glomerulosa cells NR4A2 was only minimally activated by Ang II at 6 hours (Fig. 1, panel D, and see also Fig. 2, panel E). Increasing the duration of Ang II-stimulation 1 hour to 6 hours induced a right shift of the dose response curves in all conditions. In the following experiments the Ang II concentration was selected to obtain maximal stimulation. Therefore, in protocol 3 with 1 hour of Ang II-stimulation we applied 100 nM of Ang II. Since after 6-hour stimulation the Ang II-induced gene activation did not reach its maximum level at 100 nM Ang II concentration, thus in protocols 4-6, with at least 6 hours of Ang II-stimulation, we applied 1 μM of Ang II to induce maximal stimulation.

Time-dependence of Ang II-induced expression of early transcription factors NGFIB (NR4A1) and Nurr1 (NR4A2) as well as aldosterone synthase in H295R and rat adrenal glomerulosa cells (Protocol 2)

In H295R cells Ang II (1μM) caused a rapid increase of the mRNA levels of early transcription factors NR4A1 (NGFIB) and NR4A2 (Nurr1). Maximal increase of the expression levels of these genes were obtained within 1 hour stimulation with Ang II, when NR4A1 and NR4A2 mRNA expressions were stimulated 21.8±4.4-and 26.7±11.0fold over their control levels, respectively (n=4), which was slowly decreased to 13.5±4.9-and 13.3±4.5-fold over control levels, respectively, after 6 hours of Ang II stimulation (n=4) (Fig. 2, panels A-B). Expression of CYP11B2 mRNA showed a much slower increase in H295R cells, which increased by 6.8±2.5-fold after 6 hours of Ang II stimulation (n=4) (Fig. 2,panel C). In rat adrenal glomerulosa cells, Ang II-induced upregulation of early transcription factors, NR4A1 and NR4A2, was rapid and more transient compared to H295R cells. The maximum levels of NR4A1 and NR4A2 expressions were obtained after 1 hour stimulation with Ang II, at the levels of 31.7±4.8and 33.8±6.8-fold over the control levels (n=3), respectively. After prolonged stimulation with Ang II the mRNA expression levels of these transcription factors rapidly decreased to 5.9±1.1-and 2.9±0.4-fold, respectively, after 6 hour stimulation (n=3) (Fig. 2, panel D-E). In rat cells CYP11B2 reached the maximum expression level around 2 hours of stimulation (7.4±1.8-fold, n=3) (Fig. 2, panel F). Ang II-induced nuclear translocation of Nurr1 protein, which was more pronounced in H295R cells (Fig. 3,panel A,B). Cell nucleus/cytoplasm fluorescence density ratios were increased from 3.01±0.4 to 5.34±0.5 in H295R cells and from 2.38±0.16 to 3.77±0.4 in rat glomerulosa cells, respectively (Fig. 3, panel B).

Effects of Ang II on the localization of NGFIB and Nurr1 in human and rat glomerulosa cells (Protocol 6)

Early transcription factor NGFIB localizes both to the cytoplasm and the nucleus of H295R and rat glomerulosa cells, as well (Fig. 4, panel A). Prolonged stimulation with Ang II did not change significantly the total fluorescence intensity of NGFIB in these cells, however, Ang II induced nuclear translocation of NGFIB in both types of cells. Cell nucleus/plasma fluorescence density ratios were increased from 1.96±0.02 to 3.8±0.4 in H295R cells and from 1.32±0.04 to 2.17±0.27 in rat glomerulosa cells, respectively (Fig. 4, panel B).

Effects of inhibitors on Ang II-induced expression of aldosterone synthase, early transcription factors NGFIB (NR4A1) and Nurr1 (NR4A2) in human and rat adrenocortical cells (Protocols 3 and 4)

In H295R cells 1 hour Ang II-stimulation (100 nM) enhanced expression of NR4A1 and NR4A2 genes by 14.5±2.6-and 27.6±6.4-fold (n=4), respectively (Fig. 5, panels A and B). In rat glomerulosa cells 1 hour Ang II-stimulation induced NR4A1 and NR4A2 genes by 30.4±7.2-and 22.1±3.5-fold (n=3-5) (Fig. 5, panels C and D), respectively. Since in H295R and rat glomerulosa cells after 1 hour Ang II stimulation the induction of CYP11B2 was minimal (Fig. 2, panels C and F), the mechanism of this response was not investigated at this early phase of Ang II stimulation. After pretreatment of the cells with MEK, PKC and CAMK inhibitors statistically significant reduction of the Ang II-induced NR4A1 response was not observed in H295R cells (Fig. 5, panel A), and only the CAMK inhibitor caused statistically significant partial inhibition of this response in rat glomerulosa cells (Fig. 5,panel C). The Ang II-induced early induction of NR4A2 was very effectively reduced by CAMK inhibition both in H295R cells (to 4.0±1.4-fold) and in rat glomerulosa cells (to 2.9±0.8-fold). Although MEK and PKC inhibitors did not affect the NR4A1 and NR4A2 responses in rat glomerulosa cells -however BIM1 moderately decreased Ang II-induced NR4A2 expression that did not reach statistical significance -(Fig. 5, panels C-D), these inhibitors moderately interfered with the Ang II-induced NR4A2 response in H295R cells (Fig. 5, panel B, p < 0.05, n=4).

Additional studies were performed to investigate the late phase of the Ang II response. In H295R cells prolonged (6-hour) administration of Ang II (1 μM) enhanced the expression of NR4A1, NR4A2 and CYP11B2 by 12.6±0.5-, 15.7±1.9-and 4.6±1.0fold (n=4-8), respectively (Fig. 6, panels A-C). Under these experimental conditions in the MEK inhibitor PD98059 pretreated cells increased mRNA expression levels of NR4A1, NR4A2 and CYP11B2 were detected in non-stimulated cells by 1.7±0.06-, A c c e p t e d M a n u s c r i p t 8 2.34±0.3-and 1.6±0.2-fold (n=4-8), respectively (Fig. 6, panels A-C, p<0.05). The MEK inhibitor, which significantly increased the basal activities of NR4A1, NR4A2 and CYP11B2 also statistically significantly interfered with the Ang II-induced responses of the expressions of these genes (Fig. 6, p<0.05). Basal expression of genes in response to PKC inhibition with BIM1 did not change in H295R cells (Fig. 6), and even an increased dose of BIM1 (10 μM) did not change the expression of CYP11B2 (data not shown). Although BIM1 decreased Ang II-induced expression of NR4A1 and NR4A2 significantly (p<0.05 by one-way ANOVA without interaction), the moderate decrease in CYP11B2 expression was not significant. CAMK inhibition had no effect on the Ang IIinduced NR4A1 expression (Fig. 6, panel A), but significantly interfered with the Ang IIinduced stimulation of NR4A2 (Fig. 6, panel B), similarly to the results obtained in Fig. 5B. The Ang II-induced CYP11B2 response was completely abolished by CAMK inhibitor treatment. The effect of CAMK inhibitor in non-stimulated cells was also significant (Fig. 6, panel C, p<0.05). In parallel experiments, AG1478, an EGFR kinase inhibitor, did not have significant effects on Ang II-induced gene activations (data not shown).

In contrast to the observations in H295R cells, in rat glomerulosa cells the inhibitors alone did not cause any elevation of the gene expression levels (Fig. 7, n=4-5). PKC inhibitor BIM1 slightly increased CYP11B2 expression that did not reach statistical significance (Fig. 7, panel B). Increasing the dose of BIM1 to 10 μM even did not change the expression of CYP11B2 (data not shown). 6-hour Ang II-stimulation induced expression of NR4A1 and CYP11B2 genes by 7.5±1.8-and 3.7±1.2-fold, respectively (Fig. 7). Since Ang II-induced activation of NR4A2 mRNA expression was transient and only minimal effect was detected after 6 hours of stimulation with Ang II in rat glomerulosa cells (Fig. 2, panel E), NR4A2 expression studies were excluded from this series of experiments. Ang II-induced late activation of NR4A1 was slightly reduced by MEK, PKC and CAMK inhibitors, but these effects were not statistically significant (Fig. 7, panel A). Ang II-induced CYP11B2 expression was completely inhibited by the CAMK inhibitor (p<0.05), whereas the other inhibitors had no effect on this response (Fig. 7, panel B). AG1478 did not have significant effects on Ang II-induced gene activations (data not shown).

G protein-independent signaling effects of Ang II in H295R cells (Protocol 4)

SII-Ang II was applied in order to investigate G protein-independent signaling effects of Ang II (Wei et al., 2003, Fig. 8). In H295R cells Ang II-induced expression of NR4A1, NR4A2 and CYP11B2 genes at 6-hours of stimulation (see also at Fig. 6) was compared with the effects of SII-Ang II. SII-Ang II induced the expression of NR4A1, NR4A2 genes by 2.1±0.5-, 2.0±0.8-and 2.9±0.9-fold (n=4), respectively, which was significant in case of NR4A1 and CYP11B2 (Fig. 8, panel A). In case of rat glomerulosa cells 6 hours of stimulation with Ang II induced significant increase in mRNA levels of NR4A1, NR4A2 and CYP11B2 genes to 10.8±2.8-, 2.6±0.3-and 8.5±1.2-fold (n=3), respectively. However, SII-Ang II did not cause significant changes in gene expression levels in these cells (Fig. 8, panel B).

Discussion

A c c e p t e d M a n u s c r i p t 9 Ang II is a major physiological regulator of the aldosterone secretion of adrenal glomerulosa cells. The mechanism of the regulation of aldosterone synthesis involves stimulation of the cholesterol supply to the mitochondria, increased reduction of pyridine nucleotides in the mitochondria providing NADPH for the steroid biosynthesis and induction of the proteins required for aldosterone production [START_REF] Spät | The effect of endogenous angiotensin on the biosynthesis of aldosterone in the rat[END_REF][START_REF] Spät | Stimulus-secretion coupling in angiotensin-stimulated adrenal glomerulosa cells[END_REF][START_REF] Spät | Generation and role of calcium signal in adrenal glomerulosa cells[END_REF]Bassett and White et al., 2004;[START_REF] Spät | Control of aldosterone secretion: a model for convergence in cellular signaling pathways[END_REF]Rossier and Capponi, 2004). Earlier studies have demonstrated that in the adrenal cortex transcription factors, such as NGFIB, Nurr1, SF-1, ATF-1, CREB, GATA4, GATA6 can induce promoter activation of steroidogenic genes, including CYP11B2, CYP17, CYP21, CYP11A, HSD3B2 [START_REF] Davis | Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands[END_REF]Bassett and White et al., 2004;[START_REF] Kelly | Modulation of steroidogenic enzymes by orphan nuclear transcriptional regulation may control diverse production of cortisol and androgens in the human adrenal[END_REF][START_REF] Bassett | Expression profiles for steroidogenic enzymes in adrenocortical disease[END_REF]. In zona glomerulosa cells Ang II stimulates rapidly the expression and synthesis of the StAR protein leading to increased cholesterol supply of the mitochondria, whereas induction of CYP11B2 occurs more slowly during the late phase of the Ang II-induced aldosterone response [START_REF] Enyeart | ACTH and AII differentially stimulate steroid hormone orphan receptor mRNAs in adrenal cortical cells[END_REF][START_REF] Capponi | The control by angiotensin II of cholesterol supply for aldosterone biosynthesis[END_REF][START_REF] Rainey | Adrenocortical cell lines[END_REF][START_REF] Bassett | The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production[END_REF].

The agonist-induced activation of CYP11B2 gene expression is preceded by early induction of transcription factors and other genes, which has been characterized in detail in H295R cells [START_REF] Clyne | Calcium regulates human CYP11B2 transcription[END_REF][START_REF] Clyne | Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements[END_REF][START_REF] Rainey | Adrenocortical cell lines[END_REF]. The CYP11B2 gene contains a cAMP-response element (CRE) and at least three additional cis-acting regulatory elements (NBRE-1, Ad-4 and Ad-5) in its promoter region, which serve as targets for transcription factors, including Nurr1, NGFIB, SF-1, ATF-1, COUP etc. (Bassett and White et al., 2004;[START_REF] Capponi | The control by angiotensin II of cholesterol supply for aldosterone biosynthesis[END_REF]. It has been suggested that members of the NGFIB family of orphan nuclear receptors, Nurr1 and NGFIB, have major roles in the Ang II-induced stimulation of CYP11B2 expression in adrenocortical cells [START_REF] Bassett | The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production[END_REF][START_REF] Lu | Nur-related factor 1 and nerve growth factor-induced clone B in human adrenal cortex and its disorders[END_REF][START_REF] Rainey | Adrenocortical cell lines[END_REF]. Microarray analysis of the Ang II-induced gene expression patterns has been studied in H295R human adrenocortical cells, in human adrenal glands, and primary rat and bovine glomerulosa cells [START_REF] Romero | Angiotensin II early regulated genes in H295R human adrenocortical cells[END_REF][START_REF] Rainey | Adrenocortical cell lines[END_REF][START_REF] Bassett | Expression profiles for steroidogenic enzymes in adrenocortical disease[END_REF][START_REF] Romero | Gene expression profile in rat adrenal zona glomerulosa cells stimulated with aldosterone secretagogues[END_REF][START_REF] Nogueira | Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells[END_REF]. In a recent study early gene expression after Ang II stimulation has been compared in these adrenocortical model systems [START_REF] Nogueira | Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells[END_REF]. The microarray comparison revealed 20-36% similarity in the Ang II-induced genes in bovine, human and rat cells. However, several genes were increased in all three models, including activating transcription factor 3 (ATF3), B-cell translocation gene (BTG2), c-FOS, FOSB, JUNB, NR4A1, NR4A2, NR4A3 and EGR-1. Interestingly, although members of the NGFIB family of orphan nuclear receptors were among the top genes induced by Ang II in all three cells, in the three species the quantitatively largest response occurred in different members of this family: Ang II-induced largest response was observed in NR4A1 (NGFIB), NR4A2 (Nurr1) and NR4A3 in rat glomerulosa cells, bovine glomerulosa cells and H295R cells, respectively [START_REF] Nogueira | Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells[END_REF]. The Ang II-induced genes in these adrenocortical cells, and their roles in zona glomerulosa function has been reviewed in a paper of this issue (Nogueira et al., in press, in this issue).

In the present study we compared the mechanism of Ang II-induced CYP11B2 expression in H295R cells and primary rat adrenal glomerulosa cells. The dose-and timedependent kinetics of Ang II-induced gene expression was different between human and A c c e p t e d M a n u s c r i p t 10 rat adrenocortical cells. Ang II-induced dose-responses in NR4A1 and NR4A2 expressions were maximal at 100 nM concentration of Ang II when cells were stimulated for 1 hour, however, the curves were shifted to the right showing a lower potency of Ang II when the cells were stimulated for 6 hours (Fig. 1). The apparently lower potency of Ang II in experiments when stimulation was 6 hours or longer may be caused by degradation of the peptide ligand during prolonged stimulation. Time-dependent kinetics of Ang II-stimulation indicated differences in gene activations among the two cell types. Although both in human and rat adrenocortical cells transcription factors NR4A1 and NR4A2 show an activation peak by 1 hour of stimulation followed by a decline in the response, in H295R cells the kinetics of decline is slower compared to rat glomerulosa cells (Fig. 2). The Ang II-induced NR4A2 response was particularly rapid in rat glomerulosa cells, and mRNA level returned close to the control level after 6 hours of stimulation. Ang II-induced activation of CYP11B2 gene peaked at 2-4 hours in rat cells, however in human cells there was a slower increase up to 6 hours. Similar kinetics of Ang II-induced gene expression in H295R cells was observed by Romero et al., although in that study the earliest time point the authors investigated was 3 hours [START_REF] Romero | Angiotensin II early regulated genes in H295R human adrenocortical cells[END_REF].

Immunofluorescence imaging of early transcription factors Nurr1 and NGFIB revealed different localization patterns of these transcription factors in adrenocortical cells. In resting cells Nurr1 can be found predominantly in the cell nucleus of both human and rat adrenocortical cells; however NGFIB shows a more dense appearance also in the cytoplasm. Long term stimulation with Ang II lead to a reproducible increase of Nurr1 protein density in H295R cells, but not in rat glomerulosa cells, which is consistent with the transient nature of the Nurr1 mRNA response in the rat cells. In non-stimulated human prostate cancer cells NGFIB (TR3) was also found to exhibit nuclear localization [START_REF] Li | Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3[END_REF]. However Ang II also induced nuclear translocation of NGFIB and Nurr1 both in human and rat adrenocortical cells, which is consistent with the role of these transcription factors in the induction of steroidogenic enzymes. It has been reported that NGF-induced phosphorylation of NGFI-B in PC12 phaeochromocytoma cells results in a transient translocation of the NGFI-B-RXR heterodimer complex out of the nucleus using nuclear export signals within NGFI-B [START_REF] Katagiri | Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B[END_REF]. On the other hand, it is also well-established that calcium-mediated activation of calcium/calmodulin-dependent protein kinase I (CAMKI) and possibly CAMKIV regulates human CYP11B2 expression [START_REF] Condon | Calmodulindependent kinase I regulates adrenal cell expression of aldosterone synthase[END_REF]Bassett andSuzuki et al., 2004, Bassett andWhite et al, 2004). Based on these findings it is tempting to speculate that CAMK-mediated phosphorylation of NGFIB and/or Nurr1 is responsible for the increase accumulation of these factors in the cell nucleus. It has also been suggested that NGFIB may also translocate to the cytoplasm upon activation of apoptosis stimuli [START_REF] Katagiri | Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B[END_REF][START_REF] Li | Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3[END_REF]. These findings are consistent with the proposal that agonist-induced nuclear translocation of these factors contributes to the Ang II-induced regulation of aldosterone biosynthesis.

In agreement with earlier findings suggesting that CAMKs play a predominant role in the regulation of aldosterone production stimulated by Ang II, K + and cAMP in rat and bovine glomerulosa cells [START_REF] Pezzi | Ca(2+)-regulated expression of aldosterone synthase is mediated by calmodulin and calmodulin-dependent protein kinases[END_REF][START_REF] Condon | Calmodulindependent kinase I regulates adrenal cell expression of aldosterone synthase[END_REF][START_REF] Gambaryan | Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP[END_REF], we also found, that inhibition of the activity of CAMKs eliminates the Ang IIinduced induction of CYP11B2, and inhibits the stimulation of Nurr1, and to a lesser extent NGFIB in rat glomerulosa cells and in H295R cells. However, the reduced A c c e p t e d M a n u s c r i p t 11 sensitivity of the NGFIB response to Ang II, compared to that of CYP11B2, suggests, that induction of NGFIB is not sufficient to achieve the CYP11B2 response and/or CAMK-mediate regulation is not limited to an effect on NGFIB, and other targets may play a role in this process.

The role of ERK activation in the mechanism of Ang II-induced aldosterone secretion and CYP11B2 expression response of adrenal glomerulosa cells is controversial. [START_REF] Natarajan | Key role of P38 mitogenactivated protein kinase and the lipoxygenase pathway in angiotensin II actions in H295R adrenocortical cells[END_REF] found that Ang II-induced activation of p38 MAP kinases, but not ERK, plays a role in the Ang II-induced aldosterone response in H295R adrenocortical cells. Both p42/44 and p38 MAPKs have been shown to be involved in the Ang IIinduced aldosterone production in glomerulosa cells with the involvement of the Rho-Rac pathways [START_REF] Otis | Role of MAPKs in angiotensin II-induced steroidogenesis in rat glomerulosa cells[END_REF]. However, the inhibitory effect of MEK1 inhibition on the Ang II-induced aldosterone response of bovine adrenal glomerulosa cells and H295R cells has also been reported [START_REF] Osman | Repression of DAX-1 and induction of SF-1 expression. Two mechanisms contributing to the activation of aldosterone biosynthesis in adrenal glomerulosa cells[END_REF][START_REF] Suzuki | Novel action of activin and bone morphogenetic protein in regulating aldosterone production by human adrenocortical cells[END_REF]. On the other hand, LeHoux and Lefebvre ( 2004) found, that MEK inhibitor PD98059 activated CYP11B2 promoter activity suggesting, that p44/42 MAPK kinase pathway is inhibitory for basal CYP11B2 promoter activity. Our data demonstrate that MEK1 inhibition, in agreement with the findings of LeHoux and Lefebvre, stimulates the basal expression level of CYP11B2 in H295R cells; but not in rat adrenal glomerulosa cells. However, our data also suggest that MEK1 inhibition with PD98059 also interferes with the Ang II-induced CYP11B2 induction in H295R cells, but this effect is also absent in rat glomerulosa cells. This finding is consistent with the data of [START_REF] Osman | Repression of DAX-1 and induction of SF-1 expression. Two mechanisms contributing to the activation of aldosterone biosynthesis in adrenal glomerulosa cells[END_REF] and [START_REF] Suzuki | Novel action of activin and bone morphogenetic protein in regulating aldosterone production by human adrenocortical cells[END_REF] on the inhibitory effect of MEK1 inhibition on the Ang II-induced aldosterone response. Both inhibitors U0126 and PD98059 were shown to decrease Ang II-induced aldosterone production in adrenal glomerulosa cells [START_REF] Osman | Repression of DAX-1 and induction of SF-1 expression. Two mechanisms contributing to the activation of aldosterone biosynthesis in adrenal glomerulosa cells[END_REF] and U0126 was also effective in H295R cells [START_REF] Suzuki | Novel action of activin and bone morphogenetic protein in regulating aldosterone production by human adrenocortical cells[END_REF]. However, the opposite effect of MEK1 inhibition with PD98059 on CYP11B2 expression in control and Ang IIstimulated adrenocortical cells suggests that ERK activation can lead to different consequences in control and Ang II-stimulated cells. Although the explanation for this discrepancy is not clear, it is important to mention that phospho-ERK activity can be present in different cellular compartments, such as in the cytosol and the nucleus, but accumulation of phospho-ERK on the surface of endosomal membranes has also been reported in Ang II-stimulated cells [START_REF] Luttrell | Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds[END_REF]. Inhibition of the ERK activity in different compartments may explain the different effects of MEK1 inhibition in control and Ang II-stimulated H295R cells.

Our data suggest that MEK1 inhibition also interferes with the Ang II-induced stimulation of NR4A1 and NR4A2 expression in H295R cells. The role of MAP kinases in the activation and induction of NR4As in different cells types has also been reported [START_REF] Katagiri | Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B[END_REF][START_REF] Darragh | MSKs are required for the transcription of the nuclear orphan receptors Nur77, Nurr1 and Nor1 downstream of MAPK signalling[END_REF][START_REF] Li | NR4A1, 2, 3--an orphan nuclear hormone receptor family involved in cell apoptosis and carcinogenesis[END_REF]. The effect of MEK1 inhibition on NR4A1 and NR4A2 expression may contribute to the observed inhibition of Ang II-induced CYP11B2 response in H295R cells.

Earlier studies have suggested that PKC can inhibit the steroidogenesis of adrenal glomerulosa cells. In rat adrenal glomerulosa cells PKC activation and inhibition lead to inhibition and stimulation of the Ang II-stimulated aldosterone response, respectively (Hajnoczky et al., 1992). In accordance with the earlier observations, treatment of H295R cells with BIM1 increased CYP11B2's promoter activity (LeHoux and Lefebvre, 1998) also showing a negative regulatory effect of PKC on the transcription of CYP11B2. More A c c e p t e d M a n u s c r i p t 12 PKC inhibitors were found to stimulate aldosterone output, such as GF109203X and staurosporine as well [START_REF] Lehoux | Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms[END_REF], Hajnoczky et al., 1992[START_REF] Spät | Control of aldosterone secretion: a model for convergence in cellular signaling pathways[END_REF]. [START_REF] Lehoux | Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms[END_REF] suggests, that the seemingly paradoxical effect of Ang II on stimulating aldosterone biosynthesis while activating DAG-dependent PKC that inhibits CYP11B2 expression may be explained by that Ang II exerts a positive action on CYP11B2 expression by other signaling pathway(s) yet to be discovered, and a negative action through the PKC signaling pathway. The negative regulatory action would be delayed in comparison to the positive action producing a feedback control to restore normal levels of CYP11B2 expression and to end the enhancing Ang II-action [START_REF] Lehoux | Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms[END_REF]. Based on the literature a rather negative regulatory action of PKC on acute control of aldosterone synthesis has been suggested [START_REF] Spät | Control of aldosterone secretion: a model for convergence in cellular signaling pathways[END_REF]. Several isoforms of PKC have been found in adrenocortical cells that make it more difficult and complex to understand the role of PKC in the signaling. [START_REF] Lehoux | Control of CYP11B2 gene expression through differential regulation of its promoter by atypical and conventional protein kinase C isoforms[END_REF] found in agreement with previous reports, that the rat adrenal zona glomerulosa expressed PKCα and -ε and PKC isoforms α, -ε, and -ζ were also commonly expressed in the human adrenocortical NCI-H295 cell line. They also found, that DAG-dependent PKC isoforms can suppress whereas the atypical PKC ζ can enhance the basal expression of CYP11B2 in transfected NCI-H295 cells. Lehoux and Lefebvre found, that overexpression of constitutive active PKCα and PKCε inhibited Ang II-induced CYP11B2 gene transcription, and suggested that PKCε inhibited Ang II-stimulated CYP11B2 gene expression via MEK-ERK signaling pathway in H295R cells [START_REF] Lehoux | Novel protein kinase C-epsilon inhibits human CYP11B2 gene expression through ERK1/2 signalling pathway and JunB[END_REF][START_REF] Lehoux | Angiotensin II activates p44/42 MAP kinase partly through PKCepsilon in H295R cells[END_REF].

Although, we have found that inhibition of PKC with BIM1 had no major effect on the basal and Ang II-stimulated CYP11B2 expression (there was a slight increase in basal CYP11B2 in rat glomerulosa cells, which did not reach statistical significance, Fig. 7, panel B), suggesting that PKC has no major role in the CYP11B2 response after prolonged Ang II stimulation (Fig. 6, panel C and Fig. 7, panel B). We also have found, that 1-hour inhibition of PKC with BIM1 did not alter basal CYP11B2 expression in rat glomerulosa cells as well, (data not shown), however, these cells showed some Ang IIinduced CYP11B2 expression at this time point in contrast with H295R cells (Fig. 2, panel C and 2, panel F). Regarding the role of PKC in the expression of NR4A1 and NR4A2 genes we have found, that BIM1 did not modify the basal expression level of early transcription factors, however, the Ang II-induced expression of NR4A1 and NR4A2 was reduced by BIM1 treatment in H295R cells.

Ang II-induced PKC activation can lead to ERK activation in several cell types [START_REF] Hunyady | Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II[END_REF][START_REF] Shah | Differential pathways of angiotensin II-induced extracellularly regulated kinase 1/2 phosphorylation in specific cell types: role of heparin-binding epidermal growth factor[END_REF]. Another mechanism of Ang II-induced ERK activation is EGFR transactivation, which can be detected in adrenal glomerulosa cells as well [START_REF] Shah | Differential pathways of angiotensin II-induced extracellularly regulated kinase 1/2 phosphorylation in specific cell types: role of heparin-binding epidermal growth factor[END_REF][START_REF] Shah | Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells[END_REF]. However, we did not find a consequent role of AT 1 receptor-EGFR-transactivation in the Ang II-induced expressions of CYP11B2, NR4A1 and NR4A2 in human and rat adrenocortical cells.

In addition to G protein-dependent mechanisms, Ang II can also stimulate G proteinindependent signal transduction mechanisms by directly associating with signaling molecules, such as ß-arrestins and tyrosine kinases (Hunyady andCatt, 2006, Wei et al., 2003). It has been suggested, that ß-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex A c c e p t e d M a n u s c r i p t 13 [START_REF] Luttrell | Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes[END_REF]. As in other G protein-coupled receptors, activation of the AT 1 R leads to its phosphorylation by specific receptor kinases and binding of ß-arrestin, which uncouples it from G proteins and also serves as an adaptor for clathrin-mediated internalization. ß-arrestins also serve as scaffolds to organize the formation of signaling complexes that mediate AT 1 R-mediated activation of ERK and JNK3 MAPKs [START_REF] Hunyady | Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II[END_REF]. Previous studies on a mutant AT 1 R that cannot couple to G proteins have shown that ß-arrestin2-mediated ERK activation can occur in the absence of G proteinactivation [START_REF] Hunyady | Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II[END_REF]. G protein-independent mechanisms through AT 1 R can be also studied by SII-Ang II, an AT 1 receptor ligand that cannot activate G proteinmediated signaling [START_REF] Wei | Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2[END_REF][START_REF] Daniels | Divergent behavioral roles of angiotensin receptor intracellular signaling cascades[END_REF]. We have found, that SII-Ang II-induced partial (about 2-3-fold) induction of NR4A1, NR4A2 and CYP11B2 genes in H295R cells (Fig. 8, panel A) suggesting that Ang II-induced activation of CYP11B2 and early transcription factors Nurr1 and NGFIB may be also partially mediated by G protein-independent mechanisms in H295R cells. In rat glomerulosa cells Ang II-induced G protein-independent signaling does not seem to play a role in the expression of these genes (Fig. 8, panel B). These data suggest that G-independent mechanisms may contribute to the signaling response in human cell lines (e. g. HEK cells), whereas these mechanisms are not detectable in rat adrenal glomerulosa cells.

In conclusion we found, that there are kinetic differences, as well as differences in the mechanisms of Ang II-induced gene activation patterns between H295R cells and rat adrenocortical cells. Although the previously reported calmodulin kinase-mediated stimulation of early transcription factors NGFIB and Nurr1 has a key role in Ang IIinduced CYP11B2 activation in both H295R cells and rat adrenal glomerulosa cells, this response has a much more complex mechanism in H295R cells. In addition to CAMKmediated mechanisms, ERK activation and G protein-independent mechanisms also contribute to the mechanism of Ang II-induced CYP11B2 activation in H295R cells. 1. Real time PCR primers used for the amplifications of human (h) and rat (r) genes. Primer set for human CYP11B2 was purchased from SuperArray Biosciences (Frederick, MD). Amplicon sizes are between 109 and 190 base pairs.

  culture H295R human adrenocortical cells (generously provided by Dr. William E. Rainey)

  early transcription factors, Nurr1 and NGFIB, in adrenocortical cells at the protein level was studied using confocal microscopy. Confocal imaging of Nurr1 shows its extensive localization in the cell nucleus of both H295R and rat glomerulosa cells (Fig.3, panel A). Prolonged stimulation with Ang II increased Nurr1 fluorescence intensity of H295R cells, but not of rat glomerulosa cells (Fig.3, panel A), which is consistent with the transient kinetics of mRNA expression in rat cells.

Fig. 1 .

 1 Fig. 1. Dose-response curves of Ang II (n=3-6). Expression of NR4A1 and NR4A2 genes in H295R cells (Panels A-B) and rat glomerulosa cells (Panels C-D) stimulated by Ang II for 1 hour (black dots) or 6 hours (white dots). Standardization was made against GAPDH control gene. Mean±SEM values are shown.

Fig. 2 .

 2 Fig. 2. Time-dependent effect of Ang II on the expression of genes in human and rat adrenocortical cells. H295R cells (Panels A-C, n=4) and rat glomerulosa cells (Panels D-F, n=3) were stimulated with Ang II (1 µM). Standardization was made against GAPDH control gene. Mean±SEM values are shown.

Fig. 3 .

 3 Fig. 3. Localization of Nurr1 in human and rat adrenocortical cells visualized by immunofluorescence. H295R and rat glomerulosa cells were stimulated for 24 hours with 1 µM Ang II, which stimulation was repeated after 12 hours. Immunostaining was performed and rhodamine fluorescence was detected by confocal microscopy (Panel A). Images were analyzed with Zeiss LSM Image Browser and ImageJ softwares. Cell nucleus/plasma fluorescence ratio was analyzed and compared between control and Ang II-treated cells (panel B). Several cells of each of 3-4 images for each treatments of 2-2 separate experiments were analyzed. Mean±SEM values are shown. * p < 0.05 compared to control.

Fig. 4 .

 4 Fig. 4. Localization of NGFIB in human and rat adrenocortical cells visualized by immunofluorescence. H295R and rat glomerulosa cells were stimulated with 2 µM Ang II for 24 hours. Immunostaining was performed and rhodamine fluorescence was detected by confocal microscopy (Panel A). Images were analyzed with Zeiss LSM Image Browser and ImageJ softwares. Cell nucleus/plasma fluorescence ratio was analyzed and compared between control and Ang II-treated cells (Panel B). Several cells of each of 3-4 images for each treatments of 3-3 separate experiments were analyzed. Mean±SEM values are shown. * p < 0.05 compared to control.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Ang II-induced signaling mechanisms involved in the expression of NR4A1 (NGFIB) and NR4A2 (Nurr1) genes in human and rat adrenocortical cells. H295R (panels A, B, n=4) and rat glomerulosa cells (panels C, D, n=3-5) were treated with MEK inhibitor (PD98059, 20μM), PKC inhibitor (BIM1, 3μM) and calmodulin kinase inhibitor (KN93, 10μM) half hour prior to 1-hour Ang II (100 nM) stimulation. Mean±SEM values are shown. Standardization was made against GAPDH control gene. * statistically significant interaction (p < 0.05) of the inhibitor with the Ang II response.

Fig. 7 .

 7 Fig. 7. Ang II-induced signaling mechanisms involved in the expression of NR4A1 (NGFIB, panel A) and CYP11B2 (panel B) genes in rat glomerulosa cells (n=4-5). Cells were treated with MEK inhibitor (PD98059, 20μM), PKC inhibitor (BIM1, 3μM) and calmodulin kinase inhibitor (KN93, 10μM) half hour prior to 6-hour Ang II (1 µM) stimulation. Mean±SEM values are shown. Standardization was made against GAPDH control gene. * statistically significant interaction (p < 0.05) of the inhibitor with the Ang II response.

Fig. 8 .

 8 Fig. 8. Effect of Sar 1 -Ile 4 -Ile 8 -Ang II (SII) on the expression of NR4A1, NR4A2 and CYP11B2 genes in human and rat adrenocortical cells. H295R cells (panel A, n=4) and rat glomerulosa cells (panel B, n=3) were treated with 1 μM Ang II and 30 μM SII for 6 hours in order to investigate Ang II-induced G protein-independent effects. Mean±SEM values are shown. Standardization was made against GAPDH control gene. * p < 0.05 compared to control.

  Figure1

Acknowledgements

Supported by Grants OTKA T49851 of Hungarian National Academy of Sciences and Jedlik Ányos 1/010/2005. The authors express their thanks for László Szidonya, Judit Bakacsi, Kata Szabolcsi, András Berekméri, Klára Tóth, Klára Somogyi, Jolán Józan for their expert assistance.


T Martijn Kuijper

Kirsten Ruigrok-Ritstier

Miriam Verhoef-Post

Djura Piersma

Martijn W P Bruysters

Els M J J Berns

PhD Axel P N Themmen
email: a.themmen@erasmusmc.nl

Axel P N Themmen<ce

T Martijn Kuijper

LH receptor gene expression is essentially absent in breast

Keywords: breast cancer, LH receptor, prognosis, qRT-PCR

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A c c e p t e d M a n u s c r i p t ABSTRACT Worldwide, breast cancer is the most frequently occurring malignancy in women. Early age at full term pregnancy has a protective effect against breast cancer.

Evidence coming from a rat breast cancer model suggests a possible role for the pregnancy hormone hCG, a ligand of the LH receptor, as a mediator for this effect. In a previous study, we found that a common polymorphism in the LH receptor associates with tumor progression in premenopausal breast cancer patients, as carriers of the variant receptor showed a shorter disease free survival compared to noncarriers.

How hCG and its receptor exert their effects on breast cancer, however, is unclear. One possibility is that these effects take place through LH receptors present in the ovaries, thereby influencing steroid hormone production. Another possibility is that the effects take place through LH receptors present in breast tumor cells itself, as some studies have detected the receptor in both normal and neoplastic breast tissues and in breast cancer cell lines.

To investigate whether a direct effect of LH signalling in breast cancer is likely, we measured LH receptor mRNA expression levels in 1551 breast tumors and 42 different human breast cancer cell lines using a qRT-PCR with a wide dynamic range. In addition, associations between LH receptor expression and clinicopathologic factors were investigated.

Assay validation showed that as little as ~10 copies per reaction volume of LH receptor cDNA could still be detected by our assay. We show that LH receptors are undetectable in 62% of breast tumor samples and 41 of 42 breast cancer cell lines. For the remaining samples we found expression levels to be very low. Although low, A c c e p t e d M a n u s c r i p t expression of the LH receptor appears to be associated with normal breast cells, favorable tumor characteristics and low tumor percentage.

Since expression of the LH receptor in breast cancer cells is very low, it is almost excludes the possibility of direct signaling effects. We therefore conclude that signaling effects of the LH receptor on breast cancer most likely take place by an indirect pathway through the ovaries.

A c c e p t e d M a n u s c r i p t INTRODUCTION Breast cancer is the most common malignancy in women around the world, with an estimated number of 1.5 million new cases to occur in the year 2010 [START_REF] Parkin | Global cancer statistics[END_REF].

While incidence rates are highest in western industrialized countries, an increase in incidence rates is seen in many developing countries, most likely as a consequence of the adoption of western lifestyles [START_REF] Parkin | Use of statistics to assess the global burden of breast cancer[END_REF].

Several risk factors for developing breast cancer have been identified and established over the years. Gender, older age and carriership of high risk susceptibility genes (i.e. BRCA1 or BRCA2) each give rise to a more than ten fold increase in risk [START_REF] Clemons | Estrogen and the risk of breast cancer[END_REF][START_REF] Nelson | Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility: systematic evidence review for the U.S. Preventive Services Task Force[END_REF]. In addition to these, factors related to hormone exposure, especially estrogens, and several reproductive factors are also highly associated with the disease [START_REF] Clemons | Estrogen and the risk of breast cancer[END_REF][START_REF] Yager | Estrogen carcinogenesis in breast cancer[END_REF].

A reproductive factor that has frequently been associated with breast cancer risk is a woman's age at birth of her first child [START_REF] Clemons | Estrogen and the risk of breast cancer[END_REF][START_REF] Brekelmans | Risk factors and risk reduction of breast and ovarian cancer[END_REF][START_REF] Dumitrescu | Understanding breast cancer risk -where do we stand in 2005?[END_REF][START_REF] Macmahon | Epidemiology and the causes of breast cancer[END_REF]. It was shown that a linear association exist between age at first full term pregnancy and breast cancer risk, a younger age having a protective effect against the disease [START_REF] Macmahon | Epidemiology and the causes of breast cancer[END_REF]. However, the mechanism by which this protective effect takes place is largely unknown. It has been postulated that the protective effect of pregnancy on breast cancer is mediated by the hormone hCG [START_REF] Russo | Hormonal approach to breast cancer prevention[END_REF][START_REF] Rao | Does full-term pregnancy at a young age protect women against breast cancer through hCG?[END_REF]. hCG is a glycoprotein hormone, produced by the placenta during early pregnancy. Structurally and functionally, hCG is very similar to LH and both hormones exert their effects through the common LH/CG receptor [START_REF] Themmen | Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function[END_REF].

Evidence for a protective effect of hCG on breast cancer was found by Russo and coworkers [START_REF] Russo | Hormonal approach to breast cancer prevention[END_REF][START_REF] Rao | Does full-term pregnancy at a young age protect women against breast cancer through hCG?[END_REF]. In a rat model for breast cancer, tumor induction with the chemical 7,12-dimethylbenz[α]anthracene (DMBA) was more effective in nulliparous than parous animals. Notwithstanding the fact that CG does not naturally occur in A c c e p t e d M a n u s c r i p t rats, Russo and coworkers found that a similar protective effect could be obtained when nonparous animals were pretreated with hCG for 21 days, a period equivalent to full-term pregnancy in rats [START_REF] Russo | Hormonal approach to breast cancer prevention[END_REF][START_REF] Rao | Does full-term pregnancy at a young age protect women against breast cancer through hCG?[END_REF]. These results suggest that both pregnancy and treatment with hCG have a protective effect against mammary tumors and tumor progression when given after tumor induction [START_REF] Russo | Hormonal approach to breast cancer prevention[END_REF][START_REF] Rao | Does full-term pregnancy at a young age protect women against breast cancer through hCG?[END_REF].

In addition to the findings that hCG has protective effects on breast cancer progression, evidence that the LH receptor is involved in tumor progression as well has came from studies performed by our research group [START_REF] Powell | Luteinizing hormone signaling and breast cancer: polymorphisms and age of onset[END_REF][START_REF] Piersma | A common polymorphism renders the luteinizing hormone receptor protein more active by improving signal peptide function and predicts adverse outcome in breast cancer patients[END_REF][START_REF] Piersma | GnRH and LHR gene variants predict adverse outcome in premenopausal breast cancer patients[END_REF]. In two independent cohorts, breast cancer patients that are carrier of a common polymorphism in the LH receptor, the insLQ variant, show a significantly shorter disease free survival compared to non-carriers [START_REF] Piersma | A common polymorphism renders the luteinizing hormone receptor protein more active by improving signal peptide function and predicts adverse outcome in breast cancer patients[END_REF][START_REF] Piersma | GnRH and LHR gene variants predict adverse outcome in premenopausal breast cancer patients[END_REF]. Interestingly, the adverse effect of the insLQ variant on disease free survival was observed in premenopausal women only [START_REF] Piersma | GnRH and LHR gene variants predict adverse outcome in premenopausal breast cancer patients[END_REF]. In addition to this, it was shown that the insLQ variant is a functional polymorphism, as transfection studies revealed that the variant receptor shows both a higher surface expression and a slight increase in receptor sensitivity after stimulation with LH or hCG when compared to the more common nonLQ variant [START_REF] Piersma | A common polymorphism renders the luteinizing hormone receptor protein more active by improving signal peptide function and predicts adverse outcome in breast cancer patients[END_REF].

These apparent contradictory results indicate that hCG and its target receptor play a role in breast cancer progression. However, the mechanisms of these effects remain unclear. A possibility would be that the effects are mediated by LH receptors present in the ovaries, thereby influencing the production of ovarian hormones, subsequently leading to either a protective or adverse effect on breast cancer progression. However, an effect on disease progression of LH/hCG through LH receptors present in breast cancer tissue cannot be excluded.

Although LH receptors have long been considered to be expressed exclusively in ovarian and testicular tissues, over the last decade several studies have been A c c e p t e d M a n u s c r i p t published that claim their presence also in both normal and neoplastic breast tissues [START_REF] Meduri | Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer[END_REF][START_REF] Meduri | Luteinizing hormone receptor status and clinical, pathologic, and prognostic features in patients with breast carcinomas[END_REF][START_REF] Carlson | Presence of luteinizing hormone/human chorionic gonadotropin receptors in male breast tissues[END_REF], and in breast cancer cell lines [START_REF] Meduri | Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer[END_REF][START_REF] Lojun | Presence of functional luteinizing hormone/chorionic gonadotropin (hCG) receptors in human breast cell lines: implications supporting the premise that hCG protects women against breast cancer[END_REF][START_REF] Hu | Determinants of Transcription of the Chorionic Gonadotropin /Luteinizing Hormone Receptor Gene in Human Breast Cells[END_REF][START_REF] Jiang | Alternately spliced luteinizing hormone/human chorionic gonadotropin receptor mRNA in human breast epithelial cells[END_REF][START_REF] Gebauer | Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells[END_REF][START_REF] Bodek | A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate[END_REF][START_REF] Rao Ch | Human chorionic gonadotropin decreases proliferation and invasion of breast cancer MCF-7 cells by inhibiting NF-kappaB and AP-1 activation[END_REF]. In these studies, LH receptor expression was determined using qualitative techniques such as immunohistochemistry and non-quantitative RT-PCR. In one study, on 160 breast cancer specimens, this qualitative immunohistochemical assessed LH receptor expression was associated with younger age at diagnosis, premenopausal status, lower grade tumors, lobular invasive carcinomas and the presence of estrogen receptors [START_REF] Meduri | Luteinizing hormone receptor status and clinical, pathologic, and prognostic features in patients with breast carcinomas[END_REF].

In the present study we investigated whether a direct effect of LH receptor signaling in breast cancer is probable. In order to do this, we have determined LH receptor mRNA expression levels using a specific and quantitative method on a large cohort of 1551 breast tumors and on a panel of 42 different breast cancer cell lines.

We found that expression levels are in most cases undetectable or extremely low. If present, intriguingly, these low LH receptor expression levels are associated with favorable tumor characteristics.

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

MATERIALS AND METHODS

Breast tumor samples

The study design was approved by the Medical Ethics Committee of the Erasmus MC (Rotterdam, The Netherlands; MEC 02.953). It included breast tumor tissue specimens of 1,683 female patients with primary operable breast cancer. Frozen tumor samples were processed from patients with breast cancer who entered the clinic between 1979 and 1996. Details on follow-up and analyses are given in [START_REF] Foekens | High tumor levels of vascular endothelial growth factor predict poor response to systemic therapy in advanced breast cancer[END_REF]. Tumors with specimens of poor RNA quality were excluded from analysis [START_REF] Sieuwerts | How ADAM-9 and ADAM-11 differentially from estrogen receptor predict response to tamoxifen treatment in patients with recurrent breast cancer: a retrospective study[END_REF] and as a consequence tumor specimens of 1551 patients (92%) were analyzed for LH receptor expression. The cut-off point to classify primary breast tumors as ER and/or progesterone receptor (PgR) -positive was 10 fmol / mg cytosolic protein.

Breast cancer cell lines

cDNA samples derived from 42 different human breast cancer cell lines were a kind gift of Dr. Mieke Schutte (Department of Internal Oncology, Erasmus MC, Rotterdam, the Netherlands). Detailed information about the origin of these cell lines has been described by Elstrodt et al. [START_REF] Elstrodt | BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants[END_REF].

TRex-hLHR cell line

TRex-hLHR cells were used to provide a stable cell line expressing the LH receptor and were established as described previously [START_REF] Richter-Unruh | Leydig cell hypoplasia: absent luteinizing hormone receptor cell surface expression caused by a novel homozygous mutation in the extracellular domain[END_REF]. To induce expression of the LH receptor, cells were plated in Costar six-well plates (Corning Inc., Corning, A c c e p t e d M a n u s c r i p t NY) and treated overnight with 15 ng / ml tetracycline (Sigma, St. Louis, MO), after which RNA was isolated.

LH receptor plasmid DNA

Construction of the expression plasmid pSG5-LHRWT is described by Kraaij et al. [START_REF] Kraaij | A missense mutation in the second A c c e p t e d M a n u s c r i p t transmembrane segment of the luteinizing hormone receptor causes familial malelimited precocious puberty[END_REF].

RNA isolation and cDNA synthesis

RNA isolation and cDNA synthesis from breast tumor samples and breast cancer cell lines were performed as described previously by Sieuwerts et al [START_REF] Sieuwerts | How ADAM-9 and ADAM-11 differentially from estrogen receptor predict response to tamoxifen treatment in patients with recurrent breast cancer: a retrospective study[END_REF].

For the TRex-hLHR and testis samples, total RNA was extracted with Trizol (Invitrogen, Breda, the Netherlands) according to the manufacturers protocol and treated for 30 minutes at 37 ˚C with DNase (Promega, Leiden, the Netherlands).

Thereafter, for the TRex-hLHR samples, reverse transcription was performed using 1.5 µg of total RNA aliquots in a final volume of 40 µl and subsequent RNase Htreatment was performed. For the testis samples, reverse transcription was performed using 1 µg of total RNA aliquots in a final volume of 25 µl without subsequent RNase H-treatment.

qRT-PCR

qRT-PCR reactions were performed in 25-µl reaction volume on an MX 3000P Stratagene System, in accordance with the manufacturer's protocol. A commercially available Assay-on-Demand kit (Applied Biosystems) was used for the LH receptor (Hs00174885_m1) on all samples. This Assay-on-Demand kit targets the exons 7 and 8 of the LH receptor mRNA transcripts. Although the method does not A c c e p t e d M a n u s c r i p t detect alternatively spliced LH receptor transcripts not containing exons 7 and 8, such transcripts are not relevant to the present study, since the amino acids encode by exons 7 and 8 are essential to LH receptor protein function [START_REF] Themmen | Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function[END_REF]. For the breast tumor samples and testis samples, three reference genes (ie, porphobilinogen deaminase (PBGD), hypoxanthine-guanine phospho-ribosyltransferase (HPRT), and β-2microglobulin (β2M)) were determined to allow for correction for differences in total RNA content between samples. Primer sequences and amplification protocol for these reference genes have been previously described by Sieuwerts et al [START_REF] Sieuwerts | How ADAM-9 and ADAM-11 differentially from estrogen receptor predict response to tamoxifen treatment in patients with recurrent breast cancer: a retrospective study[END_REF].

Forty-five rounds of amplification were performed according to the supplier's protocol, and at the end of every round of amplification fluorescent signals of the TaqMan probes (Applied Biosystems) were acquired and used to generate cycle threshold (Ct) values from which mRNA expression levels were calculated when applicable.

Measurements were performed in triplicate for both the TRex-hLHR cDNA and LH receptor plasmid dilution series. For the breast tumor and testis samples, expression levels of the LH receptor were determined in duplicate and triplicate respectively, while expression levels of each of the three reference genes were determined only once. A relative copy number of the LH receptor, normalized against the three reference genes, was calculated as follows: relative copy number = 2 (mean Ct

Ref -mean Ct LHR)

. We were able to reliably measure LH receptor expression up to 37 amplification rounds using a dilution series of LH receptor cDNA derived from the TRex-hLHR cell line. We therefore used a relative copy number of 2 -16 , approximating a Ct value of 37, as detection threshold for the LH receptor. Whenever LH receptor quantification rounds exceeded this value, quantities were considered to be undetectable and measurements were considered to be invalid. In case that two A c c e p t e d M a n u s c r i p t valid measurements were obtained for a breast tumor, the tumor was classified as having 'detectable' expression. In case that only one valid measurement was obtained for a tumor, expression was classified as 'inconclusive' and a relative copy number was computed as described above using only the valid measurement. In case that two invalid measurements were obtained for a tumor, no relative expression level could be calculated and expression was classified as 'not detectable'. For the breast cancer cell lines, crude Ct values were used for analysis.

Data analysis and statistics

Graphpad Prism version 3.02 for Windows (Graphpad Software, Inc., San Diego, CA) was used to generate linear fits through the data points obtained from the qRT-PCR on the dilution series of the TRex-hLHR cDNA and LHR plasmid DNA.

RESULTS

Assay Validation

The range of Ct values was defined within which LH receptor expression levels can be adequately determined by our qRT-PCR. For this purpose a 4-fold dilution series was made of cDNA derived from TRex-hLHR cells stably transfected with a wild type human LH receptor expression construct and qRT-PCR was performed (Figure 1A). A straight line with slope held constant to -1/log 2 (corresponding to a decrease of 1 Ct value per 2-fold increase in DNA content) was fitted through the data points. We concluded that the assay runs linear up to a Ct value of approximately 37, which was subsequently defined as the detection threshold of our assay.

To study the minimum number of LH receptor cDNA copies that can still be adequately detected by the LH receptor qRT-PCR, a dilution series was made of plasmid DNA encoding the LH receptor. A straight line fit as described above showed that the assay runs linear largely across the whole dilution series and that as little as ~10 copies (~6 fg) of plasmid DNA per reaction could still be detected by the qRT-PCR (Figure 1B).

For comparison expression of LH receptor mRNA in a human testis sample was determined using the identical qRT-PCR technique. A reference-corrected copy number of 5.64 . 10 -3 for the LH receptor in the testis tissue sample was found (Figure 2)

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

Expression of the LH receptor in breast cancer cell lines

Of the 42 breast cancer cell lines investigated, only five cell lines generated a Ct value after the maximum number of 45 amplification rounds. However, only one cell line, MDA-MB-415, was positive since it generated a Ct value that did not exceed the detection threshold of 37 cycles (Table 1).

LH receptor expression in breast tumors

LH receptor expression levels were determined in 1551 breast tumor samples by qRT-PCR (Figure 2). In 955 tumors (62%), LH receptor cDNA could not be detected. For 259 tumors (17%), only one of the two measurements was considered valid. Values found in the latter tumors fall in the lowest copy number category (0-0.025), indicating that LH receptor expression is very low in these tumors (Figure 2B). Moreover, 90% of the 337 remaining tumors with detectable expression also fall in the lowest expression category (Figure 2C). Only 33 tumors (approximately 2% of all breast cancer specimens studied) show a higher copy number. Remarkably, one tumor shows a relatively high expression of the LH receptor in comparison to the other tumors with a relative copy number of 4.78 . 10 -3 (Figure 2C).

LH receptor status in relation to patient and tumor characteristics

Associations between receptor status and clinico-pathologic factors are shown in Table 2. Patients were grouped based on LH receptor expression and included 1446 tumors of which LH receptor status and all clinico-pathologic data were available for further analysis. LH receptor expression is significantly associated with older age as well as postmenopausal status.

M a n u s c r i p t

No associations were found between expression status and surgical procedure, the number of lymph nodes involved or tumor grade. LH receptors are more frequently expressed by tumors that also express the estrogen and/or the progesterone receptor. Furthermore, expression of the LH receptor was significantly more often found in smaller tumors and tumor sections with lower tumor content.

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

DISCUSSION

To our knowledge this is the first study that addresses expression of the LH receptor in breast cancer specimens on such a large cohort, using 1551 breast tumor samples and 42 different human breast cancer cell lines. Moreover, it is the first to measure expression levels of LH receptor mRNA in breast tumors using a sensitive quantitative real time-PCR with a wide dynamic range.

The sensitivity of this assay to detect even low levels of LH receptor cDNA content was demonstrated by several experiments. First of all, we have shown that LH receptor cDNA levels could be adequately determined on a dilution series of cDNA derived from the TRex-hLHR cell line up to a Ct value of 37, corresponding to an approximately 8.5 . 10 6 fold dilution of cDNA obtained from the cDNA synthesis reaction. Secondly, the assay was able to adequately detect physiological levels of the LH receptor in a human testis sample. It should be noted that testicular tissue has a highly heterogeneous composition and that LH receptor-containing Leydig cells only contribute a small fraction of the total number of cells present in the human testis.

Finally, using a dilution series made of plasmid DNA containing the LH receptor, we show that the assay is able to detect a low number of approximately 10 plasmid copies per reaction volume.

These results indicate that this assay is highly sensitive and is able to detect very low levels of LH receptor mRNA. Nevertheless, we find that in about two third of the investigated breast tumors LH receptors cannot be detected at all. For the remaining tumors we find very low expression levels, generally more than 20-fold lower than the expression level measured in the testis sample. Of the 42 breast cancer cell lines investigated, only one cell line, MDA-MB-415, generated a Ct value just A c c e p t e d M a n u s c r i p t below the maximum number of 37 amplification rounds. This means that expression is detectable, but very low in this single cell line. The results obtained for the breast cancer cell lines support our findings in the breast tumors, meaning that expression is lost, or very low, in breast cancer specimens.

Few studies have been published over the last decade in which LH receptors were reported to be present both in human breast tumors [START_REF] Meduri | Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer[END_REF][START_REF] Meduri | Luteinizing hormone receptor status and clinical, pathologic, and prognostic features in patients with breast carcinomas[END_REF][START_REF] Carlson | Presence of luteinizing hormone/human chorionic gonadotropin receptors in male breast tissues[END_REF] and in breast cancer cell lines MCF-7, MBA-MB-231, T47D and ZR-75-1 [START_REF] Meduri | Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer[END_REF][START_REF] Lojun | Presence of functional luteinizing hormone/chorionic gonadotropin (hCG) receptors in human breast cell lines: implications supporting the premise that hCG protects women against breast cancer[END_REF][START_REF] Hu | Determinants of Transcription of the Chorionic Gonadotropin /Luteinizing Hormone Receptor Gene in Human Breast Cells[END_REF][START_REF] Jiang | Alternately spliced luteinizing hormone/human chorionic gonadotropin receptor mRNA in human breast epithelial cells[END_REF][START_REF] Gebauer | Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells[END_REF][START_REF] Bodek | A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate[END_REF][START_REF] Rao Ch | Human chorionic gonadotropin decreases proliferation and invasion of breast cancer MCF-7 cells by inhibiting NF-kappaB and AP-1 activation[END_REF][START_REF] Zhang | Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription[END_REF]. It is possible that in the studies that were able to detect the LH receptor, expression levels in cell lines were low, but detectable, whereas in the cell lines we investigated expression had dropped to levels that are not detectable anymore. Cell lines as they are kept in culture in different laboratories may have evolved and lost or acquired expression of certain genes, such as the LH receptor gene. Indeed, Zhang et al. [START_REF] Zhang | Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription[END_REF] found low but detectable expression levels of the LH receptor in the MCF-7 cell line using qRT-PCR. The discrepancy between our results and those reported in the literature cannot be explained by limitations in sensitivity. In most studies, less sensitive techniques than the qRT-PCR method employed in the present study were used to detect the LH receptor in breast cancer cell lines, such as conventional RT-PCR, northern and western blotting or immunostaining techniques.

Two studies have been published on the presence of LH receptor in human female breast tumors [START_REF] Meduri | Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer[END_REF][START_REF] Meduri | Luteinizing hormone receptor status and clinical, pathologic, and prognostic features in patients with breast carcinomas[END_REF], using immunohistochemical methods. In a pilot study [START_REF] Meduri | Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer[END_REF] conducted on only 19 malignant breast tumors, nine benign breast lesions and three normal breast tissue specimens, using the monoclonal LHR29 antibody for immunostaining, the majority of breast tumors and all benign breast lesions but also normal breast tissue samples were found to be positive for the LH receptor.

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

Remarkably, expression levels were found to be highest in the normal breast tissue samples rather than in the malignant breast tumor specimens. In the follow-up study of Meduri [START_REF] Meduri | Luteinizing hormone receptor status and clinical, pathologic, and prognostic features in patients with breast carcinomas[END_REF], 160 malignant breast tumors, approximately 10% of the number of tumors in our study, were investigated using the same antibody. Based on the staining pattern and an arbitrary cut-off value (18% labeled cells), 72% of the breast tumors were found to be positive for the LH receptor [START_REF] Meduri | Luteinizing hormone receptor status and clinical, pathologic, and prognostic features in patients with breast carcinomas[END_REF]. The rationale for this cut-off value was not further specified in this publication. This high prevalence is in contrast with our findings that LH receptor mRNA can be detected in maximally 38% of breast tumors. However, with mRNA levels as low as we observed, it seems highly unlikely that any specific staining of LH receptor proteins could be observed in these tumor samples. We should therefore take into consideration the possibility that in the studies performed by Meduri et al. [START_REF] Meduri | Luteinizing hormone receptor status and clinical, pathologic, and prognostic features in patients with breast carcinomas[END_REF], most of the staining observed in breast tumors is due to nonspecific staining instead of staining of LH receptor proteins present at the cellular membranes.

In the present study we found that expression of the LH receptor appears to be related to a lower percentage of tumor cells present in the tumor sections. In addition, we found that presence of the LH receptor is associated with smaller tumors. This leads us to hypothesize that the little LH receptor expression that we find in tumors may occur for the greatest part, or even completely, in the surrounding non-tumorous tissue, instead of in the tumor cells. This is in line with, Meduri et al. who found expression levels to be highest in normal breast tissue samples compared to breast tumors [START_REF] Meduri | Luteinizing hormone/human chorionic gonadotropin receptors in breast cancer[END_REF] and our findings using immunohistochemical analyses that also show higher expression in normal cells (data not shown). Another possibility would be that expression of the LH receptor by tumors reflects in some way the grade of A c c e p t e d M a n u s c r i p t differentiation of the tumor. In accordance with this is the finding that LH receptors are more often found in tumors that also express estrogen and/or progesterone receptors, features of good differentiation and less aggressive behavior of the tumor. Furthermore, we observe a trend between expression of the LH receptor and good/moderate tumor grade in this study. If expression of the LH receptor is indeed associated with less aggressive tumor behavior, then this could in turn explain the association we found between expression and smaller tumor size and low percentage of tumor cells. Further research is needed to find a definite explanation for these observations. Preclinical findings have led researchers to the idea that hCG may be used, either alone or in conjugate with a toxic compound, as a therapeutic agent that is capable to specifically target breast cancer cells [START_REF] Gebauer | Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells[END_REF][START_REF] Bodek | A novel approach of targeted ablation of mammary carcinoma cells through luteinizing hormone receptors using Hecate-CGbeta conjugate[END_REF][START_REF] Leuschner | Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo[END_REF][START_REF] Hansel | Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo[END_REF]. In addition to finding a cytotoxic effect of the Hecate-CGβ conjugate on MCF-7 and MDA-MB-435S cells in vitro, Leuschner et al. [START_REF] Leuschner | Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo[END_REF][START_REF] Hansel | Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo[END_REF] demonstrated this compound to be effective in vivo as well. After establishing MDA-MB-435S xenografts in athymic nude mice, they found that Hecate-CGβ could not only inhibit tumor growth [START_REF] Leuschner | Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo[END_REF], but was even able to cause tumor regression [START_REF] Hansel | Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo[END_REF]. This resulted in one clinical trial in which hCG alone was given as a treatment to breast cancer patients [START_REF] Janssens | Human chorionic gonadotropin (hCG) and prevention of breast cancer[END_REF]. According to the authors, treatment with hCG decreased the proliferative index (Ki67) of tumors (n=25) and was capable to slow down tumor progression in some patients with metastatic disease (n=13). This seems to be in contrast with the overt clinical effect of LHRH agonists for treatment of premenopausal breast cancer, which for a large part acts by suppression of LHR signaling. Furthermore, in the era of evidence-based medicine and of targeted Although these results may argue for an effect of hCG, either alone or in conjugated form, taking into consideration the expression levels of LH receptors in tumors as low as we find, it seems highly unlikely that any of these observed effects could be explained by the binding of hCG to the LH receptor. We must therefore consider the possibility that another factor is present at the cellular membranes of breast tumor cells that is also capable to bind hCG. Indeed, the study Zaleska et al. [START_REF] Zaleska | Growth repression in diethylstilbestrol/dimethylbenz[a]anthracene-induced rat mammary gland tumor using Hecate-CGbeta conjugate[END_REF] underscores our view. They observed in a rat model with DES/DMBA induced tumors that the Hecate-CGβ conjugate inhibited tumor growth to a greater extent than treatment with Hecate alone. However, similar to our present data, they could only detect low levels of LH receptor expression in 17% of the tumors using a nested RT-PCR method. Based on the observed almost absence of LHR on breast tumor specimens, targeted treatment of breast cancer using hCG-derivatives are unjustified.

On the other hand, a possible protective effect of hCG -high during the superphysiological condition of pregnancy-, on normal breast cells might make this a chemopreventive agent for breast cancer. Yet, elaborate studies are needed before final conclusions can be drawn.

Lastly, previous work of our group indicated that premenopausal breast cancer patients that are carrier of a common and functional polymorphism in the LH receptor, the insLQ variant, show a significant shorter disease free survival compared to non-carriers [START_REF] Piersma | A common polymorphism renders the luteinizing hormone receptor protein more active by improving signal peptide function and predicts adverse outcome in breast cancer patients[END_REF][START_REF] Piersma | GnRH and LHR gene variants predict adverse outcome in premenopausal breast cancer patients[END_REF]. It was postulated that the variant receptor may exert its effects by an indirect effect through the ovaries, affecting steroid hormone synthesis, or by a A c c e p t e d M a n u s c r i p t direct effect on breast cancer tissue itself. With expression levels in breast tumor tissues from the same cohort as low as we currently present, however, we may exclude the possibility of a direct effect of the LH receptor gene variant on breast tumor behavior. Thus, the insLQ polymorphism, causing a change in the expression level of the LH receptor in the ovary [START_REF] Piersma | A common polymorphism renders the luteinizing hormone receptor protein more active by improving signal peptide function and predicts adverse outcome in breast cancer patients[END_REF][START_REF] Piersma | GnRH and LHR gene variants predict adverse outcome in premenopausal breast cancer patients[END_REF], may exposure of the breast tissue to ovary-derived estrogens. The ovarian origin of the effect of the polymorphism is underlined by the observation that an association between breast cancer disease and the insLQ polymorphism is only seen in premenopausal women.

  Slopes were held constant to -1/log2, corresponding to the expected decrease of one Ct per two-fold increase in DNA content.Associations between LH receptor expression status and the various patient and tumor characteristics were investigated using Pearson's χ 2 test. All tests were twotailed, and statistical significance was assumed at P ≤ 0.05. Statistical analyses were performed with the STATA statistical package, release 9.2 (STATA Corp, College Station, TX).

A

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

A

  c c e p t e d M a n u s c r i p t treatment the authors failed to measure the LH receptor expression in the breast tumor specimens.

Figure 1 .

 1 Figure 1. Cycle threshold values obtained by performing the LH receptor qRT-PCR

Figure 2 .

 2 Figure 2. LH receptor expression levels in 1551 human breast tumors. Pie chart

Figure 1 A

 1 Figure 1

Figure 2 A

 2 Figure 2

Relative copy number (X 1000) Number of samples

		350
		300
	Number of samples	100 150 200 250																									T
	259	50
		0	0.00	0.25	0.50	0.75	1.00	1.25	1.50	1.75	2.00	2.25	2.50	2.75	3.00	3.25	3.50	3.75	4.00	4.25	4.50	4.75	5.00	5.25	5.50	5.75	6.00
														Relative copy number (X 1000)
	955
		350
		300
	337	250
		200
	Not detectable	150
	Inconclusive	100																									T
	Detectable	50
		0	0.00	0.25	0.50	0.75	1.00	1.25	1.50	1.75	2.00	2.25	2.50	2.75	3.00	3.25	3.50	3.75	4.00	4.25	4.50	4.75	5.00	5.25	5.50	5.75	6.00

Table 1

 1 Cycle threshold values as a measure for LH receptor mRNA expression in a panel of 42 different breast cancer cell lines. Measurement exceeds the detection threshold of 37 but was below 45 cycles, and expression should therefore be considered inconclusive. Cell lines that did not generate a Ct value after 45 rounds of amplification are indicated with 'Not detectable' (ND).

	Cell line	Ct value		Cell line	Ct value
	BT20	ND		MPE600	ND
	BT474	ND		OCUB-F	ND
	BT483	ND		OCUB-M	ND
	BT549	ND		SK-BR-3	ND
	CAMA-1	ND		SK-BR-5	ND
	DU4475	ND		SK-BR-7	ND
	EVSA-T	ND		SUM44PE	ND
	HCC1937	ND		SUM52PE	ND
	HS578T	inconclusive	a	SUM102PT	ND
	MCF-7	ND		SUM149PT	inconclusive	a
	MDA-MB-134VI	ND		SUM159PT	ND
	MDA-MB-157	inconclusive	a	SUM185PE	ND
	MDA-MB-175VII	ND		SUM190PT	ND
	MDA-MB-231	inconclusive	a	SUM225CWN	ND
	MDA-MB-330	ND		SUM229PE	ND
	MDA-MB-361	ND		SUM1315MO2	ND
	MDA-MB-415	35.89		T47D	ND
	MDA-MB-435S	ND		UACC812	ND
	MDA-MB-436	ND		UACC893	ND
	MDA-MB-453	ND		ZR75-1	ND
	MDA-MB-468	ND		ZR75-30	ND

a

Table 1 A

 1

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

Table 1

 1 LH receptor expression in relation to patient and tumor characteristics. Associations were tested using Pearson's χ 2 test. Cut-off value used was 10 fmol/mg cytosolic protein. c ER status was not known for 20 patients.

	Number of patients (%)
	LH receptor expression

a b d PgR status was not known for 101 patients.

Table 2

 2

ACKNOWLEDGEMENTS

The authors would like to thank Dr Mieke Schutte for providing the cDNA samples from the breast cancer cell lines and Drs John Foekens, Anieta Sieuwerts and Maxime Look for their contribution to this study.