Jean-Louis Louis Dacheux
email: jdacheux@tours.inra.fr

Clémence Belleannée

Russell Jones

Valérie Labas

Maya Belghazi

Benoît Guyonnet

Xavier Druart

Jean Luc Gatti

Françoise Dacheux

Mammalian Epididymal Proteome

Keywords: proteome, secretome, epididymis, sperm maturation

In all mammalian species, the final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several mammals could be an informative approach to finding common sperm modifications which are not speciesspecific. The new global biological approaches such the transcriptomes and proteomes provide considerable information which can be used for such comparative approaches. This report summarizes our proteomic studies of the epididymis of several mammals, including humans.

Introduction:

Epididymal function is essential for the fertility of male mammals because their sperm are infertile when they leave the testes and only acquire the ability to fertilize an ovum during passage through the epididymides. It is also essential that the epididymides accumulate and store sperm as, depending upon species, it takes 0.5 to 2 days for the testes to produce the number of sperm in a normal ejaculate, and in a competitive mating system males may inseminate up to 50 females in a day [START_REF] Jones | To store or mature spermatozoa? The primary role of the epididymis[END_REF][START_REF] Jones | Role of the epididymis in sperm competition[END_REF]. The efficacy of sperm storage in the epididymis is so high that fertile sperm can survive in an isolated epididymis for several days at 4°C [START_REF] Guérin | Conservation et utilisation du sperme épididymaire d'ovins et de cervidés en insémination artificielle et fécondation in vitro[END_REF] The epididymis is a very long duct which receives testicular sperm via the efferent ducts.

In eutherian and marsupial mammals the duct is differentiated into about 6 structurally distinct segments, indicating a well developed division of labor. Although all mammals have an initial segment of the epididymis with distinctive characteristics, there is variation between species in the structure and extent of the different segments, suggesting some variation in post-testicular sperm maturation and storage [START_REF] Jones | Evolution of the epididymis[END_REF]. The division of labor through the epididymis has been confirmed in studies of epididymal physiology and sperm modifications along the duct, and these findings have lead to the paradigm that the maturational changes in sperm in the epididymis are the result of sequential changes in their milieu, particularly the proteins secreted by the epididymal epithelium [START_REF] Dacheux | Contribution of epididymal secretory proteins for spermatozoa maturation[END_REF]. In view of the variations in epididymides between species mentioned above, it is considered that there must be conserved aspects and variations between species in the changes in protein composition throughout the epididymis, presumably reflecting the occurrence and relative significance of sperm maturation and storage between species. This short review therefore compares our findings regarding the proteome and secretome of epididymal fluid in mammalian species: i.e. the stallion, ram, boar and human. .

Characteristics of intra luminal epididymal proteomes

Most of the studies analyzing the proteins of epididymal fluids have been performed on samples obtained either from tubule micropuncture [START_REF] Turner | Protein synthesis and secretion by the rat caput epididymidis in vivo: influence of the luminal microenvironment[END_REF][START_REF] Turner | Obstruction of the vas deferens alters protein secretion by the rat caput epididymidal epithelium in vivo[END_REF] or from microperfusion techniques [START_REF] Druart | Analysis by two-dimensional gel electrophoresis of ram epididymal secreted proteins[END_REF][START_REF] Syntin | Characterization and identification of proteins secreted in the various regions of the adult boar epididymis[END_REF][START_REF] Fouchecourt | Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins[END_REF][START_REF] Dacheux | Human epididymal secretome and proteome[END_REF] The epididymal fluid provides a milieu for the gamete analogous to blood plasma providing a milieu for cell tissues. Due to the presence of the blood-testis and blood-epididymis barriers, most of the blood proteins are not found in epididymal plasma (albumin, transferrin and certain others being exceptions). In contrast to the stable concentration of blood plasma proteins, the protein concentration in epididymal fluid varies greatly along the duct: from 2-4 mg/ml in the initial segment of the epididymis, a maximum of 50-60 mg/ml in the distal caput and 20-30 mg/ml in the more distal regions of the organ (Fig 1C .). For most of the species studied, these variations in protein concentration follow the changes in water content of the fluid as assessed by changes in sperm concentration (Fig. 1A).

Several hundred epididymal proteins have been described electrophoretically and some have been identified. There is a wide range of dynamics in the abundance of these proteins (probably around 10 orders of magnitude). About 15-20 proteins make up more than 60 to 80% of the total protein concentration. The most common proteins found are lactoferrin, procathepsin D, NCP2 (HE1, CTP, cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, mannosidase, galactosidase, PGDS (Prostaglandin D2 synthase), clusterin, CRISP (Cystein-rich secretory protein) and E-RAPB (epididymal retinoic acid-binding protein).

The protein composition changes continuously throughout the duct, independently of the protein concentration in the fluid. The concentrations of the major common proteins cited above vary between species (Fig. 2). Lactoferrin, mannosidase, PGDS and albumin are present in high concentrations in the stallion, boar, ram and human, respectively, but GPX and PGDS are virtually absent in humans and boar, respectively.

Most of the epididymal proteins are characterized by their numerous isoforms which result from their high degree of glycosylation. The pI of these multi isoforms can vary widely, ranging from pH 3 to 8 for the same protein (i.e. RNase 10 in the boar, Fig. 3). The degree of glycosylation for the same protein can be different according to the epididymal

Dynamics of epididymal fluid proteomes

The spatial changes in the composition of luminal proteins are the result of two opposite activities of the epithelium: protein secretion and protein absorption throughout the epididymal duct. In the anterior part of the epididymis, the epididymal fluid is composed of a mixture of testicular and epididymal proteins. Most of the proteins originating from the testis, such as albumin, transferrin, testicular clusterin and PGDS [START_REF] Fouchecourt | Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins[END_REF] are reabsorbed in the efferent ducts [START_REF] Clulow | Micropuncture and cannulation studies of fluid composition and transport in the ductuli efferentes testis of the rat: comparisons with the homologous metanephric proximal tubule[END_REF] The rapidity of their absorption is species-specific and generally almost all are absent in the posterior part of the epididymis, except in humans in which albumin and transferrin are still present in large quantities.

The epididymal epithelium has high protein synthesis and secretion activity, activity being high both in the rates of protein synthesis and secretion and in the variety of proteins secreted. The anterior part of the epididymis is the most active (Figs. 1B,2). As for the protein concentration, from 70 to 80% of the secretome is composed of 10 to 20 of. the major secreted proteins present in the luminal fluid (Fig. 4). Most of the luminal proteins are secreted by the epithelium, but some, such as ACE, are released from the sperm surface by an unknown sheddase in an anterior part of the epididymis (Fig. 6) [START_REF] Gatti | A 105-to 94kilodalton protein in the epididymal fluids of domestic mammals is angiotensin Iconverting enzyme (ACE); evidence that sperm are the source of this ACE[END_REF][START_REF] Metayer | Germinal angiotensin Iconverting enzyme is totally shed from the rodent sperm membrane during epididymal maturation[END_REF][START_REF] Thimon | Shedding of the germinal angiotensin I-converting enzyme (gACE) involves a serine 27[END_REF].

Among the different proteins secreted, the same protein can be secreted in the same region of the epididymis in different species, for example PGDS, GPX and clusterin in the anterior part, and glucosidases in the middle part. Clusterin is secreted at a greater rate than the other proteins and can represent 30-40% of the all the proteins secreted. This clusterin can be sequentially secreted under different isoforms in different parts of the epididymis as in the stallion and the ram (Figs. 2,3). Some highly secreted proteins are characteristic of a species, for example, lactoferrin in the stallion, PGDS and GPXin the ram and RNase 10, mannosidase and hexosaminidase in the boar (Fig. 2).

In humans, in contrast to other species, few changes in pattern of protein secretion occur throughout the epididymis, a finding which correlates with the low degree of structural differentiation of the epididymal epithelium along the duct [START_REF] Holstein | Morphologische Studien am Nebenhoden des Menschen[END_REF].

Variations in luminal protein concentrations, controlled by secretory and absorption activites, are modulated for each species by the length of the epididymal duct involved in the two activities, i.e. the flow rate of the luminal fluid, enzyme degradation or the binding of protein on the sperm membrane. All these parameters differentially affect the concentrations of almost all proteins. Some are reabsorbed as soon as they are secreted and never reach high luminal concentrations, for example RNase 10 in the boar (Fig 5B) [START_REF] Castella | Train A, an RNase A-like protein without RNase activity, is secreted and reabsorbed by the same epididymal cells under testicular control[END_REF], and several isoforms of clusterin in the stallion (Fig. 3) [START_REF] Fouchecourt | Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins[END_REF]. For most other proteins, concentrations are high in the region where they are secreted and gradually decrease in the following region, for example GPX and PGDS in different species (Figs. 2,5A). The concentrations of some other proteins increase throughout the epididymal duct, as for example lactoferrin in the stallion and hexosaminidase and mannosidase in the boar (Fig. 2).

Relationships between sperm maturation and epididymal proteome among species

The most obvious and easily observed change in sperm directly related to acquisition of fertility is the activation of their flagella machinery. A gradual increase in the coordination of propagation of flagellar bending makes the gamete motile and progressive. In species that have been studied, this activation occurs in sperm in the corpus epididymides, after a transitory phase of uncoordinated beating. In humans, this activation occurs in sperm from the distal caput of the epididymis ( Few relationships have been observed between the major sequential modifications of the epididymal proteome and the sequential changes on the sperm surface. Furthermore, there is currently no direct evidence of a specific role for these major proteins, although several of them are known to be enzymes, inhibitors or binding proteins. For several of them, such as clusterin which is the most common epididymal protein found, gene KO in mice does not change the fertility of the animals [START_REF] Mclaughlin | Apolipoprotein J/clusterin limits the severity of murine autoimmune myocarditis[END_REF] .

It is probable that these major epididymal proteins which surround the gametes are more involved in sperm preservation than in inducing specific and localized modifications on the sperm surface. The high concentrations of proteins involved in the protection of gametes from oxidative stress, such as GPX5, thioredoxin peroxidase, glutathione S-transferase P, thioredoxin peroxidase and superoxide dismutase, probably contribute to sperm survival during epididymal storage.

However, no more than 10% of all the proteins present in the epididymal fluid have been identified. The wide range of protein concentrations makes identification of the less highly represented proteins by mass spectrometry more difficult. Furthermore, these unidentified proteins contain the most hydrophobic proteins. These proteins are almost never present in a soluble form in the epididymal luminal fluid but partly associated with membrane structures such as epididymosomes [START_REF] Frenette | Selected proteins of "prostasome-like particles" from epididymal cauda fluid are transferred to epididymal caput spermatozoa in bull[END_REF][START_REF] Gatti | Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles[END_REF], or directly transferred to the sperm surface or associated with several binding proteins such as clusterin and several lipocalins [START_REF] Ecroyd | The epididymal soluble prion protein forms a high-molecular-mass complex in association with hydrophobic proteins[END_REF].

Conclusion

New approaches, including large scale analysis, are now being applied to the study of epididymis physiology and the post-testicular differentiation of spermatozoa. Both transcriptomic [START_REF] Thimon | Shedding of the germinal angiotensin I-converting enzyme (gACE) involves a serine 27[END_REF] and proteomic approaches provide a dramatic advance to our understanding of male reproduction in different species. From the first results of these global approaches, it is evident that significant differences exist between species either in the sequential changes in the luminal proteome or sperm surface proteins. Each species appears to have developed its own strategy for sperm maturation and preservation.

A general feature of sperm maturation probably exists between mammalians but it probably involves different protein combinations. However, there is a need to identify more epididymal and sperm proteins in order to obtain greater understanding of this aspect of the male reproductive system which is fundamental for the survival of all species.

  region eg. clusterin and PGDS in the horse, where the number of isoforms is different between the anterior and the posterior part of the organ (Fig 3.), or RNase 10 (Train A) which is different between species for the boar and the ram (Fig 3).

  Fig 6).Transit of the sperm through the epididymis must be necessary for sperm maturation since sperm maturation has not been achieved in vitro to date. Sperm maturation is probably related to sequential modifications which occur mainly on the sperm surface. During the maturation process, the immature gametes progressively lose or modify most of their testicular surface proteins and gain new transient or permanent proteins in well organized membrane protein domains. The pattern of sperm surface modification is species-specific, related to the nature of the surface proteins involved or the epididymal regions where these modifications occur. However, common changes in several identified sperm surface proteins have been described, e.g. such as fertilin[START_REF] Primakoff | Fully effective contraception in male and female guinea pigs immunized with the sperm protein PH-20[END_REF], CRISP1[START_REF] Roberts | Epididymal secreted protein Crisp-1 and sperm function[END_REF], P34H[START_REF] Sullivan | Levels of P34H, a sperm protein of epididymal origin as a predictor of conventional in vitro fertilization outcome[END_REF] and ACE[START_REF] Gatti | Prion protein is secreted in soluble forms in the epididymal fluid and proteolytically processed and transported in seminal plasma[END_REF], which represent the most important sperm surface changes (Fig6). Such common changes can also be illustrated by the global surface changes of sialoglycoproteins on the sperm surface in the last part of the epididymis[START_REF] Dacheux | Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar[END_REF][START_REF] Dacheux | Motility and surface transformations of human spermatozoa during epididymal transit[END_REF].

Figure 1 :

 1 Figure 1: Spermatocrit (A), protein secretion (B) and concentrations of luminal proteins (C) in the epididymal fluid from 9 regions of the epididymis (1-4: caput; 5-6:corpus; 7-9: cauda) for three species (ram, boar, stallion) from[START_REF] Syntin | Characterization and identification of proteins secreted in the various regions of the adult boar epididymis[END_REF][START_REF] Fouchecourt | Stallion epididymal fluid proteome: qualitative and quantitative characterization; secretion and dynamic changes of major proteins[END_REF] and unpublished data.

Figure 2 :

 2 Figure 2: Epididymal secretomes and proteomes of four species. For proteomes, each plate corresponds to 1D gel electrophoresis separation of about the same quantity of epididymal protein from each region of the epididymis. The secretome diagrams represent each secreted protein from the different epididymal regions and are expressed as the percentage of total secretion of the whole organ. The plates illustrating the secretory activities of the different epididymal regions for the four animal species correspond to the autoradiograms of the same four 1D gel separations presented above for the proteome (Syntin et al., 1996; Fouchecourt et al., 2000 ; Druart et al., 1994 ; Druart, unpublished data ; Dacheux et al., 2006).

Figure 3 :

 3 Figure 3: Immunodetection of 2D electrophoresis gels of isoforms of three proteins (PGDS, clusterin and RNase 10) according to their epididymal and species origins (stallion: Fouchecourt et al., 2000; boar: Castella et al., 2004 and ram: unpublished data).

Figure 4 :Figure 5 :

 45 Figure 4: Distribution of the major proteins secreted by the human and boar epididymis (Dacheux et al., 2006; Syntin et al., 1996) Figure 5: Relationship between secretome and proteome. A) Secretion and protein concentrations for five major proteins present in the epididymal fluid of the stallion. B) Epididymal localisation of the secretion, luminal protein and corresponding RNA of RNase 10 (Train A) from the initial segment (IS), caput(1-4), corpus (5-6), cauda[START_REF] Dacheux | Sequential analysis of the epididymal sperm maturation process in the boar[END_REF][START_REF] Druart | Analysis by two-dimensional gel electrophoresis of ram epididymal secreted proteins[END_REF][START_REF] Ecroyd | The epididymal soluble prion protein forms a high-molecular-mass complex in association with hydrophobic proteins[END_REF] in the boar[START_REF] Castella | Train A, an RNase A-like protein without RNase activity, is secreted and reabsorbed by the same epididymal cells under testicular control[END_REF]).

Figure 6 :

 6 Figure 6: Sperm epididymal maturation according to epididymal region in the human (Dacheux et al., 1987) and porcine (Dacheux et al., 1984) related to ACE, sialoprotein modifications on the sperm surface and percentage of progressive motility along the epididymis.

  Figure 6


Jonna Salonen

Ralf Butzow

Jorma J Palvimo

MD Markku Heikinheimo
email: markku.heikinheimo@helsinki.fi

Oskari Heikinheimo

Oestrogen receptors and small nuclear ring finger protein in malignant ovarian germ cell tumours

Keywords: Oestradiol, oestrogen receptor, ovarian germ cell tumour, small nuclear ring finger protein (SNURF/RNF4)

The peak incidence of malignant ovarian germ cell tumours occurs soon after puberty. Thus, gonadal steroids may play a role in their development.

Oestrogen receptors (ERα and ERβ) and their co-regulators, including small nuclear ring finger (SNURF/RNF4) protein mediate oestrogen actions. While ERβ and SNURF are down-regulated in testicular germ cell tumours, their role in the ovarian germ cell tumours remains unknown.

We herein studied the different subtypes of malignant ovarian germ cell tumours, and found that they all express ERα, ERβ, and SNURF. Stimulation with oestradiol (E2), ERα, ERβ and SNURF significantly up-regulated mRNA expression in the human germinoma derived NCC-IT cells. Further, the effects of E2 were counteracted by an anti-oestrogen (ICI 182,780).

Neither E2 nor ICI 182,780 had an effect on the proliferation of NCC-IT cells as assessed by flow cytometric analysis. Our results suggest that oestrogen signalling has a role in malignant ovarian germ cell tumours.

Introduction

Malignant ovarian germ cell tumours (MOGCTs) are rare tumours representing less than 5% of all ovarian malignancies [START_REF] Pectasides | Germ cell tumors of the ovary[END_REF]. Histologically they are a heterogeneous group including various subtypes (dysgerminomas, yolk sac tumours, immature teratomas, embryonal carcinomas) [START_REF] Pectasides | Germ cell tumors of the ovary[END_REF]. They may also harbour a mixture of these different subtypes. These different germ cell tumour types are thought to develop from pluripotent primordial germ cells. However, relatively little is still known about the molecular biology of germ cell tumours. The peak incidence of MOGCTs occurs soon after puberty, at an age of less than 20 [START_REF] Tewari | Malignant germ cell tumors of the ovary[END_REF]. Thus, gonadal steroids may have a role in their tumorigenesis.

Understanding of steroid hormone action and its regulation has increased considerably in recent decades. Regarding the regulation of oestrogen signalling, two distinct oestrogen receptors (ERs) have been identified (ER and ER) [START_REF] Walter | Cloning of the human estrogen receptor cDNA[END_REF][START_REF] Mosselman | ER beta: identification and characterization of a novel human estrogen receptor[END_REF]. Knockout mouse models have revealed the roles of ERs in normal ovarian physiology and folliculogenesis [START_REF] Lubahn | Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene[END_REF][START_REF] Couse | Exploring the role of sex steroids through studies of receptor deficient mice[END_REF]. The human ovary expresses both ERs. Oestrogen receptor beta is localized to granulosa cells, whereas ERα is localized to the theca and interstitial cells [START_REF] Pelletier | Immunocytochemical localization of estrogen receptors alpha and beta in the human reproductive organs[END_REF]. Ovarian epithelial cells express both ER! and ER! [START_REF] Hillier | Expression of oestrogen receptor alpha and beta in cultured human ovarian surface epithelial cells[END_REF]. In the developing human ovary, ERs are expressed from the 20 th week of pregnancy onwards and their expression is localized to the granulosa cells and oocytes [START_REF] Vaskivuo | Estrogen receptors and estrogen-metabolizing enzymes in human ovaries during fetal development[END_REF].

In contrast to the ovary, human testicular germ cells and Sertoli cells express only ERβ [START_REF] Carreau | Aromatase and estrogen receptors in male reproduction[END_REF]. The expression of ERβ is markedly diminished in testicular seminomas, embryonal carcinomas and mixed germ cell tumours, both at transcriptional and translational levels [START_REF] Pais | Estrogen receptorbeta expression in human testicular germ cell tumors[END_REF]. In contrast, the expression of ERβ remains normal in testicular yolk sac tumours and teratomas [START_REF] Pais | Estrogen receptorbeta expression in human testicular germ cell tumors[END_REF]. However, ER expression in human MOGCTs has not been assessed previously.

Steroid receptor action is regulated by multiple co-regulators [START_REF] Hermanson | Nuclear receptor coregulators: multiple modes of modification[END_REF]. Small nuclear RING finger protein (SNURF/RNF4) is one of these co-regulators, and it can co-activate both androgen-and oestrogen-dependent transcription by interacting directly with the corresponding steroid receptors [START_REF] Moilanen | Identification of a novel RING finger protein as a coregulator in steroid receptor-mediated gene transcription[END_REF]. In murine foetal testis, SNURF is localized to gonocytes and to Leydig and Sertoli cells, whereas in murine foetal ovary it is present in the oogonia and granulosa cells [START_REF] Hirvonen-Santti | Small nuclear RING finger protein expression during gonad development: regulation by gonadotropins and estrogen in the postnatal ovary[END_REF].

The expression of ovarian SNURF is up-regulated in immature mice following oestradiol (E2) treatment. The expression of SNURF has been found to be down-regulated in human testicular germ cell cancer as compared with normal testicular tissue [START_REF] Hirvonen-Santti | Down-regulation of estrogen receptor beta and transcriptional coregulator SNURF/RNF4 in testicular germ cell cancer[END_REF]. The potential role of SNURF in MOGCTs has not been studied.

In order to study the role of oestrogen action in ovarian germ cell tumours, we characterized for the first time the expression of ER, ER and SNURF in MOGCTs. In addition, in vitro studies were carried out using the human germ cell tumour cell line NCC-IT. This cell line, obtained from a mediastinal mixed germ cell tumour, is unique in that it expresses markers of both pluripotent germ cells and embryonal stem cells [START_REF] Damjanov | Retinoic acid-induced differentiation of the developmentally pluripotent human germ cell tumorderived cell line[END_REF]. It may thus be a suitable in vitro model for ovarian dysgerminoma. andGynaecology, Helsinki University Central Hospital, between 1982 and[START_REF] Hermanson | Nuclear receptor coregulators: multiple modes of modification[END_REF] were used for the present study. The mean age was 28.9 years (range 12-49), all of the patients were postpubertal. Of the MOGCT specimens, 5 were dysgerminomas (DGs), 5 were yolk sac tumours (YSTs), 4 were immature teratomas (ITs) and there was one mixed tumour with DG and embryonal carcinoma (EC) components.

Materials and Methods

Patients

Cell lines

The NCC-IT human germinoma cell line [START_REF] Teshima | Four new human germ cell tumor cell lines[END_REF] was obtained from The American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with foetal bovine serum (10%) and penicillin-streptomycin (1%).

Immunohistochemistry

The antibody used for immunohistochemistry of ER was from DakoCytomation, (Glostrup, Denmark) (M7047), and that for ER was from Serotec, (Oxford, UK) (MCA1974s). The anti-SNURF antibody has been described previously [START_REF] Häkli | SUMO-1 promotes association of SNURF (RNF4) with PML nuclear bodies[END_REF]. The tissue sections from paraffinembedded tissue blocks were deparaffinised and rehydrated using descending concentrations of ethanol. Antigen retrieval was performed by incubating the slides in 0.1 M citric acid (pH 8.0) at approximately 100 C for 20 min.

Endogenous peroxidase activity was blocked with 3% H 2 O 2 in water for 5

min. An avidin-biotin immunoperoxidase system was used to visualize bound antibody (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA), with 3,3'-diaminobenzidine (Sigma, St. Louis, MO, USA) as

Stimulation of NCC-IT cells with oestradiol

The cells were plated on 12-well dishes at a density of 25 × 10 5 per well and incubated in RPMI-1640 (phenol red-free) supplemented (10%) with dextrancharcoal-treated foetal bovine serum (HyClone, Logan, UT, USA) for 48 h before stimulation. They were stimulated with 17!-estradiol (E2) (Sigma, St.

Louis, MO, USA) at concentrations of 1 to 1000 nM for 24 to 120 h to determine time-and dose-responses of the expression of ERs and their coactivator SNURF. For control experiments, the anti-oestrogen ICI 182,780 (Tocris Cookson Ltd, Bristol, UK) was added at a concentration of 1000 nM simultaneously with 1 nM E2. The concentrations of E2 and anti-oestrogen were based on those used in previous reports [START_REF] Martin | The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling[END_REF]. All incubations were performed in duplicate and repeated at least three times.

Untreated and vehicle-only-treated controls were included in each experiment.

Semi-quantitative RT-PCR

Cells were collected at different time points (24-120 h) after stimulation, and total RNA was isolated by using RNeasy kit (Qiagen, Hilden, Germany).

Purified RNA (1 g) was reverse-transcribed and 2 L of the reaction mixture was used for each PCR reaction. The primers and thermal cycler conditions are presented in Table 1. The number of cycles used for PCR were 32 for ER, 32 for ER, 30 for SNURF, and 30 for -actin. Agarose gel electrophoresis (2%), run in the presence of SybrSafe™ DNA gel stain (Invitrogen Corporation, Carlsbad, CA, USA) demonstrated PCR products of

Statistical analysis

Data are expressed as mean values ± SEM derived from three independent experiments, all with two replicates, and adjusted to a value of 1.0 for the mean of the first control culture. The E2 and ICI 182,780 stimulation data were analysed using Student's t test. A p value <0.05 was considered statistically significant. The statistical software used was SPSS, version 15.0 (SPSS Inc., Chicago, IL, USA). 2) and ER (Figure 1D,E,Table 2).

Results

ER, ER and SNURF (RNF4) are expressed in normal ovarian oocytes, MOGCTs and NCC-IT cells

Expression of ER! was stronger than ER! in all MOGCT specimens with an exception of one YST (Table 2). In addition, all MOGCT subtypes expressed SNURF localized into the nucleus and cytoplasm (Figure 1G, H, Table 2). In line with the expression of dysgerminomas human germinoma derived cell line (NCC-IT) expressed both ER and ER as well as their co-receptor SNURF (Figure 1A, D, G insets).

Stimulation of NCC-IT cells with E2 increased the mRNA expression of ER!,

ER! and SNURF

Expression of ER and ER mRNA was increased at all time points (24-120 h) following stimulation with E2 at all concentrations (1-1000 nM) tested, and at 48 h the increases were statistically significant (Figure 2A,B,C). The maximal increase in the expression of ER was seen at 72 h, with 100 nM E2 (Figure 2B). With the lowest concentration (1 nM) used, ERα expression reached its maximum as soon as at 48 h (p<0.05). Expression of ER mRNA reached its maximum at 48 h (p<0.005) (Figure 2A). The increased expression of ERs after stimulation with 1 nM E2 was blocked by the antioestrogen ICI 182,780 (1000 nM) at 48 h (Figure 3).

Stimulation with E2 increased SNURF mRNA expression levels during the 5-day period at all time points (24-120 h) and with all tested concentrations (1-1000 nM) of E2 (Figure 2C). The maximal increase was seen at 48 h, with 100 nM E2 (p<0.05) (Figure 2B). Increased expression of SNURF was evident even at the lowest concentration (1 nM) at 24 h (p<0.05) and 48 h (p<0.005) after stimulation (Figure 2A,B). The increased expression of SNURF induced by E2 at 48 h was inhibited by the anti-oestrogen ICI 182,780 (Figure 3). Neither E2 nor the anti-oestrogen ICI 182,780 had an effect on NCC-IT cell proliferation as assessed by flow cytometric analysis. The percent of the proliferating NCC-IT cells in the control group was 47.7% (range 43.9-54.5%) at 24 hours, and 48.3% (42.2-54.2) in E2 the stimulation group. At 48 hours, the corresponding figures were 56.7% (53.6-59.7), and 55.4% (52.8-58.8), respectively. There were no obvious morphological changes in the NCC-IT cells after exposure to E2 nor anti-oestrogen (data not shown).

Discussion

Two thirds of epithelial ovarian cancer specimens express ERs [START_REF] Lindgren | Estrogen and progesterone receptors in ovarian epithelial tumors[END_REF] and anti-oestrogen therapy with tamoxifen has resulted in modest responses in cases of recurrent ovarian cancer [START_REF] Makar | Hormone therapy in epithelial ovarian cancer[END_REF]. Thus, oestrogens might have a role in the pathogenesis of epithelial ovarian cancer.

Moreover, the use of oestrogen therapy, such as administration of combined oral contraceptives (COCs), or hormone therapy (HT) of menopausal symptoms, is associated with an altered incidence of epithelial ovarian cancer [START_REF] Rossing | Menopausal hormone therapy and risk of epithelial ovarian cancer[END_REF][START_REF] Lurie | Combined oral contraceptive use and epithelial ovarian cancer risk: time-related effects[END_REF]. The potential role of hormone therapy in the pathogenesis of MOGCTs has not been assessed. This may be a result of the rarity of these tumours and the fact that they are usually diagnosed during adolescence.

The expression of ERs, or their modulators, has not been assessed previously in malignant ovarian germ cell tumours. We herein find that all histological subtypes of MOGCTs expressed both ER and ER. In addition, these tumours expressed the ER co-regulator SNURF. Thus, oestrogens might have a role in ovarian germ cell tumour pathogenesis. We speculate that the stimulatory effect of oestrogens on primordial germ cells occurs when the Several studies have demonstrated a decrease in ER and/or an increase in the ER!/! ratio in epithelial ovarian cancer in comparison with normal ovarian tissue [START_REF] Brandenberger | Estrogen receptor alpha (ER-alpha) and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ERbeta in neoplastic tissues[END_REF][START_REF] Pujol | Differential expression of estrogen receptor-alpha and -beta messenger RNAs as a potential marker of ovarian carcinogenesis[END_REF][START_REF] Rutherford | Absence of estrogen receptor-beta expression in metastatic ovarian cancer[END_REF][START_REF] Bardin | Loss of ERbeta expression as a common step in estrogen-dependent tumor progression[END_REF]. Furthermore, an increased ER!/! ratio has been reported in breast [START_REF] Roger | Estrogen-induced growth inhibition of human seminoma cells expressing estrogen receptor beta and aromatase[END_REF], prostate [START_REF] Fixemer | Differential expression of the estrogen receptor beta (ERbeta) in human prostate tissue, premalignant changes, and in primary, metastatic, and recurrent prostatic adenocarcinoma[END_REF] and colorectal carcinoma [START_REF] Campbell-Thompson | Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer[END_REF]. The results of recent studies with ovarian, prostate and breast cancer cell lines have indicated a role of ER as a tumour suppressor in these hormone-responsive organs [START_REF] Bardin | Loss of ERbeta expression as a common step in estrogen-dependent tumor progression[END_REF][START_REF] Lazennec | ER beta inhibits proliferation and invasion of breast cancer cells[END_REF][START_REF] Cheng | Expression of estrogen receptor beta in prostate carcinoma cells inhibits invasion and proliferation and triggers apoptosis[END_REF]. In the present study, expression of ER! was stronger than that of ER! in most of the MOGCT specimens analysed. In addition, both ER and ER were expressed in NCC-IT cells. Thus, in contrast to several other types of tumours, ER! might not play a suppressor role in ovarian germ cell tumours.

Oestrogens modulate the expression of ERs in a tissue-and cell type-specific fashion. Thus, stimulation with oestrogen has diverse effects on different tumour types [START_REF] Clayton | Insulin-like growth factors control the regulation of oestrogen and progesterone receptor expression by oestrogens[END_REF][START_REF] Vladusic | Expression and regulation of estrogen receptor beta in human breast tumors and cell lines[END_REF][START_REF] Le | DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells[END_REF]. It is known, that oestrogen enhances proliferation of ER-positive ovarian epithelial cancer cell lines [START_REF] Galtier-Dereure | Estradiol stimulates cell growth and secretion of procathepsin D and a 120kilodalton protein in the human ovarian cancer cell line BG-1[END_REF][START_REF] Langdon | The regulation of growth and protein expression by estrogen in vitro: a study of 8 human ovarian carcinoma cell lines[END_REF]. Furthermore, E2 stimulates cell proliferation both in prostate and breast cancer cell lines [START_REF] Martin | The anti-oestrogen ICI 182,780, but not tamoxifen, inhibits the growth of MCF-7 breast cancer cells refractory to long-term oestrogen deprivation through down-regulation of oestrogen receptor and IGF signalling[END_REF][START_REF] Arnold | Comparative effects of DHEA vs. testosterone, dihydrotestosterone, and estradiol on proliferation and gene expression in human LNCaP prostate cancer cells[END_REF], whereas decreased proliferation has been reported in a human seminoma (a counterpart of female dysgerminoma) cell line [START_REF] Roger | Estrogen-induced growth inhibition of human seminoma cells expressing estrogen receptor beta and aromatase[END_REF]. In addition, estradiol has non-genomic actions through membrane-mediated activation of extracellular regulated kinase and protein kinase A, resulting stimulation of proliferation of seminoma cells [START_REF] Bouskine | Estrogens promote human testicular germ cell cancer through a membrane-mediated activation of extracellular regulated kinase and protein kinase A[END_REF]. We now found that E2 increased expression of both ER! and ER! in the germinoma derived NCC-IT cell line. Similarly to ERs, SNURF mRNA was increased following stimulation with E2, which is in line with augmented SNURF mRNA expression in mouse ovaries following E2 treatment [START_REF] Hirvonen-Santti | Small nuclear RING finger protein expression during gonad development: regulation by gonadotropins and estrogen in the postnatal ovary[END_REF]. However, E2 and the anti-oestrogen ICI 182,780 did not have an effect on the proliferation of the NCC-IT cells. ER has been reported to act as a suppressor of ER action. As both ER! and ER! were up-regulated following E2 treatment, the lack of oestrogen effect on proliferation of NCC-IT cells could be associated with the balance of ER/, in which the ERs might counteract each other. Unfortunately, due to the paucity of malignant ovarian germ cell tumour derived cell lines oestrogen effects cannot be studied in additional cell lines. Moreover, the results obtained with NCC-IT cell line cannot be directly related to other germ cell tumour types encountered in MOGCTs.

In conclusion, ER, ER and their co-regulator SNURF (RNF4) are all expressed in MOGCTs. In addition, in vitro studies on the human germinoma-derived NCC-IT cell line demonstrated increased ER and SNURF expression after stimulation with E2. These data suggest that oestrogen action may have a role in the regulation of MOGCT tumorigenesis.

Conflict of Interests Statement

No conflict of interest.

Primer sequences and thermal conditions for semi-quantitative PCR.

  Approval for the study was obtained from the Ethics Committee of the Department of Obstetrics and Gynaecology, University of Helsinki, and from the National Authority for Medicolegal Affairs. Tumour samples (n=14), originally collected for diagnostic purposes at the Department of Obstetrics

  negative control experiments PBS replaced the primary antibody. Scoring of the antigens was based on the staining intensity of at least 10% of the tumour cells, divided into three categories: no staining (-), weak staining (), positive staining (+, ++ or +++). The staining intensity of ER! was compared with that of ER!.

  size (Fig 2A, B) for each of the PCR primer pairs. Threshold cycles were normalized to threshold cycles for -actin. The intensities of the bands were analysed by using the Bio-Rad Gel Doc program (Bio-Rad Laboratories, Hercules, CA, USA) and quantified by using Quantity One software (Bio-Rad Laboratories). Flow cytometric analysis To analyse the proliferation of the cells APC BrdU Flow Kit (BD Biosciences, BD Biosciences, San Jose, CA, USA) was used following the staining protocol by the manufacture. After E2 or anti-oestrogen ICI 182,780 stimulation cells were labelled with BrdU (an analog of the DNA precursor thymidine) at concentration of 10M. Cells were harvested at different time points (6 to 48 h), fixed, and stained with anti-BrdU specific APC fluorochrome according to manufactures protocol. Cells with incorporated BrdU indicating proliferating cells were analysed by flow cytometry (FACSAria, BD Biosciences).

  In normal ovary, expression of ER, ER and SNURF was localized to the oocytes (Figure1C, F, I). All histological types of MOGCTs studied expressed ER! (Figure1A,B, Table

  axis is activated at the time of puberty. This may trigger the development of MOGCT.

  Figure 1.

Figure 2 .

 2 Figure 2. Effects of oestradiol on the NCC-IT cell line. A. Effect of E2 (10 nM) on ER!, ER! and SNURF mRNA following 24to 72-h stimulation. B. Effect of E2 (100 nM) on ER!, ER! and SNURF mRNA following 24to 72-h stimulation. C. Effect of E2 (1-1000 nM) on ER!, ER! and SNURF mRNA following 48h stimulation. Data are expressed as mean values  SEM derived from three independent experiments, all with two replicates per experiment. (E2 = oestradiol, ICI = anti-oestrogen ICI 182,780, C = control ,*p<0.05, **p<0.005)

  Effect of oestradiol (1 nM) + anti-oestrogen (ICI 182,780, 1000 nM) on ER!, ER! and SNURF mRNA following 48-h stimulation. Data are expressed as mean values  SEM derived from three independent experiments, all with two replicates per experiment. (E2 = oestradiol, ICI = anti-oestrogen ICI 182,780, C = control)

  FIG 1.

  Figure 1

  Figure 2.

Figure

  Figure

Table 2 .

 2 Immunohistochemical staining of ER, ER and SNURF in different histological subtypes of human malignant ovarian germ cell tumors. Percent of the positively staining tumor cells is presented in parenthesis. DGs included one mixed tumour with DG and embryonal carcinoma (EC) components. DG, dysgerminoma; YST, yolk sac tumour; IT, immature teratoma; EX, died; CR, complete response; , ER/ER ratio decreased;; , ER/ER ratio elevated; =, ER/ER ratio equal.

	Diagnosis Stage	Survival	ER!	ER!	ER/ER SNURF
	DG*	IIIC	EX	+ (80) ++		++ (15)
					(80)
	DG	IC	CR	+ (15) ++		+ (80)
					(80)
	DG	IIIC	CR	++	++	=	+ (50)
				(80)	(50)
	DG	IIA	CR	+ (80) ++		± (50)
					(80)
	DG	IC	CR	± (80) +++		++ (15)
					(80)
	YST	IIIA	CR	+++	+++	=	+++ (50)
				(50)	(80)
	YST	IA	CR	+++	++		+++ (50)
				(80)	(50)
	YST	IC	CR	++	++	=	± (15)
				(80)	(80)
	YST	IIIA	CR	+ (80) ++		± (15)
					(80)
	YST	IIIC	EX	-	++		++ (50)
					(50)
	IT	IIIC	EX	+ (15) ++		+ (15)
					(80)
	IT	IC	EX	+ (15) +++		± (15)
					(50)
	IT	IC	CR	+ (50) +++		± (15)
					(80)

*

Acknowledgments

Funding

This work was supported by the Helsinki University Central Hospital Research Funds, the Sigrid Jusélius Foundation, the Finnish Cancer Organizations, and the National Clinical Graduate School.


Hogan Am

Collins D 12

Baird Aw

Winter Dc

Aisling Hogan
email: aislinghogan@yahoo.com

Estrogen and gastrointestinal malignancy

Keywords: Estrogen, Esophagus, Stomach, Gallbladder, Colon, Malignancy

The concept that E2 exerts an effect on the gastrointestinal tract is not new and its actions on intestinal mucosa have been investigated for at least three decades. An attempt to consolidate results of these investigations generates more questions than answers, thus suggesting that many unexplored avenues remain and that the full capabilities of this steroid hormone are far from understood. Evidence of its role in esophageal, gastric and gallbladder cancers is confusing and often equivocal. The most compelling evidence regards the protective role conferred by estrogen (or perhaps ERβ) against the development and proliferation of colon cancer. Not only has the effect been described but many mechanisms of action have been explored. It is likely that, along with surgery, chemotherapy and radiotherapy, hormonal manipulation will play an integral role in colon cancer management in the very near future.

Introduction:

Only a few decades ago, it was believed that the primary (and perhaps sole) function of 17β estradiol (E2) was in the development of female secondary sexual characteristics and subsequent regulation of reproductive function. However this has been recognised as an over-simplification and there are few organs in which some effect of E2 has not been described. From a neurological perspective, E2 may confer protection against Alzheimer's disease [START_REF] Brinton | Estrogens regulation of glucose metabolism and mitochondrial function: therapeutic implications for prevention of Alzheimer's disease[END_REF] through its direct action on mitochondrial function. A protective role is described against the development of retinal degenerative disease [START_REF] Kumar | Estrogens and neuroprotection in retinal diseases[END_REF] , improvement of memory [START_REF] Hojo | Estrogens synthesis in the brain--role in synaptic plasticity and memory[END_REF] , induction of cerebro-vascular accidents 4 5 and modulation of the sleep-wake cycle [START_REF] Deurveilher | Estradiol replacement enhances sleep deprivation-induced c-Fos immunoreactivity in forebrain arousal regions of ovariectomized rats[END_REF] . The role of E2 in both development and prognostic prediction in breast [START_REF] Jick | Postmenopausal estrogen-containing hormone therapy and the risk of breast cancer[END_REF] and ovarian cancers [START_REF] Suzuki | Loss of estrogen receptor beta isoform expression and its correlation with aberrant DNA methylation of the 5'-untranslated region in human epithelial ovarian carcinoma[END_REF] is well described and many therapeutic interventions (in the form of receptor modulation) are in widespread use to this effect. Potent estrogenic effects on pulmonary vasculature have been suggested as an explanation for the poorly understood but nonetheless clinically apparent impact of gender in trauma, ischaemia/reperfusion, shock and sepsis. [START_REF] Lahm | The effects of estrogen on pulmonary artery vasoreactivity and hypoxic pulmonary vasoconstriction: potential new clinical implications for an old hormone[END_REF] Epidemiologically, men are more likely to develop colon cancer than women and hormone replacement therapy appears to confer an additive protective role. [START_REF] Kennelly | Oestrogen and the colon: potential mechanisms for cancer prevention[END_REF] A similar male predominance is apparent in esophageal and gastric cancers but evidence as to the role of E2 is less convincing.

Methods:

A literature search was conducted using the PubMed database and the search terms "estrogen", "estrogen AND cancer" "estrogen AND esophagus" "estrogen AND stomach" "estrogen AND gallbladder" and "estrogen AND gastrointestinal tract". Only studies published in English between 1967 and 2008 were included. Bibliographies of extracted studies were further cross references. In all, 157 full text articles were selected for review.

Estrogen-Mechanism of Action:

Genomic:

Members of steroid receptor superfamily (including ERα and ERβ) exert their actions through at least two diverse mechanisms-traditional transcriptional regulation of target genes in the nucleus (genomic) or alternative mechanisms initiated at the cell membrane and mediated by secondary messenger proteins (non-genomic) [START_REF] Winter | Mitogenic effects of oestrogen mediated by a non-genomic receptor in human colon[END_REF] . As with other steroid hormones, estrogens diffuse across cell membranes thus facilitating potential regulation of intracellular (cytoplasm) signalling cascades. Genomic actions involve the binding of estrogen to one of the receptor is forms, translocation to the nucleus, induction of conformational change enabling coupling with estrogen response elements and causing regulation of gene expression. [START_REF] Simoncini | Non-genomic actions of sex steroid hormones[END_REF] These transcriptional effects of E2 have a latency of onset of two to eight hours and may be sustained. [START_REF] Harvey | Nongenomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon[END_REF]

Non-Genomic:

More recently, effects of steroid hormones have been described which are too rapid to be accounted for by altered transcription or translation. These are termed non-nuclear, non-genomic or non-transcriptional effects. A single molecular mechanism has not been elucidated but actions can be considered non-genomic when: (i) they are too rapid to be compatible with RNA and protein synthesis (occur within minutes of exposure to the hormone (ii) they can be reproduced in the presence of inhibitors of RNA or protein synthesis (iii) they can be reproduced by using steroid hormones coupled to cell membrane impermeable molecules (iv) they occur in cells with highly compacted chromatin in which RNA and protein synthesis are absent (spermatozoa) or (iv) they are elicited via steroid hormones binding to receptors containing mutations which make them incapable of activating transcriptional processes. [START_REF] Moggs | Estrogen receptors: Orchestrators of pleiotropic cellular responses[END_REF] Various secondary messengers at cytoplasmic level have been described as mediators of non-genomic actions of estrogen including protein kinase C [START_REF] Alzamora | Direct binding and activation of protein kinase C isoforms by steroid hormones[END_REF] , intracellular calcium [START_REF] Muchekehu | 17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3[END_REF] , cytosolic cyclic AMP [START_REF] Doolan | Rapid non-genomic activation of cytosolic cyclic AMP-dependent protein kinase activity and [Ca(2+)](i) by 17beta-oestradiol in female rat distal colon[END_REF] and GMP [START_REF] Ropero | Non-genomic actions of 17beta-oestradiol in mouse pancreatic beta-cells are mediated by a cGMPdependent protein kinase[END_REF] nitric oxide [START_REF] Simoncini | Genomic and nongenomic mechanisms of nitric oxide synthesis induction in human endothelial cells by a fourth-generation selective estrogen receptor modulator[END_REF] and mitogen activated protein kinase [START_REF] Hennessy | 17beta-Estradiol rapidly stimulates c-fos expression via the MAPK pathway in T84 cells[END_REF] . The understanding of the molecular basis of this non-genomic signal transduction is important and may, in the future provide therapeutic windows for various pathological processes.

Estrogen and Esophageal Cancer:

Esophageal cancer occurs more frequently in men than women [START_REF] Cook | A systematic review and meta-analysis of the sex ratio for Barrett's esophagus, erosive reflux disease, and nonerosive reflux disease[END_REF] and men progress to invasive carcinoma from Barrett's esophagus at twice the rate of their female counterparts [START_REF] Yousef | The incidence of esophageal cancer and high-grade dysplasia in Barrett's esophagus: a systematic review and meta-analysis[END_REF] . While ER had been demonstrated in esophageal mucosa in 1983 [START_REF] Winborn | Autoradiographic demonstration of estrogen receptors in the esophagus[END_REF] , a second isoform of the receptor was described in 1996 [START_REF] Kuiper | Cloning of a novel receptor expressed in rat prostate and ovary[END_REF] and this warranted further characterisation and classification its presence in esophageal tissue. The presence of ER-ß was confirmed by immunohistochemistry in both esophageal adenocarcinoma and Barrett's esophagus cells with and without dysplasia 25 26 and it was later demonstrated that, when compared to Barret's metaplasia negative for dysplasia, esophageal adenocarcinoma significantly over expressed ERß. Animal studies are equivocal, however, regarding the role of E2 in esophageal carcinogenesis. While a protective role of ethinyl estradiol was suggested in the presence of 0.1% N-ethyl-N-hydroxyethylnitrosamine [START_REF] Shirai | Organ specific modifying potential of ethinyl estradiol on carcinogenesis initiated with different carcinogens[END_REF] the same paper demonstrated a decrease in esophageal hyperplasia in rats pre-treated with 0.03% Nnitrosopiperidine. The first convincing description of estrogenic inhibition of esophageal cancer proliferation was in a cell line cultured from metastatic esophageal tissue. [START_REF] Matsuoka | Sex hormone response of a newly established squamous cell line derived from clinical esophageal carcinoma[END_REF] An absolute response of this cell line (named KSE-1) was higher than that of human mammary carcinoma cells but lower than that of non-malignant human mammary epithelial cells. Inhibition of KSE-1 cell growth was dose dependant at maximum concentrations as low as 10¯¹² mg/ml oestradiol. A few years later an estradiol sensitivity assay for use in oesophageal and breast cancer cell lines was developed. [START_REF] Matsuoka | Estradiol sensitivity test using contact-sensitive plates of confluent BALB/c 3T3 cell monolayers[END_REF] Again growth inhibition of KSE-1 cells was demonstrated and an affinity of 4.2fmol/mg for estrogen receptors was described. A Swedish epidemiological study examined males undergoing hormonal manipulation for treatment of prostate cancer (increased estrogen and decreased testosterone level) and its potential reduction of risk of developing esophageal carcinoma. [START_REF] Lagergren | Do sex hormones play a role in the etiology of esophageal adenocarcinoma? A new hypothesis tested in a population-based cohort of prostate cancer patients[END_REF] A total of 100,215 patients were followed up for an average of 4 years. Relative risk was calculated. No correlation, however, was evident between hormonal manipulation and the relative risk of developing adenocarcinoma of the esophagus. Thus, despite a male prevalence in the incidence of esophageal carcinoma, no convincing evidence has been presented in animal or population studies and it appears less than likely that E2 per se is protective against esophageal cancer.

Estrogen and Gastric Cancer:

Epidemiological studies of the incidence and prevalence of gastric cancer also show a worldwide male: female predominance in the order of at least 2:1. [START_REF] Brenner | Epidemiology of stomach cancer[END_REF] It is believed that some (not yet fully elucidated) role is played by female sex hormones in the process of gastric carcinogenesis. [START_REF] Vecchia | Menstrual and reproductive factors and gastric-cancer risk in women[END_REF] It has been suggested that, not unlike breast cancer, later age at menarche [START_REF] Frise | Menstrual and reproductive risk factors and risk for gastric adenocarcinoma in women: findings from the canadian national enhanced cancer surveillance system[END_REF] , older menopause, and nulliparity [START_REF] Freedman | Menstrual and reproductive factors and gastric cancer risk in a large prospective study of women[END_REF] are associated with increased risk of development of gastric carcinoma in women. Japanese findings, however, were not consistent with this and they concluded that although early estrogen exposure may have some protective effect, female reproductive factors have no substantial influence on gastric cancer development. [START_REF] Persson | Female reproductive factors and the risk of gastric cancer in a largescale population-based cohort study in Japan (JPHC study)[END_REF] Female, castrated-male and estrogen-treated male rats do, however have a lower incidence of chemically induced gastric cancer with lower histological differentiation than non-treated male rats 36 and estrogen receptors have been isolated in many but not all gastric adenocarcinomas. 37 38 Comparison of presence of estradiol in intracellular spaces of cancerous and non-cancerous gastric epithelium using the avidin-biotin peroxidase complex and immunohistochemistry revealed that estradiol was present only in the cytoplasm of cancer cells, and most commonly in those with scirrhous growth pattern. [START_REF] Nishi | Immunohistochemical study of intracellular estradiol in human gastric cancer[END_REF] Positive nuclear estrogen receptor immunoreactivity (ER-IR) or detection of D5 antibody (an estrogen receptor related protein) confers a worse prognosis 40 41 , more widespread lymph node involvement [START_REF] Zhao | Expression of estrogen receptor and estrogen receptor messenger RNA in gastric carcinoma tissues[END_REF] , increased metastatic potential (specifically ER-ß isoform) [START_REF] Takano | Expression of estrogen receptor-alpha and -beta mRNAs in human gastric cancer[END_REF] and has been previously correlated with a significantly lower 10 year cumulative survival. [START_REF] Matsui | The prognosis of patients with gastric cancer possessing sex hormone receptors[END_REF] The true impact of the presence or absence of ER on survival has, however been challenged [START_REF] Singh | Differential expression of oestrogen receptor and oestrogen inducible genes in gastric mucosa and cancer[END_REF] and, with use of modified techniques (dextran coated charcoal assay [START_REF] Wu | Clinicopathologic comparisons between estrogen receptor-positive and -negative gastric cancers[END_REF] or flow cytometry [START_REF] Karat | Expression of oestrogen and progesterone receptors in gastric cancer: a flow cytometric study[END_REF] ), no correlation between ER positivity and grade, no difference in expression between cancerous and normal mucosa and no impact on survival was demonstrated. Geographical inconsistencies have also been reported. [START_REF] Cui | Estrogens receptors are not expressed in esophagogastric carcinomas that come from a high incidence area of China[END_REF] The true prognostic value of the presence or absence of ER in malignant gastric mucosa remains to be elucidated and it may be that presence of ER is a feature of normal mucosa rather than a function of malignancy. Perhaps available methods are not yet sufficiently refined to differentiate ER with absolute certainty but, despite this the evidence for the prognostic role of cathepsin D (an estrogen-regulated lysosomal protease) [START_REF] Ikeguchi | Clinicopathological significance of cathepsin D expression in gastric adenocarcinoma[END_REF] is convincing. The fact that ER binding sites have almost identical biochemical characteristics to those of breast and endometrial cancers is, however, highly suggestive of an equivalent hormonal dependency of gastric cancer. [START_REF] Matsui | Characterization of estrogen receptor in human gastric cancer[END_REF] Following the description of ERß isoform by Kuiper [START_REF] Kuiper | Cloning of a novel receptor expressed in rat prostate and ovary[END_REF] and presentation of unequivocal evidence of the presence of ER almost ubiquitously throughout the gastrointestinal mucosa, interest was re-ignited in its potential role in gastric adenocarcinoma and indeed, the presence of ER-ß (in normal and abnormal gastric mucosa) was confirmed and a functional role in carcinogenesis suggested. [START_REF] Matsuyama | Estrogens receptor beta is expressed in human stomach adenocarcinoma[END_REF] Perhaps it was the presence of various previously undescribed ER isoforms that created technical difficulty in their detection and so led to inconsistencies in results. An effort to translate this new found knowledge of the role of receptors to therapeutic use was unsuccessful as, not only did estrogen stimulate growth in gastric cancer cell lines but so did its partial-agonist 4-hydroxytamoxifen. [START_REF] Harrison | The effect of sex hormones and tamoxifen on the growth of human gastric and colorectal cancer cell lines[END_REF] Further evidence was provided in a randomised control trial that ER positivity conferred a worse prognosis but disappointingly the administration of tamoxifen at therapeutic concentrations did not prolong survival. [START_REF] Harrison | The effect of tamoxifen and estrogen receptor status on survival in gastric carcinoma[END_REF] Conversely, more recent evidence suggests that patients treated with tamoxifen may have an overall increased risk of development of gastric carcinoma and a shorter latency period between the diagnosis of breast and gastric cancer. [START_REF] Chandanos | Tamoxifen exposure in relation to gastric adenocarcinoma development[END_REF] A Chinese group [START_REF] Wu | Hormonal therapy for stomach cancer[END_REF] hypothesised that, since the amplification of c-erbB-2 oncogene [START_REF] Tian | Inhibitive effects of mifepristone on growth of breast cancer: experiment with rats[END_REF] and its overexpression had been implicated in the advancement of gastric carcinoma, and estrogen was known to inhibit its expression in breast cancer cell lines, perhaps estrogen itself could be administered as a reasonable therapeutic option for the treatment of gastric cancer. Soon afterwards a Swedish nationwide cohort study examined the incidence of gastric cancer in men with prostate cancer treated between 1950 and 1980 (when estrogen was the treatment of choice). [START_REF] Lindblad | Estrogens and risk of gastric cancer: a protective effect in a nationwide cohort study of patients with prostate cancer in Sweden[END_REF] Those exposed to therapeutic exogenous estrogens demonstrated a reduced incidence of gastric cancer when compared to an age and sex matched population. Much controversy and diversity exists among findings pertaining to the role of estrogen in the development of gastric cancer. However, a few certainties prevail throughout the decades: (i) Some protective role appears to be afforded to women against the development of gastric carcinoma when compared to age matched male counterparts, (ii) ER (ß) has been isolated in gastric carcinoma mucosa, (iii) Both estrogen and progesterone cause proliferation in gastric cancer cell lines. What remains to be investigated is the true gradient in ER expression between malignant and normal gastric mucosa and whether or not it carries a true clinical significance.

Estrogen and gallbladder cancer:

Because of the low incidence of gallbladder cancer worldwide, studies tend to be small and many are inconclusive. A link between a woman's age of first parity and risk of developing gallbladder cancer has been suggested (reported that 1 st parity under 25 increases risk but over 30 confers protective effects) [START_REF] Lambe | Parity and cancers of the gall bladder and the extrahepatic bile ducts[END_REF] . However, this appears to be an isolated finding in a single retrospective study and has not been validated since. Estrogen receptor positivity in gallbladder cancer, when assessed with a specific immunohistochemistry assay, was only weakly positive and felt to be of questionable clinical significance. [START_REF] Ko | Estrogens receptors in gallbladder cancer: detection by an improved immunohistochemical assay[END_REF] Poorly differentiated tumours may, however, be more likely to be receptor negative [START_REF] Malik | Immuno-histochemical analysis of estrogen receptors on the malignant gallbladder tissue[END_REF] , and evidence is increasing as to the correlation of negative expression of ER β with increased metastatic potential 61 62 but, again, the therapeutic implications are uncertain. A small Italian study demonstrated a link between the use of postmenopausal hormone replacement therapy and an increased risk of gallbladder cancer (n=31; Odds Ratio 3.2). [START_REF] Gallus | Postmenopausal hormonal therapy and gallbladder cancer risk[END_REF] However, this has not been shown elsewhere.

Estrogen and colon cancer

Colorectal cancer, like gastric and esophageal, shows considerable predilection for the male sex. Furthermore, gender differences exist between type of cancer and location within the colon with women displaying a higher relative incidence of right sided tumours compared to men. [START_REF] Jemal | Cancer statistics[END_REF] The lower incidence of colon cancer in pre-menopausal women [START_REF] Ries | The annual report to the nation on the status of cancer, 1973-1997, with a special section on colorectal cancer[END_REF] has been attributed to the protective effect of estrogen and this is supported by the widely recognised prolonged protection afforded by hormone replacement therapy in the post-menopausal age-group. [START_REF] Rossouw | Writing Group for the Women's Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial[END_REF] Kuiper's description of a novel ER isoform [START_REF] Sica | Estradiol and progesterone receptors in malignant gastrointestinal tumors[END_REF] (ER β) in 1996 incited a resurgence of interest in the previously unexplained preventative role of female sex hormones against development of colon cancer. This new isoform has been shown by in situ hybridisation techniques [START_REF] Enmark | Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern[END_REF] to be more widely expressed in colonic epithelium than ER α and is thus felt to be responsible for anti-proliferative estrogenic effects on the colonic mucosa. Higher ER β expression has been shown to confer survival advantage in patients [START_REF] Foley | Selective loss of estrogen receptor beta in malignant human colon[END_REF] and increase viability in cell lines [START_REF] Qiu | Oestrogen-induced apoptosis in colonocytes expressing oestrogen receptor beta[END_REF] and lower expression appears to mirror tumour dedifferentiation [START_REF] Konstantinopoulos | Oestrogen receptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour's dedifferentiation[END_REF] in human and animal models [START_REF] Francesca | Expression of Estrogens beta Receptors in Colon Cancer Progression[END_REF] . Many theories exist as to the mechanism of this protective action of ER β on colonic mucosa and it is probably most likely that the effect is exerted through interaction of a variety of concomitant events-both genomic and non-genomic. One hypothesis is that ER β exerts a genomic action independent of E2 and this was substantiated by a study in which human-colon-tumour-118 cells demonstrated only 20% of normal proliferation related to the expression of cyclin E and cyclin-dependant kinase inhibitor 1A in the absence of estrogen. [START_REF] Martineti | ERbeta is a potent inhibitor of cell proliferation in the HCT8 human colon cancer cell line through regulation of cell cycle components[END_REF] It was surmised that these effects may have been mediated by epidermal growth factor or insulin like growth factor 1. Much debate exists as to how ER β induces apoptosis in colon cancer cells and potential mechanisms that have been described include increased DNA fragmentation [START_REF] Xie | Expression of estrogen receptor beta in human colorectal cancer[END_REF] , increased p53 signalling [START_REF] Hsu | Apoptotic effects of over-expressed estrogen receptor-beta on LoVo colon cancer cell is mediated by p53 signalings in a ligand-dependent manner[END_REF] and induction of a caspase dependant pro-apoptotic cascade. [START_REF] Marino | Estrogens signaling multiple pathways to impact gene transcription[END_REF] Rapid non-genomic actions are the subject of much research worldwide and because uncertainty exists as to what exact cellular messaging cascades are involved in these non-genomic events, a wide variety of mediators have been examined. Whether or not classical estrogen receptors are involved in this rapid signalling is uncertain but a membrane bound receptor (which is dependant on palmitoylation for localisation) has been described [START_REF] Galluzzo | Role of ERbeta palmitoylation in the inhibition of human colon cancer cell proliferation[END_REF] and is likely involved. It is not known whether ER β migration to the cell membrane accounts for these actions or whether an entirely different receptor isoform exerts the effects. [START_REF] Doolan | A Galphas protein-coupled membrane receptor, distinct from the classical oestrogen receptor, transduces rapid effects of oestradiol on [Ca2+]i in female rat distal colon[END_REF] Furthermore, some evidence exists as to non-genomic actions that occur independently of any known receptor, as proven by a rapid calcium flux in ER negative SKBR-3 cells when exposed to E2 [START_REF] Pedram | Nature of functional estrogen receptors at the plasma membrane[END_REF] . It is felt that protein kinase C is a most likely involved in mediation of this rapid action. Further putative mechanisms of actions include p38 mitogen activated protein kinase leading to caspase-3 dependant apoptosis 79 , increased expression of vitamin D receptor mediated by mitogen activated protein kinase 1 or 3 80 or ER β activation of p38 mitogen activated protein kinase cascade 81 . Unlike esophagus, stomach or gallbladder, evidence of protective effects of estrogen against colorectal cancer is compelling. Current controversies in the literature focus primarily on the mechanism(s) of these actions. When these are fully elucidated, it is likely that hormonal manipulation will provide as vital a role in the management of colon cancer as it currently does in breast and prostate disease.

Discussion:

The concept that E2 exerts an effect on the gastrointestinal tract is not new and its actions on intestinal mucosa have been investigated for at least three decades. An attempt to consolidate results of these investigations generates more questions than answers, thus suggesting that many unexplored avenues remain and that the full capabilities of this steroid hormone are far from understood. While it appears intuitive that E2 is protective against esophageal cancer (higher incidence and worse prognosis in men), this hypothesis is supported by neither laboratory nor epidemiological studies. Undoubtedly, estrogens are somehow implicated in the development of gastric cancer (supported by both cell work and population studies) but therapeutic potential of either E2 or its antagonists remains undescribed despite much investigation. In the gallbladder, further controversy exists in that, although malignant mucosa has stained positive for ER, no significant correlation with cancer free survival or metastatic potential has been proven. (Table 1) The most convincing and compelling evidence regards the protective role conferred by estrogen (or perhaps ERβ) against the development and proliferation of colon cancer. Not only has the effect been described but many mechanisms of action have been explored. It is likely that, along with surgery, chemotherapy and radiotherapy, hormonal manipulation will play an integral role in colon cancer management in the very near future. 79

Table 1 . Summary of Estrogenic effects on gastrointestinal malignancy.

 1 Marino M, Galluzzo P, Leone S, Acconcia F, Ascenzi P. Nitric oxide impairs the 17beta-estradiol-induced apoptosis in human colon adenocarcinoma cells. Endocr Relat Cancer. 2006;13(2):559-69. 80 Gilad LA, Schwartz B. Association of estrogen receptor beta with plasmamembrane caveola components: implication in control of vitamin D receptor. J Mol Endocrinol. 2007 ;38(6):603-18. 81 Caiazza F, Galluzzo P, Lorenzetti S, Marino M. 17Beta-estradiol induces ERbeta up-regulation via p38/MAPK activation in colon cancer cells. Biochem Biophys Res Commun. 2007;359(1):102-7.

		Cells		Animals		Epidemiology	Reference
	Oesophagus	Growth		Equivocal		Male>Female	21, 27, 28.
		Inhibition of			No correlation
		KSE-1 Cells			with hormonal
						manipulation
	Stomach	Stimulation of	Lower		Male>Female	31, 36, 52
		gastric	cell	incidence in
		growth (as did	female	and
		agonist		castrated rats.
		tamoxifen)
	Gallbladder	Correlation		-		Increased risk	61, 62, 63
		between				with
		expression of			postmenopausal
		ERβ and poor			HRT use
		tumour
		differentiation.
	Colon	Decreased		Decreased		Premenopausal	65, 66, 71, 72
		proliferation	expression of	female<
		(80%)	in	ERβ in higher	Postmenopausal
		human-colon-	disease stage	female < Male
		tumour-118		in mice
		cells in the
		presence	of
		estrogen

Table


M.D. Gabor Horvath

Szilvia Vasas

Adam Wanner

Adam W Anner

Inhaled corticosteroids reduce asthma-associated airway hyperperfusion through genomic and nongenomic mechanisms

Keywords:

Inhaled corticosteroids have both genomic and nongenomic actions on the tracheobronchial (airway) vasculature in asthma. Genomic actions involve the activation or repression of target genes associated with inflammation, and reduce inflammatory hyperperfusion in the airway. In contrast, nongenomic actions are mediated by rapid cellular mechanisms, and induce transient vasoconstriction. This article reviews recent progress on the mechanisms by which inhaled corticosteroids reverse inflammatory blood flow changes in the airway in asthma.

Inhaled corticosteroids have prominent effects on inflammatory changes of the tracheobronchial (airway) vasculature in patients with bronchial asthma. These include the inhibition of hyperperfusion, microvascular hyperpermeability, mucosal oedema formation, inflammatory cell recruitment and the formation of new blood vessels (angiogenesis) [START_REF] Charan | Bronchial vascular congestion and angiogenesis[END_REF][START_REF] Horvath | Inhaled corticosteroids: effects on the airway vasculature in bronchial asthma[END_REF]. Inhaled corticosteroids are believed to exert most of their vascular effects indirectly by suppressing inflammation in the airway [START_REF] Mcfadden | A century of asthma[END_REF].

However, recent studies revealed interactions of inhaled corticosteroids and airway blood vessels by which vascular manifestations of inflammation can be rapidly reversed in patients with asthma. These recent advances in our understanding of the cellular mechanisms may open new avenues for additional interventions in the pharmacotherapy of asthma.

Corticosteroids are known to interrupt the development of inflammation and suppress ongoing inflammatory reactions through the activation or repression of target genes [START_REF] Adcock | Glucocorticoid: effects on gene transcription[END_REF]. According to this transcriptional (genomic) model of action, the lipophilic corticosteroid molecules cross the cell membrane and bind to intracellular glucocorticoid receptors, which ultimately alter transcription through direct DNA binding (transactivation) [START_REF] Hayashi | Effects of glucocorticoids on gene transcription[END_REF] or bythe inactivation of pro-inflammatory transcription factors (transrepression) [START_REF]New twists in gene regulation by glucocorticoid receptor: is DNA binding dispensable?[END_REF] such as nuclear factor-κB [START_REF] Barnes | Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases[END_REF] and activating protein-1 [START_REF] Nguyen | Chemogenomic identification of Ref-1/AP-1 as a therapeutic target for asthma[END_REF].

Corticosteroids are the most effective drugs for suppressing asthma-associated airway inflammation mainly by downregulation of pro-inflammatory proteins. In addition to genomic actions, corticosteroids have also been shown to regulate mRNA stability and thus translation of many pro-inflammatory genes [START_REF] Wilusz | Bringing the role of mRNA decay in the control of gene expression into focus[END_REF]. Since increased mRNA degradation leads to reduced production of pro-inflammatory proteins, the Some corticosteroid effects are mediated by cellular mechanisms that are too rapid to involve gene expression [START_REF] Losel | Nongenomic actions of steroid hormones[END_REF][START_REF] Norman | Steroid-hormone rapid actions, membrane receptors and a conformational ensemble model[END_REF] and have been termed nongenomic actions.

These rapid actions are initiated by actions on membrane-bound [START_REF] Johnson | Localization of glucocorticoid receptors at postsynaptic membranes in the lateral amygdala[END_REF][START_REF] Horvath | Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells[END_REF] or cytoplasmic receptors [START_REF] Tumlin | Aldosterone and dexamethasone stimulate calcineurin activity through a transcriptionindependent mechanism involving steroid receptor-associated heat shock proteins[END_REF][START_REF] Croxtall | Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism[END_REF], or nonspecific interactions with the cell me mbrane [START_REF] Whiting | Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms[END_REF]. The common features of nongenomic actions include rapid onset of action and recovery after drug removal, insensitivity to blockers of gene transcription (e.g. actinomycin D) and protein synthesis (e.g. cycloheximide), and retained activity of cell membrane impenetrable corticosteroid-protein conjugates.

In this article, we review genomic and nongenomic cellular mechanisms by which inhaled corticosteroids reverse inflammatory blood flow changes in patients with asthma.

Airway blood flow in asthma

Since the vasculature typically participates in the inflammatory responses at the tissue level, airway blood flow is expected to change in an inflammatory airway disease such as asthma. In anima l models of airway inflammation such as Ascaris suum sensitized sheep, antigen challenge has been shown to cause an increase in airway blood flow [START_REF] Long | Modification of bronchial blood flow during allergic airway responses[END_REF][START_REF] Wanner | Circulation of the airway mucosa[END_REF]. These findings have now been confirmed in human subjects using recently invented noninvasive methods for airway blood flow measurements [START_REF] Kumar | Airway mucosal blood flow in bronchial asthma[END_REF][START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF][START_REF] Brieva | Effect of an inhaled glucocorticosteroid on airway mucosal blood flow in mild asthma[END_REF][START_REF] Brieva | Adrenergic airway vascular smooth muscle responsiveness in healthy and asthmatic subjects[END_REF]. By measuring the uptake of an inhaled, soluble inert gas (dimethyl-ether, DME) [START_REF] Onorato | Airway mucosal blood flow in humans. Response to adrenergic agonists[END_REF][START_REF] Scuri | Measurement of airway mucosal blood flow with dimethylether: validation with microspheres[END_REF], mean airway mucosal blood flow values (comprising about 70% of total airway blood flow) were 24% to 77% higher in patients with stable asthma than in healthy controls [START_REF] Kumar | Airway mucosal blood flow in bronchial asthma[END_REF][START_REF] Brieva | Adrenergic airway vascular smooth muscle responsiveness in healthy and asthmatic subjects[END_REF]. Using acetylene rather than DME for A c c e p t e d m a n u s c r i p t 6 measurements, airway blood flow has been confirmed to be elevated in patients with mild persistent and moderate asthma compared with healthy subjects [START_REF] Paredi | Correlation of exhaled breath temperature with bronchial blood flow in asthma[END_REF].

In the airway of asthmatics, increased blood flow is due to dilatation of resistance arteries and increased numbers of vessels in the bronchial mucosa. In animal models, most inflammatory mediators cause vasodilation [START_REF] Widdicombe | Neuroregulation and pharmacology of the tracheobronchial circulation[END_REF]. Histamine has a triphasic effect with an initial vasodilation followed by vasoconstriction, and then a long-lasting vasodilation. Sensory neuropeptides released from afferent nerves are also strong vasodilators. A number of studies have also demonstrated increased vascularity (i.e. size and number of vessels, or cross-sectional vascular area) of the bronchial mucosa in patients with asthma compared with healthy controls [START_REF] Wilson | The bronchial microcirculation in asthma[END_REF][START_REF] Chetta | Vascular component of airway remodeling in asthma is reduced by high dose of fluticasone[END_REF].

However, the contribution of increased hypervascularity to airway hyperperfusion needs to be explored.

Rapid effects on airway blood flow

Inhaled corticosteroids are known to exert their effects on the airway vasculature through genomic and nongenomic mechanisms in asthma (Fig. 1). Among these effects, corticosteroids acutely suppress airway hyperperfusion associated with asthma [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF][START_REF] Paredi | Correlation of exhaled breath temperature with bronchial blood flow in asthma[END_REF][START_REF] Clarke | The effects of inhaled spasmogens on airway mucosal blood flo w in asthmatic subjects[END_REF]. Inhaled fluticasone propionate has been shown to decrease airway mucosal blood flow with a maximal effect about 30 min after inhalation, and a return to baseline at 90 min [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF]. The blood flow effect increased in a dosedependent manner up to 880 µg of fluticasone propionate, with a significantly greater effect in asthmatics than in healthy controls. The vasoconstrictor action has also been demonstrated after inhalation of beclomethasone dipropionate and budesonide [START_REF] Mendes | Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids[END_REF]. Since pretreatment with the selective α 1 -adrenoreceptor antagonist terazosin inhibited the effect of fluticasone propionate on blood flow [START_REF] Wanner | Nongenomic actions of glucocorticosteroids on the airway vasculature in asthma[END_REF], it is likely that Corticosteroids could increase sympathetic signal transmission and consequently vascular tone by inhibiting extraneuronal catecholamine uptake [START_REF] Iversen | Inhibition of catecholamine uptake-2 by steroids in the isolated rat heart[END_REF]. This nongenomic mechanism was demonstrated recently in dissociated rabbit aortic smooth muscle cells by showing that the inhibitory action on noradrenaline uptake occurred rapidly, was neither altered by protein synthesis nor transcription inhibitors, and could be reproduced with a corticosteroid made membrane-impermeant by conjugation to bovine serum albumin [START_REF] Horvath | Steroid sensitivity of norepinephrine uptake by human bronchial arterial and rabbit aortic smooth muscle cells[END_REF]. Furthermore, the impaired extraneuronal catecholamine uptake in atopy, which was demonstrated by comparing uptake by vascular smooth muscle cells isolated from ovalbumin-sensitized and control rabbits [START_REF] Horvath | Systemic ovalbumin sensitization downregulates norepinephrine uptake by rabbit aortic smooth muscle cells[END_REF], might lead to increased vasoconstrictor responsiveness to inhaled corticosteroids as seen in asthmatic subjects in vivo [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF].

In recent in vitro experiments, various clinically significant corticosteroids such as budesonide and methylprednisolone have been shown to inhibit the extraneuronal monoamine transporter-mediated uptake of norepinephrine by bronchial arterial smooth muscle cells [START_REF] Horvath | Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells[END_REF][START_REF] Horvath | Steroid sensitivity of norepinephrine uptake by human bronchial arterial and rabbit aortic smooth muscle cells[END_REF]. This rapid (< 5 min) effect could reduce the removal of noradrenaline released from airway sympathetic nerves and, therefore, may be responsible for the rapid vasoconstrictor actions of corticosteroids.

Alternatively, the acute vasoconstrictor effect may involve other nongenomic actions of corticosteroids on vascular smooth muscle cells (Table 1). In addition, corticosteroids could disrupt the inflammatory process through rapid inhibitory actions on cellular processes or mediators related to inflammation, which might reduce inflammatory hyperperfusion in the airway in asthma ( changes, these genomic vascular actions of inhaled corticosteroids may be of therapeutic value and have received increased interest recently [START_REF] Busse | Future research directions in asthma: an NHLBI Working Group report[END_REF].

A number of studies have demonstrated that inhaled corticosteroid therapy decreases airway mucosal blood flow in patients with asthma. In a cross-sectional study, airway mucosal blood flow was higher by about 23% in corticosteroid-naive compared with corticosteroid-treated asthmatics [START_REF] Kumar | Airway mucosal blood flow in bronchial asthma[END_REF]. In interventional studies, inhaled fluticasone propionate (440 µg/day) therapy for 2 weeks reduced blood flow by about 12% and 21% in two different groups of asthma patients [START_REF] Brieva | Effect of an inhaled glucocorticosteroid on airway mucosal blood flow in mild asthma[END_REF][START_REF] Mendes | Effect of montelukast and fluticasone propionate on airway mucosal blood flow in asthma[END_REF], and additionally showed that airway blood flow returns to the pretreatment level 2 weeks after cessation of therapy.

Corticosteroids are believed to decrease blood flow in the airway mucosa largely by suppressing inflammation and the release of vasodilator mediators. This concept was supported by the fact that the effect of inhaled fluticasone propionate (440 µg/day for 2 weeks) was similar to that of the leukotriene modifier montelukast (10 mg/day) in decreasing airway blood flow in asthma patients [START_REF] Mendes | Effect of montelukast and fluticasone propionate on airway mucosal blood flow in asthma[END_REF]. Corticosteroids may also act directly on both vascular smooth muscle and endothelial cells (reviewed in [START_REF] Horvath | Inhaled corticosteroids: effects on the airway vasculature in bronchial asthma[END_REF]), thereby increasing vascular tone and reducing blow flow in the airway vasculature (Table 3).

Inhaled corticosteroids have a potential therapeutic role of reversing airway angiogenesis in asthma. In a cross-sectional study, therapy with high doses (≥ 800

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

9 µg/day) of beclomethasone dipropionate was associated with a reduced vascular area of the lamina propria [START_REF] Orsida | Vascularity in asthmatic airways: relation to inhaled steroid dose[END_REF]. In interventional studies, beclomethasone dipropionate (800 µg/day) therapy for 6 months reduced the number of blood vessels and the vascular area [START_REF] Hoshino | Inhaled corticosteroids decrease vascularity of the bronchial mucosa in patients with asthma[END_REF], whereas a 6-week treatment with fluticasone propionate was effective at 1000 µg/day inhaled dose in reducing the number of blood vessels and the vascular area [START_REF] Chetta | Vascular component of airway remodeling in asthma is reduced by high dose of fluticasone[END_REF].

Perspective

Since vascular hyperperfusion is a consistent feature of tissue inflammation, airway hyperperfusion has recently been proposed as a biomarker of asthmaassociated inflammation. Thus, with the advent of a noninvasive method to measure airway blood flow [START_REF] Kumar | Airway mucosal blood flow in bronchial asthma[END_REF][START_REF] Scuri | Measurement of airway mucosal blood flow with dimethylether: validation with microspheres[END_REF], airway blood flow changes might be assessed independent of lung function as an index of disease severity and to quantitate the response to anti-inflammatory therapy. Reported reduction of airway blood flow by anti-inflammatory therapy in asthmatics [START_REF] Brieva | Effect of an inhaled glucocorticosteroid on airway mucosal blood flow in mild asthma[END_REF][START_REF] Mendes | Effect of montelukast and fluticasone propionate on airway mucosal blood flow in asthma[END_REF] and a positive correlation between exhaled nitric oxide and airway blood flo w levels [START_REF] Paredi | Correlation of exhaled breath temperature with bronchial blood flow in asthma[END_REF] lend support to this concept.

In vivo drug activity is known to depend on the drug's tissue bioavailability, which is the rate and extent to which the drug is absorbed and becomes available at the site of action [START_REF] Hammond | Determinants of steroid hormone bioavailability[END_REF]. Currently, the standard screening test to rank the relative antiinflammatory potencies and tissue bioavailabilities of corticosteroids is the McKenzie "skin blanching" test [START_REF] Mckenzie | Method for comparing percutaneous absorption of steroids[END_REF]. This test is based on the ability of corticosteroids to cause vasoconstriction in the skin after topical application [START_REF] Noon | A comparison of techniques to assess skin blanching following the topical application of glucocorticoids[END_REF], a phenomenon that is similar to the observed rapid vasoconstriction in the human bronchial mucosa [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF]. Airway blood flow responsiveness to inhaled corticosteroids seems to be ideal in assessing how these drugs gain access to their sites of actions in the bronchial wall [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF][START_REF] Mendes | Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids[END_REF]. The airway vasculature is known to occupy a significant portion of the inner bronchial wall [START_REF] Mariassy | Morphometry of the subepithelial circulation in sheep airways. Effect of vascular congestion[END_REF]. Thus, the hypothesis that inflammatory changes of the vasculature increase mucosal thickness enough to influence airway resistance might seem reasonable. Theoretically, a small change in airway wall thickness caused by the increased sized of the vascular compartment interior to the smooth muscle layer might potentiate airway narrowing when bronchial smooth muscle shortening occurs (airway hyperresponsiveness). Although airway blood and vascular engorgement have been proposed to contribute to airway narrowing and hyperresponsiveness in asthma, the reported data are conflicting [START_REF] Csete | Vasomotion influences airflow in peripheral airways[END_REF][START_REF] Wagner | Effects of bronchial vascular engorgement on airway dimensions[END_REF]. Despite the prominent effects of inhaled corticosteroids on airway blood flow, the immediate impact of inhaled corticosteroids on lung function appears to be marginal in patients with acute asthma [START_REF] Kumar | Transient effect of inhaled fluticasone on airway mucosal blood flow in subjects with and without asthma[END_REF][START_REF] Rodrigo | Acute asthma in adults: a review[END_REF].

Since the airway circulation has an important role in the clearance of inhaled drugs (e.g. bronchodilators) from the respiratory tract, the inflammatory increase in airway blood flow is likely to have clinically undesirable effects in asthma [START_REF] Wanner | Clinical perspectives: role of the airway circulation in drug therapy[END_REF]. With respect to airway disposal of inhaled drugs, the corticosteroid induced decrease in airway blood flow is likely to enhance the action of inhaled bronchodilators by diminishing their clearance from the airway. Thus, the rapid airway vasoconstriction induced by inhaled budesonide [START_REF] Mendes | Comparative bronchial vasoconstrictive efficacy of inhaled glucocorticosteroids[END_REF] might be responsible for enhancing the bronchodilator action of inhaled formoterol as seen in patients with chronic obstructive pulmonary disease [START_REF] Cazzola | Onset of action of formoterol/budesonide in single inhaler vs. formoterol in patients with COPD[END_REF]. Since the corticosteroid-induced vasoconstriction

Conclusions

In recent years, attention has focused on vascular components of airway inflammation because of their correlations to asthma severity, including airflow limitation and bronchial hyperresponsiveness [START_REF] Busse | Future research directions in asthma: an NHLBI Working Group report[END_REF]. Inhaled corticosteroids seem to reverse both functional and structural abnormalities of the airway vasculature through

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

 5 -transcriptional actions might also have significant implications in the pharmacotherapy of asthma[START_REF] Stellato | Post-transcriptional and nongenomic effects of glucocorticoids[END_REF].

A c c e p t e d m a n u s c r i p t 7 corticosteroids

 7 facilitate noradrenergic signal transmission to induce neurogenic vasoconstriction.

A c c e p t e d m a n u s c r i p t 8 4.

 8 Delayed effects on airway blood flowIn asthma, inhaled corticosteroids have been shown to counteract all the vascular manifestations of airway inflammation by inhibiting the release of vasoactive inflammatory mediators or by acting directly on vascular smooth muscle or the endothelium. Because disease severity seems to correlate with airway vascular

A c c e p t e d m a n u s c r i p t 10 Thus

 10 , analysis of the rapid vasoconstrictor action can provide information on the airway tissue bioavailability of inhaled corticosteroids, which might also depend on the aerosolized drug's physicochemical characteristics and the aerosol-generating device.

A c c e p t e d m a n u s c r i p t 11 peaks

 11 rapidly ( within 30 min after drug inhalation), simultaneous administration of inhaled corticosteroids and bronchodilators is likely to be of clinical significance.

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 A c c e p t e d m a n u s c r i p t 18 [A c c e p t e d m a n u s c r i p t

 18 genomic and nongenomic actions. Although the respective roles of these actions are still incomplete ly understood, the inhibitory effects on airway hyperperfusion, hyperpermeability, and angiogenesis are expected to have significant diagnostic and therapeutic implications in the management of asthma. ACKNOWLEDGEMENT G.H. is a recipient of the Bolyai Fellowship of the Hungarian Academy of Sciences, Budapest, Hungary. 53] Schmid D, Burmester GR, Tripmacher R, Kuhnke A, Buttgereit F. Bioenergetics of human peripheral blood mononuclear cell metabolism in quiescent, activated, and glucocorticoid-treated states. Biosci Rep 2000; 20(4):289-302. [54] Baccan GC, Oliveira RDR, Mantovani B. Stress and immunological phagocytosis: possible nongenomic action of corticosterone. Life Sci 2004; 75(11):1357-1368. [55] Long F, Wang YX, Liu L, Zhou J, Cui RY, Jiang CL. Rapid nongenomic inhibitory effects of glucocorticoids on phagocytosis and superoxide anion production by macrophages. Steroids 2005; 70(1):55-61. [56] Pitzalis C, Pipitone N, Perretti M. Regulation of leukocyte-endothelial interactions by glucocorticoids. Ann N Y Acad Sci 2002; 966:108-118.

A c c e p t e d m a n u s c r i p t 21 Table 3 .A c c e p t e d m a n u s c r i p t 22 LEGENDSFigure 1 .A c c e p t e d m a n u s c r i p tFigure 1 .

 2132211 Figure 1. Genomic and nongenomic actions of inhaled corticosteroids in the airway vasculature in patients with asthma

Figure 1

 1 Figure 1

Table 2 ).

 2

19 TABLES Table 1 .

 TABLES1 Rapid nongenomic actions of corticosteroids in vascular smooth muscle cells

	Tissue used Rabbit aorta, human bronchial artery Rat aorta Rat aorta Rat aorta A c c e p t e d Rapid action Inhibition of the cell membrane catecholamine transporter Activation of the cell membrane Na + /H + transporter Intracellular Ca 2+ mobilization Stimulation of phosphoinositide breakdown to increase inositol-1,4,5-triphosphate levels m a n u s c r i p t Antihormon Sensitivity Ref. RU486 No effect [14, 34] RU486 Inhibition [49] Not tested [50] RU486 Inhibition [51]
	20

Table 2 .

 2 Rapid nongenomic actions by which corticosteroids could interrupt the development of inflammation and suppress ongoing inflammatory reactions

	Cell type	Rapid action	Ref.
	Neutrophils	Inhibition of degranulation	[52]
	Lymphocytes	Inhibition of membrane cation cycling	[53]
		and respiration
	Macrophages	Inhibition of phagocytosis	[54]
	Macrophages	Inhibition of superoxide anion	[55]
		production
	Leukocytes	Inhibition of endothelial adhesion	[56]
	Lung	Inhibition of phospholipase A 2 activity	[16]
		and arachidonic acid release