Regulation of Endometrial 1 lp-Hydroxysteroid Dehydrogenase (11P-HSD) Types 1 and II
BY Andrew D. Darne1 Gradliate Prograim in Physiology
Submitted in partial fiiltilrnent of the requirements for the degree of Master of Science
Facdty of Graduate St~idies The University of Western Ontario London, Ontario January 1999
O Aiîdrew D. Darnei 1999 National Library Bibliothèque nationale I*m of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. nie Wellington Ottawa ON KIA ON4 Wwa ON KIA ON4 Canada Canada
The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Libraq of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/^ de reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othemise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation- The intracellular 1 1p-hydroxysteroid dehydrogenase type 1 and 2 ( 1 1 P-HSD 1 and 2) enzymes are responsible for the interconversion of glucocorticoids and their inactive metabolites. The present study \vas undertaken to examine the regulation of 1 1 P-HSD I and 2 in the o\ine endometrium and the Ishikawa human endornetrial ce11 Iine. respective1y.
Progesterone induced the expression of 1 1B-HSDI mRNA without affecting the level of 1 1P-HSD 1 dehydrogenase activity in the endornetriurn of ovariectoniized ewes.
Estradiol-17P. medrosyprogesterone acetate. desaniethasone and cortisol increased. while EGF decreased the level of 1 1P-HSDZ activity and mRYA in Isliikau-a cells. a \\-el1 di fferentiated hunian endometrial adenocarcinorna ce11 line that maintains functional estrogen and progesterone receptors.
The preseiit study has slio\vn tliat 11 P-HSD1 espression is regulated by progesterone in the ovine r~idoiiietriuiii. In Isliikawa cells. 1 1 P-HSD? expression is regulated b!. steroid hormones and EGF. The Isliikawa ce11 line ma) be a useful mode1 to srudy 1 I P-HSDZ regulation in human endometrial epitheliun~.
Ke~*~tm-d.s: 1 1 P-hydroxysteroid dehydrogenase. 1 1 P-HSD. endometrium. regulation. EGF. desaiiiethasone. estradiol. medrosyprogesterone acetatr. progesterone. Ishika\va. human. oi-ine.
.*. III CO-AUTHORSHIP
The following thesis contains material from rnanuscripts submitted for pubiication CO-authored by A.D. Damel. G.E. Lamming. T.K. Archer and K Yang. Al1 of the erperimental work presented in this thesis was performed by A.D. Damel. Original manuscripts. versions of which appear in Chapters 3 and 4 of rhis thesis. were \vritten by A.D. Damel and K. Yang. DEDICATION
1 would like to dedicate this thesis to my loving mother. Margarita and to Edn-ard and Gary. wlio are not only my brothers. but also my best friends. h4)- family ha\-e ailval-s been tl-iere to support and encourage me throughout my studies at Western. The'. ha1.e taught me the true value of farnily and frirndship. ACKNOWLEDGEMENTS
To stan. 1 u-ould like to express my sincerest appreciation and respect to Dr. Kaipiiig Yang. niy supervisor. mentor and colleague. uho provided me \vit11 man\- years of support. guidance and encouragement during both my undergraduzte and graduate years at Western. He has taught me the true value of solid researcli- committment. team work and communication. This thesis would never have been possible n-itliout his support.
1 sincerely tliai~kniy ad\-isors. Dr. Gabe DiMattia. Dr. David Ponierantz and Dr. Tom Kennedy. \\-ho supported me \vit11 a wealth of inspiration and c~icourapeiiierittliroug1iout my Master's pro,iTrani.
A huge tliank-?-ou goes to my friends/colleques of theYang laboratory. U'here ïvould 1 be n-itliout !-ou guys? You are: Dr. Min Yu. Dr. R. Sampatli-Kuniar. Laura Pereira. Helcn Pu. JO Mouyal. Dan Hardy. Katherine Sliearnian. and Jessica Dy.
I am also ver>-privilcgcd to bave liad the opponunity to discuss researcli and pick at the minds of sonle of rnost dedicated scientists and c1i11icians at the La\\-son Researcli Institute (LRI) and St. Josepli's HeaIth Centre. Tliank !.ou to Dr. Victor Han. Dr. Waliid Khalil. Dr. Don Killinger. Dr. Bryan Richardson. Dr. Robert Gagnoii. Dr. Tom Drysdale and Dr. Giio Foiig.
1 M-ould likc to thank Dr. Trevor Archer and the nisnibsrs of Iiis lab. espccially Dr. Harriet Kin\-aiiiu and Andrea Ricci. at the London Regional Cancer Centre (LRCC) Ibr tlieir assistancr iii various aspects relating to my project.
1 would like to acknou-ledge Dr. EIena Choleras and Ales Thomas for their patience and kindness in tutoring me in statistical analysis. Thank ~OLIto Tom Stavraky. Bruce Arppe uid the Department of Pliysiology for giving me the opponunity to teach undeqraduate Physiology. I feel privileged to ha1.e had the opportunit). to share my howledge of Physioiogy with otlier students.
A big thank !-ou also to the many friends 1 have made over the years at tlie LRI. They not only understand the life of the 2.5 hota. dny graduate student. but tliey liave been a source of inimensc inspiration and strength. Thank )-ou to Dr. Bertrand Du\.illié. Dr. Edith Arany. Dr. Madhulika Gupta. Dr. Vladimir Semino\-. Dr. Ki!-oini Seguchi. Da\\-r? Kilkemy. Brenda Strutt. Daniel Mang. Kim Handoko. Palvel Krysiak. Jin Hayatsu. Marnie Setterington. Chris Schramek. and Jarna Morrison.
The Isliika\va endometrial adenocarcinorna ce11 line used in tliis stud)- \\as gnciousl>. donated by Dr. Masato Nishida of the Depanment of Obstetrics and Gyiecolopy. Institute of C linical Medicine. Tsukuba University. Japan.
Financial support for tliis study came fro~ilthe Medical Ressarcli Council of Canada (MRC) ressarcli grant awarded to Dr. Kaiping Yang.
The 101-e and enintioiial support 1 receive from ni!- caring niorlier. Xlargarita Darnel and my brotlirrs. Gary aiid Edward Darne1 have Iielped me pull tlirougli the roucli- times. In the end. it is this thssis tliat 1 dedicate to thein.
My time speiit Iiere as a student u-ould never have been the sanie n.itliout my .4ikidu training. Jainiie Slieppard-sensei and Ashley He~essy-senseiat the Aiki Budo Centre dojo are my best friends and really tme masters of a very gracious and beautiful art. They ha~ealn-ays been there for me and have Iielped me to understand a little niore about the harniony of life. 1 u-il1 Iial-e ntany fond memories and will miss tlie niany good friends 1 lixs niade tlirough -4ikitlo. Domo wig~lfogo~~li~~~c~hii~~! OSC-!
vii CONTENTS
TITLE PAGE CERTIFICATE OF EXAMINATION ABSTRKT AND KEYWORDS CO-AUTHORSHIP DEDICATION ACKN0LC'LEDGEbfEXTS CONTENTS LIST OF TABLES sii LIST OF FIGURES LIST OF PHOTOGRAPHIC PLATES LIST OF ABBREVIATIONS
CH-APTER 1 - INTRODUCTION
CHAPTER 2 - LITERATURE REVIEW
2.1 F.4CTORS AFFECTING GLUCOCORTICOID ACTIOSS
2.1.1 Iiitroduction
3.1 -3 O\-enk\-of glucocorticoid actions
2.1 2.1 The Endocrine Orchestra Maestro: The HPA mis --3 173 .-.- Tlir Endocrine Orchestra Players - Pan 1: Glucocorticoid synthesis 2.1 2.3 The Endocrine Orchestra Players - Pan II: Binding proteins in circulation 2.I 2.4 The Endocrine Orchestra Players - Part III: Glucocorticoid receptors
2.1.2.4.1 Structure of the GR 2.1.3.4.2 GR activation and GR nlediated transcriptional modulatioil 2.1.2.4.3 GR Agonists and Antagonists
7.1 2.5 The Endocrine Orchestra Players - Part IV: Escretion and Metabolisni of Glucocorticoids
2.1 2.6 Tlis Endocrine Orchestra Ticket Master: Physiology of 1 1 P-hydrosysteroid deh~-drogenase (1 1 P-HSD) 23
2.1.2.6.1 The short chain deh~drogenase/reductase (SDR) family 24 2.1 2-62 The DISCOVERY of 1 1 P-HSD
2.2. I 1 1P-Hydrosysteroid Dehydrogenase Type 1
2.2.1.1 Structure 2.2.1.2 Tissue distribution of 1 1P-HSD 1 2.2.1.3 Function of 1 1f3-HSDl 2.3.1.4 1 1P-HSD 1 Regulation 3-3.1.5 I 1P-HSD 1 and its clinical iinplications
-.-.-333 1 1 0-H ydrosysteroid Dehydrogenase T>-peII
-.-.-.3771 Structure 34 -.-.-.-3373 Tissue distribution of 1 1P-HSD2 37 2.2.2.3 Function of 1 1P-HSD2 37 -.-.-.3374 1 1P-HSDî Regulation 38 -.-.-.>337- Clinical implications of I 1P-HSDZ expression 4 1
2.2.2.5.1 Apparent Mineralocorticoid Escess (AME) 4 1 -.--W.333-3 3.- Fetal development 42
2.3 GLL~COCORTICOIDSAND THE FEMALE REPROD~CTIVESYSTEhl
-.J9-3 .- Uterine anatomy and female reproductive endocrino1og~-
2-321 Tlie Uterus 3-37 - .-7 .- .- ~~Ieiistrualcyle -.J.--39 -7- Ses steroids. receptors and antagonists
2.3.2.3.1 Ses steroid horn~ones 2.~? .3.~1 .2 Ses steroid hormone receptors: PR and ER ? -9 2.~2~.J Progestin and estrogen antagonists
3.3 -3 Implantation and Decidualization
2.3.4 Glucocorticoid actions in the uterus
Regulation of glucocorticoid action in the endonietrium
3.4 SCOPE OF THE PRESENT STUDY
2.5 REFERENCES CHAPTER 3 - PROGESTERONE INDUCES THE EXPRESSION OF 11P- HYDROXYSTEROID DEHYDROGENASE TYPE 1 mRNA IN THE OVINE ENDOMETRIUM
Introduction
Materials and Methods
3 -2.1 Tissue collections 3.2.2 RNA extraction and Northern blot analysis 3-23 Analysis of 1 I P-HSD 1 mRNA - semi-quantitative RT-PCR 3 -2.4 Assay of 1 1P-HSD 1 activity
Results
3.3.1 11P-HSDl mKNA 3 -3.2 1 1 P-HSD 1 activity
Discussion
Acknowled,=enlents
CHAPTER 4 - REGULATION OF 1 1 P-HYDROXYSTEROID DEHYDROGENPLSE TYPE 2 BY STEROID HORMONES AND EPIDERMAL GRO\\'TH FACTOR IN THE ISHIKAC\'I\ HUMAN ENDOMETRIAL CELL
4.1 introduction 96
4.2 Materials and Methods
4.2.1 Reagents and supplies 4-32 Ce11 culture and treatments 4.2.3 Assay of 1 1P-HSDZ acirivity - radiometric conversion assay 4.1.4 Analysis of 1 1B-HSD2 mRNA - semi-quantitatke RT-PCR 3.2.5 Data analysis
4.3 Results
4.3.1 Characterization of 1 1 P-HSD in 1shikan-a cells 4.3 2 Effects of se'; steroid hormones on 1 1 P-HSDZ activity 4.33 Effects of Glucocorticoids on 1 1P-HSD2 activity 4.4.4 Effects of EGF on 1 1P-HSD2 activity 4.45 Effects of sex steroids. glucocorticoids and EGF on 1 1 P-HSDZ mRNA
Discussion
Acki~o~vledgements
Retèrences
CHAPTER 5 - DISCUSSION
1 1 P-HSD 1 gene espression in the endometriuni
1sliikan-a cells: in vitro model system for the study of Iiuman endornerrial 1 1 P-HSDî
Re~ulationof 1 1 P-HSDI b>. sex steroid hormones. glucocorticoids aiid EGF
Tlx --s\virch" hypothesis
Furiire directions
PIi!~siological significance and implications of currenr findinos
Re tki-eiices
CurricuIuiii Vitae LIST OF TABLES
Table 2.1 Hormone response elements (HE) 19
Tuble 2.3 Cl~aracteristicsof human 1 1P-hydrosysteroid dehydro,aenase type I and II (1 I P-HSD1 and 2) 26
sii LIST OF FIGURES
Figrn-c 2.1 The hypotlialamic-pituitary-adrenal asis
Figt 11-e2.2 The syntliesis of cortisol from cholesterol
Figrtr-c 2.3 The human glucocorticoid receptor (GR)
Fig1n.e 2.4 Mechanism of GR-mediated transcriptional activation
Figtire 2. j Prii~cipalurinary metabolites of cortisol and cortisone
Figri~-e2.6 Human type 1 1 1 P-hydroxysteroid dehydro,uenase ( 1 1 P-HSD 1 ) gene. mRNA and proteiii
Human type 2 1 1P-hydroxysteroid dehydrogenase ( 1 1 P-HSDî) gene. mRNA and protein
The rneiistrual cycle
Sclieniatic diagram of the fernale Iiypotlialan~ic-pituitary-ovarian(HPO) asis
Nol-tkrn blot of 1 I P-HSDI in the endonietriuim of OVX Etves follon-i-ingprogesterone replacement
RT-PCR of 1 1P-HSD 1 in the endometrium of OVS ews Treated u-ith progesterone replacement
Changes in 1 I P-HSD 1 enzyme activity in the endometriuni of OVX sheep treated with progesterone replacenient 90
Kinetic characteristics 1 1 P-HSD dehydrogeiiase activity in Isliikatva cells 1 08
RT-PCR analysis of 1 1P-HSD 1 and 3 niRNA in Isliikaw cells 109
Effects of ses steroids and their antagonists on 1 1P-HSDî activit>.in Ishikawa cells 111
Effects of glucocorticoids on 1 1P-HSDî activity in Isliikawa cells
Et'ficts of epidermal growth factor (EGF) on 1 1 P-HSD2 activit>-in Isliikawa cells 115 Figru-e 4.6 Effects of steroid hormones and EGF on 1 1 P-HSD:! niRNA (RT-PCR/Southern blot analy sis)
LIST OF PHOTOGRAPHIC PLATES
Phoro y/w 1 photo of Isliikawa cells
siv LIST OF ABBREVIATIONS
3P-HSD 3 p-l~ydrosysteroiddehydrogenase 1 1P-HSD 1 1 P-liydrosysteroid deliydropenase 17P-HSD 1 7P-hydrosysteroid dehydrogenase aa amino acids ACTH adrenocorticotropic hormone ADX adrenalectoiny AERD apparent (E) cortisonse reductase deficiency AME apparent mineralocorticoid escess -41;P arginiiie vasopressin bp base pairs CBG corticosteroid binding globulin CBX carbenosolone CHO Chinese ha~nsterovary (cells) CRH corticotropin releasing hormone Des desaiiistliasone E2 estradio1 ECh1 estracellular matris E cortisone EGF epidermal gronth factor ER estrogen receptor ERE estrogen response element F cortisol FSH follicle sti~iiulatinghormone GE -cl!.cyrrlietinic acid GnRH gonadotropin-releasing hormone GRE glucocorticoid response element C GR (KR) glucocorticoid receptor (human) hsp Iieat sliock protein HP.4 li!-potlialaniic-pituitary-adrenal (asis) HP0 hypothaiamic-pituitary-ovarian (axis) HRE hormone response eiement IL interleukin IUGR intra-uterine gron-th restriction IVF-ET in vitro fertilization and embryo transfer kb kilo bases LH luteinizing !lomiorie MPA iiiedrosypro,<'esterone acetate bi R iiiineralocorticoid receptor NAD iiicotinaniide adenine dinucleotide NIDD non-insulin dependent diabetes ORF open reading frame OVX O\-ariectomy P4 progesterone PG prostaglaiidin PLA, phospliolipase A-? PO MC pro-opiomelai~ocortin PR progesterone receptor Pm paraventricular nucleus RAR retinoic ncid receptor RT-PCR re\wss trariscription-polymerase cllain reciction SOY supraoptic nucleus T testosterone T3 tiiyroid horinone TGFu tnnsforming grou-th factor a
svi CHAPTER 1 rNTRODUCTION Glucocorticoids exen powerful biological efiects on target tissues. including the mammalian endometriurn. Within the endometrium. glucoconicoids have been show to influence endometrial ce11 apoptosis. prostaglandin and cytokine synthesis. immune function. implantation and subsequent decidualization. Since 1 L P-hydrosysteroid dehydrogenase (1 1P-HSD) enzymes regulate the bioavailability of glucocorticoids to target tissues by interconverting biologically active piucocorticoids to their inactive metabolites. the expression of 1lP-HSDs in the endometrium will undoubtedly ha1.e a major impact on the dynamic function of the endometriurn. Follo~vingthe discovey of 1 1 P-HSD enzyme acti~ityin the uterus of non-gra\.id and pregnant n-omen by Murphy (1977). there has since been a greater interest into the physiological role of this glucocorticoid metabolizing enzyme in reproductive physioIogy. Indeed studies have established the presence of 1 1 P-HSD 1 and 2 enqme activity. protein and mRNA in the uterus of numerous species including human (Smith er nl.. 1997: Arcuri et al.. 1996b). sheep (Yang er al.. 1996) and rat (Burton et d..1998; Albiston et al.. 1995). However. few studies have investigated the regulation of 1 1 P- HSD1 aiid!or 2 gene in the endometriurn. Apan from a study that in\-estigared ses sreroid regulation of 1 1 P-HSD 1 and 2 enzyme actkity in primary cultures of human endometrial stroma1 cells (Arcuri et al.. 1996b) and other studies tliat have alluded to the regulation of 1 1P-HSD 1 and/or 2 in the uterus by sex steroids (Smith et al.. 1997: Yang er al.. 1996). these studies remain ambiguous. speculative and incomplete. Altliough. Arcuri and colleagues ( 1996b) report the presence of I 1 P-HSD 1 and 2 enzyme activiry in Iiuiiian endoinetrial stroma1 cells foilowing CO-incubarion n-itli a synthetic progestin and estradiol. a separate study identified iinmunoreacti\.e 1 1P-HSD7 protein almost esclusively in the lun-iinal and glandular epithelium of the human endometrium (Smith er al.. 1997). These contradicting results and the studies done to date have yet to fully elucidate the iink between sex steroid regulation and 1 1P-HSD I and/or 2 gene espression in the endometrium. In a study done in Our laboratory. 1 1 P-HSDI mRNA \vas identified in the endonietrium of slieep only during pregnancy and in non-pregnant animals during the late luteal phase of the estrous cyIe (Yang et cd.. 1996). Since circulating levels of progesterone are elevated during both of tiiese periods. these findings suggested that progesterone may be responsible for the induction of 1 IP-HSDl gene expression obsenred in the endometrium of this species. Due to the apparent lack of definitive results sho~i-inga clear relationship between sex steroids and the expression of 1 1 P-HSD 1 and 2 gene in the endometrium, the present study was designed to address these issues. In addition to defining the effects of progesterone on 1 1 P-HSDl expression in the o\.ine endometrium. the studies described in this thesis characterized and identified an in i-irr-o mode1 system. the human Ishikawa endometrial ce11 line. for the study of human endometria1 1 1 P-HSD2. Moreover. using tliis mode1 system. these studies investigated the regulation of 1 IP-HSDî by ses steroid hormones, glucocorticoids and EGF. al1 of whicli are ho\vn to play an important role in the process of implantation and decidualizarion. CHAPTER 2
LITERATURE REVIEW 2.1 FACTORS AFFECTING GLUCOCORTICOID ACTIONS
Glucocorticoids are crucial for sunival since these steroid lionnones play important roles in maintaining a complex and dynamic homeostasis in various systenis. including the reproductive axis and during fetal development (Magiakou er cd.. 1997: Liggins 1976: Cliallis et al.. 1989). As a consequence. there has been enormous interesi in studying the factors that regulate glucoconicoid actions. and the underlying mechanisms upon n-hic11 they operate. This thesis outlines the researcli done on tlie regulation of the two glucocorticoid rnetabolizing enzymes. naniel'. 1 1 P-iiydrosysreroid
deliydrogrnase types 1 and 2 (1 1 B-HSDI and 2). iii tlie rnaiiinialian endomerriuni. Tlis followitig sections present an 01-enriew of the literature. particularl>- concerning tlie synthesis and escretion of glucocorticoids. and factors affecting glucocorticoid actions in the reproductive system.
Naiiied for tlieir glucose-regulating propenies. glucocorticoids are ubiquitous as plil-siological regulators and seme man) functions that in fact do not involve carbohydrate nietabolisni. They include the nietabolisni of lipids. proteins and nucleic acids. GI~icocorticoidsalso play a very important role in immune system response (Clirousos 1 995). cardio1-ascular tone. fluid and electro 1yte balance. bone and calciwn dytiaiiiics. central nen-ous system activity. rou-tli. developnient and reproductive function (Miller and Tyrrell. 1995). Undoubtedl),. glucocorticoids are absolutely essential for life. However. an escess of tliese liorniones. especially over a prolonged period of tinie. can lead to severe consequences. The liarn~ful repercussions of clironically elevated cortisol levels in humans. for instance. is apparent in patients diagnosed witii Cushing's Syndrome (Orth 1995). Ne\-ertheless. glucocorticoids adniinistered at physiological doses can also have man- useful therapeutic effects. Syithetic gIucocorticoids are utilized clinically by obstetricians. for esample. in accelerating lung gron-th in the fetus expecting to deliver prematurely. This is dons in an effort to prei-ent the development and onset of respiratoc- distress syndrome in rlie premature neonate (Liggins. 1976). Numerous factors influence glucocorticoid actions. The hypothalamic-pituitary-adrenal (HPA) mis occupies a pivotal position in tlie regulation of adrenal cortisol production.
Tlir li!~potlialamus. pituitary and adrenais form a neuroetidocrine asis wliose principal function is to regulate the production of cortisol. The adrenal cortex consists of three histologically distinct zones. the outer zona glomerulosa. the intermediate zona fasciculata. and tlie inner zona reticdaris. Aldosterone. a mineralocorticoid- is produced in tlie zona glomerulosa. whereas the zona fasciculata and zona reticularis are responsible for the syiitliesis of cortisol and adrenal androgens. respectively (Neville and Mackay. 1972). Stcroidogenic tissues do not store appreciable amounts of steroid hormones. tlierefore the regulation of steroid hormone secretion occurs primari1~-at the level of steroid Iior~nonesy~tl~esis. Adrenocorticotropic hormone (ACTH) binds to ACTH recrptors on tlie surface of adrenal cortical ceils and activates a protein kiiiase A mediated patiiway theventually stimulates the synthesis and release of corrisol. ACTH is a 39 amino acid peptide tliat is proteolytically cleaved froni a larger 245 amino acid precursor molecule. preproopiomelanocortin (POMC). ACTH is syitliesized and secreted by corticotropes of the anterior pituitar) under the control of Iiypothalamic conicotropi~i- releasing hormone (CRH) and arginine vasopressin (AVP). Bot11 CRH and AVP are produced by panrocellular neurons of the paraventricular nucleus (PVN) and are secreted into the l-iypophyseal-portal system (Furutani ei of.. 1983). AVP is also produced by the supraoptic nucleus (SON) and magnocellular neurons of tlie PVN and is secreted via the posterior pituita-7 into the systen~ic circulation (Lk'itnall et CI/.. 1985). The basal activity of tlie HPA asis is influenced by regulatoq- ferdback loops. circadian rhythnls. and nunierous pliysical aiid/or emotional stresses. The HPA asis is a classic example of an endocrine feedback systeiii. u-ith severai regulatory negative feedback loops that control the output of glucocorticoids from [lie adrenals. The rnost salient of negative feedback loops (see Fig 2.1) are those eserted by glucocorticoids on CRI4 and ACTH secretion from the hypotlialan~us and anterior pituitaq-. respectively. Glucocorticoid feedback on ACTH production has been described as consisting of fast. intemiediate and slow components (Keller-Wood et cd., 1984). Slow feedback is characterized by decreased ACTH production. suppression of POhIC rrene transcription and decreased CMand AVP mRi'\iA and peptide content. Both fast C and intermediate feedback appears to be rnediated by inhibition of the release of esisting CRH and ACTH rather tlian by inhibition of actual syntliesis. Increasing concentrations of glucocorticoids accelerate the progression from fast and intermediate tèedback to sloiv
feedback ( Keller-Wood et rrl.. I98-I). The diurnal rliythrii of ACTH and cortisol release is affected by niiilierous factors. The intriilsic s!-nthesis and release of CRH by tlie 11:-potlialamiis. feeding and liglit/dark cycles. and tlie inlierent rhytlmiicit). of tlie adrenals. \i-liicli iiiay possibl~.be mediated by adrenai imenvation.appear to be notable factors intlueiicing the rliytlini of
ACTH and cortisol release in liumans (Miller and Ty-reIl. 1995). It is ive11 known that the riiytliilicity of botli ACTH and cortisoi release is cliaracterized by pulsatile secretions thar are more frequent and greater in amplitude in the early niorniiig Iiours. Tlis basal acti\-it>-of the HPA asis is also sensitive to nuineroiis stressful wents. Tlis stress response is comples and involves niany inteprated systems. The HPA ais is intimatel!. involved in the response to stress (Clirousos and Gold. 1992). Stressors. including severe trauma. illness. shock. hypo&~einia. heiiiorrhage. fei-er. deh>.dra~ion.ansiet>-. and fear (Miller and Tyrrell. 1995) have been shou-n to increase cortisol \-ia tlieir effects on the HPA axis. It appears that CRH is responsible for mediating the nietabolic. circulatory. and beliavioural adaptations rsquired in tliese deiuanding situations. The effects of CRH and glucocorticoids on tlie reproducti\ve asis. in response to stress. \vil1 be addressed in more detail in a later chapter. This section has briefly descrïbed the HPA neuroendocrine mis and its regulation by intrinsic rhphrnicity. nepative glucocorticoid feedback and stresses on the system. The nest section \vil1 outline the steps in the biosynthesis of glucocorticoids. paraventricula. nucleus (PVN)
neuro hypophysis @osterior pituitary)
, adrenal cortex
Figirre 2. i The Iiypotlialamic-pituitary-adrenal (HPA) asis. (-) Iiiliibi tory pathway. (+) stimulatory patliway. Conisol is syntliesized in the zona fasciculata of the adrenal gland in response to ACTH. ACTH rapidly facilitates the transport of cholesterol. the biolo~icalsubstrate for al1 steroidogeiiesis. across the adrenal mitochondrial membrane. In the mitchondria (see Fig 2.2). cholesteroi is then converted to pregnenolone via the cgoclirome P450 side chain cleavage enzyme (PljOssc) (Syben et cil.. 1979). This is tlie first and rate-limiting step in the sythesis of a11 steroid hormones (Sybert er rd.. 1979). Several niembers of the cytocliroiue P45O superfamily of oxidases are invol\-ed in the syntliesis of elucoconicoids. An excellent review by Hall (1986) describes cytoclirome P450 in t detail. CJ-tocluome P35Oc17. which is bound to tlie sn~ootliendoplasinic reticulum. catalyzes the 1 7u-hydrosylation of both pregnenolone and progesterone into 17u- 11)-dros!-pregiie1101oioiie and 17u-liydroxyprogesterone. respectively. Tlir rnicrosonial enzyiiie. ?P-liydrosysteroid dehydrogenase. on tlie otlier Iiand. catalyzes botli the 5p- Iiydrosysteroid deliydrogenation and isonierizatioii of the double bouiid froiii tlie B ring to the A ring of the steroids. pregnenolone and 17u-ii~~dros~~pregneiioloneto yield progestrroiie and 1 7u-liydrosyprogesterone. respectively. 17a-hydres>-progesteroiie. in turn. is Iiydrosylated at tlie C-2 1 position by cytochrome PJjOclI to produce 1 1 - deos~*cortisol.P45Ocl 1 P. like P45Oscc. is found in tlie inner mitochondrial nismbrane n-hsrr ir catnlyzes tlie Iiydrosylation of 11-deosycortisol at the C-1 1 position to produce tlie bioacti\-r: glucocorticoid. cortisol. Miller ( 1988) pro\.ides a good re\.ie\v of the molccular biolog!. of steroidogenesis. The systemic name for cortisol is 1 1 p. 17.2 1 - trih~-dros~.pregii-4-e1oiie-3.70-dione(Norman and Litwack. 1997). The producrioii rate of cortisol in liumans is approsimately 8-25 nig/day (New et nl.. 1969) u-liicli results in plasma coiicentrations ranging from 85 ng/ml in females and 1 16 nghl in males (Zunioff t i 197). Free or bioncrive cortisol levels are controlled by tliree niain factors: ( 1) adrenal s!-iitliesis (2) plasnia binding proteiiis. and (3) iuerabolisin and escretion. The nest section \vil1 address tlie pliysical state of steroids in plasnia. panicularly. their binding to plasma proteins. Pregnenolone . 17a-Hydroxy- pregnenolone
Progesterone 17a-Hydroxy- I I -0eoxycortisol Cortisol progesterone
Figzire 2.2 The syithesis of cortisol frorn clioiesterol (See test for detailed description). O cytoclironie P450 side cliaiii cleavage enzyme (P45Oscc) O 3 P-hydrosysteroid deliydrogenase (3 P-HSD) O cytocliroriie P450c 17 @ cytoclironir P45Oc2 1 O cytocliro~neP45Oc 1 1 p 2.1 2.3 Tite Edocrine Orchesrrn Plciyers - Po17 II: Bi~t~~ip~ociisin circzrkurio~t.
Cortisol is produced by the adrenal glands in free or bioacti~eforrn. However. approsiniately 90 to 97 percent of the circulating cortisol is bound to plasma proteins
(Siiteri cr ol.. 1982). Corticosteroid binding globulin (CBG). also referred to as transconiii. binds to conisol (90 percent of plasnia cortisol) specifkdly and \vit11 higli affinity (K,ratiging froni 13 to 30 nM) (Ogawa el rd.. 1983). CBG also binds to a number of otlier endogenous and synthetic steroids (Pugeat er dl..1 98 1 ). Howel-er. it has been suggested tliat @\.en the affinity of many of these steroids for CBG. the estent of tlieir bindiiig under ph?sioiogical conditions is nrgligible aiid alniost al1 steroid bound to CBG is cortisol. An esception is n-ith progesterone binding during tlie tliird trimester of pregiiacy. \\hich accounts for 25 percent of total CBG-bo~indsteroid. CBG is produced primari1'- in tlie liver. but it has also been found in otlier tissues. including tlie human uterus (Siiteri er al.. 1982). Human CBG is a 338-artlino acid glycoprotein \vit11 a inoIecu1ar u-eiglit of 58.000 (Siiteri ef al.. 1982: Hanimond er 01.. 1987). Plasma conccntriitioiis of CBG vary between individuals and are regulated bl- Iiorniones aiid otlier factors (Siiteri cr (11.. 1952). For instailce. CBG levels increase as a result of increased cstrogcn levels seen during pregnancy and \\-id1 estrogeii tlierapy and oral coiitracepti\.e use. Cortisol also binds to albumin. a plasma protein \vit11 lori-sr affinity than CBG but an almost 1000-fold greater capacity for conisol than that of CBG. CBG beconirs sriturated at higlier concentrations of conisol. and a larger proportion of plasnia- bound steroid tlien beconies associated witli albumin. Despite niucl~speculation and intense rescarcli. the prccise functioii of CBG and otlier steroid-biiiding proteiiis reniain obscure ( Rosiier. 1990). Glucocorticoids are esseiitially inactive \\-lien bound to CBG and albumin (Siiteri cr O/.. 1982). In addition. CBG-bound cortisol is also protected froni enzl-matic degradation froni metabolizing enzymes sucli as 1 1 P-HSD. Therefore. one tlieory is that tliese binding proteins senre an important resenoir of glucoconicoids in tlieir inacti\-e forni. thereby influencing the availability of tlie steroid to tissues. tlieir receptors. and steroid nietabolizing enzymes. CBG is similar to several niernbers of the serine protrasc inhibitor (SERPIN) superfamily (Haminond er id.. 1987). Tlir observation that CBG-cortisol cornplex is cleaved by neutrophil-derived elastase at sites of inflammation and the similarit' of CBG witli the SERPIN fainily Iias led to the speculatioii tliat CBG ma? facilitare the delivery of glucocorticoids to sites of infection and inflammation (Hammond et 01.. 1987; IWO). This is an interesting conjecture gi\-en the role of glucocorticoids in immune function. The delivery of cortisol to giucocorticoid and/or niineralocorticoid receptors in target tissues. however. remains to be one of the more important functions of plasma binding proteins.
Most know effects of glucocorticoids at the cellular level are mediatsd by a 91 kDa intracellulnr protein. tlie glucoconicoid receptor (GR). GR is a meniber of a phylogenetical1)- consenred superfamily of nuclear hormone receptor genes. These genss encode receptors for mineralocorticoids. androgens. progestins. estrogens. l-itamin D. tliyroid li«riiioiie. retinoic acid. and a growing nun~berof so-callrd orplian receptors for
whicli no spccific ligand Iias yet been identified (Evaiis 1988: blangelsdorf er CI/.. 1995). In tlie Iiormons-bound state. these nuclear receptors specifically bind to and niodulate the activit>- of target gene promoters and are. tlierefore. also hown as ligand-dependrnt transcription factors. Tlir Iiuinan GR (hGR) gene contains a total of 10 esons tliat undergoes alternati\-e splicing (see Fig 7.3) to give rise to two n1RVA4and protein isoforiiis. GRu and GRP. ahicli differ at tlieir carbos>.l termini (Hollenbsrg o ri/.. 1985: Encio and Detera-
Wadleigh. 199 1 ). The 1iGRu is the classic ligand-bindiiig. glucoco~~icoid-acti\.ared transcription factor \vliicli. in tlie hormone-bound state. has been suggested to modulate gene espression of glucocorticoid-responsive genes by binding to specitic DNA sequences. also holm as tlie glucocorticoid response elenlent (GRE). Ho~i*e\.er.\.ery little is knon-n about the hGRP splice and onl~.recentl!. lia1.e studies begun to enierge. Studies on the ubiquitousl~~espressed non-ligand binding p-isoforiii of IiGR appear to suggest tlinr tliis splicr functions as a domiiiaiit negative inliibitor of tlie classic IiGRu. wliicli nia). participate in defining the sensith-ity of target tissues to glucocorticoids
(Barnberger cJr td.. 1995: Oakley et 1997: Oakley er cil.. 1996). GRP mRN.4 and protein (Bamberger cr ul.. 1995) were show to be espressed in numerous hurnan tissues, sugpesting that this isoform may have a physiologically relevant role in glucoconicoid sensitivity. Tlie esistence of two distinct isoforms of GR that apparentl! exen opposite effects renders the stop of GR-mediated transcription more cornples. Tliese most recent studies- tlierefore. caution us and demand that we reevaluate pre\.ious studies tliat did not distinguish betu-een the tu-O isofonits. Tlie structure of GR is discussed in niore detail in the follon-in=section. Human GR gene
u H Ligand binding domain M N-ttnninal domain - (tra.nsactivation)-
Figta-e 73 The li~imanglucocorticoid receptor (GR) geiie and its alternative spliced Ceene products (Bainberger Cr crl.. 1996). Like other members of tlie nuclear hormone receptor superfaniily. the GR can be characterized by tliree hinctional domains (see Fig 2.3): the C-terminal or ligand binding domain. a central DNA-binding domain and the N-terminal activation domain that contains sequences responsible for activating target genes by interacting with components of the transcription niacliineq- or with other transcription factors (Hollenberg rr QI.. 1987: Giguère er cri.. 1986). Studies investigating the function of the ligand-binding domain of GR have suggested. in addition to its specific binding of hormonal ligands (Giguère et rd.. 1986). the C-terniinal of GR also contains sequences importait for hear shock protein
(l~sp)binding (Dalnian L'I cd.. 199 1 ). nuclear translocation (Picard and Yaniamoto. 1987)- dimerizarion and transactivation (Hollenberg and Evans. 1 988 ). Tu-O highl y consen-ed "zinc fingers" coiistitute tlie central DNA-bindiiig domain of GR (Giguère cr id.. 1986) wliicli participates in receptor dimerization. nuclear translocatioii (Picard and Yamamoto. 1987) and traiisacti\.ation (Hollenberg er cd.. 1987). Glucocorticoids are lipopliilic and tlius are able to difkise across the ce11 membrane to interact witli the ligand-binding domain of the iiitracellular GR.
The unliganded GR is part of a multiprotein comples that consists of the receptor and a series of Iisp. This GRlisp comples is believed to function in maintainine the GR in its inactive. !-et ligand-responsive state (Hutcliison er tr1..1993). The hormone bound GR undsrgoes a conformational change that results in the dissociation of tlie hsp coniples (Hutchison er d.. 1993). liyperphosphorylation (BodweII cr al.. 1998: Orti rr 01.. 1992) and translocation of the c~oplasniicligand bound GR rnolecule into the nucleus (Picard and Yanlaiiioto. 1987). Once in the nucleus. the lionnone-activated GR cal1 either interact directly witli specific GRE or otlier transcription factors (Sec Fig 2.4). Direct binding of the Iionnone- actil-ated GR to shon. palindroiiiically arranged GRE (table 2.1) in the promotrr of glucocorticoid responsive genes represents the classical mode1 of GR action (Beato Cr al.. 1995). Once bound to the GRE. tlie GR lioniodimer interacts \vit11 coniponents of the basic transcription machinery that enhance gene transcription eitlier by direct or indirect mechanisms. Many glucocorticoid actions are also achieved by inhibition of target grnes ratlier than activation. This is especially true of the effects of glucocorticoids on genes involved in anti-intlamatory and/or immunosuppresive response (h'onlirop el cd.. 1992). Suprisingly. promotors of these genes are still able to be repressed by glucocorticoids. altliougli tliey do not contain recognizable GREs. In tliese sceliarios. it has been wrll establislied tliat the ligand-activated GR functions to suppress transcription of tarzet genes by interacting u-ith other transcription factors. sucli as mi\-ating protein-l (AP-1) and NF-KB. ratiier tlian binding directly with GREs (Karin er tri.. 1993: Jonat er tri.. 1990:
Sclieinman CI cd.. t 995). GR agonists and antagonist lia\-e been utilized estensively in dinical medicine to nliniic or counter the effects of glucocorticoids in numerous pathologies. Figzrrc 2.4 Meclianisni of GR-inediated trailscriptional activation of target genes (Barnberger er al.. 1996). Tchlc 2.1 Hormone response elements (HE) for subgroups of the steroid hormone receptor supcrfamily. The consensus hormone responsive elenients are oriented 5' to 3'. and the noiispecific "gap" nucleotids is denoted by n. foIlowed by the second half-site. The glucocorticoid receptor (GR) is the prototype of subfmlity A, while the estrogen receptor (ER) is the prototype of subfamily B (Lowe er cd.. 1992).
Subgroup Consensus HRE
Subgroup .A GR PR
Subgroup B ER k4R-u.0 Glucocorticoid agonists such as cortisol. corticosterone. and the synthetic steroids used in sreroid therapy (prednisolone, dexamethasone. triamicinolone. etc.) are able to bind speci ficall y with the GR and thereby elicit glucocorticoid responses. The activity of these compouiids depends primarily on their plasma concentrations and affinity for GR (Lan et d..1984). Glucocorticoids have long been hown for tlieir bencficial rffects oii acceieratiiig hiig niaturation in the preterni fetus. and tli~iss!-ntheric gI~~cocorticoidssuc11 as desan~ethasoneand betametathasone ha\-e been used ai-ittinatdly in preventing the onset of respiratory distress sydrome (RDS) in preternl neonates (Liggins 1976). Antagonist steroids. such as Mifepristone (RU-486). bind to the GR witli high affinity and essentially blocks agonist response by competing for GR (Baulieu. 1991: Spitz and Barden. 1993). Bindinp of RU-486 to the GR induces a conformational cliange which is distiiict froiii tliat induced by an agonist (Barnberger and Clirousos. 1995). Tlic RU 1861'GR coiiiples is stili able to dissociate from lisp and biiid to the GRE of a target gene. ).et it faiis to interact properl). wih the basal transcription iiiachinery (Mao cr al.. C 1992). S~ibstit~itioiisat C-1 I and around the D-ring of the glucocorticoid niolecule. such as alteratioils or deletions of the C-17 side chain and C-21 substitutions. increase the affinity of steroids for GR. RU 486. for instance. is cliarncterized by a (p- dinietli~-lniiiiiio)plirii>-lsubstit~ition at C-l l n-hicli greatly increasss its aftiiiit>- for GR (Ba~ilieu.199 1 : Spitz and Bardcn. 1993). Clinically. RF456 lias btisii ~issdsucccssf~illj- in treatiiiy patients diagnosed \vit11 Cusliiiig's s?iidronis (Siriiiaii d.. 1985). Glucoco~~icoidaiitagonists lia\-e proven useful not only in the cliiiical setting but also in lielping us in understanding the structures of botli ligands and the ligand-biiiding doniailis of GR. The niagnitude of a cell's response to glucoconicoids depends botli on the horiiione Ictwls it is esposed to and its ability to respond to glucoconicoids via GR. Factors affcctiiig glucocorticoid action. such as plasi~iabindiiig proteiiis and GR iiiediatsd glucocorticoid rsspoiise. lia\.e beeii reviewed. 111 tlie nest section. enzyme merabolisni and escrctioii u-il1 be addressed as a means of controlliiig the le\-els of bioactive ducocorticoids bot11 systemically and at the local level. C 2-1-23Ti~c E17doci-ine OI-chesira - The PZaye1-i - Parr IV: Evci-eriort m7d .\icraboiism of Giricoco~~ricoids
The liver and kidney are tlie major sites of glucoconicoid ~iietabolisni(Bradlow cr oi.. 1982). Glucocorticoids uodergo enzyniaric modifications that transforn theni into inactive u-ater soliible nietabolites which are more readil>-escreted by the kidneys. The microsoinal enzyme. 1 1p-liydroxysteroid dehydrogenase ( 1 1P-HSD) catalyzes tlie oxidation reaction of the bioactive form of glucocorticoids. cortisol [in humans (Tannin er oz.. 199 1 ). sheep (Yang er rd.. 1992). rabbit (Naray-Fejes-Toth and Fejes-Totli. 1995). etc.] and corticosterone [in tlie rat (Aganval er 02.. 1989) and mouse (Rajan er cd.. 1995)] to tlieir inacti1-e 1 1-keto nietabolites. cortisone and 1 1 -deos~~corticostero~~e~respectkely. Glucocorticoids are iiiodified b>- se\.eral different enqnies. Fig 2.5 outlines tlie major reactions in the metabolism of glucocorticoids.. The initial inactilxtion step in glucocorticoid nietabolisni is an irreversible reduction of the 3.5 double bound of both cortisol and cortisone. whicli yields 5a- and jp-dili)-drocortisol and diliydrocortisoiie. respectively. Dili>-droconisol sa- and 5B-isoiuers are rapidlj- comerted by 3P- h!-droq.steroid dsli>-drogenase(5 P-HSD) to al lotetrali~~drocorrisoland tetraliydrocortisol. Dihydrocorrisoiir undergoes the same reaction to yield tetrali>-drocortisoiis (\i'alker aiid
Edwards. 199 1 ). The tetrali>-dro derivatives of cortisol and cortisone i~ietabolisnican undergo further osidation at C-21 to produce cortolic and cortoloiiic acid. respectively. w1iicl.i reiiders tliese steroids el-en more \vater soluble (Monder alid Bradlow. 1980). The iiietabolism of glucocorticoids by the microsomal enzyme. 1 I P-HSD is a significanr nieans of regulating the intracellular concentrations of bioactive glucocorticoids. NADP NADPH
NADP NADPH Cortisol (F) . - Cortisone (E)
5B-dihydrocortisol Sa-dihydrocortisol dihydrocortisone
tetrahydrocortiso 1 allo-tetrahydrocortiso 1 tetrahydrocortisone (THF) (allo-THF) (THE)
Cortolic acid Cortolonic acid
Figiwc 2.5 Priiicipal uriiiar). metabolites of cortisol and cortisone (Adapted froiii Walker and Edwards. 199 1). The metaboIism of glucocorticoids in target ceils determines their effective intracehlar concentrations. accessibility to GR. and their ability to influence gene transcript ion. and thus ce11 funcrion. 1 1P-Hydroxysteroid deliydrogenase ( 1 1 P-HSD) catal y zes tlie interconversion of pliysiologically active glucocorticoids (cortisol and corticostsroiie) to their iiiactive metabolites (cortisone and 1 1 -deliydrocorticosterone). by sliuttling the liydrosyl group at the C-Il position of tlie steroid niolecule to a krto group (See figure 2.5). By mediating this conversion. Il P-HSD is capable of controlling intracellular levels of bioactive glucocorticoids in target tissues ( h4onder and White. 1993). The cofactor. NAD(P) (nicotinarnide adenine dinucleotide). is utilized by I lp- HSD in converting cortisol to cortisone (deliydrogenase activity). u-liereas tlie con\-erse cataIysis. 1 1 -osoreductase actility. employs NAD(P)H. Binding of rlie h'XD(P)(H) cofactor takes place in a ~vell-conserved motif in the N-termiiiai domain amongst members of the short cliain dehydrogenase/reductase (SDR) tàmily of proteins. of whicli 11 P-HSD is a iiien~ber(Krozowsiii, 1992). Tlie short chain dehydrogenasdreductase famil y (SDR: fomierl y short-chain alcohol dehydrogenase) is a distinct famiIy of proteins. whose 57 nienibers. aside from
1 1P-HSD. also include 3P-HSDketosteroid isomerase (3P-HSDKSI j. 7u-HSD. 1 7P-
HSD. and I 7-11)-drosyprostaglandin dehydrogenase ( 1 7-OH-PGDH) (Krozoli-ski 1 994:
Krozou-ski 1992: JornvaII er cd.. 1995). Despite relatively low Iioniology. SDR famil!. menibers sliare identical protein folds (Jomvall et al.. 1995) that include arrangements of a-helix and P-strands tliat brinp forth a fold rudirnentary in cofactor binding. In addition. thel* al1 sliare a consen-ed consensus sequence Tyr-X-X-(Ser)-Lys . which Iias been implicated b>- site-directed mutagenesis to play a role in catall-sis (Obeid and White.
1992: Ensor and Tai. 1991). For instance. \\-lien both Tyr and Lys in tliis catalj-tic motif were niutated in 1 1 P-HSD type 1. one of tn-O 1 1 P-HSD isozyrnes. enq-me acti\.it>-was abolislied (Obsid and White. 1992). The active site of SDR proteins is aIso ivelt consen-ed. and is respoiisible for the transfer of a liydride froni the stsroid substrate to the C-4 position of the nicotinaiiiide ring of the cofactor (Krozo~i-ski.1992: Fislier 1953). The discovery of the two distinct 11P-HSD isozymes Iias evolved fiom nutiierous studies ranging from a11 in qui^>. into receptor meclianisnis to the clinical status of patients \vitli alterrd yiucoconicoid response. Currently. intense in\-estigatioiis are purs~~ing~ila~iy facets of 1 1 p-HSD rrgiilation. enzyniology and related bioclieiiiistry.
Glucocorticoids have been distinguished functionally for more tlian fifiy SVears (Selye 1 946). Altliough. the anal ysis of glucocorticoid metabolites in urine alluded to the existence of a glucoconicoid metabolizing enzyme. and the presence of an 1 ID-HSD eiizl-me acti~.it~-\vas estnblislied in the 1950s. its full biological role and impact Iias only beconie more apparent in recent years. Tlie following 20 to 30 years witnessed tlie evolution of riiolecular techniques and also the growth of infomiation on 11 P-HSD. But it \vas not until Arriza and colleagues (1987) cloned tlie Iiunian mineralocorticoid (type 1) receptor (MR) tliat an unespected challenge sparked a renen-ed curiosity into the enigmatic 1 1 P-HSD. The mystery that unfolded before Arriza and others \vas the obsenatioii tl-iat the human MR was found to bind aldosterone, corticosterone. and cortisol u-itli equûl affinities. At the time, it was not certain hou- aldosterone could rssrt its tissue specitïc effects wlien the circulating levels of cortisol were an>.where fiom 100 to IO00 tiiues greater (Seckl. 1993). This apparent obstacle n-as overconie folIo~ving studies of a rare ~netabolicdisorder involving 1 1 P-HSD. Sodium retention. severe hypertension and hypokalemia are serious clinical signs of patients diagnosed with a rare autosomal recessive disease. apparent mineralocorticoid excess (AME). in \\-hich cortisol acts as a potent mineralocorticoid in the kidne?. (UIick cr IL. 19Iiitrrestiii&-. liquorice ingestion also manifestrd synptonis of sodium retention and Iiypene~~sio~i.tlie sanie syniptoi-iis obserwd in patients diapiiosrd u-itli AME. This \vas on-iiig to the poteiit properties in liquorice. tiaiiirly glycyrrliizic acid and its metabolite. glyc>-rrlietinicacid (GE). whicli have bern fouiid to iiihibit renal I 1 P-HSD actil-it?.(Ste\\-art et d.. 1987). It \vas subsequently establislied tint the espression of 1 1 P- HSD in the kidnel- u-as responsible for conferring aldosterone specificity on tlie non- specific relia1 AIR (Funder ct rrl.. 1988: Edivards et al.. 1988). Functioiial data provide e\.idsiice foi I 1 P-HSD acti~.it).in the rend taqet cells for aldostrro~is(Kara!--Fejes-Totli cl cil.. 199 1 ). Iiowe\-er. die protein and niRNA correspondi~igto 111scloncd Iiepa~icforiii of 1 1 P-HSD (Lakslimi and Monder. 1988) appeared to be absent tiom thé distal nephron. wliere iiiinernlocorticoids esert tlieir most important fuiictions (Rundle ëi d..1989). These cliiiiacteric friidings led tlie way to the disco\.erl. of a distinct rem1 II (3-HSD. or tlie type 1 1 1 P-HSD (M~inecr cd.. 1995: Obeyesekere er cri.. 1995: Sreu-an er cil.. 1996). More rsceiitl!-. mutations in the liuman 1 1 P-HSDI gene have bern ideiititied in patients diagnosed \i-itli .Ah.lE (Mune cr cil.. 1995; Stewart er cil.. 1996). To date. t\\-o isoz>.ilies of I1P-HSD. tl-pes 1 and 2. Iiave been cloned and cliaracterized in iiumeroiis species. including Ii~~niaiis(ses Table 3.3). TIie nest section \\.il1 address the structure. tissue distribution. function- reguiation and patiiogenesis of 1 1j3-HSD types 1 and II as a preamble to studies completed on riie regulatioii aiid speculati\.e function of 1 1 P-HSD in the uterus. Tobk 2.7 Characteristics of human 1 1p-hydrosysteroid deliydrogenase type 1 and II ( 1 1P-HSD I and 2) (Yang. 1 997).
Characteristics 1 1p-HSD 1 1 1P-HSDî
S ize 34 kDa 40 kDa Primary structure 292 anlino acids 405 anlino acids Co-factor prekrence NADP(H) K.AD
Direction Cortisol +-+ Cortisone Cortisol -. Cortisone Affinity for cortisoI Low (y,,= 2- 10 pniol-') High (K,, = 10-20 nmol") Dexametliasone n~etaboIism No Yes Sites of espression Widespread Tissue-speci fic Gene stnicture G esons (spans >9 kb) 5 esons (spans 6 kb) Gens locus Clvomosome 1 Cliromosome 16 Mutations in AME ' No Yes 2.2 1 1P-HSD ISOFORMS.
The u-ork of
1995: Rajan cr ri/.. 1995). sheep (Yang er ul.. 1992). human (Tannin er cd.. 199 1 ). baboon
(Davies CI d.. 1997) and in the monkey (Moore et nl.. 1 993). Hunian type I 1 1P-HSD cDNA contains an open reading frame (ORF) of 876 bp whicli encodes a 34 kDa protein consistiiig of 292 aiiiino acids. 1 1 P-HSD I cDNA-deduced protein sequencc anal>+sis reveals tlirit tiiis isoq-nie is \wll conserved ainongst species ( Wiite cr ol.. 991 1 p- HSDI. in essence. is representative of SDR proteins in that it possesses selreral ~11 consen-ed coiiseiisus sites. including those for cofactor biiiding (Krozo\\mki. 1994). cataiytic fiinctioii (Krozon-ski. 1994: Monder. 1993: Obeid and White. 1992) and S- rrlycos~-lation(Krozo\di. 1994; Ozols. 1995). C Tu-O variants of 1 1 B-HSD I mRNA encoding two distinct proteins lia\-e been identified. ho\i-ever in vitro studies suggest tliat tliess protein variants lack eiizynie actii-ity (Krozowski Cr LI/.. 1992: Yang et a/.. 1995: Mercer c.1 il.. 1993 1. It lias been suggested tliat the expression of a kidney variant of 1 1 P-HSD 1 nia!- be a iiieaiis of do\\-n- regulating the Itxel of 1 I P-HSD1 enzyme activity. Han-ever. the uiiequii-ocal lack of enqrrne actir-ity in these spliced variants of 1 1P-HSD 1 has niade it di%cult to suggest a definitive biological function. At present, the role of these proteins remains unknown. In liuinans. the 11P-HSDI gene is found on chromosonie 1 and consists of 6 esons spaniiiiig o\.er 9 kb (Tannin et cd. 1991). Fig 7.6 illustrates the geiie for Iiuman 11 P-HSDI and its niRNA and protein product. 11P-HSDI is n-idely espressed in olucocorticoid target tissues. bon-ever 1 1P-HSD I niRNA aiid protein are localizrd C predoniiilantly in the lii-zr. Gene 5' 3' 9.0 kb
mRNA 5' AAA - 3' 1.5 kb
Figure 2.6 Human type 1 1 1P-hydroxysteroid drliydropenase ( l 1 P-HSD 1) gene. inRNA and protein (Pcnning. 1997). 1 1 P-HSDI may be an important determinant of local giucocorticoid action within target tissues by regulatinp the access of glucocorticoids to tlieir intracellular receptor.
Type 1 11P-HSD gene has been identified in glucocorticoid target tissues of sheep. human. rat. and niouse (Yang er ai.. 1992: Tannin sr al.. 1991 : Rajan er cil.. 1995: Whonvood er tri.. 1991). In addition to several braiii structures. including the hypotliala~~iusand pituitary (Yang et al.. 1992; Stewart et al.. 1995: W'homood et al.. 1992). 1 I P-HSD 1 mRNA lias also been detected in several peripheral organs. suc11 as the
liver. lung. kidney. spleen and colon (Yang er cil.. 1992: Tannin er LI/.. 199 1 : Rajan er ol..
1995: Agan\.al c.r cd.. 1989: Roland et al.. 1995: Stewart er cd.. 1995: Wioru-ood cl cd.. 199'7). Moreoi-er. adipose. skin and muscle express 1 I P-HSD 1 (Yang er trl.. 1997: Hanirnanii and Siiteri. 1991: Takeda et al.. 1994). The mkN'A. protein and!or enq-nie actii-ity rspreseiitatii-s of type 1 1 IP-HSD have also been identified in severaf reproduciii-r orgaiis. iiicluding ovary. testis. endoiuetriuiii and III>-ometrium (Tannin er al.. 199 1 : Albistoii er crl.. 1995: Burton et cf/.. 1998: Aganval el tri.. 1989: Sniith er ol.. 1997: Arc~iricr d..1996: Yang er of.. 1996: Bunon rr of.. 1996). Durin2 pregnancy. 11P-HSDI gene sspression has been detected in the chorion. decidua. ni!~oi~ietriuii~.
placenta ( '\.lurpli>-.198 1 : Lopez-Benial and Craft. 198 1 : Sun cr (11.. 1 997: Burton er d.. 1996: i!'addsll er cil.. 1998: Pepe et al.. 1996: Yang. 1995: Strn-art er 1995) and
se\-eral feral organs (Steivart cr al.. 1995: Yang er O/.. 1992). The doniinant site of espressio~iof 1 1 P-HSDl is. 1toivei.er. in the lii-er. (Whorivood tri.. 1995: Tannin er tri..
199 1: Steiixt cr crf.. 1995: Roland el d.. 1995). 1 1 P-HSD 1 gene expression is de~*elopiiieiitallyregulated (Yang et al., 1992; Yang et al.. 1997b) and can be intluenced by a nuinber of liormoiies. which will be addressed in more detail later in this tliesis (Section 2.2.1 -4 Regulation of 1 1P-HSD 1). St~idirsthat have characterized the CO-expressionof 1 1 P-HSDI and GR in various tissues have suggested that this isozyme may function to modulate the access of glucoconicoids to GR (Wlionvood er d..1992: WaddeIl L'I LI/..1998). 1 IP-HSD1 is a microsomal enzyme with a low affiiiity for endogenous glucocorticoids (Y,in tlie pM range), shares less tlian 20% homolog) ~vith1 I P-HSDL and uses NADP(H) as a cosubstrate (Yang and Yu. 1994). 1 I P-HSD type 1 is reversible in tliat it possesses botli deliydrogenase (cortisol to cortisone) and reductase (cortisone to cortisol) activities (Agarwal et al.. 1989). Aganval and colleag~ies(1989) deiiionstratrd bidirectioiial activity in Cliinese hamster ovary cells transfected u-itli tlie 11P-HSDI cDNA. but traiisfection of this same cDNA into intact mamnialian COS-7 cells encoded an alrnost ssclusiïe reductase activity (Low cr al.. 1994). Numerous studies sugpest tliat
reductase actil-it>-is predoniinant iit i1ii.o (Murphy. 1980: Yang er rd.. 1997: Apanval er cd.. 1990). The regeneration of bioactive glucocorticoids from tlieir iiiacti\-e 1 1-keto steroid iiistabolites by 1 IP-HSDI could poteiitially iiicreasr sffectiw intracellular clucocorticoid conceiitratioi~sand thereby augment gliicocorticoid response in target C
tissues. To esplore tliis notion funher. Koteleme~and colleag~ies( i 997) generated mice bearing targeted disniption of tlie 11P-HSDl gene ("knockout") and espiicitly demonstratcd Iiow the cornpensatoc. effects of glucocorticoid function were significantly
altered il7 iYn). Apan from compensatory adrenal hyperpiasia and elevated sccrerion of adrenal corticosreroiir. nuIl inice had severely conipromised Iiepatic glucorieogsnesis. aiid n-hsn provoked by stress or obesity. 1 IP-HSD1 -/- rnics u-ere able to resist liypergl!-ceni ia. This study also raised the idea that cortiso~ie and 11- deh!.drocorticosterone may function as "prohorniones". siiice plasnia le\-rls of tliese 1 1- keto steroids n-ould not be subjected to the wide diurnal tluctuations and CBG sequestratioii like tlieir bioactive counterparts. It has also been suggested that these 1 1 - keto metabolites could in essence possess higher tissue specificity because of tlieir ability to act on tissiiss espressing only 1 1 P-HSDI (Kotelemev et cl/.. 1997). Taken togetlirr. tliese obsrr\-otioiis provide funlier support for the Iiypothesis tliat 1 1 P-HSDl fuiictioiis to modulate glucocorticoid access to its intracellufar receptor (Whor\vood cr d..1992). The physiological role of 1 1 P-HSD 1 has been espanded even furtlier witli studies Iooking at the regulation of tliis enzyme. In an elegant study by Escher and colleagues (1997). the potent proinflanimatoq cytokines. IL-1 P and TNFa. stimulated 1 1P-HSD 1 expression and reductase activity in cultured rat gloiiier~ilarmesangial cells (GMC). The elevated corticosteroids resulting from tliis cytokine-induced 1 1 P-HSD 1 reductase acti~itywere found to suppress the expression of group II pliopholipase-2 (PLA,). which has been bon-n to produce a potent anti-inflamniatory response (Vishxanath et 02.. 1993). The synthetic glucocorticoid. desametliasone (Des).stimulated the expression of 1 1P-HSD 1 in tlie lker of chronically- catheterizsd kta1 shesp (Yang el d.1994). 2S FAZA kepatoma celIs (Voice ci [il..
1996). rat liepatocytes (Liu Cr LI/.. 1996). and in cult~ired liun~aii skin fibroblasts (Hainiiiaiiii aiid Siiteri. 199 1). It lias been suggested that tlie Des-induced increase in 1 1 - osoreductase actil-ity obsen-ed in culrured Iiunian skin fibroblasts \\-as likely to liave been mediated tlirougli the GR. since the GR antagonist RU486 diiiiinished tlie effects observed \vit11 tliis glucocorticoid (Hammami and Siiteri. 199 1). III coiitrast to the stimulatory effects obsened with Des. th?-roid hormone. gronth horiiione (GH).and iiisulin al1 sliowed significant decreases in 1 1 f3-HSD1 espression in w-ious studirs. For instance. in an in i*ii*ostudy by Wliorwood and colleag~ies(1993). the enzyiis activity and inRNA for 1 1 P-HSDl were signiticantl!- rsduced in tlie liver and pituitary of rats treated n-ith thq-oid homione (T:) for 3 and 7 days. In motlier study. botli ii~suliii and GH decreased 1 1 P-HSDl enzyme acti~ityin a dose- and tinis- dependerit fasliion in primary culture of rat hepatocytes (Liu er CI/.. 1996). III both 2s FAZA cells and liunian ski11 fibroblasts. insulin \vas also siioun to inhibit 1 1 P-HSD I enzyme activity in a dose-dependent manner (Voice c.1 (il.. 1996: Hain~i~aniiand Siiteri.
199 1). Of interest. in the study by Voice and colleagues ( 1996). transkction of ZS FAZA cetls \vith a Iucifsrase reporter gene driven by tlie prosiriial pro~iioterof 1 1 P-HSD 1 rwealed that both Des and insulin were fiinctioning through sites within 1800 bases of 5' flanking DNA of 1 1 P-HSDl gene. Tlis ses steroids. testosterone (T). estradiol (E2) and progesterone (P4) are potent regulators of 1 1 P-HSD 1. Ili the testis of adrenalectomized rats. for instance. 1 1 P-HSD 1 dehydrogenase activity \:as potently inhibited by both T and E2. wliereas P4 stimulated corticosterone co~iversionto I 1-del~ydrocorticosterone(Nwe ci ol.. 1997). In anothrr study by Low and colleagues (1993). hepatic 1 1P-HSDI mRNA \vas significaiitly inhibited in gonadectomized male and female rats treated with E2. In primary culture of human endometrial stroma1 celIs. Arcuri and CO-n-orkers(1996) found that E2 failed to affect stroiiial cell-associated type 1 11P-HSD activitj-. In contrast. the same study discovered tliat inedrosyprogesterone acetate (MPA). a synthetic progestin. stimulated IlP-HSDl activity. and n-hen E3 and MPA were CO-incubated. type 1 IlP-HSD activity increased to a iiieari of 8-foId. compared with \.ehicle treated coi~troIs.In a separate study by Gao and associates (1997). T decreased 1lP-HSDl osidatil-e activiry in cultured Leydig cells taken fioni intact and adrenalectoniized rats (ADX). Interestingly. tliis saine study repol-tsd a significant increase in 11 B-HSD 1 mRNA and reductive enzye acti~ity in Leydig cells from both ADX and intact animals following treatiiient u-ith epidemial gro~tlifactor (EGF). wliereas osidative activity \vas significantl>.decreased. Leydig ce11 L 1B-HSDI iiiRX'X. and osidati\.e activities were also regulated separatel!. by LH and
Des (Gao CI ul.. 1997). Sswral st~idieshave deinonsrratrd tlie potent i111iibitor)- rfkcts of carbeiiosoloiie
(CBX). licorics and its actix-e metabolite. glyyrrhetinic acid. 011 botli I 1 P-HSD 1 n1RKA and enzyiiir actixitl- (Wlionvood er ol.. 1993: Latif er ol.. 1991). The regulation of 1 1 P- HSD 1 pieexpression has also been shown to be de\-eiopnientally regulated in botli fetal tissues and placenta (Yang 1992: Langlois et al.. 1995: Yang et trl.. 1997b: Pepe el rd.. 1996).
Despire intense researcli in the area of reproductive biotechnology. pregnancy rates of only 30% or less are achieved among infertile lvomen receiving in vitro fsrtilizatioii and enibryo transfer (IVF-ET) (Michael et al.. 1993a). In tliese patients. a relationsliip between tlie outconle of IVF-ET and I 1p-HSD 1 -1urdiated cortisol to cortisone coin-ersion in cultured human granulosa-lutein cslls has been rstablislisd (Michael cr crl.. 1993b). In addition to the identification of tlie mRNA encoding IIP- HSD l in o\.ariaii tissue (Tannin er cri.. 199 1). I I P-HSD l dehydrogenase activity has also been observed in ovary tissue honiogenates (Murphy. 198 1 : Benediktsson er tri.. 1992). 1 1 P-HSD 1 activity in cultured human granulosa cells has been suggested to play a pan in regulating the sensitivity of these cells to the direct inhibiton. effects of cortisol on ovarian steroid production (Michael et al.. 1993b). Specifically. cortisol has been slio\i-II to directl!- inliibit LH-stimulated steroid synthesis in cultured Iiuman granulosa lutrin cells and 1 1P-HSDI acts in tliese cells to decrease tlie potency of cortisol. It has been suggested tliat in the absence of 1 I B-HSD 1. cortisol is able to esert positive effects in the first half of [lie cycle on oocyte maturation and subsequent implantation. whereas in the lutral pliase. raised 1 1 P-HSD 1 activity "protects" owrian steroidogenesis from the inhibitory effects of cortisol. The rate of total fenilization failure \vas grearer anioiig patients \\-itli 1 1 p-HSD 1 -positive cells. It Iias been suggested th1 1 /3-HSD 1 actil-itl. iuûy prrdicr the outcoiiic: of IVF-ET in infertile wonieii and nia! tlierefore bs used as a bioclieniical paraiiieter of O\-ariari function and could have a clinical prognostic application (Michael et d..1993a). 1 I P-HSD I Iias also been iniplicated in a clinical condirioii of Iiypercortisolisiii. in tlie absence of Cusliing's syndrome-like syniptonis. In this nietabolic disorder. apparent E (cortisoiis) reductase deficieiicy (AERD). patients are uiiable to convert cortisone to cortisol ( Pl~illipo~tcr II/.. 1 996 ). These patieiits. rlierefore. are rssciitially gl~~cocorticoid resistaiit. It is believed tliat AERD is due to a defecr in the 11 P-HSDI protein (Pliillipou er cd.. 1996). The type II 1 1 P-HSD was found in kidney and placenta microson~esof sheep and humans. cDNAs encoding the kidney isozyme have been isolated from slieep and human kidney cDNA libraries (Albiston et rd., 1994: Aganval er cd.. 1994). 1 1 P-HSDî lias since been cloned in the rat (Zhou et of.. 1995), mouse (Cole. 1992) and rabbit (Nara>--Fejes- Toth and Fejes-Totli. 1995). The human type II 11P-HSD cDNA contains an open reading fraine (ORF) of 1215 bp wliich encodes a 40 kDa protein consisting of 104 amino acids (Aganval CI cd.. 1995). The 5' flanking region of tlie 11 B-HSDZ gene is notably GC-ricli and lacks a TATA box (Aganval rr nl.. 1995). In an in\-estigatioii by Agani-al and White (199G). the 5' flanking region of 1 1 P-HSD2 \vas analyed by utilizing luciferase reporter constructs transfected into JEG-3 hunian choriocarcinoma cells. In this study. two segiiients. protected by DNase 1 footprinting analysis. revealed binding sites for the Spl transcription factor. It lias been suggested tliat tliese Spl sites are essential for transcription of 1 1B-HSDZ gene in JEG-3 cells (Aganval et cd.. 1996). Tlie predicted aiiiino acid sequence of 11 P-HSDK or type II isozye is only 2 1% identical to the predictrd sequence of the Iiuman livcr (Type 1) isozyriie of 1 1P-HSD (Aganid cr ~1..1995). wliereas the 1 1f3-HSD2 aiid type II (placental. NAD--dependent. microsonial ) isozynie of 17P-HSD sliare 35% sequence idrntity (Albistoii er rd.. 1994:
Agamal cr d..1995). Tlie relatively high degree of siniilarity ber\\-eeii 1 1 P-HSD2 and the placeiital 17P-HSD suggests tliat these two enzynies niay br in the sanie pie faniil!. within tlie SDR superfamily. T\\u strongly consen-ed regions are sliared arnong SDR menibers. One region coiistitute part of the binding site for cofactor and tlie otlier site contains abso lutel!- conserved Tyr and Lys residues that function in catal ysis. Both of these regioiis are well conser~vedin the two isozymes of 1 1 P-HSD (Aganml er cri.. 1993). In a study by Krozowski and colleagues (1 995). fluorescence in situ hybridization and an 1 ID-HSD7 cDNA isolated from Iiuman kidney were used to map the gene encoding hunian 1 1 P-HSD? to the long m of chromosome 16 at band 16q22. The 1 1 P- HSDl gene consists of 5 esons spanning appros. 6.2 kb (Aganval er d.. 1993). The predicted peptide sequence for human 11P-HSDî is 83% identical to the predicted sequence of the ovine 110-HSD2 protein (Aganuil et ol.. 1994). Fig 1.7 illusrrates tlie eene for liiinian 1 1 P-HSDI and its mRNA and protein product. C 1 ID-HSD2 is more tissue specific in its espression rlian 1 I P-HSD 1. and is localized in aldosterone target tissues where it is beliwed to function to protect tlie rnineralocorticoid receptor (MR) from occupancy by glucocorticoids. Gene 6.0 kl,
mRNA 5' AAA - 3' 2.0 kb
Protein
Figzm 2.7 Huiiian type 2 1 1P-hydroxysteroid dehydrogeenase ( l 1P-HSD3) gene. mRNA and protein (Penning. 1997). T>-peII II P-HSD mRNA and/or protein have been identified predominantly in the placenta. and mineralocorticoid target tissues. such as cortical distal convoluted tubules and collecting ducts in the kidney, distal colon and parotid gland (Albiston et ol..
1994: Aganval ef QI.- 1995: Whonvood et ni.. 1994: Stewart et crl.. 1995b: kozowski er cri.. 1995: Cainpbell el crl.. 1996: Burton et cri.. 1996: Brown CI CI/.. 1996: Roland and Funder. 1996). The expression of the type II isoqme of 11P-HSD lias also been discovered in the adrenal (Mauocchi et al.. 1998: Yang and ~~~~~~~~~S. 1995: Campbell et (il-. 1996: Roland and Funder. 1996; Zhou et (il-, 1995)- and in nunierous human fera1 orgaiis (Stw-art er cd.. 1994). III a few species. lung. ovary. testes. paiicreas and brain have beeii identified to possess lou levels of 11P-HSD2 espression (Zhou er cd.. 1995:
Albiston cr rrl,. 1994: Li et CI/..1996). 1 1 P-HSDZ lias been idetitiiïed in tlie endometrium of rat and Iiuinaii (Roland and Funder, 1996: Smith et crl.. 1996: Burton el d..1998). Like 1 1P-HSD 1. tlie type II isozyme of 1 I P-HSD \vas found to be de~elopnientally regulated ( Langlois cf al.. 1995).
111 suiiiniary. tlie studies outlined above indicate rliat IlP-HSD:! is espressed predominantl! in mineralocorticoid target tissues and placenta. It has been proposed tliat tliis pattern of espression for 1 1P-HSDZ may ser1.e two very important fiinctions: ( 1) in aldosterone target tissues. 1lP-HSD3 rnay serve to protecc non-sslective mineralocoiticoid receptors froili occupancy by glucocorticoids. and (2)in the placenta. the inacti\-ation of cortisol mas safe-guard tlie fetus froni the liigh circutating leveis of materna1 glucocorticoids (Li e/ oz.. 1996).
In a sti~dyby Naray-Fejes-Toth and Fejes-Toth ( 1996). a chinieric gene construct encoding the full lengtli of rabbit 1 IP-HSDZ fused to the codinp sequence of green fluorescent protein (1 1P-HSDZ/GFP) was stably transfected into CHO cells to determine the subcellular Iocalization of 1 1 P-HSDZ. This investigation revealed that the type II isoforni of 1 ID-HSD was localized exclusively to tlie endoplasmic reticulum (ER). Although sirnilar ro 1 I P-HSDI in cellular localization. the enq-matic characteristics of 1 I P-HSDZ differ in directionality. substrate affinity and cofactor-dependency (Aganval er al.. 1995) The enqniatic characteristics of the espressed 1 1P-HSD2IGFP fusion protein. for instance. n-ere indistinguishable from those of the native enz>-nie: high affinity for cortisol and corticosterone (nanomoiar ) NAD-dependence and esclusiw deliydrogenase acti~ityunder pliysiological conditions (Naray-Fejes-Toth and Fejes-
Toth: 1996). An investigation by Obeyesekere and CO-workers( 1997) recently found that wlien assays n-ere perfornied in fihole cells and liomogenatrs of Cliinese hamster owry celis (CHOP) transfectsd n-irli 1 I P-HSDL there ti-as. in addition to the pre\.iously reponed dcli!-drogenase activity. an avid conversion of 11-deli!-dsodesamr~liasoiie to desamethasone. This has been suggested to be an esplanation for the potency of the synthetic glucocorticoids in the face of a powerful inactivator of natural glucocorticoids
(Obeyesckere ci rd.. 1997: Li er al., 1997). Hoivever. under normal physiological conditions. 1 1 P-HSD7 functions as an esclusi~e. unidirectional dehydrogenase
(Obeyeseksre Cr ci/.. 1997).
Iii addition to the critical rols I 1P-HSDZ p1aj.s in iiiodulatiiig MR occupaiici.- bi-- olucocorticoids in aldosterone target tissues such as the kidne~-(Funder. 1994: Stcivart el C (il.. 1996). tliere is niounting evidence to suggest tliat 1 1 P-HSD2 ma)- be essential in reproduction (Siiiitli et cf/.. 1997: Arcuri ar cri.. lW6a and b: Waddell er d..1996: Burton el al.. 1998) and fetal development (Yang. 1997: Stewart er cd.. 1995a: Lindsay CI d..
1996; Edivards cl O/.. 1993: Seckl. 1994: Seckl et of.. 1995: Murph>-. 198 1 : Edivards QI . 996. Hou-ei-er.to date. few studies have in\-estigated the regdation of 1 1 P-HSD2.
The regulation of 11P-HSD:! has aided scientists in the field to elucidate the function of tliis enzyme in various tissues. including the kidney. placenta and uterus. Studies in\-estigating the effects of sex steroids. nitric oxide and otlier factors on the expression of 1 1 f3-HSD2 are considered here in brief. III tlir JEG-3 choriocarci~~oniace11 line. Pasquarette ~11(1996) observed the induction of t',pe 2 1 1 P-HSD niRhTA and activity following treatiiieiir \vit11 bot11 forskolin and dibutryl CAMP. III trying to elucidate the second messenger patliway for the potent and dose-dependent inhibition of II P-HSD2 mRNA and aciti\-ity bl- nitric oside (NO) in
cultured human placenta1 trophoblast cells, Sun and colleagues ( 1997b) treated these cells witli the s ynthetic cGMP analog. 8-bromo-guanosine-3 '5 ic mono phosphate (8-br- cGMP). Indeed. they obsemed a significant decrease in the conversion of cortisol to cortisone follou-hg incubation witli 8-br-cGMP. which suggested tIiat the effect of NO ma>-Iiave been mediated. at least partially. througli the cGMP pathwy. A previous study sIiou-ed that a sustained 811 fetaI hyposaemia \vit11 onsetting acidasmia \vas associated with a seIective dou-n-regulation of the iiiRNA for type 2 1 1 P- HSD in the kidney (Yang er d.. 1997~).In a fo1lou:up srudy. susrained hyposacrnia witli increasing acidosis follou-ed by 72h recovery resulted in a significant increase in 1 I P- HSD2 niRY.4 in fetal ovine kidney (.4sano er d.. 1997). These tu-o studies indicated that the selective sfkcts of Iiyposia 011 the expression of the 1 1 P-HSD2 gene is depsiident on the severit? of the Ii)*posic insult (Asano er cd.. 1997). Siniilar to the type 1 isoform. 1 1 P- HSDî e~izynsacti\-ity is also inliibited b~.the Liquorice metabolite. gl!*c>*rrhetinicacid. and its syntlietic analog. carbenosolone (Goniez-Sanchez cr rrl.. 1996)
The tindings of several investigative groups (Burton cr cil.. 1998: Smith cr cd..
1997: .4Ibiston CI al.. 1995: Arcuri et al.. 1996) have alluded to tlie regulation of 1 1 P- HSD3 enzyiiie activit). in tlie utenis by ses steroids in the rat and Iiuman. In tlie hurnan endoiiietriu~ii. for instance. a sig~iificantincrease in 11P-HSD2 rnzyi-iis actii*it!- n-as obsen-ed during the secretor). phase of tlie cycle. a tiiiie cliaracterized by elevated progesteroile le\-sls (Smith et a/.. 1 997).
111 priniary culture of human endometrial stronial cells. .4rcuri and colleagues (1996b) d.cmoiistrated a potent 8-fold increase in 1 1 P-HSD2 bio-activity \\.lien cells xere esposed to bot11 the sp~tlieticprogestin medrosj-progesteroiie acetate (MPA). and estrogeii. I\ltho~~ghit is an interesting observation. sel-eral criticisms can be drawn from this stud!.. For one. the expression of 1 1P-HSDZ in primaq- culture of endonietrial stronial cells appear to contradict studies done on intact endometrial tissues. since tliis isozynie lias been identified oniy in the luminal and glandular epitlielium of the hurnan endonietriuiii (Smith el al.. 1997). This may also esplain the apparent lack of 1 1 P-HSD2 mRNA mal>-sisin the study by Arcuri and associates (1996). Therefore. tliis latter study leaves major gaps in both the validity of the results and in the ambiguity of the data presented. Takeii together. altliough these studies appear to suggest a role for ses steroid homiones in the coiitrol of endometrial 1 IP-HSDI expression. direct and conclusi\-e evideiice is Iacking. III patients diagnosed with apparent mineraloconicoid escess (AME) and during fetal de\.elopment. 1 1 P-HSDI play a very decisive and consequential rolr. and tlierefore the clinical iniplications of 1 1 P-HSDî are considered separatel). in the following section. ALIE is an inbom error in metabolisni tliat effects occupancy of tlie mineralocorticoid receptor (MR) in the kidney tubule. Numerous case histories describing ckildren u-itli retention of sodium (despite low mineralocorticoid levels). severe hypertension and hypokalaemia have been described (Werder er crl.. 1974: Ulick er al.. 1979: New cf cd.- 1977: Shackleton et nl.. 1985). The link berween (11s observed hypertension. Iiypokalaeniia and tlie inability of these young patients to convert cortisol to cortisone remained a mystery for a considerabk time. It \vas suggested b\- Oberfield and colleagues ( 1983) tliat cortisol was acting as the hypertensive agent, howxer at the time the meclianisin of apparent rnineralocorticoid escess (AME) \vas purely speculati\-e. For conisol to fiinction as a rnineralocorticoid it was hypotliesized tliat cortisol had to fulfil tu-O necessities: (1) an abnomial affinity for the MR. and (3) an alteration in cortisol nietabolisni at the MR. An important step fonvard in testing tliis hypotliesis u-as the recoyiitioii of patients \vit11 hypertension and hypokaiaeiiiia follo\\.ing liquorice abuse. Tlis actil-e deri\.ativr of liquorice. gl~~cyrrlietinicacid (GE). and the Iieniisuccinate derivati\.e of GE. carbenosolone (CBX), were bot11 found to inhibit 1 1 P-HSD (Stewart cf d..1987: 1990). Folloiving tlie cloning (Arriza rr cd.. 1987) and characterization of the MR. it \vas found that cortisol Iiad the same binding affinity for MR as aldosterone. The question that \vas fonvarded sooii afier was ho\\- could one esplain MR specificity for aldosteroiie ir~ i-iw ~vheii free cortisol Ievels were one Iiundred tinies tliose of aldosreroiie'? Tlir niost tenable rlieory was tliat 1 1 P-HSD \vas conferring specificity for aldosteroiie on the MR b>. local inactivation of cortisol. Follon-ing the discovery of mutations in the gene for type II 1 1P-HSD thar adversely affected enzyme activity in patients diagnosed xith AME (Mune et al.. 1995: Stewari er cd. 1996). this tlieory has since been accepted. It tlius appears that in botli congenital and acquired 1 1 P-HSD? deficiency. failure to inactivate cortisol to cortisone is associated \vit11 abiiornial access of tliis steroid to the MR and lience iiiineralocorticoid escess syndrome. The syndroriie of AME is treated \vith the aldosterone receptor antagonist. spironolactone. and a low salt diet. 1 1 P-HSD? lias also been suggested to play an important role in fetal development.
Recent epidemiological studies in humans have linked Ion- birth weight with a substantially increased risk of cardiovascular disease. h!.penensioii and non-insulin dependent diabetes mellitus (NIDD) in subsequeiit adult life (Barker. l99-I: Lindsa) Cr al.. 1996: Sten-art cl c11.. 1995a: Seckl er cri.. 1995. 1994. 1993: Edwards er ul.. 1993:
Benedicktsson cr CI!.. 1993). These studies suggest that the intrauterine en\ironment plays a crucial role in '*programming" later metabolic respoiises and. in panicular. the control of blood pressure and glucose metabolisin (Barker. 1994). It has been suggested that glucocorticoids could esert irreversible effects during a sensitive or critical period of fetal de\.elopiiient. leading to hypertension in adulr life. an effect referred to as ~lioriiioiial inipriiitiiig" (Edn-ards cr cil.. 1993). Moreover. several studies in botli liunians (Reinisli er cri.. 1978) and in aniii-ial models (Lindsay er a/.. 1996: Noir!- el cd.. 1983) have clearl). demonstrated that prenatal esposure of the fetus to glucoconicoids retards intrauterine rot1 Tlierefore. it lias been proposed that exposure of the fetus to escess matemal olucocorticoids iiiay underpin the epiderniological findings relating intra-uterine groutli C restricted (IUGR) babies and the developrnent of cardiovascular disesease. II!-psrtension and NIDD in ad~iltlife (Barker. 1994). Most of the maternal cortisol crossing the huniaii placenta is co~ivened to pl~~.siologicall!-inacti\.e cortisone (Murphy er 01.. 1974). Nornially rhe fetus is protected froni hi& materna1 levels of physiological glucocorticoids. n-hicli circulate five to ten times higlier thaii in the fetus. by placental 11 P-HSD2. Indeed. tl.pe II 11 P-HSD is well positioned in the placenta to senfe as a barrier to maternal glucocorticoids since it is localized to the syicytiotroplioblast. the site of niaternal-fetal escliange (Krozon-ski cr cil.. 1995b). 111 the Iiuman placenta. 11B-HSD2 catalyzes the rapid metabolisni of cortisol to its inert 1 I -keto deril-ati\.e. cortisone (Brown et ni.. 1993). Yang (1 997) presents an excellent revie\i- of 1lP-HSD espression in the placenta of various specirs. related patliophysiology and the role placental 1 lf3-HSD plays as a barrier to matemal glucocorticoids. Receiitly. McCalIa and colIeagues (1998) have publislied work demonstrating a significantl>. lowr 1 1 P-HSD2 osidative activity in placentas of patients ~ithpre- eclampsia. a Iiypertensive disorder ir. pregnancy. compared to normotensive patients. This reductioii in placeiital 1 1p-HSD2 activity was also accoiiipanied by an espectsd increase in riiiibilical cord blood cortisol level and a decrease in fetal weisht. III sri~i~niaq..the studies reviewed above indicate tl-iat escess glucocorticoid exposure in utero may play a critical role in the development of IUGR and in the prograniming of the fetus for long-term metabolic consequences. such as hypertension. Moreo\.er. placeiital 1 1 P-HSD2 is an important niediator of glucoconicoid action during fetal lik. Rscsntl!-. there lias been a gronhg body of evideiice to suggsst that glucoco~~ticoidsnia? also play a decisive role in reproduction. 2.3 GLUCOCORTICOIDS AND THE FEMALE REPRODUCTIVE SYSTEM
The endocrine pli~+siologyof the female reproductive axis and the interplay of tlie many hormones associated with conception. fetal development. parturition. grou-th. pubert).. the reproducti\-e years. and finally menopause beautifully illustrate the cornplesity and responsi\.eness of this highly differentiated endocrine system. A good review esplaining the comples interactions of the HPA and reproductive asis has recently been published
(Magiakoii ci cd.. 1997). The following section presents an o\.en.iew of the female reproducti\*e asis. the interplay of glucocorticoids on tliis systeiii and the possible role that 1 1 B-I-ISD plays in tlie endometrium.
The non-prsgnaiit adulr uterus (woo,ub)in Iiunians is a Iiollou-. tliick-~valled.pear-
shaped organ. It is located witliin the pelvic cavity. anterior to tlie rectum and posterior- superior to the bladder (Marieb and Mallatt. 1992). The uteriiie \\-al1 consists of three main la>-ers: ( 1 ) the outer perirnetrium; (2) the muscular m).onietriuiii: and (3) the iniier layer or riidometrium (Norman and Litwack. 1997). The myonietrium consists of bundles of non-striated muscle fibres that function primarily in tlie CO-ordinated muscle contractioiis obsen-ed during parturition. The role tliat prostaglandins pla!? in the induction of in\-ometrial contractions during parturirioii Iias been wll establislied. aiid indeed. tlis adn~inistratioiiof prostaglandins to pregnant \\-onien Iias been shon-n to induce the oiiset of labour (Enibrey. 1971). Moreover. glucocorticoids ha~ealso been shoivn to play a part iii the fine controi of prostaglandin s)-ntliesis in the gralid uterus
(Casey et CI/.. 1985). The endometrium is the mucosa lining of the uterine cal-ity which consists of simple coluinriar epitlieliuni. underlain by a connectke tissue stroma (Marieb aiid
Mallatt. 1992). The tv-Omain layers of the endometrium are the sr]-crt~rn~jïniciionalis or functional Iayer and the si;-mini busulis (basal layer) The stratum functionalis is responske to the varying ses steroid hormone levels of the meiistrual cycle and is shed during n~enstruation. whereas the stratum basalis forms the functional layer in the following ovulatory cycle (Marieb and Maflatt, 1993). The endometrium undergoes regular cyclical changes throughout the menstmal cycle so as to offer a suitable environment for the implantation of the fertilized o\.uin. These clianges are in response to the changing liormonal milieu- specifically the ses steroids. progesterone and estrogen. Estrogen induces endonietrial proliferation during the preovrilatory pliase of tlie nienstrual cycle. wliereas progesterone fi\-es rise to secretory clianges in the esrrogen-primed proliferative endometrium during the post- ovulatory pliase (Ingamells et cd.. 1996; Lessey et al.. 1988). Based on histological dating. the nienstrual cycle in humans cm be devided into five distinct tinie periods: menses (days 1-5). early proliferative (days 6-9). lare prolifenti\-e (days 10- 1 4). earll- secretory ( days 1 5-2 1 ). and late secretory (days 21-28) (Lesse!. cl ol.. 1 988 ) Estrogen up- regulatioii of borli esrrogen (ER) and progesterone receptors (PR) gene expression has been deinoiistrated in al1 species examined to date. inciudiiig rodents. pifs. sheep. primates and Iiunians (In@and Tornesi. 1997). De novo syntliesis of both estrogen and progesterone froni the tlieca interna/granulosa and corpus luteuin of the ovary during the ovarian cycle. respectil-ely. is controlled b>. toiiadotropins. The secretion of the .ronadotropiiis: tollicls-stimulating hormone (FSH) and lutsiiiizing hormone (LH) by the t anterior pituirar!. (adsnoli~~popli~~sis)is govemed b>-tlie pulsati\-e 111-potlialaiiiic-mediatsd release of goiiadotropiii-reieasi~igIiormone (GnRH) (Yussniaii cr ol.. 1970). Fig 2.8 illustrates the clianges in tlie o\.arian follicle. endometrium and the plasma concentrations of ovarian Iioriiiones and gonadotropins in women during nomial n~ensrrualcyclss (Van de bïele and D~~eiifurtii.1974). SimiIar to the regulatory feedback Ioops obsen-ed in tlie HPA asis. botli progesterone and estrogen function as negative feedback signais at bot11 tlie hypothalan~icand pi tuitaq. Ie\rels to inhi bit GiiRH and goiiadotropiii relsase (See figure 2.9: Noriilan and Litwack. 1997). Figzrre 2.5 P lasrna concentrations of ovarian hormones and gonadotropins in women during normal menstrual cycles. Values are means * SD (n=40). E and F are diagrammatic representations of the changes in the ovarian follicle and endometrium during the cycle (Vail de Wiele and Dyrenfurtli, 1974). Fi2.9 Scliematic diagram of the female hypotlialamic-pituitary-ovarian(HPO) asis. (GF) graafiaii follicle. (CL) corpus luteum. (LH) luteinizing hormone. (FSH) follicle stimulating hormone. (GnRH) gonadotropin releasing liormone (Adapted from Rang and Dale. 199 1). Naturall!. occurring estrogens are typically 18-carbon steroids that have an aromatic A ring with a phenolic hydroxyl. In the nonpregnant fende. the' are syntliesized in the graffian follicle of the ovary. The principle source of estrogen in tlie pregnant female. on tlie other hand. is the placenta (Norman and Lirwack. 1997). Although estrogens niediate significant biological actions in tlie central nenrous systern and a variet!. of visceral tissues. tlie reproductive tract is the predominant target site for estrogens. Tlis ses steroid hormone. progesterone is a 21 carbon steroid \vit11 keto groups on botli C-3 and C-20 (Norman and Litwack. 1997). Progesterone is produced in tlie de\~elopiiiyfollicles of tlie omn (theca granulosa) and tlie corpis luteuni. The female reproducti\-e tract and mammary tissue are the priniaq- target sites for progesterone. It plays an essential role in the preparation of the endometriuni for implantation of tlie blastocyst and in the maintenance of pregnancy and Lit\\.ack. 1 997). Botli estrogeiis and progestins are transported in the circulation by spccific plasiiia binding pi-oreiiis. Circulatiiig estrogen is bound to ses iiomione-bindiiig globulin (SHBG) \\.liereos. progesteroiie is bound CBG. discussed earlier in tliis tliesis. Free concentratioiis of tliese ses steroids are typically in tlie order of 1-Zoh of total plasnia ses steroids. Total plasiiia concentrations for progesterone range from 50-1 00 ng'100 ml in the folliciilar pliase of the O\-ariancycle to 1000-1500 ng/l00 1111 in the niidluteal stage. 17P-Estradiol plasma levels var). anyw-here froni 6 ng!100 ml to 20 ng/100 ml for early folliculai and niidluteal phases. respectively (Nomian and Litwack. 1997).
L'iitil recently. estrogen was believed to regulate comples programs of gene espressioii by binding to a unique nuclear receptor belonging to tlie superfamily of nuclear Iioriiioiie receptors. However. the identification of a second estrogen receptor. referred to a ERP. is lrading to a re-evaluatioii of estrogen signalling and pli>.siolog>- (Kuiper cr rd.. 1996: Mosselman et al.. 1996). While the two ER proreins (ERu and ERP) identitied to date are remarkably similar in structure and share many tùnctional propenies. the) possess indilidual characteristics that suggest tliat eacli isofomi ma? perform specific biological functions (Trembla!. et rd.. 1998). The ER. like otlier members of the nuclear hormone receptor gene superfamily. has several imponant fûnctional domains that function in the activation of gene transcription (Pfeffer er ol.. 1996: Groneme>-er. 1991). Studies with ERa have shoun that the DNA binding domain enables the receptor to bind to its cognate target site on the pronioter. referred to as estrogen responss elenient (ERE) (See table 2.1. pp 19). It lias been suggested tliat altliough ER is prsdominantly found in the nucleus. it contiii~iousl~-sliuttles betn-een the nucleus and cytoplasiriic compannients. Once bound to a ligand. ER dissociatss from i ts multiprotein kat shock protein (1isp)-ER comples and forms a Iioiiiodiiiier. Howewr. more receiitl>- it lias been suggested that ERu and ERP appear to fonn fuiictioi~al heterodiniers capable of recognizing and binding to tlieir target ERE (Giguère et al..
1998) Tlit: ligand-ER 1101110- and/or hetero-dimer coinples tlien proceed to bind to ERE to forms a DNX-receptor coiiiples. This ERE-receptor bouiid coiiiples stabilizes the formation OC a ~ranscriptioncoi~iples. \\.hich in turn initiates the trriiiscription of its target -cenes (Tsai and 0-Malle!.. 1994). III contrast. srudies on the progesterone receptor (PR) gene indicate tiiat althougli tl~ereis oiil!- one gene. tu-Odistinct receptor proteins (PR., and PR,) are translated froin alternative splicing of tn-Oseparate mRNA products (Gralialii er cd.. 1996). Moreover. these spliced mRXA variants of the PR gene are uiider the coiitrol of separare X and B promoters. bot11 of n-hicli are kno\vn to be acrivated by estradiol aiid ER. Despite a 165 amino acid deletion at the aniino terniinus of PR,. it niaii-itaiiis its hnctional transcription replaton property (Graham et cd.. 1996). The general mode1 described for ER regulatioii of gene transcription above is also applicable to the PR. In fact. the consensus sequence of the HRE for PR is similar to that of the GR HRE (See table 2.1 : Lowe el cd.. 1992). Thsre is increasiiig evidence to suggest that although bot11 PR., aiid PR,, are able to bind to ligands and associate with the HRE tiiey are hinctio~~allydifferent. Transfection studies bave demonstrated that these two PR proteins have both promoter- and cell-specific differences. Therefore. this suggests that the responskeness of progesterone in target tissues may be modulated via alterations in the ratio of PR, and PR, protein espression. Antagonists for progestins and estrogens have been utilized extensi~*elyin elucidating the mechanisrn of these sex steroids.
2.3.2.; -3 Profestin and estrogen antagonists
Tu-Osteroid Iiornione antagonists are re\-ieu-ed Iiere in briec Mifepristone (RU- 486) is a class II (pure antagonist) antiprogestin tliat is knou-n for its ability to interfere with nornial PR actions. Indeed. it is used clinically to induce abortion via its actions on PR. Several lines of evidence suggest that RU-486 induces unique conforniations of tlie PR differeiit froiii tliose mediated by progesterone (Baulieu. 1991 : Spitz and Bardin.
99Tlis actions of RU486 on GR have been discussed previously (section 2.1 2-43). Tlie ER aiitagoiiist. ICI 152.780 (also referred to as ICI 164.384) is a class II pure antiestrogeii tliat lias been sliown to inhibit the activation of traiiscriptioii (Berry er trl.. 1990). Hoivever. tlie actions of other antiestrogens such as taiiiosifen ma! produce agonistic rffscts. depending on ce11 type and ER subtype (Watanabe el cd.. 1997). III suiiiniary. progesterone and estrogen are critical hornionrs in niodulatinp target-tiss~irspecitic effects sucii as in the critical cliaiiges tliat occur iii prepnring tlir endoiiietriuiii for implaiitation of a blastocyst. Tliese etfècts are niediared via nuclrar Iioriiione 1-ecsptors for pi-ogesterone and estrogen. For insraiice. it is \\-el1 kiiown froiii clinical obser\.ation. that for progesterone to act in [lie uterus and allow iiiiplaiitation and gro\i-tli of the blastocyst ro occur. the woman must haïe had prior esposure to estrogens. so as to inducr tlie expression of PR. In the nest section. implantation. decidualization and factors affecting these processes wil1 be considered. Follo\\.ing fertilization. the embryo enters a stage of pre-implantation developnient tliat leads to a stringent screening checkpoint. iniplaiitation. It lias been estimated tliat oiily 40% of preimplantation enibryos will implant successfully in the noriual female endometrium (Kline and Stein. 1985). A nunlber of factors influence the successful implantation of the blastocyst into the endonietriuni sucii as hormone dependrnt clianges wliich directs the development of tlie "receptive" endometrium. the stage of eiiibryonic development. and signals frorn the blastocyst (Kennedy. 1983). Implantation and the ensuing decidualization reaction is characterized by an increased vascular permeability obsen-ed in the stroma1 tissue underlyiiip die conceptus follo\vcd by oedenia and coinpositional cliaiiges in tlie estracellular niarris (ECM). cliaiiges in tlie morpliolog~-of tlie stroiiial cells and a progressive sprouting aiid ingronth of capillaries (Jolmson and Everitt. 1988). The nature of the molecules im-olved in post-iniplantation and tlie subsequent iiicrease in endonietrial wscular permeability Iiave !.et to be defined. hou-ever el-idence is available to suggest tliat botli I-iistaiiiine and prostagiandi~ismay be involved (Kenriedy. 1983). Studies in rabbit. for instance. fouiid tllat iiiipiantation u-as interrupted or delayed in tlie prrsence of inhibitors of Iiistaiiiiiie and prostaglandin bios!-iitlissis (Da).. 198 1 : El-Baiina. 1980). One proposed theor?- suggests tliat prostaglaiidins produced locally in the luminal epithelium or stroiua of the rndoiiietrium ma!- be stimulatsd in response to either physical or chernical signals from the blastocyst. sucli as liistaiiiiiis (Kennedy 1983: Jolmson and Everitt. 1988). Howewr. the signal by whicli the blastocyst brings about the events follou-ing iiiipIantation lias not been definitively establislied. In tlie rat. and probably also the Iiuman. a maximal decidual response is oiily elicitrd by tlie conceptus if the endonietrial epitlieliuin is primed \vit11 the riglit milieu of horiiioiirs. specifically progesteroiie and estrogeii (see aboi-e ). in niost species. the Literus caiiiiot accomniodate an iiiiplanting blastocj-st under tlie intluence of estrogeii aloiie. and thus togetlier with progesterone. tliese steroids function to ensure an optiirial iiitrauterine environment for the newly implanted conceptus. Han-ever. sliould a tosin or an inbalance in the homional milieu be introduced tliat upsets the materna1 environment. implantation is unlikely to occur (Yu. 1996). Escess glucocorticoids have long been knou-II for thsir inhibitory effects on the reproductive asis .
Glucocorticoids inhibit al1 levels of the hypothalamic-pituitary-ovarian reproducri\-e asis (Cluousos. 1991). The inhibitory effects of glucocorticoids are esened at the le\.el of the l~pothalmus.on the release of pituitaq gonadotropins. the gonads thenisel\.es and rendsr target tissues of ses steroids resistant to tliese lior~nones. The follou.ing discussioii will focus on tlie effects of glucocorticoids in the endometriuiii. since the scope of tliis tliesis pertains to the regulation of glucocorticoids in this ducoconicoid target organ. C Stxeral lines of evideiice suggest that glucocorticoids are acti\-el?. i~ivolvedin various processes of endometrial function. sucli as endometrial enqnie remodeling
(Salanioiiseii. 1996). cellular proliferation (Bigsby. 1993). uterine ceII death (Terada Cr crl.. 199 1: .JO cr cil.. 1993). inhibition of implantation (Hoffinan L'I NI-. 1984) and in the alteration of the syntliesis of extracellular matris (ECM) coniponeiits (GulIer el cd..
1993). 111 eiidoinstrial cells (Ishikawa). Makrigiaimakis and collsagues ( 1996: 1997) denionstrattid a clear glucocorticoid-induced inhibition of CRH gene transcription. The endometriuiii in the rat and human is capable of producing CRH. It is postulated tliat CRH participates in iiiflai~~matoryphenornena and nia\- also play a role in blastocyst inlplantatioii aiid eiidometrial decidualization (b~lakrigianiiakis cv ol.. 1996: 1997).
Moreover. in tlic in>-oiiietriuiii. glucocorticoids are kiio\i-n to iiilii bi t tlir syiitiirsis of prostacl-clin (Casey CI LI!.. 1985: Richardson cl al.. 1986). hlurplil- ( 1977) suggests th _clucocorticoids in the human uterus inay also be plajing an aiiti-iiimiune role. This is a sound h!-potliesis considering that the reaction of the uterus to the in\-ading blastocyst has been obsen-ed to possess many characteristics of inflan~niation(Ps>-cho>.os. 1976). For instance. se\.eral immune mediators of the inflammatory response. such as IL- 1 and IL-6. and tlieir receptors have been shown to be espressed in endometrial cells (Tabibzadeli.
1991 ). Iiidred. glucocorticoids are also potent repressors of IL-6 griie expression (Ray. 1997). GR has been sliown to be expressed in the endonietrium (Panko cl cd.. 1982). fünher supportiiig tlie hypothesis that glucoconicoids are intiniately involved in endometrial function. It has also been suggested tliat esposure to escess glucoconicoids disturb tlie nornial pattern of growth and differentiation and esen teratogenic effects in the embryo and fetus (Guller et al.. 1993)
On tlie basis of the evidence nom the studies reponed above. it is higlily conceiwble that gl~~cocorticoidsare playing an integral role in implantation. inflamiuation and overall uterine Iiomeostasis. Given the range of glucocorticoid effects in tlie utsrus. local espression of 1 I P-HSDI andlor 2 enzymes may liave an important role in nornial uterine piiysiology by regulating access of glucoconicoids to the GR.
Tlis espression of 1 1 f3-HSD 1 and/or 2 gene in tlie endometriuni of tlie rat (Burton et CI1998). slicep (Yang ci O/.. 1996) and hurnan (Arcuri er cri.. 1996: Sniith er cri.. 1997) suggest that tliese isozymes may be functioning to precisely coiitrol the le\-els of bioactive glucocorticoids. tliereby regulating access of glucoconicoids to their receptors in tliis tiss~ie. Ha\.ing establislied models of uterine 1 I P-HSD gene and enzyme expression. the next quesiion addressed by some of these studies fias the role that ses steroids play in tlie regulatiori of I 1 P-HSD. An analysis of 1 1P-HSD 1 niRNA and protriii expression in the myometririm of tlie rat during pregnancy suggested that the riss in the expression of tliis isozj-nie inal- iia\.r besn due to the progressive rise in estrogen secrerioii over tliis period (Buno11er ni.. 1996). A foilow-up study by Burton er (ri( 1998) discoi-ered tliar in cycliiig nonpregiiant rats. 1 I P-HSD 1 was Iocalized to luminal and glandular epitlielial cells of tlie endometriuiil. whereas 1 IP-HSDî was identified in endornetriai stroma cells aiid myometriuiii. In addition to tliese observed findings. the expression of both 11P-HSD enzymes \vas obsenred to vary across the nornial estrous cycle in the endotiietrium of rats in relation to cliaiiges in ovarian steroid secretion (Burton ei d.. 1998). III the rat utenis during the estroris cycle. the expression of 1 1 /3-HSDI \vas preatest during diestrus (Albiston er oi.. 1995). In tlie sheep, the localization of i~iii~iri~ioreacti\-e1 10-HSD 1 appears to be siniilar to tlie rat mode1 (Yang et oi.. 1996). In humaiis. Iionrver. the type 2 isofom of 1 1 P-HSD protein \vas localized in the luminal and glanduiar epitheliurn of tlie
endornetriuni (Smith ei cd.. 1997). Further inquiry into the temporal patrern of expression of 1 IP-HSD1 and 2 in these species revealed that progesterone may Iiave been responsible for iiiducing the espression of these isoq-mes (Yang er d..1996: Smith ei d..
1997: Alhistoii er O/..1995). Taken together. these studies merel' suggest a role for the induction of 1 1 P-HSD2 by progesterone. but as of yet have to br confirnied by furtlier studies. A study by Arcuri and colIeagues (1996) presents work on the induction of 1 1p- HSD f and 2 enzyme acti\.ity by sex steroids in primary cultures of hunian endometrial stroma1 çslls. Althougli this study presents sonie interesting tindings. tlirre are. ne\*ertlieless. a feu- discrepencies between this stiidy and that of Siiiitli and CO-n-orkrrs (1997). For iiistance. the finding tliat 11 P-HSDÎ is localized escIusively to the lunii~ial and glaiidular epitlieliurn of Iiuiiian endometriu~nleri\-es one to question the etlkacy of 1 1 P-HSD2 snzyie acti\.ity results obtained from endonietrial stroiiial cells in the study
by Arcuri cl cd ( 1996). Of interest is tlie early work by Murpliy (1 977). who obsen-ed 1 I P-HSD osidative eiizyiiie acti\ity for the first time in the liurnan uterus during the iiienstruai cycle and
prevalriit reductase activitl- in tlie utems tlirougliout pregiiaiicy. It lias besii established in more recelil years ttiat tlie reductase enzyme activity obssrved originall!- by bIurph>- in tlie utrrus duriiig pregiiaiic!. was niost likely due ro the espressioii of 1 1 P-HSD type 1 in
tlie decidua. tlie endometriuni of pregnancy (Stewart el al.. 1995: Baggia ci (11.. 1990: Giaiinopoulos rr rd.. 1981: Murphy et al.. 1981: 1977: Lbpez-Berna1 and Craft. 198 I :
Lbpez-Berna1 CI CI/.. 1982: Sun Cr O/.. 1997). Moreover. bot11 isozpes of 1 1PHSD liave
been deiiioiistratrd tlirough enzyme activity in tlie Iiunian eiidoiiietriuiii (Sniitli Cr cd.. 1997). The transition from predominant osidative acti\.ity in the cycliiig Literus to reductase activity in the decidua is an important findiiig. especiaily because of tlie role(s) that glucocorticoids may play in implantation. decidualization and immune response. The findiiigs by Murphy and others. and more recent work in our Iaboratory on tlie pattern of I 1P-HSDl expression in the ovine uterus. lias stiniulated tlie uark tliat is presented iii this tliesis. ParticularIy. the current interest into the regiilation of 1 1 P-HSD 1 in the o\.ine endometrium evolved from the lack of material on the regulation of tliis isozynie in tlie endometrium and also from an earlier study ivhicli showed a unique pattern of 1 1P-HSD 1 expression in the endometrium of slieep (Yang cv rfl.. 1996). In this latter study. 1 1P-HSDl mRNA was found to be espressed during the late Iuteal phase of the estrous cycle and throughout pregnancy (Yang et cd.. 1996). Tliese findings. along with those of kozow-ski's group (Smith ei al.. 1997). both present data to suggest a progesteronr effect on the expression of 1 1P-HSD1 in the mamnialian endometrium. Studies on tlie regulation of 1lP-HSDl and 2 in the mammalian endometrium or represeiitativs ce11 liiiss could have a consequential iiupact in tlie tirld of reproducti\,e endocrinology. Glucocorticoids can have a \vide range of biological actions in the endoiiietrium suc11 as prostaglandin synthesis. i111111une tiintion. iniplantation and decidualizatioii. and tlius tlie local expression and regulation of 1 IP-HSDl and 2 enzyines niay significantly alter the levels of bioactil-e glucocorticoid reaching intracellular GR. 111 the nest section. a preliminay discussion \\-il1 cover the premise for the im~estigationcoi~ducted on the regulation of IIP-HSDl and 2 in the rndoiiietriuin of sheep and rlie lli1111an e~idometrialce11 line (Ishika\va). respectil-el!-. 2.4 SCOPE OF THE PRESENT STUDY
Thsre is eleidenceto suggest that ses steroids. in particular progesterone. rnay be responsible for inducing the expression of 1 IP-HSDI and 2 in the maii~n-ialian endometri~im(Siiiitli cl al.. 1997: Yang er al.. 1996). Ho\ve\-er. direct and conclusi\re evidence in support of tliis hypothesis is lacking. Moreover. there is little information on the regulation of 1 1 PHSD1 and 2 in the marnrnalian endometrium. Tlierefore. the present study \vas designrd witli tn-O priniary objectives in mind. The first esperinient \vas conducted to esamine the Iiypotl~esisthat progesterone inducss the expression of 1 1 P- HSDl in tlie ovine endometrium. To achieve this objective- tlie endometrium of o~~ariectoitiized(OVX) slieep treated with a progesterone replacement therapy u-as analyed for the espression of 1 1P-HSD1 mRNA and enzyme activity. The second set of
esperimeiits u-as carried out to identiQ an il? virro n-iodsl sysreni in uhicli tlie detailed inolecular niscliaiiisms of progesterone induced expression of endonietrial 11P-HSDl inay be srudied. The Ishikawa ce11 line (sre photo plate 1) derived froin a ~ell-
differentiated adenocarcino~iiaof Iiuman endometriuni (Nisliida LJ/ ul.. 1985: 1996) appeared to be aii excellent prototype to study nonna1 endonietrial epitlielium and n-as chosen as a niodel for tliis study for the following reasons: (1) Isliikau-a crlls are of endometrial spitlielial origin. a known site of 1 1P-HSD1 espression in sheep (Yang cr al..
1996) (2) tlirse cells maintain functional progesterone (PR. 1-16 fniol/iiig protein) and cstrogrii recrptors (ER. 574 hol/nig protein) (Nisliida er trl.. 1985: Hata and Kuramoro.
1992). aiid (3) tliis ce11 liiie expresses niany of tlie same enzyniss. lioriiionss and structural proteins found in normal endoinetrium (Cira\-anis and Gurpide. 1986: Makrigiannakis cr al.. 1996: Castelbaum et al.. 1997).
In addition to tlie ob\rious cost and time saving benefits of utilizing an N7 i9irr-O mode1 sysreni sucli as the Ishikawa ce11 line vs. animal niodsls. Isliikawa cells provide tlie freedoni ro inanipuIate a wriety of \-ariables and conditio~is. Follo\\.ing the identitication of an iu ~~i~l-omode1 of epithelial derived endometriuni. the nest objective \vas to deteniiine if 1 I b-HSD 1 \vas espressed in Isliikawa cells. TIis 1 1 PHSD activity in Ishikawa csIls \vas characteristic of 1 1P-HSD2 in tliat it onIy possessed dsliydrogenase activity (cortisol to cortisone) and had a high affinity for cortisol (apparent K,, of 34 nM). RT-PCR denionstrated the presence of the mRNA for IlP-HSD? but not that for 1 1 P- HSDl in Isliikau-a cells. Around the sarne time. Krozowski's group published work wliich localized iminunopositive 1 1P-HSD2 protein to the luniinal and glandular epitheliuni of tlie Iiunian endometrium (Smith et ai.. 1997). Moreover. the>-demonstrated 11 P-HSD1 and 2 enzyme activity in homogenates of hunian endometrial tissue. whicli was greater duriiig the secretory phase of the menstrual cycle. This finding suggested a possible role for ses steroid hormones in the regulation of 11P-HSDI and 2. Yet tliere are gaps in the knowledge on the regulation of 1 IP-HSDI and 7 in tlie human endometri~inisince the conciusions by Smith and colleagues (1996) u-ere based on speculatioii. Therefore. tliis present study \vas designed. in pan- to address tlie regulation of 1 1 P-HSD2 by ses steroids in Isliikawa cells in the hopes of slucidatiiig tlie role(s) tlint ses steroid Iiornioiies play in tlie regulation of endometrial 1 1 P-HSD2. III addition. epidermal pro~lifactor (EGF) gene lias been implicated to play an important role in the decidualization process of the Iiuman endometrium (Horowitz er cil.. 1993: Sakakibara cl ai.. 1994) and in the regulation of endonietrial recepti\.ity in Isliikau-n cslls (Sonikuti et rd.. 1997) and 1 1P-HSD1 expression in Leydig cells (Gao cl trl.. 1997). u-c tlicrefore in\-estigatcd the role tliat EGF iris). be playiiig in regularing 1 1 P-
HSDî geiir iii Ishikan-a cells.
In suiiimary. the objectives of this study were: (1 ) to investifate the role tliat progesteroiic plays in the regulation of 11B-HSD1 niRNA and enzynie activity in tlie endoinetriuiii of OVX sheep treated witli a progesterone replacement tlierapy. (2) to characterize the expression of 1 1 P-HSD in the human Isliika~vaeiidoiiietrial cell line. and (3) ideriri fi- factors regulating tlie expression of 1 1 P-HSD in Isliikan-a cells. iiicluding ses steroids. glucocorticoids and EGF. The folio\\-ing two chapters summarize work done to date in elucidating factors in the regulation of 1 1P-HSDl and 2 gene expression in tlie slieep endonietriurn and human endonietrial Isliikawa cell Iine. Photo plttrc 1 : photo of Ishikawa cells, a well-differentiated adenocarcinorna of htiman endonietrial cpitliéli~itii. Cells in photo are approxiiuatel>- 60-70% confluriit. Magnification 100s. 2.5 REFERENCES
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Progesterone Induces the Expression of 1 1P-Hydrosysteroid Deliydrogenase
Type 1 mRNA in the Ovine Endometrium'
i This chaptsr lmbeen subrnitted for publication.
Darne1 AD. Laiiiming GE. Yang K. Progesterone induces the expression of 1 l p- hydrosysreroid deliydrogenase type 1 mRhTA in the ovine endometrium. Jorlrwal of Steroid Biochcn~isrr-.md .\4o/ccilhl- BioZ0g-y. 3-1 INTRODUCTION In the mammalian uterus. glucocorticoids esert potent biological effects. largelu in an inhibiton fasliion. OB a number of dynamic functions. including cellular proliferation (Bigsbl-. 1993). apoptosis (Terada ci nt.. 199 1 :JO cr d.. 1993). and synthesis of liornloiies. gronth factors and enzymes (Makrigiamakis ci cd.. 1992: Jacobs ci d..
1994: Salamonsen. 1996). Recently. accumulating evidence suggests that glucocorticoid actions iii target tissues are determined not only by the function of glucocorticoid receptors (GRs) but also by the local expression of 1 I P-hydrosysteroid dehl-drogenase
(1 1P-HSD) eiizyiiies (%'hite cr CI/.. 1997: Kotetevtsev ci cd.. 1997). To date. tn-Odistinct isozpes of 11P-HSD. kilow~as 1 1P-HSDI and 2. ha1.e been identitied. cloned and cliaracterized (White el al.. 19971. 1113-HSDI is a Ion- affinit?. NADP(H)-dependent deliydrogenase (cortisol to cortisone) / reductase (cortisone to cortisol) eiizyile. \vit11 an apparent K,, for glucoconicoids in the [LMrange (Aganval cr al.. 1990: Moore ci ol.. 1993). This enzyme is widely espressed in glucocorticoid target tissues. iiiost riotably the li\.er (Tar-ii-iin et CI/.. 1 991: Yang ci LI/.. 1992). III intact cells. 1 1 P-HSDI fiiiictioris predominantly as a reductase generatiiig cortisol from cortisone
\vliicli circulnres largcly in the free. unbound forni (Jamieson ci cil.. 1995). ..\ltliougli it has beeii proposed tliar Il P-HSDI regulates the intracellular le\-el of bioactive glucocortiçoids. rlie precise pliysiological role of this enzyne in individual target sites remains obscure. III coiitrast. 1 1 P-HSDI is a high affinity NAD-dependent unidirectional dehydrogeiiase enzlme. nit11 an apparent KI, for glucoconicoids in the nM range (Ruslai et ol.. 1993 Albiston ci al.. 1994: Stewart ci al.. 1994: Yang el cd.. 1994). The expression of this isoz!-me is restricted to the placenta where it is proposed to sene as a barrier to protect the fetus froni Iiigh levels of materna1 glucocorticoids (Murphy. 198 1 : Seckl e1 a 1995). and to aldosterone-target organs sucli as the kidney (Albiston el crl.. 1994) where it protects the non-selecti\-e mineralocorticoid receptors froiil liigli circulatiiig levels of glucocorticoids (Edwards ef al., 1988: Funder er al.. 1988). Deficiencies in this enzyme. eiilier congenital (caused by mutalions in tlie gene encoding 11 P-HSD2) or acquired (tlirough liquorice ingestion). leads to the syndrome of apparent mineralocorticoid escess (AME) in which cortisol acts as a mineraloconicoid causing hypertension and hypokalemia (Ulick et al., 1979; Stewart el d..1987: 1988). Given the wide range of glucoconicoid effects in the uterus. local expression of
11P-HSD enzymes will likely play an important role in the function of the uterus. In a previous sr~idy.u-e established the pattern of 11P-HSD1 gene expression in tlie ovine uterus duriiig the estrous cycle and pregnancy (Yang cf cd.. 1996). We found tliat witliin the uterus the siidonietrium is always the dominant site of 1 1 P-HSD 1 mRNA expression n-liicli occurs oiily at specific times. during pregnancy and lare luteal phase. 11-lien circulatiiig le~elsof progesterone are elevated. Tliis is indicative of a causal relationship betxeen progesterone and induction of endometrial I 1 P-HSD 1 mRNA. Therefore. the present st~idywas lindertaken to esamine this hypothesis. 3.2 MATERIALS AND METHODS 3.2.1 T~SSZWC~llecrioil~ Tu-elve Dorset ewes were used in the present study. The surgical procedures and
progesterone treatment regirne have been described in detail before (Wathes er cd.. 1996). BrieIly. al1 eu-es were ovariectomized (OVX). and divided randomly into three equal eroups. Group 1 received no treatment. Groups II and III were treated with progesterone L for 4 and 1 1 days. respectively. The progesterone treatment reginle \vas dssigned to simularr the natural luteal phase. producing peak plasma progesterone concentrations in
the range 7.7 -+ 0.2 nghl (Wathes el al.. 1996). In addition to the progesterone regimen. OVX e\i-es receiwd a low-dose estradiol treatment. a protocol that produced plasnia estradiol concentrations of about 3 pg ml" (Wathes er crl.. 1996). Endometrial tissues were renia\-ed in strips and flash-frozen in liquid nitrogen. All the tissues u-ere stored at - 80 OC until aiialysis.
3 2.2 R.\:-l E.\-rrvrcrioi~and ,\orikrn Blor AIICI~JX~S Total cellular RNA \vas estracted using litliiuin clilorideiurra (Auffra?. and Rougeon. 1980). The size as xell as the relative abundance of IIP-HSDI niRNA was
assessed hy Nortliern blot analysis as described previously (Yang cr trl.. 1992). Brieil!.. denatursd RNA sniiiples (20 kig) were subjected to agaross gel ( 1 %) elcctroplioresis in the preseiice of fornialdeli>~de.and transferred overnight b>. capillary blotting to a Zeta-
Probe itie~iibraiie(Bio-Rad Canada Ltd.. Mississauga. Ontario). The RNA \vas fised by
UV cross-linking (Gene Cross-Linker. Bio-Rad) to the membrane ivbich \vas tlien baked under ixuliiii at 80 C for 60 min. The blot \vas hybridized at 12 C for 1611 in tlir preseiice of fornianiide (50%) and [jI~]-shee~1 1P-HSD 1 cDNA (Yang er cd.. 1992) prepared by randoni priniing (Feinberg and Vogelstein. 1983) with [~~PI~CTP(Du Pont Canada: 3000 Ci/iiimoI). We used a cDNA for mouse 18s rRNA as an interna1 control for gel
loading and efficienq~of RNA transfer, as described previously (Yang ci cd.. 1991). 3.2.3 A nalysis of l iPHSDi m RNA - serni-quantitative R T-PCR
The relative abundance of 1 1P-HSDI mRNA in endometrial tissue samples from OVX ewes Las assessed by an established semi-quantitative RT-PCR protocol. as described (Tremblay er al., 1999). Briefly, total RNA was extracted as described above.
Prior to use. the RNA samples (1-2 pg) were checked by agarose gel electrophoresis in the presence of formaldehyde, and the integrity of the RNA was assessed by the presence of two sharp bands representing 28s and 18s rRNA after staining with ethidium bromide. One microgram of total RNA was reverse-transcnbed using a standard oligo-dT primer in a total volume of 20 pl. An aliquot (2 pl) of the RT reaction products was then subjected to a standard PCR (94°C. 55 sec; 60°C, 55 sec; 72OC. 1 min: 30 cycles) using sequence- specific primers (fonvard primer. 5'-GGGGGGTACCTACCTCCTCCCCATTCTG; reverse primer. 5'-GGGGGGATCCCTCTCCAGCTTTGGCAAC) whicli correspond to nucleotides 76-1 03 and 401-374 in the published sheep 1 1 P-HSDI cDNA (Yang et al.. 1992). respecth-ely. GAPDH was used as a control. The same PCR conditions were used for GAPDH escept tliat an amealing temperature of 53°C rather than 60°C \vas used in PCR. The priniers for GAPDH (fonvard primer. 5'-ACCACAGTCC.4TGCCATCAC: reverse primer. 5'-TCCACCACCCTGTTGCTGTA) were obtained from Clontech Laboratories 111~.(Palo Alto. CA). and they correspond to nucleotides 586-605 and 1018-
1037 in tlie pubiished iiuman GAPDH cDNA (Arcari er ut.. 1984). To verif- tlie RT-PCR products and to assess the relative abundance of 1 1 P-HSD 1 mRNA. a fraction of the RT- PCR products \vas then subjected to a standard Çoutliem blot analysis. using ["PI-sheep
1 1P-HSD1 cDNA and ["PI-human GAPDH cDNA as probes.
To detemine the relative abundance of i 1P-HSD 1 mRNA and GAPDH mRNA. the relative optical density of the corresponding signals on autoradiographic films were measured b!. scanning with a laser densitometer (LKB 2121-020 UtraScan XL: LKB
Produkter AB. Bromma. Sweden). as described (San-ipatli-Kumar er crl.. 1998). In al1 cases. the signals were detected within the linear scan range of the densitonieter. For each RNA sample. the ratio of 11P-HSDl mRNA signal to GAPDH mRNA signal was calculated.
3.2.4 Asscg* of I lpHSDI Actiiyiiy The dehydrogenase activity of 11p-HSD 1 in the endometrium \vas detemiined by a radiometric conversion assay using cortisol as physiological substrate. as described
(LaneloisLI et al.. 1995). Briefly, endometrial tissues (0.2-0.3 g) were homogenized in 10 vol. of ice-cold 10 mM sodium phosphate buffer. pH 7.0. containing 0.25 h4 sucrose
(Buffer -4). The homogenate was used immediately in the assay as described belo~kr.
The assay tubes contained approximatelp 100.000 cpm of the labe!Ied cortisol. 1-0 pM of non-radioactive cortisol. and 250 pM of NADP. Buffer B (0.1 M sodium phosphate buffer. pH 7.5) \vas added to brinp the volume up to 0.4 ml. Afier 10 min incubation at 37 C. 100 pl of tissue homogenate containing 200-300 pg protein was added. Afier incubation for 60 min (preliminary studies indicated that the rate of reaction
\vas linear i\-itli time froin 15 to 120 min. and with tlie amount of tissue homogenates containing between 0.1-1.3 mg protein). the reaction was arrested. and the steroids were estractecl \\-ith 4 ml etliyl acetate containing 30 pg mixture of non-radioactive cortisol and cortisone as carrier steroids. The extracts were dried. and tlie residues were resuspended in 100 ILI nietlianol. A fraction of the resuspension \vas spotted on a TLC plate \\-hich \\.as developed in cl.~lorofornl/~nethanol(9:1, vh). The bands coiltainiiig the labelled cortisol and cortisoiie \\-ers identified b>-UV light of the cotd carriers. cut out iiito scintilIation vials and counted in scintisafeTM Econol 1 (Fisher Scientific. Toronto. Canada). The rate of cortisol to cortisone conversion was calculated from the specific activity of tlie labelled conisol and tlie radioactivity of cortisone. and results espressed as the amount of cortisone (picomoles) fomed per min per mg protein. 3.3 RESULTS
3 -3.1 11 PHSD I inR.Y.4
Wlien total RNA samples extracted from endometrial tissues were subjected to
Nonhern blot analysis using the ovine 1 1P-HSDI cDNA probe. a single 1.8 kb transcript was detected in 3 of the 4 samples from the animals treated witli progesterone for 1 1 dap
(Fig. 3.1). By contrast. tliis transcript was undetectable in total RNA saniples fro~iithe control and aiiiiiials treated \vit11 progesterone for 4 days (Fig. 3.1 ).
In addition. total RNA \vas subjected to RT-PCR and Southem blot analysis using the o\.ine 1 ID-HSDI cDNA and GAPDH cDNA as probes (Fig. 3.2). A single 1.4 kb transcript u-as obsen-ed in al1 sarnples from anirnals treated u-ith progesterone and in control aiiiiilals. However. the ratio of 1lP-HSD1 to GAPDH in 4 da'. treated aninials \vas substniitiall!- loiver tlian in control animals and sheep treated \vit11 progesterone for
1 1 days.
3 -3.2 l l p-HSD lcicii\Y~.
To drrrriiiine ivliether the induction of 1 1 P-HSD I niRNA by pro,cTesterone \US translated to 1 1 0-HSD 1 protein. levels of 1 1 P-HSD 1 deliydrogenase acti\-ity in endo~iirti-in1tissue Iiaiiiogeiiares \\-ere measured and coiupnrrtl hetweii the y-oups. .As slio\vii iii Fig. 3.3. there u-ere no correspondhg clianges in the lei-el of eiidonietrial 11P- HSD l deliydrogenase activity following progesterone treatiiient. altliougii rlie activity \vas liiglily i-ariable both betweeii and within the groups. 3.4 DISCUSSION The present study has demonstrated that progesterone induces the espression of Il P-HSDI mRiA in the ovine endometrium. Moreover. tliis effect appears to require more than 4 days to be manifested. since the mRNA for 1 IP-HSDI \vas detected in the aninials treated with progesterone for 11 days but not in those treated for 4 days. Altliough the underlying molecular basis for this uncon-imonlg- lengthy tirne requirement is unknoivn. the present finding is consistent uith our previous results denionstrating tliat 1 1P-HSD 1 niEWA \vas detectable in the endometrium of intact ews only afier da). I O of the estrous cycle (Yang et al.. 1996). It is noteworthy tliat 1 IP-HSDI mRNA \vas undetectable by Northern blot analysis in the endometrium of one of the 1 1-da?.- progesteronr treated anirnals. Given Our previous results. it is possible tliat tlie absence of
1 1 P-HSD 1 iiiRN.4 iii tliat animal ii-iay be due to a slight delal- in the manifestation of the progesteroiie effect. Whatever the mechanisiiis. the iiiductioii of eiidoiiietrial 1 I P-HSDI niRNA by progesteroile adds a new dimension to tlie coniples iiiteractioii betiveen tliese two steroid liorniones in the dynamic function of tlie endometrium.
To deterinine if corresponding changes occurred in tlie level of 1 1 P-HSDl enzyme activity foliowing progesterone treatiiient. e nieasiired I I P-HSD 1 dehydrogriiasr nctii-ity iii endonietrial tissue liomogsnates. The 1 I P-HSD 1 reductase activity \vas not nieasursd because it is highly unstable in cell-free preparatioils (Monder and White. 1993). Very low and indistinguishable levels of 1 1 P-HSD 1 dehydrogenase activity n-ere detected in al1 three groups. indicating tliat the induction of 1 1 P-HSDI mRNA by propesterone was not translated into 11 P-HSDI protein. Gil-en our previous findings that 1 1 P-HSD 1 mRNA was detectable in the ei-idonistriu~iionly afier day I O of the estrous cycle. it reniains possible that 1 1P-HSDI mRNA may have been induced by progesterone just prior to sacrifice. and thus there ivas no sufficient tinie for the niRNA to be translated into the protein. If confirmed by future studies. tliis nia>-iielp to esplain i\-ii!' 1 1 P-HSDl inRNA \vas undetectable in one of the 1 I -da>--propesteronetreated anii-iials. since the induction of 11P-HSD1 mRNA by progesterone niau not have been synchronized in al1 the animals. Hou-ever. the presence of 1 I P-HSD 1 dehydrogenase acti\-ity. al bei t at very low levels at dl. in the absence of appreciable arnounts of IIP-HSD1 niWrA. is intriguing.
Recently. it lias been reported tliat there may exist a new iso-me of 1 1P-HSD. n-hich has been tentatively narned 1 1B-HSD3 (Gomez-Sanchez er al.. 1996: 1997: Ge et cd.. 1997). 1 1 B-HSD3 is a higli-affinity NADP-dependent dehydrogenase. Therefore. it is possible that the ohsen-ed 1 1P-HSD 1 dehydrogenase activity. nieasured in the presence of NADP. in tlie presciit studl- ma)- be attribured to a constituti\.ely-espressed 1 1 /3-HSD3 in the ovine eiidoiiietriuiii. Obviously. the definitive confhiatioii of tliis possibilit~.a\vaits the cloiiing of I 1 P-HSD3.
Tlie 1 1 P-HSDl isozyne is know to be espressed in tlie utrrus of other ~iiamnials
(Baggia er tri.. 1990: Albiston er cd.. 1995; Smith et al.. 1997). Using primary cultures of human eiidoiiietrial stroiiial cells. Arcuri and colleagues (Arcuri cl al.. 1996) found tiiat progestrmnc stini~ilatedthe espression of 1 1P-HSDI niRNA and activity. By contrast. estradiol ;tlriiir Iiad no effect but potentiated the stiiiiulatory effects of progesteroiie. in the rat utenis. tlierr \vas a draiiiatic decrease in tlie level of 1 IP-HSDI protein follou-iiig ouu-iectoiiiy. and this effect was reversed by estrogen replacement u-itli or n-itliout progesterom (Burton er ai.. 1998). Collectively. tliese and our preseiit findings indicate that ses stcroid Iiorn~onesare potent regulators of uterine 1 I P-HSD 1 expression- Tlie etucidatioii of tlie uiiderlying ii.iolecular meclianisnis and interactioiis between these ses steroids ûiid ~lucocorticoidswill undoubtedly provide new insiglit into orir uiidristandiiip of dynaniic hiiiction of ~lianiriialianuterus. 3.5 ACKNO WLEDGMENTS
This work \vas supponed by the British Agricultural and Food Researcli Council (Group Grant to G.E.L.) and by the Canadian MRC (Grant MT-12100 to K.Y.). K. Yang is an Ontario Ministry of HeaIth Career Scientist. ControI P4 (4 days) P4 (1 1 days) I I I 1 I I I 1 I
Fig 3.1 Nortliern blot of 1 1 P-HSDl mRNA in the endometri~iinof the ovariectoniizrd ewes trentcd \\-ith progesterone for 4 (n=4) and 1 I (il+) days. aiid of tliose received no steroid treatiiieiit (11=4). Total RNA saniples (30 p)were aiialyzrd. and the top ptrrwl is an autoradiograpli of the blot probed witli [32~]-sher~1 1 P-HSD 1 cDNA (esposure tiiiie
5 days). As a control. the saine blot was stripped and reprobsd with [j2~]-mo~isr18s rRNA cDNA (horrom pnml: esposure time 2 h). Control P4 (4 days) P4 (1 1 days)
Fig 3.2 An autoradiograph of the semi-quantitative RT-PCR analysis of 1P-HSD 1 mRNA (top panel) and changes in the relative abundance of 1 1P-HSD 1 mRNA (bottom panel, k SD) in the endometrium of ovariectomized ewes treated with progesterone for 3 (n=2) and 11 (n=2) days, and of tl-iose receiving no steroid treatrnent (n=4). Total RN.4 was extracted and subjected to a semi-quantitative RT-PCR, followed by Soutllrrn blotting, as described in the Materials and Methods. Expected size of amplified PCR products for 1 1P-HSD 1 and GAPDH are 326 bp and 450 bp, respectively. OVX OVX+P (4 days) OVX+P (1 1 days'
Fig 3.3 Changes in the endornetrial I 1P-HSD I enzyme activity. Tissue homogenates of endometrium O btained from the contro 1 and animals treated with progesterone for 4 and 11 days were incubated with 1.0 pM cortisol in the presence of NADP for determining the dehydrogenase activity of 1 1P-HSD 1, as described in the Materials and Methods. Each bar represents group mean+SEM- (n=4). 3.6 REFERENCES
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Regulation of 1 1 P-Hydrosysteroid Dehydrogenase Type 2 by Steroid Hormones and Epidemial Growt11 Factor in the Ishikawa Human Endometrial CeIl ~ine'
I This cliapter has been submitted for publication.
Darne1 AD. Archer TK. Yang K. Regulation of I 1p-hydrosysteroid dehydrogenase type 2 by steroid hormones and epidermal groudrh factor in the Ishikawa human endometrial cell line. .Jorrrwrrl ofSfet-oidBioclternist?y and ,Mo/eczrlor-Biolog-r. 4.1 INTRODUCTION
In the maninialian utems. glucocorticoids exert potent effects. largely in an inhibitor-y fasliion. on a number of dynamic functions. including cellular proliferation
(Bigsby. 1993). apoptosis (Terada et al.. 1991: JO et al.. 1993). and synthesis of horniones. grontli factors and enzymes (Makrigiannakis er cil.. 1993: Jacobs el d..1991:
Salamonsen cl ol.. 1996). RecentI y. there is increasing elaidence that g lucocort ico id actions in target tissues are controlled not only by the fùnction of glucocorticoid receptors
(GRs) but also by tlie local expression of 1 1 P-hydrosysteroid dehydrogenase ( 1 1 P-HSD) enzymes (Wliite et crl.. 1997: Kotelevtsev et 01.. 1997). which detemine the intracelluiar level of bioacti\-e glucocorticoids.
To date. tu-O distinct isozymes of 1 1P-HSD. known as 1 1 P-HSD 1 and 2. have been identified. cloned and cl-iaracterized (White CI al.. 1997). 1 1 13-HSD 1 is a lou- aftiriity
NADP(H)-dependent deliydroge~iase(cortisol to cortisone) ! reductase (cortisone to cortisol) cnzyiiie. with an apparent K, for glucocorticoids in the pM range (Apanval Cr al.. 1990: h4oore et 01.. 1993). This enzyme is widely expressed in glucocorticoid target tissues. iiiost notably the lker (Tannin er al.. 1991: Yang cr ol.. 1991). hi intact cells.
1 I P-HSD 1 f~inctionspredoii-iinantly as a reductase generating cortisol froiii conisoiie wliicli circulates largeIl. in the free. unbound for111 (Jarnieson cr rd.. 1995). Altliough it lias been proposed that 1 1 P-HSD 1 regulares tlie intracellulai. levsl of bioactive glucocorticoids. the precise pliysiological role of tliis enzyme in individual target sites remains obscure. In contrast. 11P-HSDZ is a high affinity NAD-dependent unidirectional dehydrogtriiase enzyme. witli an apparent KI,,for glucocorticoids in tlie nhd range (Rusni and Naray Fejes Totli. 1993: Albiston et cil.. 1993: Stewart er d..1994: Yang er rrl..
1994). The espression of this isozyme is restricted to the placenta U-liereit is proposed to sene as a barrier to protect tlie fetus froni high levels of materna1 glucocorticoids
(Murphy. 198 1: Seckl er al.. 1995). and to aldosterone-target organs such as the kidney (Albiston er ol.. 1994) where it protects the non-selecti~emineralocorticoid receptors from hi@ circulating leveIs of glucocorticoids (Edwards er al.. 1988: Funder er al._
1988). Deficiencies in tliis enzyme. either congenital (caused by mutations in the gene encodiiig I 1 P-HSD2) or acquired (tlwough liquorice ingestion). leads to tlie syndrome of apparent ~iiineralocorticoidexcess (AME) in which cortisol acts as a niineralocorticoid
causing hypertension and hypokaieniia (Ulick et d..1979: Sten-art et cil.. 1987: 1988). Given tlie wide range of glucocorticoid effects in niamnialian uterus. local expression of 11P-HSD enzymes ~villlikely play an important role in the function of the
uterus. III a recent stiidy. Smith and colleagues (1997) shon-ed that I 1 P-HSDZ
imrnunorrncti\.it!- \\as localized in luminal and glandular epitlielia of tlic human
endon~etriiim.Furthermore. tlie level of I 1 b-HSD2-like acti\-ity in endoinetrial tissue Iioniogeiiates \vas Iiigher in tlie secretory than in rlie proliferati\-e pliase. suggrsting tliat
endometrial 1 ID-HSD2 is under the control of sex steroid horniones. Currently few. if
an!.. niodels esist for the study of Iiuman endometrial 1 1P-HSD2. The 1sliikni.a Iiiinian endometrial adenocarcinon~a ce11 l iiie lias been ussd esteiisi\.el!- as a niodel to study endometrial function sirice this ce11 line expresses niany of the sanie eiiz!-mes. steroid hormone receptors. grou-th factors and tlieir receptors as well as structural proteins found in nomal eiidometriuni (Xisliida et cd.. 1985:
Castlebauiii CI LI/.- 1997). Tlierefore. the present study \vas undertaken to determine wherlier Ishikaiva cclls express the 1 1P-HSD2 isoqrne. and if so. wlietlier its espression is regulatrd hy steroid Iiormones. Given thar epidermal gro\\tli factor (EGF) is producsd and functioiis locally \i-itliin the Iiuman endometrium (McBeaii el trl.. 1 997). we also esamined the effects of EGF on 1 1 P-HSD2 espression in Isliikaw cells. 4.2 MATERIALS AND METHODS
4 2- 1 Rec~ge~rscrnd supplies [1.2.6.7-'Hm)]-Cortisol (80 Ci/mmol) kvas purchased froni Du Pont Canada Inc. (Markhani. Ontario). Non-radioactive steroids were obtained from SteraIoids Inc. (Wilton. NH). Dexamethasone (DEX) was obtained from Sigma Cliernicals (St. Louis. MO). Recombinant human epidermal growth factor (EGF) was obtained from R&D Systems (h4inneapolis. MN). RU486 and ICI 182.780 were kindly donated by Dr. T. K Archer (Dept. of Biocliemstry and Ob/Gyn at the University of Western Ontario. London. Canada). Polyester-backed thin-layer chromatograpI-iy (TLC) plates were obtained from Fisher Scientific Ltd. (Unionville. Ontario). Al1 solvents used were OmniSolv grade froni BDH Inc. (Toronto. Ontario). General molecular biology reagents were from Gibco BRL (Burlington. Ontario) or Pliarmacia Canada Inc. (Baie D'Urte. Quebec). Ce11 culture supplies n-ere obtained from Gibco BRL or Fisher Scientific.
Isliikau-a cells wre kindly provided by Dr. M. Nisliida (Tsukuba Cniversity.
Japaii). Cells were routinely grown in DMEME- 12 (1 :1 ) suppleniented \vit11 1 0% FBS. penicillin-strepto111yci11and sodium pyrwate. Cells (0.5 - 1.0 x 10" cells~nil) were mai~iraii-isdin T-25 Corning flasks at 37 C in a 95% air - 5% CO1 Iiuniidified incubator. The mediuni \vas chaiiged every other day. The cells u-ere passed as required. To study the effects of different treatnient regimes on IlB-HSDI activity. cells were passed onto 12-\i-el1 Corniiig plates and cultured to 60-70s confluence (utilizing trypan blue exclusion at 0.5 - 1 .O x 1 O6 cells/nil). The cells were then cultured in semiri-free medium 2411 prior to treatment. and al1 the treatments in triplicate n-ells u-ere carried out under seruni-free conditions. Controls (also in triplicates) were incubated siiiiilarly but witliout the addition of treatnient. For 1 ID-HSD1 IIIRNA analysis. cells wre subjected to identical culture and treatment conditions as described above escept tliat tliey were rnaintained in T-25 flasks.
4.2 -3 Assoi- of- l l,&HSD2 oc f iviiy - rudiornerric conversion cissciy
Tlie le\-el of 11 P-HSD? activity in intact cells \vas determined by measuring the rate of cortisol to cortisone coiwersion. as described previously (Trembla! er al.. 1999). Briefly. at the end of treatment. the cells were washed 3 tinies in serum-free medium to remove the steroids or grouth factor. in an attempt to esclude their having a possible
competitke inhibition. The cells were then incubated for 4 hours at 37 C in serum-free
medium containiiig appros. 100.000 cpm ['Hl-cortisol and 10 nM unlabelled cortisol. At the end of incubation. tlie medium was collected. and steroids estracted. The estracts
were drird. and tlie residues resuspended. A fraction of die resuspension \\-as spottrd on a
TLC plate \\-hich \vas drveloped in clilorofom~n~etlianoI(9: 1. Y!\-). Tlie bands containing the labellcd cortisol aiid cortisone were identified by UV liglit of tlie cold carriers. cut out into sciiiiillation \.ials aiid counted in ScintisafeT" Econol 1 (Fisher Scientific. Toronto. Canada). The rate of cortisol to cortisone con~rersion \vas calculated from the radioacti\-it!- of cortisol and cortisone. and the blank values (defined as tlie rate of con\-ersinii in tlie absence of cells) were subtracted. and the results sspressed as tlie percentage coin-ersion. Data are presented as inean -+ SEM of 3-6 independent esperiments.
To deteniiine if changes in 1 1 P-HSD? activity followiiig tlie different rreatment reginies \wre associated \vit11 alterations in 1 1 (3-HSDZ mRNA. the relative abundance of IlP-HSDI niRNA in Isliika\va cells was assessed by an established semi-quantitative
RT-PCR protocol. as described (Tremblay el nl.. 1999). Briefly. total RNA [vas estracted from cultured cells using RNeasy kit (QIAGEN Inc.. Mississauga. Ontario. Canada) according to the maiiufacturer's instructions. Prior to use. tlie RNA samples (1-1 pg) were cliecked by agarose gel electrophoresis in the presence of formaldehyde. and the integrity of the RNA kvas assessed by the presence of two sharp bands representing 18s and 18s rRNA afier staining with ethidium bromide. One microgram of total RNA \vas reverse-transcribed using a standard oligo-dT primer in a total volume of 20 pl. An aliquot (2pi) of tlie RT reaction products \vas then subjected to a standard PCR (95 C. 55 sec: 55 C. 55 sec: 73 C- 1 min: 28 cycles) using sequence-specific primers (fonvard primer. 5.-AGTAGTTGCTGATGCGGA; rel-erse primer. j-- ACC.4CAGTCCATGCCATCAC) which correspond to nucleotides 633-64 1 and 1004-
1 02 1 iii the puhlished human 1 1 B-HSDZ cDNA (Albiston er cri.. 1 994). respectively. GAPDH n-as usrd as a control. The same PCR conditions were used for GAPDH escepr that a cycle ilumber of 23 instead of 28 was used. The primers for GAPDH (fonvard primer. 3'-ACCACAGTCCATGCCATCAC; reverse primer. 5'- TCCACCXCCCTGTTGCTGTA) were obtained from Clontech Laboratories Inc. (Palo Aito. CA). and tliey correspond to nucleotides 586-605 and 1018-1 037 in the publislied hunian GAPDH cDNA (Arcari er cri.. 1984). To verify the RT-PCR products and to assess tlir relatil-e abundance of 1 ID-HSDî mWA. a fraction of the RT-PCR products
\vas theii subjecred to a standard Southem biot analysis. usiiig [.:'PI-liunian 1 1 (3-HSD3 cDN.4 and ["PI-liiinian GAPDH cDNA as probes.
To dèter~iiinethe relative abundance of 1 IP-HSDî niRNA and GAPDH mRhrA. the relati\-s optical density of the corresponding signals on autoradiographic films were measured by scanning u-ith a Iaser densitometer (LKB 1222-020 UtraScan XL: LKB
Produkter AB. Broiilma. Slveden). as described (Sampatli-Kuiriar er rd.. 19%). In al1 cases. the sigiials were detected within the linear scan range of the densitometer. For each RNA sample. tlie ratio of 1 IP-HSD2 mRNA signal to GAPDH mRNA signal \vas calculated- and group means were obtained. Statisticai analyses of 1 I P-HSDZ mRNA and 1 1 B-HSD2 activity data were perfom~ed using one-\va>- ANOVA. follow-ed by LSD (least squares differeiice) test. Significance was set at p dehydrogeiiase activity of 11P-HSD liad a high affinity for cortisol (apparent KI,,.34.3 k 10.7 nM: Fig. 4.1 ). Collecti\~ely.these activity data indicated the presence of 1 1 P-HSDZ in Ishikaw cetls. To coiifinii the expression of IlP-HSDîin Isliikaw cells. RT-PCR u-as used to esamine the prescnce of 1 I P-HSDI mRNA. It \vas found that the niRNA for I 1 P-HSDî but not tlirit for 11P-HSDI was detected (Fig. 4.2). This indicated tiiat Isliikawa celis espress exclusi\-el>-tlie 1 1 P-HSDî isozyme. To study tlie effects of ses steroid hormones. Ishikan-a cells were treated for 7311 with 1 O nh,l estradiol-17P (E,). 100 nM medros!*progesterone acetate (MPA). or 10 nM E, plus 100 nM k1P.A. This treatnient regilne produced a classic ses steroid effect: the greatest increase in the level of 11f3-HSDî acti\-it>- \vas caused by the combined treatment. followed by MPA witli Elbeing the least effective (Fig. 4.X). To esamine the irnVolvenientof estrogen receptors (ER). Isliikau-a cells wzre treated for 7211 witli E, (10 nM). ICI 182,780 (100 nM). a pure antiestrogen. or the t\vo togetiier. \\-hile it Iiad no effect alone. the ICI compound abolislied tlie stiniulatory effects of E2 on 1 IP-HSD1 activity (Fig. 4.3B). Hoivever. when the cells v-ere treated \vit11 MPA (100 nM) plus tlie anti-progesterone RU486 (100 nM). RU186 did not counteract ivitli MPA. rather acted as an agonist: increased 1 1 P-HSDI acti\.ity (Fig. 4.X). To study the effects of glucocorticoids. Ishikawa cells u-ere treated witli 100 nb1 dexametlicisone (Des) for different lengths of time (2411. 4811, and 7211). Des resulted in a time-dependent increase in the level of II P-HSD2 activity with more than 2-fold increase afier 7211 treatment (Fig. UA). Wlien the cells were esposed to different concentrations of Des. ranging froin 1 to 1000 nM for 48h. there was a dose dependent increase in the level of 1 1 P-HSD2 activity with a significant stimulation at 10 nM (Fig. WB). To examine if tlie endogenous glucoconicoid. cortisol. was also effective in tliis regard. Ishikatva cetls LI-eretreated witli cortisol (1-1000 nh4) for 4811. This resiihed in a dose- dependent increase in the 1 1P-HSD2 activity. and the magnitude of tliis increase was comparable \kit11 that seen afier Dex treatment (Fig. 4.4C). Again. RU-186. an anti- glucocorticoid and ami-progesterone. did not block the stiniulatory effects of Dex on C 1 1P-HSDZ acti~ity(data not shown). To study the effects of EGF. Ishikan-a cells were treatsd \\-ith 10 ng/ml EGF for 24-72 II. tliere n-as a tinie-dependent decrease in tlie le\-el of 1 1P-HSD2 acrivity (Fie. -!.SA). When the ceIls xere esposed to differeiit concentrations of EGF (1. 5 and 10 ng/ml) for 4811. tliere \vas a dose dependent inhibition of IlP-HSD:! activity u-ith a significant and near masinial effect at 5 ng/ml (Fig. 3.5B). III order to deterinine if changes in 1lP-HSD2 activity follo\ving El. MPA. Dex and EGF treatment were associated with alterations in 11P-HSD2 mRNA. the reIati1.e IeveI of 11P-HSDî mRNA was assessed by a semi-quantitati~~eRT-PCR protocol. As shown in Fig. 4.6. tliere were corresponding changes in the level of 11 P-HSD:! mRN.4 following cadi treatment. In Ishikawa cells. an increase in IlP-HSD2 IIIRNA le\.el. compared to \.eliicle treated cells. was obsen~edfollou-ing treatments \vit11 E,. MPA and Dex. n-hereas a sigriificant decrease in 1 IP-HSD2 mRNA was obsen-ed in these cells following treatnient with EGF. 4.4 DISCUSSION In tlie present study. we have demonstrated for the first time that the hunian IsliiLa\w etidometrial ce11 line espresses exclusii+elytlie 1 1 P-HSD2 isozyme. Moreovcr. we liave presented the first direct elridence tfiat ses steroid hormones and glucoconicoids stimulate u-hile EGF inhibit the expression of 1 I P-HSD2 in Isliikan-a cells. suggestiiig tliat endonietrial 1 1P-HSDî is under the control of steroid hormones and EGF. Tlius. the Ishikaw ce11 line represents an excellent mode1 in which the biological function and regulatioii of endometrial 1 1 P-HSDî may be studied. Tlic driiioiistration of I 1 P-HSDZ imrnunoreacti1-ity iti ilic luniitial aiid glandular epitlielia of Iiuiiian endonietrium and the presence of 1 1P-HSD1-like enzyme activity in Iiunian eiidoiiietrial tissue homogenates sugsest an important role for 1 1 P-HSDZ in the rzlucocorticoid-mediated effects on the dynaniic function of Iiuman endonietriuiii (Smith C el LI/.. 1997). The le\-el of 11P-HSD3-like enzyme activity in the Iiunian endonietri~im xas signiticantl!- hiplier in the secretory than in the proiiferati1-e pliase of the cycle (Smith o cd.. 1997). iiidiccitiiig a stimulatory effect of progesteroile on endornetrial Il f.3- HSDl act il-il>-.Indeed. in priniac cultures of hunian endoinetrial stroma1 cells in wliich I 1P-HSDI and 2 appeared to be CO-expressed. MPA increased tlie level of both 1 l j3- HSD1- and 2-like enzyme activities. While there was a comespoiiding increase in the level of 1 I P-HSD 1 mRNA. possible changes in I 1 P-HSD2 mRNA were not studird (Arcuri c.1 td.. 1996). Tlie prescnt study demonstrated that treatnient of Isliikau-a cells i\-itli E,. MPA and El plus MPA elicited a classic sex steroid response: tlie combined treatment resulted in the greatest increase in the level of 1 IP-HSD2 activity. followed bj. MPA and witii E, being the least effective. Moreover. we have detected a correspoiiding increase in 11P- HSD7 niRNA. suggesting that the stimulatory effects of E, and MPA are mediated. at least in part. at the level of 1 I P-HSD? gene transcription. When Ishikawa cells were co- incubated witli E, and the pure anti-estrogen ICI 182.780. the ICI compound blocked the E,-induced increase in IIP-HSD2 activity. This indicated that tlie stimulatory effects of E2 on 1 1 P-HSDI activity were likely mediated througli estrogen receptors. However. the antiprogestin-aiitiplucocorticoid RU486 did not counter-act \vit11 MPA or Des. ratlier acted as an agonist: stiniulated 1 1B-HSDZ activity in Ishikau-a cells. It is note\\-onhy that RU46 lias been son previously in Ishikawa cells to function as a progrsteio~ir:'~liicoco~icoidagonist in inhibiting P-endorphin relense (Makrigiannakis LV ~1..1992) niid suppressiiig CRH gene activation (Makrigiannakis er tri.. 1996). Tlius. it is coiiceivable tliat Isliikau-a cells mal. possess an altered PR/GR n-liicli can be activated bj- RU-486. Botli cortisol and tlie syntlietic glucocorticoid. Des stiniulated tlie actil-itl-of Il P- HSDl in a dose-dependent nianner in Ishikawa cells. Funherniore. a stimulatioii of l l P- HSDl inRN.-\ n-as obsen-ed \\-lien tliese cells were treated with Des. suggesting tliat the stinmlatosy cffects of glucoconicoids were mediated. at least in pan. at [lie lewl of IlP- HSDl pie transcription. These findings. for the tirst tiiiis. suggsst a putative autoreg~i1atoi-yrole of glucocorticoids in the control of tlieir ou-n local bioactive levels by inducing tlie espression of 11 P-HSDZ. If sucli a niechanisni is operational in normal human endometrium. it is tempting to speculate tliat autoregulation of local glucocorticoid levels via IIP-HSD2 may serve to prûtect the endonietrial milieu and {lie iniplanting blastocyst froni the terarogenic effects of escessi\.e:ele\-ated glucoconicoids. It is knon-n that bot11 11P-HSDI and 2 are espressed in tlie Iiiinian endoinetriu~ii (Smith el cil.. 1997). Hoivever. following implantation and decidualization of the endometriuni. the decidua expresses only 1 1P-HSDI (Stewart er rd.. 1995: Sun er cil.. 1997). Given tliat 1 1P-HSD1 fùnctions predorninantly as a reductase. it has been suggested that 1 1 P-HSDI in the decidua may provide the fetus with an extra-adrenal source of cortisol via the arnniotic fluid (Lopez Berna1 and Craft. 198 1 ). Moreo\*er. Murphy (Miirpliy. 1977) suggested that cortisol generated from cortisone by the action of I1P-HSDI in tlie uterus during pregnancy may also play an ami-immune role in the uterine wall. However. no studies have been conducted to address the disappearance of 1 I P-HSD2 from the human endornetrium following decidualization. Tliere is accumularing evidence for the invokement of various growli factors in the implantation process and subsequent decidualization reaction. For instance. it has been suggested tliat EGF and related gro~lifactors (TGFu) ma!- play a role in rendering the endoinetriuni receptive to ernbryo implantation (Giudice, 1995). Furthemiore. EGF and its receptors are knoxn to be expressed in the endometriuni concornitantly with decidualization in Iiumans (Hofman et al.. 1993). Taken together. these findings suggest that EGF nia!- play a very important role in the endonietrium. especially at tlie time of implantation. III the present study. EGF wûs shown to inhibit the expression of Ilp- HSD2 inRNA and eiizynie actii-ity in 1shikan.a cells. suggesting tliat endoiiierrial l I P- HSD2 is under the negatii~einfluence of EGF. Thsrefore. it is possible tliat EGF iiiay bs one of the contributitig factors Ieading to the disappearance of IlP-HSD7 in the endometriiim follou-ing implantation and decidualization in huniaiis. Obviously. furtlier studies are required to test this hypothesis. Gii-en tlie potent effects of glucocorticoids on the dynarnic function of liuman endoinetriuiii and tlie cnicial role of 11P-HSDI in detenniiiiiig tlie intracellular lei-el of bioacti\.e glucoconicoids. it \vil1 be important to elucidate the underlying molecular rnechanisnis and interactions between these newly identified physiological regulators of I 1 p-HSD2. 3.5 ACKNOD'LEDGMENTS This work \vas supported by the Canadian MRC (Grant MT-171 00 to K.Y.). K. Yang is an Ontario h4inistI-y of Health Career Scientist. Fig 4. I Plot accorciing to the Michaelis-Menten equation for determining kinetic parameters of 1 1 P-HSD deliydrogeiiase activity. Data from a representative esperiment are showii. Radionietric conversion assays were conducted using intact cells. as described in the Materials and Metliods. with varying amounts of cortisol (0.035 fi 0.25 PM). V. velocity (prnohiri/well): S. substrate concentration (PM). Fig 4.2 Total crlliilar RNA was rstracted from Ishikawa crlls and subjrcted to a standard RT-PCR iitiliziiig li~iiiianspecikic primers. as described in tlir Materials and Metliods. RT-PCR prodiicts for GAPDH (espected size 450 bp). 1 I P-HSD 1 (564 bp) and 2 (512 bp) iiiRN.4 are slio\vii in lanrs 1. 2 and 3. respectively. No PCR pi-oducr \vas obserwd for 11P-MD 1 in Iane 2. Fig 4.3 Effecrs of estradiol and progesterone on 11b-HSDZ acti~ity.Ishika\\-a cells were incubated in the absence of serurn for 72h with (A) estradioi-171) (Es 10 nM). medroxyprogesrerone acetate (MPA: 100 nM). or E, (10 nM) plus MPA ( 100 nM): (B) estradiol- 1 7P (E,: 10 nM). ICI 1 82.780 (ICI: i 00 nM). or E, (10 nM) plus ICI ( 100 nM): and (C) MPA (100 nM). RU486 (RU; 100 nM). or MPA (100 nM) plus RU (100 nM). At the end of treatment. the levei of 1 LP-HSD2 activity in intact cells tvas determined by a radionletric conversion assay. as described in the Materials and Mehods. Bars represent the mean - SEM of 3-6 independent experiments. each perfornied in triplicate. * p Control Control E2 K)1 E2+lCI MPA Fig 4.4 Effects of glucocorticoids on 1 1P-HSD? activity. Ishikawa cells were incubated in the absence of semm (A) with 100 nM dexamethasone (Des)for the indicated times; (B) for 4811 witli the indicated concentrations of Des: and (C)for 4811 u-ith the indicarrd concentrations of cortisol. At tlie end of treatment. the led of i 1 P-HSD7 activity in intact cells \vas determined by a radiometric conversion assay. as described in the Materiais and Methods. Bars represent the mean +- SEM of 3-6 independent esperiments. each perforined in triplicate. * p 24 48 72 Incubation time (hours) Dexamethasone (nM) 1 1 O Cortisol (n M) Fig 4-j Effects of epidennal grouth factor (EGF) on 11P-HSD2 activity. Ishikawa cells were incubated in the absence of semm (A) uith 10 ndml EGF for the indicated tirnes: and (B) for 3811 n-ith the indicated concentrations of EGF. At the end of treatrnent. the level of 1 1 P-HSDÎ actkity in intact cells was determined b~ a radiometric conversion assay. as described in the Materials and Methods. Bars represent the mean -+ SEM of 5-6 independent esperinients. each performed in triplicate. * p EGF (nghl) Fig 4.6 Effects of steroid horn~onesand EGF on 1 1 P-HSD2 mRiï.i\. One representative autoradiograpli of the semi-quantitative RT-PCR analysis of 1 IP-HSDI m~~A(top panel) and changes in tlie relative abundance of 1 1P-HSD2 mRNA (bottom panel) in Ishikawa cells following different treatment repimes are shown. Cells were treated in semm free medium for 48h with dexarnethasone (Dex: 100 nM). EGF ( 10 ng/ml). or for 72h with estradiol-17P (E,: 10 nM). rnedroxyprogesterone acetate (MPA: 100 nM). or E, (1 0 nM) plus MPA ( 100 nM). At the end of treatmerit. total cellular RhT.4 \vas estracted and subjecrcd to a srnii-quantitative RT-PCR. followd by So~itliern blotring. as described in tlie Materials and Methods. Total RNA from hunlan endonletriuni \vas used as a positi\-e control. Bars represent the mean -+ SEM of 3-6 independent esperiments. * p<0.05. and ** pcO.0 I when compared with the control. GAPDH *** - Control Dex BS+ Control E2 MPA E2+MPA 4.6 REFERENCES Aganval AK. Tusie Luna MT. Monder C. White PC (1990). Expression of 11 beta- hydrosysteroid deh>.drogrnase using recombinant vaccinia virus. .C/ol Eïducriiwl4: 1 827- 1832. Albiston AL. Obeyesekere V. Smith R, Krozonski ZS (1991). Cloning and tissue distribution of the Iiuman 11 beta-hydroxysteroid dehydrogensae type 2 enzyme. .bfui Ce11 Eïdocrir~ol105:R 1 1 -R 1 7. Arcari P. klcirtinelli R. Salvatore F (1984). The complete sequence of a full Isngth cDNA for hunian li\-er gl>.ceraldeliyde-3-phosphate debydrogenase: el-idrnce for multiple mRN.4 species. .\i~ci~.ic.A ci& Rcs 1291 79-9 189. ArcLiri F. Monder C. Lockn-c?odCJ. Schatz F (1996). Espression of 11 P-h>.dros>-steroid deh>-drogenasç during decidualization of human endonietrial stroma1 cells. EIICIOCI-~I~OIO~~~13 7595-600. Bigsb!. 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Tannin GM. Aganvai AK. Monder C, New MI, White PC (1 99 1). The human gene for I 1p-hydrosysteroid dehydrogenase. Structure, tissue distribution. and chroniosomal localizatioii. J Biol Cher71 266: 16653- 166%. Terada N. Yamamoto R. Yamamoto T, Nishizawa Y. Tanigushi H. Terakawa N. Kitamura Y. Matsumoto K (1 991). Effect of dexarnethasone on uterine ce11 death. J Siei-oid Biochm7 Moi Bi02 38: 1 1 1-1 15. Tremblay J. Hardy DB. Pereira LE. Yang K (1999). Retinoic acid stimulates the expression of 1 1P-hydroxysteriod dehydrogenase type 2 in human clioriocarcinoma JEG- 3 cells. Biol Repmd in press. Ulick S. Levine LS. Gunczler P. Giovanni Z. Raniirez LC. Rauh Cir. Roder A. Bradlow HL. New MI (1979). A syndrome of apparent nlineralocorticoid escess associated wirh defecrs in tlie peripheral metabolism of cortisol. J ChEmlocr-iml .Iferc/b 19:757-764. White PC. Mune T. Aganval AK (1997). 1 lp-liydrosysteroid deliydrogenase and tlie syndrome of apparent mineralocorticoid excess. Endocr Rei: 1 8: 135-156. Yang K. Sinith CL. Dales D. Harnmond GL. Challis JR (1992). Cloning of an O\-ine I 1p-liydrosysteroid deliydrogenase complementary deoxyribonucleic acid: tissue and temporal distribution of its rnessenger ribonucleic acid during fetal and neonatal development. Em/ocrinolo~*1 3 1:2 120-2 126. Yang K. Yu M. (1994). Evidence for distinct isoforms of I lp-liydrosysteroid dehydrogsiiase in the ovine liver and kidney . J Smoid Bioci~cru.UJ/ Biol 49:245-25 0. CHAPTER 5 DISCUSSION The role of glucocorticoids in reproductive phyçiolog- is not fully understood: however. tliere is evidence to suggest that glucocorticoids are important in various processes of endometriai function (Salamonsen, 1996: Bigsby. 1993: Terada el cd.. 199 1 : Hoffman CI al.. 1984; GulIer et al.. 1993). Tlis action of glucocorticoids has been shown to be rnodulated by 1 1 P-HSDs. the microsonial enzymes responsible for the interconversion of bioactiw glucocorticoids to their inactive 1 1-keto metabolites. III tlie previous chapters. a series of esperirnents. aimed at elucidating the factors involved in tlie regulation of 1IP-HSDI and 2 in the ovine endometrium and Ishikawa endonietrial ce11 line. respectively. have been described. In presenting a more complete picture of 1 1 P-HSD 1 and 2 regulation in the endometrium. tliis final chapter wiI1 attempt to link togetlier tlie present findings witli those in tlie literature. Moreover. the pliysiological significaiice of EGF in the regulatioii of 11P-HSDI espression in tlie endometriiini and its possible role as a "switch" meclianisn~~iI1 be addressed. FinaIly. an outIins of potsntial future studies and summary are discussed. A prel-ious ii7 ~-iwstudy in our laboratory esamined the pattern and cellular localizatioii of 1 1 P-HSD 1 geiie expression in the oviiic utenis during rliè estrous cycle and pregnancy (Yang Cr d..1996). The mmA for 1 1 P-HSD 1 \\-as highly espressed oiily during tlir late Iuteal pliose of the estrous cycle (days 10-1 7) and tlirougliout pregnancy. In slieep. pregnaiicy and the late luteal phase of estrous cycle are characterized by elevated levels of circulating progesterone. Therefore. it was suggested by Yang and colleagues ( 1996) that the induction of 11 B-HSD 1 gene expression obseneed during botli of these periods \\-as due to the circulating progesterone. Indeed. the u-ork presented in tliis tliesis (Cliapter 3) provides evidence in support of this liypotliesis. silice the expressioii of 11B-HSD1 mRNA is induced in the endonietriuni of ovariectomizrd (OVX) sheep treated \vith progesterone for 11 days but not in tliose treated for 4 days. In a study by Arcuri and colleagues (1996). tlie synthetic progestin niedrosypro,(vzsteroiie acetate (R;IPA) u-as shown to stimulate 11P-HSD1 activity and mkWA in Iiuman endometrial stroma1 cells in vitro. especially in the presence of estrogen (Arcuri er d.. 1996). In a recent study by Burton and CO-workers(1998). the espression of 1 1P-HSD 1 in the endometrium of the rat was most certainly due to cyclic variation in ovarian steroid secretion. Tliey found tiiat 1 L P-HSDI protein (immunohistoclien~istryand Western blot analysis) \vas dramatically reduced by ovariectomy and this nas restored by estrogen with or u-itliout progesterone replacement. It was suggested. l~owever.tliat the potent effects obsen-ed witli estrogen alone (in the absence of exogenous progesterone) may have been due to the presence of endogenous progesterone in OVX rats. as the adreiial cones pro\+ides an additional source of progesterone in the rat (Burton et cd.. 1998). During the estrous c>-cle in the rat uterus. the higliest level of 1 1P-HSDI niRNA \vas found ar dicstrus (Albiston er cd.. 1995). The results of tlie esperiments drscribed witliin tliis thesis are tlierefore consistent with the previous findings in the literature tliat ses sreroid hor~iionesstitiiulate the expression of 1 1P-HSD 1 in tlie endoinetriuii~. The lack of a suitable il7 ivirr.o mode1 of buman endonietriuin lias lirnited studies on 1 1 P-HSD regdation in the hunlan endometrium. Previous work with tlie Isliika\va ceIl line lias validated its use as a model system for studies of Iiormoiially regulated eveiits in the eiidonietrial epitlielium (Somkuti et al.. 1997). Althoiigli nunisrous endometrial cancer celi lines have been establislied and characterized. the Ishikawa endometrial adenocarcinorna ce11 line appears to be an excellent ~iiodelto stiidy nomial endometrial epitlieliiini. Tliese cells. first described by Nisliida CI cd.. (1985) niaintain both ER and PR. with inducible PR identical to normal endonietrial epitheliuili (Lessey er ol., 1996: Hata and Kuraiiioto. 1992). In addition. Isliikawa cells espress many of the same enzymes and structural proteins as tlieir nornial epitlieiial counterpart (Lesse) et cd.. 1996; Makrigiannakis er nl.. 1996). The Isliika\va endometrial ceil line appeared to be an ideal candidate for our studies. since it was of epitlielial origin and possessed both PR and ER. This tliesis describes for tlie iirst time the cliarac terizrition of 1 1 P-HSD in Isl~ikawacells. Exclusive deliydrogeiiase activity aiid a Iiigli affinity for cortisol (&, - 25 nM) botli suggested tliat 1 1 P-HSD? usrspressed in this ce11 line. In tliese cells. RT-PCR analysis confirmed the espression of 1 1 P-HSD2 but not of tlie type 1 isozye of 1 1P-HSD. The present study. therefore. has identified an excellent in ~*iri-omode1 for the study of human endometrial 1 1 P-HSDZ. 5.3 Regirlorion of 1 IPHSD2 by sex sreroid hormones. glircocorricoids ortd EGF The results of the experiments describing the progestin stiiiiuiatory effects on tlie expression of 1 1 P-HSD2 in tliis thesis are consistent with the obsen-arions made by otlier croups (Sniitli er al .. 1997: Arcuri el al., 1996). However. tliose studies appear to present L contradicting results. In the study by Arcuri and colleagues ( 1996). 1 1 P-HSD1 enq-nie activity was sliown in a primary culture of endometrial stromai cells. and yet Sniith er ol. (1 997) dsiiloiistrated the localization of immunoreacti\.e I 1 P-HSDI protein almost exclusi\-el! to the luminal and glandular epitheiium of the endometrium. The present study deiiionstrates unaiiibiguously the regulation of 1 1 P-HSD2 mRNA and enzyme acti\.it!- by rstradiol and MPA in Isliikawa cells. Considering the uark by Krozon-ski's group (Siiiitli cr rd.. 1997). wliicli demonstrated ele\.ated 1 1P-HSDI rnzynir activity during tlir srcraory pliase of the menstrual cycle of bealthy u-onien. tlie role that ses steroids ph!. in regulating 1 1 P-HSD2 gene expression in Isliikan-a cells mal- lil It is houn that both IlP-HSDI and 2 are espressed in the human endometrium (Smith L>I CI/.. 1997: Arcuri er al., 1996). However. follou-ing in~plantation and decidualization of the endometrium. the decidua expresses only 1 1 P-HSDl (Stewan ri cd.. 1995). Enqme activity analysis of uterine tissues during the menstrual c)de of healthy woiiien revealed predominant oxidatiïe activity. whereas reductase activity \\-as niore premlsnt in tlie decidua during pregnancy (Murpliy. 1977: 198 1 : Lopez-Berna1 and Crafi. 198 1 : Lopez-Berna1 er rd.. 1982; Baggia el rd.. 1990: Sun er cd.. 1997). A few studies have suggested ththe prevalent reductase activity obsen-ed in the decidua ma)- pro\-ide tlie fetus witli an extra-adrenal source of cortisol in the aiiiniotic tluid (Lopez- Bernal and Crafi. 198 1 ). \vliereas Murphy (1 977) suggested tliat cortisol produced iii tlie uterus durinp pregnancy nia!- play an anti-immune roIe in the iiterine wall. This latter interpretarion appsars attractive @-en the role that tlie decidua plays in iiiimune systeni responsr duriiig pregnaiicy (Beer er al.. 1981: Beer and Sio. 1982: Pepe and Albreclit. 1995). Altliougli several theories liave been put forward witli respect to the role of 11 P- HSDl in tlie decidua. no studies to date have been conducted to address tlie disappearance of 1 1 P-HSD3 frorn the human endometnuni follou-inp decidualization. Tlirre is growing appreciation for the roles of various ron~lifactors in tlie impIantation process alid subsequent decidualization reaction. For instance. widence suggests tliat EGF and related species (TGFa) inay play a role in rendering the endometriuni receptil-e to embryo implantation (Giudice. 1995). There is accumulating evidence to suggest that EGF is important in the processes of iniplantation and decidualization in several species incIuding human (Sakakibara cr d. 1994: Sonikuti al.. 1 997: Wang et cri.. 1992). rabbit (Hofmann and Anderson. 1 990). skunk (Paria er rrl.. 1994). mouse (Das et cd.. 1994; Paria and Dey. 1990) and rat (Tamada er cd.. 1 993). EGF has also been shown to dramatically influence the HPA asis (Luger cr d.1988: Sin@- Asa and Waters. 1983). Moreover. EGF and its tyrosine-kinase receptor (EGFR) have also been shown to be espressed in the endometrium concomitan In tliis study. EGF \vas show to potently inliibit 1 IP-HSD2 expression in Isliikawa cells. To funher elucidate the regulation of 1 1P-HSD2 by EGF. an in iiiso study could be designed to investigate the switch hypothesis eluded to in the previous section. Tlie rat woiild likely be a good mode1 for tliis study for the foiloi\-ing reasoiis: (1) rat espresses bot11 isofonus of 11 P-HSD in tlie endoiiietriuiii (Burton cr rd.. 1998) (1) EGF induces decidualization in the rat (Tamada et al.. 1994) aiid (3) Ininiunoreactive 1 IP-HSDI and GR protein colocalize to the uterine epithelium in the prepanr rat (Burton et cd.. 1996). In addition. it would be interesting to address the interactions betiveen the promotor of iniman 1 1 P-HSD2 and sex steroids. glucocorticoids and EGF. Transfection of Isliilia\\-a cells \vit11 a reporter gene construct suc11 as luciferase or CAT driven by a human 1 1 P-HSD2 gene promotor could possibly identify sequences which could mediate responses to E2. MPA. Des and EGF. In conclusion. the present study has explored tlie regulation of 1 1 P-HSDI and 1 in the ovine endo~iietriumand liuman endornetrial ce11 line (Isliika\va). respectively. The folIowing tentative conclusions can be drawn froni this tliesis: 1) I 1P-HSDI iiiRiYA \vas detected in the endometrial samples froni OVX sheep treated uitli progesterone for Il days. but not from those treated for 4 days or the control aiiimals. This iiidicates that progesterone is able to induce the expression of 1 1 P-HSDI mRhTAin the ovine endometrium but requires more tlian 4 days to do so. There \vas 110 correspoiidinç induction of I 1P-HSD 1 activity by progesterone. suggssting tliat a tiiiie lag esists. 2) The Isliikau-a human endometrial adenocarcinoma ce11 linc lmbeen iised estensiwly as a iiiodel to study nomial endonietrial function silice tliis cell line espresses many of tlie sanie ciizyies. steroid receptors and structural proteins found in normal endoiiictriiiiii. The present study detemiined tlie potential utility of Isliikan-a cells as a mode1 tùr in\-estigation of tlie regulation of endonietrial 1 1 P-HSDZ. The 1 1 /3-HSD activity in tsliikan-a cells was characteristic of I lP-HSD2 in tliat it only possessed dehydroprnase activity (cortisol to cortisone) and had a higli affinity for cortisol (apparent K,, of 34 nM). RT-PCR demonstrated the presence of the ~IWAfor 1 1 P- HSD? but not tliat for I 1P-HSDI in Ishikawa cells. Taken togerlier tliese findings have establislied the presence of only 11 P-HSDZ gene in Ishi kau-a ceIls 3) In Isliikaw cells. 1 ID-HSDI enzyme activity \vas stiniulated in a dose-dependeiit nia~uierb~- Des and cortisol. These results suggest a putati\.e autoregulatory rote of glucocorticoids in the control of their ou-n 1eveIs by inducing tlie espression of 11P- 4) The ses steroids. E2 and MPA stimulated 11 P-HSDZ enzyme activity in the Ishikawa ce11 line. suggesting that endometrial I IP-HSDI is under tlie control of ses steroid hormones. 5) EGF \\.as a potenr inhibitor of I ID-HSD? enzyme activity in Isliikawa cells. It is hypotliesized that EGF may be responsible for switching off tlie expression of 1 1P- HSD2 in tlie endometrium following implantation and decidual ization in humans. 6) Tliere n-ere corresponding clianges in the mRNA for 1 1P-HSDZ follou-ing treatment of Isliikalva cells ~\*itliDex. ElMPA and EGF suggesting tliat the effects of tliese Iiorniones/factors 111ay be mediated. at least in part. at the lexl of IIf3-HSD:! gene transcriptio~i. 011tlie basis of the foregoing conclusions. it can be seen tliat this study \vil1 have a significant impact on the field of reproductive physiology both in tenus of eiucidating the role and regulation of 1 I P-HSDI and 2 gens expression in tlie mammaliaii endoiuetriuiti. and also possibl). in aidin us in understaiiding the coiiiplss inrrractions in\-oll~edin maii~iiialimi~nplantarioii and decidualization. j. 7 P~xio/ogicci/sig17@cc/17ce rmd hiiplicalion7.s of clrr-rr'17t-fiitciit7g-Y The local expression of I I P-HSDl andior 2 enzymes in the nianimalian endometrial epitlielium may play a significant role in regulating tlie level of bioactiïe ducoconicoids. Alrliough specularive. it is possible tliat such a means of regulating L glucocorticoid levels may hnction to protect an implantin: blastocyst froni the teratogenic effects of glucocorticoids. From the current study. u-e observed the regulation of 11P-HSDI and 2 by ses steroids in tlie sheep endometrium and a hunian endonietrial adenocarcino~iiace11 line. Two studies have alluded to ses steroid regulation of 11 P- HSDl and/or Z expression in the endometrium (Smith et d.. 1997: Yang er d.. 1996). Therefore. it is concei\.abIe to presume that during the luteal phase of the menstrual cyle in humans. for instance. elevated estradiol and progesterone could stimulate the espressioii 1 IP-HSD2 in the endometrium. Similar to the protective role that placenta1 I 1P-HSD2 plays in fetal de\-elopment, 1 1P-HSDZ could tlien function to inactivate bioactive glucoconicoid. and thereby act as a protective mechanism. at a rime most crucial for impIantation of the fertilized egg. 11P-HSDl and 2 in the endometrium is also expressed in cells ideally located for the necessary cross-talk and early interactions between tlie matenial and embryonic interface. Given the important role that elucocorticoids pla~in implantation and perhaps decidualization. it is likely tliat 1lP- C HSDl aiid/or 2 are involved in the local regulatioii of this steroid in the endonirltriuiu. It is not knowi u-11at role EGF plays in the regulatio~iof I IP-HSDZ espression in tlie 1iumar-i endonietrium- but tlie present study appears to suggest tlmt EGF ma' Iiaw an important role in regulating this isoqme. Wliether this occurs il7 isiiw or at a critical tiiiie of irnplantatio~iof the blastocyst is unknoun. However. one can speculate that following decidualization. EGF may function to down-regdate 1 IP-HSDZ so as to allow 1 lp- HSDl to take o\.er tlie control of glucocorticoid iiietabolism in the decidualizsd endometriiim. Further studies are. however. required to substantiate these speculations. 5.7 REFERENCES Albiston AL. Smith RE. Krozowski ZS (1995). Changes in the levels of 1 lp- hydrosysteroid deliydrogenase mRNA over the oestrus cycle in the rat. J Ster-oid Biocheni Molec Biol 5245-48 Arcuri F. Monder C. Lockwood CJ. Schatz F (1996). Espression of 1 IP-hydrosysteroid dehydrogeiiase during decidualization of human endoiiietrial stronial cells. Endoci.iirolo,q 1 3 7:595-600. Baggia S. Albreclit ED. Babischkin JS, Pepe GJ (1990). Interconversion of cortisol and cortisone in baboon trophoblast and decidual cells in culture. Ei~doci-inolo~117: 1735- 1741. Beer AE. Qiiebbeman JF. Ayers JW, Haines RF (1 98 1). 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