Oestrogens exert their actions via specific nuclear protein receptors that are members of the steroid/thyroid receptor superfamily of transcription factors. Recently, a second oestrogen receptor (ERbeta) has been cloned, and using reverse transcription-PCR and immunohistochemistry it has been shown to have a wide tissue distribution in the rat that is distinct from the classical oestrogen receptor, ERalpha. Using commercial polyclonal antisera against peptides specific to human ERbeta, we have determined the sites of ERbeta expression in archival and formalin-fixed human tissue and compared its expression with that of ERalpha. ERbeta was localised to the cell nuclei of a wide range of normal adult human tissues including ovary, Fallopian tube, uterus, lung, kidney, brain, heart, prostate and testis. In the ovary, ERbeta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea, whereas ERalpha was weakly expressed in the nuclei of granulosa cells, but not in the theca nor in the copora lutea. In the endometrium, both ERalpha and ERbeta were observed in luminal epithelial cells and in the nuclei of stromal cells but, significantly, ERbeta was weak or absent from endometrial glandular epithelia. Epithelial cells in most male tissues including the prostate, the urothelium and muscle layers of the bladder, and Sertoli cells in the testis, were also immunopositive for ERbeta. Significant ERbeta immunoreactivity was detected in most areas of the brain, with the exception of the hippocampus - a tissue that stained positively for ERalpha. In conclusion, the almost ubiquitous immunohistochemical localisation of ERbeta indicates that ERbeta may play a major role in the mediation of oestrogen action. The differential expression of ERalpha and ERbeta in some of these tissues suggests a more complex control mechanism in oestrogenic potential than originally envisioned.
AH Taylor and F Al-Azzawi
PS Leung and C Sernia
The blood-borne renin-angiotensin system (RAS) is known best for its role in the maintenance of blood pressure and electrolyte and fluid homeostasis. However, numerous tIssues show intrinsic angiotensin-generating systems that cater for specific local needs through actions that add to, or differ from, the circulating RAS. The male reproductive system has several sites of intrinsic RAS activity. Recent focus on the epididymis, by our laboratories and by others, has contributed important details about the local RAS in this tIssue. The RAS components have been localized morphologically and topographically; they have been shown to be responsive to androgens and to hypoxia; and angiotensin has been shown to influence tubular, and consequently, fluid secretion. Components of the RAS have also been found in the testis, vas deferens, prostate and semen. Angiotensin II receptors, type 1 and, to a lesser extent, type 2 are widespread, and angiotensin IV receptors have been localized in the prostate. The roles of the RAS in local processes at these sites are still uncertain and have yet to be fully elucidated, although there is evidence for involvement in tubular contractility, spermatogenesis, sperm maturation, capacitation, acrosomal exocytosis and fertilization. Notwithstanding this evidence for the involvement of the RAS in various important aspects of male reproduction, there has so far been a lack of clinical evidence, demonstrable by changes in fertility, for a crucial role of the RAS in male reproduction. However, it is clear that there are several potential targets for manipulating the activity of the male reproductive system by interfering with the locally generated angiotensin systems.
R S Guenette, H B Corbeil, J Léger, K Wong, V Mézl, M Mooibroek and M Tenniswood
After weaning, the mammary gland ceases lactation and involutes. The wet weight of the gland decreases by 70% within 4 days of weaning. This involves significant tissue remodelling as the ducts regress and return to the resting state. The presence of apoptotic bodies in the luminal epithelial compartment 2 to 3 days after weaning provides clear evidence that a substantial proportion of the regression is attributable to the induction of active cell death (ACD) of the epithelial cells.
These changes in the architecture of the gland were found to be mirrored by changes in gene expression. The steady-state level of β-casein mRNA decreased rapidly after weaning from the high levels seen during lactation to undetectable levels by 8 days after weaning. The steady-state levels of expression of a number of genes associated with ACD, including TRPM-2, tissue transglutaminase (TGase) and poly(ADP-ribose) polymerase (PARP), increased transiently during this time-frame. The steady-state level of TRPM-2 mRNA increased 2 days after weaning, reaching a peak on day 4, and decreasing to undetectable levels by day 8 after weaning. The steady-state levels of two other mRNAs, TGase and PARP, showed very similar kinetics. In contrast, the mRNA for Hsp 27, which has been shown to be induced during prostate regression, was not significantly induced in the regressing mammary gland. In-situ hybridization demonstrated that the TRPM-2, TGase and PARP genes were expressed predominantly in the luminal epithelial cells of the ducts. These cells expressed β-casein mRNA during lactation, and underwent ACD after weaning.
While the ultrastructural changes in the mammary gland after weaning, and the induction of TRPM-2, TGase and PARP mRNAs, are reminiscent of apoptosis in the prostate, several features of the process are different. Most notably, the disruption of the secretory processes and the lack of increased expression of Hsp 27 in the regressing mammary gland suggest that there may be a number of important events in ACD that are not common to all cells.
C Gutacker, R Flach, P Diel, G Klock and C Koch-Brandt
Clusterin (gp 80, apolipoprotein J, TRPM-2) is a widely expressed multifunctional glycoprotein. Its demonstrated and proposed functions include the transport of lipids and membrane fragments, the inhibition of the cytolytic action of the terminal complement complex and the modulation of cell—cell interactions. The expression of the gene is enhanced during tissue injury and remodelling and by hormone-withdrawal-induced apoptosis of prostate and mammary cells. We show here that, in the kidney-derived epithelial cell line MDCK, clusterin mRNA is repressed by glucocorticoids and by progesterone. Treatment with epidermal growth factor also represses clusterin gene expression in MDCK cells. Incubation with 12-O-tetradecanoyl-phorbol-13-acetate, which activates protein kinase C (PKC), induces clusterin mRNA, while chelerythrine, an inhibitor of PKC, represses clusterin gene expression, suggesting that the clusterin gene responds to signalling pathways involving PKC. These results open up the possibility of studying the complex regulation of the clusterin gene by multiple signal transduction pathways within a single cell type, and most importantly, of characterizing interactions between the individual signal transduction cascades.
H Hiroi, S Inoue, T Watanabe, W Goto, A Orimo, M Momoeda, O Tsutsumi, Y Taketani and M Muramatsu
In order to investigate the localization of estrogen receptor (ER) alpha and ERbeta in the reproductive organs in the rat, polyclonal antibodies were raised to each specific amino acid sequence. The Western blot with anti-ERalpha antibody showed a 66 kDa band in rat ovary and uterus, while that with anti-ERbeta antibody detected a 55 kDa band in rat ovary, uterus and prostate. The ligand-independent nuclear localization of the two receptors was verified by immunocytochemistry. By immunohistochemistry, the nuclei of glandular and luminal epithelial cells in the uterus were stained with anti-ERalpha antibody, whereas only the nuclei of glandular epithelium cells were stained with anti-ERbeta antibody. In rat ovary, positive signals were shown with anti-ERbeta antibody in the nuclei of granulosacells. No specific immunostaining was observed with anti-ERalpha antibody. Although ERbeta was immunostained at the proestrous, metestrous and diestrous stages, the immunoreactivity of ERbeta was hardly detected at the estrous stage in rat ovary. Thus, we show differential expression of ERalpha and ERbeta in rat uterus and ovary at the protein level, which may provide a clue for understanding the roles of the two receptors in reproductive organs.
CW Gregory, B He and EM Wilson
Activation domains in the 114 kDa androgen receptor (AR) NH(2)- and carboxyl-terminal regions are thought to contribute to different extents to AR-mediated transactivation. We investigated using anti-peptide antibodies whether smaller AR forms that migrate like the previously described 87 kDa AR-A occur in vivo resulting in constitutive or increased gene activation. Immunoblots of prostate cancer and fibroblast cell culture extracts revealed 114 and 84 kDa AR forms. Antibody mapping indicated the 84 kDa AR lacked the ligand-binding domain and comigrated with the constitutively active AR fragment AR1-660. AR expressed in COS cells was 114 and 92 kDa. Migration of the 92 kDa AR was slightly slower than that of a 90 kDa expressed fragment that was designed to initiate at the second methionine (residue 189) and lacked the NH(2)-terminal FxxLF interaction sequence. The 92 kDa AR did not result from alternative initiation since it was observed when the second methionine was changed to alanine. Optimization of extraction conditions indicated that both 84 and 92 kDa forms resulted from in vitro proteolytic cleavage and that cleavage by caspase-3 could account for the 92 kDa form. The results suggest that AR forms with gel mobility similar to that of the previously described 87 kDa AR-A result from in vitro proteolytic cleavage of NH(2)- or carboxyl-terminal regions during cell extraction and storage and that smaller forms with increased transcriptional activity do not occur in vivo.
JG Lemmen, RJ Arends, AL van Boxtel, PT van der Saag and B van der Burg
With the aim of developing an in vivo model that directly detects activation of estrogen receptors (ERs), transgenic mice carrying a luciferase reporter gene were generated. The luciferase reporter gene was under the control of three consensus estrogen-responsive elements (EREs) coupled to a minimal TATA-box, with or without flanking chick beta-globin insulators. By using this model in combination with the IVIS imaging system, in vivo ER activation was measured. Dose- and time-dependent luciferase activity was induced in various organs of adult transgenic male mice exposed to diethylstilbestrol (DES) (10-1000 micro g/kg) and 17beta-estradiol dipropionate (EP) (10-1000 micro g/kg), when luciferase activity was measured ex vivo. The highest (>10 000-fold) induction of luciferase was measured in bone and kidney 24 h after exposure to 1000 micro g/kg EP. Other highly responsive organs include liver, testis, pituitary, brain, prostate and colon, which show different activity profiles. This in vivo model for detecting estrogenic activity can be used to assess tissue-specific action of ER agonists and antagonists. These could include selective ER modulators and environmental estrogens. In combination with the IVIS imaging system, this in vivo model is a powerful tool for assessing the kinetics of gene activation by estrogenic compounds.
K Hofman, JV Swinnen, F Claessens, G Verhoeven and W Heyns
In search of potential androgen receptor coregulators we performed a yeast two-hybrid screening using the androgen receptor ligand-binding domain as bait and a human prostate cDNA library as prey and found that the carboxy-terminal domain of retinoblastoma-associated Kruppel protein (RbaK), a member of the Kruppel zinc finger protein family, interacts in a ligand-dependent way with the ligand-binding domain of the androgen receptor. RBaK was recently identified as a transcriptional regulator that interacts with the retinoblastoma protein and thereby influences E2F regulated transcription. The interaction of RBaK with the androgen receptor was further documented using mammalian two-hybrid experiments, in vitro binding studies and coimmunoprecipitation. Finally, we demonstrated that both RBaK and the retinoblastoma protein coactivate androgen receptor-mediated transcription in cotransfection experiments. In conclusion, our data show that RBaK interacts with the androgen receptor and increases its transcriptional activity. Moreover, the double interaction of RBaK with the retinoblastoma protein and with the androgen receptor provides a novel link between the androgen receptor and the regulation of the cell cycle.
LK Beitel, YA Elhaji, R Lumbroso, SS Wing, V Panet-Raymond, B Gottlieb, L Pinsky and MA Trifiro
The androgen receptor (AR) N-terminal domain plays a critical role in androgen-responsive gene regulation. A novel AR N-terminal-interacting protein (ARNIP) was isolated using the yeast two-hybrid system and its interaction with amino acids 11-172 of the normal or corresponding region of the polyglutamine-expanded human AR confirmed by glutathione S-transferase pulldown assays. ARNIP cDNAs cloned from NSC-34 (mouse neuroblastoma/spinal cord) or PC-3 (human prostate adenocarcinoma) mRNA encoded highly homologous 30 kDa (261 amino acids) cysteine-rich proteins with a RING-H2 (C3H2C3 zinc finger) domain; this motif is highly conserved in predicted ARNIP-homologous proteins from several other species. Expression of the approximately 1.7 kb ARNIP mRNA was detected in various tissues by Northern blotting, but was highest in mouse testes, kidney and several neuronal cell lines. In addition, the human ARNIP protein was found to be encoded by nine exons spanning 32 kb on chromosome 4q21. In COS-1 cells, coexpression of ARNIP and AR did not affect AR ligand-binding kinetics, nor did ARNIP act as a coactivator or corepressor in transactivation assays. However, AR N-terminal:C-terminal interaction was reduced in the presence of ARNIP. Intriguingly, ARNIP, and in particular its RING-H2 domain, functioned as a ubiquitin-protein ligase in vitro in the presence of a specific ubiquitin-conjugating enzyme, Ubc4-1. Mutation of a single cysteine residue in the ARNIP RING-H2 domain (Cys145Ala) abolished this E3 ubiquitin ligase activity. Fluorescent protein tagging studies revealed that AR-ARNIP interaction was hormone-independent in COS-1 cells, and suggest that colocalization of both AR and ARNIP to the nucleus upon androgen addition may allow ARNIP to play a role in nuclear processes. Thus, identification of a novel AR-interacting protein with ubiquitin ligase activity will stimulate further investigation into the role of ubiquitination and the ubiquitin-proteasome system in AR-mediated cellular functions.
Y Zhang, I Dufort, P Rheault and V Luu-The
It has been suggested that 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) is a T-cell differentiation marker in mice. In the human, this enzyme has generally been associated with types 1 and 2 17beta-HSDs, which belong to the short-chain alcohol dehydrogenase family, whereas the rat, rabbit, pig and bovine 20alpha-HSDs are members of the aldoketo reductase superfamily, which also includes the 3alpha-HSD family. In this study, we report the cloning, from a human skin cDNA library, of a cDNA that shows, after transfection into human embryonic kidney (HEK-293) cells, high 20alpha-HSD activity but negligible 3alpha- and 17beta-hydroxysteroid dehydrogenase activities. A comparison of the amino acid sequence of the human 20alpha-HSD with those of other related 20alpha- and 3alpha-HSDs indicates that the human 20alpha-HSD shares 79.9, 68.7 and 52.3% identity with rabbit, rat and bovine 20alpha-HSDs, whereas it shows 97, 84 and 65% identity with human type 3, type 1 and rat 3alpha-HSDs. In contrast, the enzyme shares only 15.2 and 15.0% identity with type 1 and type 2 human 17beta-HSDs. DNA analysis predicts a protein of 323 amino acids, with a calculated molecular weight of 36 767 Da. In intact transfected cells, the human 20alpha-HSD preferentially catalyzes the reduction of progesterone to 20alpha-hydroxyprogesterone with a K(m) value of 0.6 microM, the reverse reaction (oxidation) being negligible. In a cell cytosolic preparation, the enzyme could use both NADPH and NADH as cofactors, but NADPH, which gave 4-fold lower K(m) values, was preferred. We detected the expression of 20alpha-HSD mRNA in liver, prostate, testis, adrenal, brain, uterus and mammary-gland tissues and in human keratinocyte (HaCaT) cells. The present study clearly indicates that the genuine human 20alpha-HSD belongs to the aldoketo reductase family, like the 20alpha-HSDs from other species.