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
T Florio and G Schettini
Somatostatin is a peptide widely distributed in both the central nervous system (CNS) and peripheral tissues. It is found as two bioactive peptides of 14 and 28 amino acids, the latter being an N-terminal-extended form (Reichlin 1983a).
The name somatostatin comes from its initial discovery as an inhibitor of growth hormone (GH) release from anterior pituitary cells (Brazeau et al. 1973). Since then, numerous other physiological activities of somatostatin have been discovered associated with differing peptide and receptor localization (Reichlin 1983a,b). Besides GH, somatostatin is also able to inhibit secretion of prolactin (PRL) and thyroid-stimulating hormone (TSH) (Reichlin 1983a). In the CNS, the highest somatostatin concentrations have been detected in the hypothalamus in the tuberoinfundibular neurons where, acting as a neurohormone, the peptide regulates the hypothalamic-hypophyseal axis (Schettini 1991). Somatostatin-containing neurons are also present in many other areas of the brain, such as the cerebral cortex,
HF Vischer and J Bogerd
A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino acids, specific for the TSH-R subfamily, was also present in the carboxy terminus of the amino-terminal extracellular domain of the cfTSH-R. Next to the testis and thyroid follicles, abundant cfTSH-R expression was detected in cerebellum, brain, ovary, seminal vesicles and pituitary, while weaker expression was found in muscle, stomach, intestine, head-kidney, liver, kidney and heart. HEK-T 293 cells, transiently expressing the cfTSH-R, significantly increased intracellular cAMP levels in response to human TSH. Catfish LH, human choriogonadotropin and human FSH were also able to induce this cfTSH-R-mediated response, although with considerably lower efficiency than human TSH. These results indicated that a functional cfTSH-R had been cloned from the testis of African catfish.
JM Weitzel, S Hamann, M Jauk, M Lacey, A Filbry, C Radtke, KA Iwen, S Kutz, A Harneit, PM Lizardi, and HJ Seitz
Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPARgamma coactivator 1 family (PGC-1) are regulated by T3. Only PPARgamma and PERC/PGC-1beta exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.
KS Wang, RB Hodgetts, and WR Addison
Expression of the rat alpha 2u-globulin gene family is regulated in the adult male liver by a number of hormones, including growth hormone, thyroid hormone and several steroids. Upon injection into ovariectomized females, estrogens first induce alpha 2u-globulin expression and then suppress this gene after several days of hormone administration. To study this phenomenon, we developed a mouse L-cell line that expressed the human estrogen receptor. High levels of rat alpha 2u-globulin transcript were induced in stable transfectants of this line carrying a cloned alpha 2u-globulin gene, following exposure to 17 beta-estradiol. Since this induction was inhibited by cycloheximide, the response to estrogen, as to other steroids, appears to be secondary. Using genes with variously deleted 5'-upstream regions, sequences responsible for this induction were located between -730 bp and -223 bp relative to the start of transcription. Examination of the DNA in this region revealed that an estrogen receptor element was located at -590 bp in an area that is highly conserved in most known alpha 2u-globulin genes. Administration of both dexamethasone and estrogen produced a synergistic effect in this system. The induction of alpha 2u-globulin RNA by estrogen in L-cells may re-capitulate the initial response to estrogen in vivo, and therefore represents a good model system to seek the identity of the other factors required to effect full induction.
GS Seetharamaiah, S Kaithamana, RK Desai, and BS Prabhakar
Expression of large quantities of conformationally intact thyrotropin receptor (TSHR) is essential to understand the structure-function relationship of the receptor. We expressed three different constructs of full-length human TSHR in insect cells: (a) a TSHR cDNA lacking signal sequence (TSHR-ns), (b) a TSHR cDNA containing human TSHR signal sequence (TSHR-hs) and (c) a TSHR cDNA with baculovirus envelope protein encoded signal sequence gp-67 (TSHR-gp). No unique protein band, corresponding to any of these recombinant proteins, was visible upon Coomassie Blue staining after SDS-PAGE. However, Western blot using TSHR specific monoclonal antibody showed unique bands around 80, 100 and 100 kDa in TSHR-ns, TSHR-hs and TSHR-gp virus infected insect cells respectively. All three full-length TSHR proteins could neutralize the TSH binding inhibitory immunoglobulin (TBII) activity from sera of experimental animals. However, only glycosylated proteins (TSHR-hs and TSHR-gp) neutralized the TBII activity of sera from autoimmune thyroid patients, confirming the importance of glycosylation for patient autoantibody reactivity. Expression levels of full-length TSHR proteins were much lower than the levels of similarly produced corresponding ectodomains of TSHR proteins. Southern blot and Northern blot analyses showed that DNA and RNA levels in full-length TSHR virus infected insect cells were comparable to the levels found in cells infected with viruses encoding only the ectodomain of TSHR. These data suggest that full-length TSHR expression is very low and is regulated at the translational level.
S Costagliola, L Alcalde, J Ruf, G Vassart, and M Ludgate
The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21–414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cRl vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting.
To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes. Subsequently, immunoglobulins from mice immunized with MBP-ECD were shown to contain TSH-binding inhibiting activity and to inhibit TSH-mediated cyclic AMP production; these mice had a lower serum thyroxine level when compared with mice immunized with the MBP—β galactosidase fusion protein MBP-GAL.
The study shows the feasibility of using recombinant TSHR expressed in E. coli (i) to produce antibodies which recognize the native receptor and thus could be applied to studies of TSHR expression (e.g. in thyroid tumours), (ii) to establish animal models of autoimmune hypothyroidism and (iii) as the starting material in denaturation and refolding experiments which may help in defining structure—function relationships.
PP Dwivedi, GE Muscat, PJ Bailey, JL Omdahl, and BK May
Repression of basal transcription of a 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) responsive 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter construct as observed in kidney cells in the absence of ligand and this repression was dependent on a functional vitamin D response element (VDRE). Basal repression was also seen with a construct where a consensus DR-3-type VDRE was fused to the thymidine kinase promoter. Expression of a dominant negative vitamin D receptor (VDR) isoform that strongly bound to the VDRE motif in the CYP24 promoter ablated basal repression. This VDR isoform lacked sequence in the hinge- and ligand-binding domains implicating one or both of these domains in basal repression. It is well known that thyroid hormone and retinoic acid receptors silence basal transcription of target genes in the absence of ligands and this repressor function can be mediated by the nuclear receptor corepressor N-CoR. Two variants of N-CoR have been described, RIP13a and RIP13delta1. N-CoR and the variants contain two receptor interaction domains, ID-I and ID-II, which are identical except region ID-II in RIP13delta1 has an internal deletion. We have used the mammalian two hybrid system to investigate whether VDR, in the absence of ligand 1,25-(OH)2D3, can interact with these domains. The data showed that unliganded VDR does not interact with either ID-I or ID-II from RIP13a and RIP13delta1, but does interact strongly with a composite domain of ID-I and ID-II from RIP13delta1 (but not from RIP13a) and this strong interaction is abrogated in the presence of ligand. This finding implicates RIP13delta1 in VDR-dependent basal repression of the promoter constructs under investigation. However, over-expression of RIP13delta1 in kidney cell lines did not alter basal expression of the CYP24 promoter construct. It is concluded that either the level of endogenous RIP13delta1 in these kidney cells permits maximal repression or that repression occurs by a mechanism that is independent of RIP13delta1. Alternatively, repression may be dependent on RIP13delta1 but requires an additional cofactor that is limiting in these cells.
Amy Warner and Jens Mittag
enter the brain ( Dumitrescu et al . 2006 , Trajkovic et al . 2007 ). The final level of thyroid hormone signalling control is given by the expression of nuclear thyroid hormone receptors encoded by the genes THRA and THRB ( Yen et al . 2006
S. Harvey and J. S. Baidwan
The number, but not affinity, of binding sites for [3H]3-methyl-histidine2-TRH ([3H]Me-TRH) on chicken adenohypophysial plasma membranes was increased in chickens made hypothyroid by goitrogen (methimazole) treatment (50 mg/kg per day for 7 days), which also increased circulating GH concentrations. Daily i.p. injection of thyroxine (T4; 100 μg/kg for 7 days) had no effect on [3H]Me-TRH binding to pituitary membranes, although it suppressed endogenous GH secretion. Binding of [3H]Me-TRH to pituitary caudal lobe membranes was, however, suppressed by tri-iodothyronine (T3) injected chronically (100 μg/kg per day, i.p., for 7 days) or acutely (100 μg/kg, 2 h before being killed). The suppression of [3H]Me-TRH binding and inhibition of GH secretion following T3 administration was dose related. Binding of [3H]Me-TRH to caudal lobe membranes was also suppressed following the incubation of pituitary glands with T3 in vitro, and the response was both dose and time related.
These results suggest that T3 inhibits GH secretion in fowl by a down-regulation of pituitary TRH receptors. However, other mechanisms are involved in thyroidal inhibition of GH release in birds, since T4 had no effects on [3H]Me-TRH binding yet suppressed GH secretion in vivo.