bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional activation of cathepsin D gene expression by growth factors
Authors: Wang F , Duan R , Chirgwin J , Safe SH
Published: December 2004

Insulin-like growth factor-I (IGF-I), transforming growth factor alpha (TGFalpha) and epidermal growth factor (EGF) induced cathepsin D gene expression and reporter gene activity in MCF-7 human breast cancer cells transiently transfected with a construct (pCD1) containing a -2576 to -124 cathepsin D gene promoter insert. In contrast, IGF-I, but not TGFalpha or EGF, induced reporter gene activity in cells cotransfected with wild-type estrogen receptor (ER) expression plasmid and a construct (pCD2) containing estrogen-responsive downstream elements from -208 to -101. Promoter deletion and mutational analysis experiments identified four GC-rich sites and an imperfect palindromic estrogen responsive element required for IGF-I activation of the ER (ligand-independent). Subsequent studies with the mitogen-activated protein kinase (MAPK) inhibitor, PD98059, and a serine(118(-ER mutant confirmed the role of the MAPK pathway for IGF-I activation of the ER in MCF-7 cells. Thus, growth factor activation of ER can mediate transactivation vs ER/Sp1 binding to GC-rich sites and represents a novel pathway for ligand-independent ER action. The divergent pathways for IGF-I and TGFalpha/EGF activation of the ER observed in MCF-7 cells contrast with previous data indicating that pathways for growth factor activation of the ER are dependent on the gene and/or gene promoter and on cell context.
Androgen receptor localisation and turnover in human prostate epithelium treated with the antiandrogen, casodex
Authors: Waller AS , Sharrard RM , Berthon P , Maitland NJ
Published: December 2004

In vitro models of normal and malignant human prostate are currently limited to a few well established cell lines that, with a single exception (LNCaP), fail to express the androgen receptor (AR) - a common characteristic of prostatic epithelium grown in culture. To investigate the molecular mechanism of action of the non-steroidal antiandrogen Casodex (bicalutamide) against wild-type AR, we have established a transient AR expression model in non-tumorigenic prostate cells of both epithelial and mesenchymal origin. In this model, both dihydrotestosterone and Casodex can effectively transport the AR protein into the nucleus of prostate cells. Whereas the natural ligand, dihydrotestosterone, stabilises the receptor, the AR is rapidly degraded at a nuclear location when the transfected cells are treated with Casodex. In contrast, whereas the mutant AR in the LNCaP line is also degraded on Casodex treatment over the same time period, its intracellular targeting is defective.
Immunoelectron microscopic localization of 3beta-hydroxysteroid dehydrogenase and type 5 17beta-hydroxysteroid dehydrogenase in the human prostate and mammary gland
Authors: Pelletier G , Luu-The V , El-Alfy M , Li S , Labrie F
Published: December 2004

The subcellular distribution of steroidogenic enzymes has so far been studied mostly in classical endocrine glands and in the placenta. In the peripheral intracrine organs which synthesize sex steroids there is no indication about the organelles which contain the enzymes involved in steroid biosynthesis. We have thus investigated the subcellular localization of two enzymes involved in the production of sex steroids, namely 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and type 5 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Using specific antibodies to these enzymes, we conducted immunoelectron microscopic studies in two peripheral tissues, namely the human prostate and mammary gland. In the prostate, immunolabelling for both 3beta-HSD and type 5 17beta-HSD was detected in the basal cells of the tube-alveoli as well as in fibroblasts and endothelial cells lining the blood vessels. In all the labelled cell types, the gold particles were distributed throughout the cytoplasm. No obvious association with any specific organelle could be observed, although some concentration of gold particles was occasionally found over bundles of microfilaments. In mammary gland sections immunolabelled for 3beta-HSD or type 5 17beta-HSD localization, labelling was observed in the cytoplasm of the secretory epithelial cells in both the acini and terminal ducts. Immunolabelling was also found in the endothelial cells as well as in fibroblasts in stroma and blood vessels. The gold particles were not detected over any organelles, except with the occasional accumulation of gold particles over microfilaments. The present data on the localization of two steroidogenic enzymes leading to the synthesis of testosterone indicate that these enzymes are located not only in epithelial cells but also in stromal and endothelial cells in both tissues studied. The absence of any association of the enzymes with membrane-bound organelles appears as a common finding in the reactive cell types of two peripheral tissues.
Molecular cloning of the mouse homologue of Rab3c
Authors: Pavlos NJ , Xu J , Papadimitriou JM , Zheng MH
Published: December 2004

Small GTP-binding proteins of the Rab subfamily are key regulators of intracellular vesicle transport. Here we report the isolation of a cDNA clone encoding the complete Rab3c isoform from mouse embryo using a degenerative PCR-based approach. Multiple sequence alignment revealed that the predicted amino acid sequence was identical to the previously identified rat Rab3c isoform and 98% identical to the published bovine Rab3c GTPase from brain. Furthermore by in situ hybridisation, Rab3c mRNA was detectable within various regions of the brain, cartilage and highly enriched within intestinal villi of foetal tissues. Chondrocytes in the hypertrophic zone, but not reserve or proliferative zones, expressed high levels of Rab3c. This pattern of expression corresponds with the genesis of matrix vesicles during endochondral ossification.In all, our results suggest that in addition to its functional role during regulated secretion in brain, Rab3c may play a part in matrix vesicle trafficking during skeletal development.
Estrogen response element binding induces alterations in estrogen receptor-alpha conformation as revealed by susceptibility to partial proteolysis
Authors: Ramsey TL , Klinge CM
Published: December 2004

Genes whose expression is highly induced by estradiol (E(2)) contain multiple estrogen response elements (EREs) in their promoters. Previously we reported that estrogen receptor-alpha (ERalpha) binds cooperatively to and E(2) synergistically activates reporter gene expression from three or four tandem copies of a consensus ERE (EREc38). Here we evaluated how ERalpha binding to one, two, three or four tandem copies of EREc38 affects ERalpha conformation as detected by altered ERalpha trypsin digestion patterns in Western blots. E(2)- or 4hydroxytamoxifen (4-OHT)-occupied ERalpha bound to the pS2 ERE or to a single copy of EREc38 showed enhanced susceptibility to trypsin digestion compared to E(2)- or 4-OHT-ERalpha incubated with DNA lacking an ERE. ERalpha binding to multiple tandem copies of EREc38 further increased sensitivity to trypsin digestion. These results correlate with synergistic transcription and cooperativity of ERalpha binding to multiple tandem copies of EREc38. These observations suggest that EREc38 binding alters the overall conformation of ERalpha and that multiple tandem copies of EREc38 enhance these conformational changes. We hypothesize that ERE-induced alterations in ERalpha conformation modulate interaction with coregulatory proteins, resulting in synergistic transcriptional activation.
Pegvisomant: structure and function
Authors: Pradhananga S , Wilkinson I , Ross RJ
Published: December 2004

Pegvisomant is the pegylated form of mutant growth hormone (B2036). B2036 has increased affinity in one binding site and lowered affinity in its second binding site, it has been shown that this molecule still enables dimerisation of the growth hormone receptor at the cell surface but does not allow the necessary conformational changes for signalling. Pegylation decreases the antagonistic activity of B2036, however the rate of clearance of the pegylated B2036 is greatly reduced compared to the unpegylated form. Even though the antagonistic activity of pegvisomant is lower than B2036, the reduced rate of clearance makes it an effective clinical drug for the treatment of conditions such as acromegaly.
Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems
Authors: Bustin SA
Published: December 2004

The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.
Transient expression pattern of prolactin in Sparus aurata
Authors: Herrero-Turrion MJ , Rodriguez RE , Aijon J , Lara JM
Published: December 2004

Using reverse transcription-polymerase chain reaction and in situ hybridization, the expression of the prolactin (PRL) gene was determined during development in gilthead sea bream (Sparus aurata) for the first time. The mRNA for PRL was detected from the second day of the larval stage onwards. This transcript was also located in the adenohypophysial cells, starting at four days post-hatching and was found to be pituitary-specific. Moreover, the possible involvement of PRL in asynchronous growth in the cultivation of gilthead sea bream was also examined. No differences in the distribution of PRL cells were observed in the three sizes of juvenile gilthead sea bream studied. These results suggest that the transcription of PRL is involved in the early development stages of sea bream and that the asynchronous growth-related changes are not due to distinct distribution of PRL cells.
'Location, location, location': activation and targeting of MAP kinases by G protein-coupled receptors
Authors: Luttrell LM
Published: December 2004

A growing body of data supports the conclusion that G protein-coupled receptors can regulate cellular growth and differentiation by controlling the activity of MAP kinases. The activation of heterotrimeric G protein pools initiates a complex network of signals leading to MAP kinase activation that frequently involves cross-talk between G protein-coupled receptors and receptor tyrosine kinases or focal adhesions. The dominant mechanism of MAP kinase activation varies significantly between receptor and cell type. Moreover, the mechanism of MAP kinase activation has a substantial impact on MAP kinase function. Some signals lead to the targeting of activated MAP kinase to specific extranuclear locations, while others activate a MAP kinase pool that is free to translocate to the nucleus and contribute to a mitogenic response.
AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase
Authors: Kefas BA , Cai Y , Ling Z , Heimberg H , Hue L , Pipeleers D , Van de Casteele M
Published: December 2004

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.
A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors
Authors: Chopin-Delannoy S , Thenot S , Delaunay F , Buisine E , Begue A , Duterque-Coquillaud M , Laudet V
Published: December 2004

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.
Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus)
Authors: Vischer HF , Bogerd J
Published: December 2004

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.
Do unliganded thyroid hormone receptors have physiological functions?
Authors: Chassande O
Published: December 2004

Thyroid hormone (TH) is required for the development of vertebrates and exerts numerous homeostatic functions in adults. TH acts through nuclear receptors which control the transcription of target genes. Unliganded and liganded thyroid hormone receptors (TRs) have been shown to exert opposite effects on the transcription of target genes in vitro. However, the occurance of an aporeceptor activity in vivo and its potential physiological significance has not been clearly addressed. Several data generated using experimental hypothyroidism and thyrotoxicosis in wild type and TR knockout mice support the notion that apoTRs have an intrinsic activity in several tIssues. ApoTRs, and in particular TRalpha1, are predominant during the early stages of vertebrate development and must be turned into holoTRs for post-natal development to proceed normally. However, the absence of striking alterations of embryonic and fetal development in mice devoid of TRs indicates that apoTRs do not play a fundamental role. During development, as well as in adults, apoTRs rather appears as a system which increases the range of transcriptional responses to moderate variations of T3.
Diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type II diabetic rat model: a possible cause for erectile dysfunction in diabetes
Authors: Jesmin S , Sakuma I , Salah-Eldin A , Nonomura K , Hattori Y , Kitabatake A
Published: December 2004

Erectile dysfunction (ED) is commonly experienced in men with diabetes mellitus. Vascular endothelial growth factor (VEGF) has been extensively documented for its pathogenic significance in different complications of diabetes. We hypothesized that expressions of VEGF, its receptors and its signaling pathway Akt may be drastically altered in diabetic penile tIssues and their alterations may modulate penile expression of the molecules that are believed to play a role in diabetic ED. Otsuka Long-Evans Fatty (OLETF) rats, a type II (non-insulin-dependent) diabetes mellitus, were used at the insulin-resistant stage of type II diabetes (20 weeks of age). We determined protein and mRNA expressions of VEGF, its receptors, Akt, nitric oxide synthase isoforms, and apoptosis-related molecules in the penis using immunohistochemistry, Western blotting, in situ hybridization, and real-time quantitative PCR analyses. The penile sections were also submitted to the Tdt-mediated dUTP nick end labeling assay for apoptosis. OLETF rats showed marked reductions in penile expression of VEGF, its two receptors and Akt. In OLETF rat penises, endothelial and neuronal nitric oxide synthase isoforms were expressed less abundantly. Furthermore, while anti-apoptotic markers, Bcl-2 and phosphorylated Bad, were down-regulated, pro-apoptotic markers, active caspase-3 and Bax, were up-regulated, resulting in the appearance of apoptotic cells in the penile tIssues of OLETF rats. The VEGF signaling system would work less well in diabetic penile tIssues as a result of the reduced expression, leading to diminished endothelial production of nitric oxide and apoptosis-related erectile tIssue damage. We propose that the abnormalities of the VEGF signaling system in the penis may play a role in the pathophysiology of diabetic ED.
Cytokines and nitric oxide inhibit the enzyme activity of catalase but not its protein or mRNA expression in insulin-producing cells
Authors: Sigfrid LA , Cunningham JM , Beeharry N , Lortz S , Tiedge M , Lenzen S , Carlsson C , Green IC
Published: December 2004

Pancreatic beta-cells have low activities of the antioxidant enzyme catalase. Nitric oxide interacts with the haem group of catalase inhibiting its activity. We have studied the activity of catalase in beta-cells under conditions mimicking prediabetes and in which nitric oxide is generated from cytokine treatment in vitro. We also studied whether there is regulation of catalase enzyme activity by nitric oxide at the protein or gene expression level. RINm5F insulin-producing cells, treated for 24 h with cytokines, showed increased medium nitrite production (17+/-2.2 vs 0.3+/-0.2 pmol/ micro g protein) and significantly decreased cellular catalase activity (42.4+/-4.5%) compared with control cells. A similar reduction was seen in catalase-overexpressing RIN-CAT cells and in rat or human pancreatic islets of Langerhans. Catalase activity was also suppressed by the long-acting nitric oxide donor diethylenetriamine/nitric oxide adduct (Deta-NO) and this inhibition was reversible. The inhibition of catalase activity by cytokines in RINm5F cells was significantly reversed by the addition of the nitric oxide synthase 2 (NOS2) inhibitors nitro monomethylarginine or N-(3-(aminomethyl)benzyl)acetamidine (1400W). Protein expression was found to be unchanged in cytokine- or Deta-NO-treated RINm5F cells, while mRNA expression was marginally increased. We have shown that inhibition of catalase activity by cytokines is nitric oxide dependent and propose that this inhibition may confer increased susceptibility to cytokine- or nitric oxide-induced cell killing.
Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways
Authors: Gao XH , Dwivedi PP , Omdahl JL , Morris HA , May BK
Published: December 2004

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at -114/-101 and a CCAAT box at -62/-51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKCzeta contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKCzetaK281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKCzeta signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH)2D3 levels.
Tightly regulated and inducible expression of a yoked hormone-receptor complex in HEK 293 cells
Authors: Meehan TP , Puett D , Narayan P
Published: December 2004

We have previously reported the construction of a constitutively active luteinizing hormone receptor by covalently linking a fused heterodimeric hormone to the extracellular domain of the G protein-coupled receptor. This yoked hormone-receptor complex (YHR) was found to produce high levels of cAMP in the absence of exogenous hormone. Stable lines expressing YHR were generated in HEK 293 cells to obtain lines with different expression levels; however, in a relatively short time of continued passage, it was found that YHR expression was greatly reduced. Herein, we describe the development of clonal lines of HEK 293 cells in which the expression of YHR is under the control of a tetracycline-regulated system. Characterization of clonal lines revealed tight control of YHR expression both by dose and time of incubation with doxycycline. These experiments demonstrated a good correlation between expression levels of the receptor and basal cAMP production. Moreover, the reduction in receptor expression following doxycycline removal revealed that YHR mRNA and protein decayed at similar rates, again suggesting a strong linkage between mRNA and protein levels. The controlled expression of YHR in this cell system will allow for a more detailed analysis of the signaling properties associated with constitutive receptor activation and may prove to be advantageous in developmental studies with transgenic animals.
The manifold actions of the protein inhibitor of activated STAT proteins on the transcriptional activity of mineralocorticoid and glucocorticoid receptors in neural cells
Authors: Tirard M , Jasbinsek J , Almeida OF , Michaelidis TM
Published: December 2004

Corticosteroid actions in the brain are exerted via the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). These receptors share several structural and functional similarities but their activation in the brain triggers distinct biological actions, for instance on neuronal survival or the regulation of the hypothalamo-pituitary-adrenal axis. Like other hormone-activated receptors, the transcriptional properties of the MR and GR depend on their ability to recruit a variety of co-regulators, which modulate their activity on target promoters, in a specific manner. The N-terminal regions of the MR and GR share the smallest degree of sequence conservation, whereas they display opposite effects on the transactivation properties of these receptors; thus, they may provide surfaces suitable for receptorspecific interactions with co-regulatory proteins. Here, we employed a yeast two-hybrid system to identify molecules interacting with the N-terminal part of the MR (amino acids 170-433). This approach resulted in the isolation of representative cDNAs from all members of the protein inhibitor of activated STAT (PIAS) family of proteins as potential MR-interacting partners. In neural cells, PIAS3 exhibited a strong and specific interaction with MR, but not GR, as indicated by mammalian two-hybrid assays and co-immunoprecipitation experiments in vivo. The interaction with MR was enhanced in the presence of aldosterone, an MR agonist, and was found to occur through a conserved, serine- and acidic amino acid residue-rich domain of PIAS3. To compare the modulatory properties of PIAS proteins on MR and GR transcriptional activity in a neural environment, MMTV reporter gene assays were performed in the human neuroblastoma cell line SK-N-MC. This analysis revealed that PIAS3 can inhibit MR, but not GR, transactivation in response to their corresponding ligands. Further, it showed that PIAS1 and PIASxbeta, but not PIASy, could also inhibit MR-mediated transcription despite the lack of detected physical interaction with MR. Interestingly, PIASxbeta and PIASy dose-dependently co-activated GR, whereas PIAS1 impaired GR-induced transcription. Taken together the results reveal differential modulatory roles of the PIAS proteins on the transcriptional properties of MR and GR, thus providing new insights into the bifurcating actions of these two receptors in neural cells where they are frequently co-localized.
Analysis of the estrogen regulation of the zebrafish estrogen receptor (ER) reveals distinct effects of ERalpha, ERbeta1 and ERbeta2
Authors: Menuet A , Le Page Y , Torres O , Kern L , Kah O , Pakdel F
Published: December 2004

We have previously cloned and characterized three estrogen receptors (ER) in the zebrafish (zfERalpha, zfERbeta1 and zfERbeta2). We have also shown that they are functional in vitro and exhibit a distinct expression pattern, although partially overlapping, in the brain of zebrafish. In this paper, we have shown that the hepatic expression of these zfER genes responds differently to estradiol (E2). In fact, a 48-h direct exposure of zebrafish to E2 resulted in a strong stimulation of zfERalpha gene expression while zfERbeta1 gene expression was markedly reduced and zfERbeta2 remained virtually unchanged. To establish the potential implication of each zfER in the E2 upregulation of the zfERalpha gene, the promoter region of this gene was isolated and characterized. Transfection experiments with promoter-luciferase reporter constructs together with different zfER expression vectors were carried out in different cell contexts. The data showed that in vivo E2 upregulation of the zfERalpha gene requires ERalpha itself and a conserved transcription unit sequence including at least an imperfect estrogen-responsive element (ERE) and an AP-1/ERE half site at the proximal transcription initiation site. Interestingly, although in the presence of E2 zfERalpha was the most potent at inducing the expression of its own gene, the effect of E2 mediated by zfERbeta2 represented 50% of the zfERalpha activity. In contrast, zfERbeta1 was unable to upregulate the zfERalpha gene whereas this receptor form was able to tightly bind E2 and activate a reporter plasmid containing a consensus ERE. Altogether, these results indicated that the two ERbeta forms recently characterized in teleost fish could have partially distinct and not redundant functions.
Expression of the orphan nuclear receptor ERRalpha is under circadian regulation in estrogen-responsive tissues
Authors: Horard B , Rayet B , Triqueneaux G , Laudet V , Delaunay F , Vanacker JM
Published: December 2004

Circadian gene expression has been demonstrated in many tissues and involves both positive and negative regulatory loops. The potential interferences of circadian rhythmicity with other well-known biologic rhythms, such as the ovarian cycle, at least in part controlled by estrogens, has not been questioned. The estrogen receptor-related receptor (ERR)alpha is an orphan nuclear receptor that is widely expressed in estrogen-responsive tissues such as liver, uterus and bone. In addition, expression of the ERRalpha gene has been proposed to be transcriptionally controlled by estrogens in the uterus. Here we show that the expression of ERRalpha displays a circadian rhythmicity in liver, bone and uterus. This is in contrast to other uterine estrogen-regulated genes. Analysis of clock/clock mutant mice shows that ERRalpha is an output gene of the circadian clock oscillator. The expression of clock-control genes, such as Bmal1 and Rev-erbalpha, also displays diurnal oscillations in the uterus, but not in bone. In this tissue, however, Per2 displayed a rhythmic expression, altogether suggesting unconventional loops in the regulation of circadian rhythm in bone.
Gene expression profiling of glucocorticoid-inhibited osteoblasts
Authors: Leclerc N , Luppen CA , Ho VV , Nagpal S , Hacia JG , Smith E , Frenkel B
Published: December 2004

Glucocorticoid (GC) treatment for the management of autoimmune and inflammatory diseases is associated with decreased bone formation and increased risk for fracture. In MC3T3-E1 cell cultures, 0.1-1 microM dexamethasone (DEX) arrests development of the osteoblast phenotype when administration commences at a commitment stage around the time of confluency. To gain new insights into GC-induced osteoporosis, we performed microarray-based gene expression analysis of GC-arrested MC3T3-E1 cultures, 2.5 days after the administration of DEX. Of the >12 000 transcripts interrogated, 74 were up-regulated and 17 were down-regulated by at least 2.5-fold (P < or = 0.05). Some of these genes, such as Mmp13, Serum/GC-regulated kinase and Tieg, have previously been reported as GC-responsive. Others are shown here for the first time to respond to GCs. DEX strongly repressed Krox20/Egr2 at both the mRNA and the protein level. This is especially significant because mice lacking this transcription factor develop osteoporosis. The data also suggest that the bone morphogenetic protein (BMP) pathway, which is involved in regulating bone mass, and other pathways that influence BMP signaling, are abrogated by GCs: (i) DEX increased the mRNA levels of the BMP antagonists Follistatin and Dan; (ii) DEX increased the levels of p21 Rasgap3 and Ptpn16/MKP-1 mRNAs, negative regulators of the MAP kinase pathway; and (iii) DEX decreased Cox mRNA levels. DEX also increased thrombospondin mRNA levels, which negatively regulate bone mass in vivo, as well as the adipocytic marker Fkbp51. These and other observations disclose novel gene targets, whose regulation by GCs in osteoblasts may shed light on and provide new therapeutic approaches to osteoporosis.
Dimerization is required for transactivation by estrogen-receptor-related (ERR) orphan receptors: evidence from amphioxus ERR
Authors: Horard B , Castet A , Bardet P-L , Laudet V , Cavailles V , Vanacker J-M
Published: December 2004

The estrogen-receptor-related (ERR) receptors are orphan members of the nuclear receptor superfamily that bind to their specific DNA target sites as homodimers. However, it has not been shown whether this mode of binding is required for the transcriptional activation they drive. We here show that heterodimerization can also occur between these receptors. Furthermore, we demonstrate that the unique amphioxus ortholog of ERR genes (AmphiERR) is expressed as two isoforms differing by an in-frame insertion. While the short isoform behaves like its mammalian counterparts, the long isoform (AmphiERR(L)) displays divergent transcriptional properties according to the target site to which it binds. Indeed, AmphiERR(L) binds as a monomer but does not activate transcription through the SF1 response element (SFRE). On the contrary, this isoform binds as a homodimer and activates transcription through the classical estrogen-response element. Our results strongly suggest that dimerization is required for transactivation exerted by the ERR receptors.
Aromatase is abundantly expressed by neonatal rat penis but downregulated in adulthood
Authors: Jesmin S , Mowa C N , Sakuma I , Matsuda N , Togashi H , Yoshioka M , Hattori Y , Kitabatake A
Published: December 2004

Although synthesis of estrogen by male gonads has been well documented for over half a century, it is only recently that the role of estrogen in male reproductive events has gained appreciation. We recently reported abundant expression of estrogen receptor (ER)-α and -β in different cell types of the rat penis, whose levels diminished with advancing age. The present study, which builds on data from the ER study, was designed to determine whether the penis is capable of generating its own local estrogen by examining evidence of the expression of aromatase, a microsomal enzymatic complex which irreversibly converts androgens to estrogens, using immunohistochemistry, Western blotting, in situ hybridization and real-time PCR analyses. Secondly, the effects of sex steroid hormones on penile aromatase were examined. Discrete aromatase immunoreactive cells were localized in primordial corpus cavernosum, corpus spongiosus and os penis, blood vessels and sensory corpuscle of glans penis. In situ hybridization signals corresponded with immunohistochemical findings. Western blot, enzyme immunoassay and real-time PCR analyses of rat penile samples revealed an age-dependent expression of aromatase and estrogen, with levels at week 1 almost resembling those of the ovary, but they decreased sharply by week 8, and decreased further by week 35. This expression pattern was strikingly similar to that of ER-α reported previously. Testosterone and diethylstilbesterol administered prenatally upregulate levels of aromatase mRNA and protein, and estrogen postnatally. Dihydrotestosterone upregulated aromatase mRNA and protein, but not estrogen. We conclude that estrogen acts via ER in a paracrine and/or autocrine manner to regulate penile events, particularly during development, and that estrogen synthesis is regulated by estrogen and androgens.
Prolactin release from subpopulations of rat lactotrophs in the presence and absence of dopamine
Authors: Hu L , Lawson D M
Published: December 2004

In this study we examined the effects of dopamine (DA) and its withdrawal on in vitro prolactin (PRL) release from subpopulations of lactotrophs from two regions of the anterior pituitary obtained from untreated ovariectomized (OVX) rats or OVX rats treated with estrogen, progesterone or a combination of the two. Anterior pituitaries were cut horizontally into an inner (dorsal) zone and an outer (ventral) zone. Each of these regions was enzymatically dispersed and the resulting cells were otherwise untreated (unseparated) or centrifuged through a discontinous Percoll gradient to separate the cells into two subpopulations (light and heavy cells). Each of these types of cells was perifused for 1 h with culture medium containing 1 μm DA followed by medium devoid of DA for 1 h. Prolactin released into the perifusion medium, collected as 5-min fractions, was measured by radioimmunoassay and normalized to the number of lactotrophs in the cellular pools as determined by immunocytochemistry.

In the presence of DA, PRL release from unseparated cells of the outer zone was significantly increased by estradiol treatment compared with the release from similar cells from OVX rats. (Differences were considered significant where P<0·05.) However, no effect of estradiol treatment was observed with unseparated cells of the inner zone or light or heavy cells from either zone. Progesterone had no effect on any cell type when administered alone. However, when progesterone was given following estradiol, PRL release from unseparated cells of the inner zone was increased significantly compared with similar cells from the other steroid-treated groups. Similar significant increases were observed with light and heavy cells of the outer zone, but there was no effect of the combined steroid treatment on light or heavy cells from the inner zone. When DA was withdrawn, prolactin release was significantly increased from all cells except unseparated cells of the outer zone of OVX rat pituitaries. However, when the cells of the outer zone from OVX rats were separated into light and heavy cells, they responded to the withdrawal of DA with significant and equivalent increases in prolactin release. Light cells of the inner zone of pituitaries from OVX rats were more responsive to DA withdrawal than were heavy cells. Estradiol increased the response to the withdrawal of DA by light and heavy cells of the outer zone and heavy cells of the inner zone. Progesterone significantly reversed these effects of estradiol on separated cells.

These results suggest that lactotrophs in two regions of rat pituitaries respond differently to dopamine and to its withdrawal, that subpopulations of lactotrophs within these regions also respond differently and that steroids modulate these responses.

Journal of Endocrinology (1996) 148, 113–120
Expression of collagenase-3 in the rat ovary during the ovulatory process
Authors: Balbín M , Fueyo A , López J M , Díez-Itza I , Velasco G , López-Otín C
Published: December 2004

We have examined the expression of the murine counterpart of human collagenase-3, a matrix metalloproteinase produced by breast carcinomas, in the course of processes which involve extensive tissue remodeling. By using Northern blot analysis, we have found that collagenase-3 is expressed in the rat ovary, but not in the remaining analyzed tissues including brain, kidney, liver, lung, mammary gland, uterus, bladder, heart, intestine, prostate, spleen, testis and thymus. Collagenase-3 mRNA was detected at high levels in rat ovaries at proestrus and estrus, was at a minimum at metestrus and started to increase during diestrus through to proestrus. In addition, collagenase-3 was also detected on day 21 of pregnancy, which is approximately one day before parturition. However, no significative expression was detected in RNA from ovaries taken immediately after parturition, or on days 1, 5 or 30 postpartum. Northern blot analysis also revealed that collagenase-3 was not expressed at significant levels, compared with ovarian expression, in the uterus or in the mammary gland during pregnancy or after parturition. When follicular granulosa cells were separated from residual ovarian tissue and their RNA was analyzed by Northern blot, it was seen that collagenase-3 was not expressed by the granulosa cells but was present in the residual tissue containing interstitial and thecal tissues, growing follicles and corpora lutea. Immunohistochemical studies also confirmed, at the protein level, the localization of collagenase-3 in rat ovary. Gonadotropic stimulation of ovulation in immature rats by priming with pregnant mare's serum gonadotropin and stimulation with human chorionic gonadotropin failed to induce the expression of collagenase-3, suggesting that additional factors which are not present in the immature stimulated rats are needed for completely effective induction of the expression of this matrix metalloproteinase. On the basis of these results, together with the comparative analysis of expression of different matrix metalloproteinases in the rat ovary, we propose that collagenase-3 is a major ovarian metalloproteinase potentially involved in ovarian function during the reproductive cycle.

Journal of Endocrinology (1996) 149, 405–415
Interaction between insulin-like growth factor-I and insulin-like growth factor-binding proteins in TC-1 stromal cells
Authors: Grellier P , Feliers D , Yee D , Woodruff K , Abboud S L
Published: December 2004

IGF-I and -II play an important role in regulating bone formation. Bone marrow stromal cells, particularly those with osteoblast-like features, may act in concert with osteoblasts to increase IGF-I and -II levels in the bone microenvironment. Local bioavailability of IGFs, however, is modulated by IGF binding proteins (IGFBPs). We have previously demonstrated that murine TC-1 stromal cells constitutively secrete IGF-I and IGFBPs. In the present study, we determined the phenotype of these cells and used them as a model to explore the effect of IGFBPs on IGF-I-induced mitogenesis. The effect of IGF-I on IGFBPs expressed by TC-1 was also determined. When grown under conditions that promote osteogenic differentiation, TC-1 cells showed high alkaline phosphatase activity and mRNA levels, weakly expressed osteocalcin mRNA, and formed mineralized bone-like nodules. TC-1 cells expressed IGF-I and IGF-II mRNAs, while other stromal phenotypes preferentially expressed IGF-I. IGF-I stimulated TC-1 DNA synthesis in a dose-dependent manner and this effect was inhibited by recombinant IGFBP-1 and -4. Since IGF-I may regulate IGFBP production, the effect of IGF-I on IGFBPs expressed by TC-1 cells was determined. IGF-I increased the abundance of IGFBP-3, -4 and -5 in TC-1 conditioned medium; this correlated with induction of IGFBP-3 mRNA, but not with that of IGFBP-4 or -5 mRNAs. The findings demonstrate that most stromal cells express IGF-I which may act in an autocrine and/or paracrine fashion. The local effects of IGF-I, however, may be blocked by IGFBP-1 or -4. IGF-I regulates the relative abundance of IGFBPs in stromal cells which, in turn, may influence IGF-I-mediated effects on bone remodeling.

Journal of Endocrinology (1996) 149, 519–529
Thyroid hormone induces the synthesis of a putative protein in the rat granulosa cell which stimulates progesterone release
Authors: Bandyopadhyay A , Roy P , Bhattacharya S
Published: December 2004

125I-3,5,3′-tri-iodothyronine (T3) binds specifically to a pure nuclear preparation from rat granulosa cells. A Scatchard analysis of T3 binding showed a Kd of 0·65 × 10−9 mol/l and a Bmax of 1·57 pmol/mg DNA. The biological relevance of T3 binding to granulosa cells was evaluated by adding T3 (20 ng/incubation) to granulosa cells (1 × 106 cells/incubation) which greatly stimulated progesterone release. T3-stimulated progesterone release was significantly inhibited by actinomycin-D (P<0·01) and cycloheximide (P<0·01). T3 caused about a twofold increase in granulosa cell protein synthesis as compared with the control which was inhibited by actinomycin-D and cycloheximide. The addition of T3 to granulosa cell incubations also resulted in a more than 2·5-fold increase in mRNA. The results indicated that T3 stimulation of progesterone release is mediated via T3-induced protein(s) or TIP. TIP was located in the soluble supernatant fraction (100 000 g supernatant; 100 k sup) from T3-incubated cells but could not be detected in the 100 k sup from the control cells or LH-incubated cells. TIP was purified based on its biological activity, i.e. its addition to granulosa cell incubations stimulated progesterone release into the medium. The 100 k sup from T3-incubated granulosa cells was subjected to Sephadex G-75 gel filtration, FPLC Mono Q and FPLC Superose 6 chromatography which resulted a 273-fold purification over the starting material and a clearly homogeneous protein was obtained. SDS-PAGE of purified TIP showed it to be a 53 kDa monomer protein. Experiments conducted with radiolabelled TIP suggested internalization of TIP into the granulosa cell. The results therefore showed that T3 induces the synthesis of mRNA and proteins in rat granulosa cells and that one of the proteins is TIP which, in turn, stimulates progesterone release from the cell, suggesting thereby that this putative protein is a novel mediator of T3 function in the granulosa cell.

Journal of Endocrinology (1996) 150, 309–318