Biomedical subjects

W W Chin

Publications and source records attributed to W W Chin.

At least 37 records · Page 2Linked to original sources

PubMed1999

Identification and characterization of the gonadotropin-releasing hormone response elements in the mouse gonadotropin-releasing hormone receptor gene.

The response of the pituitary gonadotrope to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of GnRHR gene expression. Several hormones have been implicated in this regulation, most notably GnRH itself. Despite these observations and the central role that GnRH is known to play in reproductive development and function, the molecular mechanism(s) by which GnRH regulates transcription of the GnRHR gene has not been well elucidated. Previous studies in this laboratory have identified and partially characterized the promoter region of the mouse GnRHR gene and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within the 1.2-kilobase 5'-flanking sequence. By using deletion and mutational analysis as well as functional transfection studies in the murine gonadotrope-derived alphaT3-1 cell line, we have localized GnRH responsiveness of the mouse GnRHR gene to two DNA sequences at -276/-269 (designated Sequence Underlying Responsiveness to GnRH-2 (SURG-2), which contains the consensus sequence for the activating protein-1-binding site) and -292/-285 (a novel element designated SURG-1), and demonstrated that this response is mediated via protein kinase C. By using the electrophoretic mobility shift assay, we further demonstrate that a member(s) of the Fos/Jun heterodimer superfamily is responsible in part for the DNA-protein complexes formed on SURG-2, using alphaT3-1 nuclear extracts. These data define a bipartite GnRH response element in the mouse GnRHR 5'-flanking sequence and suggest that the activating protein-1 complex plays a central role in conferring GnRH responsiveness to the murine GnRHR gene.

Animals↗

PubMed1999

Human trabecular meshwork cells as a thyroid hormone target tissue: presence of functional thyroid hormone receptors.

PURPOSE: To determine whether human trabecular meshwork cells (HTM) are a potential target tissue for thyroid hormone (3,3',5-triiodothyronine, T3). METHODS: Cultured HTM were assayed for the presence of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs) by reverse-transcriptase polymerase chain reaction (RT-PCR) to detected TR and RXR mRNA, and by immunohistochemistry to detect nuclear TR and RXR proteins. Functionality of the TR was determined by analysis of 125I-T3 binding affinity and capacity in HTM nuclear extracts. Effects of T3 on modulation of hyaluronic acid (HA) levels in HTM were measured as a function of dose and duration of T3 administration. RESULTS: Analysis of RT-PCR and immunohistochemistry demonstrated that cultured HTM expressed TRalpha1, TRalpha2, and TRbeta1 but not TRbeta2; and RXRalpha but not RXRbeta and RXRgamma isoforms. Saturation binding and analysis of 125I-T3 to HTM nuclear extracts revealed Kd of 57 pM. The number of T3 binding sites extrapolated from a Scatchard plot was 7.3 x 10(10)/microg of HTM nuclear protein extract. T3 supplementation reduced the concentration of HA in the cell medium by 32-43% compared to cells grown in the absence of T3. CONCLUSIONS: Cultured HTM express three TR isoforms and one RXR isoform, bind T3 with an affinity similar to that of TR in responsive cells, and modulate their HA production in response to T3. These findings indicate that the human trabecular meshwork tissue has the capacity to respond to thyroid hormones.

Adult↗

PubMed1999

ROR alpha augments thyroid hormone receptor-mediated transcriptional activation.

This study is designed to clarify the role of an orphan nuclear hormone receptor, ROR alpha, on thyroid hormone (TH) receptor (TR)-mediated transcription on a TH-response element (TRE). A transient transfection study using various TREs [i.e., F2 (chick lysozyme TRE), DR4 (direct repeat), and palindrome TRE] and TR and ROR alpha1 was performed. When ROR alpha1 and TR were cotransfected into CV1 cells, ROR alpha1 enhanced the transactivation by liganded-TR on all TREs tested without an effect on basal repression by unliganded TR. By electrophoretic mobility shift assay, on the other hand, although ROR alpha bound to all TREs tested as a monomer, no (or weak) TR and ROR alpha1 heterodimer formation was observed on various TREs except when a putative ROR-response element was present. The transactivation by ROR alpha1 on a ROR-response element, which does not contain a TRE, was not enhanced by TR. The effect of ROR alpha1 on the TREs is unique, because, whereas other nuclear hormone receptors (such as vitamin D receptor) may competitively bind to TRE to exert dominant negative function, ROR alpha1 augmented TR action. These results indicate that ROR alpha1 may modify the effect of liganded TR on TH-responsive genes. Because TR and ROR alpha are coexpressed in cerebellar Purkinje cells, and perinatal hypothyroid animals and ROR alpha-disrupted animals show similar abnormalities of this cell type, cross-talk between these two receptors may play a critical role in Purkinje cell differentiation.

Animals↗

PubMed1999

Expression of steroid receptor coactivator-1 mRNA in the developing mouse embryo: a possible role in olfactory epithelium development.

Ligand-dependent nuclear hormone receptors (NRs), such as retinoic acid and thyroid hormone receptors, play critical roles in diverse aspects of development. They enhance or repress transcription by recruiting an array of coactivator and corepressor proteins, which function as signaling intermediates between the NRs and the basal transcriptional machinery. To study the possible involvement of these cofactors on tissue-specific regulation of gene expression by NRs, we examined the expression of the coactivator SRC-1 mRNA during mouse embryogenesis by in situ hybridization (ISH). 35S-labeled riboprobe specific for SRC-1 mRNA was used for analysis. The distribution of this transcript was studied from 8.5 to 18.5 embryonic days (E8.5-E18.5) and in postnatal day 15 (P15). The SRC-1 transcript was largely ubiquitously expressed, even on E8.5. At E14.5 and E18.5, highest levels of SRC-1 transcript was found in the olfactory epithelium. Significant SRC-1 hybridization signal was also detected in the neocortex, anterior pituitary and heart. We conclude that (1) SRC-1 mRNA is widely expressed in the developing embryo, and (2) SRC-1 mRNA is expressed at the highest level in the olfactory epithelium, suggesting that this coactivator may be involved in the development and/or function of the olfactory system.

Animals↗

PubMed1999

The gonadotropin-releasing hormone receptor gene promoter directs pituitary-specific oncogene expression in transgenic mice.

Our previous work has shown that 1.2 kb of the 5' flanking region of the mouse GnRH receptor (mGnRH-R) gene is sufficient to direct tissue-specific expression in vitro. In this study, we have used the cell-specific regulatory sequences of the mGnRH-R gene promoter to target the expression of the simian virus 40 virus T antigen (TAg) to the pituitary gland of transgenic mice. A hybrid transgene, GnRH-R/TAg, was prepared using the -1164/+52 region of the mGnRH-R gene and +2533/+5234 sequences encoding the large T antigen of the simian virus 40. Two founders developed tumors of apparent pituitary origin at 44 (M28, female) and 50 (M25, male) days of age. M28 and M25 mice were about 50% underweight, and their gonads were grossly underdeveloped compared with wild-type litter mates. A third male founder, M29, developed a tumor at a later time (109 days). M29 was able to breed successfully and stably transmit the GnRH-R/TAg transgene. Mice of the M29 transgene line developed tumors at 4-5 months of age. Gross examination showed that the tumors extend from the sella and infiltrate into the inferior surface of the brain. In small tumors collected from young transgenic animals, normal pituitary cells as well as transition areas of increasing cellular atypia are evident. Frankly malignant cells are seen in all tumors. The pituitary tumors express the alpha-, FSHbeta-, and LHbeta-subunits and the GnRH-R messenger RNA, all markers of a gonadotrope but not of other anterior pituitary cell lineages. In summary, our studies indicate that 1.2 kb of the 5'-flanking region of the mGnRH-R gene can be used to target expression specifically to the gonadotropes of the pituitary gland in transgenic mice. The GnRH-R gene promoter-directed expression appears to be cell-specific and results in the formation of tumors that are primarily of gonadotropic origin.

Animals↗

PubMed1999

Escape from the acute Wolff-Chaikoff effect is associated with a decrease in thyroid sodium/iodide symporter messenger ribonucleic acid and protein.

In 1948, Wolff and Chaikoff reported that organic binding of iodide in the thyroid was decreased when plasma iodide levels were elevated (acute Wolff-Chaikoff effect), and that adaptation or escape from the acute effect occurred in approximately 2 days, in the presence of continued high plasma iodide concentrations. We later demonstrated that the escape is attributable to a decrease in iodide transport into the thyroid, lowering the intrathyroidal iodine content below a critical inhibitory threshold and allowing organification of iodide to resume. We have now measured the rat thyroid sodium/iodide symporter (NIS) messenger RNA (mRNA) and protein levels, in response to both chronic and acute iodide excess, in an attempt to determine the mechanism responsible for the decreased iodide transport. Rats were given 0.05% NaI in their drinking water for 1 and 6 days in the chronic experiments, and a single 2000-microg dose of NaI i.p. in the acute experiments. Serum was collected for iodine and hormone measurements, and thyroids were frozen for subsequent measurement of NIS, TSH receptor, thyroid peroxidase (TPO), thyroglobulin, and cyclophilin mRNAs (by Northern blotting) as well as NIS protein (by Western blotting). Serum T4 and T3 concentrations were significantly decreased at 1 day in the chronic experiments and returned to normal at 6 days, and were unchanged in the acute experiments. Serum TSH levels were unchanged in both paradigms. Both NIS mRNA and protein were decreased at 1 and 6 days after chronic iodide ingestion. NIS mRNA was decreased at 6 and 24 h after acute iodide administration, whereas NIS protein was decreased only at 24 h. TPO mRNA was decreased at 6 days of chronic iodide ingestion and 24 h after acute iodide administration. There were no iodide-induced changes in TSH receptor and thyroglobulin mRNAs. These data suggest that iodide administration decreases both NIS mRNA and protein expression, by a mechanism that is likely to be, at least in part, transcriptional. Our findings support the hypothesis that the escape from the acute Wolff-Chaikoff effect is caused by a decrease in NIS, with a resultant decreased iodide transport into the thyroid. The observed decrease in TPO mRNA may contribute to the iodine-induced hypothyroidism that is common in patients with Hashimoto's thyroiditis.

Animals↗

PubMed1999

Promoter-specific regulation of the brain-derived neurotropic factor gene by thyroid hormone in the developing rat cerebellum.

Thyroid hormone (TH) plays a critical role in normal cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. This action could be exerted in part through brain-derived neurotropic factor (BDNF), as cerebellar BDNF messenger RNA (mRNA) expression is lower, and replacement of BDNF partially reverses the abnormal neurogenesis in the hypothyroid rat. The rat BDNF gene consists of four noncoding exons (exons I-IV), each of which is linked to a different promoter, and a protein-coding exon (exon V). To study promoter-specific regulation of the BDNF gene by TH, ribonuclease protection assay of each exon mRNA was performed using total developing rat cerebellar RNA. During cerebellar development, all exon mRNAs were detected, but with different expression patterns; among noncoding exon mRNAs, exon II mRNA was the most abundant. Daily TH replacement induced a 3-fold increase in exon II mRNA on postnatal day (P) 15. On P30, exon II mRNA was still much greater in the TH-replaced animal. Exon I mRNA was detected on P2 and P7. However, in contrast to exon II mRNA, TH treatment suppressed the expression of exon I mRNA on P2. Exon III and IV mRNAs were not detected on P2 and P7, but small amounts were observed starting on P15 in TH-replaced animals. They were not detected by P30 in hypothyroid animals. In contrast, in the cerebral cortex, although all exons are differentially regulated during development, the expression of each mRNA was not significantly altered by TH. These results indicate that TH regulates BDNF gene expression in a promoter-, developmental stage-, and brain region-specific manner, which may play an important role in region- and stage-specific regulation of brain development by TH.

Aging↗

PubMed1999

The protein kinase C system acts through the early growth response protein 1 to increase LHbeta gene expression in synergy with steroidogenic factor-1.

Expression of the LHbeta gene has been shown to be modulated by both the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1, Egr-1. It is also well known that LHbeta mRNA levels are increased after hormonal activation of the protein kinase C (PKC) signaling system, for example by GnRH; however, the mechanisms by which the PKC system exerts this effect has not been fully characterized. By transient transfection of the GH3 cell line, we demonstrate that activation of the PKC system with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases activity of region -207/+5 of the rat LHbeta gene promoter (approximately 2-fold) and markedly augments SF-1-induced stimulation (95-fold in the presence of both factors vs. 13-fold for SF-1 alone). Mutation of the two previously identified Egr-1 sites not only prevents Egr-1 effects on the LHbeta gene promoter, but also eliminates the synergistic response to PMA and SF-1 together, findings that were confirmed in a longer construct spanning region -797/+5. In the gonadotrope-derived cell line, alphaT3-1, these mutations eliminate the GnRH responsiveness of the -207/+5 LHbeta promoter construct. We next show that PMA treatment (GH3 and alphaT3-1 cells) or GnRH treatment (alphaT3-1 cells) induces expression of Egr-1, as detected by Egr-1 interaction with Egr-1 DNA-binding sites in the rat LHbeta gene promoter sequence. Furthermore, we demonstrate that PMA increases steady-state Egr-1 mRNA levels via increased Egr-1 transcription. We conclude that PMA-induced stimulation of LHbeta gene expression is achieved, at least in part, by induction of Egr-1 expression.

Animals↗

PubMed1998

Intracellular proteolytic cleavage of 9-cis-retinoic acid receptor alpha by cathepsin L-type protease is a potential mechanism for modulating thyroid hormone action.

We previously reported that the responsiveness of hepatocytes to thyroid hormone is markedly attenuated when they were cultured as monolayers rather than spheroids. To elucidate the mechanisms underlying the altered responsiveness, thyroid hormone receptor auxiliary proteins in the hepatocytes were analyzed by electrophoretic mobility shift assay. The major thyroid hormone receptor auxiliary protein was identified as 9-cis-retinoic acid receptor alpha (RXRalpha) in the hepatocytes regardless of the culture conditions. The cytoplasmic fraction was shown to contain a protease(s) that cleaves RXRalpha at its amino terminus. The presence of the protease in the cytosol, but not in the nucleus, was ascertained by incubating full-length 35S-labeled RXRalpha with each fraction. Using various protease inhibitors, it was shown that cathepsin L-type protease could participate in the cleavage of the RXRalpha. The enzyme activity was much higher in the monolayers than the spheroids. Inhibition of this enzyme activity in the monolayer hepatocyte resulted in the increase of nuclear RXRalpha protein and the augmentation of T3-dependent induction of spot 14 mRNA. These results suggest that the changes in cathepsin L-type protease activity in the cytosol may alter the turnover of RXRalpha in the nucleus and modify the function of steroid receptor superfamilies that heterodimerize with RXRalpha.

Animals↗

PubMed1998

Thyroid hormone response elements differentially modulate the interactions of thyroid hormone receptors with two receptor binding domains in the steroid receptor coactivator-1.

Ligand-dependent transcriptional activation by nuclear receptors is mediated by interactions with coactivators. Recently, a consensus interaction motif (LXXLL) has been identified in a number of coactivators such as steroid receptor coactivator-1 (SRC-1). SRC-1 contains three such motifs in the central (nuclear receptor binding domain-1, NBD-1) and a single one in the C-terminal (NBD-2) regions. To define the nature and role of the two NBDs in SRC-1, interaction studies between the two NBDs and thyroid hormone receptor (TR) were performed. Although NBD-1 and NBD-2 showed similar ligand- and AF-2-dependent interactions with TR in solution, these two NBDs possessed distinct interaction properties with TR when TR is bound to a thyroid hormone-response element (TRE). Both in vitro and in vivo interaction studies demonstrate that NBD-1, but not NBD-2, exhibits ligand-dependent interaction with TR in the presence of TREs. In addition, a natural isoform of SRC-1, SRC-1E, which lacks NBD-2, preserved TR as well as progesterone receptor-mediated coactivator function on reporter gene expression. Finally, we found that NBD-1 failed to interact with a TR and retinoid X receptor heterodimer complex on a transcriptionally inactive direct repeat +4 TRE in electrophoretic mobility shift assays. These observations indicate that DNA-induced, as well as ligand-induced, conformational change(s) of TR may influence the nature of its binding to SRC-1, and that the two NBDs of SRC-1 may play different roles to regulate ligand-dependent transactivation of TRs.

Animals↗

PubMed1998

Steroidogenic factor-1 and early growth response protein 1 act through two composite DNA binding sites to regulate luteinizing hormone beta-subunit gene expression.

Recent in vivo and in vitro studies have implicated the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1 (Egr-1) in the transcriptional regulation of the luteinizing hormone beta-subunit (LHbeta) gene. We have previously demonstrated the ability of SF-1 to bind to and transactivate the rat LHbeta gene promoter acting at a consensus gonadotrope-specific element (GSE) located at position -127. We have now identified a second functional GSE site at position -59. In addition, based on electrophoretic mobility shift assay, in vitro translated Egr-1 is shown to bind to two putative Egr-1 binding sites (positions -112 and -50), which appear to be paired with the identified GSE sites. By transient transfection assay in pituitary-derived GH3 cells, it was seen that Egr-1 increases promoter activity of region -207/+5 of the rat LHbeta gene promoter through action at both Egr-1 sites. Furthermore, LHbeta gene promoter activity is markedly augmented in the presence of both factors together relative to activity in the presence of SF-1 or Egr-1 alone (150-fold versus 14-fold and 12-fold, respectively). These data define two composite SF-1-Egr-1 response-elements in the proximal LHbeta gene promoter and suggest that SF-1 and Egr-1 act synergistically to increase expression of the LHbeta gene in the gonadotrope.

Animals↗

PubMed1998

Sp1 binds to the rat luteinizing hormone beta (LHbeta) gene promoter and mediates gonadotropin-releasing hormone-stimulated expression of the LHbeta subunit gene.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) plays a critical role in reproductive function by regulating the biosynthesis and secretion of the pituitary gonadotropins. Although it is known that GnRH induces luteinizing hormone beta (LHbeta) gene transcription, the mechanisms by which this occurs remain to be elucidated. We have shown previously that GH3 cells transfected with the rat GnRH receptor cDNA (GGH3-1' cells) support the expression of a cotransfected fusion gene composed of 797 base pairs of rat LHbeta gene 5'-flanking sequence and the first 5 base pairs of the 5'-untranslated region fused to a luciferase reporter (-797/+5LHbetaLUC) and respond to a GnRH agonist with a 10-fold stimulation of activity. Furthermore, we have shown that DNA sequences at -490/-352 confer GnRH responsiveness to the rat LHbeta gene. We have now identified two putative binding sites for Sp1, a three-zinc-finger transcription factor, within this region. Using electrophoretic mobility shift assay, DNase I footprinting, and methylation interference assays, we demonstrate that Sp1 can bind to these sites and that Sp1 is responsible for DNA-protein complexes formed using GGH3-1' and alphaT3-1 nuclear extracts. Mutations of the Sp1 binding sites, which block binding of Sp1, blunt the stimulation of the LHbeta gene promoter by GnRH. These data define GnRH-responsive elements in the LHbeta 5'-flanking sequence and suggest that Sp1 plays an important role in conferring GnRH responsiveness to the LHbeta subunit gene.

Animals↗

PubMed1998

Ligand-inducible retinoid X receptor-mediated protein: DNA interactions in the retinoic acid receptor beta2 gene promoter in vivo.

Retinoid X receptors (RXRs) are recently characterized transcription factors that are members of the nuclear hormone receptor superfamily. However, it is not known whether the endogenous RXR complex requires its ligand for access to its hormone response element (HRE) of a target gene in vivo. Hence, dimethyl sulfate-based genomic footprinting was carried out to examine occupancy of HREs in the retinoic acid (RA) receptor beta2 (RARbeta2) gene promoter in the murine melanoma cell line S91 cultured in the absence or presence of T3, all-trans-RA (atRA), or CD2624, an RXR-selective retinoid. No footprint was observed at the RA-response element (betaRARE) in the absence of ligands. However, a footprint was detected at the betaRARE and other cis-acting elements after a 6 h incubation with CD2624 and atRA. Interestingly, only the betaRARE was footprinted after 60 min incubation with CD2624. These results suggest that the endogenous RXR complex can interact with an HRE of a target gene in the presence of ligand, and subsequently may initiate additional interactions between DNA and other transcription factors.

Animals↗

PubMed1998

Lack of coactivator interaction can be a mechanism for dominant negative activity by mutant thyroid hormone receptors.

We studied the interactions of two natural thyroid hormone receptor (TR) mutants from patients with resistance to thyroid hormone (RTH) and an artificial TR mutant with a nuclear receptor corepressor, N-CoR, and a steroid receptor coactivator, SRC-1. In electrophoretic mobility shift assays, wild-type TRbeta-1 interacted with N-CoR in the absence of ligand, whereas T3 caused dissociation of the TRbeta-1/N-CoR complex and formation of TRbeta-1/SRC-1 complex. In contrast, a natural mutant (G345R) with poor T3-binding affinity formed TRbeta-1/N-CoR complex, both in the absence and presence of T3, but could not form TRbeta-1/SRC-1 complex. Another TR mutant, which bound T3 with normal affinity and containing a mutation in the AF-2 region (E457D), had normal interactions with N-CoR but could not bind SRC-1. Both these mutants had strong dominant negative activity on wild-type TR transactivation. Studies with a TR mutant that had slightly decreased T3-binding affinity (R320H) showed a T3-dependent decrease in binding to N-CoR and increase in binding to SRC-1 that reflected its decreased ligand binding affinity. Additionally, when N-CoR and SRC-1 were added to these receptors at various T3 concentrations in electrophoretic mobility shift assays, TR/N-CoR and TR/SRC-1 complexes, but not intermediate complexes were observed, suggesting that N-CoR release is necessary before SRC-1 binding to TR. Our data provide new insight on the molecular mechanisms of dominant negative activity in RTH and suggest that the inability of mutant TRs to interact with coactivators such as SRC-1, which results from reduced T3-binding affinity, is a determinant of dominant negative activity.

DNA↗

PubMed1998

Differential use of signal transduction pathways in the gonadotropin-releasing hormone-mediated regulation of gonadotropin subunit gene expression.

The regulation of LH and FSH subunit gene expression is under the control of GnRH. Physiological changes in the frequency of pulsatile GnRH release from the hypothalamus result in differential stimulation of alpha-, LHbeta-, and FSHbeta-gene expression. Previous studies indicate that the GnRH receptor couples to G proteins of the G(q/11) family, with phosphoinositide turnover and its resultant increase in intracellular calcium concentration and protein kinase C (PKC) activation, to stimulate secretion of LH and FSH. However, the molecular mechanisms by which GnRH mediates its transcriptional effects remain largely unknown. We used GH3 cells, constitutively expressing the rat GnRH receptor (GGH(3)-1' cells) and transiently transfected with a luciferase reporter gene controlled by either the alpha, LHbeta, or FSHbeta gene regulatory region (alphaLUC, LHbetaLUC, and FSHbetaLUC, respectively), to examine the roles of several signal transduction pathways in the GnRH-mediated stimulation of gonadotropin subunit gene expression. Activation of PKC by phorbol, 12-myristate, 13-acetate resulted in an increase in the luciferase activity of all three gonadotropin subunit gene reporter constructs. Phorbol, 12-myristate, 13-acetate had a greater stimulatory effect, relative to the maximal stimulation with GnRH, for the beta-subunit genes than for the alpha-subunit gene. Depletion of PKC, or inhibition of PKC by GF109203X, demonstrated that PKC-dependent pathways play a larger role in the GnRH-mediated transcriptional control of the LHbeta- and FSHbeta-genes than the alpha-subunit gene. In contrast, an L-type calcium channel agonist, Bay K 8644, was able to stimulate alphaLUC but not LHbetaLUC or FSHbetaLUC. Nimodipine, an L-type calcium channel antagonist, had a larger inhibitory effect on the GnRH response of alphaLUC, relative to LHbetaLUC or FSHbetaLUC. We conclude from these results that the differential regulation of gonadotropin subunit gene expression by GnRH is caused, in part, by differential use of signal transduction pathways, activated upon GnRH binding.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Expression and hormonal regulation of coactivator and corepressor genes.

Steroid/thyroid/retinoid receptors are members of the nuclear receptor superfamily and ligand-inducible transcription factors. These receptors modulate transcription of various cellular genes, either positively or negatively, by interacting with specific hormone-response elements located in the target gene promoters. Recent data show that nuclear receptors enhance or inhibit transcription by recruiting an array of coactivator and corepressor proteins to the transcription complex. We examined and compared the expression of four coactivator (steroid receptor coactivator-1 and E1A-associated 300-kDa protein) and corepressor (SMRT and N-CoR) genes in a number of tissues including several endocrine glands and cell lines. We also addressed whether their messenger RNA levels are hormonally regulated by studying the effects of thyroid hormone (T3) and estrogen (E2) treatment in rat pituitary cells (GH3) in vitro and in anterior pituitary in vivo. Our studies show that there are distinct tissue-specific expression patterns of these genes. We show that T3 and E2 regulate the expression of steroid receptor coactivator-1 messenger RNA in the anterior pituitary in addition to a gender-related difference. These tissue variations may have physiological implications for heterogeneity of hormone responses that are observed in normal and malignant tissues.

Animals↗

PubMed1998

Identification of cis-acting deoxyribonucleic acid elements that mediate gonadotropin-releasing hormone stimulation of the rat luteinizing hormone beta-subunit gene.

GnRH plays a critical role in reproductive development and function by regulating the biosynthesis and secretion of the pituitary gonadotropins, LH and FSH. Although it is known that GnRH induces gonadotropin subunit gene transcription, the mechanism by which this occurs has not been elucidated. Studies have been hindered by the lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have transfected the rat pituitary GH3 cell line with the rat GnRH receptor complementary DNA. These cells, when cotransfected with regulatory regions of the LH or FSH subunit genes fused to a luciferase reporter gene, respond to GnRH with an increase in promoter activity comparable to that seen in primary rat pituitary cells. In this study, we have used this cell model to identify cis-acting elements of the LHbeta gene that mediate stimulation by GnRH. Analysis of a series of 5'-deletion and internal deletion constructs has revealed two regions of the rat LHbeta gene promoter involved in mediating the response to GnRH, region A (-490/-352) and region B (-207/-82). Fusion of region A upstream of a heterologous minimal promoter linked to the luciferase gene conferred GnRH responsiveness to the promoter, whereas region B did not. However, the presence of both regions A and B conferred a greater GnRH response than region A alone. Electrophoretic mobility shift assay revealed the presence of a protein(s) binding to region A using GH3 as well as alphaT3-1 nuclear extracts. Thus, region A (-490/-352) confers GnRH responsiveness to the LHbeta subunit gene and binds to a protein(s) present in pituitary cell lines. DNA sequences in region B (-207/-82) also contribute to GnRH responsiveness. The identification of putative GnRH response elements in the rat LHbeta gene promoter will aid in elucidation of the mechanisms of regulation of gene expression by GnRH.

Animals↗

PubMed1998

ROR alpha gene expression in the perinatal rat cerebellum: ontogeny and thyroid hormone regulation.

Deficiency of thyroid hormone (TH) during the perinatal period results in severe neurological abnormalities in rodent cerebellar development. However, the molecular mechanisms of TH action in the developing cerebellum are not fully understood. Of note, a mutant mouse, staggerer, in which the orphan nuclear hormone receptor ROR alpha gene is disrupted, exhibits cerebellar abnormalities similar to those seen in the hypothyroid animals, despite normal thyroid function. We, therefore, speculated that TH (tetraiodo-L-thyronine; T4) may regulate ROR alpha gene expression, which then may regulate genes essential for normal brain development. To test this hypothesis, we studied the changes in ROR alpha gene expression in perinatal hypothyroid rat cerebellum and the effect of TH replacement using Northern blot analysis, ribonuclease protection assay and in situ hybridization histochemistry. During cerebellar development, an approximately 3-fold increase in the cerebellar content of ROR alpha messenger RNA (mRNA) was seen in both propylthiouracil-treated, and propylthiouracil-treated and T4-replaced animals. However, the increase was accelerated when T4 was injected, although the ROR alpha mRNA content was identical, with or without T4, by 30 days after birth (P30). In contrast, T4 treatment suppressed the TH receptor alpha1 and c-erbA alpha2 mRNA content by P30; retinoic acid X receptor-beta mRNA content was not influenced by thyroid status. A significant hybridization signal for ROR alpha mRNA was seen only over Purkinje cells in the cerebellar cortex by in situ hybridization histochemistry. These results indicate that TH alters the timing of expression of the ROR alpha gene in the Purkinje cells of the cerebellar cortex, which may, in turn, influence Purkinje cell differentiation.

Amino Acid Sequence↗