Biomedical subjects

W W Chin

Publications and source records attributed to W W Chin.

At least 19 recordsLinked to original sources

PubMed1992

Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor.

Rat pituitary cDNAs encoding the full peptide coding sequence of the rat gonadotropin-releasing hormone receptor were isolated and characterized. The deduced amino acid sequence encodes a protein of 327 residues with seven putative transmembrane domains characteristic of the family of G-protein coupled receptors. It is 95% identical at the amino acid level with the mouse gonadotropin-releasing hormone receptor. An mRNA of 4.5 Kb was identified in the rat pituitary, ovary, and testis, and in murine alpha T3 cells. In addition, a larger mRNA species of 5.0-5.5 Kb was present in these rat tissues, and a smaller mRNA species of 1.8 Kb was present in the rat pituitary and ovary, and in alpha T3 cells. The receptor mRNA levels were increased in the female rat pituitary after ovariectomy compared to levels in intact female rats.

Amino Acid Sequence↗

PubMed1992

Triiodothyronine (T3) differentially affects T3-receptor/retinoic acid receptor and T3-receptor/retinoid X receptor heterodimer binding to DNA.

Thyroid hormone receptor (TR) heterodimerizes with retinoic acid receptor (RAR), retinoid X receptor (RXR), and triiodothyronine receptor auxiliary protein (TRAP) on natural and synthetic hormone response elements. Recently we showed that triiodothyronine (T3) decreased TR homodimer, but not TR/TRAP heterodimer, binding to several thyroid hormone response elements (TREs). The effect of ligand on TR/RAR and TR/RXR heterodimer binding to DNA is not known. In this study, we showed that TR formed heterodimers with RAR and RXR on a retinoic acid (RA) response element and two TREs. Surprisingly, T3, but not RA, decreased TR/RAR heterodimer binding to DNA. In contrast, T3, all-trans-RA, or 9-cis-RA did not affect TR/RXR binding to DNA. This finding suggests that TR/RXR heterodimer is a stable receptor complex that remains bound to response elements in the presence of ligand and therefore may be a receptor complex involved in T3-regulated transcription.

Animals↗

PubMed1992

Triiodothyronine (T3) decreases binding to DNA by T3-receptor homodimers but not receptor-auxiliary protein heterodimers.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that bind to thyroid hormone response elements (TREs) to mediate positive and negative regulation of transcription of thyroid hormone-responsive genes. TR binding to TREs can be enhanced by interaction with a nuclear protein, triiodothyronine (T3) receptor auxiliary protein (TRAP). There are two major isoforms of thyroid hormone receptors, TR alpha-1 and TR beta-1, which are encoded on two separate genes. We studied the binding of TR alpha-1 and TR beta-1 to several TREs: the chick lysozyme TRE (F2), which is positively regulated by T3; rabbit beta-myosin heavy chain TRE, which is negatively regulated by T3; and an idealized inverted palindrome, TRElap. We demonstrate the formation of homodimers, TR alpha/TR beta dimers, and TR/TRAP heterodimers when receptor is bound to these DNA sequences. Surprisingly, we found that T3 decreased TR alpha-1 and TR beta-1 homodimer binding in a dose-dependent manner to these TREs as well as TR alpha/TR beta dimer binding to F2. In contrast, T3 did not affect TR/TRAP heterodimer binding to TREs suggesting that this heterodimer may be the stable complex occupying TREs in the presence of ligand.

Animals↗

PubMed1992

Regulation of angiotensinogen gene expression by estrogen.

OBJECTIVE: Clarification of the role of estrogen in the regulation of angiotensinogen gene expression in multiple tissues. DESIGN: The effect of 17 beta-estradiol (E2; 10 micrograms/100 mg body weight) administration in ovariectomized (OVX) rats upon angiotensinogen messenger RNA (mRNA) levels in multiple tissues was assessed. Confounding ovarian factors were thus removed by studying the animals in the castrate state. Controls consisted of OVX and intact female rats. METHODS: Adult female Sprague-Dawley rats were ovariectomized and experiments begun 21 days postsurgery. Animals were injected with E2 and studied after 0, 1, 4, and 24 h of treatment. Levels of angiotensinogen mRNA were determined by Northern blot analysis using beta-actin mRNA as an internal standard. RESULTS: A single angiotensinogen mRNA species with molecular size of approximately 1800 bp was observed in rat liver, aorta, kidney, cardiac atria, hypothalamus and whole brain. Little or no angiotensinogen mRNA was identified in the pituitary gland. Angiotensinogen mRNA was most abundant in rat liver, hypothalamus, aorta and progressively less abundant in whole brain, cardiac atria and kidney. A twofold induction of hepatic angiotensinogen mRNA levels in E2-OVX rats was observed by 4h. The angiotensinogen mRNA levels in kidney were threefold higher by 4 h compared with OVX control animals. In aorta, the angiotensinogen mRNA level was also threefold higher by 1 h after E2 treatment. No significant effect of estradiol treatment was observed in cardiac atria although the level of angiotensinogen mRNA was higher in intact female rats compared with OVX controls. CONCLUSION: These results suggest that estrogen modulates angiotensinogen gene expression in a tissue-specific manner.

Angiotensinogen↗

PubMed1992

New insights on the mechanism(s) of the dominant negative effect of mutant thyroid hormone receptor in generalized resistance to thyroid hormone.

Generalized resistance to thyroid hormone (GRTH) is a syndrome of hyposensitivity to triiodothyronine (T3) that displays autosomal dominant inheritance. The genetic defect commonly lies in the ligand-binding domain of one of the TR beta alleles. Since there are two major thyroid hormone receptor (TR) isoforms, TR alpha and TR beta, it is not known how the mutant receptor mediates a dominant negative effect. Previously, we showed that T3 caused dissociation of TR homodimers and TR alpha/TR beta dimers from several thyroid hormone response elements (TREs). Hence, we used the electrophoretic mobility shift assay to compare the effect of T3 on the DNA binding of mutant TR beta-1 (Mf-1) from a kindred with GRTH with normal TR beta. Mf-1 bound better as a homodimer than TR beta, but dissociated from DNA only at high T3 concentrations. Both receptors heterodimerized with nuclear auxiliary proteins. They also dimerized with TR alpha and with each other. Surprisingly, T3 disrupted the DNA binding of the Mf-1/TR isoform dimers. Thus, mechanisms for the dominant negative effect by mutant TRs likely involve either increased binding to TREs by mutant homodimers that cannot bind T3 (hence cannot dissociate from DNA) and/or the formation of inactive mutant TR/nuclear protein heterodimers.

Animals↗

PubMed1992

Isoform-specific thyroid hormone receptor antibodies detect multiple thyroid hormone receptors in rat and human pituitaries.

There are three known isoforms of the thyroid hormone receptor (TR) in the rat: TR alpha-1, TR beta-1, and TR beta-2. The TR alpha-1 and TR beta-1 mRNAs are found in many tissues, whereas TR beta-2 mRNA is detected only in the pituitary gland. Thus far, TR alpha-1 and TR beta-1 mRNAs have been found in humans and are highly homologous to their counterparts in rats; however, TR beta-2 mRNA has not yet been demonstrated in humans. To examine the expression of these TRs at the protein level, we have raised isoform-specific polyclonal antibodies in female New Zealand White rabbits against the rat TRs and c-erbA alpha-2, a carboxy-terminal variant of TR alpha-1 that does not bind thyroid hormone. The rabbits were immunized with synthetic peptides that contained the following amino acid sequences: TR alpha-common-(10-31), c-erbA alpha-2-(428-442), TR beta-1-(73-93), and TR beta-2-(86-101, 113-133). All immune sera could bind specifically to their respective immunizing peptides on enzyme-linked immunosorbent assay as well as immunoprecipitate specifically in vitro translated rat and human TRs. Anti-TR beta-1 and anti-TR alpha-common antibodies could immunoprecipitate TR beta-1 or TR alpha-1, respectively, in transfected COS-7 cells. We also immunostained normal adult rat and human pituitary glands. Each isoform-specific antibody could immunostain almost all of the anterior pituitary cells, suggesting that TR alpha-1, TR beta-1, TR beta-2, and c-erbA alpha-2 are most likely expressed in all anterior pituitary cell types in rats and humans. The staining of rat pituitary glands by the anti-TR beta-2 antibodies demonstrates for the first time that TR beta-2 is expressed as a protein in pituitary cells. Furthermore, the staining of human pituitary glands by the anti-TR beta-2 antibodies suggests that there is a human homolog of the rat pituitary-specific TR beta-2 that shares similar epitopes with the rat TR beta-2. In summary, we have prepared isoform-specific antibodies against TRs that can recognize in vitro translated, transiently transfected, and in situ rat and human pituitary TRs. These antibodies will be useful in examining tissue- and cell type-specific expression of rat and human TRs at the protein level.

Animals↗

PubMed1992

Follistatin gene expression in the pituitary: localization in gonadotropes and folliculostellate cells in diestrous rats.

Follistatin, a glycosylated single chain protein that was originally isolated from ovarian follicular fluid, can specifically inhibit the biosynthesis and secretion of FSH by the pituitary. Follistatin has also been isolated from bovine pituitary and shown to have activin-binding activity. We wished to determine whether the follistatin gene is expressed in the rat pituitary and, if so, to identify the specific cell types. A 337-basepair fragment of the follistatin cDNA was amplified by polymerase chain reaction from a rat ovarian cDNA library and subcloned into pGEM3. Low levels of follistatin mRNA from rat pituitary poly(A)+RNA were detected by ribonuclease protection analysis using a specific follistatin riboprobe generated from the cDNA clone. The presence of follistatin mRNA in the pituitary was confirmed using polymerase chain reaction to amplify the follistatin cDNA generated by reverse transcription from total rat pituitary RNA. Furthermore, in situ hybridization studies combined with immunostaining for pituitary hormones were used to localize follistatin mRNA within the rat pituitary. When a biotinylated oligonucleotide complementary to follistatin mRNA was used with dispersed pituitary cells from rats in diestrus II, labeling was found in 5-7% of the cells. The in situ hybridization protocol was then combined with immunolabeling protocols for LH beta, FSH beta, or S-100 protein (a marker for folliculostellate cells). Follistatin mRNA was detected in 70 +/- 5% of LH beta cells, 44 +/- 11% of FSH beta cells, and 35 +/- 2% of folliculostellate cells. These results suggest that follistatin is expressed in pituitary gonadotropes and folliculostellate cells during diestrus II, where it may have a role in the local autocrine or paracrine regulation of FSH biosynthesis and secretion, possibly by binding to and modulating the effects of activin in the pituitary.

Animals↗

PubMed1992

Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells.

Rat pituitary GH cells have been used extensively to study the biochemical actions of TRH on lactotropic cells. To investigate the structure and regulation of the rat TRH receptor (rTRHR), we have cloned its cDNA from GH4C1 cells. Using the polymerase chain reaction with degenerate primers and pools of cloned cDNAs from a GH4C1 cDNA library, a fragment sharing high similarity to the mouse thyrotrope TRHR (mTRHR) was identified. Conventional library screening with this fragment was used to isolate a single cDNA. mRNA synthesized in vitro from this cDNA was injected into Xenopus oocytes, and a characteristic conductance response to TRH was detected by voltage clamp recording. DNA sequence analysis revealed a molecule of 412 amino acid residues, with 96% similarity to the mTRHR. However, in contrast to the mTRHR, the rTRHR had an additional 19 amino acid residues at its carboxy-terminus. A mRNA of about 4 kilobases was identified in GH3 cells. Regulation of the rTRHR mRNA concentration was studied in GH3 cells. Steady state rTRHR mRNA levels were decreased to 30% of the control level by incubation with TRH for 48 h and increased 4-fold by incubation with dexamethasone for 12 h. Southern blot analysis of genomic DNA from GH3 cells gave a simple banding pattern consistent with a single copy gene. We conclude that the rTRHR shares high primary sequence similarity to the mTRHR, but the rTRHR has an extension of 19 amino acids at its carboxy-terminus, which is lacking in the mTRHR.

Amino Acid Sequence↗

PubMed1991

Ligand-binding and heterodimerization activities of a conserved region in the ligand-binding domain of the thyroid hormone receptor.

The ligand-binding domain of the thyroid hormone (3,5,3'-triiodothyronine) receptor (TR) contains poorly characterized subdomains involved with ligand binding, transactivation, and protein-protein interactions. The region between residues 288-331 of rat TR alpha-1 was analyzed by modeling and site-directed mutagenesis. Our results suggest that part of this sequence adopts an amphipathic alpha-helical conformation. The integrity of the putative helix is important for 3,5,3'-triiodothyronine binding but not necessarily for heterodimerization with nuclear factor(s). Mutants defective for both activities were found clustered in a region overlapping the C-terminal portion of the helix and further downstream. The sequence conservation of this particular region among the entire superfamily suggests a similar role in dimerization in other receptors.

Amino Acid Sequence↗

PubMed1991

Gastrin-releasing peptide gene expression in small cell and large cell undifferentiated lung carcinomas.

Gastrin-releasing peptide (GRP; mammalian bombesin) is present in the neuroendocrine cells of human fetal lung and in small cell lung carcinomas (SCLCs), where it may act as a growth factor. Considering the potential importance of GRP as a tumor marker, we have conducted a retrospective immunohistochemical analysis of 176 lung tumors for markers of GRP gene expression, as well as several other markers of neuroendocrine cell differentiation: chromogranin A, neuron-specific enolase, and calcitonin. The majority of carcinoids contained mature GRP, in contrast to only a minority of SCLCs and large cell lung carcinomas (LCLCs). However, a majority of SCLCs and LCLCs contained proGRP immunoreactivity. In situ hybridization did not add any information beyond what was obtained using proGRP antisera. In spite of sharing these neuroendocrine cell markers, SCLCs are associated with a graver prognosis than LCLCs. No prognostic significance was associated with immunostaining for GRP or several other markers of neuroendocrine cell differentiation.

Adult↗

PubMed1991

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.

Amino Acid Sequence↗

PubMed1991

Evidence for an autocrine role of activin B within rat anterior pituitary cultures.

Activins, dimers of inhibin beta subunits, are potent stimulators of FSH secretion in vivo and in vitro and of FSH beta mRNA expression in rat anterior pituitary cultures. In this study, we investigated the possibility that locally secreted activin B (beta B beta B) may function as an autocrine modulator of basal FSH secretion and expression based on the previous observation that beta B is expressed within gonadotropes. The incubation of cultured rat anterior pituitary cells with a m mouse monoclonal antibody specific for the activin B homodimer (MAb-activin B) significantly attenuated the basal secretion of FSH in a concentration- and time-dependent manner, without influencing LH secretion. Moreover, MAb-activin B selectively inhibited FSH beta mRNA accumulation without affecting either LH beta or alpha subunit mRNAs. The MAb-activin B completely blocked the stimulation of FSH secretion by exogenous activin B, but not by activin A, confirming its specificity. As previously shown, inhibin A and follistatin significantly suppressed basal FSH secretion in these cultures. This inhibitory effect, albeit of lower magnitude, was still evident even in the presence of the MAb-activin B which by itself suppressed basal FSH secretion. These data suggest that the secretion of activin B by the gonadotropes of the anterior pituitary may serve as an autocrine signal in the selective modulation of FSH expression and secretion. Furthermore, the inhibitory actions of inhibins and follistatins on gonadotropes may, in part, be explained by their ability to interfere with the actions of endogenous activin B.

Activins↗

PubMed1991

Differential regulation of thyroid hormone receptor messenger ribonucleic acid levels by thyrotropin-releasing hormone.

In addition to its well known actions in stimulating TSH and PRL synthesis and secretion, TRH has been shown to decrease the concentration of thyroid hormone receptors (TRs) in GH4C1 cells as measured by nuclear thyroid hormone (T3) binding. In the present study we have investigated the effects of TRH on the levels of mRNA encoding the different forms of TR, TR beta-1, TR beta-2, and TR alpha-1 as well as that of the non-T3-binding variant, c-erbA alpha-2. GH3 cells were incubated with 100 nM TRH in the presence or absence of 1 nM T3 for 48 h, and mRNA levels were determined by Northern blot analysis. Results revealed that there is differential regulation of the individual TRs by TRH at the pretranslational level. The mRNA for the pituitary-specific form of TR, TR beta-2, was down-regulated by 60% by TRH in GH3 cells, while that of its alternative splice product, TR beta-1, was unchanged. A modest change was observed in TR alpha-1 mRNA levels, which were down-regulated by 20%; there was no change in c-erbA alpha-2 mRNA levels. Levels of nuclear T3 binding were assessed under the same conditions, and 100 nM TRH was found to decrease binding by 40% from 0.78 to 0.46 fmol/micrograms DNA. A similar change in nuclear T3 binding was seen after incubation with 1 nM T3. The effect of TRH on the GH mRNA response to T3 was investigated. In the absence of TRH there was a 4-fold induction of GH mRNA after incubation with 1 nM T3. In the presence of 100 nM TRH, no significant induction in GH mRNA by T3 was seen, indicating that T3 responsiveness as well as receptor concentration are diminished by TRH under these conditions.

Adenoma↗

PubMed1991

Activin stabilizes follicle-stimulating hormone-beta messenger ribonucleic acid levels.

Activin, a gonadal peptide, stimulates FSH secretion in association with an increase in FSH beta messenger RNA (mRNA) levels at the level of the anterior pituitary gland. The goal of these studies was to determine whether the effects of recombinant human activin A (rhActivin A) are exerted at the post-transcriptional level by affecting the stability of FSH beta mRNA. We determined the apparent half-life of FSH beta mRNA in the presence and absence of rhActivin A using actinomycin D. The anterior pituitary glands from adult male rats were isolated and dispersed enzymatically. Cells were preincubated in the presence of rhActivin A for 24 h to increase FSH beta mRNA levels. Actinomycin D was then added and the cells were incubated for a subsequent 4, 6, 8, 12, and 24 h in the presence or absence of rhActivin A. As reported earlier, the addition of rhActivin A caused parallel increases in FSH secretion and FSH beta mRNA levels, while having no effect on alpha or LH beta mRNA levels. Actinomycin D treatment decreased FSH beta mRNA to 49, 39, and 16% of control levels at the 4, 6, and 8 h time points, respectively. In contrast, when actinomycin D was added in the presence of rhActivin A FSH beta mRNA was reduced to 80, 58, and 42% of control levels at the 4, 6, and 8 h time points, respectively. Using the least squares method of analysis, the apparent half-lives of FSH beta mRNA under these two conditions were calculated. In the presence of actinomycin D, the half-life of FSH beta mRNA was 3.1 h. The addition of activin significantly increased the half-life to 6.5 h. These results suggest that activin A stimulates FSH beta mRNA levels, at least in part, at the posttranscriptional level by increasing the stability of FSH beta mRNA.

Activins↗

PubMed1991

Triiodothyronine receptor beta-2 messenger ribonucleic acid expression by somatotropes and thyrotropes: effect of propylthiouracil-induced hypothyroidism in rats.

mRNA for a thyroid hormone receptor isoform that is unique to the pituitary gland (TR beta-2) is down-regulated by T3. Increases in the expression of this mRNA are seen in rats rendered hypothyroid by treatment with propylthiouracil (PTU). This study used dual labeling to determine which pituitary cells expressed TR beta-2 mRNA in normal and PTU-treated rats. In situ hybridization protocols localized the mRNA (with biotinylated complementary oligonucleotide probes detected by avidin-biotin-peroxidase), and immunoperoxidase protocols identified the pituitary hormone proteins. In dispersed pituitary cells, 20 +/- 2% (average +/- SD) of cells from normal rats and 30 +/- 3% of cells from PTU-treated rats were labeled for TR beta-2 mRNA. PTU caused increases in the area of the labeled cells (from 114 +/- 11 to 225 +/- 7 microns 2), the area of the label per cell (from 27 +/- 3 to 71 +/- 11 microns 2), and label density. PTU produced increases in the percentage of TSH cells from 8 +/- 1% to 19 +/- 2%, decreases in the percentage of GH cells from 27 +/- 3% to 11 +/- 2%, and no change in other cell types. After dual labeling, 73% of cells that expressed TR beta-2 mRNA stored either TSH (35 +/- 8) or GH (38 +/- 6). Less than 10% stored other hormones. When each cell type was analyzed, 56 +/- 3% of TSH cells and 43 +/- 4% of GH cells expressed TR beta-2 mRNA. When these percentages were multiplied by the percentages of each cell type in the overall population, TSH and GH cells with TR beta-2 mRNA represented 6.8 +/- 1% and 11.6 +/- 1% of the pituitary cells, respectively. Less than 1% of all pituitary cells expressed TR beta-2 and ACTH (0.9 +/- 0.06), LH (0.8 +/- 0.1), FSH (0.8 +/- 0.1), and PRL (0.9 +/- 0.04). PTU treatment increased the percentage of TSH cells with TR beta-2 mRNA to 72 +/- 4% and decreased the percentage of GH cells with TR beta-2 mRNA to 30 +/- 3%. However, some enlarged putative TSH cells could not be identified by immunolabel because the storage levels were low. Thus, changes in TR beta-2 mRNA in hypothyroid rats may be the net result of the increase in the percentage of TSH cells, the amount of mRNA per cell (measured by area and density of label), and the decrease in the percentage of GH cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

In vivo regulation of FSH synthesis by inhibin and activin.

The effect of a single sc injection of the gonadal peptide, recombinant human activin A (rhActivin A), on gonadotropin synthesis and secretion was examined in adult and immature male and female rats and the effect of recombinant human inhibin A (rhInhibin A) was examined in adult male rats. Pituitary FSH beta, LH beta and alpha messenger RNA (mRNA) levels were determined by blot hybridization. Trunk blood was collected to measure serum FSH levels. Treatment with rhInhibin A (100 micrograms/kg) resulted in a decrease in FSH beta mRNA to 2% of controls levels 6 h after injection. FSH beta mRNA levels started to rebound at 10 h, but were still significantly lower than vehicle-treated controls. Serum FSH levels were significantly reduced at 2 h and were reduced further at 6 and 10 h. There were no significant changes in alpha and LH beta mRNA levels. RhActivin A, at the highest dose (500 micrograms/kg), in immature male rats had only a modest effect (1.2- and 1.3-fold increase) on FSH beta mRNA levels and FSH secretion, respectively, at 2 h. No increase in FSH synthesis and FSH secretion was observed in adult male rats. In contrast, both immature and adult-ovariectomized E2 implanted females showed a robust response to rhActivin A. In immature females, 2 h after rhActivin A (100 and 500 micrograms/kg) administration, FSH beta mRNA levels were elevated 2.0- and 2.2-fold. At this time serum FSH was also elevated. At 6 and 10 h rhActivin A significantly reduced FSH beta mRNA levels from vehicle-treated controls. In contrast, FSH secretion was elevated at 6 h and returned to baseline at 10 h. Administration of rhActivin A (500 micrograms/kg) to adult, ovariectomized-E2 females resulted in a significant increase in FSH beta mRNA levels and FSH secretion at 2 and 6 h. There were no significant changes in alpha and LH beta mRNA levels in either males or females. Thus, these in vivo studies have shown that rhInhibin A can inhibit FSH beta mRNA levels and FSH secretion in the adult male rat. RhActivin A stimulates FSH synthesis and secretion in the immature and adult ovariectomized-E2 females, but has little or no effect in immature and adult males. Hence, there is a sexual dimorphic response to rhActivin A in vivo in the rat.

Activins↗

PubMed1991

Basal and thyroid hormone receptor auxiliary protein-enhanced binding of thyroid hormone receptor isoforms to native thyroid hormone response elements.

There are three known isoforms of the rat thyroid hormone receptor, TR alpha-1, TR beta-1, and TR beta-2. The first two are expressed in all tissues, whereas TR beta-2 appears to be expressed only in the pituitary. The differences in the roles of the three receptor isoforms are unknown, but may involve preferential interaction with different subsets of thyroid hormone-regulated genes in different tissues. We tested the binding of the three TR isoforms to putative thyroid hormone response elements (TREs) from genes that are expressed in the pituitary or other tissues and are regulated by thyroid hormone. In vitro translated 35S-labeled rat TR alpha-1, rat TR beta-2, and human TR beta-1 receptors were bound to a battery of biotinylated synthetic deoxyribonucleotides containing naturally occurring putative TREs from genes expressed either in only pituitary (rat glycoprotein hormone alpha-subunit, TSH beta-subunit, and GH) or in nonpituitary (rat alpha-myosin heavy chain, malic enzyme, and Moloney murine leukemia virus promoter) tissues. All three receptor forms bound to each of the TREs. TR beta-2 did not show preferential binding to TREs of pituitary-specific genes compared to TR beta-1. Additionally, TR alpha-1 had a similar TRE-binding pattern as the TR beta s, except for possibly less binding to rat glycoprotein hormone alpha-subunit TRE. Finally, rat pituitary and liver nuclear extracts enhanced TR binding to TREs, with the greatest enhancement seen with the alpha-subunit TRE. These studies suggest that all TR isoforms bind similarly to native TREs. Also, TR binding to TREs can be differentially enhanced by interactions with nuclear proteins.

Animals↗

PubMed1991

3,5,3'-triiodothyronine (T3) receptor-auxiliary protein (TRAP) binds DNA and forms heterodimers with the T3 receptor.

Nuclear factors enhance binding of T3 receptors (TR) to DNA, suggesting that T3 action may require a multicomponent complex bound to thyroid hormone response elements (TREs). We refer to the 65,000 Da nuclear protein in GH3 cells that enhances TR binding to DNA as the TR-auxiliary protein (TRAP) and have characterized its interaction with TR. Using a TRE-DNA affinity matrix we show that TRAP is able to bind to DNA, even in the absence of functional TR. We then used carboxyl-terminal truncations of rat TR alpha-1 and human TR beta in the avidin-biotin complex DNA-binding assay to identify regions that are important for interaction with TRAP. Removal of 34 residues of hTR beta abolishes T3-binding activity, but the ability to bind TRAP is retained. Further truncations and point mutations suggest that TRAP interacts with the ligand-binding domain of TR and with an independent region which overlaps a conserved sequence adjacent to the second Zn2+ finger (amino acids 120-149 in rTR alpha-1). A fragment of rTR alpha-1 (alpha C291) which encompasses these two regions inhibits the ability of TRAP to enhance TR binding to DNA. This is due to binding of alpha C291 to TR, demonstrating the ability of TR to form homodimers. The inability of TRAP to interact with TR dimers and the similarity of the locations of the estradiol receptor dimerization domains with the TRAP interaction regions lead us to conclude that TRAP stabilizes TR binding to DNA by formation of TRAP-TR heterodimers with both proteins bound to the DNA. TR bound to the estrogen response element is unable to respond to TRAP and unable to stimulate transcription, possibly due to the absence of TRAP in the TR-estrogen response element complex. In addition, TRAP may interact with a certain subset of the nuclear receptor superfamily, since human retinoic acid receptor-beta and vitamin D receptor show increased binding to TREs in the presence of nuclear extract, but c-erbA alpha-2, a variant TR, does not respond to TRAP.

Animals↗