Expression and regulation of c-erb-A mRNA from lymphocytes in patients with thyroid dysfunction

To investigate the expression and the regulation of nuclear triiodothyronine (T3) receptor at the gene level, cellular(c)-erb-A mRNA isolated from lymphocytes in patients with thyroid dysfunction was examined by Northern gel analysis and dot blot hybridization using viral (v)-erb-A cDNA probe. Human lymphocytes contained c-erb-A mRNA (approximately 2.0 kilobase (kb) in length), and c-erb-A mRNA, which was determined by dot blot hybridization, was observed to be increased in hypothyroid patients but unaltered in hyperthyroid patients. The high level of c-erb-A mRNA may contribute in part to the increase in nuclear T3 receptor and these results suggest the presence of up-regulation of nuclear T3 receptor at the gene level in the lymphocytes of hypothyroid patients.     

Retinoic acid decreases retinoic acid and triiodothyronine nuclear receptor expression in the liver of hyperthyroidic rats.

Retinoic acid (RA) and triiodothyronine (T3) exert many of their actions by binding to specific nuclear receptors (respectively, RA receptor (RAR) and T3) receptor (TR) belonging to a 'superfamily' of receptors. Some heterologous regulation of these receptors has been shown, and in particular regulation of the maximum binding capacity of TR by either retinol or RA. Now, using hyperthyroidic rats as a model, the effect of RA on binding capacity and on the mRNA levels of TR and RAR was investigated. The results show that the benefit of vitamin A treatment for the hyperthyroidic state, which has been described for a long time, could be the result of a down-heteroregulation of TR by RA, the active metabolite of retinol.     

视黄醇结合蛋白RBP4可与多种核受体相互作用

视黄醇结合蛋白 (retinolbindingprotein ,RBP4 )是体内一种重要的转运蛋白 ,主要负责结合、转运全反式视黄醇 (维生素A ,VitA ) .VitA及其衍生物如11 cis 视黄醛、all trans 视黄酸等 ,均是体内非常重要的疏水分子 ,与视觉循环、胚胎发育等多种过程有关 .RBP4的功能障碍会导致     

Regulation of two c-erbA messenger ribonucleic acids in rat GH3 cells by thyroid hormone

There is recent evidence suggesting that c-erbA is the thyroid hormone nuclear receptor, and that there may be multiple c-erbA genes. We investigated the effect of T3 on two c-erbA mRNAs present in GH3 cells. A partial cDNA was isolated from rat GH3 cells which is nearly identical (99.6% nucleotide identity) to rat c-erbA alpha, except for a unique 3'-region corresponding to the carboxyl terminal region of the predicted protein sequence. This cDNA (c-erbA alpha-2), like rat c-erbA alpha, hybridizes to a 2.6 kilobase (kb) mRNA which is distinct from a 6.2 kb species that hybridizes to c-erbA beta. Since nuclear T3-binding is down-regulated by T3, we hypothesized that one or both c-erbA mRNAs might be regulated by T3. GH3 cells were treated with 10 nM T3 for up to 24 h, a manipulation known to decrease nuclear T3 binding by approximately 2-fold in GH cells. Both the 6.2 kb and 2.6 kb mRNA species decreased to nearly 50% of control values at 24 h. These data indicate that these two c-erbA mRNAs are regulated by T3 and suggest that the T3 effect on T3 binding-activity in GH cells may be mediated, in part, by down-regulation of c-erbA mRNA levels.     

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.     

Lung retinol storing cells synthesize and secrete retinoic acid,an inducer of alveolus formation

Retinoids play a key role in the formation of pulmonary alveoli. Lipid interstitial cells (LICs) of the alveolar wall store retinol and are concentrated at sites of alveolus formation, suggesting they are an endogenous source of retinoids for alveolus formation. We show in cultured rat lung cells that LICs synthesize and secrete all-trans retinoic acid (ATRA); its secretion is halved by dexamethasone, an inhibitor of alveolus formation. In a second alveolar wall cell, the pulmonary microvascular endothelial cell (PMVC), ATRA increases expression of the mRNA of cellular retinol binding protein-I (CRBP-I), a protein involved in ATRA synthesis. Serum-free, exogenous ATRA-free medium conditioned by LICs rich in retinol storage granules caused a 10-fold greater increase of CRBP-I mRNA in PMVCs than media conditioned by LICs with few retinol storage granules. This action of medium conditioned by retinol storage granule-rich LICs is decreased by a retinoic acid receptor pan-antagonist and by a retinoid X receptor pan-antagonist, suggesting the responsible molecule(s) is a retinoid and that retinoid signaling occurs in a paracrine fashion.     

SDR-O: an orphan short-chain dehydrogenase/reductase localized at mouse chromosome 10/human chromosome 12

We report cloning a cDNA that encodes a novel short-chain dehydrogenase/reductase, SDR-O, conserved in mouse, human and rat. Human and mouse liver express SDR-O (short-chain dehydrogenase/reductase-orphan) mRNA intensely. The mouse embryo expresses SDR-O mRNA as early as day seven. Human SDR-O localizes on chromosome 12; mouse SDR-O localizes on chromosome 10 with CRAD1, CRAD2 and RDH4. SDR-O shares highest amino acid similarity with rat RoDH1 and mouse RDH1 (69-70%), but does not have the retinol and 3alpha-hydroxysteroid dehydrogenase activity of either, nor is it active as a 17beta- or 11beta-hydroxysteroid dehydrogenase. Short-chain dehydrogenase/reductases catalyse the metabolism of ligands that bind with nuclear receptors: the occurrence of 'orphan' nuclear receptors may imply existence of 'orphan' SDR, suggesting that SDR-O may catalyse the metabolism of another class of nuclear receptor ligand. Alternatively, SDR-O may not have a catalytic function, but may regulate metabolism by binding substrates/products and/or by serving as a regulatory factor.     

A nuclear factor that enhances binding of thyroid hormone receptors to thyroid hormone response elements

Recent studies from this laboratory have demonstrated the presence of thyroid hormone response elements (TREs) in the 5'-flanking region of the rat alpha and TSH beta subunit genes. Using an avidin-biotin complex DNA binding assay, we have shown that these TREs bind the thyroid hormone (T3) receptor present in nuclear extracts of GH3 cells, as well as the in vitro synthesized Hc-erbA beta, which has been identified as a member of the family of T3 receptors. The binding of Hc-erbA beta to the alpha subunit TRE can be enhanced 3-4-fold by including GH3 nuclear extract in the binding assay. Binding to the TRE present in the TSH beta gene or the rat growth hormone gene was similarly enhanced, although to a lesser degree. The enhanced binding activity is trypsin-sensitive and heat labile, and is not reproduced by the addition of histones, bovine serum albumin, or cytosol instead of nuclear extract. Gel exclusion chromatography suggests a molecular size of approximately 65,000 Da. This protein, which is present in several different cell types, is also able to complement binding of the rat erbA alpha-1 and the pituitary-specific erbA beta-2 forms of the receptor. These data suggest that the binding of the T3 receptor to a TRE is augmented by another nuclear protein, which may be involved in the mechanism of action of thyroid hormone.     

Vitamin A receptors. II. Characteristics of retinol binding in chick retina and pigment epithelium

Gel filtration studies demonstrate that retinol receptors of chick retinal and pigment epithelial cytosols are (1) of very similar nature (2) of small molecular size (about 18000 daltons) and are different in character from serum proteins. Citral inhibits the binding of [3H]retinol to the retinal 2 S receptor. Retinol acetate competes with retinol for binding to 2 S receptor in both retina and pigment epithelium whereas retinol palmitate is an effective competitor only in the pigment epithelium. Dithiothreitol maximizes 2 S binding in retina and pigment epithelial cytosol; its absence does not lead to receptor aggregation however. A limited number of high affinity binding sites (2 S receptor) appear to be present in retina and pigment epithelium. A 5 S binding species is also present in pigment epithelium; it is similar in character to [3H]retinol binding in serum and may arise from serum contamination of the pigment epithelial preparation. Binding affinity in retina is high with possibly two classes of retinol binding sites present of KD about 1 - 10(-9) and 4 - 10(-8).     

Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus

Cellular retinoic acid-binding protein (CRABP), a potential mediator of retinoic acid action, enables retinoic acid to bind in a specific manner to nuclei and chromatin isolated from testes of control and vitamin A-deficient rats. The binding of retinoic acid was followed after complexing [3H]retinoic acid with CRABP purified from rat testes. The binding was specific, saturable, and temperature dependent. If CRABP charged with nonlabeled retinoic acid was included in the incubation, binding of radioactivity was diminished, whereas inclusion of free retinoic acid, or the complex of retinol with cellular retinol binding protein (CRBP) or serum retinol binding protein had no effect. Approximately 4.0 X 10(4) specific binding sites for retinoic acid were detected per nucleus from deficient animals. The number of binding sites observed was influenced by vitamin A status. Refeeding vitamin A-deficient rats (4 h) with retinoic acid lowered the amount of detectable binding sites in the nucleus. CRABP itself did not remain bound to these sites, indicating a transfer of retinoic acid from its complex with CRABP to the nuclear sites. Further, CRBP, the putative mediator of retinol action, was found to enable retinol to be bound to testicular nuclei, in an interaction similar to the binding of retinol to liver nuclei described previously.     

Triiodothyronine nuclear receptor and the role of non-histone protein factors in in vitro triiodothyronine binding

The rat liver triiodothyronine (T3) nuclear receptor rapidly looses, after a partial purification from the nuclear extract, its ability to bind T3. We previously reported that histones, in the presence of DNA, could protect against inactivation enhancing the T3 binding site concentration and maintaining the high affinity for T3. A nuclear fraction discarded during the receptor purification (fraction A) was also found able to restore T3 binding and was analyzed. As histones + DNA, fraction A stabilized the T3 binding site from irreversible inactivation during incubation with T3, increasing its concentration while keeping the same high affinity for T3. It was active even at relatively high receptor concentration, appeared slightly more active than histones (+ DNA) in the same protein concentration range (up to 50-fold increment of T3 binding at the optimal concentration of 25 micrograms/ml) and was unaffected or slightly inhibited by DNA. Other proteins (ovalbumin, soybean trypsin inhibitor, RNAase) and rat liver cytosol were several times less effective, suggesting a major role of some nuclear constituents. The active factors in fraction A essentially belong to non-histone nuclear proteins. Fraction A was found heterogeneous regarding the molecular size and pHi of the active factors, the existence of subfractions more active on a protein concentration basis being suggested but not yet clearly evidenced. Efficient in vitro T3 binding to the isolated T3 nuclear receptor thus depends on the presence of several different nuclear constituents, histones + DNA or some non-histone proteins. Whether interactions with these constituents could modulate T3 binding within the nucleus remains to be elucidated.     

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.