Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.     

Molecular cloning and expression of TRα and TRβ in the protandrous cinnamon clownfish,Amphiprion melanopus during sex reversal

We divided the process of sex reversal from immature male to mature female in the protandrous cinnamon clownfish (Amphiprion melanopus) into six developmental stages as follows: I, immature male; II, mature male; III, male at 60 days after female removal; IV, male at 90 days after female removal; V, male at 120 days after female removal; and VI, mature female. Thyroid hormone receptors α (TRα) and β (TRβ) cDNAs were cloned from the ovary and mRNA expression levels were compared during the sex-reversal process. The nucleotide sequences of the TRα and TRβ cDNA were 1230 and 1188 bp in length with open reading frames encoding peptides of 409 and 395 amino acids, respectively. We observed that TRα mRNA and protein levels were high in all stages except the immature gonad, while TRβ mRNA levels were higher in the mature ovary than in any other gonadal stage. We injected gonadotropin-releasing hormone analogue to identify its effects on TRs mRNA in immature fish. The mRNA levels of TRs increased significantly. We therefore propose that TRs are related to testicular development as well as ovarian development in cinnamon clownfish. The present study also provides basic data on the role of TRs during sex reversal in fish.     

Daily patterns of clock and cognition-related factors are modified in the hippocampus of vitamin A-deficient rats

The circadian expression of clock and clock-controlled cognition-related genes in the hippocampus would be essential to achieve an optimal daily cognitive performance. There is some evidence that retinoid nuclear receptors (RARs and RXRs) can regulate circadian gene expression in different tissues. In this study, Holtzman male rats from control and vitamin A-deficient groups were sacrificed throughout a 24-h period and hippocampus samples were isolated every 4 or 5 h. RARα and RXRβ expression level was quantified and daily expression patterns of clock BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins, as well as temporal expression of cognition-related RC3 and BDNF genes were determined in the hippocampus of the two groups of rats. Our results show significant daily variations of BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins and, consequently, daily oscillating expression of RC3 and BDNF genes in the rat hippocampus. Vitamin A deficiency reduced RXRβ mRNA level as well as the amplitude of PER1, REVERB gene, and REVERB protein rhythms, and phase-shifted the daily peaks of BMAL1 and RORα mRNA, RORα protein, and RC3 and BDNF mRNA levels. Thus, nutritional factors, such as vitamin A and its derivatives the retinoids, might modulate daily patterns of BDNF and RC3 expression in the hippocampus, and they could be essential to maintain an optimal daily performance at molecular level in this learning-and-memory-related brain area.     

Localization of estrogen receptor‐α and ‐βmRNA in brain areas controlling sexual behavior in Japanese quail

Two estrogen receptors (ERs), denoted ERα and ERβ, have been identified in humans and various animal species, including the Japanese quail. Estrogens play a key role in sexual differentiation and in activation of sexual behavior in Japanese quail. The distribution of ERα in the brain of male and female adult quail has previously been studied using immunohistochemistry, whereas in situ hybridization has been employed to study the distribution of ERβ mRNA in males only. In this article, we used in situ hybridization to study the distribution of mRNAs for both ERα and ERβ in brain areas controlling sexual behavior of Japanese quail. Our results show that both ERα mRNA and ERβ mRNA are localized in areas important for sexual behavior, such as the preoptic area and associated limbic areas, in both males and females. Moreover, we found differences in distribution of mRNA for the two receptors in these areas. The results of this article support previously reported data and provide novel data on localization of ER mRNAs in adult quail brain of both sexes. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Topoisomerase II expression in osseous tissue

The molecular mechanisms that mediate the transition from an osteoprogenitor cell to a differentiated osteoblast are unknown. We propose that topoisomerase II (topo II) enzymes, nuclear proteins that mediate DNA topology, contribute to coordinating the loss of osteoprogenitor proliferative capacity with the onset of differentiation. The isoforms topo II-α and -β, are differentially expressed in nonosseous tissues. Topo II-α expression is cell cycle-dependent and upregulated during mitogenesis. Topo II-β is expressed throughout the cell cycle and upregulated when cells have plateaued in growth. To determine whether topo II-α and -β are expressed in normal bone, we analyzed rat lumbar vertebrae using immunohistochemical staining. In the tissue sections, topo II-α was expressed in the marrow cavity of the primary spongiosa. Mature osteoblasts along the trabecular surfaces did not express topo II-α, but were immunopositive for topo II-β, as were cells of the marrow cavity. Confocal laser scanning microscopy was used to determine the nuclear distribution of topo II in rat osteoblasts isolated from the metaphyseal distal femur and the rat osteosarcoma cells, ROS 17/2.8. Topo II-α exhibited a punctate nuclear distribution in the bone cells. Topo II-β was dispersed throughout the interior of the nucleus but concentrated at the nuclear envelope. Serum starvation of the cells attenuated topo II-α expression but did not modulate expression of the β-isoform. These results indicate that the loss of osteogenic proliferation correlates with the downregulation of topo II-α expression. J. Cell. Biochem. 67:451–465, 1997. © 1997 Wiley-Liss, Inc.     

Effects of steroid hormones on reproduction- and detoxification-related gene expression in adult male mosquitofish, Gambusia affinis

The molecular mechanisms that mediate fish reproduction and detoxification in response to steroid hormones were studied by using adult male western mosquitofish (Gambusia affinis) as sentinel species. The expression patterns of three vitellogenins (VtgA, VtgB and VtgC), two estrogen receptors (ERα and ERβ), two androgen receptors (ARα and ARβ), metallothionein (MT) and cytochrome P450 1A (CYP1A) in the liver and testis of adult male mosquitofish were assessed through exposure treatments with progesterone (P), testosterone (T) and 17β-estradiol (E2), alone and in combination for eight days. The results showed that expression patterns of Vtg subtype, ER subtype, AR subtype, MT and CYP1A genes in male mosquitofish varied according to tissue and specific hormone stress. Vtg subtype mRNA expression was induced in the liver in E2-added treatments, and an up-regulation of ERα mRNA expression was also observed. In addition, hormone treatments increased three Vtg subtype mRNA expression levels in the testis, at least to some extent. All hormone treatments significantly inhibited ERα, ERβ and ARβ mRNA expression in the testis. Some of hormone treatments could affect MT and CYP1A gene expression in mosquitofish. In general, multiple hormone treatments showed different effects on target gene expression compared with corresponding hormone alone. The results from the present study provided valuable information on the toxicological effects of steroid hormones in mosquitofish.     

Inhibition of PPARα/RXRα-mediated direct hyperplasia pathways during griseofulvin-induced hepatocarcinogenesis

Chronic griseofulvin (GF) feeding induces preneoplastic foci followed by hepatocellular carcinoma in the mouse liver. Our previous study suggested that GF-induced hepatocellular proliferation had a different mechanism from that of peroxisome proliferator (PP)–induced direct hyperplasia. The GF-induced hepatocellular proliferation was mediated through activation of immediate early genes such as Fos, Jun, Myc, and NFκB. In contrast, PP-induced direct hyperplasia does not involve activation of any of these immediate early genes. It has been shown that nuclear hormone receptors including peroxisome proliferator activated receptors (PPARs) and retinoid x receptors (RXRs) play important roles in mediating the pleiotropic effects of PPs. To examine the possible roles of PPARs and RXRs during non-PP-induced hepatocellular proliferation and the interaction between PP and non-PP-induced proliferation, we have studied the expression of the PPAR and RXR genes in the GF model using northern blot hybridizations and gel retardation assays. The data showed that the expression of PPARα and RXRα genes was down-regulated in the livers containing preneoplastic nodules and in the liver tumors induced by GF. The mRNA down-regulation was accompanied by a decrease in the amount of nuclear protein–bound to peroxisome proliferator and retinoic acid responsive elements. Down-regulation was also associated with the suppressed expression of the PPARα/RXRα target genes (i.e., acyl-Co oxidase and cytochrome P450 4A1) and the catalase gene. The RXRγ gene was also down-regulated, but the RARα, β, and γ and PPARβ and γ genes were up-regulated. These results indicated that the hepatocarcinogenesis induced by GF is accompanied by suppression of the PPARα/RXRα-mediated direct hyperplasia pathway. The differential expression of these nuclear hormone receptors reveals a new aspect for understanding the individual roles and intercommunication of PPAR, RXR, and RAR isoforms in the liver. J. Cell. Biochem. 69:189–200, 1998. © 1998 Wiley-Liss, Inc.     

Retinoid-related orphan receptor alpha and the regulation of lipid homeostasis

Many nuclear hormone receptors (NRs) control lipid, glucose and energy homeostasis in an organ specific manner. Concordantly, dysfunctional NR signalling results in metabolic disease. The Retinoic acid receptor-related orphan receptor alpha (RORα), a member of the NR1F subgroup, is expressed in metabolic tissues. Previous studies identified the role of this NR in dyslipidemia, apo-lipoprotein metabolism and atherosclerosis. Recent data is underscoring the significant role of this orphan NR in the regulation of phase I/II metabolism (bile acids, xenobiotics, steroids etc.), adiposity, insulin signalling, and glucose tolerance. Moreover, oxygenated sterols, have been demonstrated to function as native ligands and inverse agonists. This review focuses on the rapidly emerging and evolving role of RORα in the control of lipid and glucose homeostasis in major mass metabolic tissues. Article from the special issue orphan receptors.     

Effect of a pharmacological activation of PPAR on the expression of RAR and TR in rat liver

It has recently been shown that high-fat diets induce the expression of peroxisome proliferator-activated receptor (PPAR) with a concomitant decrease in expression of retinoic acid (RAR) and triiodothyronine (TR) receptors in rat liver. The authors have suggested that PPAR activation may be responsible for these modifications in nuclear receptor expression. With the aim of gaining further insight into a possible relationship between the patterns of expression of these receptors, we have examined, using a pharmacological model, the effect of a strong and specific PPAR activation induced by bezafibrate, a peroxisome proliferator agent. Activation of PPAR was evaluated by quantifying PPARα mRNA and acyl-CoA oxidase mRNA. The expression of RAR and TR was determined by assaying the binding properties of these nuclear receptors and by quantifying the mRNA level of RARβ and TRα₁,β₁ isoforms. After a 10 day treatment of young rats, induction of PPAR (PPARα mRNA was increased by 40% [P< 0.05 and acyl-CoA oxidase mRNA by 411% [P<0.001]) and a concomitant decrease of RAR and TR expression (Maximal Binding Capacity was decreased by 21 and 26%, respectively [P<0.05]) in the liver was observed. RXRα mRNA expression was unchanged by treatment. Cross-talk between RAR, TR and PPAR signalling pathways may be implicated in the new patterns of nuclear receptor expression observed. The decreased expression of RAR and TR reported here could provide a novel element for the understanding of the link between PPAR and tumorigenesis in rat liver.     

人SDCT2基因的两种不同转录产物选择性转录机理分析

为了克隆人高亲和力钠离子依赖性二羧酸转运蛋白 (highaffinitysodium dependentdicarboxylatetransporter,SDCT2 ,或NaDC3)基因并研究其生理功能 ,用大鼠SDCT2基因序列作为电子杂交探针对人EST数据库进行电子筛选 ,得到了一系列与大鼠SDCT2序列具有高度同源性的人EST序列 ,将它们拼接成 2个基因重叠群 ,设计特异性PCR引物通过RT PCR扩增得到 2条杂交探针用于筛选人肾cDNA文库 .从肾组织中同时克隆出了人SDCT2基因 2种mRNA变异体的全长cDNA(SDCT2α和SDCT2 β) ,两者 5′端前 3435bp序列完全一致 ,但 3′端长度不同 ,SDCT2 β在第 3435bp以后比SDCT2α多出了 5 85bp的序列 .Northern杂交和RT PCR显示 ,SDCT2α在人肾中的表达丰度最高 ,在肝、脾、胎盘、脑及结肠中也有低水平的表达 .而SDCT2 β主要在肾脏中表达 ,在脾也有低水平的表达 .基因组结构分析表明 ,虽然两种mRNAs均由 13个外显子组成 ,但是SDCT2α的第 13外显子含有 1个poly(A)加尾信号AATAAA ,而SDCT2 β的第 13外显子含有 2个poly(A)加尾信号 .这表明在肾脏和脾脏组织中 ,人SDCT2基因可能通过选择性使用位于第 13外显子不同位置的 2个poly(A)信号而转录出 2种不同长度的mRNA变异体 .