Interactions of nuclear receptor coactivator/corepressor proteins with the aryl hydrocarbon receptor complex.

MCF-7 human breast cancer cells express the aryl hydrocarbon receptor (AhR), and treatment with AhR agonists such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits estrogen receptor (ER)-mediated responses. This study investigates physical and functional interactions of the AhR complex with a prototypical coactivator (estrogen receptor associating protein 140, ERAP 140) and corepressor (silencing mediator for retinoic acid and thyroid hormone receptor, SMRT) for ER and other members of the nuclear receptor superfamily. The AhR, AhR nuclear translocator (Arnt), and AhR/Arnt proteins were coimmunoprecipitated with 35S-ERAP 140 and 35S-SMRT and, in gel mobility shift assays, AhR/Arnt binding to 32P-dioxin response element (DRE) was enhanced by ERAP-140 and inhibited by SMRT; supershifted bands were not observed. In transactivation assays, coactivator and corepressor proteins enhanced or inhibited AhR-mediated gene expression; however, these responses varied with the amount of coactivator/corepressor expression. These results confirmed functional and physical interactions of AhR/Arnt with ERAP 140 and SMRT in breast cancer cells.     

Estrogen effects on histone messenger ribonucleic acid levels in the rat uterus

RNA was isolated from uteri of immature rats before and after estrogen treatment. The concentration of histone mRNA was analyzed by Northern hybridization and compared with messenger RNA concentration of alpha-actin, beta-actin, and beta-tubulin. Steady state levels of common histone mRNAs did not change up to 9 h after hormone administration. After that time the histone mRNA levels increased significantly and reached a maximum at 18 h, several hours later than the time of maximal histone protein biosynthesis induced by estrogen. The concentration of control mRNAs (alpha- and beta-actin and beta-tubulins) increased shortly after estradiol injection and reached a peak at 9 h. These results show that the pattern of histone gene expression induced by estrogen has some features similar to those observed during embryogenesis.     

Differential Changes in Expression of Stress- and Metabolic-Related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15–30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls.     

应激性高血压犬鼠脑干及下丘脑-垂体-肾上腺轴中肾上腺髓质素及其受体mRNA表达的改变

采用逆转录- 聚合酶链式反应检测了慢性足底电击结合噪声应激致高血压大鼠下丘脑、延髓、中脑、垂体和肾上腺等组织中编码肾上腺髓质素的肾上腺髓质素前肽原(preproadrenomedullin, ppADM) 基因以及ADM 的特异性受体组件降钙素受体样受体(calcitonin-receptor-like receptor,CRLR)和受体活性调节蛋白2 和3(receptor-activity-modifying proteins, RAMP2 和RAMP3)表达的变化。我们观察到:与对照组相比,以 3- 磷酸甘油醛脱氢酶作为内参照,15 d 足底电击结合噪声应激引起下丘脑、垂体和肾上腺中ppADM mRNA表达上调,而在延髓和中脑表达明显下调(P<0.01 或 P<0.05); CRLR基因表达量正常时在下丘脑相对较高,应激15 d 后CRLR 表达在延髓、中脑和下丘脑下调(P<0.01 或 P<0.05), 而在垂体和肾上腺的表达无明显变化;应激后RAMP2 基因在延髓和下丘脑表达上调,而在肾上腺表达显著下调(P <0.01), 其他部位无明显变化;RAMP3 基因在对照组大鼠的中脑和下丘脑表达较高,在应激性高血压大鼠的下丘脑和垂体表达上调(P<0.01 或P<0.05), 而在中脑和肾上腺表达下调(P<0.05), 在延髓中的表达变化无统计学差异。上述结果提示:慢性足底电击结合噪声应激引起明显的中枢和下丘脑- 垂体-肾上腺轴ADM 及其受体组件CRLR/RAMP2 或CRLR/R     

应激性高血压大鼠脑干及下丘脑-垂体-肾上腺轴中肾上腺髓质素及其受体mRNA表达的改变

采用逆转录-聚合酶链式反应检测了慢性足底电击结合噪声应激致高血压大鼠下丘脑、延髓、中脑、垂体和肾上腺等组织中编码肾上腺髓质素的肾上腺髓质素前肽原(preproadrenomedullin,ppADM)基因以及ADM的特异性受体组件降钙素受体样受体(calcitonin-receptor-like receptor,CRLR)和受体活性调节蛋白2和3(receptor-activty-modifying proteins,RAMP2和RAMP3)表达的变化.我们观察到:与对照组相比,以3-磷酸甘油醛脱氢酶作为内参照,15 d足底电击结合噪声应激引起下丘脑、垂体和肾上腺中ppADM mRNA表达上调,而在延髓和中脑表达明显下调(P＜0.01或P＜0.05);CRLR基因表达量正常时在下丘脑相对较高,应激15 d后CRLR表达在延髓、中脑和下丘脑下调(P＜0.01或P＜0.05),而在垂体和肾上腺的表达无明显变化;应激后RAMP2基因在延髓和下丘脑表达上调,而在肾上腺表达显著下调(P＜0.01),其他部位无明显变化;RAMP3基因在对照组大鼠的中脑和下丘脑表达较高,在应激性高血压大鼠的下丘脑和垂体表达上调(P＜0.01或P＜0.05),而在中脑和肾上腺表达下调(P＜0.05),在延髓中的表达变化无统计学差异.上述结果提示:慢性足底电击结合噪声应激引起明显的中枢和下丘脑-垂体-肾上腺轴ADM及其受体组件CRLR/RAMP2或CRLR/RAMP3基因的表达变化.但慢性应激后中枢源性ADM及其受体的表达变化对应激和血压的调节以及在应激致高血压中的确切作用及机制尚待进一步研究.     

Induction of CCK mRNA levels in the limbic-hypothalamic circuit: Time course and site-specific effects of estrogen

Estrogenic regulation of cholecystokinin (CCK) and its receptors is correlated with the initiation and termination of lordosis behavior. To understand the effect of circulating estrogen concentration on the temporal aspects of CCK mRNA expression in the posterodorsal medial amygdaloid nucleus (MeApd) and the central part of the medial preoptic nucleus (MPNc) of the limbic-hypothalamic circuit, ovariectomized female rats were treated with a 10 mm Silastic™ capsule filled with estradiol, a bolus injection of 50 μg estradiol benzoate or 2 μg estradiol benzoate every 4 days for five “cycles.” In situ hybridization was used to compare the relative changes of CCK mRNA levels at 0 h to levels measured at 6, 12, 24, 48, 72, or 96 h after estrogen administration. In the MPNc and the MeApd, the 10-mm capsule significantly increased and maintained CCK mRNA levels from 6 to 96 h. The range of the increase was 3.0–5.1-fold in the MPNc and 2.8–5.0 in the MeApd. The 50-μg injections significantly increased and maintained CCK mRNA levels in the MPNc from 12 to 96 h (range of the increase 2.4–4.1-fold) and in the MeApd from 24 to 96 h (range of the increase 2.2–2.8-fold). The repeated administration of 2 μg estrogen induced a significant increase of message levels in the MPNc at 12 and 24 h that were 4.2- and 4.7-fold, respectively. In the MeApd this estrogen treatment did not significantly increase CCK mRNA. These studies demonstrate that small doses (2 μg) of estrogen that mimic the pattern and circulating levels of estrogen dramatically stimulate CCK mRNA levels in the limbic-hypothalamic circuit. To further study this steroid stimulation, ovariectomized female rats were implanted with estradiol-filled cannulae into the bed nucleus of the stria terminalis or MeA. Estrogen elevated CCK mRNA levels locally in each nucleus. Implants in the bed nucleus also elevated CCK mRNA levels in the MeApd indicating that physiologic estrogen stimulation of CCK in the MeApd is the result of both local and distal transsynaptic elevation of CCK mRNA levels. The site-specific induction of CCK mRNA levels within the limbic-hypothalamic nuclei provides another important facet of estrogenic modulation of CCK induction. © 1996 John Wiley & Sons, Inc.     

Effects of kainic acid on messenger RNA levels of IL-1 beta, IL-6, TNF alpha and LIF in the rat brain.

We examined the kainic acid-induced changes of mRNA levels of several cytokines such as IL-1 beta, IL-6, TNF alpha and LIF in the rat brain regions using semiquantitative RT-PCR method. IL-1 beta mRNA was markedly increased in the cerebral cortex (CC), thalamus (THL) and hypothalamus (HT) 2 h after the injection of kainic acid in a convulsive dose (12 mg/kg i.p.), and tended to decrease 4 h after the injection. IL-6 mRNA was weakly induced in the hippocampus (HPP) 2 h after the injection of kainic acid and was markedly increased in the CC, HPP, THL, and HT at 4 h. The level of TNF alpha mRNA was highly elevated in the CC, HPP, striatum (STR), THL and HT at 2 and 4 h after the injection. LIF mRNA apparently expressed in the CC and HPP of control rats and was increased in the CC, HPP and HT by the treatment with kainic acid. These results indicate that mRNAs of several cytokines are increased in various brain regions with different time-courses by kainic acid.     

Orexin 和P物质对顺铂诱发大鼠异食癖的影响

目的:观察食欲素(Orexin)和P物质(SP)对顺铂诱发大鼠异食癖的影响。方法:雄性Wistar大鼠被随机分为对照组和顺铂处理组,顺铂处理组给予大鼠顺铂(3或6 mg/kg,腹腔注射),对照组给予等量生理盐水。记录顺铂大鼠摄食高岭土量、摄食量的改变;Real-time PCR法观察顺铂对大鼠下丘脑Orexin和延髓中SP前体-前速激肽原A(PPT-A)m RNA表达的影响;分别和联合应用SP受体(NK1受体)拮抗剂阿瑞匹坦和Orexin-A对顺铂大鼠异食癖和摄食量的作用。结果:皮下注射3 mg/kg(低剂量组)的顺帕后大鼠高岭土摄入量和摄食量与对照组相比无明显差异(P0.05),而注射6 mg/kg(高剂量组)顺铂后,大鼠高岭土摄入量与对照组和低剂量组相比显著增加(P0.05);高剂量的顺铂作用12 h时,大鼠延髓内PPT-A的m RNA表达有轻微增加,但无统计学差异(P0.05),24 h后,延髓内PPT-A的m RNA表达量显著增加(P0.05)。在此后持续观察的5天中,顺铂可持续引起延髓中PPT-A的mRNA表达增高,在第5天时PPT-A的m RNA仍维持166.23±16.92%的高表达。高剂量顺铂抑制大鼠下丘脑中Orexin的mRNA表达,24 h时Orexin降低幅度最明显,为对照组的34.81±7.22%(P0.05)。此后检测的5天,Orexin浓度均低于对照组;将阿瑞匹坦和orexin联合应用,大鼠高岭土摄入量较单独应用阿瑞匹坦或orexin明显减少,摄食量显著增加(P0.05)。结论:P物质和orexin通路对顺铂化疗大鼠的异食癖和摄食量调控具有协同作用。     

Neonatal oxytocin treatment modulates oxytocin receptor, atrial natriuretic peptide, nitric oxide synthase and estrogen receptor mRNAs expression in rat heart

Oxytocin (OT) has been implicated in reproductive functions, induction of maternal behavior as well as endocrine and neuroendocrine regulation of the cardiovascular system. Here we demonstrate that neonatal manipulation of OT can modulate the mRNAs expression for OT receptor (OTR), atrial natriuretic peptide (ANP), endothelial nitric oxide synthase (eNOS) and estrogen receptor alpha (ERalpha) in the heart. On the first day of postnatal life, female and male rats were randomly assigned to receive one of the following treatments: (a) 50microl i.p. injection of 7microg OT; (b) 0.7microg of OT antagonist (OTA); or (c) isotonic saline (SAL). Hearts were collected either on postnatal day 1 or day 21 (D1 or D21) and the mRNAs expression of OTR, ANP, inducible NOS (iNOS), eNOS, ERalpha and estrogen receptor beta (ERbeta) were compared by age, treatment, and sex utilizing real time PCR. OT treatment significantly increased heart OTR, ANP and eNOS mRNAs expression on D1 in both males and females, ERalpha increased only in females. While there were significant changes in the relative expression of all types of mRNA between D1 and D21, there were no significant treatment effects observed in D21 animals. OTA treatment significantly decreased basal ANP and eNOS mRNAs expression on D1 in both sexes. The results indicate that during the early postnatal period OT can have an immediate effect on the expression OTR, ANP, eNOS, and ERalpha mRNAs and that these effects are mitigated by D21. Also with the exception of ERalpha mRNA, the effects are the same in both sexes.     

Effect of estradiol on expression and activation of Akt protein in rat hypothalamus exposed to chronic [D-Met2, Pro5]-enkephalinamide treatment

Adult ovariectomized rats were implanted with [D-Met2, Pro5]-enkephalinamide (ENK)-containing osmotic minipumps. Two hours prior to sacrifice, some animals were treated with estradiol-17beta (E2) at a dose 10 microg/100 g bodyweight (BW). Expression and activation of Akt proteins, nuclear [3H]estradiol binding, and the expression of estrogen receptor alpha (ERalpha) and beta (ERbeta) and of progesterone receptor (PR) were investigated. Estradiol increased the level of activated Akt protein (pAkt473) in the hypothalamus by 52 +/- 11% in comparison to the vehicle-treated controls. No such effect of E2 was observed 24 and 48 h after ENK implantation. This effect of ENK was abolished by concomitant treatment with naloxone. Time-dependent changes in nuclear [3H]estradiol binding and the expression of estrogen and progesterone receptors were also detected in the hypothalamus of ENK-implanted and E2-treated rats. At 24-48 h following ENK implantation, expression of ERalpha and high affinity [3H]estradiol binding decreased. At this time point, the PR level was also reduced, while the ERbeta level was augmented. In conclusion, these results suggest that the stimulatory effects of E2 on the expression and activation of Akt protein and the expression of ERalpha and PR are negatively regulated in rat hypothalamus exposed to chronic ENK treatment.     

Tissue-specific regulation of rat estrogen receptor mRNAs

The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for physiological estrogen responses, including estrogen-induced tissue-specific changes in gene expression. We studied the estrogen regulation of the mRNAs encoding the ER in rat uterus, liver, and pituitary. Ovariectomized (21-28 day post surgery) female CD-1 rats were injected daily with 17 beta-estradiol (E2, 10 micrograms/100 g BW) for 0, 1, or 4 h, 1, 3, or 7 days and compared with intact controls. Steady-state levels of ER mRNA were quantified using a human ER cDNA probe. Only one hybridizing species of approximately 6.2 kilobase (kb) was detected in uterine and liver RNA, similar to that observed in MCF7 human breast cancer cells. However, the ER mRNA regulation by E2 differed in direction depending on the tissue examined. In uterus, ER mRNA increased 3- to 6-fold after ovariectomy, and returned to intact levels within 24 h of E2 replacement. In contrast, liver ER mRNA declined 1.5- to 3-fold after ovariectomy and returned to intact levels after 1-3 days of E2. In pituitary tissue two hybridizing forms of ER mRNA were observed, with one species migrating at 6.2 kb, equivalent to the form in other tissues, and a second smaller species at approximately 5.5 kb. The lower molecular weight species varied somewhat in abundance from animal to animal, averaging about 20% of the intensity of the 6.2 kb band. The ER mRNA forms were regulated positively with E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organ-specific and age-dependent expression of insulin-like growth factor-I (IGF-I) mRNA variants: IGF-IA and IB mRNAs in the mouse

Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse IGF-IA and IGF-IB mRNAs using SYBR Green real-time RT-PCR. In the liver, IGF-I mRNA expression increased from 10 days of age to 45 days. In the uterus and ovary, IGF-I mRNA expression increased from 21 days of age, and then decreased at 45 days. In the kidney, IGF-I mRNA expression decreased from 10 days of age. IGF-IA mRNA levels were higher than IGF-IB mRNA levels in all organs examined. Estradiol-17beta (E2) treatment in ovariectomized mice increased uterine IGF-IA and IGF-IB mRNA levels from 3 hr after injection, and highest levels for both mRNAs were detected at 6 hr, and relative increase was greater for IGF-IB mRNA than for IGF-IA mRNA. These results suggest that expression of IGF-I mRNA variants is regulated in organ-specific and age-dependent manners, and estrogen is involved in the change of IGF-I mRNA variant expression.