Hormone-dependent Gene Expression Research Articles

Abstract P6-12-02: Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Abstract Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer “decommissioning” and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous and typically high dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation (i.e., the presence of hormone) and non-stimulation (i.e., the absence of hormone). In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating and short duration E2 exposures using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to low dose E2 treatment and E2 withdrawal. Moreover, for some E2 target genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking. Citation Format: Hyung Bum Kim, William L. Kraus. Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-02.

RF03 | PMON297 Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Abstract Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer "decommissioning" and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation and non-stimulation. In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating E2 levels using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to E2 withdrawal. Moreover, for some E2 traget genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Presentation: Saturday, June 11, 2022 1:12 p.m. - 1:17 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The Impact of Cancer‐Associated Mutations in Thyroid Hormone Receptor (TR) on TR Localization

The thyroid hormone receptor (TR)—a nuclear receptor that is essential for the proper regulation of metabolism and development, as it regulates gene expression in response to thyroid hormone—contains nuclear localization signals (NLSs) and nuclear export signals (NESs) that allow for TR transport into and out of the nucleus, respectively. Previous research has shown that nuclear import, nuclear retention, and nuclear export of TR are associated with modulation of gene expression, the alteration of which can contribute to various diseases, including Resistance to Thyroid Hormone (RTH) Syndrome, type II diabetes mellitus, and several types of cancer. There are three main subtypes of TR: TRα1, TRβ1, and TRβ2. Here, through mammalian cell transfection of expression plasmids for green fluorescent protein (GFP)‐tagged TR and fluorescence microscopy, we examined particular groups of TRα1 mutations that were observed in patients with hepatocellular carcinoma and renal carcinoma and are associated with NLSs and NESs of TRα1. More specifically, we calculated the nuclear‐to‐cytoplasmic ratios of TRα1 variants to determine the impact of TRα1 mutations on receptor localization within HeLa cells. We found that, while wild‐type TR has a primarily nuclear localization with a smooth distribution in human cells grown in culture, the TRα1 mutations S40T, K136R, L251P, V390A and A225G, D246N, G350K lead to significantly decreased nuclear‐to‐cytoplasmic TRα1 ratios (greater cytoplasmic distribution) and formation of TRα1 aggregates. We observed that the distribution of a TRα1 mutant with just one of the four mutations (L251P) is very similar to the distribution of the TRα1 mutant with all four mutations (S40T, K136R, L251P, V390A), suggesting that L251P plays a major role in the mislocalization of TRα1. In addition, we found that TRα1 with a G24E mutation had a significantly greater cytoplasmic distribution, compared to wild‐type TRα1. In conclusion, our findings suggest that certain TRα1 mutations that are associated with carcinoma lead to TRα1 mislocalization, which may alter the expression of thyroid hormone‐dependent genes. These results contribute to an improved understanding of TR structure and function, which could lead to novel strategies for discovering therapies for TR‐related illnesses.Support or Funding InformationNIH #2R15DK058028‐04This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epigenetic biomarker screening by FLIM-FRET for combination therapy in ER+ breast cancer

Hormone-dependent gene expression involves dynamic and orchestrated regulation of epigenome leading to a cancerous state. Estrogen receptor (ER)-positive breast cancer rely on chromatin remodeling and association with epigenetic factors in inducing ER-dependent oncogenesis and thus cell over-proliferation. The mechanistic differences between epigenetic regulation and hormone signaling provide an avenue for combination therapy of ER-positive breast cancer. We hypothesized that epigenetic biomarkers within single nucleosome proximity of ER-dependent genes could serve as potential therapeutic targets. We described here a Fluorescence lifetime imaging-based Förster resonance energy transfer (FLIM-FRET) methodology for biomarker screening that could facilitate combination therapy based on our study. We screened 11 epigenetic-related markers which include oxidative forms of DNA methylation, histone modifications, and methyl-binding domain proteins. Among them, we identified H4K12acetylation (H4K12ac) and H3K27 acetylation (H3K27ac) as potential epigenetic therapeutic targets. When histone acetyltransferase inhibitor targeting H4K12ac and H3K27ac was combined with tamoxifen, an enhanced therapeutic outcome was observed against ER-positive breast cancer both in vitro and in vivo. Together, we demonstrate a single molecule approach as an effective screening tool for devising targeted epigenetic therapy.

Long-Lasting Primed State in Maize Plants: Salicylic Acid and Steroid Signaling Pathways as Key Players in the Early Activation of Immune Responses in Silks.

In the present study, we investigated the induced systemic resistance (ISR) activated by the beneficial fungus Trichoderma atroviride in maize plants, and the early immunological responses triggered after challenge with the ear rot pathogen Fusarium verticillioides. By transcriptional analysis, we were able to identify the gene core set specifically modulated in silks of maize plants expressing ISR. Our results showed that the main transcriptional reprogramming falls into genes involved in five main functional categories: cell structure or cell wall, amino acid and protein metabolism, stress responses, signaling, and transport. Among these ISR-related genes, it is important to highlight novel findings regarding hormone metabolism and signaling. The expression of hormone-dependent genes was in good agreement with the abscisic acid, jasmonic acid, and salicylic acid (SA) levels detected in the plants under study. The experimental design allowed the identification of novel regulatory elements related to a heightened state of defense in silks and suggests that steroids and SA are central components of a master regulatory network controlling the immunity of silks during ISR. The results presented also provide evidence about the molecular mechanisms used by maize silks against F. verticillioides to counteract pathogenic development and host invasion, including pathogenesis-related genes, plant cell-wall reinforcement, fungal cell-wall-degrading enzymes and secondary metabolism.

Comparison of triadimefon and its metabolite on acute toxicity and chronic effects during the early development of Rana nigromaculata tadpoles

Pesticides are one of major causes for amphibian population declines and the behavior of pesticide metabolite products to amphibians has become a rising concern. In this study, the acute toxicity and the chronic effects of triadimefon and triadimenol (the metabolite of triadimefon) on Rana. nigromaculata were investigated. In the acute assay, significant differences were observed in antioxidant enzyme activities and malondialdehyde levels between the triadimefon and triadimenol. The 96 h-acute toxicity of triadimefon (25.97 mg/L) and triadimenol (34.55 mg/L) to tadpoles was low. In 28d-chronic exposure, we studied the relative expression of tadpoles genes related to thyroid hormone-dependent metamorphic development, histological examination of liver and some biological index, including wet weight, snout-to-vent length (SVL) and development stages. The results revealed that the effects of triadimefon and triadimenol on tadpole development are driven by a disruption of the hormonal pathways involved in metamorphosis. Interestingly, triadimefon was more harmful on R. nigromaculata than triadimenol at high dose, whereas the reverse result was observed at low doses. According to the relative expression of thyroid hormone-dependent genes, we also found that the two compounds may have different mechanisms of toxic action on R. nigromaculata. Our study developed a pragmatic approach for use in the risk assessment of pesticide and its metabolite，and increased the information and understanding of the impacts of fungicides and other potential endocrine disrupting environmental contaminants on amphibians.

FOXA1 acts upstream of GATA2 and AR in hormonal regulation of gene expression.

Hormonal regulation of gene expression by androgen receptor (AR) is tightly controlled by many transcriptional cofactors, including pioneer factors FOXA1 and GATA2, which, however, exhibit distinct expression patterns and functional roles in prostate cancer. Here, we examined how FOXA1, GATA2, and AR crosstalk and regulate hormone-dependent gene expression in prostate cancer cells. ChIP-seq analysis revealed that FOXA1 reprograms both AR and GATA2 cistrome by preferably recruiting them to FKHD-containing genomic sites. By contrast, GATA2 is unable to shift AR or FOXA1 to GATA motifs. Rather, GATA2 co-occupancy enhances AR and FOXA1 binding to nearby ARE and FKHD sites, respectively. Similarly, AR increases, but not re-programs, GATA2 and FOXA1 cistromes. Concordantly, GATA2 and AR strongly enhance the transcriptional program of each other, whereas FOXA1 regulates GATA2- and AR-mediated gene expression in a context-dependent manner due to its reprogramming effects. Taken together, our data delineated for the first time the distinct mechanisms by which GATA2 and FOXA1 regulate AR cistrome and suggest that FOXA1 acts upstream of GATA2 and AR in determining hormone-dependent gene expression in prostate cancer.

H4K12ac is regulated by estrogen receptor-alpha and is associated with BRD4 function and inducible transcription.

Hormone-dependent gene expression requires dynamic and coordinated epigenetic changes. Estrogen receptor-positive (ER+) breast cancer is particularly dependent upon extensive chromatin remodeling and changes in histone modifications for the induction of hormone-responsive gene expression. Our previous studies established an important role of bromodomain-containing protein-4 (BRD4) in promoting estrogen-regulated transcription and proliferation of ER+ breast cancer cells. Here, we investigated the association between genome-wide occupancy of histone H4 acetylation at lysine 12 (H4K12ac) and BRD4 in the context of estrogen-induced transcription. Similar to BRD4, we observed that H4K12ac occupancy increases near the transcription start sites (TSS) of estrogen-induced genes as well as at distal ERα binding sites in an estrogen-dependent manner. Interestingly, H4K12ac occupancy highly correlates with BRD4 binding and enhancer RNA production on ERα-positive enhancers. Consistent with an importance in estrogen-induced gene transcription, H4K12ac occupancy globally increased in ER-positive cells relative to ER-negative cells and these levels were further increased by estrogen treatment in an ERα-dependent manner. Together, these findings reveal a strong correlation between H4K12ac and BRD4 occupancy with estrogen-dependent gene transcription and further suggest that modulators of H4K12ac and BRD4 may serve as new therapeutic targets for hormone-dependent cancers.

The Expression of Histone Deacetylase 1, But Not Other Class I Histone Deacetylases, Is Significantly Increased in Endometriosis

Class I histone deacetylases (HDACs-1-3) play an important role in steroid hormone-dependent gene expression and in modulating cell survival and proliferation. We analyzed their expression in a tissue microarray including 74 endometriosis samples and 30 normal endometrium controls. The mean HDAC-1 immunoreactivity score (IRS ± standard deviation) was 7.6 ± 2.5 in endometriosis and 5.3 ± 2.3 in normal endometrium (P < .001). In contrast, the IRSs of HDAC-2 and -3 were 11.7 ± 0.7 and 11.8 ± 1.1 in endometriosis and 11.6 ± 1.0 and 11.9 ± 0.4 in normal endometrium (P = .7 and P = .2), respectively. Significant correlations were found between HDAC-1 and estrogen (-alpha/-beta) and progesterone receptor expression. In conclusion, HDAC-1, but not HDAC-2/-3, was significantly increased in endometriosis and associated with steroid hormone receptor expression that may reflect interdependence. In context with the literature, specific inhibitors of HDAC-1 may have inhibitory activities similar to those of broad-spectrum HDAC inhibitors and may be clinically tolerated, which would increase their chance as an option in the treatment of endometriosis.

3,5-di-iodothyronine stimulates tilapia growth through an alternate isoform of thyroid hormone receptor β1

Recent studies in our laboratory have shown that in some teleosts, 3,5-di-iodothyronine (T2 or 3,5-T2) is as bioactive as 3,5,3'-tri-iodothyronine (T3) and that its effects are in part mediated by a TRβ1 (THRB) isoform that contains a 9-amino acid insert in its ligand-binding domain (long TRβ1 (L-TRβ1)), whereas T3 binds preferentially to a short TRβ1 (S-TRβ1) isoform that lacks this insert. To further understand the functional relevance of T2 bioactivity and its mechanism of action, we used in vivo and ex vivo (organotypic liver cultures) approaches and analyzed whether T3 and T2 differentially regulate the S-TRβ1 and L-TRβ1s during a physiological demand such as growth. In vivo, T3 and T2 treatment induced body weight gain in tilapia. The expression of L-TRβ1 and S-TRβ1 was specifically regulated by T2 and T3 respectively both in vivo and ex vivo. The TR antagonist 1-850 effectively blocked thyroid hormone-dependent gene expression; however, T3 or T2 reversed 1-850 effects only on S-TRβ1 or L-TRβ1 expression, respectively. Together, our results support the notion that both T3 and T2 participate in the growth process; however, their effects are mediated by different, specific TRβ1 isoforms.

Jasmonate signalling: a copycat of auxin signalling?

Plant hormones regulate almost all aspects of plant growth and development. The past decade has provided breakthrough discoveries in phytohormone sensing and signal transduction, and highlighted the striking mechanistic similarities between the auxin and jasmonate (JA) signalling pathways. Perception of auxin and JA involves the formation of co-receptor complexes in which hormone-specific E3-ubiquitin ligases of the SKP1-Cullin-F-box protein (SCF) type interact with specific repressor proteins. Across the plant kingdom, the Aux/IAA and the JASMONATE-ZIM DOMAIN (JAZ) proteins correspond to the auxin- and JA-specific repressors, respectively. In the absence of the hormones, these repressors form a complex with transcription factors (TFs) specific for both pathways. They also recruit several proteins, among which the general co-repressor TOPLESS, and thereby prevent the TFs from activating gene expression. The hormone-mediated interaction between the SCF and the repressors targets the latter for 26S proteasome-mediated degradation, which, in turn, releases the TFs to allow modulating hormone-dependent gene expression. In this review, we describe the similarities and differences in the auxin and JA signalling cascades with respect to the protein families and the protein domains involved in the formation of the pathway-specific complexes.

Modulation of Thyroid Hormone-Dependent Gene Expression in Xenopus laevis by INhibitor of Growth (ING) Proteins

BackgroundINhibitor of Growth (ING) proteins belong to a large family of plant homeodomain finger-containing proteins important in epigenetic regulation and carcinogenesis. We have previously shown that ING1 and ING2 expression is regulated by thyroid hormone (TH) during metamorphosis of the Xenopus laevis tadpole. The present study investigates the possibility that ING proteins modulate TH action.Methodology/Principal FindingsTadpoles expressing a Xenopus ING2 transgene (TransING2) were significantly smaller than tadpoles not expressing the transgene (TransGFP). When exposed to 10 nM 3,5,3′-triiodothyronine (T3), premetamorphic TransING2 tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T3-treated TransGFP tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor β (TRβ) and TH/bZIP transcript levels in TransING2 tadpole tails compared to TransGFP tadpoles while TRα mRNAs were unaffected. In contrast, no difference in TRα, TRβ or insulin-like growth factor (IGF2) mRNA abundance was observed in the brain between TransING2 and TransGFP tadpoles. All of these transcripts, except for TRα mRNA in the brain, were inducible by the hormone in both tissues. Oocyte transcription assays indicated that ING proteins enhanced TR-dependent, T3-induced TRβ gene promoter activity. Examination of endogenous T3-responsive promoters (TRβ and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T3-dependent and independent ways, respectively. Moreover, ING and TR proteins coimmunoprecipitated from tail protein homogenates derived from metamorphic climax animals.Conclusions/SignificanceWe show for the first time that ING proteins modulate TH-dependent responses, thus revealing a novel role for ING proteins in hormone signaling. This has important implications for understanding hormone influenced disease states and suggests that the induction of ING proteins may facilitate TR function during metamorphosis in a tissue-specific manner.

Differential Glucocorticoid Receptor-mediated Transcription Mechanisms

Nuclear receptors such as the glucocorticoid receptor (GR) are ligand-dependent transcription factors that mediate transcription of target genes by recruiting factors that modulate chromatin structure. In this study, curcumin, a compound known to inhibit GR-mediated transcription, was used to examine the different mechanisms by which GR regulates transcription. The mechanisms of transcription regulation of metallothioneine-2A (MT2A) and solute carrier family 19 member 2 (SLC19A2), two GR target genes where the hormone-dependent gene activation is inhibited or unaffected by curcumin treatment, respectively, were analyzed by chromatin immunoprecipitation and RT-PCR experiments. The data suggest that the loss of hormone-dependent MT2A gene expression is due to the inhibition of continued transcription activity after initial assembly of the transcription machinery. In contrast, the hormone-dependent SLC19A2 gene expression is maintained because the continued transcription output after assembly of transcription machinery is unaffected by curcumin. These results suggest that the two GR target genes use alternate mechanisms to regulate expression levels at the level of continued transcription output after transcription machinery assembly.

Minireview: role of kinases and chromatin remodeling in progesterone signaling to chromatin.

Steroid hormones regulate gene expression by interaction of their receptors with hormone-responsive elements on DNA or with other transcription factors, but they can also activate cytoplasmic signaling cascades. Rapid activation of Erk by progestins via an interaction of the progesterone receptor (PR) with the estrogen receptor is critical for transcriptional activation of the mouse mammary tumor virus (MMTV) promoter and other progesterone target genes. Erk activation leads to the phosphorylation of PR, activation of mitogen- and stress-activated protein kinase 1, and the recruitment of a complex of the three activated proteins and of P300/CBP-associated factor (PCAF) to a single nucleosome, resulting in the phosphoacetylation of histone H3 and the displacement of heterochromatin protein 1γ. Hormone-dependent gene expression requires ATP-dependent chromatin remodeling complexes. Two switch/sucrose nonfermentable-like complexes, Brahma-related gene 1-associated factor (BAF) and polybromo-BAF are present in breast cancer cells, but only BAF is recruited to the MMTV promoter and cooperates with PCAF during activation of hormone-responsive promoters. PCAF acetylates histone H3 at K14, an epigenetic mark recognized by BAF subunits, thus anchoring the complex to chromatin. BAF catalyzes localized displacement of histones H2A and H2B, facilitating access of nuclear factor 1 and additional PR complexes to the hidden hormone-responsive elements on the MMTV promoter. The linker histone H1 is a structural component of chromatin generally regarded as a general repressor of transcription. However, it contributes to a better regulation of the MMTV promoter by favoring a more homogeneous nucleosome positioning, thus reducing basal transcription and actually enhancing hormone induced transcription. During transcriptional activation, H1 is phosphorylated and displaced from the promoter. The kinase cyclin-dependent kinase 2 is activated after progesterone treatment and could catalyze progesterone-induced phosphorylation of histone H1 by chromatin remodeling complexes. The initial steps of gene induction by progestins involve changes in the chromatin organization of target promoters that require the activation of several kinase signaling pathways initiated by membrane anchored PR. Because these pathways also respond to other external signals, they serve to integrate the hormonal response in the global context of the cellular environment.

Thyroid Hormone-Regulated Mouse Cerebral Cortex Genes Are Differentially Dependent on the Source of the Hormone: A Study in Monocarboxylate Transporter-8- and Deiodinase-2-Deficient Mice

Thyroid hormones influence brain development through the control of gene expression. The concentration of the active hormone T(3) in the brain depends on T(3) transport through the blood-brain barrier, mediated in part by the monocarboxylate transporter 8 (Mct8/MCT8) and the activity of type 2 deiodinase (D2) generating T(3) from T(4). The relative roles of each of these pathways in the regulation of brain gene expression is not known. To shed light on this question, we analyzed thyroid hormone-dependent gene expression in the cerebral cortex of mice with inactivated Mct8 (Slc16a2) and Dio2 genes, alone or in combination. We used 34 target genes identified to be controlled by thyroid hormone in microarray comparisons of cerebral cortex from wild-type control and hypothyroid mice on postnatal d 21. Inactivation of the Mct8 gene (Mct8KO) was without effect on the expression of 31 of these genes. Normal gene expression in the absence of the transporter was mostly due to D2 activity because the combined disruption of Mct8 and Dio2 led to similar effects as hypothyroidism on the expression of 24 genes. Dio2 disruption alone did not affect the expression of positively regulated genes, but, as in hypothyroidism, it increased that of negatively regulated genes. We conclude that gene expression in the Mct8KO cerebral cortex is compensated in part by D2-dependent mechanisms. Intriguingly, positive or negative regulation of genes by thyroid hormone is sensitive to the source of T(3) because Dio2 inactivation selectively affects the expression of negatively regulated genes.

Normalization of Circulating Nucleic Acid Results

Nucleic acids, both DNA and mRNA, have been detected in the circulation and have been demonstrated to be useful in such areas as fetal medicine, oncology, and transplantation. When mRNA is measured in circulating blood, the results are expressed in relation to a reference gene product in order to correct for any differences in extraction, volume of starting material or other differences. Many authors use beta-actin mRNA and express results as a ratio of target mRNA to beta-actin mRNA. We have used a similar approach when studying diabetic retinopathy. Recently, we planned to investigate the expression of thyroid dependent gene expression in acutely ill patients. As a control study, we examined the expression of thyroid hormone-dependent gene expression in subjects with hyperthyroidism and found that the expression of beta-actin mRNA was affected by thyroid hormone status. Blood samples were taken into PAX genetrade mark tubes from 31 healthy subjects (mean age, 43 +/- 16 yrs) and 7 patients with hyperthyroidism (mean age, 43 +/- 5 yrs). Diagnosis of hyperthyroidism was confirmed by clinical findings and biochemical results. After extraction of mRNA, cDNA was synthesized using reverse transcription. Quantification of Na/K-ATPase, T3 receptor, and beta-actin cDNA was carried out by RT-PCR. Median beta-actin levels were significantly higher in hyperthyroid subjects compared to healthy subjects (18.2 versus 2.30; P < 0.00042). When mRNA for the T3 receptor was expressed in relation to beta-actin, there was a significantly higher in hyperthyroidism (0.0168 versus 0.218, P < 0.05). However, this was significantly lower when expressed in relation to total RNA (12.2 versus 2.24, P < 0.00015). We conclude that normalizing results to beta-actin may not be appropriate in all circumstances.

Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components

In view of their different actions on thyroid hormone receptor (TR) isoforms we set out to investigate whether amiodarone (AM) and dronedarone (Dron) have different and/or component-specific effects on cardiac gene expression. Rats were treated with AM or Dron and the expression of TRalpha 1, TRalpha 2, TRbeta 1 and several tri-iodothyronine (T3)-regulated genes was studied in different parts of the heart, namely the right atrium (RA), left ventricular wall (LVW) and apex. Rats were treated for 14 days with 100 mg/kg body weight AM or Dron. The expression of TRalpha 1, TRalpha 2, TRbeta 1 and T3-regulated genes was studied using real-time PCR and non-radioactive in situ hybridisation. AM and Dron affected TR expression in the RA similarly by decreasing TRalpha 1 and beta 1 expression by about 50%. In the LVW, AM and Dron decreased TRbeta 1 and, interestingly, AM increased TRalpha 1. In the apex, AM also increased TRalpha 2. The changes seen in T3-dependent gene expression are reminiscent of foetal reprogramming. Taken together, our results indicate that AM and Dron have similar effects on the expression of TR isoforms in the RA, which could partly contribute to their ability to decrease heart rate. On the other hand, the more profound effect of AM appears on TR- and T3-dependent gene expression in the left ventricle suggests foetal reprogramming.

Hexamerin-based regulation of juvenile hormone-dependent gene expression underlies phenotypic plasticity in a social insect

Worker termites of the genus Reticulitermes are temporally-arrested juvenile forms that can terminally differentiate into adultsoldier- or reproductive-caste phenotypes. Soldier-caste differentiation is a developmental transition that is induced by high juvenile hormone (JH) titers. Recently, a status quo hexamerin mechanism was identified, which reduces JH efficacy and maximizes colony fitness via the maintenance of high worker-caste proportions. Our goal in these studies was to investigate more thoroughly the influences of the hexamerins on JH-dependent gene expression in termite workers. Our approach involved RNA interference (RNAi), bioassays and quantification of gene expression. We first investigated the expression of 17 morphogenesis-associated genes in response to RNAi-based hexamerin silencing. Hexamerin silencing resulted in significant downstream impacts on 15 out of the 17 genes, suggesting that these genes are members of a JH-responsive genomic network. Next, we compared gene-expression profiles in workers after RNAi-based hexamerin silencing to that of (i) untreated workers that were held away from the colony; and (ii) workers that were also held away from the colony, but with ectopic JH. Here, although there was no correlation between hexamerin silencing and colony-release effects, we observed a significant correlation between hexamerin silencing and JH-treatment effects. These findings provide further evidence supporting the hypothesis that the hexamerins modulate JH availability, thus limiting the impacts of JH on termite caste polyphenism. Results are discussed in a context relative to outstanding questions on termite developmental biology, particularly on regulatory gene networks that respond to JH-, colony- and environmental-cues.

Human Papillomavirus E7 Oncoprotein Dysregulates Steroid Receptor Coactivator 1 Localization and Function

High-risk human papillomaviruses (HPVs) are present in virtually all cervical carcinomas. However, the majority of women infected with high-risk HPVs do not develop cervical cancer. Therefore, cofactors must contribute to the development and progression of cervical cancer. Although numerous studies have implicated steroid hormones as cofactors in the initiation and progression of cervical neoplasia, the molecular mechanisms by which they contribute to cervical carcinogenesis are currently unknown. These observations led us to investigate a newly discovered association of the high-risk HPV type 16 (HPV16) E7 oncoprotein with steroid receptor coactivator 1 (SRC-1), an essential component of steroid hormone signaling. HPV16 E7 has been previously reported to interact with p300 and p300/CBP-associated factor (PCAF), members of some SRC-1 transcriptional complexes. We demonstrate here that HPV16 E7 associates in vivo and in vitro with SRC-1 independently of p300 and PCAF. Luciferase reporter constructs under the control of either the interleukin-8 promoter or a promoter containing multimerized synthetic estrogen response elements were used to determine the effect of high- and low-risk HPV E7 expression on SRC-1-mediated transcription. In addition, histone acetyltransferase (HAT) assays were performed to determine the effect of HPV E7 on SRC-1-associated HAT activity. These experiments reveal that HPV16 E7 expression down-regulates SRC-1-mediated transcription and SRC-1-associated HAT activity. SRC-1 localization experiments show that SRC-1 is relocalized to the cytoplasm in the presence of high- and low-risk HPV E7 proteins. Our data suggest that HPV E7 proteins dysregulate hormone-dependent gene expression by association with and relocalization of SRC-1. Dysregulation of SRC-1 localization and function by HPV E7 may provide insight into the molecular mechanisms by which steroid hormones act as cofactors in the induction and progression of cervical neoplasia.

Thyroid hormone-dependent gene expression as a biomarker of short-term 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) exposure in European common frog (Rana temporaria) tadpoles

The effects on thyroid hormone-dependent gene biomarker responses of the persistent organochlorine pesticide metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) were investigated after exposure of 4-week-old European common frog (Rana temporaria) (stage 36) tadpoles to two (0.001 and 0.01 ppm) DDE concentrations. Total body weight, total length, and tail length and width increased after 3-day exposure to DDE. Expression patterns of genes encoding for growth hormone, thyroid-stimulating hormone (TSHβ) and thyroid hormone receptor (TRα and TRβ) isoforms were evaluated in the head, body and tail regions using a validated real-time polymerase chain reaction (PCR) method. The mRNA expression of growth hormone in the body, and TSHβ in the head showed significant DDE concentration-dependent decreases. While DDE caused variable effects on TRα mRNA steady-state, the expression of TRβ was significantly decreased in the tail by DDE in a concentration-specific manner. The effect of DDE exposure on TRβ mRNA expression showed a negative correlation with tail length and width during the exposure period. The unique pattern of a DDE-induced decrease of tail TRβ expression probably reflects the significant role of this thyroid hormone receptor isoform in tail re-absorption and overall metamorphosis in anuran species. Therefore, the present study shows that the evaluation of thyroid hormone-dependent genes may represent quantitative biomarkers of acute exposure to organochlorine pesticides in anuran species during critical developmental periods such as metamorphosis. Given the widespread environmental levels of DDT and its metabolites, these pollutants will remain a subject of concern and their effects on anuran species should be studied in more detail.