GR Transcription Research Articles

The histone methyltransferase KMT2D maintains cellular glucocorticoid responsiveness by shielding the glucocorticoid receptor from degradation

Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.

Modification in the ascorbate-glutathione cycle leads to a better acclimation to high light in the rose Bengal resistant mutant of Nannochloropsis oceanica

Understanding how to adapt outdoor cultures of Nannochloropsis oceanica to high light (HL) is vital for boosting productivity. The N. oceanica RB2 mutant, obtained via ethyl methanesulfonate mutagenesis, was chosen for its tolerance to Rose Bengal (RB), a singlet oxygen (1O2) generator. Compared to the wild type (WT), the RB2 mutant showed higher resilience to excess light conditions. Analyzing the ascorbate-glutathione cycle (AGC), involving ascorbate peroxidases (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.8.1.7), in the RB2 mutant under HL stress provided valuable insights. At 250 μmol photon m−2 s−1 (HL), the WT strain displayed superoxide anion radicals (O2▪-) and hydrogen peroxide (H2O2) accumulation, increased lipid peroxidation, and cell death compared to normal light (NL) conditions (50 μmol photon m−2 s−1). The RB2 mutant didn't accumulate O2▪- and H2O2 after HL exposure, and exhibited increased APX, DHAR, and GR activities and transcript levels compared to WT and remained consistent after HL treatment. Although the RB2 mutant had a smaller ascorbate (AsA) pool than the WT, its ability to regenerate dehydroascorbate (DHA) increased post HL exposure, indicated by a higher AsA/DHA ratio. Additionally, under HL conditions, the RB2 mutant displayed an improved glutathione (GSH) regeneration rate (GSH/GSSG ratio) without changing the GSH pool size. Remarkably, H2O2 or menadione (a O2▪- donor) treatment induced cell death in the WT strain but not in the RB2 mutant. These findings emphasize the essential role of AGC in the RB2 mutant of Nannochloropsis in handling photo-oxidative stress.

Effects of cortisol and lipopolysaccharide on expression of select growth-, stress- and immune-related genes in rainbow trout liver

Many studies have shown that stress-induced cortisol levels negatively influence growth and immunity in finfish. Despite this knowledge, few studies have assessed the direct effects of cortisol on liver immune function. Using real-time PCR, the expression of three cortisol-responsive genes (GR: glucocorticoid receptor, IGF-1: insulin-like growth factor-I and SOCS-1: suppressor of cytokine signaling-I), genes involved with innate and adaptive immunity (IL-1β: interleukin-1 beta, IgM: immunoglobin-M and Lyz: lysozyme), and liver-specific antimicrobial peptides (hepcidin and LEAP-2A: liver-expressed antimicrobial peptide-2A) was studied in vitro using rainbow trout liver slices. The abundances of GR, SOCS-1 and IGF-1 mRNAs were suppressed by cortisol treatment. Abundance of IL-1β mRNA was upregulated by LPS and suppressed by cortisol treatment in a time-dependent manner. While abundance of IgM mRNA was suppressed by cortisol treatment and stimulated by LPS, there were no effects of cortisol or LPS on abundance of Lyz mRNA. Abundance of hepcidin and LEAP-2A mRNA levels were suppressed by cortisol treatment and stimulated by LPS. These results demonstrate that cortisol directly suppresses abundance of GR, IGF-1, IL-1β, IgM, hepcidin, LEAP-2A and SOCS-1 mRNA transcripts in the rainbow trout liver. We report for the first time, a suppressive effect of cortisol (within 8 h of treatment) on hepcidin and LEAP-2A mRNAs in rainbow trout liver, which suggests that acute stress may negatively affect liver immune function in rainbow trout.

Oxidative Stress in the Muscles of the Fish Nile Tilapia Caused by Zinc Oxide Nanoparticles and Its Modulation by Vitamins C and E.

The effects of zinc oxide nanoparticles (ZnONPs) on antioxidants in Nile tilapia muscles and the protective role of vitamins C and E were examined. Two hundred males of Nile tilapia were held in aquaria (10 fishes/aquarium). Fishes were divided into 5 groups: 40 fishes in each group; the first group was the control; the 2nd and 3rd groups were exposed to 1 and 2 mg/L of ZnONPs, respectively; and the 4th and 5th group were exposed to 1 and 2 mg/L of ZnONPs and treated with a (500 mg/kg diet) mixture of vitamin C and E mixture (250 mg/kg diet of each). Muscles were collected on the 7th and 15th day of treatments. Muscle malondialdehyde, reduced glutathione levels, superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) activities were measured after treatments. Relative quantification of SOD, CAT, GR, GPx, and GST mRNA transcripts was detected in the muscles. Results showed that MDA and GSH concentration; SOD, CAT, GR, GPx, and GST activities; and mRNA expression were significantly decreased in groups exposed to ZnONPs. Vitamins C and E significantly ameliorated the toxic effects of ZnONPs. In conclusion, vitamins C and E have the ability to ameliorate ZnONP oxidative stress toxicity in Nile tilapia.

A role for glutathione reductase and glutathione in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress.

The role of glutathione reductase (GR; EC 1.6.4.2) in the tolerance of Chlamydomonas reinhardtii P.A. Dangeard to high-intensity light stress (HL, 1400 μmol m-2 s-1 ) was examined. Cells survived under high light (HL) stress, although their growth was inhibited after long-term treatment (9-24 h). GR activity increased 1 h after HL treatment. The contents of total glutathione, reduced glutathione (GSH) and glutathione disulfide (GSSG) increased 1-3 h after HL treatment and then decreased after 24 h, while the GSH:GSSG ratio (glutathione redox potential) decreased after 3-9 h and recovered after 24 h. The transcript abundance of GR, CrGR1 (Cre06.g262100) and CrGR2 (Cre09.g396252) as well as glutathione synthesis-related genes, CrGSH1 (Cre02g077100.t1.1) and CrGSH2 (Cre17.g70800.t1.1), increased with a peak near 1 h after HL treatment. Except for enhanced glutathione synthesis, the GR-mediated glutathione redox machinery is also critical for the tolerance of C. reinhardtii cells to HL stress. Therefore, GR was downregulated or upregulated to investigate the importance of GR in HL tolerance. The CrGR1 knockdown amiRNA line exhibited low GR transcript abundance, GR activity and GSH:GSSG ratio and could not survive under HL conditions. Over-expression of CrGR1 or CrGR2 driven by a HSP70A:RBCS2 fusion promoter resulted in a higher GR transcript abundance, GR activity and GSH:GSSG ratio and led to cell survival when exposed to high-intensity illumination, i.e. 1800 μmol m-2 s-1 . In conclusion, GR-mediated modulation of the glutathione redox potential plays a role in the tolerance of Chlamydomonas cells to photo-oxidative stress.

Tyrosine hydroxylase of the brain and it’s regulation by glucocorticoids

Early life stress events can produce long-lasting changes in neurochemistry and behaviors related to monoamine systems, with increased risks of cardiovascular, metabolic, neuroendocrine, psychiatric disorders, generalized anxiety and depression in adulthood. Tyrosine hydroxylase (TH), the key enzyme for catecholamine synthesis, also plays an important role in the activity of the noradrenergic system and may be a target for glucocorticoids during the perinatal programming of physiological functions and behavior. Administration of hydrocortisone or dexamethasone to female rats on day 20 of pregnancy and to 3-day-old neonatal pups significantly increased TH mRNA levels (real-time PCR) and enzyme activity as well as protein levels determined by ICH in the locus coeruleus. Moreover, our treatment led to increase in TH mRNA levels in 25- and 70-day-old animals, as well as an increase in enzyme activity in the brainstem and cerebral cortex of adult rats. The long-term changes in TH expression are limited by the perinatal period of development. Administration of hormones on day 8 of life was not accompanied by changes in TH mRNA levels or enzyme activity. Glucocorticoids use several mechanisms to bring about transactivation or transrepression of genes. The main mechanism includes direct binding of the hormone-activated GRs to glucocorticoid responsive elements (GREs) in the promoter region of genes. However, despite optimistic claims made the classical GRE was not found in the TH gene promoter. Protein – protein interactions between hormone-activated GR and other transcription factors, for example, AP-1, provide an additional mechanism for the effects of glucocorticoids on gene expression. An important feature of this mechanism is its dependence on the composition of proteins formed by AP-1. Hormone-activated GRs are able to enhance gene expression when AP-1 consists of the Jun / Jun homodimer, but do not do that when AP-1 appears as the Jun / Fos heterodimer. Furthermore, as has been shown recently, the GRE / AP-1 composite site is the major site of interaction of glucocorticoids with the TH gene in the pheochromocytoma cell line. Ontogenetic variation in the expression of Fos and Jun family proteins, which affects their ratio, can be one of the reasons for the TH gene regulation by glucocorticoids at near-term fetuses and neonates. However, to date this hypothesis has been supported only by in vitro data, and the existence of this mechanism in in vivo conditions needs to be explored in further studies.

Where does transcription start? 5'-RACE adapted to next-generation sequencing.

The variability and complexity of the transcription initiation process was examined by adapting RNA ligase-mediated rapid amplification of 5′ cDNA ends (5′-RACE) to Next-Generation Sequencing (NGS). We oligo-labelled 5′-m7G-capped mRNA from two genes, the simple mono-exonic Beta-2-Adrenoceptor (ADRB2R) and the complex multi-exonic Glucocorticoid Receptor (GR, NR3C1), and detected a variability in TSS location that has received little attention up to now. Transcription was not initiated at a fixed TSS, but from loci of 4 to 10 adjacent nucleotides. Individual TSSs had frequencies from <0.001% to 38.5% of the total gene-specific 5′ m7G-capped transcripts. ADRB2R used a single locus consisting of 4 adjacent TSSs. Unstimulated, the GR used a total of 358 TSSs distributed throughout 38 loci, that were principally in the 5′ UTRs and were spliced using established donor and acceptor sites. Complete demethylation of the epigenetically sensitive GR promoter with 5-azacytidine induced one new locus and 127 TSSs, 12 of which were unique. We induced GR transcription with dexamethasone and Interferon-γ, adding one new locus and 185 additional TSSs distributed throughout the promoter region. In-vitro the TSS microvariability regulated mRNA translation efficiency and the relative abundance of the different GR N-terminal protein isoform levels.

Impairment of the cortisol stress response mediated by the hypothalamus–pituitary–interrenal (HPI) axis in zebrafish (Danio rerio) exposed to monocrotophos pesticide

In teleosts, an important component of the stress response is coordinated by the hypothalamic–pituitary–interrenal (HPI) axis. Environmental contaminants might disrupt the stress axis and consequently affect the stress response in fish. To investigate the effect of monocrotophos (MCP) pesticide on the stress response of fish and its potential mechanisms, adult zebrafish (Danio rerio) were exposed to 0, 1, 10, and 100μg/L of a 40% MCP-based pesticide for 21d, after which time fish were subjected to a 3-min air-exposure stressor. Concentrations of the whole-body cortisol were measured by radioimmunoassay and abundances of transcripts of proteins involved in the HPI axis were determined using quantitative real-time PCR. Results showed that 100μg/L of MCP pesticide decreased whole-body cortisol levels of female zebrafish in response to an acute stressor, but without any effect on the cortisol response in males. 100μg/L MCP pesticide reduced POMC and GR expression in the brain, MC2R and P45011β expression in the head kidney, but enhanced 20β-HSD2 expression in the head kidney, suggesting that MCP damaged the HPI axis involving acting at pituitary regulatory levels, inhibiting cortisol synthesis and stimulating cortisol catabolism, or disturbing the negative feedback regulation. Additionally, MCP depressed liver GR transcription but did not affect phosphoenolpyruvate carboxykinase and tyrosine aminotransferase expression in zebrafish, suggesting a role for this pesticide in reducing target tissue responsiveness to cortisol. Considered together, the reduced ability to elevate cortisol levels in response to an acute stress may be an endocrine dysfunction occurring in zebrafish subchronically exposed to MCP pesticide.

Role of the 5′-untranslated regions in post-transcriptional regulation of the human glucocorticoid receptor

GR transcripts display a remarkable heterogeneity in their 5′ untranslated regions (5′UTRs). These variable 5′UTRs are encoded by a series of alternative 1st exons, and together with their associated promoters they maintain tissue-specific GR expression levels. In this study we over-expressed GR transcripts containing individual 1st exons, and assessed their effect on RNA stability, 3′-splicing, translation initiation and protein isoform production. We showed that these alternative 5′UTRs influence the predicted mRNA structure and free energy, and were associated with differential levels of functional spliced mRNA. However, the 5′UTR had little influence on the relative levels of the two principal 3′ splice transcripts, GR-α and -β. The overall mRNA length, the free energy of the transcript and the translational efficiency directly influenced total GR levels. However, individual N-terminal protein isoform levels appeared to depend upon elements within the 5′UTR. Membrane-GR specific labelling suggested that the mGR originates from transcripts containing exon 1D and possibly 1H, although the specific trafficking sequences or structures within these transcripts remain unidentified. The role of the alternative first exons and their associated 5′UTRs has now been expanded to translational control, influencing total GR levels, individual constituent isoform levels, as well as trafficking to the cell surface.

Effects of HIP in protection of HSP70 for stress-induced cardiomyocytes injury and its glucorticoid receptor pathway.

Moderate levels of stress can be beneficial to health, while stress overload can cause injury or contribute to diseases. Despite a number of studies of adaptation or stress damage, the mechanisms of adaptation and stress damage remain far from clear. The effect and mechanisms of adaptation on cardiomyocytes damage caused by stress overload are discussed in this study. Data showed that mild repeated stress mitigated stress overload-induced cardiomyocyte injury both in an animal model of restraint stress and in H9C2 cells with GC (glucocorticoid) treatment. HSP70, HIP expression and interaction between HSP70 and HIP increased during adaptation induced by mild stress both in animals and H9C2 cells. Overexpression or inhibition of HSP70 in H9C2 cells with pCDNA-3.1-Hsp70 or KNK437 (HSP70 inhibitor) showed that HSP70 can protect H9C2 cells from GC-induced cell damage. A luciferase assay showed that Hsp70 plays its protective role through inhibition of GR transcription activity dependent on the interaction with HIP. These results indicated that HSP70 may promote adaptation with its interacting protein HIP, and increased levels of HSP70 and its interacting protein HIP during adaptation may play a protective role on stress-overload-induced cardiomyocyte injury.

Integrative genomic analyses of the histamine H1 receptor and its role in cancer prediction

The human histamine receptor H1 (HRH1) gene is located on chromosome 3p25 and encodes for a 487 amino acid G protein-coupled receptor (GPCR) with a long third intracellular loop (IL3). The HRH1 predominantly couples to Gαq/11 proteins, leading to the activation of phospholipase C (PLC) and subsequent release of the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) followed by the activation of PKC and the release of [Ca2+]i. In the present study, we identified HRH1 genes from 14 vertebrate genomes and found that HRH1 exists in all types of vertebrates including fish, amphibians, birds and mammals. We identified 88 SNPs including 4 available alleles disrupting an existing exonic splicing enhancer and 84 SNPs causing missense mutation, which may impact the effect of histamine on the HRH1 protein. We found that the human HRH1 gene was expressed in many tissues or organs, and predominant expression of HRH1 was shown in the bone marrow, whole blood, lymph node, thymus, brain, cerebellum, retina, spinal cord, heart, smooth muscle, skeletal muscle, small intestine, colon, adipocytes, kidney, liver, lung, pancreas, thyroid salivary gland, skin, ovary, uterus, placenta, prostate and testis. When searched in the PrognoScan database, human HRH1 was also found to be expressed in bladder cancer, blood cancer, brain cancer, breast cancer, colorectal cancer, eye cancer, head and neck cancer, lung cancer, ovarian cancer, skin cancer and soft tissue cancer tissues. The relationship between the expression of HRH1 and prognosis was found to vary in different types of cancers, even in the same cancer from different databases. This implies that the function of HRH1 in these tumors may be multidimensional. GR, STAT5A and c-Myb regulatory transcription factor binding sites were identified in the HRH1 gene upstream (promoter) region, which may be involved in the effect of HRH1 in tumors.

Polymorphisms at the 5′ flanking region of the HSP90AA1 gene in native Turkish sheep breeds

In this study, several polymorphisms in the gene encoding the inducible form of the cytoplasmic Hsp90 (HSP90AA1) were characterized in 10 sheep breeds reared in different climatic regions of Turkey. A new indel (AA) polymorphism located at the −704 position in the HSP90AA1 5′ flanking region was found in all investigated breeds. This polymorphism create/remove a GR transcription site that could affect heat stress response and is completely linked with the C/G transversion located at −660 in the same region of the gene. Genotype frequencies differed significantly between breeds. No clearcut relationship was found between this polymorphism and climate factors, but breeds from northern Turkey had higher frequencies of the indel AA allele than breeds from southern Turkey.

Breed-Dependent Transcriptional Regulation of 5′-Untranslated GR (NR3C1) Exon 1 mRNA Variants in the Liver of Newborn Piglets

Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P<0.05) higher in the liver of newborn piglets of Large White (LW) compared with Erhualian, a Chinese indigenous breed. Overall level of CpG methylation in the region flanking exons 1–4 and 1–5 did not show breed difference. However, nuclear content of Sp1, p-CREB and GR in the liver was significantly (P<0.05) higher in LW piglets, associated with enhanced binding of p-CREB, and higher level of histone H3 acetylation in 1–4 and 1–5 promoters. In contrast, GR binding to promoters of exons 1–4 and 1–5 was significantly diminished in LW piglets, implicating the presence of negative GREs. These results indicate that the difference in the hepatic expression of GR transcript variants between two breeds of pigs is determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3 acetylation to the promoters.

Interaction of the Glucocorticoid Receptor with the Chromatin Landscape

In model systems, nuclear receptors induce chromatin remodeling at regulatory elements. We have carried out a genome‐wide analysis of GR binding events, as well as a global characterization of DNaseI hypersensitive transitions associated with GR responsive genes. Our findings indicate that receptor/genome interactions are universally associated with local chromatin remodeling events. Furthermore, multiple remodeling systems are required for these events. The presence of these locally open chromatin regions is highly cell specific, and both GR binding and GR transcription regulation correlate well with the presence of these sites. These results indicate that the chromatin status of a specific gene locus in a given cell type is a critical determinant for the GR responsiveness of the locus.

Phosphodiesterase 4 Inhibitors Augment Levels of Glucocorticoid Receptor in B Cell Chronic Lymphocytic Leukemia but Not in Normal Circulating Hematopoietic Cells

Type 4 cyclic AMP (cAMP) phosphodiesterase (PDE4) inhibitors, a class of compounds in clinical development that activate cAMP-mediated signaling by inhibiting cAMP catabolism, offer a feasible means by which to potentiate glucocorticoid-mediated apoptosis in lymphoid malignancies such as B-cell chronic lymphocytic leukemia (B-CLL). In this study, we show that PDE4 inhibitors up-regulate glucocorticoid receptor (GRalpha) transcript levels in B-CLL cells but not T-CLL cells or Sezary cells or normal circulating T cells, B cells, monocytes, or neutrophils. Because GRalpha transcript half-life does not vary in CLL cells treated with the prototypic PDE4 inhibitor rolipram, the 4-fold increase in GRalpha mRNA levels observed within 4 h of rolipram treatment seems to result from an increase in GRalpha transcription. Rolipram treatment increases levels of transcripts derived from the 1A3 promoter to a greater extent than the 1B promoter. Treatment of B-CLL cells with two other PDE4 inhibitors currently in clinical development also augments GR transcript levels and glucocorticoid-mediated apoptosis. Washout studies show that simultaneous treatment with both drug classes irreversibly augments apoptosis over the same time frame that GR up-regulation occurs. Although treatment of B-CLL cells with glucocorticoids reduces basal GRalpha transcript levels in a dose-related manner, cotreatment with rolipram maintained GRalpha transcript levels above baseline. Our results suggest that as a result of their unusual sensitivity to PDE4 inhibitor-mediated up-regulation of GRalpha expression, treatment of B-CLL patients with combined PDE4 inhibitor/glucocorticoid therapy may be of therapeutic benefit in this disease.

The Transcriptional Coactivator PGC-1β Drives the Formation of Oxidative Type IIX Fibers in Skeletal Muscle

Skeletal muscle must perform different kinds of work, and distinct fiber types have evolved to accommodate these. Previous work had shown that the transcriptional coactivator PGC-1alpha drives the formation of type I and IIA muscle fibers, which are "slow-twitch" and highly oxidative. We show here that transgenic expression of PGC-1beta, a coactivator functionally similar to but distinct from PGC-1alpha, causes a marked induction of IIX fibers, which are oxidative but have "fast-twitch" biophysical properties. PGC-1beta coactivates the MEF2 family of transcription factors to stimulate the type IIX myosin heavy chain (MHC) promoter. PGC-1beta transgenic muscle fibers are rich in mitochondria and are highly oxidative, at least in part due to coactivation by PGC-1beta of ERRalpha and PPARalpha. Consequently, these transgenic animals can run for longer and at higher work loads than wild-type animals. Together, these data indicate that PGC-1beta drives the formation of highly oxidative fibers containing type IIX MHC.

The HPA system during the postnatal development of CD1 mice and the effects of maternal deprivation

In this study we describe in 9- and 18-day-old CD1 mice (i) the basal and stress-induced activity of markers of the HPA system in blood, brain and pituitary, (ii) the effects of a 24-h maternal deprivation and (iii) the influence of anogenital stimulation (stroking) in deprived pups on these markers. We found low basal concentrations of ACTH and corticosterone at postnatal day (pnd) 9 and no or little response to a mild stressor at this age, confirming the existence of a SHRP in mice. At pnd 18 the mice displayed an adult-like ACTH and corticosterone response following a mild stressor. Maternal deprivation resulted in enhanced basal and stress levels of corticosterone at both ages. Interestingly, basal ACTH levels were elevated following maternal deprivation at pnd 9. At pnd 18 maternal deprivation resulted in a blunted ACTH response. Maternal deprivation resulted in a down-regulation of GR, MR, CRH and POMC transcript in the brain. However, maternally deprived 18-day-old pups displayed increased levels of CRH and POMC transcript, while GR and MR mRNA was also down-regulated. Anogenital stroking could reverse maternal deprivation effects on ACTH and MR mRNA, but not CRH mRNA. We conclude that (i) at the two measured time points the HPA axis develops similar in mice as in rats, (ii) maternal deprivation has pronounced effects in mice, which are similar to that found in the rat and (iii) there are a number of significant differences, which mainly concern the central CRH–ACTH components of the axis.

Biochemical and Molecular Characterisation of Wheat Chloroplastic Glutathione Reductase

Wheat leaves contain two charge/mass-separable isoforms of glutathione reductase (GR, EC 1.6.4.2), one chloroplastic and the other probably cytosolic. The chloroplastic GR was purified to homogeneity, and its biochemical and molecular characterisation showed features very similar to the other plant GRs. In its native conformation the enzyme is composed by two subunits of 56 kDa and an associated polypeptide of 32 kDa, with an overall molecular mass of approximately 150 kDa. Optimum activity was observed at pH 8.00 and with an ionic strength between 60 to 100 mM. GR activity is highly sensitive to temperature changes, exhibiting an exponential increase up to 45 °C. It showed a high affinity for oxidised glutathione and an intermediate affinity for NADPH at pH 8.0. Inhibition tests with thiol and histidine modifiers demonstrated that -SH groups and histidine residues are essential for the catalytic properties of the enzyme. T study the origin of GR isoforms, the number of GR gene copies and the number and size of GR transcripts were determined. Southern analyses showed that wheat GR isoforms are encoded by multiple gene copies. However, a single size transcript of approximately 1.4 kb was observed, suggesting that different GR isoforms could be generated by post-transcriptional and/or post-translational modifications.

Multiple Promoters Exist in the Human GR Gene, One of Which Is Activated by Glucocorticoids

A new human GR gene sequence (hGR 1Ap/e), which is distinct from the previously identified human GR promoter and coding sequences, has been isolated and characterized. The hGR 1Ap/e sequence is approximately 31 kbp upstream of the human GR coding sequence. This sequence (2,056 bp) contains a novel promoter (the hGR 1A promoter; 1,075 bp) and untranslated exon sequence (hGR exon 1A sequence; 981 bp). Alternative splicing produces three different hGR 1A-containing transcripts, 1A1, 1A2, and 1A3. GR transcripts containing exon 1A1, 1A2, 1B, and 1C are expressed at various levels in many cancer cell lines, while the exon 1A3-containing GR transcript is expressed most abundantly in blood cell cancer cell lines. Glucocorticoid hormone treatment causes an up-regulation of exon 1A3-containing GR transcripts in CEM-C7 T-lymphoblast cells and a down-regulation of exon 1A3-containing transcripts in IM-9 B-lymphoma cells. Deoxyribonuclease I footprinting using CEM-C7 cell nuclear extract reveals four footprints in the promoter region and two intraexonic footprints. Much of the basal promoter-activating function is found in the +41/+269 sequence, which contains two deoxyribonuclease I footprints (FP5 and FP6). When this sequence is cloned into the pXP-1 luciferase reporter gene, hormone treatment causes a significant increase in luciferase activity in Jurkat T cells that are cotransfected with a GR expression vector. FP5 is an interferon regulatory factor-binding element, and it contributes significantly to basal transcription rate, but it is not activated by steroid. FP6 resembles a glucocorticoid response element and can bind GRbeta. This novel hGR 1Ap/e sequence may have future applications for the diagnosis, prognosis, and treatment of T-cell leukemia and lymphoma.

Characterization of Human Mucin Gene MUC4Promoter: IMPORTANCE OF GROWTH FACTORS AND PROINFLAMMATORY CYTOKINES FOR ITS REGULATION IN PANCREATIC CANCER CELLS

The human mucin gene MUC4 encodes a large transmembrane mucin that is thought to play important roles in tumor cell biology and that is overexpressed in human pancreatic carcinomas. In this report, we describe the structure and functional activity of the 5'-flanking region, including 1.0 kilobase of the promoter. The long 5'-untranslated region (2.7 kilobases) is characterized by a high content of GC in its 3'-end. The first TATA box was located at -2672/-2668. Multiple transcription start sites and a high density of putative binding sites for Sp1 (GC and CACCC boxes), AP-1/-2/-4, cAMP-responsive element-binding protein, GATA, GR, and STAT transcription factors were found within the 5'-flanking region. Transcriptional activity of the promoter was assessed using pGL3-luciferase deletion mutants in two MUC4-expressing (CAPAN-1 and CAPAN-2) and one nonexpressing (PANC-1) pancreatic cancer cell line. Two highly active fragments (-219/-1 and -2781/-2572) that drive MUC4 transcription in CAPAN-1 and CAPAN-2 cells were identified. Gel retardation assays indicated that Sp1 and Sp3 bind to cognate cis-elements found in the 5'-flanking region and that Sp1 transactivates, whereas Sp3 inhibits the GC-rich region (-464/-1) in CAPAN-2 cells. Activation of protein kinase C with phorbol ester and treatment of cells with epidermal growth factor and transforming growth factor-alpha resulted in up-regulation of the promoter. Tumor necrosis factor-alpha and interferon (IFN)-gamma inflammatory cytokines had no or mild effect on MUC4 transcriptional activity when used alone. However, a very strong synergistic effect (10-12-fold activation) between IFN-gamma and tumor necrosis factor-alpha or IFN-gamma and transforming growth factor-alpha was obtained in CAPAN-2 cells. Altogether these results demonstrate that the 5'-flanking region of MUC4 contains epithelial cell-specific, positive, and negative regulatory cis-elements, that Sp1/Sp3 are important regulators of MUC4 basal expression, and that its regulation in pancreatic cancer cells involves complex interplay between several signaling pathways.