Strong iron demand during hypoxia-induced erythropoiesis is associated with down-regulation of iron-related proteins and myoglobin in human skeletal muscle

[EN] Iron is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin. An interaction between iron homeostasis and oxygen regulation is further suggested during hypoxia, in which hemoglobin and myoglobin syntheses have been reported to increase. This study gives new insights into the changes in iron content and iron-oxygen interactions during enhanced erythropoiesis by simultaneously analyzing blood and muscle samples in humans exposed to 7 to 9 days of high altitude hypoxia (HA). HA up-regulates iron acquisition by erythroid cells, mobilizes body iron, and increases hemoglobin concentration. However, contrary to our hypothesis that muscle iron proteins and myoglobin would also be up-regulated during HA, this study shows that HA lowers myoglobin expression by 35% and down-regulates iron-related proteins in skeletal muscle, as evidenced by decreases in L-ferritin (43%), transferrin receptor (TfR; 50%), and total iron content (37%). This parallel decrease in L-ferritin and TfR in HA occurs independently of increased hypoxia-inducible factor 1 (HIF-1) mRNA levels and unchanged binding activity of iron regulatory proteins, but concurrently with increased ferroportin mRNA levels, suggesting enhanced iron export. Thus, in HA, the elevated iron requirement associated with enhanced erythropoiesis presumably elicits iron mobilization and myoglobin down-modulation, suggesting an altered muscle oxygen homeostasis.

Disparity in regional and systemic circulatory capacities: do they affect the regulation of the circulation?

[EN] In this review we integrate ideas about regional and systemic circulatory capacities and the balance between skeletal muscle blood flow and cardiac output during heavy exercise in humans. In the first part of the review we discuss issues related to the pumping capacity of the heart and the vasodilator capacity of skeletal muscle. The issue is that skeletal muscle has a vast capacity to vasodilate during exercise [approximately 300 mL (100 g)(-1) min(-1)], but the pumping capacity of the human heart is limited to 20-25 L min(-1) in untrained subjects and approximately 35 L min(-1) in elite endurance athletes. This means that when more than 7-10 kg of muscle is active during heavy exercise, perfusion of the contracting muscles must be limited or mean arterial pressure will fall. In the second part of the review we emphasize that there is an interplay between sympathetic vasoconstriction and metabolic vasodilation that limits blood flow to contracting muscles to maintain mean arterial pressure. Vasoconstriction in larger vessels continues while constriction in smaller vessels is blunted permitting total muscle blood flow to be limited but distributed more optimally. This interplay between sympathetic constriction and metabolic dilation during heavy whole-body exercise is likely responsible for the very high levels of oxygen extraction seen in contracting skeletal muscle. It also explains why infusing vasodilators in the contracting muscles does not increase oxygen uptake in the muscle. Finally, when approximately 80% of cardiac output is directed towards contracting skeletal muscle modest vasoconstriction in the active muscles can evoke marked changes in arterial pressure.

Arterial O2 content and tension in regulation of cardiac output and leg blood flow during exercise in humans

[EN] A universal O2 sensor presumes that compensation for impaired O2 delivery is triggered by low O2 tension, but in humans, comparisons of compensatory responses to altered arterial O2 content (CaO2) or tension (PaO2) have not been reported. To directly compare cardiac output (QTOT) and leg blood flow (LBF) responses to a range of CaO2 and PaO2, seven healthy young men were studied during two-legged knee extension exercise with control hemoglobin concentration ([Hb] = 144.4 +/- 4 g/l) and at least 1 wk later after isovolemic hemodilution ([Hb] = 115 +/- 2 g/l). On each study day, subjects exercised twice at 30 W and on to voluntary exhaustion with an FIO2 of 0.21 or 0.11. The interventions resulted in two conditions with matched CaO2 but markedly different PaO2 (hypoxia and anemia) and two conditions with matched PaO2 and different CaO2 (hypoxia and anemia + hypoxia). PaO2 varied from 46 +/- 3 Torr in hypoxia to 95 +/- 3 Torr (range 37 to >100) in anemia (P < 0.001), yet LBF at exercise was nearly identical. However, as CaO2 dropped from 190 +/- 5 ml/l in control to 132 +/- 2 ml/l in anemia + hypoxia (P < 0.001), QTOT and LBF at 30 W rose to 12.8 +/- 0.8 and 7.2 +/- 0.3 l/min, respectively, values 23 and 47% above control (P < 0.01). Thus regulation of QTOT, LBF, and arterial O2 delivery to contracting intact human skeletal muscle is dependent for signaling primarily on CaO2, not PaO2. This finding suggests that factors related to CaO2 or [Hb] may play an important role in the regulation of blood flow during exercise in humans.

Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activation

Transcription is controlled by promoter-selective transcriptional factors (TFs), which bind to cis-regulatory enhancers elements, termed hormone response elements (HREs), in a specific subset of genes. Regulation by these factors involves either the recruitment of coactivators or corepressors and direct interaction with the basal transcriptional machinery (1). Hormone-activated nuclear receptors (NRs) are well characterized transcriptional factors (2) that bind to the promoters of their target genes and recruit primary and secondary coactivator proteins which possess many enzymatic activities required for gene expression (1,3,4). In the present study, using single-cell high-resolution fluorescent microscopy and high throughput microscopy (HTM) coupled to computational imaging analysis, we investigated transcriptional regulation controlled by the estrogen receptor alpha (ERalpha), in terms of large scale chromatin remodeling and interaction with the associated coactivator SRC-3 (Steroid Receptor Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a steroid-dependent transcriptional factor (16) that belongs to the NRs superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2), regulates transcription of distinct target genes involved in development, puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the nucleus and undergoes a rapid (within minutes) intranuclear redistribution following the addition of either agonist or antagonist (17,18,19). We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated reporter gene array (PRL-array) containing multicopy hormone response-binding elements for ERalpha that are derived from the physiological enhancer/promoter region of the prolactin gene. Following GFP-ER transfection of PRL-HeLa cells, we were able to observe in situ ligand dependent (i) recruitment to the array of the receptor and associated coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout of the reporter gene. Addition of E2 causes a visible opening (decondensation) of the PRL-array, colocalization of RNA Polymerase II, and transcriptional readout of the reporter gene, detected by mRNA FISH. On the contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI), a dramatic condensation of the PRL-array was observed, displacement of RNA Polymerase II, and complete decreasing in the transcriptional FISH signal. All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family member and a known oncogenic protein (4,34). SRC-3 is regulated by a variety of posttranslational modifications, including methylation, phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These events have been shown to be important for its interaction with other coactivator proteins and NRs and for its oncogenic potential (37,39). A number of extracellular signaling molecules, like steroid hormones, growth factors and cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by a wide range of kinases, including extracellular-regulated kinase 1 and 2 (ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs) (41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein, whose cellular localization is regulated by phosphorylation and interaction with ERalpha. Using a combination of high throughput and fluorescence microscopy, we show that both chemical inhibition (with U0126) and siRNA downregulation of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by EGF signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known partecipants in the regulation of SRC-3 activity (39). Accordingly, the cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further supports these results. In the presence of ERalpha, U0126 also dramatically reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell lysates; localization of SRC-3 at the ER-targeted prolactin promoter array (PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of estrogen receptor. While a wealth of studies have revealed the molecular functions of NRs and coregulators, there is a paucity of data on how these functions are spatiotemporally organized in the cellular context. Technically and conceptually, our findings have a new impact upon evaluating gene transcriptional control and mechanisms of action of gene regulators.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Study on the regulation of mRNA expression of GABA transporters (GAT-1 and GAT-3) in rat brain

The submitted work concentrated on the study of mRNA expression of two distinct GABA transporters, GAT-1 and GAT-3, in the rat brain. For the detection and quantification of the chosen mRNAs, appropriate methods had to be established. Two methods, ribonuclease protection assay (RPA) and competitive RT-PCR were emloyed in the present study. Competitive RT-PCR worked out to be 20 times more sensitive as RPA. Unlike the sensitivity, the fidelity of both techniques was comparable with respect to their intra- and inter-assay variability.The basal mRNA levels of GAT-1 and GAT-3 were measured in various brain regions. Messenger RNAs for both transporters were detected in all tested brain regions. Depending on the region, the observed mRNA level for GAT-1 was 100-300 higher than for GAT-3. The GAT-1 mRNA levels were similar in all tested regions. The distribution of GAT-3 mRNA seemed to be more region specific. The strongest GAT-3 mRNA expression was detected in striatum, medulla oblongata and thalamus. The lowest levels of GAT-3 were in cortex frontalis and cerebellum.Furthermore, the mRNA expression for GAT-1 and GAT-3 was analysed under altered physiological conditions; in kindling model of epilepsy and also after long-term treatment drugs modulating GABAergic transmission. In kindling model of epilepsy, altered GABA transporter function was hypothesised by During and coworkers (During et al., 1995) after observed decrease in binding of nipecotic acid, a GAT ligand, in hippocampus of kindled animals. In the present work, the mRNA levels were measured in hippocampus and whole brain samples. Neither GAT-1 nor GAT-3 showed altered transcription in any tested region of kindled animals compared to controls. This leads to conclusion that an altered functionality of GABA transporters is involved in epilepsy rather than a change in their expression.The levels of GAT-1 and GAT-3 mRNAs were also measured in the brain of rats chronically treated with diazepam or zolpidem, GABAA receptor agonists. Prior to the molecular biology tests, behavioural analysis was carried out with chronically and acutely treated animals. In two tests, open field and elevated plus-maze, the basal activity exploration and anxiety-like behaviour were analysed. Zolpidem treatment increased exploratory activity. There were observed no differencies between chronically and acutely treated animals. Diazepam increased exploratory activity and decresed anxiety-like behaviour when applied acutely. This effect disappeard after chronic administration of diazepam. The loss of effect suggested a development of tolerance to effects of diazepam following long-term administration. Double treatment, acute injection of diazepam after chronic diazepam treatment, confirmed development of a tolerance to effects of diazepam. Also, the mRNAs for GAT-1 and GAT-3 were analysed in cortex frontalis, hippocampus, cerebellum and whole brain samples of chronically treated animals. The mRNA levels for any of tested GABA transporters did not show significant changes in any of tested region neither after diazepam nor zolpidem treatment. Therefore, changes in GAT-1 and GAT-3 transcription are probably not involved in adaptation of GABAergic system to long-term benzodiazepine administration and so in development of tolerance to benzodiazepines.

The regulation of Satellite Cells during skeletal muscle regeneration and neuromuscular disease.

Skeletal muscle possesses the remarkable capacity to complete a rapid and extensive regeneration, even following severe damage. The regenerative ability of skeletal muscle relies on Satellite Cells (SCs), a population of muscle specific adult stem cells. However, during aging or under several pathological conditions, the ability of skeletal muscle to fully regenerated is compromised. Here, a morphological and molecular study on SCs from patients affected by ALS is described. Moreover, the role of the cell cycle regulator P16Ink4a during skeletal muscle regeneration and aging has been investigated.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.