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Objectives: To investigate the effects of interleukin 1beta (IL1beta) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro. Methods: Mouse articular chondrocytes in primary culture were challenged with IL1beta, alone or combined with Notch1 and IL1beta pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q)PCR. IL1beta-responsive genes were assessed by real-time qPCR and a specific siRNA against Hes1 was used to identify Hes1 target genes. Results: Notch1 labelling remained nuclear and stable in intensity irrespective of treatment, suggesting a steady state activation of this pathway in our model. IL1beta transiently increased Hes1 mRNA (2.5-fold) and protein expression in treated versus naive chondrocytes. Hes1 mRNA level then decreased below control and its cyclic pattern of expression was lost. This was associated with nuclear translocation of the cytoplasmic Hes1 protein. IL1beta induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)kappaB activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the gamma-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), which suggested its independence from novel Notch1 activation. Hes1 expression was efficiently silenced by a specific siRNA. This experiment revealed that Hes1 did not mediate IL1beta-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1beta induction of matrix metalloproteinase (MMP)13 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). The Hes1-related repressor Hey1 was expressed at a very low level and was not inducible by IL1beta. Conclusion: Hes1 is a novel IL1beta target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation. [ABSTRACT FROM AUTHOR]

Trans-resveratrol (t-Res) is a natural compound of a family of hydroxystilbenes found in a variety of spermatophyte plants. Because of its effects on lipids and arachidonic acid metabolisms, and its antioxidant activity, t-Res is considered as the major cardioprotective component of red wine, leading to the "French Paradox" health concept. In the past decade, research on the effects of resveratrol on human health has developed considerably in diverse fields such as cancer, neurodegenerative and cardiovascular diseases, and metabolic disorders. In the field of rheumatic disorders, in vitro evidence suggest anti-inflammatory, anti-catabolic, anti-apoptotic and anti-oxidative properties of t-Res in various articular cell types, including chondrocytes and synoviocytes, along with immunomodulation properties on T and B lymphocytes. In preclinical models of osteoarthritis and rheumatoid arthritis, resveratrol has shown joint protective effects, mainly mediated by decreased production of pro-inflammatory and pro-degradative soluble factors, and modulation of cellular and humoral responses. Herein, we comprehensively reviewed evidence supporting a potential therapeutic interest of t-Res in treating symptoms related to rheumatic disorders., Competing Interests: The authors declare no conflict of interest.

Several endocrine disrupting compounds (EDC) elicit skeletal dysgenesis at pharmacological doses. We have investigated the impact of doses below the "No Observed Adverse Effect" (NOAEL) for vinclozolin (V), an anti-androgenic fungicide, alone or associated with xenoestrogens (Genistein, G and bisphenol-A, BPA). V, G, BPA and their combinations were administered orally to female Wistar rats during gestation and lactation. F1 and F2 offspring were investigated for skeletal anomalies at post-natal days 30, 110 (d30, d110). Skeletal development was monitored by measuring caudal vertebrae and long bones dimensions by X-ray micro-CT-scan. A significant increase in Inter Transverse Apophysis (ITA) distance at the upper head of caudal vertebrae, associated with a reduction in vertebral body height was observed in treated F1 females, but not males. Histometrical analysis of vertebral body growth plate cartilage was performed on serial sections of caudal vertebrae. F1 females but not males showed a diminution in growth plate thickness, with greater impact on the hypertrophic zone. All effects were maximal at d30. Effects on ITA width persisted until d110 while effects on growth plate disappeared. These effects were essentially vinclozolin or BPA-dependent. F2 animals were not affected. Our data suggest that vinclozolin and xenoestrogens act as cartilage developmental disruptors. We suggest that present NOAEL values for these compounds, and EDC at large, might be reconsidered using gestational exposure models. Finally, micro CT-scan appears a valuable non-invasive technique to detect EDC effects on live fauna., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

We have developed an innovative soluble galenic form to overcome the low absorption of trans-Resveratrol (t-Res) as a dry powder. We present here data on pharmacokinetics, bioavailability, and toxicity of t-Res in human volunteers treated with this soluble form, plus additional data on biological effects in rodents. Fifteen healthy volunteers of both sexes received 40 mg of t-Res in two forms, the soluble formulation (caplets) and the original powder (capsules), in a crossover design. Blood samples were collected at 15 min, 30 min, and every hour for 5 h. Plasma concentrations of t-Res and its metabolites were analyzed by liquid chromatography and mass spectrometry. The single dose (40 mg) of the soluble t-Res was well absorbed and elicited biologically efficient blood levels (0.1-6 μM) for several hours, despite metabolization into glucuronide and sulfate conjugates coupled to renal elimination. In contrast, t-Res administered as a dry powder barely elicited efficient blood levels for a short duration. The new formulation led to 8.8-fold higher t-Res levels in plasma versus the powder. t-Res metabolism was not modified and neither intolerance nor toxicity were observed during the study and the following week. The soluble formulation elicited a robust anti-inflammatory effect in various tissues of mice fed a high-fat diet, while dry powder t-Res was almost inactive. Our data suggest that significant improvements in t-Res bioavailability and efficiency can be obtained by this soluble galenic form, also allowing lower doses. The use of t-Res in human therapy is thus greatly facilitated and the toxicity risk is reduced., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme, synthesized in the liver and secreted into the blood. PON1 displays antioxidant properties and is involved in organophosphorous compounds and oxidized lipids degradation. Because of these beneficial effects, pharmacological regulation of PON1 appears to be highly relevant in toxicology and cardiology. Recent studies undertaken on the regulation of the PON1 promoter in our laboratory have identified resveratrol, through its activation of the Aryl hydrocarbon Receptor (AhR), as a putative inducer of PON1. We have tested a new modulator of AhR, (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile, and established that it is a more potent inducer of PON1 at the mRNA, protein and enzymatic activity as compared to resveratrol. It also acts by activating the AhR. However, in contrast with traditional AhR agonists, it does not induce cyp1A1 transcription. (Z)-2,3-bis (4-nitrophenyl)-acrylonitrile is therefore a specific AhR modulator targeting PON1., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The plant phytoalexin resveratrol was previously demonstrated to inhibit the differentiation and bone resorbing activity of osteoclasts, to promote the formation of osteoblasts from mesenchymal precursors in cultures, and inhibit myeloma cell proliferation, when used at high concentrations. In the current study, we screened five structurally modified resveratrol analogues for their ability to modify the differentiation of osteoclasts and osteoblasts and proliferation of myeloma cells. Compared to resveratrol, analogues showed an up to 5,000-fold increased potency to inhibit osteoclast differentiation. To a lesser extent, resveratrol analogues also promoted osteoblast maturation. However, they did not antagonize the proliferation of myeloma cells. The potency of the best-performing candidate in vitro was tested in vivo in an ovariectomy-induced model of osteoporosis, but an effect on bone loss could not be detected. Based on their powerful antiresorptive activity in vitro, resveratrol analogues might be attractive modulators of bone remodeling. However, further studies are required to establish their efficacy in vivo.

We demonstrate that intestinal inflammation caused by high-fat diet is increased by the environmental contaminant benzo[a]pyrene. Our in vivo results indicate that a high-fat diet (HFD) induces a pre-diabetic state in mice compared with animals fed normal chow. HFD increased IL-1betamRNA concentration in the jejunum, colon, and liver, and TNFalpha was increased in the colon and strongly increased in the liver. HFD also increased the expression of other genes related to type 2 diabetes, such as the uncoupling protein UCP2, throughout the bowel and liver, but not in the colon. The treatment of HFD with BaP enhanced the expression of IL-1beta in the liver and TNFalpha throughout the bowel and in the liver. Adding BaP to the diet also caused a significant decrease in the expression of the incretin glucagon-like peptide 1, which plays an important role in insulin secretion. Our results suggest that intestinal inflammation may be involved in the onset of type 2 diabetes and that chronic exposure to environmental polycyclic aromatic hydrocarbons can increase the risk of type 2 diabetes by inducing pro-inflammatory cytokine production., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Oxidative and inflammatory processes are elicited during hepatic post-ischemic reperfusion and generate liver damage. This study investigated the early anti-inflammatory effect of trans-resveratrol (T-res) and its consequences on the late self-aggravating inflammatory process in liver ischemia-reperfusion (I/R). Partial hepatic ischemia was initiated in rats for 1 h and T-res (0.02 and 0.2 mg/kg) was administered intravenously 5 min before starting reperfusion for 3 h. Plasma levels of aminotransferases and cytokines (tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6) and hepatic neutrophil recruitment were assessed. Hepatic expression of stress protein (heat-shock protein (HSP-70), heme oxygenase-1(HO-1)) and cytokine (TNF-alpha, IL-1beta, keratinocyte chemoattractant (KC)) mRNA was investigated. I/R caused an increase in aminotransferase levels and increased polymorphonuclear cell infiltration. Post-ischemic treatment with T-res (0.02 and 0.2 mg/kg) resulted in a significant decrease in aminotransferase, IL-1beta and IL-6 plasma levels by about 40%, 60% and 40%, respectively, compared to the vehicle I/R group. Post-ischemic treatment with T-res (0.02 mg/kg) also significantly decreased hepatic neutrophil recruitment. TNF-alpha, IL-1beta, KC and HO-1 hepatic mRNA expression was reduced by T-res without any change in HSP-70 mRNA. This T-res mediated decrease in early release of cytokines and neutrophil recruitment led to a reduction in the late inflammatory process. T-resveratrol might be useful in the prevention of inflammation secondary to hepatic surgery or liver transplantation., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERalpha and only express the cytoplasmic beta isoform. ERbeta localization was not modified by either steroid. The threshold level was surprisingly low, around 10(-16) M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.

Objective: To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers., Methods: Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry., Results: Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged., Conclusion: The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.

Objective: To assess the interleukin 1 receptor antagonist (IL-1Ra)/IL-1beta ratio in synovial fluid (SF) of patients with knee osteoarthritis (OA) or rheumatoid arthritis (RA) to determine a possible relation between cytokine level and disease activity., Methods: IL-1beta and IL-1Ra concentrations were measured by ELISA in knee SF from patients with OA (n = 42) or RA (n = 11). For OA patients, pain and disability were assessed by a visual analog scale (VAS) and the Lequesne index. RA disease activity was assessed using the Disease Activity Score 28 Joint Count (DAS28)., Results: Patients with OA showed lower median levels of IL-1beta and IL-1Ra in SF than patients with RA (p < 0.001) but a higher IL-1Ra/IL-1beta ratio: 1793 (584-6221) versus 773.5 (187.64-1570.5) (p = 0.05). For patients with OA, the IL-1Ra/IL-1beta ratio was not associated with pain or disability. For patients with RA, the IL-1Ra/IL-1beta ratio and IL-1Ra and IL-1beta levels were related to SF white blood cell count., Conclusion: High endogenous IL-1Ra/IL-1beta ratio occurs in SF from knee OA and does not correlate with pain or Lequesne index. Our results suggest that intraarticular injection of IL-1Ra might be self-limited in patients with knee OA and a naturally high SF ratio.

Objective: To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesized sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1 beta., Methods: Primary cultured rabbit articular chondrocytes were treated or not with IL1 beta alone or with concentrations of CS for 20 h. Neosynthesized PGs were labelled by incorporation of [35SO(4)]-sulphate and analysed by chromatography on Sepharose 2B columns. Gelatinolytic activity was measured by zymography, and matrix metalloproteinase (MMP)1 mRNA level in chondrocytes underwent real-time PCR. Expression of ADAMTS (for "a disintegrin and metalloproteinase with thrombospondin motifs") -4 and -5 was analysed by real-time PCR and western blotting., Results: The production of [35SO(4)]-labelled PGs was significantly increased with 10 microg/ml CS in the cellular pool rather than in the incubation medium. The addition of CS to IL1 beta-treated cells inhibited in part the disaggregation of sulphated PGs induced by IL1 beta. This inhibitory effect of CS is associated with a significant decrease in ADAMTS-5 expression at the mRNA and protein levels. No effect of CS was observed on IL1 beta-induced gelatinolytic activity, MMP1 mRNA expression or ADAMTS-4 expression., Conclusion: CS increases the production of functional sulphated PGs in the direct environment of chondrocytes in vitro. This beneficial effect of CS in IL1 beta-treated cells is associated with decreased expression of ADAMTS-5.

Liver ischemia-reperfusion (I/R) injury occurs in many clinical conditions, including liver surgery and transplantation. Oxygen free radicals generated during I/R reduce endogenous antioxidant systems and contribute to hepatic injury. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is reported to have antioxidant properties. We investigated the effect of trans-resveratrol on liver injury induced by I/R. After 1 hour of ischemia, administered 5 minutes before 3 hours of reperfusion, trans-resveratrol was hepatoprotective at a low dose (0.02 mg/kg). It significantly decreased aminotransferase levels by about 40% and improved sinusoidal dilatation. trans-Resveratrol preserved antioxidant defense by preventing total and reduced glutathione depletion caused by I/R. At 0.2 mg/kg, trans-resveratrol significantly increased glutathione reductase, Cu/Zn-superoxide dismutase, and catalase activities. However, at a high dose (20 mg/kg), trans-resveratrol became prooxidant with an aggravation of liver injury evaluated by aminotransferase release and histological analysis and associated with a depletion of total and reduced glutathione levels and a decrease of antioxidant enzyme activities. In conclusion, a prereperfusion treatment by trans-resveratrol only at low doses decreases liver injury induced by I/R by protecting against antioxidant defense failure. This administration protocol could reduce liver damage during surgery or transplantation.

The destruction of articular cartilage represents the outcome of most inflammatory and degenerative rheumatic diseases and leads to severe disability. Articular cartilage being unable to repair spontaneously, alterations of the joint surface often results in end-stage osteoarthritis, requiring surgical intervention and total joint replacement. This makes damaged tissues repair a major challenge in our aging society. Cartilage harbors only one cell type, the chondrocyte, which synthesizes and secretes specific matrix proteins such as type II collagen and high molecular weight proteoglycans. Matrix proteins are responsible for the conservation of the chondrocyte phenotype and the maintenance of the mechanical functions of cartilage. Development of therapeutic strategies for cartilage repair should thus comprise not only the replacement of lost cartilage cells but also that of extracellular matrix with cartilage-like properties. Different protocols are under investigation. The most commonly employed materials include transplantation of autologous osteochondral tissue. More recently, cell-based therapies using autologous mature chondrocytes or pre-chondrogenic stem cells have drawn particular attention. Tissue-engineering procedures represent the actual trend in cartilage repair. This approach combines biodegradable polymeric three-dimensional matrixes and isolated prechondrogenic stem cells. The cells are seeded within the biocompatible matrix and then implanted into the joint. Numerous non-degradable and degradable polymers, which efficiently "mimic" the natural surroundings of cartilage cells, are currently under investigation.

Objective: To determine the consequences of pharmacologic up-regulation of heme oxygenase 1 (HO-1), and inhibition of HO-1 by injection of an anti-HO-1 small interfering RNA (siRNA), in vivo in the acute phase of a mouse model of nonautoimmune arthritis., Methods: In the K/BxN mouse serum transfer model, which mimics human inflammatory arthritis without lymphocyte influence, HO-1 was up-regulated by intraperitoneal injection of cobalt protoporphyrin IX (CoPP), a potent pharmacologic inducer, and was inhibited using a specific siRNA. The clinical progress of arthritis was monitored by measurement of paw thickness. Interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNFalpha), serum antioxidant, and nitric oxide (NO) levels, prostaglandin E(2) (PGE(2)) production, and matrix metalloproteinase 9 (MMP-9) activity were measured in serum. At the end of the experiments, joints were examined for immunohistopathologic changes., Results: Intraperitoneal injection of CoPP alleviated disease symptoms, such as joint swelling, cartilage degradation, and proliferation of inflammatory tissue in joints, in the acute phase of inflammatory arthritis. The CoPP-induced expression of HO-1 in the joints and liver was associated with marked decreases in IL-1beta, IL-6, and TNFalpha levels, PGE(2) secretion, and MMP-9 activity in serum, and with a marked increase in systemic antioxidant activity. In contrast, NO production in serum and inducible NO synthase expression in chondrocytes were not affected by HO-1 induction. Specific inhibition of HO-1 by in vivo delivery of anti-HO-1 siRNA repressed the protective effects., Conclusion: Our data provide the first evidence that pharmacologically induced up-regulation of HO-1 triggers a robust protective antiinflammatory response in a model of nonautoimmune arthritis in mice. This suggests that exogenously induced HO-1 may have potential as therapy in the acute phase of inflammatory arthritis in humans.

Objectives: To investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression., Methods: The presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry., Results: ERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2., Conclusions: Our data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

In Europe, endocrine disruptors (EDs) have been defined as substances foreign to the body that have deleterious effects on the individuals or their descendants, due to changes in endocrine function. In the United States, EDs have been described as exogenous agents that interfere with the production, release, transport, metabolism, binding, action or elimination of the natural ligands responsible for maintaining homeostasis and regulating body development. These two definitions are complementary, but both indicate that the effects induced by EDs probably involve mechanisms relating in some way to hormonal homeostasis and action. EDs are generally described as substances with anti-oestrogenic, oestrogenic, anti-androgenic or androgenic effects. More recently, other targets have been evidenced such as the thyroid and immune system. Many different EDs are present in the various compartments of the environment (air, water and land) and in foods (of plant and animal origin). They may originate from food packaging, combustion products, plant health treatments, detergents and the chemical industry in general. In addition to the potential effects of these compounds on adults, the sensitivity of embryos and fetuses to many of the xenobiotic compounds likely to cross the placenta has raised considerable concern and led to major research efforts. With the exception of the clearly established links between diethylstilbestrol, reproductive health abnormalities and cancers, very little is known for certain about the effects of EDs on human health. Given the lack of available data, current concerns about the possible involvement of EDs in the increase in the incidence of breast cancer, and possibly of endometriosis and early puberty in girls, remain hypothetical. Conversely, the deterioration in male reproductive health is at the heart of preoccupations and progress in analyses of the relationship between EDs and human health. This literature review aims to describe the current state of knowledge about endocrine disruption, focusing in particular on the problem of food contaminants.

Aryl hydrocarbon receptor (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo(a)pyrene interfere with hormonal regulatory pathways, leading to endocrine disruption. Notably, the activated AhR exerts complex effects on estrogens and retinoids at both levels of their metabolism and regulation of cognate genes. Our current investigation of these AhR effects revealed the TCDD-dependent activation of a subset of retinoid-dependent genes (tissue-transglutaminase, IGF binding protein-3, AhR) in MCF-7 breast cancer cells. A collection of in vitro hormone-dependent reporter gene models showed that AhR activation by TCDD stimulated transactivation by several class I heteromeric receptors (retinoic and thyroid hormone receptors) while it antagonized homodimeric nuclear receptors (estrogen and progesterone receptors, ER and PR). TCDD exerted a dose-dependent effect on a retinoic acid-dependent reporter gene expressed in MCF-7 cells. AhR was shown to be involved in a mutual antagonism with RARalpha corepressor SMRT (silencing mediator of retinoid and thyroid receptors). This, and the documented physical interaction between AhR and SMRT suggested that SMRT sequestration by AhR might activate RARalpha in the absence of ligand. Immunocytochemical studies of AhR and SMRT strongly suggested they colocalized in nuclear bodies during this sequestration. Concurring with this interpretation, we observed an interaction in vitro between AhR and the PML protein, the core component of nuclear bodies. This ability of AhR to elicit spurious activation of retinoid receptors expands the scope of AhR ligands influence beyond ER antagonism and specific Dioxin-responsive genes. Unknown AhR endogenous ligands may also elicit gene transactivation by class I receptors, while being inactive on classic xenobiotic-responsive genes.

Proteoglycan production is one of the major extracellular matrix components implicated in the dynamic process of intervertebral disc degeneration. Mechanical stress is an important modulator of the degeneration, but the underlying molecular mechanism at the proteoglycan level remains unclear. The aim of this work was to study the regulation of proteoglycan production by cyclic tensile stretch applied to intervertebral disc annulus fibrosus cells. Matrix metalloproteinases do not seem to be implicated in the regulation of proteoglycan production. By contrast, nitrite oxide production is induced by cyclic tensile stretch, in a time, intensity, and frequency dependant manner. Using a non-specific nitric oxide synthases inhibitor [NG-methyl-L-arginine (L-NMA)], we suppress totally the inhibition of proteoglycan production induced by cyclic tensile stretch suggesting the implication of nitric oxide synthases in the observed phenomenon. Introducing the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or a more specific inhibitor of nitric oxide synthases II [N-iminoethyl-L-lysine (L-NIL)] did not affect the decreased proteoglycan production, which suggests a post-translational regulation. In contrast, N-omega nitro-L-arginine (L-NNA) a more specific inhibitor of NOS I and III abrogated the cyclic tensile stretch-dependant inhibition of proteoglycan production. These results suggest that cyclic tensile stretch regulates proteoglycan production through a post-translational mechanism involving nitrite oxide. This result could be of interest in the development of local therapeutic strategies aimed at controlling intervertebral disc degeneration.

We developed new stilbene derivatives of resveratrol (E)-1-(4'-hydroxyphenyl)-2-(3,5-dihydroxyphenyl)ethene) selective for AhR and devoid of affinity for ER. Among the 24 stilbenes synthesized, all display a higher affinity than resveratrol for AhR. (E)-1-(4'-Trifluoromethylphenyl)-2-(3,5-ditrifluoromethylphenyl)ethene (4e), (E)-1-(4'-methoxyphenyl)-2-(3,5-dichlorophenyl)ethene (4j), and (E)-1-(4'-chlorophenyl)-2-(3,5-dichlorophenyl)ethene (4b) are selective, high-affinity AhR antagonists with, respective, K(i)s of 2.1, 1.4, and 1.2 nM. (E)-1-(4'-Trifluoromethylphenyl)-2-(3,5-dichlorophenyl)ethene (4i) displays a K(i) of 0.2 nM and is a selective and high-affinity agonist on AhR.

Background and Aims: Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction., Methods: The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay., Results: We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer., Conclusions: We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

Interleukin-1beta (IL-1beta) induces degradation via hyperexpression of an array of genes, including metalloproteinases (MMP), in cartilage cells during articular degenerative diseases. In contrast, natural ligands for peroxisome proliferator-activated receptors (PPARs) display protective anti-cytokine effects in these cells. We used the PPAR agonist rosiglitazone (Rtz) to investigate PPAR-gamma isotype on IL-1beta-target genes. Immunocytochemistry, electrophoretic mobility shift, and transient transfection assays revealed a functional PPAR-gamma in chondrocytes in vitro. Rtz displayed significant inhibition of IL-1beta effects in chondrocytes. Low Rtz concentrations (close to K(d) values for PPAR-gamma, 0.1 to 1 microm) inhibited the effects of IL-1beta on (35)S-sulfated proteoglycan production and gelatinolytic activities and downregulated MMP1 expression at mRNA and protein levels. We have investigated the mechanism of action of Rtz against IL-1beta-mediated MMP1 gene hyperexpression. Rtz effect occurs at the transcriptional level of the MMP1 promoter, as observed in transiently transfected cells with pMMP1-luciferase vector. Transient expression of wild type PPAR-gamma enhanced Rtz inhibitory effect in chondrocytes, whereas a mutated dominant negative PPAR-gamma abolished it, supporting the role of PPAR-gamma in this effect. MMP1 gene promoter analysis revealed the involvement of a cis-acting element located at -83 to -77, shown to be a composite PPRE/AP1 site. Gel mobility and supershift assays demonstrated that PPAR-gamma and c-Fos/c-Jun proteins bind this cis-acting element in a mutually exclusive way. Our data highlight a new PPAR-gamma-dependent inhibitory mechanism on IL-1beta-mediated cartilage degradation occurring through DNA binding competition on the composite PPRE/AP1 site in the MMP1 promoter.

The aryl hydrocarbon receptor (AhR) is involved in various processes such as cytochrome P450 (P450) 1A induction after xenobiotic exposure. It is also considered to play a major role in cell proliferation and differentiation. Recent evidences have suggested a cross-talk between AhR functions and the mitogen-activated protein kinase (MAPK) cascade. We now report that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a specific inhibitor of MAPK kinase (MEK) MEK1/2, elicits a marked increase in CYP1A1 expression at both mRNA and protein levels associated with a significant increase of enzyme activity in primary rat hepatocytes and a human hepatoma cell line. This induction occurred independently of MEK/extracellular signal-regulated kinase (ERK) activation and in the absence of ERK1 and ERK2 expression. The effect of U0126 was mediated by its ability to transactivate xenobiotic responsive element (XRE)-driven genes, as demonstrated by transfection assays with an XRE-driven luciferase construct in the human B16A2 hepatoma cell line. CYP1A1 modulation was abolished by a cotreatment with resveratrol, an established AhR antagonist, arguing for AhR activation by U0126. Such an effect was demonstrated by direct in vitro ligand binding competition assays using rabbit liver cytosol, showing that this compound binds AhR with an EC(50) = 25 x 10(-6) M. Moreover, we demonstrated that U0126 is a substrate for several P450s including human CYP1A2, -1A1, and -1B1. We conclude that the widely used specific inhibitor of MEK/ERK, U0126, also acts as a potent AhR activator and an inducer of related genes. Such effects on the AhR may have an impact on biological functions attributed previously to MAPK inhibition.

Background and Aims: The molecular mechanism of hereditary persistence of alpha-fetoprotein (HPAFP) has been previously described in a large Scottish family, consisting of a -119G>A substitution in the distal hepatocyte nuclear factor 1 (HNF-1) binding site of the alpha-fetoprotein (AFP) gene promoter. We report here the molecular mechanisms of HPAFP in 2 new unrelated families., Methods: Family 1 was of Bengali origin, and family 2 was Italian. Four of 5 subjects (family 1) and 3 of 9 (family 2) showed HPAFP. The AFP gene promoter was studied in all available family members., Results: All subjects with high AFP levels had mutated promoter sequences. Family 1 showed the reported -119G>A substitution. Family 2 showed -55C>A and -65C>T substitutions in the proximal putative HNF-1 binding region of the promoter. The -55C>A mutation increased the similarity of the proximal HNF-1 binding region to a consensus binding region. Gel shift assays confirmed its increased affinity toward HNF-1, and transfection experiments revealed an increased level of gene transcription. The -65C>T substitution theoretically created a CCAAT box. However, gel shift and transfection experiments failed to show any biological effect of this substitution that is associated with the -55C>A mutation., Conclusions: Two different mutations localized in either HNF-1 binding sites of the AFP gene promoter may result in HPAFP. This highlights the importance of HNF-1 in AFP gene expression. Unexplained persistent AFP should lead to family study and/or AFP gene promoter sequencing to avoid inappropriate explorations and treatment decisions.

Benzo[a]pyrene (BaP) is an agonistic ligand for the aryl hydrocarbon receptor (AhR) and a major environmental carcinogen implicated in the aetiology of lung cancer through the induction of benzo[a]pyrene diol epoxidation (BPDE) and BPDE-DNA adducts. Because BaP metabolization requires cytochrome P-450 1A1 (CYP1A1) induction through activation of the AhR, we hypothesized that resveratrol, a natural competitive inhibitor of AhR, could prevent these adverse effects of BaP on the lung. Balb-C mice were injected for 5 weeks with corn oil, BaP (5 mg kg(-1) week(-1)), resveratrol (50 mg kg(-1) week(-1)) or BaP + resveratrol. Immunohistochemistry was performed on lung sections for the determination of CYP1A1 protein, BPDE-DNA adducts and apoptosis. A semi-quantitative immunohistochemistry score (H score) was used for data analysis. Mice exposed to BaP had a significant induction of lung BPDE-DNA adducts when compared with controls (H scores: control, 26, interquartile range 18-33; BaP, 276, interquartile range 269-288; P < 0.01). The BPDE-DNA adduct induction by BaP was abrogated significantly by resveratrol (H score: BaP + resveratrol, 103, interquartile range 96-113). A similar pattern was found by immunohistochemistry for apoptosis (H scores: control, 121, interquartile range 102-137; BaP, 288, interquartile range 282-292, P < 0.05; BaP + resveratrol, 132, interquartile range 121-141, P = NS) and CYP1A1 (H scores: control, 170.3, interquartile range 164-175; BaP, 302.3, interquartile range 291-315, P < 0.05; BaP + resveratrol, 200.7, interquartile range 174-215, P = NS). Western blotting confirmed that resveratrol prevented BaP-induced CYP1A1 expression. This increase in CYP1A1 expression in response to BaP administration most likely causes BaP metabolism, BPDE-DNA adduct formation and subsequent apoptosis. All BaP-induced effects could be prevented by resveratrol, suggesting a possible chemopreventive role for this natural phytoalexin against the development of lung cancer., (Copyright 2003 John Wiley & Sons, Ltd.)

This review reconsiders a major cause of cardiovascular diseases, tobacco smoking, as the activation of the Aryl hydrocarbon Receptor (AhR), also known as the dioxin receptor, by aryl hydrocarbons from the tar fraction of tobacco in various organs of the cardiovascular domain. This concept sheds new light on well-known albeit controversial epidemiological concepts such as the Mediterranean diet and the French paradox. We also review the discovery that resveratrol, a natural AhR antagonist, may be of interest in the prevention and treatment of cardiovascular diseases.

The industrial contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is associated with inflammatory disorders in women and other mammals. The current studies were performed to investigate the effect of TCDD on RANTES expression in an in-vitro model of endometriosis. The biochemical effects of dioxins are mediated by binding to aryl hydrocarbon receptors (AhR). This study showed that both normal and endometriotic endometrial stromal cells express AhR protein, which was observed to be down-regulated by 40-60% after exposure to TCDD. Treatment with TCDD for 24 h increased the luciferase activity of the RANTES promoter by 2.5 +/- 1.0-fold in stromal cells derived from normal endometrium and endometriotic implants. When AhR were over-expressed in these cells, luciferase activity increased 6.1 +/- 1.4-fold, and RANTES protein secretion increased from undetectable to 31 +/- 10 pg/100,000 cells. TCDD failed to activate a RANTES construct with a mutated dioxin response element. Other AhR ligands had similar effects to TCDD on RANTES transcription and secretion. Control transfections using tumour necrosis factor (TNF)-alpha and nuclear factor (NF)-kappaB response element reporters indicated that these pathways are not activated by TCDD in endometrial stromal cells. This study has demonstrated that functional AhR are present in endometrial and endometriotic stromal cells and that TCDD up-regulates the expression of RANTES, providing a possible mechanistic link between dioxin exposure and chemokine expression in endometriosis.

The various properties of the stilbene phytoalexin Resveratrol provide interesting new avenues of research in the field of chemoprevention and chemotherapy. A particular emphasis is given on xenobiotic-related carcinogenesis.

New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or beta were cotransfected, CAT activity was induced in the presence of 17beta-estradiol, and NET tetrahydro-reduced derivatives. The results indicate that this AGMK-CV1-PR cell assay system appears to be suitable for measuring the effects of different synthetic progestins at the transcriptional level. In this assay system, NET, LNG, and GSD exhibit potent progestational effects at the transcriptional level. In the particular case of NET, the assay system allowed us to determine that the single or multiple hormonal transcriptional effects of this compound are partially mediated by its A-ring-reduced derivatives.

Benzo(a)pyrene (BaP), an aryl hydrocarbon receptor (AhR) ligand present in cigarette smoke and car exhaust, is thought to have negative effects on male reproduction. We hypothesized that BaP damages sperm through AhR activation, phase I enzyme induction, DNA adduct formation, and increased germ cell apoptosis in the testis, and that resveratrol, a natural competitive inhibitor of the AhR found in some red wines, could prevent the adverse effects of BaP on sperm. Male Balb C mice were injected subcutaneously (s.c.) for 5 weeks with a range of BaP doses (0.5 mg/kg to 50 mg/kg). Live sperm were obtained from the vas deferens, counted, and stained to measure annexin-V positive (apoptotic) cells. In a subsequent study, mice were injected for 5 weeks with corn oil (control), BaP (5 mg/kg/week), or BaP plus resveratrol (50 mg/kg/week) (n = 3 per group). Immunohistochemistry (IHC) was performed on testis sections for the determination of CYP1A1, BaP diol epoxide (BPDE) DNA adducts, and apoptosis and the results quantified by using the HSCORE, a semiquantitative scoring system. Our results demonstrated that sperm counts after 5 weeks were inversely correlated to BaP dosage. BaP (0.5 to 5 mg/week) positively correlated with sperm apoptosis while higher doses increased sperm necrosis. CYP1A1 protein was observed mainly in interstitial cells of some testis sections, but there was no significant induction by BaP. BPDE DNA adducts were induced in all components of the seminiferous tubules by BaP and suppressed by resveratrol: median HSCORE (interquartile range) control 61 (52-71.5); BaP 213 (192-248), P = 0.01 compared to control; BaP plus resveratrol 83 (70-90). BaP significantly increased apoptosis, mainly in spermatogonia: medain HSCORE (interquartile range) BaP 189 (161-223) versus control 83 (57-93), P < 0.01; and this effect was abrogated by resveratrol. Median HSCORE for BaP plus resveratrol was 112 (range 99-121). In summary, BaP caused increased sperm cell BPDE DNA adduct formation and apoptosis in the mouse. The natural AhR antagonist, resveratrol diminished BaP-induced DNA adducts and apoptosis in seminiferous tubules.

The effect of resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, known to inhibit inducible cyclooxygenase-2 (COX2) and its transcription were examined in a model of hyperalgesia induced by carrageenan in the rat. Pretreatment with resveratrol did not reverse swelling and edema, but reversed the hyperalgesia induced by local tissue injury provoked by carrageenan. This reversal, occurring at resveratrol concentrations as low as 2 mg/kg, lasted for at least 48 hours. The link with COX2 activity inhibition and COX2 gene transcription, as well as a potential AhR inhibitory effect, remain to be established.

We have identified 7-ketocholesterol (7-KC) as an endogenous modulator that inhibits transactivation by the arylhydrocarbon receptor (AhR) through competitive binding against xenobiotic ligands. 7-KC binds AhR and displaces labeled dioxin (2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD)). IC(50) is 5 x 10(-7) m in vivo and 7 x 10(-6) m in vitro. These figures are consistent with its concentration in human blood plasma and tissues. Association with 7-KC prevents AhR binding to DNA. 7-KC blocks the TCDD-mediated transactivation of stably expressed reporter gene constructs in T47-D cells as well as the expression of the endogenous CYP 1A1 gene in HepG2 cells and in primary porcine aortic endothelial cells. Injection of 7-KC to rats blocks the induction of CYP 1A1 messenger RNA and protein in endothelial cells from myocardial blood vessels. The differential sensitivity of mammalian species to toxic effects of AhR ligands, especially dioxin (TCDD), correlates with the expression of 7-hydroxycholesterol dehydrogenase, which synthesizes 7-KC from 7-hydroxycholesterol. The documented involvement of AhR ligands in cardiovascular diseases through lipid peroxidation and endothelium dysfunction can now be examined in the context of displacement of this protective modulator.

Aryl hydrocarbon receptor (AhR) ligands such as dioxin and benzo[a]pyrene are environmental contaminants with many adverse health effects, including immunosuppression, carcinogenesis, and endothelial cell damage. We show here that a wine component, resveratrol (3,5,4'-trihydroxystilbene), is a competitive antagonist of dioxin and other AhR ligands. Resveratrol promotes AhR translocation to the nucleus and binding to DNA at dioxin-responsive elements but subsequent transactivation does not take place. Resveratrol inhibits the transactivation of several dioxin-inducible genes including cytochrome P-450 1A1 and interleukin-1beta, both ex vivo and in vivo. Resveratrol has adequate potency and nontoxicity to warrant clinical testing as a prophylactic agent against aryl hydrocarbon-induced pathology.

One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.

Gonadotrophin and thyrotrophin receptors belong to a subgroup of G-protein-coupled receptors. These receptors are characterized by a large extracellular domain that is responsible for the binding of the hormone. Soluble receptors, such as some luteinizing hormone receptors, arise from premessenger RNA alternative splicing, or, in the case of thyroid-stimulating hormone (TSH) receptors, by the cleavage and shedding of the ectodomain. Follicle-stimulating hormone and TSH receptors are restricted to the basolateral domain of their target cells. These receptors are also present in endothelial cells of target organ vessels and are involved in hormone transcytosis. Various genetic abnormalities of these receptors have been described.

PML (promyelocytic leukemia) is a protein involved in the t (15;17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML, because when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. The use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys-rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR which is not localized in nuclear bodies, also enhanced the transactivating activity of PR, but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by retinoic acid.

Norethisterone (NET) and levonorgestrel (LNG) are synthetic progestins used as contragestational agents. Both compounds are biotransformed at target tissues into A-ring reduced metabolites which possess different pharmacological properties. The aim of this study was to determine the molecular mechanisms of the progestational and antiprogestational effects of NET, LNG and their metabolites by using a highly efficient, sensitive in vitro molecular assay based on the detection of a reporter gene expression (the bacterial chloramphenicol acetyltransferase (CAT) inserted downstream of a minimal promoter containing two progesterone responsive elements (PRE2) and the TATA box. For this purpose we used CV-1 monkey kidney cells, which do not possess steroid receptors. These cells were cotransfected with a progesterone receptor expression vector and the reporter vector PRE2-TATA-CAT. Data obtained using this model showed that NET and LNG induced CAT activity in a manner similar to that of the potent progestin R5020. NET and LNG metabolites exhibited a weak progestational activity; however, when 5 alpha-NET metabolite was simultaneously administered with R5020, a clear antiprogestational effect similar to that of the antiprogestin RU486 was observed. Therefore, the results clearly demonstrate that the use of the reporter CAT vector containing hormone responsive elements is a suitable assay for the screening and evaluation of new synthetic steroids with agonist or antagonist progestational activities in transfected CV-1 cell line.

Transcriptional regulation of the progesterone receptor gene involves induction by estrogens and down-regulation by progestins, retinoic acid, and AP-1 proteins. We have previously identified an intragenic (+698/+723) estrogen-responsive element present in the progesterone receptor gene, which binds the estradiol receptor and mediates estrogen and 4-OH tamoxifen induction. Progesterone receptor gene expression was equally stimulated by estradiol and 4-OH tamoxifen in the presence of a NH2 terminally deleted estrogen receptor mutant lacking activation function 1, suggesting that activation function 2 was the predominant activation domain. This was confirmed by the lack of activity of an estrogen receptor mutant deleted of activation function 2. Repression by progestins, retinoic acid, and AP-1 was mediated by the same estrogen responsive element although retinoic and progesterone receptors as well as AP-1 proteins did not bind to this element. Repression by these proteins appears to involve different transactivating regions of the estrogen receptor. Repression by retinoic receptors involved only activation function 2 whereas repression by progesterone receptor and AP-1 necessitated both functional domains. Since these proteins act without directly contacting the DNA, it seems likely that repression may be achieved by protein-protein interactions among different domains of the estrogen receptor and/or the transcriptional machinery.

The progesterone receptor displays the typical three-domains structure of the steroid-thyroid receptor family. The central domain contains two 'zinc finger' structures responsible for the specific recognition of the cognate DNA sequences. The carboxy-terminal domain contains the hormone and anti-hormone binding site. Progesterone and synthetic progestins (R5020, Org 2058) activate the receptor, provoke its phosphorylation and DNA-binding ability and induce its regulatory activities. The antagonist RU38486 elicits the same sequence of events but leads to an abortive conclusion without specific gene transactivation. The progesterone receptor is down-regulated by its own ligand at the transcriptional level through inhibition of oestrogen receptor-mediated induction through protein-protein interactions. This mechanism is also inhibited by RU38486.

The rabbit progesterone receptor undergoes dual regulation at the level of transcription: positive by estrogens and negative by progestins. The two aspects of this regulation are mediated by a single intragenic estrogen-responsive element. Estrogen receptor binding to this element has been demonstrated but progestin down-regulation does not proceed through DNA binding of the progesterone receptor. This result suggests some kind of protein-protein interaction--direct or indirect--between estrogen and progesterone receptors. At the post-transcriptional level, the progesterone receptor undergoes a hormone-dependent hyperphosphorylation of serine residues localized in the N-terminal region. Studies of progesterone receptor mutants have determined the influence of the different receptor domains in the phosphorylation mechanism. A casein kinase copurifies with the receptor. The role of this phosphorylation remains to be determined.

The transcription of the progesterone receptor gene is induced by estrogens and decreased by progestins. Studies were performed to define the regions of the gene and the molecular mechanisms involved. No hormonal regulation could be observed using 5' flanking regions of the gene up to -2762 in front of a heterologous gene. Estrogen and progestin regulation could be observed only when using fragments of the gene extending down to +788. Progressive deletions from the 5' and 3' ends, site-directed mutagenesis and DNase protection experiments with purified estrogen receptor suggested that the biologically active estrogen responsive element (ERE) is present at +698/+723, overlapping the initiation of translation. An oligonucleotide was synthesized bearing this ERE and shown to impart estrogen inducibility to a heterologous gene. Its regulation by anti-estrogens corresponded to that of the in situ progesterone receptor gene since tamoxifen was a partial agonist whereas ICI 164384 was a full antagonist. This ERE also mediated down-regulation by progestins in the presence of the progesterone receptor, even though it has no progesterone receptor binding ability. DNase footprinting showed that this effect was not due to a decrease of estrogen receptor affinity for the ERE in the presence of progesterone receptor. Finally, use of deletion mutants of the progesterone receptor showed that the steroid binding and the DNA binding domains were necessary for down-regulation whereas deletions of various parts of the N-terminal domain were without effect.

The T47-D breast cancer cell line constitutively expresses high levels of progesterone receptor (PR). This does not appear to be related to an anomaly in the estrogen receptor (ER) as shown by cloning of the ER cDNA from T47-D cells and its insertion into the expression vector pKSV-10. When transfected into heterologous Cos-7 and L cells this receptor exerts a normal biological activity, stimulating the transcription of a reporter gene only in the presence of estrogen. Moreover, normal estrogen regulation of the transcription of the reporter gene was also observed in situ in T47-D cells. Southern blot experiments showed the presence of four copies of the progesterone receptor gene in T47-D cells. This was related to the existence of four copies of chromosome 11 in these cells. The most likely explanation of the anomalous regulation of progesterone receptor expression in T47-D cells is thus the presence of at least one copy of the PR gene bearing an anomaly in its regulatory region(s).

Several vectors were used to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS2) in Escherichia coli. The best results were obtained by using the pUR 290 expression vector after deletion of the sequence encoding the signal peptide of the protein. In these conditions, beta-galactosidase-BCEI/pS2 fusion protein accounted for approximately 20% of total proteins in bacterial extracts. It was purified by chromatography on DEAE-Trisacryl or by gel electrophoresis and electroelution. Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native protein and was very efficient for the immunoprecipitation and immunopurification of the protein from breast cancer cells; a second class recognized the denatured protein and was especially effective for immunoblot studies. BCEI/pS2 could be detected by immunocytochemistry in breast cancer biopsies using monoclonal antibodies on frozen or paraffin-embedded sections. One of the antibodies (mBCEI11) exhibited high affinity for the protein and could be used at 1.9 micrograms/ml concentration for immunolabeling of histological sections. The mBCEI11 antibody was used in immunoaffinity chromatography to purify the peptide in a single step from culture media of estrogen-treated MCF-7 cells.