Vitamin D deficiency in childhood obesity is associated with high levels of circulating inflammatory mediators, and low insulin sensitivity.

HYPOTHESIS: Childhood obesity is accompanied by low-grade systemic inflammation, which contributes to the development of insulin resistance and cardiovascular complications later in life. As vitamin D exhibits profound immunomodulatory functions and vitamin D deficiency is highly prevalent in childhood obesity, we hypothesized that vitamin D deficiency in childhood obesity coincides with enhanced systemic inflammation and reduced insulin sensitivity. METHODS: In a cross-sectional study of 64 obese and 32 healthy children aged 6-16 years, comprehensive profiling of 32 circulating inflammatory mediators was performed, together with assessment of 25-hydroxyvitamin D (25(OH)D) levels and measures for insulin sensitivity. RESULTS: Severe vitamin D insufficiency, which is further referred to as vitamin D deficiency, was defined as a 25(OH)D level ≤37.5 nmol l(-1), and was highly prevalent in obese (56%) versus healthy control children (16%). Throughout the study, 25(OH)D-deficient children were compared with the other children, including 25(OH)D insufficient (37.5-50 nmol l(-1)) and 25(OH)D sufficient children (≥50 nmol l(-1)). First, 25(OH)D-deficient obese children showed a lower insulin sensitivity than other obese children, as measured by a lower quantitative insulin sensitivity check index. Second, the association between 25(OH)D deficiency and insulin resistance in childhood obesity was confirmed with multiple regression analysis. Third, 25(OH)D-deficient obese children showed higher levels of the inflammatory mediators cathepsin S, chemerin and soluble vascular adhesion molecule (sVCAM), compared with the other obese children. Finally, hierarchical cluster analysis revealed an over-representation of 25(OH)D deficiency in obese children expressing inflammatory mediator clusters with high levels of cathepsin S, sVCAM and chemerin. CONCLUSION: 25(OH)D deficiency in childhood obesity was associated with enhanced systemic inflammation and reduced insulin sensitivity. The high cathepsin S and sVCAM levels may reflect activation of a pro-inflammatory, pro-diabetic and atherogenic pathway, which could be inhibited by vitamin D supplementation.

Invariant natural killer T cells in adipose tissue: novel regulators of immune-mediated metabolic disease.

Adipose tissue (AT) represents a microenvironment where intersection takes place between immune processes and metabolic pathways. A variety of immune cells have been characterized in AT over the past decades, with the most recent addition of invariant natural killer T (iNKT) cells. As members of the T cell family, iNKT cells represent a subset that exhibits both innate and adaptive characteristics and directs ensuing immune responses. In disease conditions, iNKT cells have established roles that include disorders in the autoimmune spectrum in malignancies and infectious diseases. Recent work supports a role for iNKT cells in the maintenance of AT homeostasis through both immune and metabolic pathways. The deficiency of iNKT cells can result in AT metabolic disruptions and insulin resistance. In this review, we summarize recent work on iNKT cells in immune regulation, with an emphasis on AT-resident iNKT cells, and identify the potential mechanisms by which adipocytes can mediate iNKT cell activity.

Systemic inflammation in childhood obesity: circulating inflammatory mediators and activated CD14++ monocytes.

AIMS/HYPOTHESIS: In adults, circulating inflammatory mediators and activated CD14(++) monocytes link obesity to its metabolic and cardiovascular complications. However, it is largely unknown whether these inflammatory changes already occur in childhood obesity. To survey inflammatory changes during the early stages of obesity, we performed a comprehensive analysis of circulating inflammatory mediators, monocyte populations and their function in childhood obesity. METHODS: In lean and obese children aged 6 to 16 years (n = 96), 35 circulating inflammatory mediators including adipokines were measured. Hierarchical cluster analysis of the inflammatory mediator profiles was performed to investigate associations between inflammatory mediator clusters and clinical variables. Whole-blood monocyte phenotyping and functional testing with the toll-like receptor 4 ligand, lipopolysaccharide, were also executed. RESULTS: First, next to leptin, the circulating mediators chemerin, tissue inhibitor of metalloproteinase 1, EGF and TNF receptor 2 were identified as novel inflammatory mediators that are increased in childhood obesity. Second, cluster analysis of the circulating mediators distinguished two obesity clusters, two leanness clusters and one mixed cluster. All clusters showed distinct inflammatory mediator profiles, together with differences in insulin sensitivity and other clinical variables. Third, childhood obesity was associated with increased CD14(++) monocyte numbers and an activated phenotype of the CD14(++) monocyte subsets. CONCLUSIONS/INTERPRETATION: Inflammatory mediator clusters were associated with insulin resistance in obese and lean children. The activation of CD14(++) monocyte subsets, which is associated with increased development of atherosclerosis in obese adults, was also readily detected in obese children. Our results indicate that inflammatory mechanisms linking obesity to its metabolic and cardiovascular complications are already activated in childhood obesity.

Adipose tissue quantity and composition contribute to adipokine concentrations in the subclavian vein and the inferior mesenteric vein.

BACKGROUND: Adipose tissue dysfunction is associated with inflammation, type 2 diabetes mellitus and vascular diseases. Visceral adipose tissue (VAT)-derived adipokines, which are released in the portal circulation may influence liver metabolism. OBJECTIVES: (1) To estimate the contribution of VAT and subcutaneous adipose tissue (SAT) on adipokine levels by measuring differences in adipokine concentrations between the portal draining inferior mesenteric vein and the subclavian vein. (2) To determine the relation of both VAT and SAT quantity and composition to mesenteric and systemic concentrations of adipokines. DESIGN: Cross-sectional cohort study. SUBJECTS: A total of 32 patients undergoing abdominal aortic surgery. MEASUREMENTS: A panel of 18 adipokines was measured in perioperatively obtained blood samples from the subclavian vein and the inferior mesenteric vein. Adipocyte size, macrophage infiltration and capillary density were measured in subcutaneous and mesenteric adipose tissue biopsies; SAT and VAT areas were measured on computed tomography images. RESULTS: Serum interferon-γ-inducible protein 10 (IP-10) and hepatocyte growth factor (HGF) concentrations were significantly higher in the inferior mesenteric vein vs the subclavian vein. SAT area (ß -18; 95% confidence interval (CI) -35 to -2), subcutaneous adipocyte size (ß -488; 95% CI -938 to -38) and SAT macrophages quantity (ß -1439; 95% CI -2387 to -491) were negatively associated with adiponectin levels in the systemic circulation. SAT area was related to systemic concentrations of leptin. Mesenteric adiponectin concentrations were related to VAT area (ß -20; 95% CI -35 to -5) and visceral adipocyte size (ß -1076; 95% CI -1624 to -527). VAT area, adipocyte size and capillary density were related to systemic adiponectin concentrations. CONCLUSION: SAT and VAT quantities as well as morphologic characteristics of both adipose tissue depots are related to systemic and mesenteric adipokine concentrations. There were no differences in adipokine concentrations between the mesenteric and subclavian vein, except for higher IP-10 and HGF concentrations in the inferior mesenteric vein, indicating a possible contribution of VAT to IP-10 and HGF levels.

Characterisation of non-obese diabetic patients with marked insulin resistance identifies a novel familial partial lipodystrophy-associated PPARγ mutation (Y151C).

AIMS/HYPOTHESIS: Familial partial lipodystrophy (FPLD) is a rare metabolic disorder with clinical features that may not be readily recognised. As FPLD patients require a specific therapeutic approach, early identification is warranted. In the present study we aimed to identify cases of FPLD among non-obese patients with type 2 diabetes mellitus and marked insulin resistance. METHODS: We searched the databases of three diabetic outpatient clinics for patients with marked insulin resistance, arbitrarily defined as the use of ≥100 U insulin/day, and BMI ≤ 27 kg/m(2). In all patients, metabolic variables and anthropomorphic measurements were evaluated and DNA was sequenced for mutations in the genes encoding lamin A/C (LMNA), peroxisome proliferator-activated receptor γ (PPARγ) and cell death-inducing DFFA-like effector c (CIDEC). RESULTS: Out of 5,221 diabetic individuals, 24 patients fulfilled all criteria. Twelve patients were willing to participate, of whom five showed clinical features of lipodystrophy. In three of these patients the clinical diagnosis of FPLD was confirmed by the presence of mutations in LMNA or PPARG; one patient harboured a novel heterozygous mutation (Y151C) in PPARG. The Y151C mutant displayed impaired DNA-binding capacity and hence reduced transcriptional activity compared with wild-type PPARγ. Dominant-negative activity was absent. CONCLUSION/INTERPRETATION: The combination of BMI ≤ 27 kg/m(2) and the use of >100 U insulin/day increases the chance of identifying lipodystrophy. Thus careful assessment of clinical features of FPLD should be considered in these patients, allowing earlier therapeutic interventions.

Gene-gene and gene-environment interactions in lipodystrophy: Lessons learned from natural PPARγ mutants.

Monogenic lipodystrophies are a heterogeneous group of rare disorders characterized by a lack of adipose tissue (AT), all of which predispose patients to the development of insulin resistance and its related metabolic sequelae. The extent of AT loss ranges from partial, as in familial partial lipodystrophy (FPLD), to a total absence of metabolically active AT in congenital generalized lipodystrophy (CGL) and is generally associated with the severity of metabolic complications. Significant genetic, allelic, phenotypic, and clinical heterogeneity exists among the lipodystrophies. Patients with FPLD3 due to mutations in the PPARG gene, which encodes a key transcriptional regulator of adipocyte development and function, provide a particularly striking example of this heterogeneity. We will present several gene-gene and gene-environment factors and mechanisms that are critical for adequate PPARγ expression and activity in AT and discuss how these interactions potentially contribute to the observed spectrum of FPLD3 phenotypes. Comparable mechanisms may play a role in other types of lipodystrophies too, and their elucidation may further improve our molecular understanding of AT dysfunction.

AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors.

Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.

Monocyte gene expression in childhood obesity is associated with obesity and complexity of atherosclerosis in adults.

Childhood obesity coincides with increased numbers of circulating classical CD14++CD16- and intermediate CD14++CD16+ monocytes. Monocytes are key players in the development and exacerbation of atherosclerosis, which prompts the question as to whether the monocytosis in childhood obesity contributes to atherogenesis over the years. Here, we dissected the monocyte gene expression profile in childhood obesity using an Illumina microarray platform on sorted monocytes of 35 obese children and 16 lean controls. Obese children displayed a distinctive monocyte gene expression profile compared to lean controls. Upon validation with quantitative PCR, we studied the association of the top 5 differentially regulated monocyte genes in childhood obesity with obesity and complexity of coronary atherosclerosis (SYNTAX score) in a cohort of 351 adults at risk for ischemic cardiovascular disease. The downregulation of monocyte IMPDH2 and TMEM134 in childhood obesity was also observed in obese adults. Moreover, downregulation of monocyte TMEM134 was associated with a higher SYNTAX atherosclerosis score in adults. In conclusion, childhood obesity entails monocyte gene expression alterations associated with obesity and enhanced complexity of coronary atherosclerosis in adults.

Expression of the glucocorticoid receptor gene is regulated during early embryogenesis of Xenopus laevis.

To study the possible role of the glucocorticoid receptor (GR) in early embryogenesis, we isolated a Xenopus glucocorticoid receptor cDNA from an embryonic stage 17 cDNA library. Overexpression of this Xenopus GR in COS cells confers the ability to transactivate a GRE-tk CAT promoter construct in a ligand dependent manner. Expression of the Xenopus GR gene at the RNA level was analyzed by Northern blot hybridization. Transcripts of 4 and 6 kb are present in oocytes. The 4 kb mRNA is abundant and is degraded together with the 6 kb mRNA during cleavage stages of early development. Between stages 17 and 24, GR messengers are extremely rare. From stage 32 onwards, both GR transcripts start to be expressed again at intermediate levels. These results provide the first evidence that expression of the GR gene is regulated during early embryonic development.

Steroid receptor coactivator-1 splice variants differentially affect corticosteroid receptor signaling.

The mechanisms of receptor- and cell-specific effects of the adrenal corticosteroid hormones via mineralo- (MRs) and glucocorticoid receptors (GRs) are still poorly understood. Because the expression levels of two splice variants of the steroid receptor coactivator-1 (SRC-1) 1a and 1e, can differ significantly in certain cell populations, we tested the hypothesis that their relative abundance could determine cell- and receptor-specific effects of corticosteroid receptor-mediated transcription. In transient transfections, we demonstrate three novel types of SRC-1a- and SRC-1e-specific effects for corticosteroid receptors. One is promoter dependence: SRC-1e much more potently coactivated transcription from several multiple response element-containing promoters. Mammalian 1-hydrid studies indicated that this likely does not involve promoter-specific coactivator recruitment. Endogenous phenylethanolamine-N-methyltransferase mRNA induction via GRs was also differentially affected by the splice variants. Another type is receptor specificity: responses mediated by the N-terminal part of the MR, but not the GR, were augmented by SRC-1e at synergizing response elements. SRC fragment SRC(988-1240) by the MR but not the GR N-terminal fragment in a 1-hybrid assay. The last type, for GRs, is ligand dependence. Due to effects on partial agonism of RU486-activated GRs, different ratios of SRC-1a and 1e can lead to large differences in the extent of antagonism of RU486 on GR-mediated transcription. Furthermore, we show that SRC-1e but not SRC-1a mRNA expression was regulated in the pituitary by corticosterone. We conclude that the cellular differences in SRC-1a to SRC-1e ratio demonstrated in vivo might be involved in cell-specific responses to corticosteroids in a promoter- and ligand-dependent way.

The mouse homeobox gene, S8, is expressed during embryogenesis predominantly in mesenchyme.

The murine S8 gene, originally identified by Kongsuwan et al. [EMBO J. 7(1988)2131-2138] encodes a homeodomain which resembles those of the paired family. We studied the expression pattern during mid-gestation embryogenesis of S8 by in situ hybridization. Expression was detected locally in craniofacial mesenchyme, in the limb, the heart and the somites and sclerotomes all along the axis, and was absent from the central and peripheral nervous system, splanchnopleure, and endodermal derivatives. This pattern differs considerably from that of most previously described homeobox containing genes. By genetic analysis, the gene was located on chromosome 2, about 20 cM from the HOX-4 cluster.

Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the alpha-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the gamma-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.

Synthetic progestins induce proliferation of breast tumor cell lines via the progesterone or estrogen receptor.

Proliferation of the human breast tumor cell lines T47D and MCF7 was stimulated by high concentrations (10(-6) M) of the synthetic progestins gestodene and 3-ketodesogestrel, but not by Org2058, comparable to the stimulation by low dosages of estradiol (10(-10) M). At physiological concentrations of the progestins (10(-10) M) only T47D cells responded. Using specific antihormones it was shown that the effect at pharmacological dosages is mediated by a crossreaction of these compounds with the estrogen receptor (ER), while the stimulation of T47D cells at physiological concentrations seems progesterone receptor (PR) mediated. This was further substantiated using transient transfection assays with ER- and PR-inducible reporter constructs and mRNA induction of the ER- and PR-target genes pS2 and fatty acid synthetase, respectively. Using a whole cell ligand binding assay, 20-fold higher amounts of PR were measured in T47D compared to MCF7 cells. This was in line with a much higher PR-dependent transactivation in T47D cells and suggests that the level of transcriptionally active PR is a major determinant for the response to physiological concentrations of progestins in human breast cancer cells.

Effects of progestins on the proliferation of estrogen-dependent human breast cancer cells under growth factor-defined conditions.

The effect was studied of four different synthetic progestins (Org 30659, gestodene, 3-ketodesogestrel and levonorgestrel) on the proliferation of the 17 beta estradiol (E2)-dependent human breast cancer cell line MCF7. All progestins were found to stimulate proliferation, but only at high pharmacological dosages. Moreover, like estrogens the progestins at high concentrations synergistically stimulated MCF7 cell proliferation together with low concentrations of insulin. This stimulatory effect could be blocked by antiestrogens, but not by antiglucocorticoids and antiprogestins. This suggests that growth stimulation by these progestins (or their metabolites) occurs through crossreaction with the E2 receptor (ER). This is confirmed by the observation that the strong synthetic progestin Org 2058 does not stimulate proliferation. The absence of a progesterone receptor (PR)-mediated growth response seems not to be due to aberrant PR expression in these cells; 27,000 receptors (Kd 1.7 x 10(-10)M) per cell were present under growth-assay conditions. Growth stimulation by E2 in the absence or presence of insulin, is slightly inhibited or unaffected by the progestins, respectively. Our data do not support a role for the recently identified gestodene binding sites [Colletta et al., J. Steroid Biochem. 33 (1989) 1055-1061] in mediating gestodene effects on breast cancer cells: gestodene and 3-ketodesogestrel, a compound that does not bind to these gestodene binding sites, showed a similar biological activity. The effects of the progestins on the MCF7 breast cancer cell line, indicate that the use of these compounds at very high concentrations may be unfavourable, but do not support a role for them in directly stimulating breast tumor proliferation at the low progestin concentration which are reached in the serum in oral contraceptive users.

Conditioned media from (pre)adipocytes stimulate fibrinogen and PAI-1 production by HepG2 hepatoma cells.

BACKGROUND: Obesity is associated with a prothrombotic state, which may contribute to the increased risk of thrombotic events. OBJECTIVE: To assess the effects of (pre)adipocyte-derived adipokines on fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) production by hepatocytes. METHODS: HepG2 hepatocytes were incubated with conditioned media (CM) derived from preadipocytes and adipocytes, which had been untreated or prestimulated with tumor necrosis factor (TNF)-α, interleukin (IL)-1ß or IL-6. After 24 h, supernatants and cell lysates were harvested for measurement of fibrinogen, PAI-1 and TF. RESULTS: (Pre)adipocyte CM significantly enhanced the production of PAI-1 by HepG2 cells 2.5- to 4.4-fold. CM from cytokine-stimulated (pre)adipocytes significantly induced fibrinogen secretion 1.5- to 4.2-fold. TF production was not affected by the CM. After specific depletion of TNF-α, IL-1ß or IL-6 from the CM, IL-6 was shown to be the most prominent stimulus of fibrinogen secretion and IL-1ß of PAI-1 secretion. In addition, fibrinogen, PAI-1 and tissue factor production was evaluated by direct stimulation of HepG2 cells with TNF-α, IL-1ß or IL-6. IL-6 enhanced fibrinogen synthesis 4.3-fold (P<0.01), whereas IL-1ß induced PAI-1 production 5.0-fold (P<0.01). Gene expression analyses showed that TNF-α and IL-1ß stimulate the adipocyte expression of TNF-α, IL-1ß and IL-6. Cytokine stimulation of adipocytes may thus have induced an inflammatory response, which may have stimulated fibrinogen and PAI-1 production by HepG2 cells more potently. CONCLUSIONS: SGBS (pre)adipocytes release cytokines that increase the production of fibrinogen and PAI-1 by HepG2 cells. IL-6 and IL-1ß produced by (pre)adipocytes were the strongest inducers of fibrinogen and PAI-1 secretion, respectively.

Altered plasma adipokine levels and in vitro adipocyte differentiation in pediatric type 1 diabetes.

CONTEXT: Type 1 diabetes (T1D) is considered a proinflammatory condition. Adipose tissue involvement seems evident because adiponectin levels correlate with disease remission and administration of leptin suppresses the low-grade systemic inflammation in mice with T1D. Whether adipose tissue involvement in T1D already occurs at a young age is yet unknown. OBJECTIVE: The aim was to explore the extent of adipokine alterations in pediatric T1D and gain more insight into the mechanisms underlying the involvement of adipose tissue. DESIGN AND PARTICIPANTS: First, plasma adipokine profiling (24 adipokines) of 20 children with onset T1D, 20 children with long-standing T1D, and 17 healthy controls was performed using a recently developed and validated multiplex immunoassay. Second, the effects of diabetic plasma factors on preadipocyte proliferation and differentiation were studied in vitro. RESULTS: In children with onset and long-standing T1D, plasma adipokine profiling showed increased levels of various adipokines acting at the crossroads of adipose tissue function and inflammation, including CCL2/monocyte chemoattractant protein-1 and the novel adipokines cathepsin S, chemerin, and tissue inhibitor of metalloproteinase-1 (P < 0.05). Furthermore, onset and long-standing diabetic plasma significantly induced preadipocyte proliferation and adipocyte differentiation in vitro (P < 0.05). Two candidate plasma factors, glucose and the saturated fatty acid palmitic acid, did not affect proliferation or adipocyte differentiation in vitro but were found to increase CCL2 (monocyte chemoattractant protein-1) secretion by adipocytes. CONCLUSIONS: The adipogenic effects of diabetic plasma in vitro and the altered adipokine levels in vivo suggest adipose tissue involvement in the low-grade inflammation associated with T1D, already in pediatric patients.

Adipose tissue dysfunction and hypertriglyceridemia: mechanisms and management.

Elevated plasma triglyceride levels, as often seen in obese subjects, are independently associated with an increased risk of cardiovascular diseases. By secreting adipokines (such as adiponectin and leptin) and other proteins (such as lipoprotein lipase and cholesteryl ester transferase protein), adipose tissue affects triglyceride metabolism. In obesity, adipocyte hypertrophy leads to many changes in adipocyte function and production of anti- and pro-inflammatory cytokines. Furthermore, free fatty acids are released into the circulation contributing to insulin resistance. Adipose tissue dysfunction will eventually lead to abnormalities in lipid metabolism, such as hypertriglyceridemia (due to increased hepatic very-low-density lipoprotein production and decreased triglyceride hydrolysis), small dense low-density lipoprotein particles, remnant lipoproteins and low high-density lipoprotein cholesterol levels, all associated with a higher risk for the development of cardiovascular diseases. The clinical implications of elevated plasma triglycerides are still a matter of debate. Understanding the pathophysiology of adipose tissue dysfunction in obesity, which is becoming a pandemic condition, is essential for designing appropriate therapeutic interventions. Lifestyle changes are important to improve adipose tissue function in obese patients. Pharmacological interventions to improve adipose tissue function need further evaluation. Although statins are not very potent in reducing plasma triglycerides, they remain the mainstay of therapy for cardiovascular risk reduction in high-risk patients.

Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription.