Inflammatory Markers and Incident Heart Failure Risk in Older Adults: The Health, Aging, and Body Composition Study

Background: Inflammation is associated with heart failure (HF) risk factors and also directly affects myocardial function. However, the association between inflammation and HF risk in older adults has not been adequately evaluated. Methods: The association of baseline serum concentrations of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF- ), and C-reactive protein (CRP) with incident HF was assessed with Cox proportional hazards models among 2610 older persons without prevalent HF enrolled in the Health, Aging, and Body Composition (Health ABC) Study (age, 73.6±2.9 years; 48.3% men; 59.6% white). Results: Median (interquartile range) baseline concentrations of IL-6, TNF- , and CRP were 1.80 (1.23, 2.76) pg/mL, 3.14 (2.41, 4.06) pg/mL, and 1.64 (0.99, 3.04) µg/mL, respectively. On follow-up (median, 9.4 years), 311 participants (11.9%) developed HF. In models controlling for clinical predictors of HF and incident coronary heart disease, doubling of IL-6, TNF- , and CRP concentrations was associated with 34% (95% CI, 18 -52%; P<.001), 33% (95% CI, 9 - 63%; P=.006), and 13% (95% CI, 3-24%; P=.01) increase in HF risk, respectively. In models including all 3 markers, IL-6 and TNF- , but not CRP, remained significant. Findings were similar across sex and race. Post-HF ejection fraction (EF) was available in 239 (76.8%) cases. When only cases with preserved EF were considered (n=105), IL-6 (HR per doubling, 1.57; 95% CI, 1.28 -1.94; P<.001), TNF- (HR per doubling, 1.59; 95% CI, 1.12-2.26; P=.01), and CRP (HR per doubling, 1.23; 95% CI, 1.05-1.44; P=.01) were all associated with HF risk in adjusted models. In contrast, when only cases with reduced EF (n=134) were considered, only IL-6 attained marginal significance in adjusted models (HR per doubling, 1.20; 95% CI, 0.99 -1.46; P=.06). Participants with 2 or 3 markers above median had pronounced HF risk in adjusted models (HR, 1.66; 95% CI, 1.12-2.46; P=.01; and HR, 1.76; 95% CI, 1.16 -2.65; P=.007, respectively). Addition of IL-6 to the clinical Health ABC HF model improved discrimination (C index from 0.717 to 0.734; P=.001) and fit (decreased Bayes information criterion by 17.8; P<.001). Conclusions: Inflammatory markers are associated with HF risk among older adults and may improve HF risk stratification.

Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release.

Microglial cells react early to a neurotoxic insult. However, the bioactive factors and the cell-cell interactions leading to microglial activation and finally to a neuroprotective or neurodegenerative outcome remain to be elucidated. Therefore, we analyzed the microglial reaction induced by methylmercury (MeHgCl) using cell cultures of different complexity. Isolated microglia were found to be directly activated by MeHgCl (10(-10) to 10(-6) M), as indicated by process retraction, enhanced lectin staining, and cluster formation. An association of MeHgCl-induced microglial clusters with astrocytes and neurons was observed in three-dimensional cultures. Close proximity was found between the clusters of lectin-stained microglia and astrocytes immunostained for glial fibrillary acidic protein (GFAP), which may facilitate interactions between astrocytes and reactive microglia. In contrast, immunoreactivity for microtubule-associated protein (MAP-2), a neuronal marker, was absent in the vicinity of the microglial clusters. Interactions between astrocytes and microglia were studied in cocultures treated for 10 days with MeHgCl. Interleukin-6 release was increased at 10(-7) M of MeHgCl, whereas it was decreased when each of these two cell types was cultured separately. Moreover, addition of IL-6 to three-dimensional brain cell cultures treated with 3 x 10(-7) M of MeHgCl prevented the decrease in immunostaining of the neuronal markers MAP-2 and neurofilament-M. IL-6 administered to three-dimensional cultures in the absence of MeHgCl caused astrogliosis, as indicated by increased GFAP immunoreactivity. Altogether, these results show that microglial cells are directly activated by MeHgCl and that the interaction between activated microglia and astrocytes can increase local IL-6 release, which may cause astrocyte reactivity and neuroprotection.

The forgotten face of regular physical exercise: a 'natural' anti-atherogenic activity.

Humans are not programmed to be inactive. The combination of both accelerated sedentary lifestyle and constant food availability disturbs ancient metabolic processes leading to excessive storage of energy in tissue, dyslipidaemia and insulin resistance. As a consequence, the prevalence of Type 2 diabetes, obesity and the metabolic syndrome has increased significantly over the last 30 years. A low level of physical activity and decreased daily energy expenditure contribute to the increased risk of cardiovascular morbidity and mortality following atherosclerotic vascular damage. Physical inactivity leads to the accumulation of visceral fat and consequently the activation of the oxidative stress/inflammation cascade, which promotes the development of atherosclerosis. Considering physical activity as a 'natural' programmed state, it is assumed that it possesses atheroprotective properties. Exercise prevents plaque development and induces the regression of coronary stenosis. Furthermore, experimental studies have revealed that exercise prevents the conversion of plaques into a vulnerable phenotype, thus preventing the appearance of fatal lesions. Exercise promotes atheroprotection possibly by reducing or preventing oxidative stress and inflammation through at least two distinct pathways. Exercise, through laminar shear stress activation, down-regulates endothelial AT1R (angiotensin II type 1 receptor) expression, leading to decreases in NADPH oxidase activity and superoxide anion production, which in turn decreases ROS (reactive oxygen species) generation, and preserves endothelial NO bioavailability and its protective anti-atherogenic effects. Contracting skeletal muscle now emerges as a new organ that releases anti-inflammatory cytokines, such as IL-6 (interleukin-6). IL-6 inhibits TNF-α (tumour necrosis factor-α) production in adipose tissue and macrophages. The down-regulation of TNF-α induced by skeletal-muscle-derived IL-6 may also participate in mediating the atheroprotective effect of physical activity.

Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.

Fasting is associated with significant changes in nutrient metabolism, many of which are governed by transcription factors that regulate the expression of rate-limiting enzymes. One factor that plays an important role in the metabolic response to fasting is the peroxisome proliferator-activated receptor alpha (PPARalpha). To gain more insight into the role of PPARalpha during fasting, and into the regulation of metabolism during fasting in general, a search for unknown PPARalpha target genes was performed. Using subtractive hybridization (SABRE) comparing liver mRNA from wild-type and PPARalpha null mice, we isolated a novel PPARalpha target gene, encoding the secreted protein FIAF (for fasting induced adipose factor), that belongs to the family of fibrinogen/angiopoietin-like proteins. FIAF is predominantly expressed in adipose tissue and is strongly up-regulated by fasting in white adipose tissue and liver. Moreover, FIAF mRNA is decreased in white adipose tissue of PPARgamma +/- mice. FIAF protein can be detected in various tissues and in blood plasma, suggesting that FIAF has an endocrine function. Its plasma abundance is increased by fasting and decreased by chronic high fat feeding. The data suggest that FIAF represents a novel endocrine signal involved in the regulation of metabolism, especially under fasting conditions.

Role of estrogen receptor signaling in mammary gland development

Abstract : Breast cancer incidence rates have increased over the past hundred years, in particular, in Western industrial countries and they continue to rise worldwide. Breast cancer risk has been linked to life exposure to endogenous and exogenous estrogens, and there is increasing concern that exposure to endocrine disruptors which are increasingly accumulating in our environment may also have a role. Using the mouse as model, I have analyzed the physiological role of estrogen signaling in mammary gland development. I have shown that estrogen signaling through the estrogen receptor alpha (ERα) in the mammary epithelium is required for ductal morphogenesis during puberty. Moreover, I have demonstrated that estrogens induce proliferation of mammary epithelial cells through a paracrine mechanism. The presence of estrogen signaling is essential cell intrinsically via ERα or ER^6; for the terminal differentiation into milk secreting cells during pregnancy. Furthermore, I have examined how perinatal exposure to the estrogenic plasticizer bisphenol A (BPA) found ubiquitously in consumer goods such as baby bottles formula and beverage containers affects the normal mammary gland development and possibly predispose the mammary gland to tumorigenesis. I have found that C57b16 mice that were exposed, via their drinking water, to several BPA doses ranging from 0.025µg/kg/day to 250µg/kg/day exhibits delayed terminal end bud formation and consequently the ductal outgrowth. Later in life, the mice that were exposed in utero to BPA displayed an increased number of mammary epithelial cells. Acute exposure of 3-week-old mice to BPA can alter gene expression levels of an important estrogen target gene, amphiregulin. Taken together these data are compatible with a scenario in which perinatal BPA exposure may alter mammary gland development by affecting developmental signaling pathways. Résumé : Les taux d'incidence des cancers du sein ont augmenté au cours des cent dernières années en particulier dans les pays industriels occidentaux et ils continuent d'augmenter dans le monde entier. Le risque du cancer du sein a été corrélé à l'exposition au cours de la vie aux oestrogènes endogènes et exogènes. Il y a une préoccupation croissante concernant l'exposition aux perturbateurs endocriniens qui ne cessent de s'accumulent dans notre environnement et qui peuvent également avoir un rôle dans l'augmentation des cancers du sein. En utilisant le modèle de souris, j'ai analysé le rôle physiologique de la voie de signalisation à l'oestrogène dans le développement mammaire. J'ai prouvé que l'oestrogène par l'intermédiaire de son récepteur alpha (ERα) est indispensable dans l'épithélium pour la morphogénèse du système canalaire pendant la puberté. De plus, j'ai démontré que les oestrogènes induisent la prolifération des cellules épithéliales mammaires par un mécanisme paracrine. La présence de la voie de signalisation à l'oestrogène est essentielle de manière intrinsèque à la cellule par l'intermédiaire d'ERα ou ER^6; pour la différentiation terminale des cellules épithéliales en cellules sécrétrices de lait pendant la grossesse. En outre, j'ai examiné comment l'exposition périnatale au bisphénol A (BPA), un plastifiant présentant des propriétés ostrogéniques et omniprésent dans divers produits d'usage courant tels que les biberons des bébés et les récipients en plastique, affecte le développement de la glande mammaire et prédispose probablement celle-ci à la tumorigénèse. J'ai constaté que l'exposition périnatale à BPA retarde la formation des bourgeons terminaux et par conséquent la croissance du système canalaire. Plus tard dans la vie, les souris qui ont été exposées dans l'utérus au BPA ont montré un plus grand nombre de cellules épithéliales mammaires. L'exposition aiguë de souris âgées de 3 semaines au BPA perturbe le niveau d'expression d'un gène cible important de l'oestrogène, l'amphiregulin. Ces données sont compatibles avec un scénario dans lequel l'exposition périnatale au BPA peut changer le développement de la glande mammaire en affectant des voies de signalisation développementales.

Characterization of an interaction between fetal hemoglobin and lipid A of LPS resulting in augmented induction of cytokine production in vivo and in vitro.

A previously described extract of sheep fetal liver was reported to reverse many of the cytokine changes associated with aging in mice, including an augmented spleen cell ConA-stimulated production of IL-4 and decreased production of IL-2. Similar effects were not seen with adult liver preparations. These changes were observed in various strains of mice, including BALB/c, DBA/2 and C57BL/6, using mice with ages ranging from 8 to 110 weeks. Preliminary characterization of this crude extract showed evidence for the presence of Hb gamma chain, as well as of lipid A of LPS. We show below that purified preparations of sheep fetal Hb, but not adult Hb, in concert with suboptimally stimulating doses of LPS (lipid A), cooperate in the regulation of production of a number of cytokines, including TNFalpha and IL-6, in vitro. Furthermore, isolated fresh spleen or peritoneal cells from animals treated in vivo with the same combination of Hb and LPS, showed an augmented capacity to produce these cytokines on further culture in vitro. Evidence was also obtained for a further interaction between CLP, LPS and fetal Hb itself in this augmented cytokine production. These data suggest that some of the functional activities in the fetal liver extract reported earlier can be explained in terms of a novel immunomodulatory role of a mixture of LPS (lipid A) and fetal Hb.

Markers of atherosclerosis and inflammation for prediction of coronary heart disease in older adults.

Although both inflammatory and atherosclerosis markers have been associated with coronary heart disease (CHD) risk, data directly comparing their predictive value are limited. The authors compared the value of 2 atherosclerosis markers (ankle-arm index (AAI) and aortic pulse wave velocity (aPWV)) and 3 inflammatory markers (C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha)) in predicting CHD events. Among 2,191 adults aged 70-79 years at baseline (1997-1998) from the Health, Aging, and Body Composition Study cohort, the authors examined adjudicated incident myocardial infarction or CHD death ("hard" events) and "hard" events plus hospitalization for angina or coronary revascularization (total CHD events). During 8 years of follow-up between 1997-1998 and June 2007, 351 participants developed total CHD events (197 "hard" events). IL-6 (highest quartile vs. lowest: hazard ratio = 1.82, 95% confidence interval: 1.33, 2.49; P-trend < 0.001) and AAI (AAI </= 0.9 vs. AAI 1.01-1.30: hazard ratio = 1.57, 95% confidence interval: 1.14, 2.18) predicted CHD events above traditional risk factors and modestly improved global measures of predictive accuracy. CRP, TNF-alpha, and aPWV had weaker associations. IL-6 and AAI accurately reclassified 6.6% and 3.3% of participants, respectively (P's </= 0.05). Results were similar for "hard" CHD, with higher reclassification rates for AAI. IL-6 and AAI are associated with future CHD events beyond traditional risk factors and modestly improve risk prediction in older adults.

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin-1beta: role of neuropeptide Y and nitric oxide.

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1beta (IL-1beta) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1beta on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1beta and NPY were also investigated. We observed that IL-1beta increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1beta. Moreover, IL-1beta regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1beta-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1beta induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1beta, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.

Haemorrhagic shock and encephalopathy syndrome: report of two cases with special reference to hypoglycaemia.

Haemorrhagic shock and encephalopathy syndrome (HSES) is a devastating disorder affecting infants. So far no cases have been reported in Switzerland. It is characterised by the abrupt onset of hyperpyrexia, shock, encephalopathy, diarrhoea, disseminated intravascular coagulation (DIC) and renal and hepatic failure in previously healthy infants. Severe hypoglycaemia has been repeatedly reported in association with HSES. However, the pathophysiology of the hypoglycaemia is not clear. We report on two infants (2 and 7 months old) with typical HSES, both of whom were presented with nonketotic hypoglycaemia. In the first case, plasma insulin was 23 pmol/l at the time of hypoglycaemia (0.1 mmol/l). In the second case, increased values for interleukin-6 (IL-6) (319 pg/ml) and IL-8 (1382 pg/ml) were found 24 hours after admission, whereas IL-1 and tumour necrosis factor-alpha (TNF-alpha) were not measurable. Alpha-1-antitrypsin was decreased (0.6 g/l). In hyperpyrexic, unconscious and shocked infants, HSES should be considered and hypoglycaemia should be specifically looked for. Hypoglycaemia is not caused by hyperinsulinism but may be secondary to the release of cytokines.

Elevated serum uric acid is associated with high circulating inflammatory cytokines in the population-based Colaus study.

BACKGROUND: The relation of serum uric acid (SUA) with systemic inflammation has been little explored in humans and results have been inconsistent. We analyzed the association between SUA and circulating levels of interleukin-6 (IL-6), interleukin-1beta (IL-1beta), tumor necrosis factor- alpha (TNF-alpha) and C-reactive protein (CRP). METHODS AND FINDINGS: This cross-sectional population-based study conducted in Lausanne, Switzerland, included 6085 participants aged 35 to 75 years. SUA was measured using uricase-PAP method. Plasma TNF-alpha, IL-1beta and IL-6 were measured by a multiplexed particle-based flow cytometric assay and hs-CRP by an immunometric assay. The median levels of SUA, IL-6, TNF-alpha, CRP and IL-1beta were 355 micromol/L, 1.46 pg/mL, 3.04 pg/mL, 1.2 mg/L and 0.34 pg/mL in men and 262 micromol/L, 1.21 pg/mL, 2.74 pg/mL, 1.3 mg/L and 0.45 pg/mL in women, respectively. SUA correlated positively with IL-6, TNF-alpha and CRP and negatively with IL-1beta (Spearman r: 0.04, 0.07, 0.20 and 0.05 in men, and 0.09, 0.13, 0.30 and 0.07 in women, respectively, P<0.05). In multivariable analyses, SUA was associated positively with CRP (beta coefficient +/- SE = 0.35+/-0.02, P<0.001), TNF-alpha (0.08+/-0.02, P<0.001) and IL-6 (0.10+/-0.03, P<0.001), and negatively with IL-1beta (-0.07+/-0.03, P = 0.027). Upon further adjustment for body mass index, these associations were substantially attenuated. CONCLUSIONS: SUA was associated positively with IL-6, CRP and TNF-alpha and negatively with IL-1beta, particularly in women. These results suggest that uric acid contributes to systemic inflammation in humans and are in line with experimental data showing that uric acid triggers sterile inflammation.

T cell receptor beta chain gene rearrangements and V beta gene usage in horse cytochrome c-specific T cell hybridomas.

Comparison of T cell receptor alpha and beta-chain genes in murine major histocompatibility complex (MHC) class I and class II-restricted T cell clones and hybridomas recognizing different antigens indicates that no simple correlation exists between the observed antigen/MHC specificity and the expression of certain alpha and beta-chain heterodimers. We have attempted to establish a possible correlation by analyzing T cell receptor beta chain gene rearrangements and V beta gene usage in five T cell hybridomas with identical antigen/MHC specificity and another hybridoma recognizing a different antigenic determinant in association with the same restriction molecule. We report here that in each of the five clones a uniquely rearranged beta chain gene is expressed in combination with at least two different V beta gene segments. The presence of the differently rearranged T cell receptor beta chain genes correlated with the finding of distinct fine specificity pattern of antigen recognition in each of the hybridomas. Interestingly, two hybridomas specific for different epitopes showed identical beta chain D-J rearrangements indicating that the differences might be encoded by the alpha chain gene or/and the V beta gene element.

Not all inflammatory markers are linked to kidney function: results from a population-based study.

BACKGROUND: Several studies have reported increased levels of inflammatory biomarkers in chronic kidney disease (CKD), but data from the general population are sparse. In this study, we assessed levels of the inflammatory markers C-reactive protein (hsCRP), tumor necrosis factor α (TNF-α), interleukin (IL)-1^6; and IL-6 across all ranges of renal function. METHODS: We conducted a cross-sectional study in a random sample of 6,184 Caucasian subjects aged 35-75 years in Lausanne, Switzerland. Serum levels of hsCRP, TNF-α, IL-6, and IL-1^6; were measured in 6,067 participants (98.1%); serum creatinine-based estimated glomerular filtration rate (eGFR(creat), CKD-EPI formula) was used to assess renal function, and albumin/creatinine ratio on spot morning urine to assess microalbuminuria (MAU). RESULTS: Higher serum levels of IL-6, TNF-α and hsCRP and lower levels of IL-1^6; were associated with a lower renal function, CKD (eGFR(creat) <60 ml/min/1.73 m(2); n = 283), and MAU (n = 583). In multivariate linear regression analysis adjusted for age, sex, hypertension, smoking, diabetes, body mass index, lipids, antihypertensive and hypolipemic therapy, only log-transformed TNF-α remained independently associated with lower renal function (^6; -0.54 ±0.19). In multivariate logistic regression analysis, higher TNF-α levels were associated with CKD (OR 1.17; 95% CI 1.01-1.35), whereas higher levels of IL-6 (OR 1.09; 95% CI 1.02-1.16) and hsCRP (OR 1.21; 95% CI 1.10-1.32) were associated with MAU. CONCLUSION: We did not confirm a significant association between renal function and IL-6, IL-1^6; and hsCRP in the general population. However, our results demonstrate a significant association between TNF-α and renal function, suggesting a potential link between inflammation and the development of CKD. These data also confirm the association between MAU and inflammation.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.