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Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn’s disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teffcells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teffcells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1−/−or Rag2−/−mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn’s disease and defines lymphocyte sub-specialization in the small intestine.

ABSTRACTAlthough macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3deletion in pink1knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7KO mice showed significantly lower expression of Nlrp3in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases.Abbreviations:ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.

As a key regulator of metabolism and inflammation, the orphan nuclear hormone receptor, liver receptor homolog-1 (LRH-1), has potential as a therapeutic target for diabetes, nonalcoholic fatty liver disease, and inflammatory bowel diseases (IBD). Discovery of LRH-1 modulators has been difficult, in part due to the tendency for synthetic compounds to bind unpredictably within the lipophilic binding pocket. Using a structure-guided approach, we exploited a newly discovered polar interaction to lock agonists in a consistent orientation. This enabled the discovery of the first low nanomolar LRH-1 agonist, one hundred times more potent than the best previous modulator. We elucidate a novel mechanism of action that relies upon specific polar interactions deep in the LRH-1 binding pocket. In an organoid model of IBD, the new agonist increases expression of LRH-1-controlled steroidogenic genes and promotes anti-inflammatory gene expression changes. These studies constitute major progress in developing LRH-1 modulators with potential clinical utility.

Pathogenic sequence variants in the nuclear bile acid receptor FXR, encoded by NR1H4, have been reported in a small number of children with low-?-glutamyl transferase (GGT) cholestasis progressing to liver failure. We describe 3 additional children from 2 unrelated families with cholestasis and liver failure because of pathologic variants in NR1H4. One patient underwent liver transplantation and has had good clinical outcomes in 6 years of follow-up. Although that patient has biochemical evidence of increased bile acid synthetic activity, he has not experienced post-transplant diarrhea or allograft steatosis, as has been reported among other transplanted patients.

Progressive familial intrahepatic cholestasis (PFIC) is a genetically heterogeneous disorder of bile flow disruption due to abnormal canalicular transport or impaired bile acid (BA) metabolism, causing excess BA accumulation and liver failure. We previously reported an intrahepatic cholestasis mouse model based on loss of function of both farnesoid X receptor (FXR; NR1H4) and a small heterodimer partner (SHP; NR0B2) [double knockout (DKO)], which has strong similarities to human PFIC5. We compared the pathogenesis of DKO livers with that of another intrahepatic cholestasis model, Bsep-/-, which represents human PFIC2. Both models exhibit severe hepatomegaly and hepatic BA accumulation, but DKO showed greater circulating BA and liver injury, and Bsep-/- had milder phenotypes. Molecular profiling of BAs uncovered specific enrichment of cholic acid (CA)-derived BAs in DKO livers but chenodeoxycholate-derived BAs in Bsep-/- livers. Transcriptomic and proteomic analysis revealed specific activation of CA synthesis and alternative basolateral BA transport in DKO but increased chenodeoxycholic acid synthesis and canalicular transport in Bsep-/-. The constitutive androstane receptor (CAR)/pregnane X receptor (PXR)-CYP2B/CYP2C axis is activated in DKO livers but not in other cholestasis models. Loss of this axis in Fxr:Shp:Car:Pxr quadruple knockouts blocked Cyp2b/Cyp2c gene induction, impaired bilirubin conjugation/elimination, and increased liver injury. Differential CYP2B expression in DKO and Bsep-/- was recapitulated in human PFIC5 and PFIC2 livers. In conclusion, loss of FXR/SHP results in distinct molecular pathogenesis and CAR/PXR activation, which promotes Cyp2b/Cyp2c gene transcription and bilirubin clearance. CAR/PXR activation was not observed in Bsep-/- mice or PFIC2 patients. These findings provide a deeper understanding of the heterogeneity of intrahepatic cholestasis.

Asprosin is a recently discovered fasting-induced hormone that promotes hepatic glucose production. Here we demonstrate that asprosin in the circulation crosses the blood–brain barrier and directly activates orexigenic AgRP+neurons via a cAMP-dependent pathway. This signaling results in inhibition of downstream anorexigenic proopiomelanocortin (POMC)-positive neurons in a GABA-dependent manner, which then leads to appetite stimulation and a drive to accumulate adiposity and body weight. In humans, a genetic deficiency in asprosin causes a syndrome characterized by low appetite and extreme leanness; this is phenocopied by mice carrying similar mutations and can be fully rescued by asprosin. Furthermore, we found that obese humans and mice had pathologically elevated concentrations of circulating asprosin, and neutralization of asprosin in the blood with a monoclonal antibody reduced appetite and body weight in obese mice, in addition to improving their glycemic profile. Thus, in addition to performing a glucogenic function, asprosin is a centrally acting orexigenic hormone that is a potential therapeutic target in the treatment of both obesity and diabetes.

Circadian rhythm regulates multiple metabolic processes and in turn is readily entrained by feeding-fasting cycles. However, the molecular mechanisms by which the peripheral clock senses nutrition availability remain largely unknown. Bile acids are under circadian control and also increase postprandially, serving as regulators of the fed state in the liver. Here, we show that nuclear receptor Small Heterodimer Partner (SHP), a regulator of bile acid metabolism, impacts the endogenous peripheral clock by directly regulating Bmal1. Bmal1-dependent gene expression is altered in Shpknockout mice, and liver clock adaptation is delayed in Shpknockout mice upon restricted feeding. These results identify SHP as a potential mediator connecting nutrient signaling with the circadian clock.

Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GRLysM) or HSC (GRhGFAP): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GRLysMmice, whereas the suppression of fibrotic gene expression was diminished in GRhGFAPmice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis.

Insulin resistance and type 2 diabetes mellitus (T2DM) result from an inability to efficiently store and catabolize surplus energy in adipose tissue. Subcutaneous adipocytes protect against insulin resistance and T2DM by coupling differentiation with the induction of brown fat gene programs for efficient energy metabolism. Mechanisms that disrupt these programs in adipocytes are currently poorly defined, but represent therapeutic targets for the treatment of T2DM. To gain insight into these mechanisms, we performed a high-throughput microscopy screen that identified ubiquitin carrier protein 9 (Ubc9) as a negative regulator of energy storage in human sc adipocytes. Ubc9 depletion enhanced energy storage and induced the brown fat gene program in human sc adipocytes. Induction of adipocyte differentiation resulted in decreased Ubc9 expression commensurate with increased brown fat gene expression. Thiazolidinedione treatment reduced the interaction between Ubc9 and peroxisome proliferator-activated receptor (PPAR)γ, suggesting a mechanism by which Ubc9 represses PPARγ activity. In support of this hypothesis, Ubc9 overexpression remodeled energy metabolism in human sc adipocytes by selectively inhibiting brown adipocyte-specific function. Further, Ubc9 overexpression decreased uncoupling protein 1 expression by disrupting PPARγ binding at a critical uncoupling protein 1 enhancer region. Last, Ubc9 is significantly elevated in sc adipose tissue isolated from mouse models of insulin resistance as well as diabetic and insulin-resistant humans. Taken together, our findings demonstrate a critical role for Ubc9 in the regulation of sc adipocyte energy homeostasis.

Impairment of drug disposition in the liver during inflammation has been attributed to downregulation of gene expression of drug-metabolizing enzymes (DMEs) and drug transporters. Inflammatory responses in the liver are primarily mediated by Toll-like receptors (TLRs). We have recently shown that activation of TLR2 or TLR4 by lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, leads to the downregulation of gene expression of DMEs/transporters. However, the molecular mechanism underlying this downregulation is not fully understood. The xenobiotic nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR), regulate the expression of DMEs/transporter genes. Downregulation of DMEs/transporters by LTA or LPS was associated with reduced expression of PXR and CAR genes. To determine the role of CAR, we injected CAR+/+and CAR−/−mice with LTA or LPS, which significantly downregulated (∼40%–60%) RNA levels of the DMEs, cytochrome P450 (Cyp)3a11, Cyp2a4, Cyp2b10, uridine diphosphate glucuronosyltransferase 1a1, amine N-sulfotransferase, and the transporter, multidrug resistance-associated protein 2, in CAR+/+mice. Suppression of most of these genes was attenuated in LTA-treated CAR−/−mice. In contrast, LPS-mediated downregulation of these genes was not attenuated in CAR−/−mice. Induction of these genes by mouse CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene was sustained in LTA- but not in LPS-treated mice. Similar observations were obtained in humanized CAR mice. We have replicated these results in primary hepatocytes as well. Thus, LPS can downregulate DME/transporter genes in the absence of CAR, whereas the effect of LTA on these genes is attenuated in the absence of CAR, indicating the potential involvement of CAR in LTA-mediated downregulation of DME/transporter genes.

Elevated bile acid levels increase hepatocellular carcinoma by unknown mechanisms. Here, we show that mice with a severe defect in bile acid homeostasis due to the loss of the nuclear receptors FXR and SHP have enlarged livers, progenitor cell proliferation, and Yes-associated protein (YAP) activation and develop spontaneous liver tumorigenesis. This phenotype mirrors mice with loss of hippokinases or overexpression of their downstream target, YAP. Bile acids act as upstream regulators of YAP via a pathway dependent on the induction of the scaffold protein IQGAP1. Patients with diverse biliary dysfunctions exhibit enhanced IQGAP1 and nuclear YAP expression. Our findings reveal an unexpected mechanism for bile acid regulation of liver growth and tumorigenesis via the Hippopathway.

Mixed background SHP–/–mice are resistant to diet-induced obesity due to increased energy expenditure caused by enhanced PGC-1α expression in brown adipocytes. However, congenic SHP–/–mice on the C57BL/6 background showed normal expression of PGC-1α and other genes involved in brown adipose tissue thermogenesis. Thus, we reinvestigated the impact of small heterodimer partner (SHP) deletion on diet-induced obesity and insulin resistance using congenic SHP–/–mice. Compared with their C57BL/6 wild-type counterparts, SHP–/–mice subjected to a 6 month challenge with a Western diet (WestD) were leaner but more glucose intolerant, showed hepatic insulin resistance despite decreased triglyceride accumulation and increased β-oxidation, exhibited alterations in peripheral tissue uptake of dietary lipids, maintained a higher respiratory quotient, which did not decrease even after WestD feeding, and displayed islet dysfunction. Hepatic mRNA expression analysis revealed that many genes expressed higher in SHP–/–mice fed WestD were direct peroxisome proliferator-activated receptor alpha (PPARα) targets. Indeed, transient transfection and chromatin immunoprecipitation verified that SHP strongly repressed PPARα-mediated transactivation. SHP is a pivotal metabolic sensor controlling lipid homeostasis in response to an energy-laden diet through regulating PPARα-mediated transactivation. The resultant hepatic fatty acid oxidation enhancement and dietary fat redistribution protect the mice from diet-induced obesity and hepatic steatosis but accelerate development of type 2 diabetes.

Mixed background SHP−/−mice are resistant to diet-induced obesity due to increased energy expenditure caused by enhanced PGC-1α expression in brown adipocytes. However, congenic SHP−/−mice on the C57BL/6 background showed normal expression of PGC-1αand other genes involved in brown adipose tissue thermogenesis. Thus, we reinvestigated the impact of small heterodimer partner (SHP) deletion on diet-induced obesity and insulin resistance using congenic SHP−/−mice. Compared with their C57BL/6 wild-type counterparts, SHP−/−mice subjected to a 6 month challenge with a Western diet (WestD) were leaner but more glucose intolerant, showed hepatic insulin resistance despite decreased triglyceride accumulation and increased β-oxidation, exhibited alterations in peripheral tissue uptake of dietary lipids, maintained a higher respiratory quotient, which did not decrease even after WestD feeding, and displayed islet dysfunction. Hepatic mRNA expression analysis revealed that many genes expressed higher in SHP−/−mice fed WestD were direct peroxisome proliferator-activated receptor alpha (PPARα) targets. Indeed, transient transfection and chromatin immunoprecipitation verified that SHP strongly repressed PPARα-mediated transactivation. SHP is a pivotal metabolic sensor controlling lipid homeostasis in response to an energy-laden diet through regulating PPARα-mediated transactivation. The resultant hepatic fatty acid oxidation enhancement and dietary fat redistribution protect the mice from diet-induced obesity and hepatic steatosis but accelerate development of type 2 diabetes.

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/-mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/-mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/-islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/-mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/-islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/-islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.

Balancing glucose homeostasis is crucial to maintain appropriate energy and metabolic state. Chronic hyperglycemia with insulin resistance and development of type II diabetes mellitus is a growing health and health-economic threat. The unfolded protein response (UPR) is a mechanism by which the endoplasmic reticulum copes with diverse physiological and pathophysiological stress stimuli. Unresolved and chronic endoplasmic reticulum stress are important features in the development of diabetes mellitus. Understanding how the UPR impacts glucose balance and what disrupts this balance is critical for development of future therapies.

The stability of attenuated virus vaccines has traditionally been assessed by a plaque assay to measure the virus’s loss of replication competency in response to a variety of environmental perturbations. Although this method provides information regarding the impact of the vaccine formulation, it involves an empirical approach to evaluate stability. Biophysical studies on the other hand have the potential to provide insight into the mechanisms of inactivation of a viral vaccine in response to a variety of stressed conditions. Herein, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy, and dynamic light scattering) for a comprehensive examination of the thermal stability of three live-attenuated human-bovine reassortant rotavirus strains (G1, G3, and G4) in the 5-8 pH range. The spectroscopic methods employed are not specific and response changes reflect an average change over the entire virus structure. The present work, however, suggests the utility of these methods in early formulation of rotaviral vaccines due to their ability to identify regions of marginal stability over which high throughput excipient screening assays can be designed. We have further shown that these methods are sufficiently sensitive to differentiate the stability of the three homologous G-subtypes differing only in the composition of their surface antigenic proteins. The data from these spectroscopic methods are also compared to biological activity using a tissue culture viral infectivity assay. Partial correlation between the structural alterations and losses in activity are observed, further suggesting the utility of biophysical studies in early formulation studies of rotavirus vaccines.

Adenovirus serotype 4 (Ad4) is a major cause of Ad-associated human diseases. Ad4 is also considered to be a potential delivery vector for gene therapy. In this study, multiple spectroscopic techniques together with transmission electron microscopy (TEM) were employed to probe viral stability and to improve pharmaceutical formulations of Ad4-based vaccines and DNA carriers. Perturbations of secondary, tertiary and quaternary structure of Ad4 proteins induced by elevated temperatures over a wide pH range (3-8) were analyzed using circular dichroism, UV absorption and intrinsic and extrinsic fluorescence spectroscopy as well as static and dynamic light scattering. The spectroscopic results obtained indicate a decrease in Ad4 stability as pH increases from 4 to 8, similar to the behavior reported previously for Ad2 and Ad5, although the Ad4 virion appears to possess slightly more tolerance to thermal stress. An empirical phase diagram (EPD) approach was used to summarize the data in the form of a colored map. In addition, the different physical states of Ad4 identified by the EPD were confirmed by TEM images. The results obtained in this study reveal both structural similarities among three commonly employed Ad subtypes (2, 4 and 5) as well as unique properties of Ad4.

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR–/–) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR–/–mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR–/–mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.

Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2–related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2-/-mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR-/-mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively.

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR−/−) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR−/−mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR−/−mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.

Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) by xenobiotic inducers of cytochromes P450 is part of a pleiotropic response that includes liver hypertrophy, tumor promotion, effects on lipid homeostasis, and energy metabolism. Here, we describe an acute response to CAR and PXR activators that is associated with induction of Insig-1, a protein with antilipogenic properties. We first observed that activation of CAR and PXR in mouse liver results in activation of Insig-1 along with reduced protein levels of the active form of sterol regulatory element binding protein 1 (Srebp-1). Studies in mice deficient in CAR and PXR revealed that the effect on triglycerides involves these two nuclear receptors. Finally, we identified a functional binding site for CAR and PXR in the Insig-1 gene by in vivo, in vitro, and in silico genomic analysis. Our experiments suggest that activation Insig-1 by drugs leads to reduced levels of active Srebp-1 and consequently to reduced target gene expression including the genes responsible for triglyceride synthesis. The reduction nuclear Srebp-1 by drugs is not observed when Insig-1 expression is repressed by small interfering RNA. In addition, observed that Insig-1 is also a target of AMP-activated kinase, the hepatic activity of which is increased by activators of CAR and PXR and is known to cause a reduction of triglycerides. The fact that drugs that serve as CAR or PXR ligands induce Insig-1 might have clinical consequences and explains alterations lipid levels after drug therapy.

Phytosterols, components of soy-derived lipids, are among the proposed exacerbants of parenteral nutrition–associated cholestasis (PNAC). We investigated whether phytosterols contribute to bile acid (BA)–induced hepatocyte damage by antagonizing a nuclear receptor (NR) critically involved in hepatoprotection from cholestasis, FXR (farnesoid X receptor, NR1H4). In HepG2 cells, stigmasterol acetate (StigAc), a water-soluble Stig derivative, suppressed ligand-activated expression of FXR target genes involved in adaptation to cholestasis (i.e. BSEP, FGF-19, OSTα/β). Furthermore, StigAc antagonized BA-activated, FXR target genes SHPand BSEPin FXR/, but not in FXR−/− mouse hepatocytes. Both Stig and StigAc inhibited BA-activated, FXR-dependent reporter gene expression in transfected HepG2 cells, whereas the most prevalent phytosterol in lipids, β-sitosterol, had no inhibitory effect. Finally, among six ligand-activated NR-ligand binding domains (LBDs) tested, antagonism by StigAc was specific to only two (FXR and PXR, pregnane X receptor, NR1I2). We demonstrate that Stig, a phytosterol prevalent in soy-derived PN lipid solutions, is a potent in vitroantagonist of the NR for bile acids FXR.

Phytosterols, components of soy-derived lipids, are among the proposed exacerbants of parenteral nutrition–associated cholestasis (PNAC). We investigated whether phytosterols contribute to bile acid (BA)–induced hepatocyte damage by antagonizing a nuclear receptor (NR) critically involved in hepatoprotection from cholestasis, FXR (farnesoid X receptor, NR1H4). In HepG2 cells, stigmasterol acetate (StigAc), a water-soluble Stig derivative, suppressed ligand-activated expression of FXR target genes involved in adaptation to cholestasis (i.e. BSEP, FGF-19, OSTa/ß). Furthermore, StigAc antagonized BA-activated, FXR target genes SHP and BSEP in FXR+/+, but not in FXR-/- mouse hepatocytes. Both Stig and StigAc inhibited BA-activated, FXR-dependent reporter gene expression in transfected HepG2 cells, whereas the most prevalent phytosterol in lipids, ß-sitosterol, had no inhibitory effect. Finally, among six ligand-activated NR-ligand binding domains (LBDs) tested, antagonism by StigAc was specific to only two (FXR and PXR, pregnane X receptor, NR1I2). We demonstrate that Stig, a phytosterol prevalent in soy-derived PN lipid solutions, is a potent in vitro antagonist of the NR for bile acids FXR.

The CYP3As are broad-spectrum drug-metabolizing enzymes that are collectively responsible for more than 50% of xenobiotic metabolism. Unlike other CYP3As, murine CYP3A44 is expressed predominantly in the female liver, with much lower levels in male livers and no detectable expression in brain or kidney in either gender. In this study, we examined the role of nuclear hormone receptors in the regulation of Cyp3a44 gene expression. Interestingly, we observed differential effects of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) -mediated activation of Cyp3a44 gene expression, which was gender-specific. For example, activation of PXR by pregnenolone-16α-carbonitrile (PCN) and dexamethasone (DEX) induced CYP3A44 mRNA levels in a PXR-dependent fashion in male mice, whereas no induction was detected in female mice. In contrast, PCN and DEX down-regulated CYP3A44 expression in female PXR null animals. Similar to PXR, CAR activation also showed a male-specific induction with no effect on CYP3A44 levels in females. When PXR knockout mice were challenged with the CAR activator phenobarbital, a significant up-regulation of male CYP3A44 levels was observed, whereas levels in females remained unchanged. We conclude that gender has a critical impact on PXR- and CAR-mediated effects of CYP3A44 expression.

The nuclear receptors of the NR1H and NR1I subgroups include the constitutive androstane receptor, pregnane X receptor, farnesoid X receptors, liver X receptors, and vitamin D receptor. The newly emerging functions of these related receptors are under the control of metabolic pathways, including metabolism of xenobiotics, bile acids, cholesterol, and calcium. This review summarizes results of structural, pharmacologic, and genetic studies of these receptors.

Although some of the transcription factors that are required for expression of crystallins during lens development have been identified, the molecular interactions that contribute to enhanced crystallin expression are not yet well defined. In this study, we designed experiments to test whether the co-activators CREB-binding protein (CBP) and/or p300 interact with c-Maf, Prox-1, or Sox-1 to enhance transcription of crystallin genes. Promoter regions from the mouse αA-, βB2-, and γF-crystallin genes were linked to a luciferase reporter. Expression of c-Maf transactivated each of these promoters. Of particular interest, co-expression of CBP or p300 with c-Maf was found to synergistically co-activate each promoter. CBP and p300 were less effective or ineffective at co-activation with Prox-1 or Sox-1. Co-immunoprecipitation and mammalian two-hybrid experiments revealed that CBP and p300 bind to c-Maf and Prox-1 but not to Sox-1. The co-activation of c-Maf by CBP/p300 requires histone acetyltransferase activity. Our results suggest that c-Maf recruits CBP and/or p300 to crystallin promoters leading to up-regulation of crystallin gene expression through localized histone acetylation and consequent chromatin re-modeling. In a promoter-specific fashion, co-activation can be modulated by Prox-1 and/or Sox-1. This modulation may help to specify the endogenous levels of crystallin gene expression.

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like OrphanReceptor) because it shares the highest amino acid identity with the mammalian FXR. We also identified a variant of FOR1, called FOR2, which has 15 additional C-terminal amino acids. Both variants include an unusual insertion of 33 amino acids in the helix 7 region of the canonical ligand binding domain sequence, suggesting a unique structure for FOR. Northern blot analysis demonstrates that the FORgene is highly expressed in adult and tadpole liver, kidney, and tail bud stage of the embryo. Detailed expression analysis using in situhybridization indicates that FOR expression is first detectable at stage 30/31 in the presumptive liver region lasting until stage 41 with a peak level evident at stage 35/36. FOR forms heterodimeric complexes with retinoid X receptor (RXR) as demonstrated by biochemical and mammalian two-hybrid approaches. Gel mobility shift assays demonstrate that FORs form specific DNA-protein complexes on an FXR binding element consisting of an inverted repeat DNA element with 1 nucleotide spacing (IR1) from the phospholipid transfer proteingene promoter. Finally, although FORs do not exhibit constitutive transcriptional activity, frog gallbladder extract significantly augments the transcriptional activities of FORs.

The orphan nuclear hormone receptor liver receptor homologous protein-1 (LRH-1; NR5A2, also known as FTF), an unusual receptor that binds DNA as a monomer, is an essential regulator of expression of a rate-limiting enzyme in bile acid formation, cholesterol 7-α-hydroxylase. In a classic negative feedback loop that is a crucial component of the complex regulation of cholesterol metabolism, cholesterol 7-α-hydroxylase expression is decreased when bile acid levels are high. This repression is thought to be based on the bile acid-dependent induction of expression of the orphan receptor small heterodimer partner (SHP) NR0B2, which inhibits the activity of LRH-1. We have explored the molecular basis for this important regulatory effect by characterizing the mechanisms by which mouse and human SHP inhibit LRH-1-mediated transactivation. Both SHP proteins specifically interact with the AF-2 transactivation domain of LRH-1 both in vivoand in vitro. This domain is a common target for coactivator interaction, and the SHP proteins can compete with p160 coactivators for binding to LRH-1. In addition to the N-terminal receptor interaction domain, SHP includes a C-terminal domain with autonomous repression function. Neither a deletion nor a point mutation specifically affecting this domain blocked the ability to interact with LRH-1 to compete for coactivator binding or to repress LRH-1 transactivation. However, the relative ability of these mutants to inhibit LRH-1-mediated transactivation was markedly decreased. We conclude that the proposed central role of SHP in cholesterol metabolism is based on a two-step mechanism that is dependent on both coactivator competition and direct transcriptional repression.

The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) interacts with a wide array of nuclear receptors and represses their transcriptional activity. SHP expression is regulated by several other members of the nuclear receptor superfamily, including the orphan receptors SF-1 and LRH-1, and the bile acid receptor FXR. We have found that the SHPpromoter is also activated by the estrogen receptor-related receptor γ (ERRγ) but not the related ERRα and ERRβ isoforms. SHP and ERRγ mRNAs are coexpressed in several tissues, including pancreas, kidney, and heart, confirming the potential relevance of this transactivation. ERRγ transactivation is dependent on only one of five previously characterized DNA-binding sites for SF-1, and this element differs from previously reported ERR response elements. However, treatment with the histone deacetylase inhibitor trichostatin A significantly increased ERRα and ERRβ activity on this element indicating that the lack of activity of ERRα and -β may depend on their association with co-repressor in vivo. Furthermore, using protease sensitivity assays on DNA bound receptors it was demonstrated that DNA sequence of different response elements may cause allosteric modulation of ERR proteins, which in turn may be responsible for the differential activities of these receptors on different response elements. SHP inhibits ERRγ transactivation and physically interacts with all three members of ERR subfamily, as demonstrated by both yeast two-hybrid and biochemical assays. As with other SHP targets, this interaction is dependent on the AF-2 coactivator-binding site of ERRγ and the previously described N-terminal receptor interaction domain of SHP. Several recently described SHP mutations associated with moderate obesity in humans block the inhibition of ERRγ activity. Overall, these results identify a new autoregulatory loop controlling SHPgene expression and significantly extend the potential functional roles of the three ERRs.

Small heterodimer partner (SHP, NR0B2) is an atypical orphan nuclear receptor that inhibits transcriptional activation by several other nuclear receptors. We recently reported that mutations in the SHP gene are associated with insulin resistance. In the present study, we demonstrated that the SHP gene is expressed in adipose tissues. A reporter gene assay showed that a gene product of SHP increased the transcriptional activation of peroxisome proliferator-activated receptor (PPAR) gamma. SHP-mediated activation of PPARgamma was observed both in the presence and absence of the ligand of PPARgamma. Immunoprecipitation and glutathione S-transferase pull-down assay showed that SHP directly bound to PPARgamma and competed with nuclear receptor corepressor for binding to PPARgamma. Serial deletion studies indicated that the C terminus of SHP is important for PPARgamma activation. Mutant SHP proteins, which are found in naturally occurring mutation, showed less enhancing activity for PPARgamma than wild-type SHP. Our results suggest that SHP may act as an endogenous enhancer of PPARgamma.

Bile acids facilitate the absorption of dietary lipids and fat-soluble vitamins and are physiological ligands for the farnesoid X-activated receptor (FXR), a member of the nuclear hormone receptor superfamily. FXR functions as a heterodimer with the retinoid X receptor and in the presence of ligand, the heterodimer binds to specific DNA sequences in the promoters of target genes to regulate gene transcription. Phospholipid transfer protein (PLTP) has been identified as a possible target gene for FXR because the human promoter contains a potential FXR response element, an inverted repeat in which consensus receptor-binding hexamers are separated by one nucleotide (inverted repeat-1). PLTP is essential in the transfer of very low density lipoprotein phospholipids into high density lipoprotein (Jiang, X. C., Bruce, C., Mar, J., Lin, M., Ji, Y., Francone, O. L., and Tall, A. R. (1999) J. Clin. Invest.103, 907–914). Here we report the regulation of PLTP gene expression by FXR and bile acids. In CV-1 cells, cotransfection of FXR and the retinoid X receptor resulted in bile acid-dependent transactivation of a luciferase reporter construct containing the human PLTP promoter. Mutation analysis demonstrated that the inverted repeat-1 (IR-1) in the PLTP promoter is required for this transactivation. Finally, we demonstrate that bile acids are able to regulate PLTP gene expression in vivo. Mice fed a chow diet supplemented with bile acid showed increased hepatic PLTP mRNA levels. These results suggest that FXR may play a role in high density lipoprotein metabolism via the regulation of PLTP gene expression.

A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

ABSTRACTA wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.

The farnesoid X-activated receptor (FXR; NR1H4) is a member of the nuclear hormone receptor superfamily and functions as a heterodimer with the 9-cis-retinoic acid receptor (RXR). In order to determine the optimal DNA binding sequence for the FXR/RXR heterodimer, we have utilized the selectedand amplified binding sequence imprinting technique. This technique identified a number of related sequences that interacted with FXR/RXR in vitro. The consensus sequence contained an inverted repeat of the sequence AGGTCA with a 1-base pair spacing (IR-1). This sequence was shown to be a high affinity binding site for FXR/RXR in vitroand to confer ligand-dependent transcriptional activation by FXR/RXR to a heterologous promoter. Electrophoretic mobility shift assays and transient transfection assays were used to investigate the importance of the core half-site sequences, spacing nucleotide, flanking sequences, and orientation and spacing of the core half-sites on DNA binding and ligand-dependent transcriptional activation by FXR/RXR. These studies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, although FXR/RXR can bind to and activate through a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, FXR/RXR can bind to and transactivate through direct repeats. Three genes were identified that contain IR-1 sequences in their proximal promoters. These elements were shown to bind FXR/RXR in vitroand to confer FXR/RXR-dependent transcriptional activation to a heterologous promoter in response to a bile acid or synthetic retinoid. The endogenous mRNA levels of one of these genes, phospholipid transfer protein, were shown to be induced by FXR and FXR ligands. The identification of the IR-1 and related elements as high affinity binding sites and functional response elements for FXR/RXR and the identification of a target gene for FXR/RXR should assist in the identification of additional genes regulated by FXR/RXR.

The orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function.

ABSTRACTThe orphan nuclear hormone receptor SHP interacts with a number of other nuclear hormone receptors and inhibits their transcriptional activity. Several mechanisms have been suggested to account for this inhibition. Here we show that SHP inhibits transactivation by the orphan receptor hepatocyte nuclear factor 4 (HNF-4) and the retinoid X receptor (RXR) by at least two mechanisms. SHP interacts with the same HNF-4 surface recognized by transcriptional coactivators and competes with them for binding in vivo. The minimal SHP sequences previously found to be required for interaction with other receptors are sufficient for interaction with HNF-4, although deletion results indicate that additional C-terminal sequences are necessary for full binding and coactivator competition. These additional sequences include those associated with direct transcriptional repressor activity of SHP. SHP also competes with coactivators for binding to ligand-activated RXR, and based on the ligand-dependent interaction with other nuclear receptors, it is likely that coactivator competition is a general feature of SHP-mediated repression. The minimal receptor interaction domain of SHP is sufficient for full interaction with RXR, as previously described. This domain is also sufficient for full coactivator competition. Functionally, however, full inhibition of RXR transactivation requires the presence of the C-terminal repressor domain, with only weak inhibition associated with this receptor interaction domain. Overall, these results suggest that SHP represses nuclear hormone receptor-mediated transactivation via two separate steps: first by competition with coactivators and then by direct effects of its transcriptional repressor function.

Small heterodimer partner (SHP) is an orphan nuclear receptor that lacks a conventional DNA binding domain. It interacts with several other members of the nuclear receptor superfamily and inhibits receptor transactivation. In order to characterize the regulation of SHP expression, a number of receptors and other transcription factors were tested for effects on the SHP promoter. Among these, the orphan receptor steroidogenic factor-1 (SF-1) was found to potently transactivate the SHP promoter. Detailed footprinting studies show that the SHP promoter contains at least five SF-1 binding sites, and mutagenesis studies demonstrate each of the three strongest binding sites is required for SF-1 transactivation. SHP is coexpressed with SF-1 in adrenal glands, but is also expressed in tissues that lack SF-1, including liver. However, liver expresses a close relative of SF-1, the orphan fetoprotein transcription factor (FTF), and FTF can also transactivate the SHP promoter. These results suggest that alterations in the levels or activities of SF-1 or FTF could modulate SHP expression in appropriate tissues and thereby affect a variety of receptor dependent signaling pathways.

The THRA gene encoding thyroid hormone receptor α shares an unusual partial overlap with the NR1D1 gene encoding the orphan receptor Rev-ErbAα. Though THRA and NR1D1 have close relatives in THRB and NR1D2, which encode TRβ and Rev-ErbAβ, these β isoforms do not share an analogous overlap. Here we report that the human THRB and NR1D2 genes are separated by approximately 1 Mb on chromosome 3 and that these two genes are also linked to the RARB gene, which encodes retinoic acid receptor β. Since previous results indicate that the THRA/NR1D1 locus is also linked to the RARA gene, these results suggest that the two receptor gene clusters were generated by a single large-scale duplication.

Activating signal cointegrator 1 (ASC-1) harbors an autonomous transactivation domain that contains a putative zinc finger motif which provides binding sites for basal transcription factors TBP and TFIIA, transcription integrators steroid receptor coactivator 1 (SRC-1) and CBP-p300, and nuclear receptors, as demonstrated by the glutathione S-transferase pull-down assays and the yeast two-hybrid tests. The ASC-1 binding sites involve the hinge domain but not the C-terminal AF2 core domain of nuclear receptors. Nonetheless, ASC-1 appears to require the AF2-dependent factors to function (i.e., CBP-p300 and SRC-1), as suggested by the ability of ASC-1 to coactivate nuclear receptors, either alone or in cooperation with SRC-1 and p300, as well as its inability to coactivate a mutant receptor lacking the AF2 core domain. By using indirect immunofluorescence, we further show that ASC-1, a nuclear protein, is localized to the cytoplasm under conditions of serum deprivation but is retained in the nucleus when it is serum starved in the presence of ligand or coexpressed CBP or SRC-1. These results suggest that ASC-1 is a novel coactivator molecule of nuclear receptors which functions in conjunction with CBP-p300 and SRC-1 and may play an important role in establishing distinct coactivator complexes under different cellular conditions.

ABSTRACTActivating signal cointegrator 1 (ASC-1) harbors an autonomous transactivation domain that contains a putative zinc finger motif which provides binding sites for basal transcription factors TBP and TFIIA, transcription integrators steroid receptor coactivator 1 (SRC-1) and CBP-p300, and nuclear receptors, as demonstrated by the glutathioneS-transferase pull-down assays and the yeast two-hybrid tests. The ASC-1 binding sites involve the hinge domain but not the C-terminal AF2 core domain of nuclear receptors. Nonetheless, ASC-1 appears to require the AF2-dependent factors to function (i.e., CBP-p300 and SRC-1), as suggested by the ability of ASC-1 to coactivate nuclear receptors, either alone or in cooperation with SRC-1 and p300, as well as its inability to coactivate a mutant receptor lacking the AF2 core domain. By using indirect immunofluorescence, we further show that ASC-1, a nuclear protein, is localized to the cytoplasm under conditions of serum deprivation but is retained in the nucleus when it is serum starved in the presence of ligand or coexpressed CBP or SRC-1. These results suggest that ASC-1 is a novel coactivator molecule of nuclear receptors which functions in conjunction with CBP-p300 and SRC-1 and may play an important role in establishing distinct coactivator complexes under different cellular conditions.

We have determined the sequences of the oriregion DNA of several phage λ mutants and hybrids, which shed light on the mechanism of DNA replication in the lambdoid phages. These include the heterologous substitution hybrids λrep82:λ and λrep80:λ, a pseudorevertant of the ori−r93 mutant λr93 mutant λr93hot5, and the insertion mutant λpk35. The oriregions of the three lambdoid phages, λ, φ80 and 82, all have repeated sequences, termed iterons, and A·T-rich zones. We note that a similar arrangement of DNA is also found in several other prokaryotic origins of replication. λ and φ80 have four iterons, and 82 has five. The origin of λr93hot5 is unusual in that contains only three iterons, yet the phage grows normally. Analysis of this mutant indicates that the spacing of iterons is crucial to orifunction, whereas their number is not. This argues against the cloverleaf model for λ oristructure (Hobom et al., 1979). In λpk35 the drug resistance element Tn903is inserted into the “inceptor” (ice) site, proposed to be crucial for λ replication initiation (Hobom et al., 1979); yet this phage grows normally.

ABSTRACTTo release transcription factor NF-κB into the nucleus, the mammalian IκB molecules IκBα and IκBβ are inactivated by phosphorylation and proteolytic degradation. Both proteins contain conserved signal-responsive phosphorylation sites and have conserved ankyrin repeats. To confer specific physiological functions to members of the NF-κB/Rel family, the different IκB molecules could vary in their specific NF-κB/Rel factor binding activities and could respond differently to activation signals. We have demonstrated that both mechanisms apply to differential regulation of NF-κB function by IκBβ relative to IκBα. Via alternative RNA processing, human IκBβ gives rise to different protein isoforms. IκBβ1 and IκBβ2, the major forms in human cells, differ in their carboxy-terminal PEST sequences. IκBβ2 is the most abundant species in a number of human cell lines tested, whereas IκBβ1 is the only form detected in murine cells. These isoforms are indistinguishable in their binding preferences to cellular NF-κB/Rel homo- and heterodimers, which are distinct from those of IκBα, and both are constitutively phosphorylated. In unstimulated B cells, however, IκBβ1, but not IκBβ2, is found in the nucleus. Furthermore, the two forms differ markedly in their efficiency of proteolytic degradation after stimulation with several inducing agents tested. While IκBβ1 is nearly as responsive as IκBα, indicative of a shared activation mechanism, IκBβ2 is only weakly degraded and often not responsive at all. Alternative splicing of the IκBβ pre-mRNA may thus provide a means to selectively control the amount of IκBβ-bound NF-κB heteromers to be released under NF-κB stimulating conditions.