Deletion of hepatic small heterodimer partner ameliorates development of nonalcoholic steatohepatitis in mice.

Small heterodimer partner (SHP, Nr0b2) is an orphan nuclear receptor that regulates bile acid, lipid, and glucose metabolism. Shp-/- mice are resistant to diet-induced obesity and hepatic steatosis. In this study, we explored the potential role of SHP in the development of nonalcoholic steatohepatitis (NASH). A 6-month Western diet (WD) regimen was used to induce NASH. Shp deletion protected mice from NASH progression by inhibiting inflammatory and fibrotic genes, oxidative stress, and macrophage infiltration. WD feeding disrupted the ultrastructure of hepatic mitochondria in WT mice but not in Shp-/- mice. In ApoE-/- mice, Shp deletion also effectively ameliorated hepatic inflammation after a 1 week WD regimen without an apparent antisteatotic effect. Moreover, Shp-/- mice resisted fibrogenesis induced by a methionine- and choline-deficient diet. Notably, the observed protection against NASH was recapitulated in liver-specific Shp-/- mice fed either the WD or methionine- and choline-deficient diet. Hepatic cholesterol was consistently reduced in the studied mouse models with Shp deletion. Our data suggest that Shp deficiency ameliorates NASH development likely by modulating hepatic cholesterol metabolism and inflammation.

Identification of bile acid-CoA:amino acid N-acyltransferase as the hepatic N-acyl taurine synthase for polyunsaturated fatty acids.

N-acyl taurines (NATs) are bioactive lipids with emerging roles in glucose homeostasis and lipid metabolism. The acyl chains of hepatic and biliary NATs are enriched in polyunsaturated fatty acids (PUFAs). Dietary supplementation with a class of PUFAs, the omega-3 fatty acids, increases their cognate NATs in mice and humans. However, the synthesis pathway of the PUFA-containing NATs remains undiscovered. Here, we report that human livers synthesize NATs and that the acyl-chain preference is similar in murine liver homogenates. In the mouse, we found that hepatic NAT synthase activity localizes to the peroxisome and depends upon an active-site cysteine. Using unbiased metabolomics and proteomics, we identified bile acid-CoA:amino acid N-acyltransferase (BAAT) as the likely hepatic NAT synthase in vitro. Subsequently, we confirmed that BAAT knockout livers lack up to 90% of NAT synthase activity and that biliary PUFA-containing NATs are significantly reduced compared with wildtype. In conclusion, we identified the in vivo PUFA-NAT synthase in the murine liver and expanded the known substrates of the bile acid-conjugating enzyme, BAAT, beyond classic bile acids to the synthesis of a novel class of bioactive lipids.

Microbial products linked to steatohepatitis are reduced by deletion of nuclear hormone receptor SHP in mice.

Deletion of the nuclear hormone receptor small heterodimer partner (Shp) ameliorates the development of obesity and nonalcoholic steatohepatitis (NASH) in mice. Liver-specific SHP plays a significant role in this amelioration. The gut microbiota has been associated with these metabolic disorders, and the interplay between bile acids (BAs) and gut microbiota contributes to various metabolic disorders. Since hepatic SHP is recognized as a critical regulator in BA synthesis, we assessed the involvement of gut microbiota in the antiobesity and anti-NASH phenotype of Shp-/- mice. Shp deletion significantly altered the levels of a few conjugated BAs. Sequencing the 16S rRNA gene in fecal samples collected from separately housed mice revealed apparent dysbiosis in Shp-/- mice. Cohousing Shp-/- mice with WT mice during a Western diet regimen impaired their metabolic improvement and effectively disrupted their distinctive microbiome structure, which became indistinguishable from that of WT mice. While the Western diet challenge significantly increased lipopolysaccharide and phenylacetic acid (PAA) levels in the blood of WT mice, their levels were not increased in Shp-/- mice. PAA was strongly associated with hepatic peroxisome proliferator-activated receptor gamma isoform 2 (Pparg2) activation in mice, which may represent the basis of the molecular mechanism underlying the association of gut bacteria and hepatic steatosis. Shp deletion reshapes the gut microbiota possibly by altering BAs. While lipopolysaccharide and PAA are the major driving forces derived from gut microbiota for NASH development, Shp deletion decreases these signaling molecules via dysbiosis, thereby partially protecting mice from diet-induced metabolic disorders.

The Mitochondrial mitoNEET Ligand NL-1 Is Protective in a Murine Model of Transient Cerebral Ischemic Stroke.

PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC50 of 5.95 µM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

Farnesoid X receptor activation increases reverse cholesterol transport by modulating bile acid composition and cholesterol absorption in mice.

UNLABELLED: Activation of farnesoid X receptor (FXR) markedly attenuates development of atherosclerosis in animal models. However, the underlying mechanism is not well elucidated. Here, we show that the FXR agonist, obeticholic acid (OCA), increases fecal cholesterol excretion and macrophage reverse cholesterol transport (RCT) dependent on activation of hepatic FXR. OCA does not increase biliary cholesterol secretion, but inhibits intestinal cholesterol absorption. OCA markedly inhibits hepatic cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) partly through inducing small heterodimer partner, leading to reduced bile acid pool size and altered bile acid composition, with the α/ß-muricholic acid proportion in bile increased by 2.6-fold and taurocholic acid (TCA) level reduced by 71%. Overexpression of Cyp8b1 or concurrent overexpression of Cyp7a1 and Cyp8b1 normalizes TCA level, bile acid composition, and intestinal cholesterol absorption. CONCLUSION: Activation of FXR inhibits intestinal cholesterol absorption by modulation of bile acid pool size and composition, thus leading to increased RCT. Targeting hepatic FXR and/or bile acids may be useful for boosting RCT and preventing the development of atherosclerosis. (Hepatology 2016;64:1072-1085).

Structure-activity and in vivo evaluation of a novel lipoprotein lipase (LPL) activator.

Elevated triglycerides (TG) contribute towards increased risk for cardiovascular disease. Lipoprotein lipase (LPL) is an enzyme that is responsible for the metabolism of core triglycerides of very-low density lipoproteins (VLDL) and chylomicrons in the vasculature. In this study, we explored the structure-activity relationships of our lead compound (C10d) that we have previously identified as an LPL agonist. We found that the cyclopropyl moiety of C10d is not absolutely necessary for LPL activity. Several substitutions were found to result in loss of LPL activity. The compound C10d was also tested in vivo for its lipid lowering activity. Mice were fed a high-fat diet (HFD) for four months, and treated for one week at 10mg/kg. At this dose, C10d exhibited in vivo biological activity as indicated by lower TG and cholesterol levels as well as reduced body fat content as determined by ECHO-MRI. Furthermore, C10d also reduced the HFD induced fat accumulation in the liver. Our study has provided insights into the structural and functional characteristics of this novel LPL activator.

Farnesoid X Receptor Agonist Represses Cytochrome P450 2D6 Expression by Upregulating Small Heterodimer Partner.

Cytochrome P450 2D6 (CYP2D6) is a major drug-metabolizing enzyme responsible for eliminating approximately 20% of marketed drugs. Studies have shown that differential transcriptional regulation of CYP2D6 may contribute to large interindividual variability in CYP2D6-mediated drug metabolism. However, the factors governing CYP2D6 transcription are largely unknown. We previously demonstrated small heterodimer partner (SHP) as a novel transcriptional repressor of CYP2D6 expression. SHP is a representative target gene of the farnesoid X receptor (FXR). The objective of this study is to investigate whether an agonist of FXR, 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), alters CYP2D6 expression and activity. In CYP2D6-humanized transgenic mice, GW4064 decreased hepatic CYP2D6 expression and activity (by 2-fold) while increasing SHP expression (by 2-fold) and SHP recruitment to the CYP2D6 promoter. CYP2D6 repression by GW4064 was abrogated in Shp(-/-);CYP2D6 mice, indicating a critical role of SHP in CYP2D6 regulation by GW4064. Also, GW4064 decreased CYP2D6 expression (by 2-fold) in primary human hepatocytes, suggesting that the results obtained in CYP2D6-humanized transgenic mice can be translated to humans. This proof of concept study provides evidence for CYP2D6 regulation by an inducer of SHP expression, namely, the FXR agonist GW4064.

The fatty acid omega hydroxylase genes (CYP4 family) in the progression of metabolic dysfunction-associated steatotic liver disease (MASLD): An RNA sequence database analysis and review.

Fatty acid omega hydroxylase P450s consist of enzymes that hydroxylate various chain-length saturated and unsaturated fatty acids (FAs) and bioactive eicosanoid lipids. The human cytochrome P450 gene 4 family (CYP4) consists of 12 members that are associated with several human diseases. However, their role in the progression of metabolic dysfunction-associated fatty liver disease (MASLD) remains largely unknown. It has long been thought that the induction of CYP4 family P450 during fasting and starvation prevents FA-related lipotoxicity through FA metabolism to dicarboxylic acids that are chain-shortened in peroxisomes and then transported to the mitochondria for complete oxidation. Several studies have revealed that peroxisome succinate transported to the mitochondria is used for gluconeogenesis during fasting and starvation, and recent evidence suggests that peroxisome acetate can be utilized for lipogenesis and lipid droplet formation as well as epigenetic modification of gene transcription. In addition, omega hydroxylation of the bioactive eicosanoid arachidonic acid to 20-Hydroxyeicosatetraenoic acid (20-HETE) is essential for activating the GPR75 receptor, leading to vasoconstriction and cell proliferation. Several mouse models of diet-induced MASLD have revealed the induction of selective CYP4A members and the suppression of CYP4F during steatosis and steatohepatitis, suggesting a critical metabolic role in the progression of fatty liver disease. Thus, to further investigate the functional roles of CYP4 genes, we analyzed the differential gene expression of 12 members of CYP4 gene family in datasets from the Gene Expression Omnibus (GEO) from patients with steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. We also observed the differential expression of various CYP4 genes in the progression of MASLD, indicating that different CYP4 members may have unique functional roles in the metabolism of specific FAs and eicosanoids at various stages of fatty liver disease. These results suggest that targeting selective members of the CYP4A family is a viable therapeutic approach for treating and managing MASLD.

Effect of aerobic exercise and particulate matter exposure duration on the diversity of gut microbiota.

Inhalation of ambient particulate matter (PM) can disrupt the gut microbiome, while exercise independently influences the gut microbiome by promoting beneficial bacteria. In this study, we analyzed changes in gut microbial diversity and composition in response to combined interventions of PM exposure and aerobic exercise, extending up to 12 weeks. This investigation was conducted using mice, categorized into five groups: control group (Con), exercise group (EXE), exercise group followed by 3-day exposure to PM (EXE + 3-day PM), particulate matter exposure (PM), and PM exposure with concurrent treadmill exercise (PME). Notably, the PM group exhibited markedly lower alpha diversity and richness compared to the Con group and our analysis of beta diversity revealed significant variations among the intervention groups. Members of the Lachnospiraceae family showed significant enhancement in the exercise intervention groups (EXE and PME) compared to the Con and PM groups. The biomarker Lactobacillus, Coriobacteraceae, and Anaerofustis were enriched in the EXE group, while Desulfovibrionaceae, Mucispirillum schaedleri, Lactococcus and Anaeroplasma were highly enriched in the PM group. Differential abundance analysis revealed that Paraprevotella, Bacteroides, and Blautia were less abundant in the 12-week PM exposure group than in the 3-day PM exposure group. Moreover, both the 3-day and 12-week PM exposure groups exhibited a reduced relative abundance of Bacteroides uniformis, SMB53, and Staphylococcus compared to non-PM exposure groups. These findings will help delineate the possible roles and associations of altered microbiota resulting from the studied interventions, paving the way for future mechanistic research.

Ultra-Thin Lithium Silicide Interlayer for Solid-State Lithium-Metal Batteries.

All-solid-state batteries with metallic lithium (LiBCC ) anode and solid electrolyte (SE) are under active development. However, an unstable SE/LiBCC interface due to electrochemical and mechanical instabilities hinders their operation. Herein, an ultra-thin nanoporous mixed ionic and electronic conductor (MIEC) interlayer (≈3.25 µm), which regulates LiBCC deposition and stripping, serving as a 3D scaffold for Li0 ad-atom formation, LiBCC nucleation, and long-range transport of ions and electrons at SE/LiBCC interface is demonstrated. Consisting of lithium silicide and carbon nanotubes, the MIEC interlayer is thermodynamically stable against LiBCC and highly lithiophilic. Moreover, its nanopores (<100 nm) confine the deposited LiBCC to the size regime where LiBCC exhibits "smaller is much softer" size-dependent plasticity governed by diffusive deformation mechanisms. The LiBCC thus remains soft enough not to mechanically penetrate SE in contact. Upon further plating, LiBCC grows in between the current collector and the MIEC interlayer, not directly contacting the SE. As a result, a full-cell having Li3.75 Si-CNT/LiBCC foil as an anode and LiNi0.8 Co0.1 Mn0.1 O2 as a cathode displays a high specific capacity of 207.8 mAh g-1 , 92.0% initial Coulombic efficiency, 88.9% capacity retention after 200 cycles (Coulombic efficiency reaches 99.9% after tens of cycles), and excellent rate capability (76% at 5 C).

CYP4V2 fatty acid omega hydroxylase, a druggable target for the treatment of metabolic associated fatty liver disease (MAFLD).

Fatty acids are essential in maintaining cellular homeostasis by providing lipids for energy production, cell membrane integrity, protein modification, and the structural demands of proliferating cells. Fatty acids and their derivatives are critical bioactive signaling molecules that influence many cellular processes, including metabolism, cell survival, proliferation, migration, angiogenesis, and cell barrier function. The CYP4 Omega hydroxylase gene family hydroxylate various short, medium, long, and very-long-chain saturated, unsaturated and polyunsaturated fatty acids. Selective members of the CYP4 family metabolize vitamins and biochemicals with long alkyl side chains and bioactive prostaglandins, leukotrienes, and arachidonic acids. It is uncertain of the physiological role of different members of the CYP4 omega hydroxylase gene family in the metabolic control of physiological and pathological processes in the liver. CYP4V2 is a unique member of the CYP4 family. CYP4V2 inactivation in retinal pigment epithelial cells leads to cholesterol accumulation and Bietti's Crystalline Dystrophy (BCD) pathogenesis. This commentary provides information on the role CYP4V2 has in metabolic syndrome and nonalcoholic fatty liver disease progression. This is accomplished by identifying its role in BCD, its control of cholesterol synthesis and lipid droplet formation in C. elegans, and the putative function in cardiovascular disease and gastrointestinal/hepatic pathologies.

Bile acid conjugation deficiency causes hypercholanemia, hyperphagia, islet dysfunction, and gut dysbiosis in mice.

Bile acid-CoA: amino acid N-acyltransferase (BAAT) catalyzes bile acid conjugation, the last step in bile acid synthesis. BAAT gene mutation in humans results in hypercholanemia, growth retardation, and fat-soluble vitamin insufficiency. The current study investigated the physiological function of BAAT in bile acid and lipid metabolism using Baat-/- mice. The bile acid composition and hepatic gene expression were analyzed in 10-week-old Baat-/- mice. They were also challenged with a westernized diet (WD) for additional 15 weeks to assess the role of BAAT in bile acid, lipid, and glucose metabolism. Comprehensive lab animal monitoring system and cecal 16S ribosomal RNA gene sequencing were used to evaluate the energy metabolism and microbiome structure of the mice, respectively. In Baat-/- mice, hepatic bile acids were mostly unconjugated and their levels were significantly increased compared with wild-type mice. Bile acid polyhydroxylation was markedly up-regulated to detoxify unconjugated bile acid accumulated in Baat-/- mice. Although the level of serum marker of bile acid synthesis, 7α-hydroxy-4-cholesten-3-one, was higher in Baat-/- mice, their bile acid pool size was smaller. When fed a WD, the Baat-/- mice showed a compromised body weight gain and impaired insulin secretion. The gut microbiome of Baat-/- mice showed a low level of sulfidogenic bacteria Bilophila. Conclusion: Mouse BAAT is the major taurine-conjugating enzyme. Its deletion protected the animals from diet-induced obesity, but caused glucose intolerance. The gut microbiome of the Baat-/- mice was altered to accommodate the unconjugated bile acid pool.

Forkhead box transcription factor O1 inhibits cholesterol 7alpha-hydroxylase in human hepatocytes and in high fat diet-fed mice.

The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a approximately 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.

Modulation of human nuclear receptor LRH-1 activity by phospholipids and SHP.

The human nuclear receptor liver receptor homolog 1 (hLRH-1) plays an important role in the development of breast carcinomas. This orphan receptor is efficiently downregulated by the unusual co-repressor SHP and has been thought to be ligand-independent. We present the crystal structure at a resolution of 1.9 A of the ligand-binding domain of hLRH-1 in complex with the NR box 1 motif of human SHP, which we find contacts the AF-2 region of hLRH-1 using selective structural motifs. Electron density indicates phospholipid bound within the ligand-binding pocket, which we confirm using mass spectrometry of solvent-extracted samples. We further show that pocket mutations reduce phospholipid binding and receptor activity in vivo. Our results indicate that hLRH-1's control of gene expression is mediated by phospholipid binding, and establish hLRH-1 as a novel target for compounds designed to slow breast cancer development.

Calendering-Compatible Macroporous Architecture for Silicon-Graphite Composite toward High-Energy Lithium-Ion Batteries.

Porous strategies based on nanoengineering successfully mitigate several problems related to volume expansion of alloying anodes. However, practical application of porous alloying anodes is challenging because of limitations such as calendering incompatibility, low mass loading, and excessive usage of nonactive materials, all of which cause a lower volumetric energy density in comparison with conventional graphite anodes. In particular, during calendering, porous structures in alloying-based composites easily collapse under high pressure, attenuating the porous characteristics. Herein, this work proposes a calendering-compatible macroporous architecture for a Si-graphite anode to maximize the volumetric energy density. The anode is composed of an elastic outermost carbon covering, a nonfilling porous structure, and a graphite core. Owing to the lubricative properties of the elastic carbon covering, the macroporous structure coated by the brittle Si nanolayer can withstand high pressure and maintain its porous architecture during electrode calendering. Scalable methods using mechanical agitation and chemical vapor deposition are adopted. The as-prepared composite exhibits excellent electrochemical stability of >3.6 mAh cm-2 , with mitigated electrode expansion. Furthermore, full-cell evaluation shows that the composite achieves higher energy density (932 Wh L-1 ) and higher specific energy (333 Wh kg-1 ) with stable cycling than has been reported in previous studies.

Scalable Synthesis of Hollow ß-SiC/Si Anodes via Selective Thermal Oxidation for Lithium-Ion Batteries.

Silicon for anodes in lithium-ion batteries has received much attention owing to its superior specific capacity. There has been a rapid increase of research related to void engineering to address the silicon failure mechanism stemming from the massive volume change during (dis)charging in the past decade. Nevertheless, conventional synthetic methods require complex synthetic procedures and toxic reagents to form a void space, so they have an obvious limitation to reach practical application. Here, we introduce SiCx consisting of nanocrystallite Si embedded in the inactive matrix of ß-SiC to fabricate various types of void structures using thermal etching with a scalable one-pot CVD method. The structural features of SiCx make the carbonaceous template possible to be etched selectively without Si oxidation at high temperature with an air atmosphere. Furthermore, bottom-up gas phase synthesis of SiCx ensures atomically identical structural features (e.g., homogeneously distributed Si and ß-SiC) regardless of different types of sacrificial templates. For these reasons, various types of SiCx hollow structures having shells, tubes, and sheets can be synthesized by simply employing different morphologies of the carbon template. As a result, the morphological effect of different hollow structures can be deeply investigated as well as the free volume effect originating from void engineering from both a electrochemical and computational point of view. In terms of selective thermal oxidation, the SiCx hollow shell achieves a much higher initial Coulombic efficiency (>89%) than that of the Si hollow shell (65%) because of its nonoxidative property originating from structural characteristics of SiCx during thermal etching. Moreover, the findings based on the clearly observed different electrochemical features between half-cell and full-cell configuration give insight into further Si anode research.

Redundant pathways for negative feedback regulation of bile acid production.

The orphan nuclear hormone receptor SHP has been proposed to have a key role in the negative feedback regulation of bile acid production. Consistent with this, mice lacking the SHP gene exhibit mild defects in bile acid homeostasis and fail to repress cholesterol 7-alpha-hydroxylase expression in response to a specific agonist for the bile acid receptor FXR. However, this repression is retained in SHP null mice fed bile acids, demonstrating the existence of compensatory repression pathways of bile acid signaling. We provide evidence for two such pathways, based on activation of the xenobiotic receptor PXR or the c-Jun N-terminal kinase JNK. We conclude that redundant mechanisms regulate this critical aspect of cholesterol homeostasis.

Liver receptor homolog-1, an emerging metabolic modulator.

The liver receptor homolog-1 (LRH-1; NR5A2) belongs to nuclear hormone receptor superfamily, and is expressed mainly in the liver, intestine, exocrine pancreas, and ovary. It binds DNA as a monomer, and is best known as a regulator of hepatic expression of the key bile acid biosynthetic enzyme cholesterol 7alpha hydroxylase (Cyp7A1). It is also expressed in embryonic stem cells and the initial stages of embryonic development, and the very early lethality of LRH-1 knockout mice highlights its essential developmental role. Recent crystal structures of LRH-1 and its closest relative steroidogenic factor-1 (SF-1; NR5A1) identified phospholipids as potential ligands. This intriguing discovery raises the possibility of an unexpected new class of nuclear receptor signaling molecules, but the broader functional roles of LRH-1 and these new ligands remain to be established.

The orphan receptor SHP and the three-hybrid interference assay.

The three-hybrid interference method described here is similar to previous interference assays based on the two-hybrid system. In these approaches the presence of a third protein disrupts the interaction between the two different hybrid proteins. The three-hybrid interference assay circumvents a potential problem that arises when the third protein is itself a direct transcriptional repressor by preventing the decrease in transcriptional readout that could occur if the third protein is recruited indirectly to the two-hybrid protein complex. This three-hybrid method should be applicable to any third protein that is a transcriptional repressor. To use this method to explore the effects of such repressors on protein-protein interactions, however, it is important to confirm that the VP16-repressor fusion does not retain active repressor function. It is also appropriate to compare the effects of the VP16-repressor fusion to those with the repressor alone. Finally, it is essential to confirm any conclusions from this or any other single method to study protein-protein interactions with alternative independent approaches.

Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation.

The nuclear receptor LRH-1 (NR5A2) functions to regulate expression of a number of genes associated with bile acid homeostasis and other liver functions, but mechanisms that modulate its activity remain unclear. We have found that mitogenic stimuli, including treatment with phorbol myristate (PMA), increase LRH-1 transactivation. This response maps to the hinge and ligand binding domains of LRH-1 and is blocked by the mitogen-activated protein kinase ERK1/2 inhibitor U0126. LRH-1 is a phosphoprotein and hinge domain serine residues at 238 and 243 are required for effective phosphorylation, both in vitro and in cells. Preventing phosphorylation of these residues by mutating both to alanine decreases PMA-dependent LRH-1 transactivation and mimicking phosphorylation by mutation to positively charged aspartate residues increases basal transactivation. Although serine phosphorylation of the hinge of SF-1 (NR5A1), the closest relative of LRH-1, confers a similar response, the specific targets differ in the two closely related orphan receptors. These results define a novel pathway for the modulation of LRH-1 transactivation and identify specific LRH-1 residues as downstream targets of mitogenic stimuli. This pathway may contribute to recently described proliferative functions of LRH-1.