Deficiency of Procollagen C-Proteinase Enhancer 1 in Mice has No Major Impact on Cardiac Collagen and Function Under Basal Conditions.

ABSTRACT: Maturation of fibrillar collagen is known to play a crucial role in the pathophysiology of myocardial fibrosis. Procollagen C-proteinase enhancer 1 (PCPE1) has a key role in procollagen maturation and collagen fibril formation. The phenotype of both male and female PCPE1 knock-out mice was investigated under basal conditions to explore the potential of PCPE1 as a therapeutic target in heart failure. Global constitutive PCPE1-/- mice were generated. Serum procollagen I C-terminal propeptide, organ histology, and cutaneous wound healing were assessed in both wild type (WT) and PCPE1-/- mice. In addition, the cardiac expression of genes involved in collagen metabolism was investigated and the total and insoluble cardiac collagen contents determined. Cardiac function was evaluated by echocardiography. No differences in survival, clinical chemistry, or organ histology were observed in PCPE1-/- mice compared with WT. Serum procollagen I C-terminal propeptide was lower in PCPE1-/- mice. Cardiac mRNA expression of Bmp1, Col1a1, Col3a1, and Loxl2 was similar, whereas Tgfb and Loxl1 mRNA levels were decreased in PCPE1-/- mice compared with sex-matched WT. No modification of total or insoluble cardiac collagen content was observed between the 2 strains. Ejection fraction was slightly decreased in PCPE1-/- male mice, but not in females. Finally, wound healing was not altered in PCPE1-/- mice. PCPE1 deficiency does not trigger any major liabilities and does not affect cardiac collagen content nor its function under basal conditions. Further studies are required to evaluate its role under stressed conditions and determine its suitability as a therapeutic target for heart failure.

G Protein-Coupled Receptors Targeting Insulin Resistance, Obesity, and Type 2 Diabetes Mellitus.

G protein-coupled receptors (GPCRs) continue to be important discovery targets for the treatment of type 2 diabetes mellitus (T2DM). Many GPCRs are directly involved in the development of insulin resistance and ß-cell dysfunction, and in the etiology of inflammation that can lead to obesity-induced T2DM. This review summarizes the current literature describing a number of well-validated GPCR targets, but also outlines several new and promising targets for drug discovery. We highlight the importance of understanding the role of these receptors in the disease pathology, and their basic pharmacology, which will pave the way to the development of novel pharmacological probes that will enable these targets to fulfill their promise for the treatment of these metabolic disorders.

Characterization of GLPG0492, a selective androgen receptor modulator, in a mouse model of hindlimb immobilization.

BACKGROUND: Muscle wasting is a hallmark of many chronic conditions but also of aging and results in a progressive functional decline leading ultimately to disability. Androgens, such as testosterone were proposed as therapy to counteract muscle atrophy. However, this treatment is associated with potential cardiovascular and prostate cancer risks and therefore not acceptable for long-term treatment. Selective Androgen receptor modulators (SARM) are androgen receptor ligands that induce muscle anabolism while having reduced effects in reproductive tissues. Therefore, they represent an alternative to testosterone therapy. Our objective was to demonstrate the activity of SARM molecule (GLPG0492) on a immobilization muscle atrophy mouse model as compared to testosterone propionate (TP) and to identify putative biomarkers in the plasma compartment that might be related to muscle function and potentially translated into the clinical space. METHODS: GLPG0492, a non-steroidal SARM, was evaluated and compared to TP in a mouse model of hindlimb immobilization. RESULTS: GLPG0492 treatment partially prevents immobilization-induced muscle atrophy with a trend to promote muscle fiber hypertrophy in a dose-dependent manner. Interestingly, GLPG0492 was found as efficacious as TP at reducing muscle loss while sparing reproductive tissues. Furthermore, gene expression studies performed on tibialis samples revealed that both GLPG0492 and TP were slowing down muscle loss by negatively interfering with major signaling pathways controlling muscle mass homeostasis. Finally, metabolomic profiling experiments using 1H-NMR led to the identification of a plasma GLPG0492 signature linked to the modulation of cellular bioenergetic processes. CONCLUSIONS: Taken together, these results unveil the potential of GLPG0492, a non-steroidal SARM, as treatment for, at least, musculo-skeletal atrophy consecutive to coma, paralysis, or limb immobilization.

Long-term use of investigational ß-Hydroxybutyrate salts in children with multiple acyl-CoA dehydrogenase or pyruvate dehydrogenase deficiency.

Several disorders of energy metabolism have been treated with exogenous ketone bodies. The benefit of this treatment is best documented in multiple acyl-CoA dehydrogenase deficiency (MADD) (MIM#231680). One might also expect ketone bodies to help in other disorders with impaired ketogenesis or in conditions that profit from a ketogenic diet. Here, we report the use of a novel preparation of dextro-ß-hydroxybutyrate (D-ßHB) salts in two cases of MADD and one case of pyruvate dehydrogenase (PDH) deficiency (MIM#312170). The two patients with MADD had previously been on a racemic mixture of D- and L­sodium hydroxybutyrate. Patient #1 found D-ßHB more palatable, and the change in formulation corrected hypernatraemia in patient #2. The patient with PDH deficiency was on a ketogenic diet but had not previously been given hydroxybutyrate. In this case, the addition of D-ßHB improved ketosis. We conclude that NHS101 is a good candidate for further clinical studies in this group of diseases of inborn errors of metabolism.

A new NRF2 activator for the treatment of human metabolic dysfunction-associated fatty liver disease.

Background & Aims: Oxidative stress triggers metabolic-associated fatty liver disease (MAFLD) and fibrosis. Previous animal studies demonstrated that the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2), the master regulator of antioxidant response, protects against MAFLD and fibrosis. S217879, a next generation NRF2 activator has been recently shown to trigger diet-induced steatohepatitis resolution and to reduce established fibrosis in rodents. Our aim was to evaluate the therapeutic potential of S217879 in human MAFLD and its underlying mechanisms using the relevant experimental 3D model of patient-derived precision cut liver slices (PCLS). Methods: We treated PCLS from 12 patients with varying stages of MAFLD with S217879 or elafibranor (peroxisome proliferator-activated receptor [PPAR]α/δ agonist used as a referent molecule) for 2 days. Safety and efficacy profiles, steatosis, liver injury, inflammation, and fibrosis were assessed as well as mechanisms involved in MAFLD pathophysiology, namely antioxidant response, autophagy, and endoplasmic reticulum-stress. Results: Neither elafibranor nor S217879 had toxic effects at the tested concentrations on human PCLS with MAFLD. PPARα/δ and NRF2 target genes (pyruvate dehydrogenase kinase 4 [PDK4], fibroblast growth factor 21 [FGF21], and NAD(P)H quinone dehydrogenase 1 [NQO1], heme oxygenase 1 [HMOX1], respectively) were strongly upregulated in PCLS in response to elafibranor and S217879, respectively. Compared with untreated PCLS, elafibranor and S217879-treated slices displayed lower triglycerides and reduced inflammation (IL-1ß, IL-6, chemokine (C-C motif) ligand 2 [CCL2]). Additional inflammatory markers (chemokine (C-C motif) ligand 5 [CCL5], stimulator of interferon genes [STING], intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) were downregulated by S217879. S217879 but not elafibranor lowered DNA damage (phospho-Histone H2A.X [p-H2A.X], RAD51, X-ray repair cross complementing 1 [XRCC1]) and apoptosis (cleaved caspase-3), and inhibited fibrogenesis markers expression (alpha smooth muscle actin [α-SMA], collagen 1 alpha 1 [COL1A1], collagen 1 alpha 2 [COL1A2]). Such effects were mediated through an improvement of lipid metabolism, activated antioxidant response and enhanced autophagy, without effect on endoplasmic reticulum-stress. Conclusions: This study highlights the therapeutic potential of a new NRF2 activator for MAFLD using patient-derived PCLS supporting the evaluation of NRF2 activating strategies in clinical trials. Impact and implications: Oxidative stress is a major driver of metabolic-associated fatty liver disease (MAFLD) development and progression. Nuclear factor (erythroid-derived 2)-like 2, the master regulator of the antioxidative stress response, is an attractive therapeutic target for the treatment of MAFLD. This study demonstrates that S217879, a new potent and selective nuclear factor (erythroid-derived 2)-like 2 activator, displays antisteatotic effects, lowers DNA damage, apoptosis, and inflammation and inhibits fibrogenesis in human PCLS in patients with MAFLD.

Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice.

Background & Aims: Oxidative stress is recognized as a major driver of non-alcoholic steatohepatitis (NASH) progression. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and thus appear to be attractive targets for the treatment of NASH. Methods: Molecular modeling and X-ray crystallography were used to design S217879 - a small molecule that could disrupt the KEAP1-NRF2 interaction. S217879 was highly characterized using various molecular and cellular assays. It was then evaluated in two different NASH-relevant preclinical models, namely the methionine and choline-deficient diet (MCDD) and diet-induced obesity NASH (DIO NASH) models. Results: Molecular and cell-based assays confirmed that S217879 is a highly potent and selective NRF2 activator with marked anti-inflammatory properties, as shown in primary human peripheral blood mononuclear cells. In MCDD mice, S217879 treatment for 2 weeks led to a dose-dependent reduction in NAFLD activity score while significantly increasing liver Nqo1 mRNA levels, a specific NRF2 target engagement biomarker. In DIO NASH mice, S217879 treatment resulted in a significant improvement of established liver injury, with a clear reduction in both NAS and liver fibrosis. αSMA and Col1A1 staining, as well as quantification of liver hydroxyproline levels, confirmed the reduction in liver fibrosis in response to S217879. RNA-sequencing analyses revealed major alterations in the liver transcriptome in response to S217879, with activation of NRF2-dependent gene transcription and marked inhibition of key signaling pathways that drive disease progression. Conclusions: These results highlight the potential of selective disruption of the NRF2-KEAP1 interaction for the treatment of NASH and liver fibrosis. Impact and implications: We report the discovery of S217879 - a potent and selective NRF2 activator with good pharmacokinetic properties. By disrupting the KEAP1-NRF2 interaction, S217879 triggers the upregulation of the antioxidant response and the coordinated regulation of a wide spectrum of genes involved in NASH disease progression, leading ultimately to the reduction of both NASH and liver fibrosis progression in mice.

Procollagen C-Proteinase Enhancer-1 (PCPE-1) deficiency in mice reduces liver fibrosis but not NASH progression.

BACKGROUND AND AIMS: Nonalcoholic Steatohepatitis (NASH) is a major cause of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma resulting ultimately in increased liver-related mortality. Fibrosis is the main driver of mortality in NASH. Procollagen C-Proteinase Enhancer-1 (PCPE-1) plays a key role in procollagen maturation and collagen fibril formation. To assess its role in liver fibrosis and NASH progression, knock-out mice were evaluated in a dietary NASH model. METHODS: Global constitutive Pcolce-/- and WT male mice were fed with a Choline Deficient Amino acid defined High Fat Diet (CDA HFD) for 8 weeks. Liver triglycerides, steatosis, inflammation and fibrosis were assessed at histological, biochemical and gene expression levels. In addition, human liver samples from control and NASH patients were used to evaluate the expression of PCPE-1 at both mRNA and protein levels. RESULTS: Pcolce gene deficiency prevented diet-induced liver enlargement but not liver dysfunction. Furthermore, liver triglycerides, steatosis and inflammation were not modified in Pcolce-/- male mice compared to WT under CDA HFD. However, a significant decrease in liver fibrosis was observed in Pcolce-/- mice compared to WT under NASH diet, associated with a decrease in total and insoluble collagen content without any significant modifications in the expression of genes involved in fibrosis and extracellular matrix remodeling. Finally, PCPE-1 protein expression was increased in cirrhotic liver samples from both NASH and Hepatitis C patients. CONCLUSIONS: Pcolce deficiency limits fibrosis but not NASH progression in CDA HFD fed mice.

Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs.

Background: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Göttingen minipig. Methods: First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results: Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions: These results, taken together, indicate that the diet-induced NASH in the Göttingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development.

Tissue-specific liver X receptor activation promotes macrophage reverse cholesterol transport in vivo.

OBJECTIVE: We previously reported that a systemic liver X receptor (LXR) agonist promoted macrophage reverse-cholesterol transport (mRCT) in vivo. Because LXR are expressed in multiple tissues involved in RCT (macrophages, liver, intestine), we analyzed the effect of tissue-specific LXR agonism on mRCT. METHODS AND RESULTS: In initial studies, the systemic LXR agonist GW3965 failed to promote mRCT in a setting in which LXR was expressed in macrophages but not in liver or intestine. To evaluate the effect of LXR activation specifically in small intestine on mRCT, wild-type mice were treated with either intestinal-specific LXR agonist (GW6340) or systemic LXR agonist (GW3965). Both GW3965 and GW6340 significantly promoted excretion of [(3)H]-sterol in feces by 162% and 52%, respectively. To evaluate the requirement for macrophage LXR activation, we assessed the ability of GW3965 to promote mRCT in wild-type mice using primary macrophages deficient in LXR alpha/beta vs wild-type macrophages. Whereas GW3965 treatment promoted fecal excretion compared with vehicle, its overall ability to promote mRCT was significantly attenuated using LXR alpha/beta knockout macrophages. CONCLUSIONS: We demonstrate that intestinal-specific LXR agonism promotes macrophage RCT in vivo and that macrophage LXR itself plays an important, but not predominant, role in promoting RCT in response to an LXR agonist.

MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.

In type 2 diabetes (T2D), both muscle and liver are severely resistant to insulin action. Muscle insulin resistance accounts for more than 80% of the impairment in total body glucose disposal in T2D patients and is often characterized by an impaired insulin signaling. Mitsugumin 53 (MG53), a muscle-specific TRIM family protein initially identified as a key regulator of cell membrane repair machinery has been suggested to be a critical regulator of muscle insulin signaling pathway by acting as ubiquitin E3 ligase targeting both the insulin receptor and insulin receptor substrate 1 (IRS1). Here, we show using in vitro and in vivo approaches that MG53 is not a critical regulator of insulin signaling and glucose homeostasis. First, MG53 expression is not consistently regulated in skeletal muscle from various preclinical models of insulin resistance. Second, MG53 gene knock-down in muscle cells does not lead to impaired insulin response as measured by Akt phosphorylation on Serine 473 and glucose uptake. Third, recombinant human MG53 does not alter insulin response in both differentiated C2C12 and human skeletal muscle cells. Fourth, ectopic expression of MG53 in HEK293 cells lacking endogenous MG53 expression fails to alter insulin response as measured by Akt phosphorylation. Finally, both male and female mg53 -/- mice were not resistant to high fat induced obesity and glucose intolerance compared to wild-type mice. Taken together, these results strongly suggest that MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.

S62798, a potent TAFIa inhibitor, accelerates endogenous fibrinolysis in a murine model of pulmonary thromboembolism.

Enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Venous thrombosis and thromboembolism risks are associated with increased plasma levels of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) as well as its active form TAFIa. A new TAFIa inhibitor, namely S62798 has been identified. Its ability to enhance fibrinolysis was investigated both in vitro and in vivo in a mouse model of pulmonary thromboembolism, as well as its effect on bleeding. S62798 is a highly selective human, mouse and rat TAFIa inhibitor (IC50 = 11; 270; 178 nmol/L, respectively). It accelerates lysis of a human clot in vitro, evaluated by thromboelastometry (EC50 = 27 nmol/L). In a rat tail bleeding model, no effect of S62798 treatment was observed up to 20 mg/kg. Enhancement of endogenous fibrinolysis by S62798 was investigated in a mouse model of Tissue Factor-induced pulmonary thromboembolism. Intravenous administration of S62798 decreased pulmonary fibrin clots with a minimal effective dose of 0.03 mg/kg. Finally, effect of S62798 in combination with heparin was evaluated. When treatment of heparin was done in a curative setting, no effect was observed whereas a significantly decreased pulmonary fibrin deposition was observed in response to S62798 alone or in combination with heparin. This study demonstrates that S62798 is a potent TAFIa inhibitor with minimal risk of bleeding. In vivo, curative S62798 intravenous treatment, alone or associated with heparin, accelerated clot lysis by potentiating endogenous fibrinolysis and thus decreased pulmonary fibrin clots. S62798 is expected to be a therapeutic option for pulmonary embolism patients on top of anticoagulants.

Deletion of GPR21 improves glucose homeostasis and inhibits the CCL2-CCR2 axis by divergent mechanisms.

INTRODUCTION: A potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are complicated by non-selective deletion of Gpr21. RESEARCH DESIGN AND METHODS: We hypothesized that a Gpr21-selective knockout mouse model, coupled with type 2 diabetes patient samples, would clarify these issues and enable clear assessment of GPR21 as a potential therapeutic target. RESULTS: High-fat feeding studies in Gpr21-/- mice revealed improved glucose tolerance and modest changes in inflammatory gene expression. Gpr21-/- monocytes and intraperitoneal macrophages had selectively impaired chemotactic responses to monocyte chemoattractant protein (MCP)-1, despite unaltered expression of Ccr2. Further genotypic analysis revealed that chemotactic impairment was due to dysregulated monocyte polarization. Patient samples revealed elevated GPR21 expression in peripheral blood mononuclear cells in type 2 diabetes, which was correlated with both %HbA1c and fasting plasma glucose levels. CONCLUSIONS: Collectively, human and mouse data suggest that GPR21 influences both glucose homeostasis and MCP-1/CCL2-CCR2-driven monocyte migration. However, a Gpr21-/- bone marrow transplantation and high-fat feeding study in mice revealed no effect on glucose homeostasis, suggesting that there is no (or limited) overlap in the mechanism involved for monocyte-driven inflammation and glucose homeostasis.

Multi-omics profiling of living human pancreatic islet donors reveals heterogeneous beta cell trajectories towards type 2 diabetes.

Most research on human pancreatic islets is conducted on samples obtained from normoglycaemic or diseased brain-dead donors and thus cannot accurately describe the molecular changes of pancreatic islet beta cells as they progress towards a state of deficient insulin secretion in type 2 diabetes (T2D). Here, we conduct a comprehensive multi-omics analysis of pancreatic islets obtained from metabolically profiled pancreatectomized living human donors stratified along the glycemic continuum, from normoglycemia to T2D. We find that islet pools isolated from surgical samples by laser-capture microdissection display remarkably more heterogeneous transcriptomic and proteomic profiles in patients with diabetes than in non-diabetic controls. The differential regulation of islet gene expression is already observed in prediabetic individuals with impaired glucose tolerance. Our findings demonstrate a progressive, but disharmonic, remodelling of mature beta cells, challenging current hypotheses of linear trajectories toward precursor or transdifferentiation stages in T2D. Furthermore, through integration of islet transcriptomics with preoperative blood plasma lipidomics, we define the relative importance of gene coexpression modules and lipids that are positively or negatively associated with HbA1c levels, pointing to potential prognostic markers.

Liver receptor homolog 1 is a negative regulator of the hepatic acute-phase response.

The orphan nuclear receptor liver receptor homolog 1 (LRH-1) has been reported to play an important role in bile acid biosynthesis and reverse cholesterol transport. Here, we show that LRH-1 is a key player in the control of the hepatic acute-phase response. Ectopic expression of LRH-1 with adenovirus resulted in strong inhibition of both interleukin-6 (IL-6)- and IL-1beta-stimulated haptoglobin, serum amyloid A, and fibrinogen beta gene expression in hepatocytes. Furthermore, induction of the hepatic inflammatory response was significantly exacerbated in HepG2 cells expressing short hairpin RNA targeting LRH-1 expression. Moreover, transient-transfection experiments and electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that LRH-1 regulates this cytokine-elicited inflammatory response by, at least in part, antagonizing the CCAAT/enhancer binding protein beta signaling pathway. Finally, we show, by using LRH-1 heterozygous mice, that LRH-1 is involved in the control of the inflammatory response at the hepatic level in vivo. Taken together, our results outline an unexpected role for LRH-1 in the modulation of the hepatic acute-phase response.

Intestine-specific regulation of PPARalpha gene transcription by liver X receptors.

Liver X receptor-alpha (LXRalpha) and LXRbeta are ligand-activated transcription factors belonging to the nuclear receptor superfamily. They have been identified as key players in cholesterol homeostasis and lipid and glucose metabolism as well as immune and inflammatory responses. In the small intestine, LXRs have been shown not only to regulate cholesterol absorption and excretion but also to promote high-density lipoprotein biogenesis via the ATP-binding cassette A1 signaling pathway. Here, using gene expression assays, we identified PPARalpha as an intestine-specific LXR target gene. Chronic administration of LXR synthetic agonists led to a significant increase of PPARalpha mRNA levels in the small intestine but not in the liver. In addition, this specific PPARalpha gene up-regulation occurred in the duodenum, jejunum, and ileum in a dose-dependent manner and translated at the protein level as demonstrated by Western blot analysis. Furthermore, PPARalpha gene induction was completely abolished in LXR-deficient mice. Finally, the physiological relevance of LXR-mediated PPARalpha up-regulation in the small intestine was assessed in PPARalpha-deficient mice. Administration of a synthetic LXR agonist to wild-type mice led to the induction of several PPARalpha target genes including PDK4 and CPT1. Those effects were completely abolished in PPARalpha-deficient mice, demonstrating the biological relevance of this LXR-PPARalpha transcriptional cascade. Taken together, these results demonstrate that PPARalpha is an intestine-specific LXR target gene and suggest the existence of a transcriptional cross talk between those members of the nuclear receptor superfamily.

Comparative genotypic and phenotypic analysis of human peripheral blood monocytes and surrogate monocyte-like cell lines commonly used in metabolic disease research.

Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, IDO1, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.

Identification of small molecule agonists of the orphan nuclear receptors liver receptor homolog-1 and steroidogenic factor-1.

We report the identification of substituted cis-bicyclo[3.3.0]-oct-2-enes as small molecule agonists of subfamily V orphan nuclear receptors (NR5A), liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). Using fluorescence resonance energy transfer (FRET)-based biochemical assays, compound 5a (GSK8470) was identified as a high-affinity ligand for LRH-1 and SF-1. In liver cells, 5a increased the expression of the LRH-1 target gene small heterodimer partner (SHP). Synthesis of analogues modified at three positions led to the development of compounds with functional selectivity between LRH-1 and SF-1.

Rosiglitazone, a peroxisome proliferator-activated receptor-gamma, inhibits the Jun NH(2)-terminal kinase/activating protein 1 pathway and protects the heart from ischemia/reperfusion injury.

This study was conducted to evaluate whether treatment of normal and diabetic rat hearts with rosiglitazone, a high-affinity ligand of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) used for the treatment of type 2 diabetes, improves postischemic functional recovery. The effects of acute rosiglitazone administration were investigated using working hearts isolated from normal rat or rats diabetic for 4 weeks after streptozotocin (STZ) injection. Hearts were subjected to 30 min of normothermic, zero-flow ischemia followed by 30-min reperfusion. Rosiglitazone (1 micromol/l) administered before ischemia had no effect on cardiac function during baseline perfusion, but it significantly improved aortic flow during reperfusion in both normal and diabetic hearts. In a chronic protocol in which rosiglitazone was given by daily gavage (10 micromol/kg body wt) immediately after STZ injection, rosiglitazone also prevented postischemic injury and significantly improved functional recovery. Using Western immunoblotting, it was demonstrated that the acute cardioprotective effect of rosiglitazone is associated with an inhibition of Jun NH(2)-terminal kinase phosphorylation in both normal and diabetic rat hearts. Furthermore, rosiglitazone also inhibited activating protein-1 DNA-binding activity. These data, demonstrating that rosiglitazone limits postischemic injury in isolated hearts, suggest an important function for PPAR-gamma in the heart.

DNA binding-independent induction of IkappaBalpha gene transcription by PPARalpha.

PPARs are ligand-activated transcription factors that regulate energy homeostasis. In addition, PPARs furthermore control the inflammatory response by antagonizing the nuclear factor-kappaB (NF-kappaB) signaling pathway. We recently demonstrated that PPARalpha activators increase IkappaBalpha mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPARalpha controls IkappaBalpha expression. Using transient transfection assays, it is demonstrated that PPARalpha potentiates p65-stimulated IkappaBalpha transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPARalpha activation of IkappaBalpha transcription requires the NF-kappaB and Sp1 sites within IkappaBalpha promoter. Chromatin immunoprecipitation assays demonstrate that PPARalpha activation enhances the occupancy of the NF-kappaB response element in IkappaBalpha promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPARalpha-mediated IkappaBalpha promoter induction, suggesting that cAMP response element binding protein-binding protein/p300 is not involved in this mechanism. By contrast, a dominant-negative form of VDR-interacting protein 205 (DRIP205) comprising its two LXXLL motifs completely abolished PPARalpha ligand-mediated activation. Furthermore, cotransfection of increasing amounts of DRIP205 relieved this inhibition, suggesting that PPARalpha requires DRIP205 to regulate IkappaBalpha promoter activity. By contrast, DRIP205 is not involved in PPARalpha-mediated NF-kappaB transcriptional repression. Taken together, these data provide a molecular basis for PPARalpha-mediated induction of IkappaBalpha and demonstrate, for the first time, that PPARalpha may positively regulate gene transcription in the absence of functional PPAR response elements.

Identification of liver receptor homolog-1 as a novel regulator of apolipoprotein AI gene transcription.

The orphan nuclear receptor liver receptor homolog-1 (LRH-1) has been reported to play a role in bile acid biosynthesis and reverse cholesterol transport. In this study, we examined the role of LRH-1 in the regulation of the apolipoprotein AI (APOAI) gene. Using RNA interference and adenovirus-mediated overexpression, we show that LRH-1 directly regulates APOAI gene transcription. Transient transfection experiments and EMSAs revealed that LRH-1 directly regulates APOAI transcription by binding to an LRH-1 response element located in the proximal APOAI promoter region. Chromatin immunoprecipitation experiments revealed that LRH-1 binds to the human APO AI promoter in vivo. Finally, we show that the transcriptional repressor SHP (small heterodimer partner) suppressed APOAI gene expression by inhibiting LRH-1 transcriptional activity. Taken together, our results demonstrate that LRH-1 is a novel regulator of APOAI transcription and underscore the role of this receptor in cholesterol homeostasis.