Ellen H. Jeninga

Role of ADP Receptor P2Y12 in Platelet Adhesion and Thrombus Formation in Flowing Blood

ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y1 and P2Y12. We have investigated the role of P2Y12 in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y12. Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient’s thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y12 antagonist, N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-&bgr;,&ggr;-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y1 antagonist (adenosine-3′,5′-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y12 interaction is essential for normal thrombus buildup on collagen. The patient’s blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3′,5′-diphospate. This suggested that P2Y12 and P2Y1 were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase.

Functional implications of genetic variation in human PPARγ

The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism. Human genetic evidence supporting this view comes from the study of both common (e.g. the Pro12Ala polymorphism) and rare (loss-of-function mutations) variants in the gene encoding PPARgamma. Indeed, patients harbouring mutant PPARgamma exhibit familial partial lipodystrophy type 3 and an extreme monogenic form of the metabolic syndrome. The recent elucidation of the crystal structure of the full-length PPARgamma-RXRalpha heterodimer bound to DNA has shed new light on the functional consequences of these genetic PPARgamma alterations and provides novel insights as to why different perturbations of receptor function unite in a common pathway of metabolic dysfunction.

Peroxisome proliferator-activated receptor gamma regulates expression of the anti-lipolytic G-protein-coupled receptor 81 (GPR81/Gpr81).

The ligand-inducible nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in the differentiation, maintenance, and function of adipocytes and is the molecular target for the insulin-sensitizing thiazoledinediones (TZDs). Although a number of PPARγ target genes that may contribute to the reduction of circulating free fatty acids after TZD treatment have been identified, the relevant PPARγ target genes that may exert the anti-lipolytic effect of TZDs are unknown. Here we identified the anti-lipolytic human G-protein-coupled receptor 81 (GPR81), GPR109A, and the (human-specific) GPR109B genes as well as the mouse Gpr81 and Gpr109A genes as novel TZD-induced genes in mature adipocytes. GPR81/Gpr81 is a direct PPARγ target gene, because mRNA expression of GPR81/Gpr81 (and GPR109A/Gpr109A) increased in mature human and murine adipocytes as well as in vivo in epididymal fat pads of mice upon rosiglitazone stimulation, whereas small interfering RNA-mediated knockdown of PPARγ in differentiated 3T3-L1 adipocytes showed a significant decrease in Gpr81 protein expression. In addition, chromatin immunoprecipitation sequencing analysis in differentiated 3T3-L1 cells revealed a conserved PPAR:retinoid X receptor-binding site in the proximal promoter of the Gpr81 gene, which was proven to be functional by electromobility shift assay and reporter assays. Importantly, small interfering RNA-mediated knockdown of Gpr81 partly reversed the inhibitory effect of TZDs on lipolysis in 3T3-L1 adipocytes. The coordinated PPARγ-mediated regulation of the GPR81/Gpr81 and GPR109A/Gpr109A genes (and GPR109B in humans) presents a novel mechanism by which TZDs may reduce circulating free fatty acid levels and perhaps ameliorate insulin resistance in obese patients.

The Multiple Endocrine Neoplasia Type 1 (MEN1) Tumor Suppressor Regulates Peroxisome Proliferator-Activated Receptor γ-Dependent Adipocyte Differentiation

ABSTRACT Menin, the product of the MEN1 (multiple endocrine neoplasia type 1) tumor suppressor gene, is involved in activation of gene transcription as part of an MLL1 (mixed-lineage leukemia 1)/MLL2 (KMT2A/B)-containing protein complex which harbors methyltransferase activity for lysine 4 of histone H3 (H3K4). As MEN1 patients frequently develop lipomas and peroxisome proliferator-activated receptor γ (PPARγ) is expressed in several MEN1-related tumor types, we investigated regulation of PPARγ activity by menin. We found that menin is required for adipocyte differentiation of murine 3T3-L1 cells and PPARγ-expressing mouse embryonic fibroblasts. Menin augments PPARγ target gene expression through recruitment of H3K4 methyltransferase activity. Menin interacts directly with the activation function 2 transcription activation domain of PPARγ in a ligand-independent fashion. Ligand-dependent coactivation, however, is dependent on the LXXLL motif of menin and the intact helix 12 of PPARγ. We propose that menin is an important factor in PPARγ-mediated adipogenesis and that loss of PPARγ function may contribute to lipoma development in MEN1 patients.

Nuclear Receptor-Coregulator Interaction Profiling Identifies TRIP3 as a Novel Peroxisome Proliferator-activated Receptor γ Cofactor

Nuclear receptors (NRs) are major targets for drug discovery and have key roles in development and homeostasis as well as in many diseases such as obesity, diabetes, and cancer. NRs are ligand-dependent transcription factors that need to work in concert with so-called transcriptional coregulators, including corepressors and coactivators, to regulate transcription. Upon ligand binding, NRs undergo a conformational change, which alters their binding preference for coregulators. Short α-helical sequences in the coregulator proteins, LXXLL (in coactivators) or LXXXIXXXL (in corepressors), are essential for the NR-coregulator interactions. However, little is known on how specificity is dictated. To obtain a comprehensive overview of NR-coregulator interactions, we used a microarray approach based on interactions between NRs and peptides derived from known coregulators. Using the peroxisome proliferator-activated receptor γ (PPARγ) as a model NR, we were able to generate ligand-specific interaction profiles (agonist rosiglitazone versus antagonist GW9662 versus selective PPARγ modulator telmisartan) and characterize NR mutants and isotypes (PPARα, -β/δ, and -γ). Importantly, based on the NR-coregulator interaction profile, we were able to identify TRIP3 as a novel regulator of PPARγ-mediated adipocyte differentiation. These findings indicate that NR-coregulator interaction profiling may be a useful tool for drug development and biological discovery.

PPARγ regulates expression of the anti-lipolytic G-protein-coupled receptor 81 (GPR81/Gpr81)

The ligand-inducible nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in the differentiation, maintenance, and function of adipocytes and is the molecular target for the insulin-sensitizing thiazoledinediones (TZDs). Although a number of PPARγ target genes that may contribute to the reduction of circulating free fatty acids after TZD treatment have been identified, the relevant PPARγ target genes that may exert the anti-lipolytic effect of TZDs are unknown. Here we identified the anti-lipolytic human G-protein-coupled receptor 81 (GPR81), GPR109A, and the (human-specific) GPR109B genes as well as the mouse Gpr81 and Gpr109A genes as novel TZD-induced genes in mature adipocytes. GPR81/Gpr81 is a direct PPARγ target gene, because mRNA expression of GPR81/Gpr81 (and GPR109A/Gpr109A) increased in mature human and murine adipocytes as well as in vivo in epididymal fat pads of mice upon rosiglitazone stimulation, whereas small interfering RNA-mediated knockdown of PPARγ in differentiated 3T3-L1 adipocytes showed a significant decrease in Gpr81 protein expression. In addition, chromatin immunoprecipitation sequencing analysis in differentiated 3T3-L1 cells revealed a conserved PPAR:retinoid X receptor-binding site in the proximal promoter of the Gpr81 gene, which was proven to be functional by electromobility shift assay and reporter assays. Importantly, small interfering RNA-mediated knockdown of Gpr81 partly reversed the inhibitory effect of TZDs on lipolysis in 3T3-L1 adipocytes. The coordinated PPARγ-mediated regulation of the GPR81/Gpr81 and GPR109A/Gpr109A genes (and GPR109B in humans) presents a novel mechanism by which TZDs may reduce circulating free fatty acid levels and perhaps ameliorate insulin resistance in obese patients.

Central players in inherited lipodystrophies

Common obesity and inherited lipodystrophies, rare disorders characterized by a partial (familial partial lipodystrophy; FPLD) or complete (congenital generalized lipodystrophy; CGL) lack of adipose tissue, are both associated with metabolic complications such as insulin resistance and type 2 diabetes. Mutations in the transcription factor peroxisome proliferator activated receptor (PPAR)γ and a number of its downstream target genes result in lipodystrophy. We hypothesize that signalling by another transcription factor, sterol response element binding protein (SREBP)1c, also needs to be intact to prevent lipodystrophy. The future challenge is to understand how inactivation of such central players or of their upstream regulators or downstream effectors can affect adipose tissue in a depot-specific fashion.

A Patient with Congenital Generalized Lipodystrophy Due To a Novel Mutation in BSCL2: Indications for Secondary Mitochondrial Dysfunction

BACKGROUND Congenital generalized lipodystrophy (CGL) results from mutations in AGPAT2, encoding 1-acyl-glycerol-3-phosphate-acyltransferase 2 (CGL1; MIM 608594), BSCL2, encoding seipin (CGL2; MIM 269700), CAV1, encoding caveolin1 (CGL3; MIM 612526) or PTRF, encoding polymerase I and transcript release factor (CGL4; MIM 613327). This study aims to investigate the genotype/phenotype relationship and search for a possible pathogenic mechanism in a patient with CGL. DESIGN Case report. PATIENTS AND SETTING A 7-day-old child of consanguineous Turkish parents presented with a generalized loss of subcutaneous fat. He had a strikingly enlarged liver, high serum triglycerides, and hyperglycaemia, suggestive for CGL. RESULTS A novel homozygous mutation in the acceptor splice site of exon 5 of the BSCL2 gene was found in the genome of the proband. This mutation causes a complex RNA splicing defect and results in two different aberrant seipin proteins, which were normally expressed and localized to the endoplasmic reticulum like wild type protein. Analysis of the patients urine showed intermittent elevations of citric acid intermediates and persistently high concentrations of ethylmalonic acid, suggestive of a disturbance of the mitochondrial respiratory chain. CONCLUSION Here we report abnormal urinary organic acid levels, indicative of mitochondrial dysfunction, in a patient with CGL resulting from a novel mutation in BSCL2. Our findings suggest for the first time an association between CGL and secondary mitochondrial dysfunction.

Nuclear receptor-coregulator interaction profiling identifies TRIP3 as a novel PPARγ cofactor

Nuclear receptors (NRs) are major targets for drug discovery and have key roles in development and homeostasis as well as in many diseases such as obesity, diabetes, and cancer. NRs are ligand-dependent transcription factors that need to work in concert with so-called transcriptional coregulators, including corepressors and coactivators, to regulate transcription. Upon ligand binding, NRs undergo a conformational change, which alters their binding preference for coregulators. Short α-helical sequences in the coregulator proteins, LXXLL (in coactivators) or LXXXIXXXL (in corepressors), are essential for the NR-coregulator interactions. However, little is known on how specificity is dictated. To obtain a comprehensive overview of NR-coregulator interactions, we used a microarray approach based on interactions between NRs and peptides derived from known coregulators. Using the peroxisome proliferator-activated receptor γ (PPARγ) as a model NR, we were able to generate ligand-specific interaction profiles (agonist rosiglitazone versus antagonist GW9662 versus selective PPARγ modulator telmisartan) and characterize NR mutants and isotypes (PPARα, -β/δ, and -γ). Importantly, based on the NR-coregulator interaction profile, we were able to identify TRIP3 as a novel regulator of PPARγ-mediated adipocyte differentiation. These findings indicate that NR-coregulator interaction profiling may be a useful tool for drug development and biological discovery.

Nuclear receptor-coregulator profiling identifies TRIP3 as a novel PPARγ cofactor

Nuclear receptors (NRs) are major targets for drug discovery and have key roles in development and homeostasis as well as in many diseases such as obesity, diabetes, and cancer. NRs are ligand-dependent transcription factors that need to work in concert with so-called transcriptional coregulators, including corepressors and coactivators, to regulate transcription. Upon ligand binding, NRs undergo a conformational change, which alters their binding preference for coregulators. Short α-helical sequences in the coregulator proteins, LXXLL (in coactivators) or LXXXIXXXL (in corepressors), are essential for the NR-coregulator interactions. However, little is known on how specificity is dictated. To obtain a comprehensive overview of NR-coregulator interactions, we used a microarray approach based on interactions between NRs and peptides derived from known coregulators. Using the peroxisome proliferator-activated receptor γ (PPARγ) as a model NR, we were able to generate ligand-specific interaction profiles (agonist rosiglitazone versus antagonist GW9662 versus selective PPARγ modulator telmisartan) and characterize NR mutants and isotypes (PPARα, -β/δ, and -γ). Importantly, based on the NR-coregulator interaction profile, we were able to identify TRIP3 as a novel regulator of PPARγ-mediated adipocyte differentiation. These findings indicate that NR-coregulator interaction profiling may be a useful tool for drug development and biological discovery.