A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

BACKGROUND: The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. OBJECTIVES: To determine the functional consequences of the R122C substitution for P2Y(12) function. PATIENT/METHODS: We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. RESULTS: ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. CONCLUSIONS: Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait.

Mitotic clonal expansion during preadipocyte differentiation: calpain-mediated turnover of p27.

Evidence is presented that calpain, a calcium-activated protease, degrades the cyclin-dependent kinase inhibitor, p27, during the mitotic clonal expansion phase of 3T3-L1 preadipocyte differentiation. Calpain activity is required during an early stage of the adipocyte differentiation program. Thus, inhibition of calpain with N-acetyl-Leu-Leu-norleucinal (ALLN) blocks clonal expansion and acquisition of the adipocyte phenotype only when added between 12 and 24 h after the induction of differentiation. Likewise, inhibition of calpain by overexpression of calpastatin, the specific endogenous inhibitor of calpain, prevents 2-day post-confluent preadipocytes from reentering the cell cycle triggered by the differentiation inducers. Inhibition of calpain with ALLN causes preadipocytes to arrest just prior to S phase and prevents phosphorylation of the retinoblastoma gene product, DNA replication, clonal expansion, and subsequent adipocyte differentiation but does not affect the expression of immediate early genes (i.e. fos, jun, C/EBPbeta, and C/EBPdelta). Inhibition of calpain by either ALLN or by overexpression of calpastatin blocks the degradation of p27. p27 is degraded in vitro by cell-free extracts from clonally expanding preadipocytes that contain "active" calpain but not by extracts from pre-mitotic preadipocytes that do not. This action is inhibited by calpastatin or ALLN. Likewise, p27 in preadipocyte extracts is a substrate for purified calpain; this proteolytic action was inhibited by heat inactivation, EGTA, or ALLN. Thus, extracellular signals from the differentiation inducers appear to activate calpain, which degrades p27 allowing density-dependent inhibited preadipocytes to reenter the cell cycle and undergo mitotic clonal expansion.

Role of calpain in adipocyte differentiation.

Evidence is presented that the calcium-activated protease, calpain, is required for differentiation of 3T3-L1 preadipocytes into adipocytes induced by methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin. Calpain is expressed by preadipocytes and its level falls during differentiation. Exposure of preadipocytes to the calpain inhibitor N-acetyl-Leu-Leu-norleucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, blocks expression of adipocyte-specific genes, notably the CCAAT/enhancer-binding protein (C/EBP)alpha gene, and acquisition of the adipocyte phenotype. The inhibitor disrupts the differentiation-inducing effect of methylisobutylxanthine (by means of the cAMP-signaling pathway), but is without effect on differentiation induced by dexamethasone or insulin. N-acetyl-Leu-Leu-norleucinal, or overexpression of calpastatin, inhibits reporter gene expression mediated by the C/EBPalpha gene promoter by preventing C/EBPbeta, a transcriptional activator of the C/EBPalpha gene, from binding to the promoter. These findings implicate calpain in the transcriptional activation of the C/EBPalpha gene, a process required for terminal adipocyte differentiation.

Environmental endocrine disruption: an effects assessment and analysis.

This report is an overview of the current state of the science relative to environmental endocrine disruption in humans, laboratory testing, and wildlife species. Background information is presented on the field of endocrinology, the nature of hormones, and potential sites for endocrine disruption, with specific examples of chemicals affecting these sites. An attempt is made to present objectively the issue of endocrine disruption, consider working hypotheses, offer opposing viewpoints, analyze the available information, and provide a reasonable assessment of the problem. Emphasis is placed on disruption of central nervous system--pituitary integration of hormonal and sexual behavioral activity, female and male reproductive system development and function, and thyroid function. In addition, the potential role of environmental endocrine disruption in the induction of breast, testicular, and prostate cancers, as well as endometriosis, is evaluated. The interrelationship of the endocrine and immune system is documented. With respect to endocrine-related ecological effects, specific case examples from the peer-reviewed literature of marine invertebrates and representatives of the five classes of vertebrates are presented and discussed. The report identifies some data gaps in our understanding of the environmental endocrine disruption issue and recommends a few research needs. Finally, the report states the U.S. Environmental Protection Agency Science Policy Council's interim position on endocrine disruption and lists some of the ongoing activities to deal with this matter.

Metabolic regulation of gene transcription.

The impact of nutrients on gene expression has become an area of considerable interest as the number of genes coding for key regulatory proteins in metabolic pathways are studied in detail. This has been greatly aided by a number of new techniques developed to study gene transcription in animals. We will use as an example studies on the regulation of transcription of the gene coding for P-enolpyruvate carboxykinase, a key enzyme in hepatic and renal gluconeogenesis. The promoter for P-enolpyruvate carboxykinase contains a number of regulatory elements within 500 bp of the start-site of gene transcription that are required for the response of the gene to metabolic signals. These elements bind tissue-specific transcription factors in complex patterns of interactions, which result in the coordinate control of P-enolpyruvate carboxykinase gene expression. An analysis of the regulation of transcription of this gene involves the use of a number of techniques ranging from gene transfection into cells in culture to the introduction of chimeric genes containing the P-enolpyruvate carboxykinase promoter into transgenic mice. This review presents a progress report on the current status of research on the nutritional and hormonal regulation of transcription of the P-enolpyruvate carboxykinase gene.

An analysis of regulatory elements in the phosphoenolpyruvate carboxykinase (GTP) gene which are responsible for its tissue-specific expression and metabolic control in transgenic mice.

Sequences in the gene for P-enolpyruvate carboxykinase (PEPCK) which are responsible for its complex pattern of transcriptional control were determined using transgenic mice containing a chimeric PEPCK-bovine growth hormone (bGH) gene consisting of a segment of the PEPCK promoter from -460 to +73, with mutations in specific regulatory domains. A mutation in the cAMP response element (CRE) (-87 to -74), which binds CCATT/enhancer-binding protein beta (C/EBP beta) and/or cAMP response element-binding protein (CREB), resulted in a 4- and 20-fold elevation in the level of bGH mRNA in the liver and kidney of transgenic mice, respectively. Expression of the PEPCK-bGH gene in the liver was reduced 60% by a mutation in the P3 (I) region (-248 to -230), whereas expression in the kidney was increased 10-fold by the same mutation. A mutation in the P2 region (-200 to -164) greatly reduced expression of the PEPCK-bGH gene in the kidney but not in the liver. Induction of hepatic PEPCK-bGH gene expression by Bt2cAMP was eliminated by mutations in the CRE, P1, P3(I), or by a double mutation of the CRE and P3(I). Mutations in the CRE or P3(I) regions of the PEPCK promoter did not interfere with the expected induction of the PEPCK-bGH gene in the liver at birth. None of the mutations in the PEPCK promoter interfered with the induction of transcription of the PEPCK-bGH gene in the liver when mice were fed a carbohydrate-free diet or the deinduction of transcription from the PEPCK promoter caused by a diet high in carbohydrate, whereas a mutation in P2 (an HNF-1 binding domain) eliminated dietary regulation of transcription of the transgene in the kidney. A model to explain the role of the various elements in the PEPCK promoter on the control of PEPCK gene transcription in the liver and kidney is presented.

Glucocorticoids regulate the induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes.

The hormonal regulation of transcription of the phosphoenolpyruvate carboxykinase (GTP) (4.1.1.32) (PEPCK) gene during diabetes was studied using transgenic mice containing a chimeric gene consisting of segments of the PEPCK promoter (-2000/+73, -460/+73, -355/+73) linked to bovine growth hormone (bGH) reporter gene. The effect of diabetes and insulin on transgenic mice containing a mutation in cAMP regulatory sequences at -90/-82 and -250/-234 was also studied. In addition, we analyzed the transcriptional response of the PEPCK gene to adrenalectomy, the administration of glucocorticoids, and alterations in dietary protein and carbohydrate. Our results indicate that deletion of the insulin regulatory sequence of the PEPCK promoter did not affect dietary control of PEPCK gene expression. However, glucocorticoids and the glucocorticoid regulatory unit appear to be essential for induction of PEPCK gene transcription by diabetes. By contrast, mutation of cAMP regulatory elements of the PEPCK promoter did not limit induction of PEPCK transcription by diabetes, nor did it affect negative regulation of transcription by insulin. These results provide evidence for the interaction of insulin and glucocorticoid regulatory elements in the control of PEPCK gene transcription and suggest an important role of glucocorticoids as a gluconeogenic activator during diabetes.

Metabolic control of gene expression: in vivo studies with transgenic mice.

Transgenic animals provide a comprehensive model for investigating genes encoding inducible enzymes involved in metabolism, since the molecular mechanisms regulating gene transcription can be studied in the whole animal. Studies on the promoters of the genes encoding two key enzymes in the gluconeogenic and glycolytic pathways--phosphoenol-pyruvate carboxykinase and pyruvate kinase are described as examples of this approach. Work on the phosphoenolpyruvate carboxykinase promoter using transgenic mice has been particularly informative: the cis-acting elements involved in hormonal regulation, tissue specificity and developmental inhibition of gene expression have been identified and their function in vivo examined.