Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDA-MB-231 cells.

BACKGROUND: Factor VIIa (FVIIa) binding to tissue factor (TF) induces cell signaling via the protease activity of FVIIa and protease-activated receptor 2 (PAR2). OBJECTIVE: We examined how the gene-expression profile induced by FVIIa corresponds to the profiles induced by protease-activated receptor 1 (PAR1) or PAR2 agonists using MDA-MB-231 breast carcinoma cells that constitutively express TF, PAR1 and PAR2. RESULTS AND CONCLUSIONS: Out of 8500 genes, FVIIa stimulation induced differential regulation of 39 genes most of which were not previously recognized as FVIIa regulated. All genes regulated by FVIIa were similarly regulated by a PAR2 agonist peptide confirming FVIIa signaling via PAR2. An appreciable fraction of the PAR2-regulated genes was also regulated by a PAR1 agonist peptide suggesting extensive redundancy between FVIIa/PAR2 signaling and thrombin/PAR1 signaling. The FVIIa regulated genes encode cytokines, chemokines and growth factors, and the gene repertoire induced by FVIIa in MDA-MB-231 cells is consistent with a role for TF-FVIIa signaling in regulation of a wound healing type of response. Interestingly, a number of genes regulated exclusively by FVIIa/PAR2-mediated cell signaling in MDA-MB-231 cells were regulated by thrombin and a PAR1 agonist, but not by FVIIa, in the TF-expressing glioblastoma U373 cell line.

The role of signal transducer and activator of transcription 5 in the inhibitory effects of GH on adipocyte differentiation.

GH inhibits primary rat preadipocyte differentiation and expression of late genes required for terminal differentiation. Here we show that GH-mediated inhibition of fatty acid-binding protein aP2 gene expression correlates with the activation of the Janus kinase-2/signal transducer and activator of transcription (STAT)-5 signalling pathway. Within minutes of treatment, GH induced the tyrosine phosphorylation, nuclear localization and DNA binding of STAT5. Importantly, there was no evidence that STAT5 acted via an interaction with peroxisome proliferator-activated receptor gamma. To further understand the mechanism of STAT5 action, we reconstituted the inhibition of aP2 in a non-adipogenic cell line. Using this system, we showed that the ability of GH to inhibit a 520 bp aP2 reporter was largely dependent upon the presence of either STAT5A or STAT5B. Mutant analysis confirmed that the tyrosine phosphorylation of STAT5 was essential for this signalling. However, STAT5's C-terminal transactivation domain was fully dispensable for this inhibition. Taken together, these data confirm a key regulatory role of STAT5 in adipose tIssue and point to STAT5 as the repressing modulator of GH-mediated inhibition in primary preadipocytes.

Identification of a novel integral plasma membrane protein induced during adipocyte differentiation.

Adipocyte differentiation is co-ordinately regulated by several transcription factors and is accompanied by changes in the expression of a variety of genes. Using mRNA differential display analysis, we have isolated a novel mRNA, DD16, specifically induced during the course of adipocyte differentiation. DD16 mRNAs are present in several tissues, but among the tissues tested, a remarkably higher level of expression was found in white adipose tissue. The DD16 cDNA encoded a polypeptide of 415 amino acids containing a single N-glycosylation site and an N-terminal hydrophobic stretch of 19 amino acids forming a transmembrane segment, indicating that DD16 is a glycosylated membrane-bound protein. Polyclonal antibodies raised against the DD16 peptide detected immunoreactive DD16 in membrane fractions, notably the plasma membrane. Association of DD16 with the plasma membrane was further confirmed by biotinylation studies of cell surface proteins, suggesting that DD16 is an integral plasma membrane protein. Therefore we propose to give DD16 the name APMAP (Adipocyte Plasma Membrane-Associated Protein). Although the biological function of this polypeptide is presently unknown, our data suggest that APMAP may function as a novel protein involved in the cross-talk of mature adipocytes with the environment.

The transcription factor Fos-related antigen 1 is induced by thiazolidinediones during differentiation of 3T3-L1 cells.

Thiazolidinediones (TZDs) are a new class of compounds that improve the insulin sensitivity in patients with non-insulin-dependent diabetes mellitus (NIDDM) as well as in rodent models of NIDDM. These compounds act as high-affinity ligands for a member of the nuclear hormone receptor superfamily PPARgamma, which has been shown to play an important role in adipocyte differentiation. The strong correlation between the antidiabetic activity of TZDs and their ability to activate PPARgamma has led to suggestions that PPARgamma or downstream regulated genes mediate the effects of TZDs. To identify novel genes that potentially mediate the effects of TZDs, we have isolated genes that are differentially expressed during thiazolidinedione-stimulated differentiation of 3T3-L1 cells. Using mRNA differential display, we have compared 3T3-L1 cells treated to differentiate in the presence of BRL49653 with untreated 3T3-L1 cells and identified Fos-related antigen 1 (Fra-1), a member of the Fos protein family, as a novel molecular target for BRL49653 action in 3T3-L1 cells. Analysis of all members of the Fos-Jun family of transcription factors showed that Fra-1 was the only member that was specifically up-regulated by BRL49653. The only other member of the Fos-Jun family expressed in differentiated 3T3-L1 cells was JunD and a complex of Fra-1 and JunD was formed on a consensus AP-1 binding element in differentiated 3T3-L1 cells, suggesting that the complex of Fra-1 and JunD may play a role in the stimulation of the differentiation process of 3T3-L1 cells observed after treatment of the cells with insulin sensitizers.

Modulation of metabolism through transcriptional control has created new treatment opportunities for type 2 diabetes.

The discovery of the important metabolic and physiological role played by a family of transcription factors, the peroxisome proliferator activated receptors (PPAR), has opened up for a new understanding of the mode of action for the lipid lowering drugs known as fibrates and for the new glucose lowering compounds described as insulin sensitizers. Both of these classes of compounds have demonstrated significant efficacy in both animal models of the metabolic derangements characteristic for type 2 diabetes and in human clinical studies. The recognition of the role of these drugs as ligands for PPAR transcription factors and the development of new molecular and cellular tools to select and characterise new PPAR selective compounds will open up for the development of even better new drug candidates for the treatment of metabolic disorders associated with type 2 diabetes. With the combined strength of new transcriptional mapping technologies developed in the field of molecular biology, such as differential mRNA display and DNA microarray hybridisations, it will be possible to perform a detailed molecular characterisation of the transcriptional events involved in drug actions in cellular and tissue systems, and information gathered from such types of analysis will lead to an enormous amount of data, from which detailed knowledge of drug actions at the gene regulatory level will emerge.

Rat peroxisome proliferator-activated receptors and brown adipose tissue function during cold acclimatization.

Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARalpha and PPARgamma mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal beta-oxidation enzymes. In contrast, PPARdelta mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT.

A regulatory role for RIP140 in nuclear receptor activation.

Transcriptional regulation of gene expression by nuclear receptors requires negatively and positively acting cofactors. Recent models for receptor activation propose that certain receptors in the absence of ligands can recruit corepressors while ligand binding results in conformational changes leading to the recruitment of coactivators. Previous work has established a coactivator role for the SRC-1 family members as well as an involvement of the coactivators CBP/p300 in nuclear receptor signaling. However, in addition to coactivators, ligand-activated nuclear receptors bind a number of different proteins that possibly serve other functions. Using peroxisome proliferator-activated receptor-alpha (PPAR alpha) as bait in a yeast two-hybrid screening, we have isolated nuclear factor RIP140 whose function in receptor activation is unclear. We now report a detailed characterization of RIP140 action with a focus on the retinoid X receptor (RXR) heterodimeric receptors PPAR and thyroid hormone receptor (TR). We show that putative PPAR ligands enhance the interaction of RIP140 with the rat PPAR subtypes alpha and gamma in solution but not with PPAR/RXR heterodimers on DNA. However, RIP140 forms ternary complexes in the presence of RXR ligands. Similar experiments with TR support the high affinity of RIP140 to the RXR subunit and also suggest that either partner in the TR/RXR heterodimer can independently respond to ligand. Coactivation experiments in yeast and mammalian cells confirm the coactivator role for SRC-1, but not for RIP140. We provide important evidence that the in vitro binding of RIP140 and SRC-1 to nuclear receptors is competitive. Since RIP140 generally down-regulates receptor activity in mammalian cells and specifically down-regulates coactivation mediated by SRC-1, we propose a model in which RIP140 indirectly regulates nuclear receptor AF-2 activity by competition for coactivators such as SRC-1.

Effects of 100 mg of controlled-release metoprolol and 100 mg of atenolol on blood pressure, central nervous system-related symptoms, and general well being.

Central nervous system (CNS)-related symptoms and quality of life during treatment with controlled-release (CR) metoprolol and a standard formulation of atenolol were compared in a double-blind crossover study in 60 patients with mild to moderate hypertension. After a 4-week placebo run-in period, each beta 1-adrenoceptor blocker was administered at a dosage of 100 mg once daily for 6 weeks. Quality of life was assessed regularly during the active treatment phases by use of two standardized self-administered questionnaires, the minor symptom evaluation (MSE) profile, and the psychologic general well-being (PGWB) index. Both questionnaires have previously been shown to be effective in detecting CNS symptoms and changes in well being produced by beta-blockers. Blood pressure and heart rate were monitored to assess the antihypertensive efficacy of the two drugs. Metoprolol CR and atenolol produced equivalent, clinically effective reductions in systolic and diastolic blood pressures measured 24 hours after administration. The drugs were found to exert similar effects on general well being, as assessed by the PGWB index, and there were no significant differences between the two treatments with regard to the three dimensions of the MSE profile, contentment, vitality, and sleep. Thus, at equivalent antihypertensive dosages, metoprolol CR and atenolol are clinically comparable with regard to the degree of CNS-related symptoms produced and effects on general well being. Because these agents differ markedly in lipophilicity, other factors, such as beta 1-selectivity/nonselectivity, may be more important determinants of whether these subjective symptoms occur during therapy with beta-blockers.

Comparison of the acceptability of the Ventolin metered-dose inhaler and the Bricanyl Turbuhaler.

One hundred fifty-nine asthmatic patients aged 19 to 76 years completed an open crossover-designed study comparing the acceptability of the Bricanyl Turbuhaler (BTH) and the Ventolin metered-dose inhaler (VMDI). The doses given were presumed to be equiactive. Each treatment period lasted for 14 days. Patients registered morning and evening peak expiratory flow rate (PEFR) before and five minutes after medication. Asthma symptoms as well as adverse events were registered on diary cards. After each study period a questionnaire containing acceptability questions was completed. At the end of the study the patients indicated their preference for one of the two treatments. The two treatments were equal concerning bronchodilation and asthma symptoms. There was an overall preference for the Bricanyl Turbuhaler.

Pilocarpine drops do not reduce intraocular pressure sufficiently in pseudoexfoliation glaucoma.

The reason for the poorer prognosis of pseudoexfoliation syndrome glaucomas (PXSG) compared with primary open angle glaucomas (POAG) is not fully understood. An open, comparative, cross-over study was performed in 15 patients (= eyes) with POAG and 15 patients (= eyes) with PXSG. Two different pharmacokinetic principles of drug administration were applied to uncover possible differences in short-term (hours) response to topical antiglaucomatous treatment. Intermittent pilocarpine drop medication (2%) and continuous low-dose pilocarpine delivery by a membrane-controlled Ocusert unit (P40) were used. The 'carry-over' pressure reduction of an ordinary four times a day drop medication was significantly less effective in controlling the morning intraocular pressure (9 a.m.) in PXSG than in POAG. The duration of action of pilocarpine drops was reduced in PXSG. Defining 'normotensive' pressure as < or = 20 mmHg, only 1 of the 15 PXSG eyes (6.7%) reached a normotensive level in the morning, compared with 8 of the 15 POAG eyes (53.3%). Using a continuous supply of pilocarpine (Ocusert), no differences between POAG and PXSG eyes were found. The study demonstrates the insufficient control of intraocular pressure in PXSG, compared with POAG, by identical antiglaucomatous drop medications. This may suggest an insufficient depot function of topical drugs in PXSG. In consequence, pseudoexfoliation material (PXM) must be sought in eyes with glaucoma, as PXM eyes will probably benefit from a more intense medical treatment compared with eyes without PXM.