Tatjana Albrektsen

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

Summary.  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.

Peroxisome proliferator-activated receptor expression and activation in normal human colonic epithelial cells and tubular adenomas

Objective Peroxisome proliferator-activated receptor (PPAR) ligands, widely used in type 2 diabetes treatment, have variably been shown to promote or prevent colon tumor formation in animal models and cell lines, but their role in normal human colon is unknown. The aim of this study was to determine PPARδ expression and function in normal human colonic epithelial cells and tubular adenomas. Material and methods Short-term cultures of normal human colonic epithelial cells were established from biopsies obtained in 42 patients with normal colonoscopy. PPAR and adipophilin mRNA expression was assessed by real-time RT-PCR. PPARs were activated by ligands for PPARα (Wy-14643), PPARδ (GW-501516) and PPARγ (rosiglitazone or troglitazone). Cell viability was measured using the methyltetrazoleum assay, proliferation by thymidine incorporation, and DNA profiles by flow cytometry. PPAR mRNA levels in tubular adenomas or metaplastic polyps (n=12) were compared with those in controls. Results PPARα and γ were consistently expressed in normal colonocytes while no PPAR expression could be detected. PPARγ activation induced a 7.5-fold increase in adipophilin expression (a PPAR-activated gene). PPARγ activation had no effect on viability or DNA profiles, but led to a 25% significant decrease in cell proliferation. Finally, a selective and significant 2.5-fold decrease in PPARα expression was observed in tubular adenomas, but not in metaplastic polyps, compared to controls. Conclusions Our findings support the view that PPARγ ligands act as anti-proliferative agents rather than as promoters of tumorigenesis in normal human colon. Moreover, they raise interest in investigation of PPARα as a therapeutic target to prevent adenoma formation.

Design and synthesis of novel PPARα/γ/δ triple activators using a known PPARα/γ dual activator as structural template

Abstract Using a known dual PPARα/γ activator (5) as a structural template, SAR evaluations led to the identification of triple PPARα/γ/δ activators (18–20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARα/γ/δ activation.

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.

Factor VIIa/tissue factor-dependent gene regulation and pro-coagulant activity: Effect of factor VIIa concentration

Factor VIIa/tissue factor-dependent gene regulation and pro-coagulant activity: Effect of factor VIIa concentration -

Health Care Discovery, Novo Nordisk A/S, DK-2880 Bagsværd, Denmark

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.