Tissue factor (TF), the cellular receptor for factor VIIa (FVIIa), besides initiating blood coagulation, is believed to play an important role in tissue repair, inflammation, angiogenesis, and tumor metastasis. Like TF, the chemokine interleukin-8 (IL-8) is shown to play a critical role in these processes. To elucidate the potential mechanisms by which TF contributes to tumor invasion and metastasis, we IntroductionCells that express tissue factor (TF) are usually not exposed to the blood. However, in normal response to vessel injury, TF exposure is an initial event of a strictly regulated process resulting in fibrin deposition, inflammation, angiogenesis, and tissue repair. Carcinomas exploit a normal physiologic response in a way that allows tumor growth and dissemination. It has long been presumed that tumors may take advantage of the hemostatic system. A relationship between increased clotting and malignancy was recognized more than a century ago. 1 Numerous clinical observations suggest that the hemostatic system is frequently activated in cancer patients. 2-5 Many tumor types have been shown to express TF. 6,7 Further, the level of TF expression in various tumor types has been shown to correlate with their metastatic potential. [8][9][10] Studies carried out with mouse tumor metastasis models establish that TF plays a critical role in tumor metastasis. 11,12 TF is the cellular receptor for coagulation factor VIIa (FVIIa). TF-induced metastasis requires participation of the cytoplasmic tail of TF and assembly of an active TF-FVIIa complex, 13,14 indicating a dual function for TF in tumor metastasis. The TF cytoplasmic domain, through its specific interaction with ABP-280, has been shown to support cell adhesion and migration. 15 At present it is unclear how TF on tumor cells contributes to tumor metastasis and whether the TF-FVIIa complex plays a direct role or whether its sole requirement is for the downstream generation of active coagulation factors, particularly thrombin, which have been implicated in tumor metastasis. [16][17][18] Recent studies show that proteolytic hydrolysis mediated by the TF-FVIIa complex induces cell signaling through G-proteincoupled receptors in a number of cell types (for reviews, see Prydz et al, 19 Pendurthi and Rao, 20 Ruf et al 21 ). TF-FVIIa-induced signaling in various cell types was shown to alter the expression of specific genes that encode transcription factors, growth factors, and proteins related to cellular reorganization. [22][23][24][25][26][27] These studies suggest that TF-FVIIa-induced signaling may play a role in growthpromoting settings, such as wound healing and cancer. However, it has yet to be shown how TF-FVIIa-induced regulation of gene expression actually affects cell phenotype or pathophysiologic processes. Moreover, a considerable overlap in signaling induced by TF-FVIIa and various other proteases, especially a highmagnitude response generated by thrombin, raises a valid question about the potential significance of TF-FVIIa-induced signaling in path...

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.

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.

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals. In vitro studies indicate that ACBP may regulate the availability of acylCoA esters for various metabolic and regulatory purposes. The protein is particularly abundant in cells with a high level of lipogenesis and de novo fatty acid synthesis and is significantly induced during adipocyte differentiation. However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown. Here we report that ACBP is a novel peroxisome proliferator-activated receptor (PPAR)␥ target gene. The rat ACBP gene is directly activated by PPAR␥/retinoid X receptor ␣ (RXR␣) and PPAR␣/RXR␣, but not by PPAR␦/RXR␣, through a PPAR-response element in intron 1, which is functionally conserved in the human ACBP gene. The intronic PPAR-response element (PPRE) mediates induction by endogenous PPAR␥ in murine adipocytes and confers responsiveness to the PPAR␥-selective ligand BRL49653. Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPAR␥/RXR in its natural chromatin context in adipocytes. Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPAR␥-response element.

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 proliferatoractivated 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 PPAR␣ and PPAR␥ 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 ␤-oxidation enzymes. In contrast, PPAR␦ 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.