The binding of advanced glycation end-products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. In the present study, we show that the cellular constituents of small vessels, endothelial cells (EC) and pericytes express novel splice variants of RAGE mRNA coding for the isoforms that lack the N-terminal V-type immunoglobulin-like domain (N-truncated) or the C-terminal transmembrane domain (C-truncated), as well as the known full-length mRNA. The ratio of the expression of the three variants was different between EC and pericytes; the content of the C-truncated form was highest in EC, whereas the full-length form was the most abundant in pericytes. Transfection experiments with COS-7 cells demonstrated that those variant mRNAs were translated into proteins as deduced; C-truncated RAGE was efficiently secreted into the culture media, and N-truncated RAGE was located mainly on the plasma membrane. The three isoforms were also detected in primary cultured human EC and pericytes. Further, full-length and C-truncated forms of RAGE bound to an AGE-conjugated column, whereas N-truncated RAGE did not. The AGE induction of extracellular-signal-related kinase phosphorylation and vascular endothelial growth factor in EC and of the growth and cord-like structure formation of EC was abolished completely by C-truncated RAGE, indicating that this endogenous secretory receptor (endogenous secretory RAGE) is cytoprotective against AGE. The results may contribute to our understanding of the molecular basis for the diversity of cellular responses to AGE and for individual variations in the susceptibility to diabetic vascular complications.

Environmental contaminants affect a wide variety of biological events in many species. Dioxins are typical environmental contaminants that exert adverse oestrogen-related effects. Although their anti-oestrogenic actions are well described, dioxins can also induce endometriosis and oestrogen-dependent tumours, implying possible oestrogenic effects. However, the molecular mechanism underlying oestrogen-related actions of dioxins remains largely unknown. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix-loop-helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Here we show that the agonist-activated AhR/Arnt heterodimer directly associates with oestrogen receptors ER-alpha and ER-beta. This association results in the recruitment of unliganded ER and the co-activator p300 to oestrogen-responsive gene promoters, leading to activation of transcription and oestrogenic effects. The function of liganded ER is attenuated. Oestrogenic actions of AhR agonists were detected in wild-type ovariectomized mouse uteri, but were absent in AhR-/- or ER-alpha-/- ovariectomized mice. Our findings suggest a novel mechanism by which ER-mediated oestrogen signalling is modulated by a co-regulatory-like function of activated AhR/Arnt, giving rise to adverse oestrogen-related actions of dioxin-type environmental contaminants.

The binding of advanced glycation end products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. Here we show that AGE, tumor necrosis factor-␣ (TNF-␣), and 17␤-estradiol (E 2 ) up-regulated RAGE mRNA and protein levels in human microvascular endothelial cells and ECV304 cells, with the mRNA stability being essentially invariant. Transient transfection experiments with human RAGE promoter-luciferase chimeras revealed that the region from nucleotide number ؊751 to ؊629 and the region from ؊239 to ؊89 in the RAGE 5-flanking sequence exhibited the AGE/TNF-␣ and E 2 responsiveness, respectively. Site-directed mutation of an nuclear factor-B (NF-B) site at ؊671 or of Sp-1 sites at ؊189 and ؊172 residing in those regions resulted in an abrogation of the AGE/TNF-␣-or E 2 -mediated transcriptional activation. Electrophoretic mobility shift assays revealed that ECV304 cell nuclear extracts contained factors which retarded the NF-B and Sp-1 elements, and that the DNA-protein complexes were supershifted by antip65/p50 NF-B and anti-Sp-1/estrogen receptor ␣ antibodies, respectively. These results suggest that AGE, TNF-␣, and E 2 can activate the RAGE gene through NF-B and Sp-1, causing enhanced AGE-RAGE interactions, which would lead to an exacerbation of diabetic microvasculopathy.

Summary Obesity-induced inflammation mediated by immune cells in adipose tissue appears to participate in the pathogenesis of insulin resistance. We show that natural killer (NK) cells in adipose tissue play an important role. High fat diet (HFD) increases NK cell numbers and the production of pro-inflammatory cytokines, notably TNFα, in epididymal, but not subcutaneous, fat depots. When NK cells were depleted either with neutralizing antibodies or genetic ablation in E4bp4+/− mice, obesity-induced insulin resistance improved in parallel with decreases in both adipose tissue macrophage (ATM) numbers and ATMs and adipose tissue inflammation. Conversely, expansion of NK cells following IL-15 administration or reconstitution of NK cells into E4bp4−/−mice increased both ATM numbers and adipose tissue inflammation and exacerbated HFD-induced insulin resistance. These results indicate that adipose NK cells control ATMs as an upstream regulator potentially by producing pro-inflammatory mediators including TNFα and thereby contribute to the development of obesity-induced insulin resistance.