Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling and Up-regulation of Egr-1 in Hypoxic Macrophages

Abstract

Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligandbinding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C ␤II, ERK1/2, and c-Jun NH 2 -terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1.The occlusion of blood vessels or insufficient blood flow to diseased tissues occurs with the onset and progression of many pathological states (1-6). Macrophages accumulate in large numbers in such ischemic/hypoxic areas and respond to oxygen signaling mechanisms involving a number of transcription factors (7-9). One such transcription factor, early growth response-1 (Egr-1), 2 an inducible zinc finger transcription factor, is rapidly up-regulated in macrophages in coordinating inflammatory and procoagulant response to hypoxia (8, 9).The generation of advanced glycation end products (AGEs) has been implicated in ischemia/reperfusion injury in the heart (10 -14). AGE-modified proteins are able to activate macrophages and stimulate secretion of cytokines and inflammatory factors (15-17). A major mechanism by which AGEs exert their cellular effects is by ligation of the multiligand receptor for AGE (RAGE) (18). In addition, our previous findings demonstrated that AGE-RAGE-dependent membrane translocation of protein kinase C (PKC) ␤II and consequent activation of JNK signaling in the heart and in endothelial cells subjected to hypoxia directly impact on regulation of Egr-1 (19). However, ligands of RAGE are not simply tethered to this receptor.Studies in vivo and in vitro revealed that cytoplamic domain of RAGE is essential for RAGE ligand-triggered signal transduction because deletion of the cytoplasmic domain of RAGE blocks ligands from inducing signaling and modulating gene expression (20). In addition, the cytoplasmic domain of RAGE interacts with a member of the formin homology domain proteins, diaphanous or mDia-1, which has been identified as a binding partner of the RAGE cytoplasmic domain (21). Previous...

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