Divergent Pathways of Gene Expression Are Activated by the RAGE Ligands S100b and AGE-BSA Jessica V. Valencia, 1,2 Manisha Mone, 1 Jin Zhang, 1 Marla Weetall, 3 Frank P. Buxton, 1 and Thomas E. Hughes 1 Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers a variety of proin- flammatory responses. However, our previous work re- vealed that RAGE-binding AGEs free of endotoxin were incapable of inducing vascular cell adhesion molecule-1 (VCAM-1) or tumor necrosis factor- (TNF-) expres- sion. Thus, the objective of this study was to clarify the role of AGEs in cell activation through gene expression profiling using both in vitro and in vivo model systems. Endothelial cells treated with AGE-BSA, previously shown to bind RAGE with high affinity, did not show gene expression changes indicative of an inflammatory response. In contrast, the alternate RAGE ligand, S100b, triggered an increase in endothelial mRNA ex- pression of a variety of immune-related genes. The effects of AGEs were studied in vivo using healthy mice exposed to two different treatment conditions: 1) intra- venous injection of a single dose of model AGEs or 2) four intraperitoneal injections of model AGEs (once per day). In both cases, the liver was extracted for gene expression profiling. Both of the short-term AGE treat- ments resulted in a moderate increase in liver mRNA levels for genes involved in macrophage-based clear- ance/detoxification of foreign agents. Our findings using AGEs with strong RAGE-binding properties indicate that AGEs may not uniformly play a role in cellular activation. Diabetes 53:743–751, 2004 A dvanced glycation end products (AGEs) are a heterogeneous group of irreversibly bound, complex structures that form nonenzymatically when reducing sugars react with free amino groups on macromolecules (rev. in 1). AGEs are highly reactive and continue to react with nearby amino groups to produce both intra- and intermolecular crosslinks (2). The formation of AGEs has been found to occur in aging and at an accelerated rate in diabetic patients (rev. in 3). The deposition of these covalent adducts on various macromolecules has been reported to contribute to the development of the complications of aging and diabetes through both direct chemical- (covalent crosslink forma- tion) and cell surface receptor–mediated pathways (4). The most characterized AGE binding protein is the receptor for AGEs (RAGE). RAGE, a 45-kDa protein belonging to the immunoglobulin superfamily, is present on the cell surface of a variety of cells, including endothe- lial cells, mononuclear phagocytes, and hepatocytes (5,6). RAGE is a multiligand receptor that has also been shown to bind to several proteins in the S100 family including S100A12 (EN-RAGE) and S100b (7,8). S100b and S100A12 are calcium binding proteins with inflammatory properties (rev. in 9). Activation of RAGE by its various ligands reportedly induces a variety of proinflammatory and pro- coagulant cellular responses, resulting from the activation of nuclear factor-B (NF-B) (10), including the expres- sion of vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor- (TNF-), interleukin (IL)-6, and tissue factor (TF) (7,11–14). Chronic infusion of model AGEs into normal/healthy animals has been reported to elicit pathologies similar to those observed in diabetes. For example, several studies reported that injection of healthy mice with 6 mg/day of model AGEs for 4 weeks resulted in an increase in the expression of several genes implicated in diabetic ne- phropathy, including TGF-, type IV collagen, and laminin (15–17). Another group reported an increase in vascular permeability and defective vasodilatory responses in rats and rabbits injected with model AGEs for 4 weeks (18). Administration of model AGEs into healthy animals was also reported to increase VCAM-1 and ICAM-1 expression, intimal proliferation, and lipid deposits, all of which are implicated in atherosclerosis (19,20). Only a few studies have examined the effects of acute administration of model AGEs. Stern and colleagues (10,13) reported that within hours of infusion of various amounts of model AGEs (0.1–1.0 mg/mouse), increases in liver IL-6 mRNA, lung heme oxygenase mRNA, lung staining for VCAM-1, NF-B activation in liver, and tissue TBARS were observed. Previously, we have found that RAGE binding AGEs can be created reproducibly using the reducing sugars— glu- cose, fructose, or ribose (21). Interestingly, we also found that those AGE preparations, which were essentially en- dotoxin free (0.2 ng/mg protein), were incapable of From the 1 Novartis Institutes for BioMedical Research, Cambridge, Massa- chusetts; the 2 Department of Molecular Genetics, Microbiology and Immunol- ogy, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey; and 3 PTC Therapeutics, South Plainfield, New Jersey. Address correspondence and reprint requests to Thomas E. Hughes, Novar- tis Institutes for BioMedical Research, 100 Technology Square, Bldg. 601/Rm. 5155, Cambridge, MA 02139. E-mail: thomase.hughes@pharma.novartis.com. Received for publication 1 August 2003 and accepted in revised form 12 November 2003. AGE, advanced glycation end product; Ctrl BSA, BSA incubated in the absence of modifying agent; EC, endothelial cell; HC, hydrocortisone; HMEC, human microvascular EC; hsRAGE, human soluble RAGE; ICAM-1, intercel- lular adhesion molecule-1; IB, inhibitor of nuclear factor-B; IL, interleukin; LPS, lipopolysaccharide/endotoxin; MHC, major histocompatibility complex; RAGE, receptor for AGE; Rib BSA, BSA incubated with ribose; TF, tissue factor; TGF-, transforming growth factor-; TNF-, tumor necrosis factor-; VCAM-1, vascular cell adhesion molecule-1. © 2004 by the American Diabetes Association. DIABETES, VOL. 53, MARCH 2004 743