title: Screening system of blocking agents of the receptor for advanced glycation endproducts in cells using fluorescence.
abstract: Activation of the receptor for advanced glycation endproducts (RAGE) triggers cellular responses implicated in the pathogenesis of diabetic complications; blockade of RAGE has been shown to inhibit the development of diabetic complications. To develop a screening system to identify novel disruptors of advanced glycation endproducts (AGE)-RAGE binding, we used an AGE-RAGE binding system in RAGE-overexpressing cells; test compounds were screened using this system. To construct human RAGE-overexpressing cells, mouse mesangial cells (MMCs) were stably transfected with the pcDNA-human RAGE (hRAGE) vector and selected under 1 mg/mL gentamicin (G418). RAGE expression in hRAGE-overexpressing MMCs was analyzed by Western blotting with specific RAGE antibody. To identify novel disruptors of AGE-RAGE binding, 50 single compounds and AGE-bovine serum albumin (BSA)-Alexa 488 (AGE-BSA labeled with Alexa 488) were treated to the hRAGE-overexpressing MMCs. Nonbinding AGE-BSA-Alexa 488 was washed and fluorescence measured by microtiter plate reader (excitation wavelength, 485 nm; emission wavelength, 528 nm). In hRAGE-overexpressing cells, only treatment with AGE-BSA-Alexa 488 significantly increased fluorescence intensity in a dose-dependent manner. Of 50 compounds tested, genistein disrupted AGE-RAGE binding in a dose-dependent manner. This AGE-RAGE binding system using AGE-BSA-Alexa 488 in hRAGE-overexpressing cells was suitable for screening of agents that disrupt AGE-hRAGE binding.
authors.affiliation: Korean Medicine-Based Herbal Drug Research Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine (KIOM), 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 305–811, Korea.
authors.forename: Dong Ho
authors.initials: DH
authors.lastname: Jung
authors.forename: Young Sook
authors.initials: YS
authors.lastname: Kim
authors.forename: Jin Sook
authors.initials: JS
authors.lastname: Kim
mesh_terms: D000818:Animals; D002460:Cell Line; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D051379:Mice; D061848:Optical Imaging; D011485:Protein Binding; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D017127:Glycation End Products, Advanced; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; C081628:advanced glycation end products-bovine serum albumin; D012710:Serum Albumin, Bovine