Soluble receptor for advanced glycation end products (sRAGE), shed from cell surfaces, is found in human circulation and has been implicated in cardiovascular disease. Its pathophysiological regulation and underlying mechanisms are scarcely understood. In endothelium-specific human RAGE transgenic mice, human sRAGE was detected in circulation, whereas its level was markedly increased after LPS treatment. That increase was preceded by a rapid rise in TNF-α level. Treatment with TNF-α also significantly increased serum sRAGE. In human microvascular endothelial cells or human umbilical vein endothelial cells with RAGE overexpression, TNF-α markedly induced RAGE shedding, which was dependent on MMP9 and ADAM10. TNF-α-stimulated MMP9 expression was completely dependent on JNK activation, with its inhibition partially effective in suppressing TNF-α-induced RAGE shedding. In contrast, TNF-α transiently induced activation transcription factor (ATF)4, a major component in unfolded protein response (UPR), whereas knockdown of ATF4 abrogated TNF-α-stimulated RAGE shedding. Protein levels of the pro and activated forms of ADAM10 were also decreased by ATF4 knockdown, whereas inhibition of other components of UPR, including XBP1 and ATF6, failed to block TNF-α-stimulated RAGE shedding. Although the endoplasmic reticulum stressors thapsigargin and tunicamycin induced markedly and sustained expression of ATF4 and XBP-1, they did not induce RAGE shedding to the same level as TNF-α, suggesting that ATF4 is necessary but not sufficient alone for TNF-α-mediated RAGE shedding. ATF4 inhibition did not affect TNF-α-stimulated MMP9 expression, whereas inhibition of JNK activity did not influence ADAM10 activation. Thus, inflammatory cascades including TNF-α induced RAGE shedding in endothelial cells in vivo and in vitro. JNK and ATF4 may be 2 platforms for regulation of TNF-α-stimulated RAGE shedding.-Miyoshi, A., Koyama, S., Sasagawa-Monden, M., Kadoya, M., Konishi, K., Shoji, T., Inaba, M., Yamamoto, Y., Koyama, H. JNK and ATF4 as two important platforms for tumor necrosis factor-α-stimulated shedding of receptor for advanced glycation end products.