|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
Nitric oxide (NO) is a multifunctional biomolecule involved in a variety of physiological and pathological processes. In pathological conditions, NO functions as a bactericidal or tumoricidal agent. Excess NO induces apoptosis in some cell types including pancreatic β cells and macrophages, however, the cascade of NO-mediated apoptosis is not fully understood. We investigated the initial steps of NO-mediated apoptosis, and found that the ER stress pathway is involved in NO-mediated apoptosis.
No induces apoptosis in pancreatic β cells and mouse macrophage-like RAW 264.7 cells. Under these conditions, p53 accumulation was not observed, indicating that DNA damage is not the main trigger of NO-mediated apoptosis. In fact, NO induced apoptosis in microglia from p53 knockout mice. On the other hand, CHOP, a transcription factor known to be induced by endoplasmic reticulum (ER) stress, was induced. RAW 264.7 cells and COS-7 cells transfected with an expression plasmid for CHOP, underwent apoptotic cell death. Pancreatic β cells and peritoneal macrophages from CHOP knockout mice showed resistance to NO-induced apoptosis. Then we analyzed the upstream of the CHOP induction. In NO-mediated apoptosis, p90ATF6, an ER membrane-bound transcription factor involved in ER stress response, was cleaved to its active soluble form p50ATF6. The latter was transported to nucleus and bound to the ER-stress-response element (ERSE) of the CHOP gene. The induction of CHOP was preceded by ATF6 activation. When cells were transfected with a p50ATF6 plasmid, apoptosis occurred. This apoptosis induced by p50ATF6 was prevented by a CHOP dominant negative form as well as by an ATF6 dominant negative form. Therefore, CHOP induction is mediated by activation of ATF6. All these results indicate that the ER stress pathway involving ATF6 and CHOP plays a key role in NO-mediated apoptosis. We also found that hsp70/DnaJ (hsp40) chaperone pairs prevent CHOP-induced apoptosis.