Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in cancer cells and this effect is involved in their antitumor activity. We recently demonstrated that NSAIDs upregulate GRP78, an endoplasmic reticulum (ER) chaperone, in gastric mucosal cells in primary culture. In the present study, induction of ER chaperones by NSAIDs and the effect of those chaperones on NSAID-induced apoptosis were examined in human gastric carcinoma cells. Celecoxib, an NSAID, upregulated ER chaperones (GRP78 and its cochaperones ERdj3 and ERdj4) but also C/EBP homologous transcription factor (CHOP), a transcription factor involved in apoptosis. Celecoxib also upregulated GRP78 in xenograft tumors, accompanying with the suppression of tumor growth in nude mice. Celecoxib caused phosphorylation of eukaryotic translation initiation factor 2 kinase (PERK) and eukaryotic initiation factor-2a (eIF2a) and production of activating transcription factor (ATF)4 mRNA. Suppression of ATF4 expression by small interfering RNA (siRNA) partially inhibited the celecoxib-dependent upregulation of GRP78. Celecoxib increased the intracellular Ca 2 þ concentration, while 1,2-bis(2-aminophenoxy) ethane-N,N,N 0 N 0 -tetraacetic acid, an intracellular Ca 2 þ chelator, inhibited the upregulation of GRP78 and ATF4. These results suggest that the Ca 2 þ -dependent activation of the PERK-eIF2a-ATF4 pathway is involved in the upregulation of ER chaperones by celecoxib. Overexpression of GRP78 partially suppressed the apoptosis and induction of CHOP in the presence of celecoxib and this suppression was stimulated by coexpression of either ERdj3 or ERdj4. On the other hand, suppression of GRP78 expression by siRNA drastically stimulated cellular apoptosis and production of CHOP in the presence of celecoxib. These results show that upregulation of ER chaperones by celecoxib protects cancer cells from celecoxib-induced apoptosis, thus may decrease the potential antitumor activity of celecoxib.

Amyloid-␤ peptide (A␤) plays an important role in the pathogenesis of Alzheimer's disease (AD). A␤ is generated by proteolysis of ␤-amyloid precursor protein (APP) and is cleared by enzyme-mediated degradation and phagocytosis by microglia and astrocytes. Some cytokines, such as TGF-␤1, stimulate this phagocytosis. In contrast, cellular upregulation of HSP70 expression provides cytoprotection against A␤. HSP70 activity in relation to inhibition of A␤ oligomerization and stimulation of A␤ phagocytosis has also been reported. Although these in vitro results suggest that stimulating the expression of HSP70 could prove effective in the treatment of AD, there is a lack of in vivo evidence supporting this notion. In this study, we address this issue, using transgenic mice expressing HSP70 and/or a mutant form of APP (APPsw). Transgenic mice expressing APPsw showed less of an apparent cognitive deficit when they were crossed with transgenic mice expressing HSP70. Transgenic mice expressing HSP70 also displayed lower levels of A␤, A␤ plaque deposition, and neuronal and synaptic loss than control mice. Immunoblotting experiments and direct measurement of ␤-and ␥-secretase activity suggested that overexpression of HSP70 does not affect the production A␤. In contrast, HSP70 overexpression did lead to upregulation of the expression of A␤-degrading enzyme and TGF-␤1 both in vivo and in vitro. These results suggest that overexpression of HSP70 in mice suppresses not only the pathological but also the functional phenotypes of AD. This study provides the first in vivo evidence confirming the potential therapeutic benefit of HSP70 for the prevention or treatment of AD.

Inflammatory bowel disease (IBD) involves infiltration of leukocytes into intestinal tissue, resulting in intestinal damage induced by reactive oxygen species (ROS). Pro-inflammatory cytokines and cell adhesion molecules (CAMs) play important roles in this infiltration of leukocytes. The roles of heat shock factor 1 (HSF1) and heat shock proteins (HSPs) in the development of IBD are unclear. In this study, we examined the roles of HSF1 and HSPs in an animal model of IBD, dextran sulfate sodium (DSS)-induced colitis. The colitis worsened or was ameliorated in HSF1-null mice or transgenic mice expressing HSP70 (or HSF1), respectively. Administration of DSS up-regulated the expression of HSP70 in colonic tissues in an HSF1-dependent manner. Expression of pro-inflammatory cytokines and CAMs and the level of cell death observed in colonic tissues were increased or decreased in DSS-treated HSF1-null mice or transgenic mice expressing HSP70, respectively, relative to control wild-type mice. Relative to macrophages from control wildtype mice, macrophages prepared from HSF1-null mice or transgenic mice expressing HSP70 displayed enhanced or reduced activity, respectively, for the generation of pro-inflammatory cytokines in response to lipopolysaccharide stimulation. Suppression of HSF1 or HSP70 expression in vitro stimulated lipopolysaccharide-induced up-regulation of CAMs or ROS-induced cell death, respectively. This study provides the first genetic evidence that HSF1 and HSP70 play a role in protecting against DSS-induced colitis. Furthermore, this protective role seems to involve various mechanisms, such as suppression of expression of pro-inflammatory cytokines and CAMs and ROS-induced cell death. Inflammatory bowel disease (IBD),2 Crohn disease, and ulcerative colitis have become substantial health problems with an actual prevalence of 200 -500 cases/100,000 people in western countries, which almost double every 10 years (1). Although the etiology of IBD is not yet fully understood, recent studies suggest that IBD involves chronic inflammatory disorders in the intestine because of "a vicious cycle." Infiltration into intestinal tissues causes intestinal mucosal damage induced by reactive oxygen species (ROS) that are released from the activated leukocytes, and this intestinal mucosal damage further stimulates the infiltration of leukocytes (2). To understand the molecular mechanism underlying the pathogenesis of IBD and to develop new types of clinical drugs for IBD, identification of endogenous factors that positively or negatively affect the development of IBD is important. For this purpose, various experimental animal colitis models, in particular the dextran sulfate sodium (DSS)-and trinitrobenzenesulfonic acid-induced colitis models, have been used (3).Pro-inflammatory cytokines play an important role in the activation and infiltration of leukocytes that are associated with IBD. This conclusion is supported by a range of evidence. Increases in the levels of various pro-inflammatory cytokines (such as tumor necr...

We recently reported that nonsteroidal anti-inflammatory drug (NSAID)-induced gastric lesions involve NSAID-induced apoptosis of gastric mucosal cells, which in turn involves the endoplasmic reticulum stress response, in particular the up-regulation of CCAAT/enhancer-binding protein homologous transcription factor (CHOP). In this study, we have examined the molecular mechanism governing this NSAID-induced apoptosis in primary cultures of gastric mucosal cells. Various NSAIDs showed membrane permeabilization activity that correlated with their apoptosis-inducing activity. Various NSAIDs, particularly celecoxib, also increased intracellular Ca 2؉ levels. This increase was accompanied by K ؉ efflux from cells and was virtually absent when extracellular Ca 2؉ had been depleted. These data indicate that the increase in intracellular Ca 2؉ levels that is observed in the presence of NSAIDs is due to the stimulation of Ca 2؉ influx across the cytoplasmic membrane, which results from their membrane permeabilization activity. An intracellular Ca 2؉ chelator partially inhibited celecoxib-induced release of cytochrome c from mitochondria, reduced the magnitude of the celecoxib-induced decrease in mitochondrial membrane potential and inhibited celecoxib-induced apoptotic cell death. It is therefore likely that an increase in intracellular Ca 2؉ levels is involved in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis. An inhibitor of calpain, a Ca 2؉ -dependent cysteine protease, partially suppressed mitochondrial dysfunction and apoptosis in the presence of celecoxib. Celecoxib-dependent CHOP-induction was partially inhibited by the intracellular Ca 2؉ chelator but not by the calpain inhibitor. These results suggest that Ca 2؉ -stimulated calpain activity and CHOP expression play important roles in celecoxib-induced apoptosis in gastric mucosal cells. Nonsteroidal anti-inflammatory drugs (NSAIDs)1 account for nearly 5% of all prescribed medications (1). The anti-inflammatory action of NSAIDs is mediated through their inhibitory effect on cyclooxygenase (COX)

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

SUMMARY Resolved ER stress response is essential for intracellular homeostatic balance, but unsettled ER stress can lead to apoptosis. Here, we show that a pro-apoptotic p53 target, CDIP1, acts as a key signal transducer of ER stress-mediated apoptosis. We identify BAP31, B-cell receptor-associated protein 31, as an interacting partner of CDIP1. Upon ER stress, CDIP1 is induced and enhances an association with BAP31 at the ER membrane. We also show that CDIP1 binding to BAP31 is required for BAP31 cleavage upon ER stress and for BAP31-Bcl-2 association. The recruitment of Bcl-2 to the BAP31-CDIP1 complex, as well as CDIP1-dependent tBid and caspase-8 activation, contributes to BAX oligomerization. Genetic knockout of CDIP1 in mice leads to impaired response to ER stress-mediated apoptosis. Together, our data demonstrate that the CDIP1/BAP31-mediated regulation of mitochondrial apoptosis pathway represents a novel mechanism for establishing an ER-mitochondrial cross-talk for ER stress-mediated apoptosis signaling.

The endoplasmic reticulum (ER) is composed of large membrane-bound compartments, and its membrane subdomain appears to be in close contact with mitochondria via ER-mitochondria contact sites. Here, I demonstrate that the ER membrane protein, BAP31, acts as a key factor in mitochondrial homeostasis to stimulate the constitution of the mitochondrial complex I by forming an ER-mitochondria bridging protein complex. Within this complex, BAP31 interacts with mitochondria-localized proteins, including Tom40, to stimulate the translocation of NDUFS4, the component of complex I from the cytosol to the mitochondria. Disruption of the BAP31-Tom40 complex inhibits mitochondrial complex I activity and oxygen consumption by the decreased NDUFS4 localization to the mitochondria. Thus, the BAP31-Tom40 ER-mitochondria bridging complex mediates the regulation of mitochondrial function and plays a role as a previously unidentified stress sensor, representing a mechanism for the establishment of ER-mitochondria communication via contact sites between these organelles.

Ulcerative colitis (UC) involves intestinal mucosal damage induced by reactive oxygen species (ROS), in particular, superoxide anion. Superoxide dismutase (SOD) catalyzes dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxified by catalase. Lecithinized SOD (PC-SOD) is a new modified form of SOD that has overcome previous clinical limitations of SOD. In this study, we examined the action of PC-SOD using an animal model of UC, dextran sulfate sodium (DSS)-induced colitis. DSS-induced colitis was ameliorated by daily intravenous administration of PC-SOD. Unmodified SOD produced a similar effect but only at more than 30 times the concentration of PC-SOD. In vivo electron spin resonance analysis confirmed that the increase in the colonic level of ROS associated with development of colitis was suppressed by PC-SOD administration. The dose-response profile of PC-SOD was bell-shaped, but simultaneous administration of catalase restored the ameliorative effect at high doses of PC-SOD. Accumulation of hydrogen peroxide was observed with the administration of high doses of PC-SOD, an effect that was suppressed by the simultaneous administration of catalase. We also found that either a weekly intravenous administration or daily oral administration of PC-SOD conferred protection. These results suggest that PC-SOD achieves its ameliorative effect against colitis through decreasing the colonic level of ROS and that its ineffectiveness at higher doses is because of the accumulation of hydrogen peroxide. Furthermore, we consider that intermittent or oral administration of PC-SOD can be applied clinically to improve the quality of life of UC patients.