1. Protein kinase C signaling during T cell activation induces the endoplasmic reticulum stress response
Steven C Pino, et al. Cell Stress Chaperones. 2008 Dec;13(4):421-34. doi: 10.1007/s12192-008-0038-0. Epub 2008 Apr 17.
T cell receptor (TCR) ligation (signal one) in the presence of co-stimulation (signal two) results in downstream signals that increase protein production enabling naïve T cells to fully activate and gain effector function. Enhanced production of proteins by a cell requires an increase in endoplasmic reticulum (ER) chaperone expression, which is accomplished through activation of a cellular mechanism known as the ER stress response. The ER stress response is initiated during the cascade of events that occur for the activation of many cells; however, this process has not been comprehensively studied for T cell function. In this study, we used primary T cells and mice circulating TCR transgenic CD8(+) T cells to investigate ER chaperone expression in which TCR signaling was initiated in the presence or absence of co-stimulation. In the presence of both signals, in vitro and in vivo analyses demonstrated induction of the ER stress response, as evidenced by elevated expression of GRP78 and other ER chaperones. Unexpectedly, ER chaperones were also increased in T cells exposed only to signal one, a treatment known to cause T cells to enter the 'nonresponsive' states of anergy and tolerance. Treatment of T cells with an inhibitor to protein kinase C (PKC), a serine/threonine protein kinase found downstream of TCR signaling, indicated PKC is involved in the induction of the ER stress response during the T cell activation process, thus revealing a previously unknown role for this signaling protein in T cells. Collectively, these data suggest that induction of the ER stress response through PKC signaling is an important component for the preparation of a T cell response to antigen.
2. Effect of tauroursodeoxycholic acid on endoplasmic reticulum stress-induced caspase-12 activation
Qing Xie, Vladimir I Khaoustov, Charles C Chung, Joohyun Sohn, Bhuvaneswari Krishnan, Dorothy E Lewis, Boris Yoffe Hepatology. 2002 Sep;36(3):592-601. doi: 10.1053/jhep.2002.35441.
Activation of death receptors and mitochondrial damage are well-described common apoptotic pathways. Recently, a novel pathway via endoplasmic reticulum (ER) stress has been reported. We assessed the role of tauroursodeoxycholic acid (TUDCA) in inhibition of caspase-12 activation and its effect on calcium homeostasis in an ER stress-induced model of apoptosis. The human liver-derived cell line, Huh7, was treated with thapsigargin (TG) to induce ER stress. Typical morphologic changes of ER stress preceded development of apoptotic changes, including DNA fragmentation and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP), as well as activation of caspase-3 and -7. Elevation of intracellular calcium levels without loss of mitochondrial membrane potential (MMP) was shown using Fluo-3/Fura-red labeling and flow cytometry, and confirmed by induction of Bip/GRP78, a calcium-dependent chaperon of ER lumen. These changes were accompanied by procaspase-12 processing. TUDCA abolished TG-induced markers of ER stress; reduced calcium efflux, induction of Bip/GRP78, and caspase-12 activation; and subsequently inhibited activation of effector caspases and apoptosis. In conclusion, we propose that mitochondria play a secondary role in ER-mediated apoptosis and that TUDCA prevents apoptosis by blocking a calcium-mediated apoptotic pathway as well as caspase-12 activation. This novel mechanism of TUDCA action suggests new intervention methods for ER stress-induced liver disease.
3. Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12
Tomoyasu Nakano, Hiroshi Watanabe, Mariko Ozeki, Masayoshi Asai, Hideki Katoh, Hiroshi Satoh, Hideharu Hayashi Cardiovasc Res. 2006 Mar 1;69(4):908-15. doi: 10.1016/j.cardiores.2005.11.023. Epub 2005 Dec 27.
Objective: Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells. Methods: Cytosolic Ca(2+) concentration ([Ca(2+)](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques. Results: Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased [Ca(2+)](i), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca(2+)](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis. Conclusions: Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.
