Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43°C for 20 min) resulted in inhibition of H 2 O 2 -induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H 2 O 2 -induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homooligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H 2 O 2 -induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.Apoptosis signal-regulating kinase 1 (ASK1) is a widely expressed serine-threonine kinase that was initially discovered as a mitogen-activated protein kinase kinase kinase (MAPKKK) in the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 MAPK signaling cascades (12, 13). ASK1 functions in apoptosis induced by diverse stimuli, including tumor necrosis factor alpha (TNF-␣), Fas, and many apoptotic stresses (12,13,30). Overexpression of a dominantnegative mutant of ASK1 thus prevents apoptosis induced by withdrawal of growth factors, DNA-damaging agents, TNF-␣, or agonistic antibodies to Fas (4,13,16,30). Activation of ASK1 is reported to induce apoptotic cell death by triggering mitochondrial events that include the release of cytochrome c from mitochondria and the subsequent activation of caspase 9 and caspase 3 (11).Many cellular stresses that stimulate the stress-activated MAPK pathways can also induce expression of heat shock proteins. Heat shock protein 72 (Hsp72) is the major inducible heat shock protein (35). It plays a role in many cellular activities including protein synthesis, folding, and translocation into organelles as well as the assembly of multiprotein complexes (2,5,24,25,33). Hsp72 contains two conserved domains, an ATP-binding domain (ABD) and a peptide-binding domain (PBD), that are important for its chaperon function (7, 21). Hsp72 also prevents cell death initiated by various apoptotic stresses including heat shock, ceramide, ionizing irradiation, TNF-␣, and ischemia (8,14,20,23,32). Hsp72 suppresses several apoptotic signaling pathways, including caspase cascades and stress-activated MAPK pathways that include the JNK and p38 signaling cascades (3,8,9,15,17,19,22,23,29,31). Furthermore, Hsp72 has been shown to physically interact with and inhibit Apaf-1 and apoptosis-inducing factor, resulting in suppression of caspase-dependent and -independent apoptosis, respectively ...

Diverse stimuli initiate the activation of apoptotic signaling pathways that often causes nuclear DNA fragmentation. Here, we report a new antiapoptotic protein, a caspase-activated DNase (CAD) inhibitor that interacts with ASK1 (CIIA). CIIA, by binding to apoptosis signal-regulating kinase 1 (ASK1), inhibits oligomerization-induced ASK1 activation. CIIA also associates with CAD and inhibits the nuclease activity of CAD without affecting caspase-3–mediated ICAD cleavage. Overexpressed CIIA reduces H2O2- and tumor necrosis factor-α–induced apoptosis. CIIA antisense oligonucleotides, which abolish expression of endogenous CIIA in murine L929 cells, block the inhibitory effect of CIIA on ASK1 activation, deoxyribonucleic acid fragmentation, and apoptosis. These findings suggest that CIIA is an endogenous antagonist of both ASK1- and CAD-mediated signaling.

The hypothalamic-pituitary-adrenal (HPA) axis is the primary endocrine system to respond to stress. The HPA axis may be affected by increased level of corticotrophin-releasing factors under chronic stress and by chronic administration of monosodium glutamate (MSG). The purpose of this study was to investigate whether chronic MSG administration aggravates chronic variable stress (CVS)-induced behavioral and hormonal changes. Twenty-four adult male Sprague-Dawley rats, weighing 200∼ 220 g, were divided into 4 groups as follows: water administration (CON), MSG (3 g/kg) administration (MSG), CVS, and CVS with MSG (3 g/kg) administration (CVS＋ MSG). In addition, for the purpose of comparing the effect on plasma corticosterone levels between chronic stress and daily care or acute stress, 2 groups were added at the end of the experiment; the 2 new groups were as follows: naïve mice (n=7) and mice exposed to restraint stress for 2 h just before decapitation (A-Str, n=7). In an open field test performed after the experiment, the CVS＋ MSG group significant decrease in activity. The increase in relative adrenal weights in the CVS and CVS＋ MSG group was significantly greater than those in the CON and/or MSG groups. In spite of the increase in the relative adrenal weight, there was a significant decrease in the plasma corticosterone levels in the CVS＋ MSG group as compared to all other groups, except the naïve group. These results suggest that impaired HPA axis function as well as the decrease in the behavioral activity in adult rats can be induced by chronic MSG administration under CVS rather than CVS alone.

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Proinflammatory molecule tumor necrosis factor alpha (TNF-α) is predominantly elevated in cytokine storm as well as worsening cardiac function. Here we model the molecular and functional effects of TNF-α in cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC). We found that treatment of hiPSC-CMs with TNF-α increased reactive oxygen species (ROS) and caspase 3/7 activity and caused cell death and apoptosis. TNF-α treatment also resulted in dysregulation of cardiomyocyte function with respect to the increased abnormal calcium handling, calcium wave propagation between cells and excitation–contraction coupling. We also uncovered significant changes in gene expression and protein localization caused by TNF-α treatment. Notably, TNF-α treatment altered the expression of ion channels, dysregulated cadherins, and affected the localization of gap-junction protein connexin-43. In addition, TNF-α treatment up-regulated IL-32 (a human specific cytokine, not present in rodents and an inducer of TNF-α) and IL-34 and down-regulated glutamate receptors and cardiomyocyte contractile proteins. These findings provide insights into the molecular and functional consequences from the exposure of human cardiomyocytes to TNF-α. Our study provides a model to incorporate inflammatory factors into hiPSC-CM-based studies to evaluate mechanistic aspects of heart disease.