Hyperosmotic shock induces cytochrome c release and capase-3 activation in Xenopus oocytes, but the regulators and signaling pathways involved are not well characterized. Here we show that hyperosmotic shock induces rapid calpain activation and high levels of Smac/DIABLO release from the mitochondria before significant amounts of cytochrome c are released to promote caspase-3 activation. Calpain inhibitors or EGTA microinjection delays osmostress-induced apoptosis, and blockage of Smac/DIABLO with antibodies markedly reduces cytochrome c release and caspase-3 activation. Hyperosmotic shock also activates the p38 and JNK signaling pathways very quickly. Simultaneous inhibition of both p38 and JNK pathways reduces osmostress-induced apoptosis, while sustained activation of these kinases accelerates the release of cytochrome c and caspase-3 activation. Therefore, at least four different pathways early induced by osmostress converge on the mitochondria to trigger apoptosis. Deciphering the mechanisms of hyperosmotic shock-induced apoptosis gives insight for potential treatments of human diseases that are caused by perturbations in fluid osmolarity.
Some properties of signaling systems, like ultrasensitivity, hysteresis (a form of biochemical memory), and all-or-none responses at a single cell level, are important to understand the regulation of irreversible processes. Xenopus oocytes are a suitable cell model to study these properties. The p38 MAPK (mitogen-activated protein kinase) pathway is activated by different stress stimuli, including osmostress, and regulates multiple biological processes, from immune response to cell cycle. Recently, we have reported that activation of p38 and JNK regulate osmostress-induced apoptosis in Xenopus oocytes and that sustained activation of p38 accelerates cytochrome c release and caspase-3 activation. However, the signaling properties of p38 in response to hyperosmotic shock have not been studied. Here we show, using Xenopus oocytes as a cell model, that hyperosmotic shock activates the p38 signaling pathway with an ultrasensitive and bimodal response in a time-dependent manner, and with low hysteresis. At a single cell level, p38 activation is not well correlated with cytochrome c release 2 h after hyperosmotic shock, but a good correlation is observed at 4 h after treatment. Interestingly, cytochrome c microinjection induces p38 phosphorylation through caspase-3 activation, and caspase inhibition reduces p38 activation induced by osmostress, indicating that a positive feedback loop is engaged by hyperosmotic shock. To know the properties of the stress protein kinases activated by hyperosmotic shock will facilitate the design of computational models to predict cellular responses in human diseases caused by perturbations in fluid osmolarity.
Hyperosmotic shock induces early calpain activation, Smac/ DIABLO release from the mitochondria, and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here, we show that JNK1-1 and JNK1-2 are activated early by osmostress, and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated, JNK1-2 is proteolyzed at Asp-385 increasing the release of cytochrome c and caspase-3 activity, thereby creating a positive feedback loop. Expression of Bcl-x L markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-x L co-expression. In these conditions a significant amount of Bid in the cytosol is mono-and bi-ubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp-52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.Hyperosmotic shock induces cytochrome c release and caspase-3 activation in Xenopus oocytes (1). Recently, we have shown that hyperosmotic shock also induces rapid calpain activation and Smac/DIABLO (second mitochondrion-derived activator of caspases/direct IAP-binding protein with low PI Smac/DIABLO) release from the mitochondria, as well as p38 and JNK (c-Jun N-terminal kinase) activation. These four pathways, induced early by osmostress, converge on the mitochondria to trigger late cytochrome c release and caspase-3 activation (2). Moreover, we have found that caspase-3 activation induces rapid phosphorylation of p38, thus creating a positive feedback loop in osmostress-induced apoptosis (3). However, the role of Bcl-2 family members in osmostress-induced apoptosis was not addressed in previous studies. It is not clear which specific p38 and JNK isoforms are activated by osmostress and how they regulate the apoptotic program. Bid is a member of the BH3 2 -only proteins that plays a crucial role in regulating the permeability of the outer mitochondrial membrane. The BH3 region is required for interaction with both pro-apoptotic Bax or anti-apoptotic protein Bcl-x L (4). Bid also contains a large unstructured loop (amino acids 42-79) with a variety of sites that are subjected to post-translational modifications, regulating Bid localization and apoptotic function (5).Bid is a caspase-8 substrate, and the resulting tBid translocates to mitochondria and initiates mitochondrial protein release. The cleavage site of caspase-8 in human Bid is Asp-60 (6) and in Xenopus laevis is Asp-52 (7). Xenopus Bid can also be proteolyzed by caspase-10 (7). In addition, it has been reported that caspase-3 can cleave human Bid at Asp-60 (8), and cleavage sites for non-caspase proteases have been detected, such as Gly...
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