Apoptosis- and Bcl-2- Induced Alterations of the Nucleocytoplasmic Barrier

Abstract

This work was aimed at elaborating a detailed knowledge on the regulation of the nuclear envelope permeability barrier in cell death. Therefore, the effects of opposing pathways, apoptosis-induction via TRAIL and staurosporine (STS) and apoptosis-inhibition mediated by overexpression of the anti-apoptotic protein Bcl-2, on nuclear envelope permeability were investigated. In both models the underlying mechanisms mediating the influence on nuclear envelope permeability were characterized in detail. Apoptosis-induced alterations of the nucleocytoplasmic permeability barrier To elucidate the alterations of nuclear envelope permeability, live cell imaging experiments were performed in a confocal setup using a reporter system established in our group. Kinetics of apoptosis and alterations of nuclear envelope permeability were assessed by fluorescently labeled marker proteins. As chromatin condensation is a hallmark of apoptosis, it was defined as time point of apoptosis execution. Our results reveal that caspase-mediated disruption of nuclear envelope permeability barrier and chromatin condensation occurs simultaneously in both models of apoptosis. However, the two employed apoptotic models differ in permeability barrier function prior to caspase activation. While in TRAIL induced apoptosis nuclear permeability is unaltered prior to caspase activation and chromatin condensation, in STS-induced cell death an early, caspase-independent nuclear leakage can be detected. To elucidate the mechanisms responsible for this latter effect, the involvement of two different types of proteases was investigated. The first one was serine proteases, which have been proposed to play a role in STS-induced, caspase-independent apoptosis. In addition, preliminary data of our group indicated a role for serine proteases in influencing the nuclear permeability barrier. However, inhibition of serine proteases had no effect on STS-induced early nuclear leakage, excluding a possible role of serine proteases in mediating nuclear permeabilization. The second investigated proteases were calpains, Ca2+-dependent proteases. These have recently been reported to be responsible for nuclear envelope permeabilization in a neuronal model of excitotoxiicity. Interestingly, calpain inhibition did not decrease early STS-induced nuclear leakage but even enhanced it. Endoplasmic reticulum (ER) Ca2+-level have been shown to influence the nuclear pore and nuclear envelope permeability. To investigate the role of Ca2+ in STS-induced apoptosis, confocal live cell imaging experiments in the presence of the Ca2+-chelator BAPTA-AM were performed. This treatment resulted in the complete inhibition of early STS-induced nuclear leakage, indicating a pivotal role of Ca2+ in this system. This hypothesis was further strengthened by the finding that treatment with STS but not with TRAIL reduces ER Ca2+ content. Furthermore, the dependence of active transport on nuclear envelope permeability alterations was investigated. The data show that active nuclear uptake and passive permeability are largely uncoupled as active transport is functional in both apoptotic models until chromatin condensation, despite the STS-induced early increase in nuclear envelope permeability. Bcl-2-induced alterations of the nucleocytoplasmic permeability barrier As Bcl-2 is an anti-apoptotic protein and a proto-oncogene, it was expected to counteract STS-induced alterations of nuclear envelope permeability. Surprisingly, preliminary data of our group indicated that Bcl-2 overexpression results in a constitutively increased nuclear permeability. It has been shown, that (ER) Ca2+-level, which can be altered by Bcl-2, influence the nuclear envelope permeability barrier. Determination of the Ca2+-level in the ER revealed that Bcl-2 overexpressing cells indeed show reduced ER Ca2+-content and diminished capacitive Ca2+ uptake. The Bcl-2 induced effect on increased nuclear envelope permeability could be mimicked by adapting cells to a medium with reduced Ca2+ content. Furthermore, replenishment of ER Ca2+-content by overexpression of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase SERCA reversed the Bcl-2 mediated increase of nuclear envelope permeability. An unexpected and remarkable result of this work is the finding that in two opposing cellular contexts, apoptosis-induction and apoptosis-inhibition, the same mechanism to increase nuclear envelope permeability is engaged. For STS-induced apoptosis, an early increase in nuclear permeability is very plausible, sinces it has been proposed that increased nuclear permeability might be important for enhanced transport of pro-apoptotic factors. In light of this, it seems surprising that the anti-apoptotic protein Bcl-2 also increases nuclear envelope permeability. Possible interpretations of this unexpected result are presented in the discussion section of this thesis.