3 MATERIALS AND METHODS
5.5 Active nuclear transport is essential for survival following TNFα treatment
Ligation of TNFα to TNF-R1 elicits both pro- and anti-apoptotic responses, with the latter pathway relying on de novo synthesis of proteins such as c-IAP1, c-IAP2 and XIAP 183,184. Following an appropriate stimulus, cytoplasmic anchoring of the transcription factor NF-κB is abolished by unmasking of an NLS. NF-κB is then transported into the nucleus in an importin β dependent manner 19,185.
Since transcriptional activation takes place in the nuclear compartment, the effect of blockage of the importin β dependent import pathways on TNFα mediated apoptosis was determined.
Microinjection of ∆impβ sensitized HeLa cells to apoptosis in response to CHX/ TNFα treatment, as judged by an increased apoptosis rate when compared to uninjected cells. In addition to the blockage of import pathways, ∆impβ binding to nucleoporins is known to shut down NES-dependent and mRNA export 148. As a result, cellular homeostasis is presumably negatively affected.
Demonstrating the importance of nuclear translocation for NF-κB for cell survival, the rate of apoptosis was not diminished in the absence of the translational inhibitor for
∆impβ injected cells. Therefore, TNFα and ∆impβ displayed a synergistic effect in promoting cell death in microinjected HeLa cells. This is in agreement with the finding
that an NLS-containing peptide competing for importin α binding of NF-κB specifically inhibits NF-κB mediated inflammatory responses 186.
In the presence of this inhibitory peptide, TNFα and IL-6 production in response to LPS treatment was abolished in RAW macrophages. The action of the peptides was selective for NF-κB translocation since the response via the NF-κB independent Jak/
STAT transcription factor pathway was not affected.
In summary, the inhibition of active transport acts as an efficient substitute for translational inhibition. The absence of NF-κB induced transcription of anti-apoptotic factors results in a sensitization of HeLa cells to TNFα induced apoptosis.
Apart from the previous finding, the proper execution of nuclear apoptosis relies on active transport processes, as demonstrated in this work. Although caspase-3 serves as the trigger for the breakdown of chromatin structure, the induction does not rely on its presence in the nucleus. Nevertheless, caspase-3 lacking the prodomain faithfully translocates into the nucleoplasm, presumably to promote cellular demise by the cleavage of several substrates.
6 SUMMARY
The subcellular localization of caspase-3 in a healthy cell was determined to be mainly cytosolic. In response to apoptotic stimuli, the apoptotic protease enters the nucleus. The mechanism employed remains the subject of controversy. In the presented study, the subcellular localization and the mechanism of nuclear entry of caspase-3 was studied employing a microinjection model.
For this purpose, recombinant caspase-3 was overexpressed in E. coli and isolated in high purity. Fusion of GFP did not affect the the proteolytic properties of the enzyme as demonstrated by strong DEVD-afc cleavage by the recombinant protease.
Injection of recombinant caspase-3 elicited the morphological features associated with apoptosis, accompanied by rapid equilibration of the apoptotic effector across the nuclear envelope. Both the inhibition of caspase-9 and caspase-3 activity failed to result in retention of the protease in the cytoplasm. Additionally, the rearrangement of the catalytic subunits following proteolytic activation of caspase-3 did not influence its cellular distribution. At the same time, the significantly smaller 40 kDa dextran was excluded from the nuclear compartment. Therefore, passive diffusion can be ruled out as the mode for caspase-3 entry into the nucleus.
The observed accumulation of caspase-3 in the nuclear compartment relied on active transport processes, as demonstrated by the requirement for interaction with nucleoporins and energy dependence. The hypothetical import factors utilized by caspase-3 do not belong to the family of importins or transportins, as inhibition of these pathways failed to block caspase-3 accumulation in the nucleoplasm.
Under conditions of import inhibition nuclear apoptosis is altered, as demonstrated by impaired chromatin condensation and DNA cleavage. The presence of caspase-3 was not required in the nucleus for the induction of these morphological features, indicating that the effectors are imported via an importin β dependent pathway from the cytosol following initiation of apoptosis.
Inhibition of the importin β pathway sensitized HeLa cells to apoptosis, probably by preventing NF-κB dependent activation of transcription. Therefore, active transport processes are indispensable for mediating cellular resistance in response to TNFα ligation, as well as the proper execution of nuclear apoptosis.
7 ZUSAMMENFASSUNG
Die Effektorcaspase-3 nimmt in der Exekution der Apoptose eine zentrale Stellung ein.
Als Antwort auf apoptotische Stimuli findet eine teilweise Umverteilung der primär cytoplasmatisch lokalisierten Protease in den Zellkern statt. Inwieweit dies auf passive Diffusion oder aktive Transportprozesse zurückzuführen ist, konnte bisher nicht beantwortet werden. Um diese Kontroverse zu klären, wurden in der vorliegenden Arbeit Mikroinjektionsexperimente dazu benutzt, die subzelluläre Lokalisation der Caspase-3 und den Mechanismus für den Übertritt in das Nukleoplasma zu untersuchen.
Dazu wurde Caspase-3 in E. coli exprimiert und aufgereinigt. Da das rekombinante Protein hohe DEVD-afc spaltende Aktivität aufwies, lag keine Beeinflussung der Protease durch die Fusion an GFP vor.
Die Mikroinjektion aktiver Caspase-3 allein war ausreichend, apoptotische Veränderungen in der Zelle zu induzieren. Gleichzeitig zeigte ein schneller Anstieg der nukleären Fluoreszenz den Übertritt des Fusionsproteins in den Kern an. Die Beeinträchtigung der Selektivität der Kernporen konnte als Auslöser für die Translokation ausgeschlossen werden. Die Inhibition der Aktivität von Caspase-9 und Caspase-3 führte nicht zur Retention von Caspase-3 im Cytoplasma. Desweiteren war die Prozessierung der Caspase-3 nicht notwendig für eine effektive Anreicherung im Kern.
Gleichzeitig blieb aber das signifikant kleinere Dextran40 aus dem Kern ausgeschlossen, was passive Diffusion von Caspase-3 unwahrscheinlich macht.
Die Abhängigkeit der Caspase-3 Akkumulation von Energie und der Interaktion mit Nukleoporinen wies auf eine Beteiligung von aktiven Transportprozessen hin. Dieser Import erfolgte unabhängig von Importin β und Transportin. Eine Blockade dieser Transportwege konnte die Anreicherung der Caspase-3 im Kern nicht verhindern.
Gleichzeitig zog die Inhibition des aktiven Imports eine Reduktion der Chromatinkondensation und der DNA Degradation nach sich. Für die Einleitung der Kernapoptose ist damit die Präsenz der Caspase-3 im Kern nicht essentiell notwendig.
Stattdessen werden die benötigten Effektoren Importin β abhängig aus dem Cytosol importiert.
Die Inhibition des Importin β vermittelten Transports führte zu einer Sensitivierung von HeLa Zellen auf TNFα. Damit wird deutlich, dass aktive Transportvorgänge sowohl für die Resistenz gegen TNFα vermittelte Apoptose als auch für die Durchführung der Kernapoptose unentbehrlich sind.
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