PDF《HtrA2/Omi》

Cell Signaling), JNK/SAPK-phospho Thr183/Tyr185, phospho-p38 MAPK Thr180/Tyr182, phospho- p44/42 MAPK Thr202/Tyr204 (all

1 : 1000;

Cell Signaling) and Lamin B1 (1 : 1000;

Santa Cruz). Secondary antibodies used were mouse-HRP, rabbit-HRP (both

1 : 2000;

Amersham) and goat-HRP (1 : 5000;

Pierce), and these were detected using enhanced chemiluminescence (Amersham). (e) NF-kB is not activated in response to Ucf-101. Primary antibodies were against phospo-IkBa (1 : 2000;

Cell Signaling) and IkBa (1 : 1000;

Santa Cruz). As a control for NF-kB activation, cells were treated with TNFa for

5 min Letter to the Editor

2158 Cell Death and Differentiation signalling pathways are activated in a time- and concentra- tion-dependent manner by Ucf-101 in wild-type and HtrA2- knockout MEFs (Figure 1d) and in Neuro-2A cells (data not shown). We conclude that HtrA2 is not essential for the stress responses induced by tunicamycin and thapsigargin. This may not be unexpected as these agents act primarily to cause ER, rather than mitochondrial stress. However, even at low concentrations, the HtrA2 inhibitor Ucf-101 seems to have a broad effect on the activation of cellular stress-response pathways that is independent of its ability to inhibit HtrA2 proteolytic activity. It is not clear how Ucf-101 elicits such a pronounced overall stress response. The most important implication of these ?ndings is that the inhibitor Ucf-101 should be used with great care and not regarded as an entirely speci?c inhibitor of HtrA2. Several reports have assumed that all the biological effects of Ucf-101 re?ect its inhibition of HtrA2 protease activity.14C19 As Ucf-101 can induce activation of ERK (Figure 1d), it is possible that its survival-promoting actions could be mediated by this known antiapoptotic pathway. Alternatively, moderate activation of various stress-induced pathways has been associated with cellular adaptation, or conditioning, and protection from subsequent challenge with death stimuli;

so it is possible that the ability of Ucf-101 to activate these pathways might also contribute to its reported protective effects.21 A number of agents that induce CHOP are also known to activate the NF-kB pathway. However, Ucf-101 is not able to activate this pathway: it fails to induce phosphorylation of IkBa under circumstances where TNFa gives a robust response (Figure 1e). Is it possible that the effects of Ucf-101 in HtrA2-knockout cells are mediated by other closely related proteases, such as HtrA1,

3 or 4?24 Although the similarity of the serine protease domains in these proteins would suggest that they may also be targeted by Ucf-101, it is likely that they have very distinct function from HtrA2. None of these proteins are known to be mitochondrial or to have consensus mitochondrial import signals;

HtrA1 is reported to be secreted from cells.25 None have been implicated in the regulation of cell death or stress signalling. If Ucf-101 is eliciting biological effects on cells through the inhibition of HtrA1,

3 or 4, it seems unlikely that it will be able to provide meaningful information about HtrA2 function, given the evident functional divergence within this family. We recommend considerable caution in the use of Ucf-101 to investigate HtrA2 function and that it should always be backed up by data from other approaches to the ablation of HtrA2 activity, such as gene knockout or RNA interference. Acknowledgements We thank Miguel Martins and He........