PDF《HtrA2/Omi》

1 h incubation with

20 mM Ucf-101, CHOP was already induced two-fold, rising to 5- to 8-fold at

2 h. ATF3 is another transcription factor known to be stress inducible (for review see Hai and Hartman22 ): it was also induced by Ucf-

101 in both wild-type and HtrA2-knockout MEFs (Figure 1c). The increase in CHOP and ATF3 mRNA levels in response to UCF-101 treatment does not seem to be cell type speci?c, because similar results were seen in the murine neuroblas- toma cell line Neuro-2A (data not shown). The induction of two transcription factors that are known to be responsive to stress, CHOP and ATF3, suggests that Ucf-101 might trigger activation of stress pathways in cells. This action is independent of its ability to inhibit HtrA2, as the response is seen as strongly in HtrA2-knockout as in wild-type MEFs. Therefore, we looked at the activation of various cellular stress and other signalling pathways using phospho- speci?c antibodies. Phosphorylation of the alpha subunit of eukaryotic initiation factor

2 (eIF2a) is a well-documented mechanism of downregulating protein synthesis under a variety of stress conditions.23 The stress-activated protein kinase/Jun-N-terminal kinase SAPK/JNK is potently and preferentially activated by diverse environmental stresses and can also be phosphorylated following stimulation of a member of the germinal centre kinase family. p38 MAP kinase Cell Death and Differentiation (2006) 13, 2157C2159 &

2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00 www.nature.com/cdd participates in a signalling cascade controlling cellular responses to cytokines and stress. Both p44 and p42 MAP kinases (ERK1 and ERK2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation, and are phosphorylated in response to a wide range of extracellular signals. All of these a [Ucf-101] ? ? M

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1 0 vehicle vehicle vehicle vehicle 20?M 50?M 20?M 50?M 20?M 50?M 20?M 50?M 20?M 50?M 20?M 50?M 20?M 50?M 20?M 50?M CHOP Fold mRNA expression ~20 ~35 2.5?g/ml Tunic 2.5?g/ml Tunic 2.5?g/ml Tunic 2.5?g/ml Tunic 2h 1h 2h 1h HtrA2 KO WILD TYPE HtrA2 KO WILD TYPE Ucf-101 Ucf-101 Ucf-101 Ucf-101 c

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1h 2h Tunic 2.5 ? g/ml Thapsi 300nM eIF2α -P (S51) JNK/SAPK-P (T183/Y185) p38-P (T180/Y182) p44/42 MAPK-P (T202/Y204) WILD TYPE Lamin B1 HtrA2 KO eIF2α -P (S51) JNK/SAPK-P (T183/Y185) p38-P (T180/Y182) p44/42 MAPK-P (T202/Y204) Lamin B1 d e Iκ κ B-α Iκ κ B-α 50?M 20?M Ucf-101 WT HtrA2 KO vehicle 30'

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100 ng/ml

10 ng/ml vehicle IκBα -P (Ser32/36) IκBα -P (Ser32/36) TNFα Figure

1 (a) Ucf-101 inhibits HtrA2 protease activity. Mature carboxy-terminal His6-tagged D133HtrA2 was expressed in and puri?ed from Escherichia coli.

100 nM HtrA2 were incubated with

10 mM optimal substrate (Mca-IRRVSYSF(Dnp)KK) at 301C in PBS containing

1 mM DTT. Fluorescence was monitored on a CytoFluor multiwell plate reader (PerSeptive Biosystems). Protease activity (arbitrary ?uorescence units/min) was determined by linear regression analysis of the data points corresponding to the maximum reaction rates for each assay condition. (b) Ucf-101 induces CHOP and (c) ATF3 mRNA. Total RNA was isolated from HtrA2 wild-type and knockout MEFs treated with vehicle, Ucf-101 (Calbiochem) or tunicamycin (Sigma) using the RNeasy system in combination with the RNase-free DNase kit (Qiagen). Complementary DNA was synthesized with the SuperScript III kit (Invitrogen) and used as a template for quantitative PCR analysis monitoring the real-time increase in ?uorescence of SYBR Green (Applied Biosystems) on a Chromo4 detector system (MJ Research). Gene-speci?c primers were designed using PrimerExpress software (Applied Biosystems) and are available on request. Relative transcript levels of target genes are normalized to Actin Gamma RNA levels. (d) Ucf-101 induces various cellular stress pathways. Whole-cell lysate was prepared from HtrA2 wild-type and knockout MEFs following treatment with vehicle, Ucf-101, tunicamycin or thapsigargin (Sigma). Proteins were separated on a NuPAGE 4C12% Bis-Tris gel (Invitrogen) and transferred onto a PVDF membrane by wet transfer. Primary antibodies were against eIF2a-phospho Ser51 (1 : 1000;