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. 2011 Apr 28;2(4):e149.
doi: 10.1038/cddis.2011.31.

Induction of ER stress in response to oxygen-glucose deprivation of cortical cultures involves the activation of the PERK and IRE-1 pathways and of caspase-12

N Badiola  1 , C Penas, A Miñano-Molina, B Barneda-Zahonero, R Fadó, G Sánchez-Opazo, J X Comella, J Sabriá, C Zhu, K Blomgren, C Casas, J Rodríguez-Alvarez
Affiliations

Induction of ER stress in response to oxygen-glucose deprivation of cortical cultures involves the activation of the PERK and IRE-1 pathways and of caspase-12

N Badiola et al. Cell Death Dis. .

Abstract

Disturbance of calcium homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) are considered contributory components of cell death after ischemia. However, the signal-transducing events that are activated by ER stress after cerebral ischemia are incompletely understood. In this study, we show that caspase-12 and the PERK and IRE pathways are activated following oxygen-glucose deprivation (OGD) of mixed cortical cultures or neonatal hypoxia-ischemia (HI). Activation of PERK led to a transient phosphorylation of eIF2α, an increase in ATF4 levels and the induction of gadd34 (a subunit of an eIF2α-directed phosphatase). Interestingly, the upregulation of ATF4 did not lead to an increase in the levels of CHOP. Additionally, IRE1 activation was mediated by the increase in the processed form of xbp1, which would be responsible for the observed expression of edem2 and the increased levels of the chaperones GRP78 and GRP94. We were also able to detect caspase-12 proteolysis after HI or OGD. Processing of procaspase-12 was mediated by NMDA receptor and calpain activation. Moreover, our data suggest that caspase-12 activation is independent of the unfolded protein response activated by ER stress.

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Figures

Figure 1
Figure 1
OGD activates PERK–eIF2α pathway. (a) Western blot analysis of total eiF2α and phosphorylated eIF2α (p-eIF2α) and PERK after OGD. (b) Western blot analysis of ATF4 levels at indicated times after OGD. α-tubulin was used as loading control. (c) RT-PCR products for gadd34 obtained from cortical cultures at indicated times after OGD (top). Histogram quantification of the fold change in gadd34 expression respect to actin levels (bottom). *P<0.001 versus control. (d) Western blot analysis of GADD34 levels at indicated times in normoxia (N) and after OGD (O). Actin was used as loading control
Figure 2
Figure 2
OGD activates IRE1–XBP1 pathway. (a) RT-PCR products for grp78 and grp94 obtained from cortical cultures at indicated times after OGD (top). Quantification of grp78 and grp94 mRNAs, normalized to actin levels (bottom). *P<0.01 and +P<0.001 versus control. (b) Time course of GRP78 and GRP94 protein expression in cortical cultures exposed to OGD. Membranes were also immunoblotted with an anti-actin antibody as loading control (top). Quantification of protein levels of GRP78 and GRP94 normalized to actin levels (bottom). *P<0.05 and +P<0.001 versus control. (c) ATF6 protein expression was study by western blot at different times after OGD. ATF6 (f) is ATF6 form that migrates faster. A lysate of Tg-treated cells (5 μ) was loaded as a positive control. (d) RT-PCR products for xbp1 and edem2 obtained from cortical cultures at indicated times after OGD (top). The processed xbp1 was obtained as explained in materials and methods section. Quantification of spliced xbp1 and edem2 mRNAs, normalized to actin levels (bottom). *P<0.001 versus control
Figure 3
Figure 3
OGD-mediated activation of caspase-12 is calpain dependent. (a) Western blot analysis of caspase-12 at indicated times in cultures exposed to OGD. A lysate of Tg-treated cells (5 μ) was loaded as a positive control of caspase-12 activation. Arrows indicate pro-caspase-12 cleavage products. Asterisk indicates a nonspecific band. (b) Procaspase-12 proteolysis analysis by western blot in cultures exposed to normoxia (N) or OGD (O) in presence of caspase pan-inhibitor, z-VAD-fmk(100 μ), calpain inhibitor, ALLN (1 μ) or NMDA receptor agonist, MK-801 (10 μ). (c) Caspase-12 activity was determined at 6 h by a fluorimetric assay as described in materials and methods section. Histograms represent the activity in normoxia (Nor) or after OGD in cultures treated with the indicated inhibitors. Activity observed in normoxia control cultures (normoxia without inhibitors) was taken as reference and considered to be 1. Results are mean±S.E.M. of three independent experiments performed in triplicate.+P<0.01 versus normoxia and *P<0.01 versus OGD (d) Procaspase-12 proteolysis in cortical cultures treated for 6 or 12 h with NMDA (100 μ) in the presence or absence of MK-801. All the membranes were also immunoblotted with an anti-actin antibody as a loading control
Figure 4
Figure 4
HI induces ER stress. (a) Western blot analysis of total eiF2α and phosphorylated eIF2α (p-eIF2α) at different times after HI. Pooled samples of homogenates from contralateral (CL) or ipsilateral (IL) cortex were loaded. Quantification of the p-eiF2 versus eIF2 ratio (bottom). *P<0.05 versus CL. (b) Western blot analysis of GRP78 and GRP94 protein expression after HI (left). Histogram quantification of pooled samples normalized with respect to GAPDH levels and showed as fold change versus CL at 0 h (*P<0.05). (c) Pro-caspase-12 proteolysis analysis by western blot, in cortex homogenates of animals exposed to HI. Lysates of cortical neurons culture exposed to OGD (O) and control (C) were use to confirm the proteolysis of procaspase-12. GAPDH was used as loading control
Figure 5
Figure 5
ER stress-activated signal transduction pathways and caspase-12 activation in OGD. OGD activates PERK, which phosphorylates eIF2α leading to inhibition of protein synthesis, but also increases the translation of ATF4 transcription factor. ATF4 mediate the increase of gadd34 expression, which is involved in the desphophorylation of eIF2α. OGD-mediated IRE1 activation allows the processing of xbp1 mRNA, resulting in enhancement of XBP1 (54 kDa) translation. XBP1 protein binds to ERSE enhancing the expression of grp78 and grp94, and binds to UPRE increasing the edem2 expression. OGD induces an increase of underglycosilated ATF6 form, this increase can be related with the translocation of ATF6 to Golgi to be cleaved. The cleaved ATF6 could translocate to nucleus, but it is not related to the upregulation of grp78 and grp94 expression. OGD activation of NMDAR also causes calcium influx and the subsequent activation of calpain. Calpain proteolyses pro-caspase-12 generating the active form of caspase-12
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