Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis

Abstract

The endoplasmic reticulum (ER) has the ability to signal organelle dysfunction via a complex signaling network known as the unfolded protein response (UPR). In this work, hamster fibroblast cells exhibiting moderate levels of ER stress were compared to those exhibiting severe ER stress. Inhibition of N-linked glycosylation was accomplished via a temperature-sensitive mutation in the Dad1 subunit of the oligosaccharyltransferase (OST) complex or by direct inhibition with tunicamycin (Tm). Temperature shift (TS) treatment generated weak activation of ER stress signaling when compared to doses of Tm that are typically used in ER stress studies (500-1000 nM). A dose-response analysis of key ER stress signaling mediators, inositol-requiring enzyme 1 (IRE1) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), revealed 20-40 nM of Tm to generate activation intensity similar to TS treatment. In parental BHK21 cells, moderate (20-40 nM) and high doses (200-1000 nM) of Tm were compared to identify physiological and signaling-based differences in stress response. Inhibition of ER Ca release via ITPR activity with 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) was sufficient to protect against apoptosis induced by moderate but not higher doses of Tm. Analysis of kinase activation over a range of Tm exposures revealed the p38 stress-activated protein kinase (SAPK) to display increasing activation with Tm dosage. Interestingly, Tm induced the extracellular regulated kinases (Erk1/2) only at moderate doses of Tm. Inhibition of ER transmembrane stress sensors (IRE1, PERK) or cytosolic signaling mediators (p38, Jnk1, Erk1/2) was used to evaluate pathways involved in apoptosis activation during ER stress. Inhibition of either PERK or p38 was sufficient to reduce cell death and apoptosis induced by moderate, but not high, doses of Tm. During ER stress, cells exhibited a rapid decline in anti-apoptotic Mcl-1 and survivin proteins. Inhibition of PERK was sufficient to block this affect. This work reveals moderate doses of ER stress to generate patterns of stress signaling that are distinct from higher doses and that apoptosis activation at moderate levels of stress are dependent upon PERK and p38 signaling. Studies exploring ER stress signaling should recognize that this signaling acts as a rheostat rather than a simple switch, behaving distinctively in a dose-dependent manner.

The effect of Cyanidin-3-o-glucoside on UVA-induced damage in human dermal fibroblasts

“…Inhibition of P38 activation was sufficient to reduce hamster fibroblast cells death and apoptosis induced by moderate tunicamycin. 31 In our study, C3G reduced the high levels of p-P38 induced by UVA.…”

“…Huang et al and Hsu et al found that p‐P38 was significantly increased in HDFs after UVA irradiation, consistent with the findings of this study. Inhibition of P38 activation was sufficient to reduce hamster fibroblast cells death and apoptosis induced by moderate tunicamycin . In our study, C3G reduced the high levels of p‐P38 induced by UVA.…”

Functional expression of TLR5 of different vertebrate species and diversification in intestinal pathogen recognition

“…S5 ). The lack of expression was accompanied by a massive phosphorylation of p38, suggesting cellular endosomal stress activation, possibly due to a trafficking impairment that can lead to downstream stress signaling 37 . Two sequence polymorphisms in the ectodomain of the cloned full-length chicken TLR5 and the ch/h chimera were identified by Sanger sequencing in comparison to the reference sequence in the NCBI database (NP_001019757.1) (Suppl.…”

Cross organelle stress response disruption promotes gentamicin-induced proteotoxicity

“…UPR is situated to mediate both adaptive and maladaptive mechanisms that account for the diverse phenotype observed during gentamicin-induced proximal tubule cell death. UPR normally limits cell stress by increasing protein refolding and mitigating CHOP activation, a major contributor to cell death 69,70 . However, if cell stress is prolonged or irreversible, the balance between misfolded proteins and available chaperones is perturbed.…”