Genome‐wide mRNA profiling identifies RCAN1 and GADD45A as regulators of the transitional switch from survival to apoptosis during ER stress

Abstract

Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol‐requiring protein 1, protein kinase RNA‐like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human‐immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18‐h time course and utilized real‐time monitoring of cell survival, next‐generation sequencing, and quantitative real‐time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage‐inducible alpha (GADD45A), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT‐enhancer‐binding protein homologous protein (a.k.a DDIT3), BCL2‐binding component 3 (a.k.a. BBC3), and phorbol‐12‐myristate‐13‐acetate‐induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor.

IRE1 Endoribonuclease Activity Modulates Hypoxic HIF-1α Signaling in Human Endothelial Cells

“…These results that confirm the hypoxic activation of HIF-1 signaling in HUVECs are in good agreement with previous studies including our own [ 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Surprisingly, the luminal endoplasmic reticulum protein BiP ( HSPA5) mRNA levels, a UPR pro-adaptive activation marker [ 41 , 42 , 43 , 44 ], were reduced after 12 h of exposure to hypoxia ( Figure 1 D), while the mRNA levels of apoptotic C/EBP homologous protein ( CHOP ( DDIT3 )) [ 41 , 43 , 44 , 45 ] were elevated only after 24 h of exposure to hypoxia ( Figure 1 E). Thus, the exposure of HUVECs to hypoxia did not result in ER stress and the subsequent activation of UPR signaling during the earlier time points of up to 12 h.…”

“…Furthermore, it needs to be noted that most ER stress and UPR studies are based on the use of high concentrations of pharmacological ER stressors that result in the potent activation of all UPR pathways at both the transcriptional and posttranscriptional levels [ 2 , 41 , 66 ]. This is in contrast to the biological role and the extent of the biological activity of PERK and IRE1 in low stress conditions which are relatively unknown.…”

SARS-CoV-2 may regulate cellular responses through depletion of specific host miRNAs

“…Both the increased ER membrane and the protein folding demand can result in coronavirus-related ER stress and activation of a dedicated signaling pathway, the unfolded protein response (UPR). Importantly, the UPR has the ability to promote cellular survival by increasing ER membranes and folding capacity, but also has the ability to induce cell death if the stress is persistent ( 5 , 8 , 10 , 37 , 45 , 51 , 104 ).…”

“…PERK gains control on the cellular translation processes to reduce ER load ( 38 , 41 ), and eventually facilitates autophagy. If the stress is persistent, PERK and ATF6 induce cell death via accumulation of CCAAT/enhancer binding homologous protein (CHOP) ( 2 , 8 , 49 , 80 ). IRE1α uses its endoribonuclease properties to selectively reduce ER mRNA ( 40 , 72 ) and to produce the active isoform of the X-box binding protein transcription factor (XBP1s) ( 118 ).…”

Triazoloacridone C-1305 impairs XBP1 splicing by acting as a potential IRE1α endoribonuclease inhibitor

“…propyl]amino}-8-hydroxy-6H- [1,2,3]triazolo[4,5,1-de]acridin-6 one (C 18 H 19 N 5 O 2 , M W = 337.38 g/mol) (compound C-1305) was synthesized by the Faculty of Chemistry, University of Gdansk, Poland, and verified for purity and identity with RP-HPLC, elemental analysis, 1 H NMR spectroscopy and HR MS spectrometry as previously described in [21,23,24]. The C-1305 chemical formula is provided in Fig.…”