The unfolded protein response to PI*Z alpha‐1 antitrypsin in human hepatocellular and murine models

Abstract

Alpha‐1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 ( SERPINA1 , also known as AAT ) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full‐length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R‐like ER kinase and inositol‐requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro‐apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.

Reply

“…We agree that the mechanisms of up‐regulation of C/EBP homologous protein (CHOP) in this model remain unclear. [ 1 , 2 ] Interestingly, we saw highly variable CHOP expression even in litter mates. The up‐regulation of CHOP is not related to mouse age and activating transcription factor 6α (ATF6α) protein level in this model.…”

Liver CHOP‐and‐change stress response

“…

To the editor, We read with interest the paper Lu and colleagues, who investigated the unfolded protein response (UPR) in livers expressing the Z allele of α1-antitrypsin (Z-AAT). [1] Activation of the UPR in response to hepatic expression of Z-AAT has been controversial, largely because the available PiZ transgenic mouse was lacking an adequate control (i.e., a mouse transgenic for the wild-type human AAT). Brantly's laboratory filled this gap by generating brand-new transgenic mouse models expressing either the human wild-type AAT or Z-AAT, which they called PI*M and PI*Z mice, respectively.

…”

“…This is suggested by normal CHOP expression in a subset of PI*Z mice with reduced Z‐AAT storage. [ 1 ] Among the three branches of the UPR, Lu et al found that the activating transcription factor 6 (ATF6) was activated, whereas the other two branches were repressed. [ 1 ] ATF6 can drive CHOP up‐regulation, but the authors did not mention whether PI*Z mice with CHOP overexpression and larger Z‐AAT storage also have increased ATF6 activation.…”

“…[ 1 ] Among the three branches of the UPR, Lu et al found that the activating transcription factor 6 (ATF6) was activated, whereas the other two branches were repressed. [ 1 ] ATF6 can drive CHOP up‐regulation, but the authors did not mention whether PI*Z mice with CHOP overexpression and larger Z‐AAT storage also have increased ATF6 activation. Moreover, the increased expression of ATF4 found by Lu et al is puzzling because ATF4 is regulated by the UPR at the translational level rather than transcriptionally, and PERK (PKR‐like endoplasmic reticulum kinase) was found to be repressed.…”

“…Moreover, the increased expression of ATF4 found by Lu et al is puzzling because ATF4 is regulated by the UPR at the translational level rather than transcriptionally, and PERK (PKR‐like endoplasmic reticulum kinase) was found to be repressed. [ 1 ] In summary, the mechanism underlying CHOP up‐regulation remains elusive.…”