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.

FIGURE 1

AAT accumulation in Huh7.5Z cells. (A) Direct DNA sequencing showed a point mutation (G > A) in the human SERPINA1 gene at position 342 in Huh7.5Z cells. (B) Western blot analysis of AAT in cell lysate showed a significantly high level of intracellular AAT in Huh7.5Z cells compared with Huh7.5 cells (6.7‐fold). Results were normalized to GAPDH levels. Western blot quantification data are presented as mean ± SD. Significance was determined by the Student t test. **p < 0.01. (C) Enzyme‐linked immunosorbent assay results showed low AAT concentration in the cell culture medium of Huh7.5Z cells compared with that of Huh7.5 cells (0.1‐fold). Data are presented as mean ± SD. Significance was determined by the Student t test. **p < 0.01. (D) Immunofluorescence images (magnification, 40×) showed the colocalization of AAT (green) with golgin‐97 (red), a Golgi marker, in Huh7 and the colocalization of AAT (green) with BiP (red), an ER marker, in Huh7.5Z cells. AAT was mainly distributed in a Golgi pattern in Huh7.5 cells but an ER pattern in Huh7.5Z cells. (E) Immunofluorescence images (magnification, 40×) showed AAT polymer (red) in Huh7.5Z cells but not in Huh7.5 cells. AAT, alpha‐1 antitrypsin; BiP, binding protein; DAPI, 4′,6‐diamidino‐2‐phenylindole; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; hAAT, human alpha‐1 antitrypsin; SERPINA1, serpin family A member 1

FIGURE 2

Endoplasmic reticulum stress and unfolded protein response in Huh7.5Z cells. (A) Representative western blot and (B) quantification graph of PERK (n = 4), p‐PERK (n = 4), eIF2α (n = 3), p‐eIF2α (n = 3), and CHOP (n = 4) in Huh7.5Z and Huh7.5 cells. Results demonstrated increased activation of the PERK signaling branch in Huh7.5Z cells. The changes in the fold increase of expression levels in Huh7.5Z cells compared with Huh7.5 cells were: PERK 3.25‐fold, p = 0.049; p‐PERK 1.47‐fold, p = 0.018; p‐eIF2α 1.86‐fold, p = 0.049; CHOP 2.27‐fold, p = 0.033. Western blot quantification data are presented as mean ± SD. Significance was determined by the Student t test. *p < 0.05. (C) Representative western blot and (D) quantification graph of IRE1α (n = 4), p‐IRE1α (n = 4), ATF6α (n = 4), and ATF6αf (n = 4). Results showed significantly increased levels of p‐IRE1α and ATF6αf in Huh7.5Z cells compared with Huh7.5 cells, indicating the activated IRE1α and ATF6α signaling branches. The fold increases were: p‐IRE1α 1.69‐fold, p = 0.018; ATF6α 1.41‐fold, p = 0.01; ATF6αf 2.64‐fold, p = 3 × 10⁻⁴. Western blot quantification data are presented as mean ± SD. Significance was determined by the Student t test; *p < 0.05, ***p < 0.001. (E) qPCR analysis showed elevated Eif2ak3 mRNA (encodes PERK) levels in Huh7.5Z cells compared with Huh7.5 cells; qPCR results are presented as mean ± SD. The change in fold increase was 2.62‐fold. Significance was determined by the Student t test; **p < 0.01. ATF6α, activating transcription factor 6α; ATF6αf, the cytosolic fragment of ATF6α; CHOP, CCAAT‐enhancer‐binding protein homologous protein; eIF2α, eukaryotic initiation factor 2α; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IRE1α, inositol‐requiring transmembrane kinase/endoribonuclease 1α; mRNA, messenger RNA; p‐eIF2α, phosphorylated eIF2α; PERK, protein kinase R‐like endoplasmic reticulum kinase; p‐IRE1α, phosphorylated IRE1; p‐PERK, phosphorylated PERK; qPCR, quantitative polymerase chain reaction; RQ, relative quantification; UPR, unfolded protein response

FIGURE 3

Up‐regulated ER molecular chaperones and the activation of autophagy and apoptosis in Huh7.5Z cells. (A) Representative western blot and (B) quantification graph of the ER molecular chaperones showed significantly increased levels of BiP (n = 3), Hsp70 (n = 3), Hsp60 (n = 3), Hsp40 (n = 3), CALR (n = 3), and PDI (n = 3) in Huh7.5Z cells compared with Huh7.5 cells. The changes of fold increase were: BiP, 3.21‐fold; Hsp70, 1.58‐fold; Hsp60, 1.39‐fold; Hsp40, 1.89‐fold; CALR, 1.48‐fold; and PDI, 2.17‐fold. (C) Representative images of western blot and (D) quantification graph showed elevated ATG5 (n = 3) and LC3 (LC3I and LC3II, n = 3). ATG5 was increased by 1.60‐fold, whereas LC3I and LC3II were increased by 1.71‐ and 1.41‐fold, respectively. (E) Representative western blot and (F) quantification graph showed increased levels of caspase‐4 (n = 3), 9 (n = 3), 7 (n = 3), and 3 (n = 3) in cell lysates of Huh7.5Z cells compared with Huh7.5 cells. Increased levels of full‐length caspase‐3 were observed, but no cleaved caspase‐3 was detected. The increase of full‐length caspase‐4 did not reach statistical significance. The fold increases were: cleaved caspase‐4, 2.01‐fold; full‐length caspase‐9, 1.59‐fold; cleaved caspase‐9, 7.69‐fold; caspase‐7, 1.34‐fold; cleaved caspase‐7, 4.65‐fold; full‐length caspase‐3, 1.77‐fold. Quantification results were normalized to GAPDH levels and are presented as mean ± SD. Significance was determined by the Student t test; *p < 0.05, **p < 0.01, ***p < 0.001. ATG, autophagy‐related protein; BiP, binding protein; CALR, calreticulin; Casp, caspase; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; Hsp, heat shock protein; LC3, autophagy marker light chain 3; PDI, protein disulfide isomerase

FIGURE 4

PERK pathway was suppressed in tunicamycin‐induced prolonged endoplasmic reticulum stress in Huh7.5 cells. Huh7.5 cells were treated with 10 μg/mL of tunicamycin for 48 hours. Representative western blots showed that the levels of PERK and p‐PERK increased within 4 hours after the initiation of treatment and then decreased at 24‐ and 48‐hour time points compared with the control group; meanwhile, ATF6α levels were increased. The deglycosylation of ATF6α presented twice—first at 2 hours after treatment and again at 24 and 48 hours after the initiation of treatment. Cleaved ATF6αf increased at the 24‐hour time point. The levels of IRE1α were increased later than that of PERK but lasted longer than PERK. This experiment was repeated 3 times independently. ATF6α, activating transcription factor 6α; ATF6αf, the cytosolic fragment of ATF6α; ctrl, control; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; h, hours; IRE1α, inositol‐requiring transmembrane kinase/endoribonuclease 1α; PERK, protein kinase R‐like endoplasmic reticulum kinase; p‐IRE1α, phosphorylated IRE1α; p‐PERK, phosphorylated PERK; TM, tunicamycin

FIGURE 5

Endoplasmic reticulum stress and unfolded protein response in the liver of PI*Z transgenic mice. Western blot analysis was performed on protein extracted from transgenic and WT mouse livers. The results were normalized to GAPDH levels and are presented as mean ± SD. Significances were determined by t test; *p < 0.05, **p < 0.01. Western blot showed low levels of (A) PERK, p‐PERK, and (B) p‐IRE1α and (C) high ATF6αf/ATF6α ratio although the levels of ATF6α were low in PI*Z mice compared with PI*M and WT mice. Fold decreases were: PERK, PI*Z/WT, 0.61‐fold; PI*Z/PI*M, 0.58‐fold; p‐PERK, PI*Z/WT, 0.41‐fold; PI*Z/PI*M, 0.45‐fold; p‐IREα, PI*Z/WT, 0.12‐fold; fold increase of the ATF6αf/ATF6α ratio in PI*Z mice compared to PI*M is 3.58‐fold. ATF6, activating transcription factor 6; ATF6αf, cytoplasmic fragment of ATF6α; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IRE1α, inositol‐requiring transmembrane kinase/endoribonuclease 1α; PERK, protein kinase R‐like endoplasmic reticulum kinase; PI, protease inhibitor; p‐IRE1α, phosphorylated IRE1α; p‐PERK, phosphorylated PERK; WT, wild type

FIGURE 6

LPS treatment up‐regulated BiP, PERK, and ATF6α in PI*Z but not in PI*M and WT mice. PI*Z, PI*M, and WT mice were treated with 0.33 μg/g body weight LPS by IP. Liver tissues were collected at designated time points. Western blotting was performed on protein extracted from mouse liver tissue. Quantification results (quantification graphs not shown) were normalized to GAPDH levels and are presented as mean ± SD. Significance was determined by the Student t test. (A) PERK, IRE1α, and ATF6α proteins were increased 16 hours after LPS treatment in the liver in PI*Z mice (n = 3) compared to PI*M (n = 3) and WT (n = 3) mice. The t test p values were: BiP, PI*Z versus WT, p = 0.002; PI*Z versus PI*M, p = 0.008; PERK, PI*Z versus WT, p = 0.027; PI*Z versus PI*M, p = 0.012; ATF6α, PI*Z versus WT, p = 0.007; PI*Z versus PI*M, p = 0.012. There were no statistically significant differences in the levels of p‐PERK, IRE1α, p‐IRE1α, or ATF6αf in PI*Z mice compared to PI*M and WT mice. (B) PERK, IRE1α, and full‐length ATF6 levels had normalized at 72 hours after LPS treatment. Levels of p‐PERK, p‐IRE1α, and full‐length ATF6α were low in PI*Z mice (n = 5) compared with PI*M (n = 5) as the fold differences between PI*Z and PI*M mice were: pPERK, 0.13‐fold; p‐IRE1α, 0.10‐fold; ATF6α, 0.25‐fold; however, ATF6αf was 5.51‐fold higher in PI*Z mice compared with PI*M mice. (C) The PI*Z, PI*M, and WT mice were treated with 0.33 μg/g body weight LPS by IP injection. Liver tissues were collected before and at 8 hours, 16 hours, 48 hours, and 7 days after LPS treatment, and each time point had three mice in each strain. Representative western blot images showed that LPS treatment led to increased levels of BiP and PERK in PI*Z mice but not in PI*M and WT mice. ATF6, activating transcription factor 6; ATF6αf, cytoplasmic fragment of ATF6α; BiP, binding protein; d, days; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; h, hours; IP, intraperitoneal; IRE1α, inositol‐requiring transmembrane kinase/endoribonuclease 1α; LPS, lipopolysaccharide; PERK, protein kinase R‐like endoplasmic reticulum kinase; PI, protease inhibitor; p‐IRE1α, phosphorylated IRE1α; p‐PERK, phosphorylated PERK; WT, wild type

FIGURE 7

CHOP protein and mRNA and ATF4 mRNA in the liver of PI*Z transgenic mice. Protein and RNA were extracted from mouse liver tissues. Western blot analyses of CHOP as well as qPCR analyses of Ddit3 (which encodes CHOP) and ATF4 mRNA were performed. PI*Z mice were separated into two subsets according to their PAS‐D scores: Pi*Z (LP) and Pi*Z (HP). (A) qPCR analysis of Ddit3 mRNA levels in the livers of Pi*Z (HP), PI*Z (LP), and PI*M transgenic and WT mice. Ddit3 mRNA was significantly higher in all Pi*Z (HP) mice, whereas only two of the PI*Z (LP) mice had mildly increased Ddit3 mRNA. (B) Western blot analysis of CHOP in the livers of PI*Z and PI*M transgenic mice and C57BL6/J WT mice. Five of the 13 PI*Z mice had increased CHOP protein and they were all in the PI*Z (HP) subset. (C–E) Correlation analysis between Ddit3 mRNA and human SERPINA1 (which encodes hAAT) genomic DNA, human SERPINA1 mRNA, and mouse liver tissue PSA‐D scores. Ddit3 levels did not correlate with SERPINA1 genomic DNA levels but correlated with SERPINA1 mRNA levels and PAS‐D scores. (F) qPCR assay results of ATF4 mRNA. qPCR results showed increased transcriptions of the ATF4 gene in the PI*Z (HP) subset but not in the Pi*Z (LP) subset and other groups. qPCR results are presented as mean ± SD. Significance was determined by one‐way analysis of variance (Dunnett's multiple comparison test); **p < 0.01, ***p < 0.001. AAT, alpha‐1 antitrypsin; ATF4, activating transcription factor 4; CHOP, CCAAT‐enhancer‐binding protein homologous protein; Ddit3, DNA damage inducible transcript 3; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; gDNA, genomic DNA; hAAT, human alpha‐1 antitrypsin; HP, high PAS‐D subset; LP, low PAS‐D subset; mRNA, messenger RNA; PAS‐D, periodic acid–Schiff–diastase stain; PI, protease inhibitor; qPCR, quantitative polymerase chain reaction; RQ, relative quantification; SERPINA1, serpin family A member 1; WT, wild type