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1-Antitrypsin Deficiency1
Respiratory Research Division, Royal College of Surgeons in Ireland, Education and Research Center, Beaumont Hospital, Dublin, Ireland
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Abstract |
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1-antitrypsin (A1AT) deficiency is a genetic disease associated with accumulation of misfolded A1AT in the endoplasmic reticulum (ER) of hepatocytes. We sought to identify intracellular events involved in the molecular pathogenesis of Z A1AT-induced liver disease using an in vitro model system of Z A1AT ER accumulation. We investigated ER stress signals induced by Z A1AT and demonstrated that both the ER overload response and the unfolded protein response were activated by mutant Z A1AT, but not wild-type M A1AT. Interestingly, activation of the unfolded protein response pathway required an additional insult, whereas NF-
B activation, a hallmark of the ER overload response, was constitutive. These findings have important implications for the design of future therapeutics for Z A1AT liver disease and may also impact on drug design for other conformational diseases. |
Introduction |
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1-antitrypsin (A1AT)3 deficiency is an autosomal recessive disorder. In recent years there has been much emphasis on the lung disease associated with A1AT deficiency, in particular with regard to augmentation therapy and the delayed progression of emphysema. Over 90 polymorphic alleles (1) of A1AT have been identified; however, the mutant Z form occurs in >95% of all individuals with A1AT deficiency. The Z allele in particular is associated with liver disease, with 
10% of all homozygous neonates developing hepatitis and cholestasis. A proportion of these children progress to liver failure, resulting in liver transplantation (2, 3). Cirrhosis in A1AT-deficient adults can occur without a preceding history of childhood liver disease. Currently liver transplantation is the only therapy for severe liver disease associated with A1AT deficiency (4, 5), partly because the pathogenesis of this condition remains unknown.
Z A1AT deficiency results in the accumulation of A1AT within hepatocytes and colangiocytes. The Z A1AT protein differs from the normal M variant by a single amino acid substitution (Glu342
Lys) (6). This mutation destroys a salt bridge and affects the secondary structure of A1AT, a perturbation involving a unique molecular interaction between the reactive center loop of one molecule and the A sheet of another (7). Z A1AT accumulates in the endoplasmic reticulum (ER) as a result of this polymerization; some of it is degraded, but the remainder aggregates to form insoluble intracellular inclusions with only 15% of Z A1AT being secreted (8, 9).
Accumulation of mutant proteins in the ER, such as Z A1AT, can lead to ER stress, and cells respond to this perturbation by inducing the expression of novel genes whose products might restore proper ER function (10), but, in turn, can be proinflammatory. Regulation of these genes can occur as a result of two discrete signal transduction pathways: the ER overload response (EOR) and the unfolded protein response (UPR). The EOR culminates in activation of the transcription factor NF-B (11). The UPR involves up-regulation of glucose responsive genes (e.g., grp78 (Ig H chain binding protein (BiP)) and grp94) (12, 13). In this study we examined the mechanism of ER stress elicited by Z A1AT accumulation. We generated a model cell system of Z A1AT-induced stress and examined EOR by NF-B-luciferase reporter gene assay and EMSA, and UPR by grp78-luciferase reporter gene assay and glucose-responsive protein 78 (GRP78)/BIP and GRP94 protein production. We also assessed the proinflammatory effects of Z A1AT by measuring cytokine production. We found that Z A1AT activated both pathways, and the EOR pathway seems to be the predominant of the two. The UPR pathway only became activated when a secondary stress was applied. The data presented in this study provide new information regarding the pathogenesis of the liver disease associated with Z A1AT.
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Materials and Methods |
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To determine the intracellular stress mechanism associated with Z A1AT production and retention, we used Chinese hamster ovary (CHO) cells (American Type Culture Collection, Manassas, VA) transiently transfected with eukaryotic expression vectors containing a normal M A1AT or mutant Z A1AT cDNA. The M A1AT cDNA was cloned on a 1256-bp EcoRI/XhoI fragment into pZeoSV2+ (Invitrogen, Carlsbad, CA) to generate pMA1AT. Z A1AT was constructed by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) and mutagenic primers encoding the Glu342Lys mutation (bold) and a ClaI restriction site (underlined) for screening (forward primer, 5'-GCT GTG CTG ACC ATC GAT AAG AAA GGG ACT GAA GCT GCT G-3'). Putative mutants were isolated, and their sequences were verified. This expression plasmid was named pZA1AT. The cDNA for green fluorescent protein S65T was amplified by PCR and cloned on a 714-bp XhoI/ApaI fragment into pMA1AT and pZA1AT to generate plasmids expressing fluorescent-tagged versions of M A1AT and Z A1AT.
Transfection
CHO or HEK293 cells were seeded at 5 x 106 on six-well plates 24 h before transfection. Transfections were preformed with TransFast transfection reagent (Promega, Madison, WI) in a 1:1 ratio according to the manufacturer’s instructions for 1 h with 10 µg pZeoSV2+ empty vector, pMA1AT or pZA1AT. After supplementation with complete medium, cells were left for further time periods (as indicated). Uniform transfection efficiencies were verified by measuring 
-galactosidase activity using the substrate o-nitrophenyl--D-galactopyranoside from a cotransfected -galactosidase expression plasmid.
A1AT protein production measurement by sandwich ELISA
A1AT protein production in cell supernatants and lysates was measured by sandwich ELISA using goat anti-human IgG A1AT (ICN Biomedicals, Aurora, CA), rabbit anti-human IgG (Roche, Indianapolis, IN), goat anti-rabbit IgG HRP (DAKO, Carpenteria, CA), and ABTS substrate (Zymed Laboratories, San Francisco, CA) (14). After absorbance reading at 405 nm on a multilabel counter (Victor2; Wallac, Gaithersburg, MD), A1AT levels were calculated from an A1AT (plasma-purified M A1AT) standard curve. Values are expressed as nanograms per 5 x 106 cells.
Reporter gene assays
CHO cells were seeded at 1 x 106/well and were cotransfected with 1 µg of pZeoSv2+ (empty vector), pMA1AT, or pZA1AT and either 1 µg of grp78 promoter-linked luciferase plasmid (a gift from K. Mori (Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan)) (13) or an (NF-B)5-luciferase reporter gene plasmid. Transfections were incubated for 1 h at 37°C. Cells were then supplemented with additional growth medium (4 ml/well) for 24 h at 37°C before being left untreated or treated with 2.5 µM thapsigargin or heat (42°C) for 24 h. Cells were lysed with reporter lysis buffer (Promega; 250 µl/well), protein concentrations were determined (15), and reporter gene activity was quantified by luminometry (Victor 1420 multilabel counter; Wallac) using the Promega luciferase assay system according to the manufacturer’s instructions. Reporter gene expression was expressed as light units per microgram of total protein.
EMSA
Nuclear extracts (5 µg of protein) were incubated with 10,000 cpm of a 22-bp oligonucleotide containing the NF-B consensus sequence (Santa Cruz Biotechnology, Santa Cruz, CA) that was labeled with [
-32P]ATP (10 mCi/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) by T4 polynucleotide kinase (Promega). Incubations were performed for 30 min at room temperature in binding buffer (4% (v/v) glycerol, 1 mM EDTA, 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM DTT, and 0.1 mg/ml nuclease-fresh BSA) and 2 µg of poly(dI-dC):poly(dI-dC) (Sigma-Aldrich, St. Louis, MO). In some experiments, unlabeled mutant or wild-type oligonucleotides or Abs to p50, p65, or c-Rel (Santa Cruz Biotechnology) were added to the extracts before incubation with the labeled oligonucleotide. All incubations were subjected to electrophoresis on native 4% (w/v) polyacrylamide gels that were dried, analyzed on a Molecular Dynamics Storm 820 phosphorimagery scanner for quantification, and autoradiographed.
Western blot analysis
Cytoplasmic extracts (5 µg of protein) were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (Sigma-Aldrich) in 20 mM Tris, 150 mM glycine, 0.01% SDS, and 20% (v/v) methanol at 75 mA for 2 h using a semidry electrophoretic blotting system. Nonspecific binding was blocked with 0.2% I-Block (Tropix, Bedford, MA) and PBS containing 0.1% Tween 20 (Sigma-Aldrich). Immunoreactive proteins were detected by incubating the membrane with a specific Ab (GRP78, GRP94 (N-20), and IB (from Santa Cruz Biotechnology); IB (from Cell Signaling Technologies, Beverly, MA); or eukaryotic initiation factor 2 (eIF2; from Abcam, Cambridge, U.K.)). After six 5-min washes with PBS containing 0.1% Tween 20, immunoreactive proteins were detected using an appropriate alkaline phosphatase-conjugated secondary Ab (Promega) and CDP-Star chemiluminescent substrate solution (Sigma-Aldrich) according to the manufacturer’s instructions.
IL-6 and IL-8 protein production
The human cell line 16HBE14o- was transfected as described under reporter gene assays. IL-6 and IL-8 protein concentrations in cell supernatants were determined by ELISA (R&D Systems, Minneapolis, MN). Protein concentrations were determined by the method of Bradford (15). Cell viability, assessed by trypan blue exclusion, was <95% in all studies. Results are expressed as picograms per microgram of total protein.
Statistical analysis
Data were analyzed with the PRISM 3.0 software package (GraphPad, San Diego, CA). Results are expressed as the mean ± SE and were compared by t test. Differences were considered significant at p 
0.05.
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Results |
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We designed a model system to investigate intracellular stress mechanisms associated with Z A1AT production and retention. We selected CHO cells because these do not produce their own native A1AT. Cells were transiently transfected with M A1AT or Z A1AT expression plasmids. Cellular accumulation and secretion of green fluorescent protein-tagged M A1AT and Z A1AT were visualized using fluorescence microscopy. Z A1AT, but not M A1AT, was retained in inclusions within the cells (data not shown).
A1AT production was measured in cell supernatants and lysates (Fig. 1). M A1AT-transfected cells secreted 65% of their A1AT, whereas Z A1AT-transfected cells secreted 10-fold lower levels of A1AT (p = 0.05), <10% of its A1AT complement. This indicated that Z A1AT was not secreted from these cells as readily as M A1AT. Lysates from Z A1AT-transfected cells also contained less A1AT than lysates from M A1AT-expressing cells (p = 0.05). This was not due to differences in transfection efficiencies, but is probably due to degradation of the incorrectly folded Z A1AT within the cells (16) due to decreased protein synthesis associated with ER stress.
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To examine the effects of Z A1AT on the EOR pathway, NF-B activation by Z A1AT was measured by NF-B luciferase reporter gene assay and EMSA (Fig. 2, A and B). NF-B promoter activation was measured in control, M A1AT-transfected, and Z A1AT-transfected cells 24 h post-transfection using an NF-B-linked luciferase reporter gene (Fig. 2A). Z A1AT induced significantly higher levels of luciferase activation compared with those in control or M A1AT-transfected cells (p = 0.001), suggesting that overexpression of Z A1AT leads to activation of NF-B.
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B nuclear localization and DNA binding capacity by EMSA 24 h post-transfection (Fig. 2B). Compared with control cells, M A1AT expression led to partial activation of NF-B, but this was less potent than the activation induced by Z A1AT, as shown by densitometry (Fig. 2C; p < 0.05). Competition studies with unlabeled mutant and wild-type NF-B probes demonstrated that Z A1AT activated NF-B specifically (data not shown). Antisera to NF-B components identified the subunit composition of the Z A1AT-induced NF-B complexes as containing p65, but not c-Rel or p50 (data not shown).
NF-B activation induced by Z A1AT does not induce phosphorylation of eIF2
We next investigated the mechanism by with Z A1AT-induced EOR activates NF-B. It has been reported that IB degradation does not occur in response to certain ER stress stimuli, but that the subunit of eIF2 can become phosphorylated by eIF2 kinases (e.g., pancreatic eIF2 kinase, heme-regulated eIF2 kinase, eIF2 protein kinase 2, and dsRNA-activated protein kinase) and that this leads not only to a general reduction in protein translation, but specifically to NF-B activation (17). We investigated whether phosphorylation of eIF2 occurred in response to Z A1AT overexpression by Western immunoblot analysis using a polyclonal Ab specific for eIF2 phosphorylated at Ser51 (eIF2-P). No difference were detected in eIF2-P at 24 h post-transfection among control, M A1AT-expressing, and Z A1AT-expressing cells (Fig. 3A), suggesting that phosphorylation of eIF2 is not required for activation of NF-B in response to Z A1AT-induced cellular stress.
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B and IB degradation in the same cells, also by Western immunoblotting. IB or IB were not degraded 4 h post transfection (data not shown); however, 24 h post-transfection both IB and IB were degraded in Z A1AT-transfected cells compared with control or M A1AT-transfected cells (Fig. 3, B and C). Z A1AT activates the UPR pathway
We next examined the effects of Z A1AT expression on UPR activation by investigating its effect on the grp78 promoter and GRP78/BiP and GRP94 protein production. Similar to control and M A1AT-transfected cells, Z A1AT had no effect on the expression of a grp78-promoter-linked luciferase reporter gene (Fig. 4A). Thapsigargin, a known ER agonist, increased grp78 promoter-linked luciferase reporter gene expression in all cells (p 0.05); however, in the presence of thapsigargin, Z A1AT induced significantly more grp78 promoter activation than M A1AT or control cells (p < 0.05 compared with M A1AT cells). We also tested the effect of heat as a stress that might potentiate the effect of Z A1AT on UPR, as it has been hypothesized that episodes of elevated temperature may contribute to liver manifestations of Z A1AT deficiency (18). Fig. 4A shows that compared with untreated cells or M A1AT cells in the presence of heat, the grp78 promoter was more strongly activated in ZA1AT cells (p 0.05).
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Z A1AT stimulates IL-6 and IL-8 protein production
To measure the effect of Z A1AT on inflammation, IL-6 and IL-8 protein production was measured in a human cell line. Basal and Z A1AT-induced IL-6 protein levels in cell supernatants from 16HBE14o- cells were quantified by ELISA (Fig. 5). 16HBE14o- cells produced a mean basal level of 160 ± 21 pg/µg of IL-6 protein and 267 ± 17 pg/µg of IL-8. M A1AT did not significantly increase either IL-6 or IL-8 levels; however, Z A1AT induced maximal IL-6 protein production from 16HBE14o- cells at 24 h (time-course experiments data not shown), increasing IL-6 levels to 335 ± 34 pg/µg of protein (p 0.05) and IL-8 levels to 482 ± 26 pg/µg of protein (p 0.05).
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Discussion |
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B activity and degradation of IB and IB, indicating activation of the EOR pathway, whereas an increase in GRP78 and GRP94 expression was also observed in the Z A1AT-transfected cell line, indicating activation of the UPR pathway. However, this only occurred with application of a secondary stress. The ER is a multifunctional signaling organelle that controls a wide range of cellular processes. In particular, it has an important role in protein folding and the handling of misfolded proteins. Specific signaling pathways (19, 20) and effector mechanisms have evolved to deal with the temporal and developmental variation in the ER load. The upstream signal that activates these pathways is referred to as ER stress and is defined functionally as an imbalance between the load of proteins facing the ER and the organelle’s ability to process that load. The cellular response to ER stress has four main functional components: EOR, UPR, a decrease in protein synthesis, and programmed cell death (21). In this study we investigate in detail the EOR and UPR pathways, as ER stress signals specifically activated by Z A1AT.
EOR leads to activation of the transcription factor NF-B (11). In a great variety of cell types, NF-B is found in an inactive cytoplasmic complex with IB. Upon stimulation of cells with a number of agents, NF-B activity is induced. This involves phosphorylation and degradation of IB and translocation of NF-B to the nucleus, where it activates transcription of proinflammatory target genes. Thus, the major role of the NF-B system is to mount a rapid and coordinated induction of proinflammatory genes in response to specific stimuli. In this study we found that accumulation of Z A1AT in the ER provided an NF-B-activating stimulus, which also resulted in a marked increase in cytokine production of IL-6 and IL-8 from the Z A1AT-expressing cell line. An alternative mechanism by which NF-B can become activated in response to specific intracellular stresses is via phosphorylation of eIF2. This moiety binds to IBs, and concomitant NF-B activation occurs in the absence of IB degradation. Specifically, thapsigargin and tunicamycin have been shown to induce this response in mouse embryo fibroblasts. A similar response was not observed in our model of ER stress induced by Z A1AT accumulation, suggesting that different mechanisms of NF-B activation occur in response to different ER stresses.
The UPR involves up-regulation of the secretory pathway’s capacity to process proteins and entails the transcriptional up-regulation of a coordinately expressed set of genes encoding ER chaperones, enzymes, and structural components of the ER. The UPR pathway culminates in the expression of glucose-responsive genes (grp) such as, grp78 and grp94 (12, 13). Transcription of the ER chaperone grp78/BiP is a classical marker for UPR activation in mammalian cells. The grp78 promoter contains a consensus binding site called the ER stress response element; this is bound by ATF6, a transcription factor specifically activated by ER stress (13). We found a marked increase in grp78 promoter activity and both GRP78 and GRP94 protein expression in the Z A1AT-transfected cell lines compared with control or M A1AT-transfected cell lines, but only after treatment with additional stresses such as thapsigargin or elevated temperature, indicating activation of the UPR pathway under these conditions. Thapsigargin is a commonly used ER stress stimulus that causes the depletion of calcium and, in turn, has a direct effect on chaperone function in the ER (22). It has previously been demonstrated that Z A1AT is degraded less efficiently in those that have liver disease than in those individuals that do not (23); therefore, activation of the UPR pathway may occur as a result of a genetic or environmental insult in those individuals that develop liver disease. Although an increase in temperature results in increased secretion of Z A1AT, this is offset by decreased intracellular degradation and increased polymerization of Z A1AT (24). This may result in activation of proteins such as GRP78 and GRP94 in an attempt to ensure that only correctly folded proteins are processed before entering the Golgi apparatus for further processing and secretion. A1AT synthesis rises during episodes of inflammation as part of the acute phase response. At these times the increased formation of polymers of Z A1AT is likely to overwhelm the degradative pathway, leading to the accumulation of polymerized Z A1AT that is associated with hepatocellular damage. This mirrors what has been shown in vivo in Z A1AT homozygous neonates, who develop marked derangement of liver function in association with inflammation (18).
The findings in this study impact not only on A1AT deficiency but also on other conformational diseases caused by inherited or acquired modifications in protein structure, where a specific protein undergoes a conformational rearrangement causing aggregation and deposits within tissues or cellular compartments. How cells respond to the production of these abnormal protein conformers and how these misfolded proteins cause cytotoxicity are important unanswered questions. These often devastating disorders, including Alzheimer’s and other neurodegenerative diseases such as Parkinson’s and Huntington’s, as well as cystic fibrosis, all involve the aberrant accumulation of proteins. In Alzheimer’s disease, for instance, the accumulation in the brain of insoluble aggregates of the amyloid- peptide, also known as senile plaques, is thought to be a key pathological event underlying the loss of brain cells (25). It has been shown in neuronal degenerating diseases that under ER stress, activation of NF-B was observed, followed by up-regulation of GRP78 protein levels (26). Z A1AT deficiency serves as an excellent model for conformational disease, because detailed structural data are available on both the wild-type and mutant proteins.
The potential therapeutics for conformational diseases such as Z A1AT deficiency include therapies directed at preventing polymerization of the aberrant protein, chemical chaperones, and anti-inflammatories. Polymerization may be prevented by the development of small peptide inhibitors, as shown by recent in vitro work in which Z A1AT polymerization was prevented by a 6-mer peptide that selectively anneals to the pathogenic serpin conformation (27). A number of chemical chaperones have been tested in vivo, including 4-phenyl butyric acid (PBA). It has been shown that the effects of chemical chaperones, in particular PBA, satisfy many of the criteria required for potential chemoprophylaxis for liver injury (24). However, it is not clear whether long term administration of PBA will reduce the load of A1AT retained in the ER of liver cells. Modulation of NF-B may also provide a target therapy by possible inhibition of the inflammation. These findings of intracellular stress mechanisms associated with Z A1AT production and retention elucidate in more detail the pathogenesis of the disease and as a model system may serve as a platform to test the effects of various interventions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Catherine M. Greene, Respiratory Research Division, Royal College of Surgeons in Ireland, Education and Research Center, Beaumont Hospital, Dublin 9, Ireland. E-mail address: cmgreene{at}rcsi.ie
3 Abbreviations used in this paper: A1AT, 1-antitrypsin; eIF2, eukaryotic initiation factor 2; EOR, endoplasmic reticulum overload response; ER, endoplasmic reticulum; grp/GRP, glucose-responsive protein; PBA, 4-phenyl butyric acid; UPR, unfolded protein response; BiP, Ig H chain binding protein.
Received for publication July 25, 2003. Accepted for publication February 13, 2004.
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