Alpha-1 antitrypsin (AT) is the most important anti-elastase in the lung. Z-AT (342Glu>Lys) polymerises within the hepatocyte resulting in severe plasma deficiency and is the commonest genetic reason for the development of COPD. The F α1-antitrypsin variant (223Arg>Cys) has been associated with mild plasma deficiency, and when found in association with the Z allele linked to emphysema and liver cirrhosis. We investigated the properties of F-AT in a cell-model.
Human F-AT cDNA was generated by site-directed mutagenesis and overexpressed into hepatocytes (HepG2 cells). Supernatants, lysates and inclusion bodies were assessed for total AT. F-AT cells were assessed for polymeric-AT, PERK, NF-kB, AP-1, TNF-α and IL-6 by ELISA, immunoblot or RT-PCR in comparison with normal (non-polymerising) M-AT and Z AT (polymerising control).
F-AT cells had no cell cytotoxicity or apoptosis upto 72h. At 24h (unless stated) F-AT secretion was slightly lower, but comparable to M-AT secretion (1479.3 ± 142pg/ml vs. 1745.5 ± 102.3pg/ml, P = 0.413) Secreted F-AT had significantly reduced elastase inhibitory capacity compared to M-AT (0.972 ± 0.069(OD at 405nm) vs. 0.449 ± 0.085, P < 0.001). F-AT formed insoluble aggregates of polymeric-AT (375 ± 32pg/ml vs. undetectable), upregulated PERK mRNA (at 3h) and significantly increased NF-κB (at 16h), AP-1, TNF-α (34.5 ± 5.3pg/ml vs. 10.53 ± 3.2, P = 0.023) and IL-6 (at 48h) (132.3 ± 20.8pg/ml vs. 23.9 ± 16) (P = 0.006). All of which were inhibited by treatment with an inhibitor of polymerisation (P < 0.001 for all). In comparison to Z-AT, F-AT secretion was greater (P < 0.001) but had significantly reduced anti-elastase activity (P = 0.032) and lower ER accumulation of polymeric-F-AT (P < 0.001). In the F-AT cell, PERK mRNA was upregulated at 3h compared to 0.5h in Z-AT. Although elevated compared to M-AT cells, F-AT cells had lower NF-κB activity (P < 0.001), TNF-a production (P = 0.046) and IL-6 production (P = 0.012) compared to Z-AT.
In conclusion, F-AT secretion was comparable to M-AT. However, secreted F-AT was defective as an anti-elastase. F-AT was found to polymerise and aggregate in inclusion bodies. ER accumulation of F-AT activated the ER overload response; PERK-dependant-NF-κB mediated inflammatory response, greater than M-AT but to a lesser degree than Z-AT. This data indicate that FZ phenotype may be at risk for liver and lung disease.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.