Calcium signalling in mammalian cell lines expressing wild type and mutant human α1-Antitrypsin

Abstract

A possible role for calcium signalling in the autosomal dominant form of dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), has been proposed, which may point towards a mechanism by which cells could sense and respond to the accumulation of mutant serpin polymers in the endoplasmic reticulum (ER). We therefore explored possible defects in Ca²⁺-signalling, which may contribute to the pathology associated with another serpinopathy, α₁-antitrypsin (AAT) deficiency. Using CHO K1 cell lines stably expressing a wild type human AAT (MAAT) and a disease-causing polymer-forming variant (ZAAT) and the truncated variant (NHK AAT), we measured basal intracellular free Ca²⁺, its responses to thapsigargin (TG), an ER Ca²⁺-ATPase blocker, and store-operated Ca²⁺-entry (SOCE). Our fura2 based Ca²⁺ measurements detected no differences between these 3 parameters in cell lines expressing MAAT and cell lines expressing ZAAT and NHK AAT mutants. Thus, in our cell-based models of α1-antitrypsin (AAT) deficiency, unlike the case for FENIB, we were unable to detect defects in calcium signalling.

Figure 1

Characterisation of human alpha-1-antitrypsin (AAT) protein in CHO K1 cell lines expressing human wild type and disease variants of AAT. (a) Cartoon representation of the doxycycline inducible pTRE2hyg constructs encoding for human AAT proteins expression used in generating the stably AAT expressing CHO K1 cells line. Single amino acid substitution from Glutamate (Glu) to Lysine (Lys) in the AAT sequence at position 342 resulted in the polymerogenic ZAAT (E342K) mutant (blue, 395 aa) that is retained in the ER. Frameshift mutation in the AAT sequence generating a premature stop codon at amino acid position 334 resulted in the NHK AAT mutant. Unlike the ZAAT, the NHK AAT mutant is cleared from the ER through the ER-associated degradation (ERAD) process. Human AAT protein expression in stably AAT expressing CHO K1 and control cells (non-AAT expressing) was evaluated by western blotting using an anti-antitrypsin 2G7 antibody that recognises both monomer and polymer forms of AAT and GAPDH as loading control. Arrows indicate the non-polymerogenic wild type MAAT and polymerogenic disease variant ZAAT proteins (~52 kDa) and the truncated form of the AAT protein, NHK AAT variant (~45 kDa). Full image of the blot is included in this paper as Supplemental Fig. 1. (b) Densitometry quantification of AAT detection in CHO K1 cells lines, expressed relative to the level of GAPDH. Data points plotted are means ± SEM. *P < 0.05; **P < 0.01 (n = 8 independent experiments). Controls are CHO K1 cells transfected with empty plasmids and otherwise treated in the same way as the AAT lines.

Figure 2

Subcellular localisation of human AAT in the stably AAT expressing CHO K1 cell lines. (a,b) Representative confocal images displaying control and CHO K1 cells stably expressing wild type MAAT, disease variant ZAAT and a truncated disease variant NHK AAT upon exposure to 1 μg/ml Doxycycline for 48 h, fixed in 4% paraformaldehyde (PFA) followed by fluorescence immunostaining. Cells were counterstained using DAPI nuclear stain. Insets show enlarged images of the association between expressed AAT and either an endoplasmic reticulum (ER) marker calreticulin (a) or a Golgi marker giantin (b). Scale bar represents 10 μm (n = 3 independent experiments). Controls are CHO K1 cells transfected with empty plasmids and otherwise treated in the same way as the AAT lines. Our results show AAT polymer formation in the endoplasmic reticulum of the AAT-expressing stable CHO K1 cells lines expressing the ZAAT and NHK AAT variants.

Figure 3

Time-course of changes in intracellular calcium signaling in CHO K1 cell lines stably expressing WT and disease variants of human alpha-1-antitrypsin. (a) Fluorescence images of Fura2-AM stained AAT-stably expressing CHO K1 cells (F_{340 nm}/F_{380 nm}) in grayscale (t = 0 s) and pseudo-coloured showing intracellular calcium at rest, in calcium free, at peak response to 1 μM thapsigargin (TG); a blocker of the ER calcium ATPase, and finally after restoration of normal extracellular calcium by exposure to 2 mM CaCl₂, which reveals store-operated calcium-entry (SOCE). Scale bar represents 20 μm. (b) Representative traces of calcium measurements obtained at 3 s intervals during exposure to Ca²⁺-free medium, addition of TG and the restoration of external calcium (2 mM CaCl₂) for control cells and AAT-expressing cells. (c) Boxplots summarising the basal intracellular calcium levels (top panel), calcium levels following TG exposure (middle panel)) and following restoration of external calcium (bottom panel) for control cells and 3 genotypes MAAT, ZAAT, NHK AAT (n = 11 independent experiments; 30 cells/genotype/experiment). Controls are CHO K1 cells transfected with empty plasmids and otherwise treated in the same way as the AAT lines.