Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha1-antitrypsin deficiency - PubMed (original) (raw)
Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha1-antitrypsin deficiency
Emer Kelly et al. J Biol Chem. 2009.
Abstract
Z alpha(1)-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFkappaB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFkappaB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFkappaB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.
Figures
FIGURE 1.
Confirmation of expression of SEPS1 and ZAAT by HepG2 transfectants. A, HepG2 cells (1 × 106) were transfected with empty vector (lane 1) or pSEPS1 (lane 2). Protein extracts (40 μg) were analyzed by Western immunoblot. B, SEPS1 expression was quantified by quantitative reverse transcription-PCR. C, HepG2 cells were cotransfected with pRLSV40 and an empty vector (CMV) or pZAAT. ZAAT expression in cell lysates was quantified by an enzyme-linked immunosorbent assay. Absorbance at 405 nm per light unit (L.U.) indicates relative expression of ZAAT. The dashed line indicates background absorbance readings. Data shown are representative of three experiments.
FIGURE 2.
SEPS1 inhibits ZAAT-induced activation of UPR. A, triplicate samples of HepG2 cells (3 × 105) were cotransfected with an empty vector (pCMV) or pSEPSI; pZAAT; an inducible grp78 promoter-linked (firefly) luciferase reporter plasmid; and pRLSV40. Cells were treated with tunicamycin (Tm; 10 μg/ml) or DMSO for 16 h. Lysates were prepared, and luciferase production from both plasmids was quantified by luminometry using specific substrates. Relative grp78 promoter activity is shown (*, versus CMV; #, versus minus SEPS1). B, HepG2 cells (3 × 105) were transfected with pCMV or pZAAT, treated with tunicamycin (Tm; 10 μg/ml) or DMSO. Western immunoblotting for ATF6 was carried out on cell lysates, and densitometry of the resulting blot is also shown. Lane 1, empty vector (CMV); lane 2, pZAAT; lane 3, CMV + tunicamycin; lane 4, pZAAT + pSEPS1; lane 5, CMV + tunicamycin + pSEPS1. Data shown are representative of three experiments.
FIGURE 3.
SEPS1 reverses the effect of ZAAT and IL-1β on NFκB activation in HepG2 cells. A, triplicate samples of HepG2 cells (3 × 105) were co-transfected with an empty vector (pCMV) or pZAAT, pSEPS1 (as indicated), an inducible NFκB (firefly) luciferase reporter plasmid, and pRLSV40. B, following overnight incubation, cells were treated with IL-1 (10 ng, 24 h). Lysates were prepared using reporter lysis buffer (Promega). Luciferase production from both plasmids was quantified by luminometry using specific substrates. Relative NFκB luciferase activity is shown (*, versus CMV; #, versus minus SEPS1). Data shown are representative of three experiments.
FIGURE 4.
Serum selenium levels in ZZ and MM AAT individuals. Individuals were screened to determine their AAT phenotype. Serum selenium levels (μg/liter) were measured in 12 ZZ and 12 MM individuals. The normal selenium range (shaded area) and optimal levels for serum glutathione peroxidase activity (dashed lines) are shown.
FIGURE 5.
Glutathione peroxidase activity and SEPS1 expression in control and selenium supplemented cells. HepG2 cells were grown in complete medium containing 0 n
m
, 40 n
m
, or 150 μ
m
seleno-
dl
-methionine, as indicated, for 72 h. A, a glutathione peroxidase cellular activity assay kit (Sigma) was used to measure total GPx activity. The result shown is representative of three repeated experiments. B, RNA was isolated and used in quantitative reverse transcription-PCRs to detect SEPS1 and glyceraldehyde-3-phosphate dehydrogenase expression. Relative SEPS1 expression is shown. C, protein extracts were prepared, and samples (10 μg) were immunoblotted for SEPS1 or stained with Ponceau S for total protein loading.
FIGURE 6.
Selenium increases 15-deoxy-Δ12,14-prostaglandin J2 production in HepG2 cells. HepG2 cells (9 × 105) were grown in complete medium (−Se) or medium supplemented with 150 μ
m
seleno-
dl
-methionine (+Se) for 48 h. 15d-PGJ2 (pg/ml) was measured in whole cell lysates by an enzyme-linked immunosorbent assay. Data are representative of three experiments.
FIGURE 7.
Selenium enhances the effect of endogenous and overexpressed SEPS1 on grp78 promoter activity and NFκB activity. A, triplicate samples of HepG2 cells (3 × 105) were transfected with an empty vector (pCMV or pZAAT) and co-transfected with an inducible grp78 promoter-linked (firefly) luciferase reporter plasmid and pRLSV40 and grown in the absence or presence (+Se) of 150 μ
m
seleno-
dl
-methionine for 16 h and then left untreated or stimulated with tunicamycin (Tm; 10 μg/ml, 24 h) or DMSO (A) or IL-1β (10 ng/ml, 24 h) (B). Lysates were prepared, and luciferase production from both plasmids was quantified by luminometry. Relative grp78 promoter (#, versus tunicamycin; *, versus ZAAT) and NFκB activity (#, versus IL-1; #, versus ZAAT) are shown.
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