Professor Cornell University Ithaca, New York, United States
Disclosure(s):
Chenhan Feng, Bachelor of Science: No relevant financial relationship(s) with ineligible companies to disclose.
Objectives: Our previous study revealed that mice overexpressing glutathione peroxidase-1 (GPX1) developed fatty liver, along with elevated endoplasmic reticulum (ER) stress and altered expression of ER-resident selenoproteins K and S (SELENOK and SELENOS). This study was to unravel the metabolic cascade and intricate mechanism among these proteins or factors.
Methods: In Expt. 1, the human GPX1 was inserted into pRetroX-Tight-Pur and transfected into HepG2 cells grown in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and supplemental Se (Na₂SeO₃, 200 nM). Cells were collected after 3, 6, 9, 12, 24, and 48 h, respectively, to assay for mRNA and(or) protein levels of GPX1, SELENOK, SELENOS, and ER stress-related genes. In Expt. 2, cells transfected with the GPX1 plasmid (600ng DNA, 0.5h) were treated with 20μM MKC8866 (an ER stress inhibitor) for 6 and 9 h before the assays as described above. In Expt. 3, HepG2 cells were first silenced their SELENOK and SELENOS gene expression by RNA interference for 48 h, and then transfected with the GPX1 plasmid. After 9 h, the cells were collected for the same assays as in Expt. 1 and 2.
Results: Compared with the control, the Gpx1 transfection caused up to 102 fold (P < 0.05) increases in mRNA levels of GPX1, SELENOK, SELENOS, IRE1a, and XBP1 at 6 h. The transfection led to (70% to 3-fold, P< 0.05) increases in protein levels of GPX1 and SELENOK at 48 h. Treating the cells with MKC8866 for 6 and 9 h reduced (39-54%, P< 0.05) the GPX1 overexpression-induced mRNA levels of IRE1a, XBP1, XBP1s, SELENOK and SELENOS. After silencing the SELENOK gene expression, the GPX1 transfection resulted in greater increases (P < 0.05) in mRNA levels of SELENOK and ER stress-related genes. In contrast, silencing the SELENOS gene expression failed to affect the GPX1 overexpression-mediated changes of SELENOS or ER stress-related gene expression. Silencing SELENOK and SELENOS elevated (P < 0.05) GPX1 mRNA levels by 52% and 2789-fold, respectively.
Conclusions: Overexpression of GPX1 elevated the mRNA and protein levels of SELENOK, SELENOS, and ER stress-related genes. That up-regulation was partially impaired by silencing SELENOK, but not SELENOS.
Funding Sources: This research did not receive external funding. We would like to acknowledge Dr. Alan Diamond for sharing the pRetroX-Tight-Pur, pRetroX-Tight-Pur-GPX1 and pRetroX-Tet-On plasmids with us.
