Effects of selenium depletion and selenoprotein knockdown on inflammatory signalling in a gut epithelial cell line

Abstract

Selenium is a micronutrient essential for human health. Low Se intake versus Se supplementation have been reported to elevate and lower mortality from colorectal cancer. Se is present in selenoproteins including glutathione peroxidases (GPx) 1-4. GPxs are antioxidant enzymes that protect cells from excessive oxidative stress and they may have a role in maintaining innate immunity homeostasis against inflammatory stimulation from exposure to luminal bacteria. This thesis describes studies of the regulatory effects of Se depletion and selenoprotein knockdown in the gastrointestinal cell line Caco-2 in relation to inflammatory responses. A luciferase reporter model was developed in which Caco-2 cells were stably transfected with gene constructs in which luciferase expression was under the control of regulatory elements that bind the Nuclear Factor-kappa B (NF

B), a transcription factor central to inflammatory signaling pathways (Chapter 3). As a control reporter luciferase coding sequences were linked to a TATA box. When Caco-2 cells expressing these constructs were grown in Se-deficient conditions, a 30% increase of reporter activity and a 50% increase in endogenous interleukin 8 mRNA levels were observed after stimulation with TNF . In comparison, no changes in reporter activity were found in Se-deficient cells after stimulation with flagellin (Chapter 4). Small interfering RNA was used to knockdown expression of individual selenoproteins including GPx1, GPx4, SelW and SelH (Chapter 5). Knockdown of GPx1 expression by ~55% led to a 25% decrease of reporter activity and a 17% decrease of IL8 mRNA level after stimulation with TNF ✁ . Knockdown of SelW or SelH expression had no observable effect on reporter activity. In addition, Se depletion elevated cellular ROS production as assessed by Carboxy-2’7’-dichlorodihydrofluorescein diacetate staining whereas GPx1 knockdown had no significant effect (Chapter 5). Knockdown of GPx4 by 85%, as assessed by RT-PCR and Western blotting, had little effect on TNF -driven luciferase activity. However, GPx4 knockdown lowered flagellin-induced reporter activity by 20% and interleukin 8 mRNA levels by 40% (Chapter 6). It is concluded that 1] low Se supply affects NF

B inflammatory response in Caco-2 cells, 2] the exact role of different selenoproteins in this effect remains to be elucidated, and 3] the endogenous and exogenous inflammatory responses in Caco-2 cells are differentially regulated by Se supply and by antioxidant selenoproteins.