Date of Award
Dissertation (Access Restricted)
Doctor of Philosophy
Thomas Robert Sutter
Carrie Hayes Sutter
Keratinocyte differentiation is the process by which basal stem cell progeny ultimately form the epidermal permeability barrier (EPB) which is required for mammalian survival. The EPB protects the organism from dehydration and invasion from foreign bodies. Differentiation is regulated through signal transduction and gene expression mechanisms. Several factors, both exogenous and endogenous, are known to disrupt differentiation, changing gene expression and upsetting EPB homeostasis. Two examples are 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and epidermal growth factor (EGF). In order to elucidate the mechanisms by which each of these factors affects keratinocyte differentiation, we integrated the results of functional genomic, metabolomic, and biochemical analyses. Microarray analysis showed TCDD altered the expression of numerous genes that are regulated during EPB formation, including 40% of the genes of the epidermal differentiation complex and 75% of those involved in de novo ceramide biosynthesis. Lipid analysis showed TCDD increased eight of nine ceramide classes of the lipid matrix. Additionally, TCDD decreased the expression of the glucose transporter SLC2A1, five glycolytic enzymes, and several glycolytic intermediates, as well as NADH, and the Kreb cycle intermediate, citrate. TCDD decreased mitochondrial glutathione reductase activity and the GSH/GSSG ratio, ultimately leading to mitochondrial dysfunction and increased production of the reactive oxygen species (ROS), hydrogen peroxide. Co-treatment of cells with antioxidants blocked the TCDD-mediated acceleration of keratinocyte cornified envelope (CE) formation, a marker of terminal differentiation. Thus, TCDD-mediated ROS production is a critical intermediate in the mechanism to accelerate keratinocyte differentiation. In a similar manner we identified 2,676 genes that are regulated by EGF, and further confirmed by functional assays that EGF abrogates all of the known essential processes of differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, leading to the reduction of free fatty acids and five of nine ceramide classes of the lipid matrix; 2) regulating numerous genes forming the CE; and 3) suppressing the expression of tight junction proteins. EGF impaired barrier function as measured by transcriptional water loss, a common feature of skin diseases. These results provide both a mechanism through which TCDD is accelerating differentiation and a systems-level understanding of how EGF inhibits differentiation.
Dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.
Kennedy, Lawrence Henry, "Modulation of Keratinocyte Differentiation by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Epidermal Growth Factor" (2012). Electronic Theses and Dissertations. 2268.