Stress induced transcriptional regulation of the glycine transporter type 1A (GlyT-1A/SLC6A9) in human intestinal epithelia

Abstract

There is mounting experimental evidence demonstrating protection by free glycine against stress in several cell types. The glycine transporter type 1 (GlyT-1) mediates the high affinity supply of glycine, which together with cysteine is required for the synthesis of the antioxidant glutathione. Previous work in this laboratory has established that GlyT-1 is expressed on the apical and basal membranes of intestinal epithelial cells and that its mRNA levels are regulated by stress. In the present study exactly how stress signals to transcriptional induction of GlyT-1 was investigated. Caco-2 cells transfected with reporter constructs of sequences of the GlyT-1a proximal promoter and 5’UTR cloned upstream of a β-galactosidase coding sequence, showed increased reporter activity following treatment with thapsigargin (Tg), tunicamycin (Tu), amino acid (AA) starvation, tert-Butylhydroquinone (tBHQ) or Diethyl maleate (DEM). Despite no changes in Nrf-2 mRNA levels, a significant increase in total Nrf-2 protein abundance was evident on western-blots following DEM treatment of Caco-2 cells. However, gel shift showed no protein-DNA complexes between Nrf-2 protein and a DNA probe sequence of the putative antioxidant response element (ARE) identified in the GlyT-1a 5’ flank. Despite a significant siRNA mediated knock-down of Nrf-2 mRNA and protein, there was no further effect on GlyT-1a expression. Unlike Nrf-2, the knock-down of Atf-4 diminished the basal and stressed induced expression of GlyT-1a. Atf-4 was detected bound to DNA probes containing a potential amino acid response element (AARE) located in the first exon of the GlyT-1a gene by gel shift and super shift assays. QPCR assays performed on DNA isolated from Caco-2 cells by chromatin immunoprecipitation (ChIP) using antibodies against Atf-4, demonstrated 9, 5 and 2-fold enrichment of the GlyT-1a AARE following Tu, AA starvation and DEM treatment respectively. Site directed mutation of the GlyT-1a AARE showed a 75% reduction in reporter activity as well as attenuated protein-DNA interaction with a representative probe. It is evident from the data presented in this thesis that the direct interaction of Atf-4 at the proposed GlyT-1a AARE contributes to its transcriptional up-regulation following endoplasmic reticulum stress, nutrient stress and oxidative stress.