Differential regulation of γ-glutamyltransferase and glutamate cysteine ligase expression after mitochondrial uncoupling: γ-glutamyltransferase is regulated in an Nrf2- and NFκB-independent manner.

The enzymes γ-glutamyltransferase (GGT) and glutamate cysteine ligase (GCL) have important roles in glutathione (GSH) homeostasis, and both are frequently upregulated after acute oxidative stress. Mitochondria are major producers of ROS, and incubating the colorectal adenocarcinoma cell line HT-29 cells with mitochondrial uncouplers significantly increased endogenous ROS as well as mRNA for both GGT and GCLC (the catalytic subunit of GCL). However, no elevation in GGT protein or activity was detected, in contrast to the increased levels of GCLC protein found. The uncouplers initiated endoplasmic reticulum (ER) stress, as demonstrated by highly increased levels of CHOP and GRP78 mRNA. Using inhibitors of proteasomes and ER-associated degradation (ERAD) together with a mitochondrial uncoupler, increased GGT protein and activity levels were obtained indicating that GGT may be a substrate for ERAD. Uncoupling increased the mRNA levels of the two redox-regulated transcription factors Nrf2 and NFκB. Using siRNA to suppress Nrf2 and NFκB expression, downregulation of GCLC expression both at the basal level and after mitochondrial uncoupling was achieved. In contrast, the expression level of GGT was not affected by this treatment. These data strongly indicate a discrepancy between the regulation of GCLC and of GGT following the oxidative stress situation due to mitochondrial uncoupling. Both the enzymes are considered to be part of the cellular antioxidant system; however, the role of GGT as a consistent oxidative response parameter needs to be reevaluated.