Differential regulation of gamma-glutamyltransferase mRNAs in four human tumour cell lines.

Human gamma-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-alpha (TNF-alpha), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-alpha and TPA did not alter these parameters. In LNCap cells, TNF-alpha, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-alpha, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.

Structure of the 5' sequences of the human gamma-glutamyltransferase gene.

In humans, five distinct mRNAs code for gamma-glutamyltransferase (GGT). Their coding regions are identical and their 5' untranslated regions exhibit both common and type-specific sequences. To elucidate the mecanisms that generate these different mRNAs, we cloned and determined the structure of the 5' region of the human GGT gene. The common regions of the 5' UTR are encoded by five exons, localized within a 2.4-kb region of the genomic DNA. Three of them are separated only by intron-donor or intron-acceptor sites at their boundaries. Alternative splicing of these exons may determine the unique pattern of the different GGT mRNA 5' UTRs in a tissue-specific manner. In addition, we have isolated a genomic fragment containing the most distal 5' sequences of the major GGT mRNA in HepG2 cells. Primer extension analysis revealed one major transcription initiation site while 5' RACE indicated that one more distal initiation site could be present. In the putative promoter sequence neither classical TATA or CAAT boxes were found. However, sites for AP1, AP2, CREB, GRE and SP1 transcription factors were identified. Chimeric plasmids, containing this genomic region fused to the luciferase gene, were transiently expressed in three cell lines of different origin: HeLa cells, ovarian carcinoma A2780 cells and V79 lung fibroblasts. The significant promoter activities obtained indicate a transcription start within this region. However, differences in the level of expression were found between the different cell lines used. These data suggest that the human GGT gene employs regulatory sequences and alternative splicing, and gene expression may therefore be regulated in tissue specific and cell-type-specific manners.

Gamma-glutamyltranspeptidase-dependent glutathione catabolism results in activation of NF-kB.

gamma-glutamyltranspeptidase (GGT) is a key enzyme implicated in the homeostasis of intracellular reduced glutathione (GSH) and hence in the regulation of the cellular redox state. Besides, the extracellular cleavage of GSH by GGT leads to reactive oxygen species (ROS) production, depending on the generation and enhanced reactivity of cysteinylglycine (CysGly). Using a model cell line, the V79 GGT, which highly expresses a human GGT transgene, we examined whether the GGT induced oxidant stress could modulate intracellular transcription factors. For the first time, we show that GGT-dependent ROS production induces the NF-kB-binding and transactivation activities. This induction mimicked the one observed by H(2)O(2) and was inhibited by catalase, suggesting the involvement of H(2)O(2) in the NF-kB activation.