Cell-Type Specific Differences in Glutamate Cysteine Ligase Transcriptional Regulation Demonstrate Independent Subunit Control

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Abstract
Glutamate cysteine ligase (GCL; also referred to as γ-glutamylcysteine synthetase, GCS) catalyzes the rate-limiting step of glutathione synthesis. The GCL holoenzyme is composed of a catalytic (GCLC; also called GCSh) and a modifier (GCLM; also called GCSl) subunit, each encoded by a unique gene. Wild-type and mutant promoter/luciferase reporter transgenes containing the promoter region of each GCL subunit gene were transfected into A549 (lung carcinoma), HEK 293 (transformed embryonic kidney), HepG2 (hepatocellular carcinoma), and RD (skeletal muscle rhabdomyosarcoma) cells to examine potential cell-type related differences in transcriptional regulation. In A549, HepG2, and RD cells, maximal basal expression of the GCLC transgene required the full-length (–3802 bp) promoter. Maximal expression in HEK 293 cells was uniquely directed by cis-elements contained within the –2752 to –1286 bp fragment of the promoter. No differences in GLCM promoter function were detected among these 4 cell lines. GCL subunit induction in each cell line by pyrrolidine dithiocarbamate (PDTC), phenethyl isothiocyanate (PEITC), and β-naphthoflavone (β-NF) was examined by RNAse protection assays. Although both genes were similarly induced in HepG2 cells by β-NF, PDTC, and PEITC, neither was induced by β-NF in A549, HEK 293, and RD cells. PDTC and PEITC induced GCLM to a much greater extent than GCLC in HEK 293 cells and failed to induce GCLC in RD cells. Neither subunit was induced by any of the agents in A549 cells. These studies indicate that the GCL subunit genes are independently regulated and display cell-type specific differences in both basal and inducible expression.

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