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

Abstract

Glutamate cysteine ligase (GCL; also referred to as gamma-glutamylcysteine synthetase, GCS) catalyzes the rate-limiting step of glutathione synthesis. The GCL holoenzyme is composed of a catalytic (GCLC; also called GCS(h)) and a modifier (GCLM; also called GCS(l)) 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 beta-naphthoflavone (beta-NF) was examined by RNAse protection assays. Although both genes were similarly induced in HepG2 cells by beta-NF, PDTC, and PEITC, neither was induced by beta-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.

Replication of a Cytopathic Strain of Bovine Viral Diarrhea Virus Activates PERK and Induces Endoplasmic Reticulum Stress-Mediated Apoptosis of MDBK Cells

“…In MDBK cells, two GCLC transcripts of 4.1 kb and 3.2 kb were detected, consistent with results found for human GCLC (8). The Northern blot data show that BVDV infection caused a progressive decrease in both GCLC transcripts (Fig.…”

“…Consistent with this observation, BVDV infection led to a reduction in GCLC mRNA, the catalytic subunit of glutamate-cysteine ligase and the rate-limiting enzyme in glutathione biosynthesis (8). Concomitant with the loss of GCLC mRNA was a dramatic decrease in intracellular GSH levels which corresponded with the phenotypic onset of apoptosis seen at 20 to 25 h postinfection (Fig.…”

Quercetin‐induced upregulation of human GCLC gene is mediated by cis‐regulatory element for early growth response protein‐1 (EGR1) in INS‐1 beta‐cells

“…The transcription of the GCLC gene was independently regulated and displayed cell-type specific differences in both basal and inducible conditions [Dahl and Mulcahy, 2001]. Our promoter analysis revealed that the proximal GC-rich region from À90 to À34 contained the cis-elements for transactivation of the GCLC gene in response to quercetin as well as for the maintenance of basal activity of the gene.…”

Efeito da estreptozotocina sobre os perfis glicêmico e lipídico e o estresse oxidativo em hamsters

“…A proteína Keap1 contém resíduos conservados de cisteína, os quais desempenham um papel crí-tico na manutenção do Nrf2 no citoplasma. Entretanto, um aumento na produção de EROS no organismo promove no citoplasma a dissociação desse complexo, fazendo com que o Nrf2 fique ativado, sendo transportado para o núcleo (34,35). No núcleo das células, esse fator de transcrição se liga a elementos de resposta a antioxidantes (ARE) na região promotora dos genes que transcrevem enzimas antioxidantes endógenas.…”