Journal
BIOCHEMICAL JOURNAL
Volume 390, Issue -, Pages 521-528Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20050439
Keywords
Chang cells; cloning; 5 '-flanking region; glutathione synthetase; NFE2; Nrf1
Categories
Funding
- NIDDK NIH HHS [R56 DK045334, P30 DK48522, R01 DK045334, DK-45334, P30 DK048522] Funding Source: Medline
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GSH synthesis occurs through a two-step enzymatic reaction driven by GCL (glutamate-eysteine ligase; made up of catalytic and modifying subunits) and GSS (glutathione synthetase). In humans, oxidative stress regulates GCL expression in an antioxidant response element-dependent manner via Nrf2 [NFE (nuclear factor erythroid)-related factor 2]. In the rat, GSS and GCL are regulated co-ordinately by oxidative stress, and induction of GSS further increases GSH synthetic capacity. Transcriptional regulation of the human GSS has not been examined. To address this, we have cloned and characterized a 2.2 kb 5'-flanking region of the human GSS. The transcriptional start site is located 80 nt upstream of the translation start site. The human GSS promoter efficiently drove luciferase expression in Chang cells. Overexpression of either Nrf1 or Nrf2 induced the GSS promoter activity by 130 and 168% respectively. Two regions homologous to the NFE2 motif are demonstrated to be important for basal expression of human GSS, as mutation of these sites reduced the promoter activity by 66%. Nrf1, Nrf2 and c-Jun binding to these NFE2 sites under basal conditions was demonstrated using chromatin immunoprecipitation assays. In summary, two NFE2 sites in the human GSS promoter play important roles in the basal expression of GSS and, similar to the GCL subunits, the human GSS gene expression is also regulated by Nrf2.
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