MECHANISMS OF GLUTAMATE-CYSTEINE LIGASE MODIFIER (Gclm) AND METALLOTHIOENIN-1 (Mt1) REGULATION BY OXIDATIVE STRESS
The first part of this dissertation focuses on the cellular antioxidant glutathione (GSH). In GSH de novo GSH synthesis , the first and rate-limiting step is catalyzed by glutamate-cysteine ligase (GCL). In this step, glutamate is ligated to cysteine to form Y-glutamylcysteine. GCL is a heterodimer composed of a 73-kDa subunit with catalytic properties (GCLC), and a 31-kDa modifer subunit (GCLM) that improves the efficiency of the holoenzyme. Specifically, this thesis focuses on the mechanisms of transcriptional regulation of mouse Gclm by oxidative stress. Treatment of mouse hepatoma (Hepa-1) cells with tert-butylhydroquinone (tBHQ) produced a robust (8-fold) increase in GCLM mRNA accumulation and this response was at the transcriptional level. Gclm has two main transcription initiation clusters, high GC content, and no canonical TATA box in its proximal promoter. Promoter deletion analysis (4.7 to 0.5 kb) showed high basal and 2-fold inducibility by tBHQ. An electrophile response element (EpRE) is located near the distal transcription start site and is hypothesized to act as a cis-element for Gclm induction. EPRE site-directed mutagenesis and transfection with NRF2 (a transcription factor that transactivates through the EPRE) plasmids demonstrated that this EPRE is only partially required for fBHQ-induction. It is concluded that the Gclm gene has a TATA-less promoter with complex regulation, and may include enhancers located further upstream form the studied constructs. The second part of this dissertation addresses the role of the metal response element binding transcription factor 1 (MTF1) on metallothionein 1 (Mt1) and Gclm gene expression by metals and oxidative stress. For this purpose, Mtf1 was retrovirally-transduced into dko7 or Mtf1(-/-) cells showing a functional protein. The transduced (MTF1dko7) cells showed a restoration of MT1 mRNA expression by Zn ^2+ or Cd ^2+ treatment. In constrast, MT1 protein levels were induced only with Zn ^2+ but not Cd ^2+ . Consequently, MTF1dko7 showed resistance to Zn ^2+ , but not Cd ^2+ -induced toxicity. In parallel experiments Zn ^2+ and Cd ^2+ induced GCLM mRNA accumulation in MTF1dko7 and dko7 cells. This suggests that Gclm regulation is MTF1-independent. These cells lines can be used to identify novel MTF1 downstream targets.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:glutamate cycsteine ligase modifier metallothiorein 1 gene regulation oxidative stress glutathione
Date of Publication:01/01/2001