Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

doi:10.1016/j.abb.2003.11.004

Archives of Biochemistry and Biophysics

Volume 423, Issue 1, 1 March 2004, Pages 116-125

https://doi.org/10.1016/j.abb.2003.11.004 Get rights and content

Abstract

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.

Section snippets

Chemicals and reagents

4HNE was purchased from Cayman Chemical (Ann Arbor, MI). TRIzol reagent was purchased from Life Technologies (Grand Island, NY). DMNQ was purchased from Oxis Research (Portland, OR). All other chemicals were of at least analytical grade and purchased from Sigma Chemical, unless otherwise noted.

Cell culture and treatments

HBE1 cells are immortalized human bronchial epithelial cells from a normal individual [26]. Cells were grown in serum-free Ham’s F-12 growth medium supplemented with six additives (5 μg/mL insulin, 3.7

4HNE, DMNQ, and tBHQ treatments alter intracellular GSH content in HBE1 cells

4HNE and tBHQ exposure have been shown to increase intracellular GSH content in L2 cells [6], a rat cell line that maintains many of the characteristics of primary type II lung epithelial cells [31]. Furthermore, previous work from our laboratory has established that 4HNE can increase intracellular GSH in the HBE1 cells [8]. To elucidate the mechanisms by which 4HNE, DMNQ, and tBHQ increase intracellular GSH, we have employed HBE1 cells because of their relevance to human physiology. It was

Discussion

Although numerous studies have indicated increases in the mRNA for GCLC and/or GCLM in response to oxidative stress and a few studies have shown increases in the corresponding proteins, the questions of the relationship between mRNA abundance and translated protein as well as the actual ratio of the two subunit proteins remained unresolved. Therefore, we approached these issues by using three compounds that produce oxidative stress by different modes. Our goal was neither to study those modes

Acknowledgements

This work was supported by Grants ES05511 to H.J.F., CA75316, and CA90473 to C.C.F. from the National Institutes of Health.

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Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Abstract

Section snippets

Chemicals and reagents

Cell culture and treatments

4HNE, DMNQ, and tBHQ treatments alter intracellular GSH content in HBE1 cells

Discussion

Acknowledgements

J. Biol. Chem.

Protein Expr. Purif.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Free Radic. Biol. Med.

J. Biol. Chem.

Free Radic. Biol. Med.

Chem. Biol. Interact.

Toxicology

Free Radic. Biol. Med.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Comm.

FEBS Lett.

Methods Enzymol.

Biochim. Biophys. Acta