DIFFERENTIAL REGULATION OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE HEAVY AND LIGHT SUBUNIT GENE-EXPRESSION

gamma-Glutamylcysteine synthetase (GCS) is the rate-limiting enzyme in the biosynthesis of glutathione and is composed of a heavy and a ligh t subunit. Although the heavy subunit is enzymically active alone, the light subunit plays an important regulatory role by making the holoen zyme function more efficiently, In the current study we examined wheth er conditions which are known to influence gene expression of the heav y subunit also influence that of the light subunit, and the mechanisms involved. Treatment of cultured rat hepatocytes with hormones such as insulin and hydrocortisone, or plating hepatocytes under low cell den sity increased the steady-state mRNA level of the heavy subunit only, Treatment with diethyl maleate (DEM), buthionine sulphoximine (BSO) an d t-butylhydroquinone (TBH) increased the steady state mRNA level and gene transcription rates of both subunits. These treatments share in c ommon their ability to induce oxidative stress and activate nuclear fa ctor kappa B (NF-kappa B). Treatment with protease inhibitors 7-amino- 1-chloro-3-tosylamido-2-heptanone (TLCK) or L-1-tosylamido-2-phenyleth yl chloromethyl ketone (TPCK) had no influence on the basal NF-kappa B and GCS subunit mRNA levels, but blocked the activation of NF-kappa B by DEM, BSO and TBH, and the increase in GCS heavy subunit mRNA level by BSO and TBH. On the other hand, the DEM-, BSO-and TBH-induced incr ease in GCS light-subunit mRNA level was unaffected by TLCK and TPCK. Thus only the heavy subunit is hormonally regulated and growth sensiti ve, whereas a both subunits are regulated by oxidative stress. Signall ing through NF-kappa B is involved only in the oxidative-stress-mediat ed changes in the heavy subunit gene expression.