Alteration of Transcriptional and Post-Transcriptional Expression of Gamma-Glutamylcysteine Synthetase by Diethyl Maleate

Abstract

Gamma-glutamylcysteine synthetase (gamma-GCS), also known as glutamate-cysteine ligase (EC 6.3.2.2), is the rate-limiting enzyme in the synthesis of glutathione (GSH). The gene GLCLC encodes the catalytic subunit while GLCLR encodes the regulatory subunit. Although it has been shown that GLCLC can respond to a variety of stresses by increased transcription, it is not known whether a similar response occurs for GLCLR. Nor is it known whether post-transcriptional regulation of either gene product is altered during stress. The present investigation was undertaken to explore transcriptional and post-transcriptional regulation of GLCLC and GLCLR gene products when HepG2 cells were challenged with the radiation sensitizer diethyl maleate (DEM). Expression of steady-state GLCLC and GLCLR mRNA was enhanced 5-20-fold after DEM challenge. Nuclear run-off assays were performed on unstressed and stressed cells to determine whether the increased expression of GLCLC and GLCLR mRNA was due to altered transcriptional activity of these genes. The DEM treatment increased the transcription rates of both genes 2-5-fold. In unstressed HepG2 cells, the half-life of GLCLC mRNA transcripts was approximately 4 h. In contrast, the half-life of GLCLR transcripts was approximately 8 h. In cells treated with DEM, the half-lives of all transcripts were increased, indicating that message stabilization contributed to the increased expression of gene products. Finally, a PEST algorithm has identified a PEST (proline, glutamate, serine, threonine) motif within the catalytic subunit of gamma-GCS, suggesting that this subunit might exhibit conditional proteolytic regulation. These results imply that regulation of the products of the GLCLC and GLCLR genes may be altered at multiple levels during exposure to stress.

Regulation of glutathione synthesis

“…In addition to increased gene transcription, DEM and 4-hydroxy-2-nonenal treatments have also been found to stabilize the GCLC mRNA (Sekhar et al, 1997a;Liu et al, 1998). The molecular mechanism of this effect remains unknown.…”

Replenishment of Glutathione Levels Improves Mucosal Function in Experimental Acute Colitis

“…Consistent with this effect we report here the induction of ␥-GCS-HS, the catalytic subunit of the rate limiting enzyme in GSH biosynthesis which determines a greater GSH biosynthetic capacity of mucosa 7 days after TNBS treatment. Since the gut is highly dependent on GSH synthesis (Martensson et al, 1990;Sido et al, 1998), the increase in ␥-GCS in mucosa from TNBS-treated rats can be interpreted as an adaptive response to control the consequences of the stress induced by TNBS as ␥-GCS-HS is known to be upregulated in response to divergent stressful conditions including oxidative stress or GSH depletion (Morales et al, , 1998Mulcahy et al, 1997;Rahman et al, 1996;Sekhar et al, 1997;Tian et al, 1997). However, despite almost normalization of mucosal GSH levels in the chronic phases of colitis (1-3 weeks) (Fig.…”

Human placenta sphingomyelinase, an exogenous acidic pH-optimum sphingomyelinase, induces oxidative stress, glutathione depletion, and apoptosis in rat hepatocytes

“…Because of the critical role of GSH in protection against apoptosis, 19,21 we examined the effect of GSH depletion in the survival of hepatocytes following incubation with the SMases. Cells were first preincubated with DEM to cause a rapid elimination of cell GSH stores through GST catalysis, followed by BSO to prevent GSH recovery caused by the induction of ␥-GCS by DEM, 25 thus ensuring a sustained depletion of GSH levels during the course of incubation. As seen, although in hepatocytes not depleted of GSH levels hSMase caused a moderate cell killing after 12 hours of incubation, GSH depletion accelerated and increased the magnitude of cell killing by hSMase treatment (Fig.…”

“…The former catalyzes the rate-limiting step in de novo GSH synthesis, and has been shown to be up-regulated in multiple cell types in response to a diverse array of stimuli. [25][26][27][28][29][30] Recent studies in a human acute lymphoblastic cell line have indicated that NSMase is inhibited by GSH, whereas GSH depletion did not affect the activity of the acid pHoptimum ASMase, suggesting that cellular GSH selectively controls the activation of NSMase. 31 However, because the regulation of cellular GSH by either NSMase or ASMase has not been previously reported, we have examined the regulation of GSH and induction of oxidative stress in cultured rat hepatocytes incubated with exogenous SMases, including the commonly used Mg 2ϩ -dependent, neutral pH optimun bacterial SMase from Bacillus cereus (bSMase).…”