Ceramide Negatively Regulates GlutathioneS-transferase Gene Transactivation via Repression of Hepatic Nuclear Factor-1 That Is Degraded by the Ubiquitin Proteasome System

Abstract

The level of cellular ceramide, an apoptotic rheostat, is increased by sphingomyelinase or de novo synthesis. The expression of the glutathione S-transferase (GST) gene, whose induction accounts for cell viability, is regulated by activation of CCAAT/enhancer binding protein-␤ (C/EBP␤) and NF-E2-related factor-2 (Nrf2). Hepatic nuclear factor-1 (HNF1) is a transcription factor necessary for cell survival. This study investigated the role of HNF1 in GSTA2 gene transactivation, the ubiquitin proteasomal degradation of HNF1, and the inhibition of activating HNF1 by ceramide for GSTA2 repression. C2-ceramide (C2), a cell-permeable analog, repressed the GSTA2 expression in H4IIE cells, whereas dihydro-C2, an inactive analog, had no effect. Immunoblot, immunocytochemical, and gel shift analyses revealed that C2 decreased the level of nuclear HNF1 and protein binding to the HNF response element (HRE). Deletion of the HRE or the GSTA2 gene promoter region containing the HRE reduced luciferase reporter expression. Immunoprecipitation and immunoblot analyses showed that C2 decreased the level of ubiquitinated HNF1, which was reversed by treatment with MG132, a proteasome inhibitor. C2 suppressed GSTA2 induction by oltipraz via inhibition of inducible HNF1 DNA binding. The functional role of HRE for C2 repression of GSTA2 gene transactivation by oltipraz was verified by both the luciferase reporter gene expression and the transfection experiment with ⌬HNF-pGL-1651 lacking the HRE. C2 similarly repressed the induction of GSTA2 promoter-luciferase by tertbutylhydroquinone via HNF1 suppression, suggesting that constitutive HNF1 activation is required for GSTA2 induction. C2 also inhibited GSTA3/5 expression. In conclusion, the HRE in the GSTA2 promoter region is functionally active for the constitutive and inducible gene expression, and ceramide inhibits GST gene transactivation through decrease in nuclear HNF1, which is degraded by the ubiquitin proteasome system.

Glucocorticoid Receptor (GR)-Associated SMRT Binding to C/EBPβ TAD and Nrf2 Neh4/5: Role of SMRT Recruited to GR in GSTA2 Gene Repression

“…We observed that deletion of the promoter region containing the C/EBP response element or the ARE had a significant inhibitory effect on both oltipraz-and t-BHQ-inducible gene expression (13,28). The constitutive expression of GSTA2 is regulated by the basal activation of the transcription factors including C/EBP␤, Nrf2, and HNF1 (13,15,29). Ineffectiveness of the mutant promoters, in which the region comprising either the C/EBP response element or the ARE was deleted, in mediating gene expression strongly suggests that C/EBP␤ and Nrf2 are both required as transcription factors for the GSTA2 gene transactivation.…”

“…To determine the activities of the deleted GSTA2 promoter constructs, we used the dual luciferase reporter assay system (Promega, Madison, WI) as described previously (13,29). For some experiments, cells were cotransfected with pCMX-SMRT, pCDNA-SMRT-RID, or pCDNA-SMRT-SD plasmid (1 g each) in addition to GSTA2 promoter-luciferase construct (1 g).…”

Repression of HumanGSTA1by Interleukin-1β Is Mediated by Variant Hepatic Nuclear Factor-1C

“…For example, HNF-1 plays a significant role in the down-regulation of GSTA2 in rats by IL-6 and dexamethasone (Voss et al, 2002;Whalen et al, 2004). Moreover, reduction of human GSTA2 gene expression by ceramide is mediated by HNF-1 (Park et al, 2004). It has been reported that different isoforms determine whether the regulatory function of HNF-1 is transactivation or repression (Bach and Yaniv, 1993).…”

“…Previous evidence from our laboratory has implicated HNF-1 in IL-1␤-mediated down-regulation of GSTA1 (Romero et al, 2002). HNF-1 is a dimeric transcriptional activator required for basal expression of a wide range of genes, including enzymes involved in drug metabolism and detoxification (Park et al, 2004). For example, transcription of the UDP-glucuronosyltransferase-1A6, -1A8, -1A9, and -1A10 genes, is activated by HNF-1 binding to their proximal promoters (Auyeung et al, 2003;Gregory et al, 2004).…”

Chemical Inducers of Rodent GlutathioneS-Transferases Down-Regulate HumanGSTA1Transcription through a Mechanism Involving Variant Hepatic Nuclear Factor 1-C

“…The fact that overexpression of HNF-1␣ prevented transcriptional repression of GSTA1 by rodent GST inducers in luciferase reporter assays further implicates HNF-1␣ in the mechanism of transcriptional repression. Ceramide, a key regulator of apoptosis, represses rat GSTA2 transcription in H4IIE cells by reducing nuclear HNF1␣ levels via ubiquitination (Park et al, 2004). Ceramide also prevents induction of GSTA2 by oltipraz and tert-butylhydroquinone by inhibiting HNF1 DNA binding.…”