Interaction of Ethanol with Inducers of Glucose-regulated Stress Proteins

Abstract

GRP78, a molecular chaperone expressed in the endoplasmic reticulum, is a "glucose-regulated protein" induced by stress responses that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking. Previously we showed that chronic ethanol exposure increases the expression of GRP78. To further understand the mechanism underlying ethanol regulation of GRP78 expression, we studied the interaction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma ؋ glioma cells. We found that, in addition to increasing basal levels of GRP78 mRNA ("induction"), ethanol produced greater than additive increases in the induction of GRP78 mRNA by the "classical" GRP inducers A23187, brefeldin A, and thapsigargin ("potentiation"). Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfection analyses with the rat grp78 promoter. Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied. In contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promoter activity. Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences required for ethanol induction differ from those mediating responses to classical GRP inducers. Furthermore, linker-scanning mutations of the grp78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in induction by ethanol or classical inducing agents. While the ethanol induction response required 16 -24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h. The potentiation response also decayed rapidly after ethanol removal. In addition, the protein kinase A inhibitor R p -cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while S p -cAMPS, an activator of protein kinase A, decreased ethanol potentiation. Taken together, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP inducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classical GRP inducers. In contrast, GRP78 induction by ethanol involves promoter sequences and a mechanistic pathway separate from that of the ethanol potentiation response or classical GRP78 inducers. These studies show that ethanol produces a novel and complex regulation of grp78 transcription which could be of particular importance during neuronal exposure to GRP-inducing stressors as might occur with central nervous system injury.

Pharmacogenomic Analysis of Mechanisms Mediating Ethanol Regulation of Dopamine β-Hydroxylase

“…Previous studies by our laboratory found increased DBH mRNA abundance in neural cell cultures and mice exposed to ethanol (12). Prior work from our laboratory and others showed that ethanol can cause selective increases in mRNA abundance by either increasing gene transcription (4,5,(62)(63)(64) or altering mRNA stability (63). In this study, transient transfection analysis showed that ethanol treatment increased DBH gene transcription.…”

Ethanol upregulates the expression of p21WAF1/CIP1 and prolongs G1 transition via a p53-independent pathway in human epithelial cells

“…A preliminary study shows that ethanol does not enhance the expression of TGF-b1 in RKO cells (data not shown), suggesting that the overexpression of p21 WAF1/ CIP1 by ethanol is not via the induction of TGF-b1. It has been reported that ethanol can also increase the transcription of several stress responsive genes such as GRP78, GRP90 and Hsc70 in neuronal cells (Miles et al, 1991;Wilke et al, 1994;Hsieh et al, 1996). The GRP78 gene is up-regulated by ethanol through the SP1 site.…”

Involvement of p38 Mitogen-activated Protein Kinase Signaling Pathway in the Rapid Induction of the 78-kDa Glucose-regulated Protein in 9L Rat Brain Tumor Cells

“…The above changes lead ultimately to accumulation of underprocessed or misfolded proteins in the endoplasmic reticulum and result in the induction of GRP78 (9). This protein is also induced by ethanol via a mechanistic pathway different from that of the "classical inducers" (10). By contrast, GRP78 is usually not induced by heat shock (HS) alone (11,12).…”