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 x 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 Rp-cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while Sp-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.
Highlights
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
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”)
Ethanol Increases the Action of Classical Inducers of GRP78 mRNA—Using subtractive hybridization cloning, we recently showed that GRP78 and GRP94 are members of a group of ethanol-responsive genes in NG108-15 neuroblastoma ϫ glioma cells [1]
Summary
Plasmid pI10, used for the majority of stable transfection studies, contains 1.25 kilobases of the rat grp promoter coupled to a chloramphenicol acetyltransferase (CAT) reporter gene [13]. Twenty-four hours after transfection, cells were treated with ethanol or other drugs as described under “Results.” CAT activity was assayed in cell lysates prepared by freeze-thawing in 0.25 M Tris, pH 8.0. Transfected NG108-15 cells were isolated by co-transfection of cells with pSV3neo [17] and a grp promoter-CAT construct (pI10 or as indicated in text). Cells were transfected, selected for resistance to G418 (Life Technologies, Inc.) and clonal isolates were screened for CAT activity as described previously [15]. Northern Blot Analysis—Total RNA from NG108-15 cells was analyzed by Northern hybridization as described previously [15]. Multiple exposures were done to ensure linearity of the film response
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