INTERACTION OF ETHANOL WITH INDUCERS OF GLUCOSE-REGULATED STRESS PROTEINS - ETHANOL POTENTIATES INDUCERS OF GRP78 TRANSCRIPTION

GRP78, a molecular chaperone expressed in the endoplasmic reticulum, i s a ''glucose-regulated protein'' induced by stress responses that dep lete glucose or intracisternal calcium or otherwise disrupt glycoprote in trafficking. Previously we showed that chronic ethanol exposure inc reases the expression of GRP78. To further understand the mechanism un derlying ethanol regulation of GRP78 expression, we studied the intera ction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma x glioma cells. We found that, in addition to i ncreasing 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 (''p otentiation''), Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfectio n analyses with the rat grp78 promoter, Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied, I n contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promo ter activity, Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences requi red for ethanol induction differ from those mediating responses to cla ssical GRP inducers, Furthermore, linker-scanning mutations of the grp 78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in inducti on by ethanol or classical inducing agents, While the ethanol inductio n response required 16-24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h, The potentiation response also decay ed rapidly after ethanol removal, In addition, the protein kinase A in hibitor R(p) cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while S-p-cAMPS, an ac tivator of protein kinase A, decreased ethanol potentiation, Taken tog ether, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP i nducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classica l GRP inducers, In contrast, GRP78 induction by ethanol involves promo ter sequences and a mechanistic pathway separate from that of the etha nol potentiation response or classical GRP78 inducers, These studies s how that ethanol produces a novel and complex regulation of grp78 tran scription which could be of particular importance during neuronal expo sure to GRP-inducing stressors as might occur with central nervous sys tem injury.