Establishment of a chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses A23187 induction of both GRP78 and GRP94

Abstract

GRP78, a 78,000 dalton protein residing in the endoplasmic reticulum, is postulated to play important roles in protein folding and cell survival during calcium and other physiological stress. Here we describe the construction of an eukaryotic expression vector for the constitutive expression of grp78 antisense RNA and the creation of a CHO cell line, 78WO, which expresses high levels of the grp78 antisense RNA through amplification of the stably transfected antisense vector. We observed that whereas 78WO maintains a basal level of GRP78 similar to that of control cells, GRP78 is no longer inducible by A23187. The 78WO cells have undergone a compensatory increase in grp78 transcription such that the effects of antisense are cancelled out at the protein level under nonstressed conditions. In these same cells, GRP94, a 94,00 dalton ER protein, is also rendered noninducible by A23187. This provides the first evidence that the regulation of two ER proteins might be coupled such that the failure to induce GRP78 results in the down-regulation of GRP94. The 78WO cell line grows with a doubling time of about 26 hr and exhibits decreased tolerance to A23187, suggesting the GRPs contribute to cell viability under calcium stress. The establishment of this cell line, which can be stably maintained, will provide a useful tool for testing whether the induction of the GRPs is important for protein folding or transport and whether their enhanced synthesis is the cause or consequence of a variety of physiological adaptations.

A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells

“…Conversely, inhibition of GRP induction increases sensitivity to death in response to calcium release from the ER (Li et al 1991(Li et al , 1992, oxidative stress (Gomer et al 1991), hypoxia (Koong et al 1994b), and T cell mediated cytotoxicity (Sugawara et al 1993). Therefore, elucidating the signaling mechanism(s) by which cells respond to ER stress may have important therapeutic implications.…”

Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls

“…Preventing the induction of GRPs through transfection of BiP antisense RNA expression constructs, transfection of anti-BiP ribozymes, or transfection with multiple copies of the BiP promoter element to titrate out positively acting transcription factors increased sensitivity to ionophore, oxidative stress, hypoxia, and cell-mediated toxicity (Gomer et al 1991;Li and Lee 1991;Li et al 1992;Sugawara et al 1993;Koong et al 1994). In contrast, overexpression of BiP itself prevented transcriptional induction in response to ER stress and protected cells from death in response to calcium depletion from the ER (Dorner et al 1992;Morris et al 1997).…”

Mutation (D472Y) in the Type 3 Repeat Domain of Cartilage Oligomeric Matrix Protein Affects Its Early Vesicle Trafficking in Endoplasmic Reticulum and Induces Apoptosis

“…Recently, it has been shown that a variety of toxic insults, including calcium ionophores, inhibitors of glycosylation, chemical toxicants, and oxidative stress, can cause ER stress and ultimately lead to cell death. [31][32][33][34][35][36][37][38][39] We found that significant ER stress and resultant apoptosis occurred in cells expressing mutated COMP compared with wild-type COMP and discuss the significance of this in PSACH.…”