Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells

Abstract

Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein ('GRP78/BiP') and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.

Regulation of Protein Synthesis in Ventricular Myocytes by Vasopressin

“…5). As anticipated, polysomes almost completely disappeared in response to thapsigargin, which slows initiation relative to peptide chain elongation in non-muscle cells (17). Vasopressin also reduced polysomal content, but not as dramatically as thapsigargin.…”

“…Findings were reproduced on at least two separate occasions. [ 35 S]Methionine labeling, one-dimensional 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) of detergent-solubilized extracts of methionine-labeled cells, and autoradiography were conducted as described previously (17). Ribosomal and polyribosomal size distributions were measured by density gradient centrifugation as described previously (18).…”

Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease

“…104 It is well established that activation of the UPR by ER stress leads to decreased rates of protein synthesis, 105 a process mediated by the ER-resident kinase, PERK. [106][107][108] Once activated, PERK phosphorylates Ser51 of eukaryotic initiation factor-2a (eIF2a), thereby inhibiting cellular mRNA translation and inducing transcriptional activation of a wide range of ER stress-inducible genes, including GRP78, GADD153, and TDAG51.…”

Induction of the unfolded protein response and cell death pathway in Alzheimer's disease, but not in aged Tg2576 mice

“…ER stress, accumulation of unfolded protein in the endoplasmic reticulum, can be provoked primarily by imbalance in homeostasis, proteasome activity during degeneration and differentiation (Kozutsumi et al, 1988;Wong et al, 1993;Friedlander et al, 2000;Cho et al, 2009). ER stress induces an adaptive signaling pathway called the UPR that involves activation of transcription factors, XBP-1 and activating transcription factor 6 (ATF6) (Yoshida et al, 2000;Lee et al, 2003).…”