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

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.

Inhibition of translational initiation by metalloendoprotease antagonists. Evidence for involvement of sequestered Ca2+ stores.

Synthetic oligopeptide inhibitors of metalloendoprotease activity have been shown to block membrane fusion events, to slow transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi, and to perturb Ca2+ homeostasis. Effects of such agents on translational activity, which requires Ca2+ sequestered putatively within the ER, were examined in this study. Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) provoked rapid inhibition of amino acid incorporation into a broad spectrum of proteins in GH3 pituitary, C6 glial, and Neuro-2a cells but not in reticulocytes, which lack ER. Polysome accumulation and incorporation were reduced concurrently, indicating that the dipeptide acted to slow translational initiation. Inhibitions were largest at low extracellular Ca2+, were reversed by increasing extracellular Ca2+, were comparable to those achieved in the presence of EGTA or Ca2+ ionophores, and were observed with assorted metalloendoprotease antagonists but not with leupeptin. At concentrations inhibitory to protein synthesis Cbz-Gly-Phe-NH2 mobilized cell-associated 45Ca, lowered cytosolic free Ca2+, and did not generate inositol phosphates. Cells treated for 3-4 h with Cbz-Gly-Phe-NH2 reacquired the ability to synthesize proteins at nearly normal rates; a phorbol ester or cAMP-elevating agent was necessary for such recovery in GH3, but not C6 or Neuro-2a, cells. GRP78, which may function in the folding and assembly of secretory proteins and in translational accommodation to agents that deplete sequestered Ca2+ stores, was induced during such treatments. Accumulation of GRP78 mRNA in treated preparations was reduced as extracellular Ca2+ was increased. Extended exposure to dipeptide followed by brief recovery in its absence rendered protein synthesis resistant to inhibition by Ca2+ ionophore. It is concluded that metalloendoprotease antagonists suppress translational initiation as a consequence of their capacity to mobilize sequestered Ca2+ stores.

Accommodation of protein synthesis to chronic deprivation of intracellular sequestered calcium. A putative role for GRP78.

Mobilization of sequestered intracellular Ca2+ with EGTA or Ca2+ ionophores severely depresses rates of translational initiation in various mammalian cell types including C6 glial, GH3 pituitary and P3X63Ag8 myeloma cells. Within 2-3 h of continuous exposure to either chelator or ionophore, cells adapt or accommodate such that their rates of amino acid incorporation are restored to 40-70% of those of untreated controls. In GH3 and P3X63Ag8 cells, treatment with either a phorbol ester or a cAMP-elevating agent was required to obtain maximal degrees of accommodation of translational initiation. Following the development of accommodation, cells restored with optimal Ca2+ exhibited rates of amino acid incorporation identical with those of nontreated controls but remained resistance to inhibition on subsequent challenge with EGTA or ionophore. Development of translational tolerance to agents depleting Ca2+ stores did not involve alterations in cellular capacity or affinity for the cation. Invariably, the development of tolerance was preceded by transcriptionally dependent, preferential synthesis of the reticuloplasmin GRP78/BiP. In Ca2(+)-deprived GH3 cells, the synthesis of GRP78 was promoted by phorbol ester and cAMP with the extent of induction correlating directly with the degree of translational tolerance to ionophore. Cells pretreated with dithiothreitol, an alternate inducer of GRP78, also became tolerant to translational inhibition by Ca2+ ionophore or EGTA. Amino acid incorporation in nonsecreting NS-1-cloned myeloma cells, which constitutively express high levels of GRP78 and its mRNA, resisted inhibition by EGTA, ionophore, and dithiothreitol. Antisense oligodeoxynucleotides directed against GRP78 mRNA reduced amino acid incorporation in tolerant, but not in non-tolerant, preparations. These results predicate the existence of a mechanism whereby mammalian cells are capable of rapidly developing translational cross-tolerance to either depletion of sequestered Ca2+ or a reducing environment. A role for nascent GRP78 is strongly implicated in this accommodation mechanism.