thapsigargin and castanospermine

Accumulation of misfolded proteins within the lumen of the mammalian endoplasmic reticulum (ER) activates the unfolded protein response (UPR). ATF6 and Ire1p are ER-associated proteins that control UPR-specific transcription systems in mammals; UPR signaling involves cleavage of ATF6 and splicing of XBP1 mRNA initiated by Ire1p. We tested the hypothesis that activation of ATF6 and/or Ire1p determines the levels of mRNAs derived from target genes encoding GRP78/BiP and EDEM. By subjecting dermal fibroblasts to multiple stresses, strong correlations were found between ATF6 activation and XBP1 splicing, and between GRP78/BiP mRNA and EDEM mRNA accumulation. Surprisingly, there was no reasonable correlation between activation of either signal transducer with accumulation of either target transcript. Thus, ATF6 and Ire1p signaling do not define the magnitude of UPR-dependent mRNA increases, even though they may be necessary for gene activation, suggesting the existence of additional stress-sensitive factors acting as "coincidence detectors" for transcript accumulation.

Topics: Activating Transcription Factor 6; Azetidinecarboxylic Acid; Cells, Cultured; Dithiothreitol; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; Fibroblasts; Humans; Indolizines; Membrane Proteins; Oxidative Stress; Protein Folding; Protein Serine-Threonine Kinases; Regulatory Factor X Transcription Factors; Signal Transduction; Skin; Thapsigargin; Transcription Factors; Transcriptional Activation; X-Box Binding Protein 1

Mobilization of Ca2+ from the endoplasmic reticulum (ER) suppresses translational initiation and inhibits post-translational processing and secretion of glycoproteins. This study explores the mechanism whereby ionomycin, a Ca2+ ionophore, and thapsigargin, an ER Ca(2+)-ATPase inhibitor, promote retention of alpha 1-antitrypsin (alpha 1-AT) bearing high mannose, endoglycosidase H (Endo H)-sensitive oligosaccharide side chains within the ER of HepG2 cells. Arrest occurred at the removal of mannose residues such that intermediates with Man7-9GlcNAc2 side chains accumulated with the Man8-9GlcNAc2 structures predominating. Maturation of alpha 1-AT bearing Man5-6GlcNAc2 side chains was unaffected. Inhibition of alpha 1-AT processing by ionomycin occurred independently of translational suppression. Forms of alpha 1-AT identical to those retained with ionomycin or thapsigargin were observed upon treatment with the alpha-1,2-mannosidase inhibitor 1-deoxymannojirimycin whereas castanospermine, an inhibitor of ER alpha-glucosidase I, produced different forms of the glycoprotein. Neither inhibitor impaired transport or secretion of alpha 1-AT. With brefeldin A, which causes redistribution of Golgi enzymes to the ER, alpha 1-AT was retained intracellularly but acquired resistance to Endo H. With ionomycin, thapsigargin, or 1-deoxymannojirimycin-treated cells, however, brefeldin A failed to promote further processing of the glycoprotein. Possible mechanisms for the suppression of alpha 1-AT processing at the alpha-1,2-mannosidase step by Ca(2+)-mobilizing agents are discussed. Excepting tunicamycin, traditional inhibitors of protein processing did not affect amino acid incorporation.

Topics: 1-Deoxynojirimycin; alpha 1-Antitrypsin; alpha-Mannosidase; Brefeldin A; Calcium; Calcium-Transporting ATPases; Carbohydrate Conformation; Carcinoma, Hepatocellular; Cyclopentanes; Endoplasmic Reticulum; Glycoside Hydrolase Inhibitors; Hexosaminidases; Humans; Indolizines; Ionomycin; Liver Neoplasms; Mannose; Mannosidases; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Oligosaccharides; Terpenes; Thapsigargin; Tumor Cells, Cultured