thapsigargin and sodium-arsenite

Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) are sensitive techniques used for detecting nucleic acids and proteins in cultured cells. However, these techniques are rarely applied together, and standard protocols are not readily compatible for sequential application on the same specimen. Here, we provide a user-friendly step-by-step protocol to perform multicolor RNA-FISH in combination with IF to simultaneously detect the subcellular localization of distinct RNAs and proteins in cultured cells. We demonstrate the use of our protocol by analyzing changes in the subcellular distribution of RNAs and proteins in cells exposed to a variety of stress conditions.

Topics: Antibodies; Arsenites; Aurintricarboxylic Acid; Base Sequence; Cytoplasmic Granules; DNA Helicases; Fluorescent Antibody Technique; Genes, Reporter; Green Fluorescent Proteins; HEK293 Cells; Hot Temperature; Humans; Hydrogen Peroxide; In Situ Hybridization, Fluorescence; Oligonucleotide Probes; Poly-ADP-Ribose Binding Proteins; Protein Binding; RNA Helicases; RNA Recognition Motif Proteins; RNA, Ribosomal, 18S; RNA, Ribosomal, 28S; Sodium Compounds; Stress, Physiological; Thapsigargin

The secretory pathway of neurons and endocrine cells contains a variety of mechanisms designed to combat cellular stress. These include not only the unfolded protein response pathways but also diverse chaperone proteins that collectively work to ensure proteostatic control of secreted and membrane-bound molecules. One of the least studied of these chaperones is the neural- and endocrine-specific molecule known as proSAAS. This small chaperone protein acts as a potent anti-aggregant both in vitro and in cellulo and also represents a cerebrospinal fluid biomarker in Alzheimer's disease. In the present study, we have examined the idea that proSAAS, like other secretory chaperones, might represent a stress-responsive protein. We find that exposure of neural and endocrine cells to the cell stressors tunicamycin and thapsigargin increases cellular proSAAS mRNA and protein in Neuro2A cells. Paradoxically, proSAAS secretion is inhibited by these same drugs. Exposure of Neuro2A cells to low concentrations of the hypoxic stress inducer cobalt chloride, or to sodium arsenite, an oxidative stressor, also increases cellular proSAAS content and reduces its secretion. We conclude that the cellular levels of the small secretory chaperone proSAAS are positively modulated by cell stress.

Topics: Animals; Arsenites; Cell Hypoxia; Cell Line; Cobalt; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Mice; Molecular Chaperones; Neuropeptides; Oxidative Stress; Protective Agents; Rats; RNA, Messenger; Sodium Compounds; Stress, Physiological; Thapsigargin; Tunicamycin; Up-Regulation

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

Topics: 3' Untranslated Regions; Amino Acids; Antigens, Surface; Arsenites; Cationic Amino Acid Transporter 1; Cell Line, Tumor; Culture Media; Cytoplasmic Structures; ELAV Proteins; ELAV-Like Protein 1; Humans; MicroRNAs; Oxidative Stress; Protein Binding; Protein Biosynthesis; RNA Stability; RNA Transport; RNA-Binding Proteins; Sodium Compounds; Thapsigargin; Up-Regulation

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arsenites; B-Lymphocytes; Calcimycin; Calcium; Cell Line; Cell-Free System; Enzyme Activation; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Hypertonic Solutions; Imidazoles; Interleukin-1; Intracellular Signaling Peptides and Proteins; Ionophores; Leukotrienes; Lipoxygenase Inhibitors; Mitogen-Activated Protein Kinases; Osmotic Pressure; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinases; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Pyridines; Sodium Compounds; Sorbitol; Subcellular Fractions; Thapsigargin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vanadates

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 microM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

Topics: Amino Acid Sequence; Animals; Arachidonate 5-Lipoxygenase; Arsenites; Calcimycin; Cell Line; Cricetinae; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Mice; Mitogen-Activated Protein Kinases; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Rats; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Sodium Compounds; Tetradecanoylphorbol Acetate; Thapsigargin; Tumor Necrosis Factor-alpha