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The role of inorganic metals and metalloporphyrins in the induction of haem oxygenase and heat-shock protein 70 in human hepatoma cells.

ABSTRACT: The role of inorganic metals and metalloporphyrins in the induction of mRNAs for haem oxygenase and heat-shock protein 70 (hsp70), the two heat-shock proteins, was examined in human HepG2 and Hep3B hepatoma cells. SnCl2, but not Sn-protoporphyrin, was found to be a potent inducer of both haem oxygenase and hsp70 mRNAs. In contrast, CoCl2, ZnCl2 and FeCl2 caused little induction of haem oxygenase and hsp70 mRNAs, whereas the porphyrin complexes of these metals strongly induced haem oxygenase mRNA, without influencing the level of hsp70 mRNA. The induction process was largely transcriptional, as judged by the inhibition of induction by actinomycin D, but not by cycloheximide, and by increased transcription demonstrated by nuclear run-off analysis. Since CoCl2 is a potent inducer of haem oxygenase in vivo in animals, the possibility of the biosynthesis of Co-protoporphyrin was examined in human hepatoma cells by incubating them with CoCl2 and protoporphyrin, or delta-aminolaevulinate (ALA), the precursor of protoporphyrin. Both types of treatment led to a potent induction of haem oxygenase mRNA. Co-protoporphyrin formation was also spectrally demonstrated in cells incubated with the metal and ALA. The results of this study indicate that certain metals, e.g. SnCl2, may directly induce haem oxygenase mRNA, whereas with other elements, incorporation of the metal into the porphyrin macrocycle is necessary for induction. Therefore CoCl2, like haemin, may activate the haem oxygenase gene via a haem-responsive transcription factor, whereas SnCl2 may exert its effect via a metal-responsive transcription factor.

SUBMITTER: Mitani K

PROVIDER: S-EPMC1132355 | biostudies-other | 1993 Mar

REPOSITORIES: biostudies-other

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Project description:HO-1 (haem oxygenase 1) is an essential antioxidant enzyme in the cell that exerts its effects through removal of pro-oxidant haem groups and the formation of antioxidant molecules and carbon monoxide. The electrophilic cyclopentenone 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2) induces the expression of HO-1 protein through the covalent modification of protein thiols. It has been shown that specific thiol residues of the redox-sensor Keap1 (Kelch-like ECH-associated protein 1) are modified by 15d-PGJ2, leading to activation of the transcription factor Nrf-2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) and up-regulation of genes under control of the electrophile-response element, including HO-1. However, 15d-PGJ2 has also been shown to modify other proteins which comprise the electrophile-responsive proteome. Since 15d-PGJ2 has been shown to localize to the mitochondria in endothelial cells, we hypothesized that mitochondrial protein modification may also be important in Keap1/Nrf-2 signal transduction, leading to HO-1 up-regulation. In order to determine the role of mitochondrial protein thiol modification in HO-1 induction, we used the mitochondrial-targeted thiol-reactive compound IBTP [(4-iodobutyl)triphenylphosphonium]. IBTP had no effect on basal HO-1 levels, but effectively blocked HO-1 induction by a variety of reagents including haemin, iodoacetamide and 15d-PGJ2. Mechanistically, IBTP did not prevent the covalent modification of Keap1 by 15d-PGJ2. However, IBTP prevented the 15d-PGJ2-dependent increases in HO-1 mRNA and protein. Furthermore, IBTP prevented the nuclear accumulation of Nrf-2, suggesting cross-talk between mitochondria and antioxidant-response signal transduction. This effect was independent of reactive oxygen species formation or mitochondrial membrane potential. In addition, IBTP significantly enhanced the toxicity of high concentrations of 15d-PGJ2, suggesting that loss of mitochondrial control of HO-1 leads to increased susceptibility to electrophilic stress in endothelial cells. The implications for these studies in understanding the balance between cytoprotection and cytotoxicity in the context of diseases such as atherosclerosis is discussed.

Project description:Heat shock proteins (Hsps) are constitutively expressed in cells and involved in protein folding, assembly, degradation, intracellular localization, etc, acting as molecular chaperones. However, their overexpression represents a ubiquitous molecular mechanism to cope with stress. Hsps are classified into families, and among them the Hsp70 family appears to be the most evolutionary preserved and distributed in animals. In this study, the expression of Hsp70 and the related messenger ribonucleic acid (mRNA) has been studied in Ostrea edulis after exposure to heat and heavy metals; moreover, levels of metallothioneins (MTs), another class of stress-induced proteins, have contemporaneously been assessed in the same animals. Thermal stress caused the expression of a 69-kDa inducible isoform in gills of O edulis but not in the digestive gland. Northern dot blot analysis confirmed that the transcription of Hsp69-mRNA occurs within 3 hours of stress recovery after oyster exposure at 32 and 35 degrees C. Hsp69-mRNA transcripts were not present in the gills of animals exposed to 38 degrees C after 3 hours of poststress recovery, but they were detected after 24 hours. The expression of the 69-kDa protein in O edulis exposed to 38 degrees C was rather low or totally absent, suggesting that the biochemical machinery at the base of the heat shock response is compromised. Together with the expected increase in MT content, the oysters exposed to Cd showed a significant enhancement of Hsp70, although there was no clear appearance of Hsp69. Interestingly, the levels of MT were significantly increased in the tissues of individuals exposed to thermal stress. Unlike oysters, heat did not provoke the expression of inducible Hsp isoforms in Mytilus galloprovincialis, Tapes philippinarum, and Scapharca inaequivalvis, although it significantly enhanced the expression of constitutive proteins of the 70-kDa family. The expression of newly synthesized Hsp70 isoforms does not seem therefore a common feature in bivalves exposed to thermal stress.

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The role of inorganic metals and metalloporphyrins in the induction of haem oxygenase and heat-shock protein 70 in human hepatoma cells.

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