At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

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Free Radicals in the Physiological Control of Cell Function

Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both servoregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fasebj.6.12.1381691

Nitric oxide as a secretory product of mammalian cells.

The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of “oxidative stress” is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.

Reactive oxygen species in cell signaling

Reactive oxygen species, antioxidants, and the mammalian thioredoxin system.

TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 29,79-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 59-upstream region, the CArG element in the 59 flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.

/pdf/production-of-hydrogen-peroxide-by-transforming-growth-jh6q7zmj8v.pdf

Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells.

H2O2, like other oxidants, is known to act as a mitogen at low concentrations in resting Balb/3T3 or mouse epidermal JB6 cells. We described previously that H2O2 induces some early response genes in Balb/3T3 cells. We extended these observations using another cell line, MC3T3 (mouse osteoblastic) cells by examination of transcriptional activity of these genes and by using inhibitors of protein kinases. H2O2 increased the expressions of c-fos, c-jun, egr-1 and JE genes which are known to be early response genes and are induced by mitogenic stimuli in many types of cells. Exogenous addition of H2O2 increased the mRNA levels of these genes, the kinetics of increase being similar to those of their inductions by a phorbol ester or serum. Nuclear run-on transcription showed that this induction occurred at the transcriptional level. H2O2 at 0.1–0.2 mM induced maximal expressions of c-fos and c-jun, whereas 0.3 mM H2O2 was required for induction of stress-induced heme oxygenase mRNA. The inductions of c-fos and were inhibited by 50 μM H7, a protein kinase inhibitor that is relatively specific for protein kinase C, but were not affected by H9, relatively specific for cAMP-dependent protein kinase. In cells pretreated with 12-O-tetradecanoylphorbol 13-acetate, however, in which protein kinase was supposed to be downregulated, H2O2 induced c-fos and heme oxygenase as efficiently as in untreated cells. H2O2 did not increase the phosphorylation of p80 protein, which is known to be a substrate for protein kinase C. Thus, H2O2 seemed to induce c-fos and c-jun by activating protein kinases distinct from protein kinase C. Activity of the chloramphenicol acetyltransferase gene under control of the serum-response element of human c-fos genes was increased by H2O2 treatment, whereas that under control of cAMP-response element was not affected. These results indicate that the inductions by H2O2 of c-fos and possibly other early response genes are mediated through activation of the serum-response element in their enhancer.

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1432-1033.1991.tb16261.x

Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line.

Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors.

Growth factors and tumor-promoting phorbol esters induce various types of "early response" genes, most of which encode transcription factors. To identify possible target genes for these early response gene products, we isolated complementary DNA clones of genes that were induced later by 12-O-tetradecanoylphorbol-13-acetate. A complementary DNA library was constructed using mRNA from mouse osteoblastic cells (MC3T3-E1) treated with 12-O-tetradecanoylphorbol-13-acetate for 4 h, and the library was screened by differential hybridization. Five independent clones were isolated. All of these genes were induced maximally 4 to 8 h after 12-O-tetradecanoylphorbol-13-acetate addition. Sequencing showed that three of them (OTS-1, -3, and -4) were those of the metallothionein II gene, and one (OTS-5) was identical to the osteopontin/2ar gene. The other (OTS-8) had no significant homology with known sequences and was considered to be a novel gene. The induction of OTS-5 and OTS-8 was decreased in the presence of cycloheximide. In mice, OTS-8 mRNA was detected in the lungs but not at appreciable levels in other organs. Open reading frame of OTS-8 complementary DNA and its in vitro transcription/translation product suggested that this gene encodes an Mr 26,000 protein. OTS-8 mRNA was induced by epidermal growth factor but not by serum. The steady-state levels of OTS-5 and OTS-8 mRNAs were significantly elevated in v-ras-transformed MC3T3 cells.

http://cgd.aacrjournals.org/cgi/reprint/1/11/511.pdf

Isolation of a gene sequence induced later by tumor-promoting 12-O-tetradecanoylphorbol-13-acetate in mouse osteoblastic cells (MC3T3-E1) and expressed constitutively in ras-transformed cells.

Kiyoshi Nose

Papers

Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells.

Transcriptional activation of early-response genes by hydrogen peroxide in a mouse osteoblastic cell line.

Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors.

Isolation of a gene sequence induced later by tumor-promoting 12-O-tetradecanoylphorbol-13-acetate in mouse osteoblastic cells (MC3T3-E1) and expressed constitutively in ras-transformed cells.

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts.