EXPERIMENTAL CELL RESEARCH 237, 434–444 (1997) ARTICLE NO. EX973825 Cooperative Induction of c- fos and Heme Oxygenase Gene Products under Oxidative Stress in Human Fibroblastic Cells Satoshi Numazawa, 1 Hiroyuki Yamada, Azusa Furusho, Tadashi Nakahara, Takiko Oguro, and Takemi Yoshida Department of Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, Shinagawa, Tokyo 142, Japan teins in most cells, the enzyme consumes glutathione Heme oxygenase-1 is a stress responsive enzyme and when a mass of the substrate fluxed resulting in its implicated in a protective function of cellular damage. depletion and eventually cells to commit into the oxida- We investigated cellular events leading to the heme tive stress. We have shown that glutathione S-trans- oxygenase-1 gene expression induced by sublethal ferase substrates such as phorone, stilbene compounds, concentrations of glutathione depletors, phorone and and diethyl maleate induce a decrease in glutathione diethyl maleate, in human fibroblastic cells. Accumula- contents and a concomitant increase in heme oxy- tion of heme oxygenase-1 mRNA by glutathione deple- genase (HO) activity in the liver of rats [1–3]. HO is a tors was canceled by simultaneous treatment with rate-limiting enzyme in heme degradation that cleaves cycloheximide, an inhibitor of protein synthesis; how- heme to form biliverdin, carbon monoxide, and iron [4]. ever, the inhibitory effect decreased when the inhibi- Biliverdin is subsequently converted to bilirubin by an tor was added 30 min later. Among the inducible early abundant cytosolic enzyme biliverdin reductase in response genes, the c-fos expression was significantly mammals. Two isoforms of HO, the inducible HO-1 and elevated with a peak at 30 min after the agents. Accu- the constitutive HO-2, have been characterized so far mulation of heme oxygenase-1 and c-fos transcripts [5]. In addition to glutathione depletion-induced oxida- was abrogated in cells pretreated with 1,4-diazabi- tive stress, HO-1 is induced by a variety of exogenous cyclo[2.2.2]octane, an oxygen-free radical quencher. and environmental stimuli such as ultraviolet light, Decrease in glutathione levels preferentially activated hydrogen peroxide, heat shock, heavy metals, cyto- extracellular-signal regulated kinases rather than kines, and lipopolysaccharide [5]. Although a precise other stress-activated protein kinases such as c-Jun N- function of HO induction is not fully understood, in- terminal kinases and p38 MAP kinase. Pretreatment creases in HO activity have been implicated in tissue of cells with PD 98059, an inhibitor of the extracellular- and cellular protection against oxidative stress since signal regulated kinase cascade, or the c-fos antisense several lines of evidence suggest that bilirubin acts as oligodeoxynucleotide inhibited the heme oxygenase- 1 induction elicited by glutathione depletion. These a physiological antioxidant in vivo [6, 7]. HO-1 is thus observations indicated that c-Fos protein plays a role considered to be a stress protein. in heme oxygenase-1 gene expression induced by glu- It is becoming increasingly clear that the mitogen- tathione depletion-mediated oxidative stress in hu- activated protein kinase (MAP kinase) family of protein man fibroblasts.  1997 Academic Press kinases involves stress-responded signaling cascades. Those include recently discovered c-Jun N-terminal ki- nases (JNK) [8, 9] and p38 MAP kinase, [10] as well as INTRODUCTION classical extracellular-signal-regulated kinases (ERK) that respond primarily to mitogenic stimuli via Ras Glutathione is a major cellular sulfhydryl source in [11]. These MAP kinase cascades are activable to vary- nonprotein matter and plays a pivotal role in redox ing extents by cellular stresses, such as heat, inflam- control in mammalian cells. It is also involved in the matory cytokines, ischemia-reperfusion, and metabolic phase II drug metabolizing reaction as a cofactor of poisons, and transduce signals to distinct nuclear tran- glutathione S-transferase. Since glutathione S-trans- scription factors [12, 13]. Significant overlaps have ferase is one of the abundantly expressed cytosolic pro- been shown to exist between stimuli responded to HO-1 induction and activation of the MAP kinases; however, little attention has been paid to cellular signaling lead- 1 To whom correspondence and reprint requests should be ad- ing to HO induction. In other words, most studies have dressed. Fax: /81-3-3784-8246. E-mail: numazawa@pharm. showa-u.ac.jp. focused on the cellular signaling from the membrane 434 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.