ELSEVIER Free RadicalBiology & Medicine,Vol. 20, No. 1, pp. 63-73, 1996 Copyright© 1995 Elsevier ScienceInc. Printed in the USA. All rightsreserved 0891-5849/96 $15.00 + .00 0891-5849(95)02024-5 Original Contribution NITRIC OXIDE DONORS MODULATE FERRITIN AND PROTECT ENDOTHELIUM FROM OXIDATIVE INJURY MARK B. JUCKETI~, * MARC WEBER, t JOZSEF BALLA,t HARRY S. JACOB, t and GREGORY M. VERCELLOTTIt • Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, USA; and *University of Minnesota, Minneapolis, MN, USA (Received 14 October 1994; Revised 29 March 1995; Accepted l0 July 1995) Abstract Ferritin protects endothelial cells from the damaging effects of iron-catalyzed oxidative injury. Regulation of ferritin occurs through the formation of an iron-sulfur cluster within a cytoplasmic protein, the iron regulatory protein (IRP) that controls ferritin mRNA translation. Nitric oxide has been shown to inhibit iron-sulfur proteins and is present at vascular sites of inflammation; therefore, we undertook a study to examine the influence of nitric oxide on changes in endothelial cell ferritin content in response to iron exposure, and the subsequent effects on susceptibility to oxidative injury. Iron-loaded endothelial cells (EC) exposed to nitric oxide donors synthesize markedly less ferritin. Treatment of EC with a nitric oxide donor increases IRP affinity for ferritin mRNA concomitant with a loss of cytoplasmic aconitase activity in iron-laden EC. Iron-treated EC exposed to NO donors were resistant to oxidative injury despite their low ferritin content when examined 1 h after the treatment period. In contrast, 24 h later, these same cells become sensitive to oxidants, whereas iron-treated EC that are ferritin-rich continue to be resistant. In conclusion, NO inhibits the increase of EC ferritin after exposure to iron but provides short-term protection against oxidants; ferritin, in turn, provides durable cytoprotection by inactivating reactive iron. Keywords---Ferritin, Nitric oxide, Endothelium, Oxidative injury, Free radicals ~TRODUCTION Investigations of atherogenesis have discerned two mechanisms by which endothelial cells may defend against damage: nitric oxide (NO), which inhibits platelet aggregation L: and leukocyte adhesion, 3 and ferritin, which sequesters catalytic iron. This latter pro- tein, ferritin, is a 24 subunit multimeric intracellular molecule that is critical for the storage and detoxifica- tion of excess cellular iron. After exposure to iron, cells rapidly increase ferritin content and downregulate transferrin receptor expression, largely through post- transcriptional mechanisms. The importance of ferritin to endothelial cell homeostasis is suggested by our previous studies demonstrating that endothelial cells treated with hemin initially are sensitive to oxidative injury, 4 but become resistant after a 12-16-h period, because of an increase in ferritin content. We have Address correspondence to: Mark B. Juckett, John L. Doyne Hospital, Box 133, 8700 W. Wisconsin Ave., Milwaukee, WI 53226, USA. E-mail address: MJUCKETT@HEMONC.MCW.EDU demonstrated that ferritin-rich endothelial cells are protected from oxidant injury mediated by oxidized LDL, 5 hydrogen peroxide, and activated neutrophils. 6 These findings implicate ferritin in the defense of the vessel wall from oxidative injury. Ferritin synthesis is regulated by a posttranscrip- tional mechanism that depends on the interaction be- tween an iron-sulfur cluster containing protein, called the iron regulatory protein (IRP), and a 5' stem-loop on the ferritin mRNA, called the iron responsive element (IRE). 7'8 The IRP binds to ferritin mRNA when in its apoprotein form, 9 thus preventing initiation of transla- tion. The IRP "senses" iron through the formation of a cubane iron-sulfur cluster that seems to result in a conformational change, decreasing its affinity for ferri- tin mRNA and thus allowing protein translation to oc- cur. to Interestingly, the iron-sulfur cluster containing IRP has aconitase enzymatic activity capable of cata- lyzing the interconversion of isocitrate and citratet~; it has been shown that the IRP is responsible for cyto- plasmic aconitase activity distinct from mitochondrial aconitase. J 2 63