Free Radical Reduction by Thioredoxin Reductase at the Surface of Normal and Vitiliginous Human Keratinocytes* Karin U. Schallreuter, M .D ., Mark R. Pittelkow, M.D., and John M. Wood, Ph.D. Departm ents of Dermatology (KUS) an d Biochemistry (JMW) , University of Minneso ta School of Medicine, Minnea polis, and · Department of Dermatology, Mayo Clinic (MRP), Roches ter, Minnesota, U.S.A. Cell cultures of human keratinocytes contain m embrane­ ulated by calcium concentrations of the cell culture m edium. associated thioredoxin reductase that is extremely active in Stratified keratinocytes are half as active in medium con­ reducin g radicals on the outer plasma membrane. This en­ taining 2 mM Ca + + compared with 0.1 mM Ca + + con­ zyme activity was confirmed by its purification from cul­ centration. (4) Product inhibition of the enzyme occurs tures of stratified human keratinocytes by affinity column with oxidized coenzyme NADP + (i. e., 87% inhibition of chro m atography. The enzym e was assayed both in vivo enzyme activity over 30 min). The enzyme is heat stable and in vitro usin g a spin-labeled quaternary ammonium at temperatures of70°C for 10 min. It is inactivated at 75°C . compound as the substrate, under saturating conditions in A comparative study of thioredoxin reductase activity on free radical substrate. Specific activities were determined stratified differentiated and undifferentiated rapidly grow­ by monitoring the sequential decrease in the amplitude of ing celI s was performed . Also, enzyme activity was quan­ the electron spin resonance signal per unit of cell protein . titated for cultured keratinocytes isolated from vitiliginous The following properties were found: (1) Cultures of adult and normal skin of the same donor. The results of this stratified cells have approximately twice the thioredoxin study, and the connection between this enzyme activity reductase activity of neonatal cells. (2) The enzyme is in­ and UV- generated free radicals are reconciled in terms of hibited by thioprotein inhibitors (i .e., parachloromecuri­ the m echanism of action and m etabolic activity of thio­ benzoate and ·dinitrochlorobenzen e). (3) The activity is reg- redoxin reductase. J Invest Dermatol 87:728-732, 1986 hioredoxin reductase and thioredoxin are ubiquitous preventing inactivation of sulfur-containing amino acids in pro­ electron-transfer systems common to a variety of or­ teins; (3) to function as electron donor for reduction of sulfate to ganisms and tissues [1,2]. In higher organisms these sulfite; (4) the reduction of protein disulfide linkages; and (5) to 2 proteins have been shown to function coordinately participate in insulin degradation [2] . in most cases, but sometimes they are found sepa­ Recently we synthesized a spin-labeled quaternary ammonium Trately in different organelles and tiss ues [3] . The widespread dis­ salt (quat) that serves as a free radical substrate for thioredoxin tribution of these 2 thioproteins, together with the observation reductase. We have shown that this enzyme acts as a free radical that they can exist separately, suggests a number of metabolic scavenger at the outer membrane surface of guinea pig and human functions. To date 5 independent functions have been described skin [4-6] and on the surface of cultured human keratinocytes for these electron-transfer systems: (1) thioredoxin/thioredoxin [7] . The enzyme can be purified from plasma membranes ofhu­ reductase as electron donors to the ribonucleotide reductases of man keratinocyte cultures by affinity column chromatography. bacteria, yeast, mammalian cell s, and plants; (2) to function as The recognition that thioredoxin reductase can reduce radicals at antioxidants in the reduction of methionine sulfoxide back to the membrane surface ofkeratinocytes documents a new function methionine, where they probabl y perform a protective role by for this fl avin-adenine dinucleotide containing thioprotein that utilizes NADPH as electron donor. The enzyme is ideally suited Manu scri pt received March 14, 1986; accep ted for publica tion June 17, for this purpose because both the thioprotein- and the fl avopro­ 1986. tein-active sites function as independent reductases [8,9]. The dual Supported by a grant from the National Institutes of Hea lth (AM 18101) function can catalyze the reduction of oxygen radicals to peroxide and by grants from th e Mayo and J. W. Kieckhefer Foundations. ions which are then reduced to water. · Part of this work was presented at the Joint International Meetin g of The spin-labeled substrate that we used to assay membrane­ the European Society for Dermatological Resea rch an d The Society for bound thioredoxin reductase has many advantages over super­ Investigative Dermatology, Inc., Geneva, Switzerland, June 22-26, 1986. oxide anion radicals . It is an excellent surfactant and reacts only Reprint requests to: Karin U. Schallreuter, M.D., Universi ty of Min­ at the outer membrane surface [4] . There it is reduced via its neso ta , P.O. Box 100, Navarre, Minnesota 55392. Abbreviations: hydroxylamine to a stable secondary amine product [5]. Hence, DNCB: dillitrochl orobenzene its reduction by the enzyme has identical stoichiometry to that EGF: epidermal growth factor expected for superoxide anion radical [5] . Confirmation that thio­ EPR: electron paramagnetic resonance spectroscopy redoxin reductase is active at the outer surface of cultured human PCMB: parachloromercuribenzoa te keratinocytes indica tes that this enzyme may function as a first quat: quatern ary am monium co mpound .line of defense against radical mediated cell damage. 0022-202X/86/S03.50 Copyright © 1986 by The Society for Investi ga tive Dermatology, In c. 728 VOL. 87, N O.6 DEC EMBER 1986 T HIOREDO X IN REDUCTASE IN HUMAN KERATINO CYTES 729 W e studied the in vivo and in vitro properties of this membrane­ bound free radical reductase in ce ll cultures from neonatal and adult human keratinocytes of healthy donors. In addition we examined thioredoxin reductase activity of cultured keratinocytes derived from vitiliginous and normal pigmented skin of 1 donor. Ker a tinocyte cultures have been grown in serum-free medium containing different Ca + + conce ntrations in the presence and ab­ sence of growth factors. MATERIALS AND METHODS Chemicals Aceta m ido-2, 2, 6, 6-tetrameth yl pi perid ine-N-oxyl benzyl dimethyl ammonium bromide (spin labeled quat) was syn­ thesized by reacting 4-bromoacetamido-2,2,6,6-tetramethylpi­ peridine-~-oxyl with dimethyl benzylamine [4,5]. All other re­ agents used in this study were obtai ne·d from Sigma C hemica l Co. (St. Louis, Missouri) . Fetal bovine serum was purchased from K . C. Biologicals (Lenexa, Kansas). Keratinocyte Cell Cultures N eonatal and adult keratinocytes wer e isolated from fores kin and breast skin, respectively, ac­ cording to procedures previously described in deta il [10] . Briefly, specimens of skin were trypsinized overnight at 4°C and the epidermis was separated from dermis. Primary cultures of ke­ ratinocytes were initiated by seeding disaggregated and washed n cells at approximately 5 X 103 cells/cm 2 into 75-cm2 tissue culture flasks. 60 Serum-free cultures were established utilizin g MCDB 153 basa l medium containing supplements and growth factors, epidermal growth factor (EGF) 10 ng/ml, insulin 5 J.L g/ml, and bovine pi­ 50 tuitary extract 25 J.L g/ml. Medium ca lcium conce ntration was 0.1 mM. Cultures routinely displayed population doubling times of appr oximately 24 h and maintained a monola yer, undiffe rentiated morphology. In the presence of growth factor-containing me­ 40 w dium, increased ca lcium concentration of 2 mM ca used cells to 0 cluster more tightly within coloni es, but the monolayer, undif­ :::> ferentiated morphology persisted. However, removal of growth I- 30 -.J factors induced keratinocyte cultures to stratify and differentiate 0... as previously reported [10]. Confluent cultures of keratinocytes ~ were obtained by the addition of selected amino acids to the <! medium to prevent subconfluent growth arrest [11] . 20 N onnal and vitiliginous skin was obtained from the forearm of a 52-year-old man with longstanding vitiligo (classified as gen­ eralized large macular type) . Normal and vitiliginous sites se­ 10 lected for epidermal harves t were readily distinguishable by visual exan1ination. Epidermal blister roofs were obtained using a suc­ tion blister vacuum device [12] , Cell s were disaggregated from epidermal sheets by brief trypsiniza tion, washed, seeded into fl asks, 40 50 and cultured utilizing the sa me techniques that were employed for propagating normal neonatal and adult keratinocytes. b TIME (min) In Vitro Human Keratinocyte Assay Cells from confluent undifferentiated and differentiated keratinocytes were harves ted Figure 1. n, Reduction of spin-labeled quat (50 JLm o l) by pure thiore­ with a rubber po li ce man and the initial velocity of the nitroxide doxin reductase from E. coli . The reaction was started by the addition of 50 JLm ol NADPH and followed by measuring the decrease in amplitude radical reduction was measured in quartz cell s usin g a Varian E4 of the nitroxide radical signal (line width 11. 0 gauss) over time. b, T he electron paramagneti c resonance spectroscopy (EPR) at 25°C after rates for the reduction of spin- labeled quat (50 JLmol) by thioredoxin placing the cells prior to the assay in spin-labeled quat under reductase (0 .1 mg/ll1l) (0-0) and by thioredoxin reductase (0 . 1 mg/ ll1l) satu ratin g substrate conditions. Detailed EPR parameters for this plus thioredoxin (3.0 Il1 g/ ll1l) from E. coli (e - e ). T hioredoxin acts as spin label were previously reported [4] . Following the enzyme an electron acceptor competing with spin-labeled quat. Reaction volume assay, cells were completely di gested in trypsin (1 % w/v) at pH as described in " Materi als and Methods." Reactions were started by add­ ing 50 JLll1 0l of NADPH.