Regulated Synthesis of Low-Molecular-Weight Antigens in Keratinocyte Cell Cultures*
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Regulated Synthesis of Low-Molecular-Weight Antigens in Keratinocyte Cell Cultures* Pamela Hawley-Nelson, Ph.D., Dennis R. Roop, Ph.D., Christina K. Cheng, M.S., and Stuart H . Yuspa, M .D. Laboratory of Cellul ar Ca rcin ogcnes is and T umor Promotion (PH-N, DRI"l., C KC, SHY) , National Can cer In stitute, Beth esda, Maryland, and Department of Biology (PH-N), Catholic Uni versity of America , Washin gton, D.C., U.S. A. Proteins from mouse epidermis cytosol extracts react on cell s. When proliferating keratinocytes in low Ca + + m.e iml11unobl ots with a polyclonal rabbit antiserum raised dium are exposed to the tumor promoter 12-0-tetradec against rat skin calcium-binding protein (SCaBP), a parv anoylphorbol-13-acetate (TPA), differentiation is induced albumin of the panniculus carnosus. Three mouse epider in a subpopulation of cel ls, and specific antigen synthesis mal proteins with molecular weights between 10-12K, which is transiently inhibited. The inhibition correlates with the are di stinct from SCaBP, are recogni zed by the antiserum. time when many cell s are differentiating in response to T he synthesis of these protein s in keratinocyte culture is TP A. When proliferating keratinocytes are pulse-labeled m odulated by Ca + +, as is the differentiation o f the kerati with 32PO", the 11K antigen is phosphorylated and the nocytes . Proliferating m ouse keratinocytes in medium phosphorylation is not enhanced by TPA exposure. All 3 conta ining 0.07 111M Ca + + (l ow Ca + + ) undergo terminal :lIltigens are synthesized in a reticul ocyte lysate preparation differentiati on w hen the Ca + + conce ntration is elevated to with added newborn m ouse epidermis messenger RNA or 1. 8 mM (hi gh Ca + +). Synthesis of the 3 antigens ca n be mRNA frol11 keratinocytes cultured in low Ca + + medium. demonstrated when soluble extra cts of keratinocytes la T hus, these antigens are likel y to represent unique pro teins beled w ith r35 S]methionine in low Ca + + medium are im rather than processed or degraded ones. T he coordinately muno precipitated w ith anti-SCaEP serum. T hese antigens regul ated expression of these antigens associated with the are not synthes ized in cultures of dermal fibroblasts. When differentiation state of the keratinocy tes suggests that these keratinocytes are switched to hi gh Ca + + medium, s'yn proteins are impo rtant in keratinocyte proliferation and thesis of these antigens is grea tly diminished over the course differentiati on. J Inves t Derlllato f 87:454-459, 1986 of 48-72 h. However, the antigens persist in differentiatin g edium ionic calcium concentration has been shown tratio n , o nl y minor alteratio ns of the genera l pattern of protein to m odulate diffe rentiation in l1I o use keratino synthesis have been demonstrated 11] . H owever, synthesis and cyte cell cultures rn. Growth under culture con in tracellular locali za tion o f specifi c m arkers are m odulated during ditions w ith reduced Ca + + (less than 0. 1 mM) Ca "'+-induced differentiati o n 14 - 11]. selects fo r basal cell s w hich ca n be induced to A skin ca lcium-binding protein (SCaEP) purified fro l11 whole Mdiffen:ntiate by raisin g the Ca "' " concentratio n to m o re than 0.1 adult rat skin 1121 was repo rted to be locali zed by indirect il11- mM . Som e m ali gnantl y transfo rmed [2] o r carcin ogen-treated [3] munoflu orescence to the basa l cell s of the epidermis [1 3]. H ow kcratin ocyte cell lines no lo nger respo nd to this signal. D espite ever, it has recently been dem o nstrated that SCaEP is immu the m arked bio logic effects of changes in m edium Ca + + concen- no logicall y [14, 15] and structurall y [1 6] indisting uishable from the protein parvalbumin (PV), and is restricted within the skin to the panniculus carnosus [14,15, 17]. T he reactivity of the anti Manu script rcccivcd Novcmber 19, 1985 ; ~ ccc pt e d fo r publi c~ ti o n Feb SCaEP serum with epidermis is explain ed because the anti-SCaBP ru ary 12, 1986. serum also reacts with cytoplasmic epidermal antigens [1 3,15] • A p r e l imi n ~ry report of this work h ~ s becn published U In vest Der that are distinct fro l11 SCaBP/ PV r1 5]. m~t o l 80:304-305, 1(83). Keratinocytes cultured in m edium containing 0.07 mM Ca + + This work w~s performed in pa rtial ful fi ll ment of the req uirements fo r (low Ca '" +) have cytoplasmic antigens recogni zed by anti-SCaBP th c Doctor of Philosophy degree of th e Biology D e p~rtm e nt , Ca th oli c University of Am eri ca. o n immuno bl ots of sodium dodecyl sulfate (SDS)-polyacrylamide Reprint requests to: Pamela Haw ley-Nelso n, Ph. D., National Cance r gel s of cultured cell extracts 115], and by indirect immuno flu o In sti tute, Buildin g 37, Room 3A23, Nati onal In stitutcs of Hea lth , llc rescence of whole cell s [1 8]. Preliminary studies indica ted that t h esd~, Maryland 20892. these antigens were synthesized in basal cell s in low Ca + +, but Abbreviations: when these cultures were changed to m edium containing 1.8 m M hi gh Ca ++ : mcd ium containing > 0. 1 mM C~ ++ Ca + + (high Ca + +), the level of immuno precipitable m aterial low Ca· +: mediulll co ntai ning < 0. 1 mM Ca'" + diminished 11 9]. T he number of cell s labeled by immunofluores NP-40: Nonidit 1'-40 cent staining is also reduced in hi gh Ca + ... 11 8]. However, when PV: parv~ l bum in S aB P: ski n calcium-binding protein a different fixatio n protocol was employed [1 5], antigen was de SDS: sodium dodecyl sul fate m o nstrable in all li ving layers of epidermis. It is the purpose of TCA: tri chl oroacetic acid this present work to clarify the distributio n and regulation of TE: 20 111 M Tris, pH 7.4; I mM EDTA synthesis of the epiderm al antigens recognized by anti-SCaBP T PA : 1 2-0- t e tr adcca n oy l phorb o l - 1 3-~ce tat e serulll . 0022-202X/86/$03. 50 Copyri ght © 1986 by T he Society for In ves ti ga tive Dermatology, In c. 454 VOL. 87. N O.4 OCTOBm 1986 REG U L ATED ANTIGEN S IN K ERATINOCYTE C U LTU RES 455 MATERIALS AND METHO DS antiserum per sa mple. In each experiment, eq ual cpm of labeled TCA-precipitabl e protein were added to each reaction m ixture. Cell Cultures C ultures of primary newborn BALB/c mo use epiderm al cells were prepared as described [20] from trypsin Gel Electrophoresis Electrophoresis was perfo rmed in the separated epidermal sheets. C ultures were plated and maintained presence of 0. 1 % SDS and 2-merca ptoethanol (5.5% in the sa m ple in m edium consistin g of Eagle's ME M witho ut CaCI2 (MA Bio buffe r), in 15% discontinuous polyacrylamide gels according to products), containing 8% fetal ca lf serum (Reheis, treated w ith Laemmli (22). Molecular weight standards were prestain ed un B io Had C helex to remove Ca + +), 1 % Antibioti c-Antimycoti c labeled protei n markers from Bethesda Research Laboratories, (G LBCO), and supplemented with CaCI2 to 0.07 mM Ca -I- + as including lysozyme (14.3K) and /3-l actoglobulin (1 8.4K), and 14 det e rmined by atomi c absorbance spectroscopy 11] (l ow Ca -I- + [methyl- C ]methylated protein s from N ew England Nuclear, medium). C ulture medium was changed every 2 days. After 3- 6 including cytochrome C (12.3K) and lactoglobulin A (18.4K). days, cultures were exposed to the sa me medium containing 1.8 Gels were subjected to flu orography by using EN "HANCE (N ew mM C a " (hi gh Ca + -I- medium) or to low Ca -I- + medium con England Nuclea r) fo ll owed by exposure to x-ray fiJm (Kodak tain ing 16-162 nM 12- 0 -tetradecanoylphorbol-1 3-acetate (TPA) X AR-5) at -70°C. Where indica ted, instead of flu orography, (Ch emica ls fo r Carcin ogenes is Research, Eden Prairie, Minne protein bands were electrophoreticall y transferred to nitrocell u sota) in 0. 1 % DM SO (Pierce). Dermal fib ro blas t cultures were lose (Schleicher and Schuell , BA 83) in 25 mM T ri s pH 8.3, 192 prep ared fro m newborn mo use dermises separated by trypsi n m M glycine, 20% methanol fo r 14 h at 60 V. Some nitrocellulose flo tation as described above. T he derlllises were digested in 0.35% strips were stained with 0. 1 % amido black [23]. Others were collagenase (type I, Worthington), in medium M-1 99 (NIH media reacted w ith anti-SCaBP at a 1: 100 dilution. Positive bands were unit) w itho ut serum , w ith stirring fo r 30 min at 37°C. T he digest visuali zed using the BioRad Immun-blot peroxidase assay kit as was filtered through nylon gauze, diluted w ith medium contain described (15) . ing 8% feta l ca lf se rum, and centrifuged at 1000 rpm for 5 min. Messenger RNA Preparation and In Vitro T rans T h e pell et was res uspended in low Ca -1- .' medium and was twice lation Poly(A) + RN A was prepared from trypsin-separated cen t ri fuged li ghtly (400 rpm for 3 min).