Regulated Synthesis of Low-Molecular-Weight Antigens in Keratinocyte 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 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 -binding (SCaBP), a parv­ anoylphorbol-13-acetate (TPA), differentiation is induced albumin of the carnosus. Three mouse epider­ in a subpopulation of cel ls, and specific antigen synthesis mal 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 [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). T he supernatant con­ newborn mo use epidermis or cell s cul tured in low Ca + + medium tained sing le fi broblasts w hi ch were plated at 6 X 104/cm2. T he as described [24], using guanidine-H C I fo r the initial extraction. con centration of Ca + -I- was adjusted as described in the figure Poly(A) + RNAs were tra nslated in a ce ll -free rabbit reticul ocyte legends. Cell s were la beled w ith 80 ILC i/ ml 35S1methionine (1000 r lysa te system containing [35S]methionine (New England Nu­ C i / m M) or 250 ILC i/ ml carrier-free 32 P-labeled inorga nic phos­ clear). ph ate (both fro m N ew England Nuclea r) in l11 ethi onine- or phos­ phate- free medium without serum and w ith the appro pria te Ca + RESULT concentrations fo r ti mes indicated in the fig ure legends. Regulated Synthesis of Low-Molecular-Weight Keratino­ Extracts After labelin g, cell s were washed 3 times with phys­ cyte Antigens Extracts fro m mouse keratinocyte and dermal 35 iologic sa lin e and scraped from the dish in 20 mM Tris-H C I pH fibrobl ast cell cultures metaboli ca ll y labeled w ith r S]methionine 7.4 w ith 1 111M EDTA (TE buffe r) and 1 mM phenylmethyl­ for times indica ted in the fi gure legend were immuno precipitated sulfonylflu oride (Boehringer Mannheim). T he ce ll s were ho­ with anti-SCaBP serum. Fi g 1A shows that, in low Ca ++ cul­ mogeni zed w ith 20 strokes of a Tefl on homogenizer and subjected tures, 3 antigens with M" o f approximatel y 12K, 11 K, and 10K to 3 cycles of alternate freezing and thawing. T he extracts were w ere recognized by the antiserum. T he preimmune serum did centri fuged at 15,000 g for 1 min and the supernatant was retained not precipitate these antigens (not shown). T he antigens were not as th e cytosol fr action. After 2 more washes of the pell et in TE synthesized in the keratinocyte cultures m ai ntained in high Ca + + buffer, membranes were extracted by resuspending the pell et in TE b uffer containing 2% N onidet P-40 (NP-40). After vortexing and centrifugin g as above, the supernatant was retained as the NP-40 fr acti on. T he pell et w as w ashed twice more with T E buffer A B CD E F G H with N P-40, and then resuspended in 2% SDS w ith 10 mM di­ thio threito l. T hi s mixture w as vortexed and incubated at 37°C fo r 15 min and thell at 100°C for 2-3 min, fo ll owed by centrif­ ugation as above. T he supernatan t w as retained as the cytoskel­ eton fracti on. In corporated label in each fracti on was determined by counting fil ter-retained protein label after precipita tion in 10% trichloroaceti c acid (TCA) at 4°C in the presence of 0.5 mg/ml bovine serum albumin. Protein concentrati on was estimated by -12.3 the B ioHad protein assay kit. 12.3- _ ~ Antisera Six di ffe rent rabbit anti-SCaBP sera and purified SCaBP 1- 12.3 were a gift of D r. J ana Pavlovitch, Paris, France, and were pre­ pared as previo usly described [1 2]. Except w here otherw ise noted, anti-SCaBP was fro m rabbit no . 3. Antiserum to PV, prepared Figure 1. Synthes is of low-molecul ar-weight antigens by ocyces. as d escribed [1 7], was a gift of D r. M artin Berchtold, H o uston, Cultures of primary 111 0use keratinocytes or fib ro bl asts were labeled with Texas. Adsorbed antiserum was prepared by in cubating 50 mg [35S ll11 cthi onin c, extracted and immunopreci pi ta tcd as described in Mn­ antigen with 1 ml antiserum fo ll owed by centrifuga ti on to remove terinls nlld Methods. La/ Ie A, Kcratinocytcs cultured in low Ca ++ fo r 2 im J1'llll1 e com plexes, repea ting w ith incubati ons usin g fres h an­ days , labeled overni ght, iml11un oprecipitated with anti-SCaBP. Lnll c B, tigen 3 times, fo ll owin g each incuba ti on with centri fugation at Ke ratin ocytes cultured as in (A), then J add itional days in hi gh Ca + + mcd iul11, labeled overni ght, ex tracted and iml11un oprccipitated with ami­ 15,000 g fo r 5 min. The first incubation was at 37°C fo r 1 h , the SCa BP. Lall es C-E, Keratin ocyte prim ary cu ltures in low Ca + + Ill edi ulll second was overnight at 4°C, the last at 3rC for 1 h. After J days, labeled J h, and fractionated as dcs cri bed in Materia ls alld Methods adsorption, the antiserum no longer reacted with the adsorbing (lane C, solu ble; lane D, NP-40; lan e E, cytoskeleton). Lall es F all d G, antigen [1 5]. Primary mouse derm al fi broblasts cultured J days in hi gh Ca + + (F) or low a + + (G) l11 ed ium, labeled J h with 135Sjl1l cthionine, il11l1lu nopre­ Irnrnunoprecipitation Soluble radioactively labeled extract cipitated with anti-SCaBl). Lall es H all d 1, Kera tin ocytes in low Ca + -I- 4 con taining 5 X 105 to 3 X lO r, T e A- precipitable cpm was im­ days, Iabelcd J h, immunoprecipitated with anti-SCaBP (H) or SCaBP­ muno precipitated as described by Stanley et al [21] using 10 ILl adsorbed anti-SCaBP (1) . 456 HAWLEY-NELSON lOT AI. T H E JO UHNAL OF IN VESTIG ATIV E DE I~MA TOLOGY fo r 3 days (Fig 1 B). Hig h-mo lecular-weight labeled bands, com ­ cultures with pro lo nged incubatio n (F ig 2H ,J). Whcther this rep­ m o n ly seen in immunoprecipitations, are no t m odul ated during resents a g radual change in the phenotype o f the keratinocytes in Ca +; -induced differentiatio n and arc no t specifi c as previously culture o r selectio n o f a tl o nsynthesizing cell type remains to b e discussed 11 5]. The 1 0-12 K antigens were to tall y locali zed in the established. solu ble fraction, as shown in Fig 1 C-E, and were not synthesized Althoug h synthesis of these antigens is dramaticall y dec reased by mouse dermal fibro blasts in low Ca + + o r hig h Ca ' + m edium undcr hi g h Ca -I- -I- g rowth conditio ns, the to tal am o unt of antigen (Fi g 1 F,G). The low-mo lecul ar- weig ht antigens were distinct from in the cells m ay no t be g rea tl y altered. Immuno blo t analysis of SCaPB/PV, since antiserum to PV, w hich cross-reacts with SCaBP SDS-po lyacrylamide gels of soluble extracts from cell cultures 1' 5], fail ed to immunoprecipitate low-mo lecular-weig ht antigens detected no dramatic difference between the amount of antigens fro m keratinocytes o r dermal fibroblasts in low Ca ; + or hi g h present in lo w C a -I- -I- cultures and equivalent cultures after 3 days Ca ' + (no t shown) . The ami-SCaBP scrum w hi ch had becn ad­ in hi g h Ca ; + m cdium (Fig 3A,B). When 3-day- o ld low Ca+ + sorbed w ith purified SCaBP was undiminishcd in its ability to cultures were pulse- labeled with r35S Imethio nine and cultured in immunoprecipitate the triplet of antigcns from low C a + + ke rat­ low Ca + ., m edium fo r 3 additio nal days, the labeled antigen s inocyte extracts (Fig 1 H , I) , althoug h it no lo nger reacted w ith were diminished (Fig 3C); however, when the cell s were Switch ed SCaBP/ PV (1 51. Taken together, these findings indica te that the to hi g h C a ; + m edium for 3 additional days afte r the pulse-la­ anti-SCaBP serum recogni zes (in addition to SCaBP/ PV) 3 new ly beling, the antigens were retained (Fi g 3D). T hese results suggest synthesized po ly peptides extracted fro m the cytosol of epidermal that during terminal differentiatio n, an ti gen synthesis decreases, cell s g rown in low Ca +. medium, but that these po lypeptides but antigen half-li fe increases, o r that antigen-contai ning cell s are are not synthesized b y differentiating epiderm al cell s in hi gh Ca + + retain ed o n the dish lo nger in differentiating cultures than in m edium. proliferating cultures. Synthes is of the triplet of antigens decreased, aftcr a lag perio d The tumor promoter TPA induces epidermal differentiation of at least 24 h, when keratinocyte cultures w ere switched to even in lo w C a -I- -t- medium 125], althoug h this occurs in o nly a growth in hig h Ca ,. ; m edium (Fig 2). This lag period indicates subpopulation of cells. When low Ca -I- -t- cul tures ofkeratinocytes that the decrease of synthesis o f these antigens is not an early w ere exposed to 16 nM TPA for 18 h, synthesis of the low­ change in the differentiation program. It is notable that the de­ molecular-weig ht antigcn triplet decreased, compared with DMSO creasc in synthesis was coordinately regulated for all 3 antigens. controls. T his is sho wn in Fig 4A and B where immunoprecipi_ There was a general decrease in amount of [3SSjmethio nine in­ tation was performed o n extracts from cell s receiving a 30-min corporated per dish into soluble protein in hi g h Ca + + m edium pulse-label of r35Sjmethionine at 24 h after TPA or DMSO ex­ (not shown). The sa m e number of T C A-precipitabl e cpm was posure. H o wever, the decrease was transient after TPA exposure added to each sample fo r immunoprecipitatio n , hence the decrease o bserved is selecti ve and not due to the general decrease in protein synthesis. Synthes is of keratin proteins III and laminin 14] do es no t diminish in hig h Ca + + m edium under these conditions, w hile synthes is of other specifi c protcins has becn shown to in crease o r A B c o decrease in rcspo nse to hi g h Ca+ + m edium [4] . Syl thesis of the thrce 1 0-12K polypeptidcs also diminished graduall y in low Ca + +

ABCDEFGH JKLM

-18.4 - -12.3 14kd-

Figure 2. Decrcascd sy nthcs is of low-molecular-wcight antigc ns in hi gh Ca r ' mcdiulll. Primary keratinocytcs wcre culturcd in low Ca" .. mc­ Figure 3. Persistencc oflow-molecul ar-wcight antigcns in diffcrentiating diulll (or 3 days thcn half th e cultures wcre switched to hi gh Ca -I- + and keratinocytc cultures. A allli B, Primary keratinocytcs werc cultured in th c rest co ntinucd in low Ca .,. .. . C ulturcs wcre pu lse-Iabci cd with low Ca -1-'" medium for 3 da ys and then half the plates were switched to 1·15 Slm ethi onine for 3 h at th e times indicatcd. Solublc extracts wcrc as­ hi gh Ca+ .,. medium. After 3 days all cultures were harvcsted, and soluble saycd fo r co unts in corporatcd in to protcin , and eq ual amounts of in cor­ extra cts co ntaining 100 J-L g protcin were e1 cc trophorescd on reducin g SDS porated cpm (3 X 106) were immunoprecipitated. Lall cs A-D, Primary 15% polyac rylamide gel s and tra nsferred to nitrocellulose ci cctroph or- cul tures in low Ca' ; medium (A, I day; B, 2 days; C, 3 days; D, 4 .eti ca ll y. Thc nitrocellulose was reacted with anti-SCaB I' no. 3 and vi­ da ys). Call e E, T hrce days in low Ca ' 0\- + I day in hi gh a H - . Lillie F, suali zed with a peroxidase rea cti on as described in Milteria ls illld Meth ods Six da ys in low Ca + + . L nIIC C, Threc days in low Ca ' -,- + 3 days in (A, low Ca++ culture; B, hi gh Ca +-'- culture). C alld D, Primary kerat­ hi gh Ca ; -1- . La ll e H, Sevcn days in low Ca++ Lalle J, Three days in low inocytes were cultured in low Ca + + medium for 3 days and pulse-labeled Ca -' ; + 4 da ys in hi gh Ca ++ . Lallc) , Nine days in low Ca +". Lalli' K, for 5 h with /35SI mcthi onine. Cel ls wcre washed with sterile sa lin e and T hrcc days in low Ca ; + + 6 days in hi gh a + +. Control immunoprc­ cultured 3 additional da ys in unlabeled medium (C rece ived low a + + cipitations with normal rabbit se rum were performed 0 11 ex tracts [ro l11 3 med ium , D rece ived hi gh Ca -I- + med ium). Harvested cells were extracted and 9 days in low c,, ++ (L , M). and immunoprecipitated with anti-ScaBP serum no. 3. VO L. 87. N O.4 OCTOI3 ER 1986 REGULATED ANTIGEN S IN KERATINO C YTE ULTURES 457

ABC D orescent studies previously repo rted [13, 18], in w hich antigen was demonstrated in the basa l layer of the epidermis and in prolif­ erating keratinocytes in lo w Ca + + 111 edium. Lanes Band Care immuno precipitates using serum fro m rabbit no. 3, previously shown in Fi gs 1- 4, w hich recogni zed 3 antigens in epidermal cell s. Sera 5-8 had different precipitati o n patterns fo r low-mo­ lecul ar-weight epidermal antigens. O nl y the M,. 11 K band was C0 111111 0n to all antisera. The regulation of synthesis of in dividual antigens has onl y been studied with antiseru111 3. It is not known how th e additio nal bands seen with antisera fro 111 rabbits nos. 5-8 are rel ated to the antigens repo rted in this study.

Antigen Phosphorylation When extracts of low Ca + + cul­ -12.3 tures labeled overnight w ith 40 ,uC i/ ml of 32 P04 were immu­ no precipitated with anti-SCaB P se ru111 no . 3, onl y 1 ba nd was seen at lVf, '11 K (Fi g 6 B), correspondin g to the central band of the 135 Sjmethi onine-labcled triplet (Fig 6A). T he simulta neous ad­ di tio n o f 162 nM T PA to the culture at the time 0f32P04 la beling decreased this in corpo rati o n in to the 11 K antigen and did not stimulate J2 PO ., labeling of the other antigens (F ig 6C).

Figure 4. Decreased synthesis of low-mo lccubr- weight antigens after Presence ofmRNA for the Antigens in Newborn Epidermis and Cultured Keratinocytes Poly(A) 4 mRNA was prepared pulse exposure to TI'A. Prilllary keratin ocytes were cultured in low Ca + 4 mediul11 . and on day 2 half the cul tures were exposed to 16 nM TPA in fr0 111 newborn 111 0use epidermis o r frolll keratinocytes cultured 0. 1 % DMSO for 18 h. T he controls recei ved O. I % DMSO al one. C ul­ in low C a" ;. medium. This m RN A was translated in vitro in a tures were washed with sa lin e and maintain ed 1-2 additional da ys in low rabbit reti cul ocyte translati on system , and the reacti on products Ca 4-" mediulll. Cell s were metaboli call y bbcled for 30 min with were iml11un oprecipitated w ith anti-SCaBP no. 3 or the preim- [35S]methionine and il11l11l1nopn:cipitated with anti-SCaUP. Lll m' A , DMSO. 1l111n e serulll fr o m th at rabbit. T he res ults (F ig 7) show all 3 1 additio nal day. L illiI' 13 , TPA, I additional day. L (/II C C, D M SO. 2 antigens were represented in the I1lRNA frolll both the epiderm is addit io nal days. L nllc D, TPA, 2 additional days. 3ll d . D ISCU SS IO N and synthes is was recovered to le vels identica l to those of the In previo us reports r 13. '18,'\ 9]. the detecti on of epidermal cell D M SO control by 48 h (Fi g 4C, D ). antigens recogni zed by SCaBP antisera was m odulated by the Different Antigens Are Recognized by Different SCaBP differentiati o n statu s of the cell s. C urrent evidence suggests that Sera Extracts fro m 3-da y-old lo w Ca" + keratinocyte cultures those antigens arc not S aBP 11 4,15]. T he antigens that arc lo­ labeled fo r 3 h with 135S1methionine were immunoprccipitated calized to the epidermis arc multiple, of low molecul ar weig ht. with SCaBP antisera fr o m 6 diffe rent rabbits. The results (Fig 5) and are coordinately regulated. T he majo r antigens of this study indicate that different sets of epidermal antigens arc recognized are 10K, 11 K , and 12K. Preliminary experiments have in dica ted by the sera from different rabbits. Lane A represents the antigen that they have isoelectric points between 5-6.5 (not shown). T hese recognized by the serul11 fr o m rabbit no. 1. O nl y 1 band w as antigens arc synthesized in acti vely proliferating cultured kerat­ precipitated at M , 11 K. This is the serul11 used fo r illll11uno flu - inocytes and synthesis ceases o r is grea tl y diminished d uring ter-

A B C 0 E F G ABC

-12.3 12.3- - -

Figure 6. Phospho rylation of low-molecula r-weight antigen in cul ture. Figure 5. Iml11uno precipitatio n of different sets o flo w- mo lecular-weight Two-day-old cultures of primary keratinocytes in low Ca + + were ex­ antigens by va ri ous anti-SCaUP se ra. Keratinocytes cultured 3 days in posed to either 0. 1% DMSO or 162 nM TPA in 0.1 % D M SO simulta­ 35 32 low Ca+" were labeled 3 h w ith 1 SIm ethi onine an d soluble extracts neously with 40 ,u i/ 1111 pO. in phosphate-free mediulll fo r 18 h. Soluble were iml11uno precipi ta ted w ith anti-SCaBP se ra : L IIIIC A, rabbit no. I: extra cts were equalized fo r cpm of TCA-precipita ble activity and il11- Lanes B IIl1d C, rabbit no. 3; L allc D, rabbit no. 5; Lillie E, rabbit no. 6; ITllll10 precipitatcd with anti-SCaBP serul11. Lalle A, 135SIMethionine-la­ Lane F, rabbit n().7; L IIIIC G, rabbit no. 8. bel cd cu lture as in Fig l A. Lalle B, D MSO. LIIIIC C , TPA. 458 HAWLEY-N ELSON ET AL T H E JO UHNAL OF INVESTIGATIVE DEHMATOLOGY

ABC D protein . T he phosphorylation state does no t account for the ap­ pea rance of3 antigens w ith different apparent mo lecular weig hts, however, since all 3 were synthes ized in an in vitro translation

assay using epidermal or low Ca + -I- cultured cell mRN A. Phos­ phorylation of a 10K protein is stimulated in mouse epidermis by T PA treatment [26), but its identity is not es tablished . It is unlikely that our 11 K antigen is the same as this 10K pro tein since TPA exposure stimulated the phospho rylati o n of the 10K protein, but reduced the level of phosphorylation of o ur 11 K antigen in culture. T he identities of the epidermal antigens described in this report remain obscure. T hey m ay be cl osely related, as suggested by

their coordinate regulati o n by Ca -I- -I- and TPA, and the increased -12.3 half-lifc for all 3 in differentiating cell s. Low-molecul ar-weigh t proteins detected in the epidermis have been repo rted by o thers. E pidermal cysteine-rich pro teins of 12.5-13.5K have been de­ scribed [27-29], and o ne report suggested these may be cysteine inhibito rs [29]. Keratoli ni n, a soluble substrate for trans­ glu taminase, w hich mig rates at 6-10K o n SOS-polyacrylamide gels, is also limited to epidermis [30-32]. H owever, it is associated w ith the cornified envelope in m ore differentiated cell s w hile the Figure 7. In vitro trans lation of low-molecul ar-weight anti gens from antigens we described arc soluble . Phospholipase A 2 , w hi ch cat­ keratinocyte mRNA. Poly(A) + ml~ NA isolated from whole newborn mouse epidermis (A a/ld B) and from primary cultures of epidermal cells alyses hydrolysis at the C 2 position of phospho li pids, has a mo­ grown for 4 days in low Ca + + medium (C a/ld D), translated in vitro in lecular weight of 13.8K \33]. It is a soluble enzymc w hi ch binds a reticul ocyte lysa te system with [35S]methionine. (A) and (C) were im­ calcium and may be phosphorylated by protein kinase C in re­ munoprecipitated with anti-SCaBl) no. 3; (B) and (D) we re immuno­ sponse to TPA ex posure of epidermis in vivo \26]. While phos­ precipitated with the 'preimmun e serum from rabbit no. 3. pholipase A2 activity is detectable in m ost tiss ues, immunologic cross-reactivity between the epidermal and those of o ther ti ssues has not been determined. O ther low-molecular-weigh t minal differentiation. Two differentiation sig nals, high Ca + + me­ antigens to be considered are the ubiquitous cellular retinoic acid dium [1 ] o r TPA [25], depress synthesis of these antigens. Thus and retinol- binding proteins of molecul ar weig ht 14K \34,35], it is pro bable that m aturation, rather than Ca + + alone, is the and ly mphokines, peptide ho rmones o f 10-25K w hi ch arc syn­ modulating influencc. However, the kinetics of the modulation thesized and secreted by epidermal cells [36,37]. by the 2 signals for the synthes is of these antigens is different. T he discovery of these epidermal antigens, w hi ch arc modu­ T he depressio n of synthesis is sustained in high Ca + + medium, lated during differentiati on, provides new markers associated with in which the differentiati o n prog ram proceeds to the terminal the differentiation program. Because of their coordinate regula­ stage in the majority ofkeratinocytes. The depression is transient tion th ey may represent a related fa mily of pro teins. We have in the response to TPA exposure. Previously we have shown that preli minary evidence that the regul ation of expressio n of these only a subpopulatio n of basal keratinocytes is induced to differ­ antigens is modified in preneoplas ti c and neopl astic keratinocytes. entiate by TPA while the remainder continue to proliferate and T hus, they may be impo rtant markers fo r studies of neoplasia, repopulate the culture dish after a growth lag of 24 h [25]. T he and perhaps other epidermal diseases. Efforts are under way to transient decrease in antigen synthesis after TPA exposure is tem­ purify these antigens and to identify cDN A clones fo r their mRNAs. porall y consistent with the induction of differentiation in a sub­ Studies of the pure antigens and clones will help determine their population of antigen-synthesizin g cell s and the consequent loss functio n and role in epidermal di ffe rentiatio n. of the differentiating population. While synthesis of the antigen triplet recogni zed by anti-SCaBl) no. 3 is associated with epidermal cell proli feration, the antigens Tli e all rli ors gra refidl), ackll olllledge rli e gifts ofm/lise rn jrlJl l/ Drs. Jalla Pa ll loilircli persist in cell s in high Ca + + m edium, as shown by immunoblot tl /ld MlIrrill Berclir old mId rli e crirical I'eadillg of rllis mallllscripr mul li elpfid analysis and immunoprecipitation of prelabeled antigens after a sll,ggesriol/ s of Dr. JOIIII Sral/ley . cold chase period. In fact, it appears that antigen degradation is diminished in differentiating cul tures. In concert with these cell RE FERE N CES culture fin dings, when antiserum no. 3 was used for immuno­ flu orescence in vivo, it stain ed all livin g layers of the epidermis 1. Henni ngs 1-1 , Mi chael D, Cheng C, Stein ert P, Holbrook K, Yuspa [1 5). SH: Calcium regul ation of growth and differentiati on of mouse epidermal cell s in culture. Cell 19:245-254, 1980 Antigen persistence in differentiating cell s is not consistent w ith the immunofluorescent studies reported by Saurat et al [1 3), in 2. Yu spa SH , Haw ley-Nelson P, Koehler B, Stanle y JR: A survey of w hi ch anti-SCaBP no. 1 was shown to stain only the basal layer. transformation markers in differentiating epidermal cell li nes in cu lture. Cancer Res 40:4694-4703, 1980 Si nce antiserum no. 1 im munoprecipita tes only an 11K protein from epidermis, it must have diffe rent specificity fro m antiserum 3. Kulesz-Martin MF , Koehler B, Hennings H, Yu spa SI-I : Q uantita­ tive assay for ca rcinogen altered differentiat ion in mouse epidermal no. 3. It is possible that the 11K proteins are different or the cell s. Ca rcinogenesis 1:995- 1006, 1980 antigenic determinant for antiserum no . 1 is masked in the dif­ 4. Sta nley JR, Yu spa SI-I: Specific ep iderm al protein markers arc mod­ ferentiating cell layers in vivo. Sim il ~ rl y, the other 4 antisera ul ated during ca lcium-induced terminal differentia tion. J Cell BioI examined recognized unique sets of low-molecular-weight ke­ 96: 1809-1814, 1983 ratinocyte antigens. T he protein preparations used to inoculate 5. Hennings H, Holb rook KA : Calcium reg ulation of cell -cell contact the rabbits to produce these sera may have contained different and differentiation of epidermal cell s in culture. An ultrastructural proteins from epidermis. Alternatively, each rabbit m ay have study. Exp ell Res 143: 127- 142, 1983 rccognized different determinants on the epidermal protein in the 6. Hennings H, Stein ert P, Buxman MM: Calcium inducti on of rrans­ inoculum w hich were shared by different sets of epidermal pro­ glu taminase and the form ation of E (I'-glutamyl) lysin e cross links teins not present in the inoculum. in cultured mouse epiderm al cells. Biochem Biophys Res Com­ The 11 K antigen recognized by antiserum no. 3 is a phospho- nlLln 102:739-745, 198 1 VOL. 87. NO.4 OCTOBER 1986 REGULATED ANTIGEN S IN K ERATINOCYTE CULTURES 459

7. Watt FM , Green H: Stratificat ion and terminal differentiation of 23. Gershoni JM. Paladc GE: Electrophoretic transfer of proteins from cultured epidermal cell s. N ature 295:434-436, 1982 sodium dodecyl sul f.lte-polyacrylamide gels to a positively charged 8. Brysk MM , Snider JM: T he effe ct of the state of differentiation on membrane fi lter. Anal Biochem 124:393-405, 1982 labeling of epidermal cell surface glycoprotein s. J In vest Dermatol 24. Roop DR, Hawley-Nelson P, C heng C K. Yuspa SH: Keratin gene 78:366-370, 1982 expression in mouse epid ermis and cultured epid ermal cell s. Proc 9. O'Keefe EJ, Payne RE: Modulation of the epidermal growth facto r N atl Acad Sci USA 80:7 16-720, 1983 receptor ofhul11 an keratinocytes by calciul11 ion. J In vest Dermatol 25. Yuspa SH , Den TB, Hennings H, Li chti U: Phorbol es ter tumor 8 1:23 1-235. 1983 promoters induce epidermal activity. Biochelll 10. Watt FM. 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Takahashi M: Cystine-ri ch protein from human stratUIll 13. Saurat JH, Didicrj ea n L, Pavlovitch JH, Lao uari D, Da lsa n S: Sk in corneu m as a marker of kcratinocyte diftcrentiation (abstr) . J ca lciuJll-binding protein is localized in the of the b:lsa l In vest Dermatol 84:3 17, 1985 layer of the epid ermis. J In ves t Dermatol 76:221-223, 198 1 29. Cook WS Jr, Nakano S, Yabe K, N akagawa M. Epstein JH, Fu­ 14. Hawley-Nelson P, Yuspa SH: Tissue locali za tion of skin ca lcium­ ku yama K: Cysteine proteinase inhibitor (CPI) in chemica ll y in­ bind in g protein and antigeni c cross-reactivity with parva lbumin. duced squamous ce ll ca rcin o ma (S C) (abstr). J In vest Dermatol J Cell Bioi 99:A430, 1984 84:36 1, 1985 15. Hawley-Nelson P, Berchtold MW, Huitfcldt H, Spiegel J, Yuspa 30. Buxlllan MM, Lobitz Cj: New findings on epidermal transglutam­ SH: Skin ca lcium- binding protein is a parvalbumin of the pan­ inase substrates . C urr Probl Dermatol 10:245- 254, 1980 niculus ca rnosus. J In vest Dermatol 86: 157-162. '1986 3 1. Buxman MM, Lobitz Cj, Wuepper KD: Epid ermal transglutami­ 16. MacManus JP, Watson DC , Yaguchi M : Rat skin calcium-binding na se: identification and purification of a solu ble substrate with protein is parvalbumin. Biochem J 229:39-45, ! 985 I studies of in vitro cross linking. J Bioi C hem 255: 1200-1203, 1980 17. Berchtold MW, Celio MR, Heizmann W: Parva lbumin in non­ 32. Goldsmith LA: Human epid ermal transglutaminase. J Inves t Der­ muscle ti ss ues of the rat. J Bioi hem 259:5 189-5 196. 1984 matal 80:395-415, 1983 18. D idierj ea n L, Pavlovitch JH. Fuseni g NE, Samat JH: Ex trace llular 33. Van den Bosch H : Intracellular phospholipases A. Biochim Biophys ca lcium concentration 'regulates the expression of skin calcium­ Acta 604: 19 1-246, 1980 binding protein (SCaBP) in mouse kcratinocytc cultu res. Arch 34. C hytil F, O ng DE: C ellular retin oid bindin g proteins, T he Hetinoids, Dermatol Hes 275:202-203, 1983 vol 2. Edited by MB Sporn , Roberts AB, Goodman DS. New 19. Hawley-Nelson p, Pa vlovitch J, Yuspa SH: Regul ati on of skin ca l­ York. Academic Press , 1984. pp 89- 123 cium-binding protein synthesis in normal and malignant epider­ 35. Siegenthaler G. Sa urat JH, Morin C, Hotz R: ell ular retinol and mal cell cultures. J Inves t Dermatol 80:304-305, 1983 retinoic acid binding proteins in the epidermis and of nor­ 20. Y uspa SH, Hawley-Nelson P, Stanley JR, Hennings H: Epidermal mal . Dr J Derlnatol III :647-654. 1984 cell culture. Transplant Proc 12 (s uppll): 11 4-122, 1980 36. Luger TA, tadler BM. Katz 51. Oppenhei m JJ : Epid ermal cell (ke­ 21. Stanl ey JR, Hawley-Nelson P, Yuspa SH, Shevach EM, Katz SI: ratin ocyre) deri ved th ymocyte activa ting facto r. J Imlllunol C haracteri za ti on of antigen: a unique base­ 127 :1 493-1498, 198 1 ment membrane protein of stratified squamous epithcli;l. Cell 37. Micksche M. Colot M, Koch A, Lu gcr TA: Human epidermal cell 24:897-903, 198 1 production of a natural killer ce ll activity au gmenting factor 22. Laemmli UK: C lea va ge of structural proteins during the asscmbly (ENKAF) w hi ch is distin ct fro m ETAFIILJ, IL2, IFN (a bstr). J of the head of bacteriophage T4. Nature 227:680-685, 1970 In vcst Dermatol 84:303, 1985