Skin Calcium-Binding Protein Is a Parvalbumin of the Carnosus*

Pam ela Hawley-Nelson, M.S. , M artin W. Berchtold, Ph.D., H em·ik Huitfeldt, M .D., Jack Spi egel , Ph.D., and Stuart H . Yusp a, M.D. Laboraco ry of Cellular Ca rcinogenes is and T umor Promotion, Nati onal Cancer Institute (PI-1 -N , HI-I , SHY), lkthcsda, Ma rybnd; Depart ment of Cell Biology. Baylor College of Med icine (MWB) , Houston, Texas; and Department of Biology. Catholi c University of Ameri ca (PI-1 -N , JS) , Was hington, D.C. , U.S.A.

Skin calcium-binding protein (SCaBP) is a ca lcium binding that the l\1,. 13,000 PV/SCaBP cross-reacting antigen was protein purified from w hole rat skin. It has a molecul ar res tricted to the hypodermal tiss ue removed by scrapin g. weig ht o f approx11nately 12,000 daltons but migrates at l\1,. Immunoflu orescent stain ing of Bouin-fixed skin sections 13,000 on sodium dodecyl sul fa te (S D S)-polyacrylamJde w ith these antisera confirmed the locali za ti on ofPV /SCaBP gels. On nitrocellulose blots of SDS-polyacrylamide gels, to the panniculus ca rnosus, a h ypodermal m uscle layer. 6 different antisera to SC aBP reacted equally wel l with N ewborn mouse skin does no t conta in this antigen. Ad­ SCaBP and parvalbumin (PV), an 11 ,500-dalton calcium­ ditional polypeptides of M ,. 10, 500 and 12,000 on SDS gel s bin ding pro tein purifi ed from rat skeletal muscle, which of extracts from the of newborn and adult rats also migrates at M ,. 13, 000 on SDS-polyacrylamide gels. and n'li ce were found to be immunoreactive with anti­ Rabbit antiserum to muscl e PV also recognized both PV SCaBP serum. T hese polypeptides were not recognized by and SCaBP, and either protein absorbed specific antibodies the PV antise rum, and the reacti vity o f anti-SCaBP fo r against either antigen fro m bo th types of antise ra. Soluble these antigens was not absorbed by purified PV o r SCaBP. protein extracts fr01n whole adult rat and m ouse skm con­ O ur results indica te that SCaBP is antigeni ca ll y indistin­ tain ed a M ,. 13,000 protein w hi ch w as recogni zed on ni­ gui shable from PV and is locali zed in the ad ult rodent trocellulose bl o ts of SDS gels by both antisera . Blots of panniculus ca rnosus, and th at antisera to SCaBP are poly­ extracts from epidermis, , whole skin, and skin scraped specifi c, recognizin g epidermal proteins in addition to on the dermal side to remove hypodermal tissue revealed SCaBP/PV. ) ln lJcst Dcnnatol 86:157-162, 1986

alcium io n concentrati on has been shown to contro l T he present study was undertaken to com pa re SCaBP and PV, the differentiati o n of m o use epiderm al and to determ ine their tiss ue distributio n by immunohistochem­ 111 culture [1] and this regul a n o n can be altered m istry and immuno bl o t analysis, the latter technique having the ca rcinogen-treated o r m ali gnantly transformed ker­ adva ntage o f providing in fo rmation o n m o lecul ar weig ht and atinocytes [2,3]. It w as of great interest, therefo re, antigenicity simultaneously. We presen t da ta dem o nstra tin g that Cwhen a skin ca lcium-binding p rotein (SCal3 P) was identified anci PV and SCaBP are antigen ica ll y indistinguishable and that the purified fro m w ho le adult rat skin [4,5], and an tisera to this pro­ PV / SCaB P antigen is locali zed in the panniculus ca rnosus of adult tein were raised in rabbits. The locali za ti on of SC:~ BP anngen, roden t skin. Additio nal antigens of unknown nature, with M,. determined b y indirect immuno flu o rescence of frozen skin sec­ 10, 500-12,000 an sodium dodecyl sul fate (SDS)-polyacrylam ide tions, was rep orted to be in basal cells of the epidermis [6]. Its gels, are recognized b y an ti-SCaBP serum and are localized in molecular weight (1 2,000 daltons), isoe.le ctric po in t (5.0), and the epidermis of newbo rn as well as adul t rodent skin. T hese amino acid composition were similar to ano ther calcium binding antigens m ay account fo r previo us reports [6] of SCJBP in epi­ protein, p arvalbumin (PV) IS]. PV was known to be a maj o r dermis, and of regulated syn thesis of low-m olecular weight an­ compo n ent o f fast-tw itch skeletal muscle (7] and has been shown tigens in epidermal ce ll cultures r<.>. l OJ. to be present in relative abundance in adult rat skin [8], locahzed to the h y po dermal muscl e, the panniculus ca rnosus. MATERIALS AN D M ETH ODS Proteins Purified adult rat skin SCal3 J> was a gift of D r. J ana Pavlovitch, Paris, France, and was p repared as descri bed previ­ Manuscript received April 4, 1985; acce pted for publica ti on Au gust 26, o usly [4,5]. Purified adult rat muscle PV an d bovin e b rain c:t l­ 1985. This work was perfo rm ed in pa rtial ful fi ll me nt of the req uirements fo r m odulin were from Calbioch em. Adul t Sprague-Dawlcy rat and the Doctor of Philosophy deg ree of the Biology Departmcm, Ca tholic B ALB/ c m o use skin was fractionated by the procedure of Ka­ University of Ameri ca (PH-N). MWB is supported by a Swiss Nati onal wamura ct al [11] . Shaved skin was treated w ith N air and then Foundati on Fellowship for Youn g Adva nced Scicmis ts (83.0 11 .082). rem oved fro m the anim al. T he dermal side was scr:t ped vigor­ *A preliminary report of this work has been published (] Cell Bioi ously w ith a scalpel to remove the hypodermal materia l; the scraped 99: A430, 1984). skin was fl oated on 1% trypsin in sa line for 1 h at 37°C. T he Reprint reques ts to: Pa mela Hawley-Nelson, M.S., Natiomtl Ca nce r epiderm is was rem oved fro m the dcrm is by sera ping on the ep­ Institute, Building 37, Room 3A23, Bethes da, Marylan d 20892. Abbreviati ons: idermal side w ith a scalpel. N ewbo rn m o use skin was no r scraped PV : parva lbumin but was fl oated on 0. 25% try psin overnig ht at 4°C, and the ep­ SCaB P: sk in ca lcium-bin di ng protein idermis and dermis were separated w ith fo rceps. Cytosol extracts SDS: sodiu m dodecyl sul fa te were prepared fro m full-thickness skill, skin fro m w hi ch the hy-

0022-202X/86/$03.50 Copyri ght © 1986 by T he Society for In vestiga ti ve De rmatology, Inc.

157 158 HAWLEY-N ELSON ET AL THE JOUHNAL OF INVESTIGATIVE DERMATOLOGY podermal material was removed, the hypodermal material,_epi ­ A B C 0 E F G H s p PSPSPS p s p s s p c p dermis, dermis, and leg muscle by ho m ogem zmg the ti ssue 111 20 c mM T ri s, pH 7.4, w ith 1 mM EDTA and 1 mM ph.enylmethyl­ sulfonyl fluoride, freezi ng and thawing 3 times and centnfugmg

at 100,000 g for 30 min, retaining the supernatant. Cell cultures - 25kd of primary newborn mouse epiderm al cells were prepared as de­ -25kd- scribed J1 2]. After 3 days of cu lture in 0.07 mM calcium medium,

cell s were scraped from the dish and homogenized in the sa me - 13kd- - 13kd buffer as described for tiss ue fractions. All cytosol preparations were as sa yed for protein concentrati on by the BioRad protein assay kit. Antisera Six different unfracti o nated rabbit anti-SCal3P sera were a gift of Dr. Jana Pavlovitch, Paris, France, and were pre­ pared as previously described J4 ]. Except w here otherw ise noted, Figure 1. Parvalbumin and SCa BP cross-rea ct antigenicall y. One mi­ anti-SCaBP was from rabbit no. 3. Unfractionatcd antiserum to crogram each of purif1ed calcium-binding proteins parvalbumm (P), SC~P (5), and ca lm odulin (C) we re el ectrophoresed on reducmg SDS 15 Yo PV as well as anti-PV lgG purified from the antiserum on a PV polyac rylamide gels and transferred (blotted) to nitrocellulose. Pa11el ,4 affi nity column were prepared as described [8]. Affinity-purified was stai ned with amido bla ck. SCal31' and PV C01111 gratc at M, 13,000. anti-PV lgG was reconstituted and used at 2.2 p.g/m l_ (final di­ Pa nel s B- 1-1 we re rea cted with eq ua l dilu tions of antise ra and visualized lution). In the undiluted anti-PV serum the concentratiOn of this wi th a peroxidase rea ction as described in Mnrc ria ls and Meth ods. B =an~­ specific lgG was 1.6 m g/ml. Absorbed antisera were prepared by SCaBP, C=anti -SCal3P wh ich had been absorb ed w1t h PV D=ann­ in cubatin g 50 p.g antigen w ith 1 ml antiserum followed by cen­ SCaBP whi ch had been absorbed with SCaBP. E=anti -PV, F=anti-PV trifugatio n at 15,000 g to remove immune complexes, repeatmg which had been abso rbed with PV , G=anti-SCaBP, 1-/ = anti-PV. Thr the in cubations 3 times: firs t at 3rC fo r 1 h, then overnight at M,. 25,000 band seen in this and subse qu ent figures probably rep resents aggrega tes or proteins dimcrs. 4°C, then again at 37°C for l h. Electrophoresis, Blotting, and lmmunostaining Elec­ trophoresis was performed in the presence of 0. 1% SDS and 2- of the sera to PV or SCaBP. After absorption by either protein, mercaptoethanol (5 .5% in sample buffer), in1 5% polyacrylamide reactivity toward both antigens was reduced (F ig 1C,D,F).These gels according to La emmli [1 3]. Protein bands were electro­ antisera were not recognizin g the w ho le class of calclllm bmdmg phoretica ll y transferred (blotted) to nitrocellulose (from Schleicher proteins, as both failed to react w ith purified ca lmodulin (Fig and Schuell , BA83) in 25 mM Tris (pH 8.3), 192 mM glycme, 1C, H ), and also fai led to react w ith PV from rabbit (not shown). 20% methanol for 14 hat 60 V. Where indicated, nitrocellulose Antisera to SCaBP produced in 6 different rabbits all reacted strips were stain ed for 1 min in 0.1 o;,, amide bl ack in 25% iso­ equall y well w ith SCaB!' and PV (F ig 2) . Affinity-purified anti­ propanol and 10% acetic acid [1 4]. Nitrocellulose strips we~e serum to PV also reacted w ith both PV and SCaBP. Complete reacted with the unfracti onated antisera at a standard 1:100 di­ cross-reactivity was also seen with purified PV and SCaBP spm­ lution or anti-PV lgG at 2.2 p.g/ ml. Positive bands were visualized ted directl y onto nitrocellulose witho ut denaturation by exposure using the BioRad "lmmun-blot peroxidase assay kit. to SDS (not shown). Immunohistochemistry Fu ll-thi ckness adult rat skin was fixed Immunohistochemical Localization of PV /SCaBP Anti­ in Bouin 's fix ative (7 1% saturated aqueous pi cri c acid, 24% form­ gen Adult rat skin contains a muscular component, the pan­ aldehyde, 5% acetic acid) as described for visuali zation of PV J7 J. niculus ca rnosus, associated with the underside of the dermiS. Fig Tissue was embedded in paraffin, sectioned , mounted on slides, 3 shows a secti o n of w hole adult rat skin including the panniculu and deparaffinized J7 ]. Immuno flu orescent staining was per­ ca rnosus (labeled H for its hypodermal location) and a secti on of formed as described [1 5], except that an indirect technique was adult rat skin w hi ch had been scraped with a scalpel o n the dermal appli ed. Briefly, sections were in cubated w ith un fractionated anti­ side to remove the h ypodermal material. Fu ll -thickness adult rat sera di lu ted 1:100 or anti-PV lgG at 2.2 p.g/m l. After washm g, sections were exposed to fluorescein-conjugated swin e antirabbit lg (Dakopatts) at 1:40 dilution , for 30 min at room temperature. All di lu tions were made in phosphate-butTered sa lin e w ith 12.5% A B c 0 E F G H bovine serum albumin and 0.01% thimerosal. Washed secti o ns PS PS PS PS PS PS PS PS were mo unted and examined on a Niko n Labophot microscope w ith an epifluorescent attachment (H MX-1-l BO 100 W Lamp­ house) with a B2 filter bl ock w ith 460-484 interference excitation filter and 515-545 barrier fi lte r. Ektachrome ASA 400 dayli ght fi lm was used. - - 25kd

RESULTS Immunochemical Cross-Reactivity of PV and SCaBP Purified preparations of PV and SCaBP were electrophoresed in SDS polyacrylamide gels, transferred to nitrocellulose, and stained w ith amide black. Both proteins yielded identical bands at M,. 13,000 (Fig 1/l). Faint bands at M, 25,000 ca n be seen in these preparations, possibl y representing dimers of the M, 13,000 pro­ teins. When identica l ni trocellulose blots of electrophoresed PV Figure 2. Anti-l'V and 6 different anti-SCaBP se ra rea ct eq uall y well with parvalbumin and SCa i3P . Purified proteins were ckctrophoresN or SCaBP were exposed to an tise ra to PV or SCaBP and visuali zed and blotted as descri bed for Fig I. Proteins arc pa rva lbumin (P) and SCaBP usi ng a peroxidase-conjugated second antibody, both proteins (5). Pa11els A-1-1 we re reacted wi th antisera as in Fig I: A = an ti-P\' were recognized equall y well by both antisera (Fig 1 B, E). N o rmal serum , 13 = aff111i ty-pu ri ficd anti- I' V lgG. C = anti-SCaJ31> from rabbit rabbit serum did not react w ith PV /SCaB P (not shown). T he no . 2, D = anti-SCaBP no. 3, E = anti-SCaBP no. 5, F = an ti-SCaBP specific antibodies in these antisera were absorbed by exposure no. 6, C = anti-SCaBP no. 7, 1-1 = anti-SCaB P no. 8. VOL. 86. NO. 2 FEI3 n UA RY 198(, S C :~li P IS A I' AHVALIJUMIN OF THE PANNICULUS CA nNOSUS 159

A. B.

Figure 3. Histologic secti ons o f ad ulr rar skin fra cti o ns. Pa11 el A is full-thickness skin. fixed in Bouin' fi xa tive and stained w ith hem atoxylin and eosin . N o re presence of (labeled /-1 due ro irs hypodermal locati on). Pa11 el B is ad ul t rat skin afrer scraping of the dnmal side w ith a scalpel, fixing and staining as in A. Note absence of the panniculus ca rnosus, and presence onl y of dermis (0), epidermis (E), and a thin b ycr o f far fro m rhe hypodermis.

skin , fixed in Bo uin 's fi xa ti ve, was secti oned, mo unted o n slides, stai ned o nl y in extracts fr o m w hole skin , hypodermal material, and reacted w ith affinity-purified :mti-PV lgG diluted to 2.2 J.L gl ml and adult leg muscle. Extracts from skin lacking the hypodermal (Fig 4A, B) . T he staining pattern of the panniculus ca rn osus (Fig material, as well as trypsin-separated adult rat epidermis and der­ 4B) is simil ar to that previo usly repo rted fo r skeletal muscle [7). mis were negative for reactivity with affmity-purified anti­ No staining is seen in epid ermis (F ig 4A). Secti o ns stain ed w ith PV IgG. Lanes containing cytosol preparations often exhibited an ti-SCaBP serum di lu ted I: I 00 showed a similar pJttern of stain­ stained bands ofhi g h mo lecular weig ht, possibly due to nonspecific ing in the panniculus carnosus (Fi g 40), w hi ch was diminished s ta ining . by absorptio n w ith SCaBP (F ig 4F). Staining of the li ving layers of the epidermis is seen w ith both anti-SCaBP and absorbed anti­ Localization of Additional Antigens Recognized by SCaBP SCaBP (Fig 4C, E). Normal co ntro l rabbit serum diluted 1: I 00 Antiserum Nitrocellulose strips similar to those used in Fi g 5 shows no staining (Fig 4C, H). were blotted from gels of cytosol extra cts from adult rat skin fractions o r muscle and were reacted with antiserum to SCaBP Tissue Localization ofPV /SCaBP Antigen Cytosol extracts (Fig 6). T his antiserum recogni zed the M, 13,000 band extracted were prepared fr o m whole ad ult rat skin , adult rat skin scraped fr o m w hole skin, hypodermal material , and muscle, as was ob­ to remove hypodermal mJte ria l, the hypodermal material. der­ served with anti-PV lgG. In additi o n, this antise rum recognized mis, epidermis, o r leg muscle. Equal amo un ts of th ese cytosol a .M, 12,000 antigen which was present in cytosol extracts of w hole protein preparati o ns were electrophoresed and transferred to ni­ skin, scraped skin, and epidermis. It was not apparent in extra cts trocellulose alo ng w ith standards of purified PV. Fi g 5 shows the of hy podermal material, muscle, o r dermis. Adult mouse skin distribution of the M , 13,000 cytosol antigen in adult rat skin fract io ns reacted w ith PV and SCaBP antisera in a pattern similar fracti o ns recognized by affinity-purified anti-PV lgG. In the low­ to th at of t·a t, althoug h the adul t mo use epidermis stained to a molecul ar weig ht reg io n o nl y a M, 13,000 band stained, and it Jesser extent with anri-SCaBP (not sho wn).

Figure 4. PV /SCaBP cross-reacting anti­ gen is loca li zed in the pa nniculus ca(nosus. Bo uin-fixed adult rat skin secti ons were pre­ pared and dcparaffinized as descri bed in Ma­ Terials mul l'vlerh ods. Sections were reacted with an tisera as fo ll ows: A atr d B = affi nity-pu­ rified anri- PV lgG, C ar rd D = anri-SCai3P, E arrd f = anti-SCaBP absorbed with SCaBP. G a11 d /-1 = no rmal rabbit serum controL All sera were diluted 1: 100. Anti­ PV lgG was used at 2.2 J.L gl ml. Pairs o f fi g ures were present in the sa n1 l' tissue sec­ ti on but were pho tographed separately (i.e., A a11d B, C a11d D, etc.) Photographs were all at the sa me magni ficatio n (ori gin al mag­ nifi ca ti on 25 x ). E pidermis and dermis w ere photographed in A , C, E, and G w hi le pan­ niculus ca rnosus was pho tog raphed in B, D, F, and /-/. Exposure times were 8 s for A, B, G, and H and 4 s for C , D, E, :md F. T HE JOURNAL O F INVESTIGATI VE D ERMATOLOGY 160 H A WLEY-N ELSON ET AL

WS SS H M E 0 A B c D E WS M P D E WS M P

Figure 5. PV in adult rat skin is limited to the hypodermal material. Figure 7. Newborn mouse skin has epidermal antigens at M, 10,500-12:000 Equal aliquots of 100 J.L g cytosol protein or 1 J.L g purified PV were el ec­ and lacks the PV /SCaBP M, 13,000 antigen. Experimental cond1t1ons trophoresed and blotted as in Fi g 1. Strips were reacted with affinit y­ were as desc ri bed in Fig 5. Cytosol extracts were pre pared from dennis purifi.ed anti-PV IgG di luted to 2.2 J.Lg/ml , and visuali zed w ith peroxid ase. (D) and epidermis (E) of newborn (0-3 day) mouse skin after fl otation Tissue fractions were prepared as described in Ma teri als a11d Me tli ods. Lanes on trypsin . Skins were not scraped to remove hypodermal material. Ex­ were as foll ows: WS = whole adult rat skin, SS = adult rat skin scraped tract of whole newborn mouse skin ( WS ) w as also included. Adult mouse on the dermal side with a scalpel to remove the hypodermal materi al, H muscle (M) extracts and purified rat parvalbumin (P) were included as = hypodermal materi al, M = adult rat leg muscl e, P = purified par­ controls. Pmtel A = anti-PV se rum; Panel B = anti-SCaBP serum; Pauel va lbumin, E = epidermis removed by scraping the epidermal side of C = anti-SCaBP serum w hich had been absorbed with SCaBP. adult rat skin aft er fl otati on on trypsin, D = dermis fro m sc raped, fl oated skin. Some higher- molecular weight proteins stain faintly in lanes con- • rain ing cytosol ex tracts, but this staining may be due to hi gh amounts antigens extracted from newborn skin , and these antigens are (1 00 J.Lg) of protein per lane staining nonspecifica ll y. enriched in epidermis (Fig 7B) . It also recognizes the M, 13,000 antigen in adult m ouse muscle cytosol. When anti-SCaBP is ab­ sorbed with purified SCaBP (Fig 7C) , the reactivity with PV and Absence ofSCaBP and Presence ofLow-Molecular Weight muscle M, 13,000 antigen is reduced, but the reactivity to the Antigens in Newborn Skin and Cultured Newborn lower-molecular weight antigens is not affected. These antigens Keratinocytes Newborn (l ess than 3 days) mouse epidermal are, therefore, distinct from the PV /SCaBP antigen in molecular cell s have been shown to contain antigens recognized by anti­ weight, tissue distribution, immunoreactivity, and develop­ SCaBP sera (9, 10). PV is not present in rat muscl e until later than mental regulati on. 3 days after birth [7]; therefore it w as o f interes t to determine C yrosol extracts prepared from cultured newborn mouse ep­ whether these antigens were PV /SCaBP or the other low-mo­ idermal cells were electrophoresed and transferred to nitrocellu­ lecular w eight epidermal an tigens. Cytosol extracts were prepared lose. Fig 8 shows that anti-SCaBP recognizes M,.10, 500 and 12,000 from newborn mouse skin fr ac tions and adult m ouse leg muscl e. antigens in epidermal ce ll cultures . No band is seen at M,. 13,000 Equal amounts of protein fro m these extrac ts w ere electro­ and, in addition, affi nity-purified anti-PV IgG does not bind any phoresed and transferred to nitrocellulose. Fig 7 A shows that PV antiserum does not recognize antigens in cytosol extracts of new­ born mouse skin although it does recognize adult mouse muscle M, 13,000 antigen. Anti-SCaBP reacts w ith M , 10,500-12, 000 A B E p E p

WS SS H M p D

- 13kd - 12kd

Figure 6. Anti-SCaBP se rum recognizes the panniculus ca rnosus antigen Figure 8. C ultured newborn mouse epidermal cells contain M, PV /SCaBP and also an epidermal antigen in adult rat skin. Extracts and 10,500-12,000 antigens but not M , 13,000 PV /SCaBP antigen. Experi­ experimental conditions were as described in Fi g 5, except that the anti­ mental conditions were as described in Fi g 5. Cytosol extracts were pre­ SCaBP serum was used. N ote the distribution of the M, 13,000 band is pared from newborn mouse epidermal cells after 3 days in culture (E). identica l to that seen with anti-PV serum, and that an additional ban d at Purified parvalbumin (P) was included as a control. Pm tel A = anti­ M , \2,000 is present in w hole skin, scraped skin, and epidermis. SC al3P, pan el B = affmit y-purified anti-PV lgG. VOL. 86, N O. 2 FEURUARY 1986 SCa BP IS A PA!lVALl:!UM IN OF THE PANN ICULUS C ARNOSUS 161 bands. Thus, PV /SCaBP canno t be identified in extracts of cul­ O ur results contradict as pects of previous reports on SCaBP. tured newbo rn m ouse epid ermal cell s. The o ri gin al repo rt using anti-SCaBP serum locali zed SCaBP to the basal layer of the epidermis [6). It now seems li kely that the D ISCU SS ION antigens visuali zed in that study were the low- molecular weight We have shown that the proteins PV and SCaBP are immuno­ antigens identified in epidermis cytosol extracts w ith anti-SCaBP logicall y indistinguishable. B y immunoblot analysis, PV and SCaBP serum but these antigens were no t SCaBP itself. The best expla­ both react equall y well With a sen es of 7 polyclonal ant1sera, 6 nation fo r the res ults w hich have been previously and currently raised against SCal3P and 1 raised against PV . The reactivity of repo rted is that various SCaBP preparations were primaril y PV th ese antisera toward PV o r SCal3P ca n be diminished b y ab­ from the panniculus carnosus but also contained minor contam­ sorption with either purified protein. D idicrjcan and Saurat (per­ inatin g proteins with m o lecul ar weights similar to PV. The an­ sonal communication) have also o bserved Immuno logiC cross­ ti gens recognized by the various anti-SCaBP sera must have been reacti vity between PV and SCaBP. Previously IS]. skeletal muscle present in the preparations of SCaBP used to immunize rabbits. PV and SCaBP have been reponed to differ in the affin ity of their Purified SCaBP used to absorb SCaBP antiserum no. 3 in our calcium- binding sites for calcium. Such differen ces, if confirmed, studies m ay have been m ore highly purified, or contained dif­ could be due to minor differences among the proteins not detected ferent contaminants, since it did no t absorb the activity recog­ by the antisera we have used. The PV and SCaBP antisera do no t nizing the additional low-molecular weight epidermal pro teins. react w ith a very closely related protem fro m a d1fferent speCies, In studies using labeled epiderm al cell culture extracts, immu­ rabbit muscle PV; hence the PV proteins from skin and muscle no precipitatio n w ith different SCaBP antisera yielded several dis­ in rat must be very similar, if no t identica l, in structure. In agree­ tinct patterns of electro pho reti c bands of molecular weight ment w ith these findings MacM anus et al (1 6) have recently de­ 8,000-16,000 proteins (data no t shown) . H ence different epider­ termined that the amino acid sequence of rat SCaBP is identica l mal pro teins m ay have been present in the pro tein prepara tions ro that of PV fro m rat skeletal muscle. used to inoculate the different rabbits. Both PV and SCaBP have identica l distributions within the Further study of the low-mo lecular weight regulated epidermal skin, bo th arc locali zed to the panniculus ca rnosus. Affinit y-pu­ antigens is in progress in order to determine their identity and rified anti-PV serum reacts o nl y with protein in the panniculus relatio nship to epidermal cell pro li ferati on and differentiati on. carnosus of skin sections or in cytosol extracts of scraped hy­ podermal m ateri al. The presence of PV in skin had n ~t been reported at the nmc of the o n g mal punfi_ca no n of SCaB[. It has T il l' mlllwrs grn tc)itlly acklw ll•il'dgc th e help of Drs. }111 10 Pa11ioll itch, A1llho 11 y since been shown with quant1tat1ve h1 g h-performance llqllld MeaiiS, C la11de Klee, tllld ) oh11 R . Sta11 lcy. The a11th ors also apprecia te th e chro m atography techniques that PV is present in adult rat skin coopcra tio11 o.f Drs. Li/ia11 e Didie1ju 111 , ). H . Sa 111·at, m1d j o/111 Mactvlm 111 s ill in relatively hig h concentration compared w ith o ther ti ss ues, and CO II S11/tario11 a11 d co 111 pariso 11 a( data prio r to p11blicatio11 . that it is locali zed by immunohistochemistry in Bouin-flxed skm sectio ns to the musculature of the dermis [8]. N o PV was detected in the epidermis. U sin g a rat eDNA pro be for PV, Berchtold and M eans [1 71 found that PV mRNA is present in whole skin REFERE N CES w ith the same m olecular size (700 and 1000 bp) as it was fo und I . Hennings H. Mi chael D. Cheng C, Stei nert P, Holbrook K, Yuspa in the muscle, indicating identity of muscle and skin PV. How­ SH: Calcium regulation of growth and diffe remiation of mouse ever, no hy bridization signals were detected in epidermis or sev­ cpidern1Jl cells in cul ture. Cell 19:245-254, 1980 eral cell lines of epidermal o ri gin (Berchtold and H awley-Nelson, 2. Ku lesz-Martin MF, Koehler B. Hennings H, Yuspa Sl-1: Q uan tita­ unpublished observ:nions). MacM anus et al (1 6] have also re­ ti ve assay for ca rcin ogen altered differentiation in mouse epidermal ported the dermal locali zatio n of PV /SCaBP. cells. Carcin ogenesis I :995-1006, 1980 Previous repo rts !6 ) had fai led to detect SCaBP antigen in der­ 3. Yuspa SH. Hawley-Nelson P, Koehler B. Stanley JR. : A survey of mal ti ssue o r muscle b y immuno flu o rescence. H owever Celi o and transformation markers in differentiating epidermal cel l lin es in H eizm ann [7] have shown that fi xatio n techniques w hi ch result culture. Cance r Res 40:4694-4703, '1980 in p ro tein cross-linking are required fo r immuno histochemical 4. La ouari D, Pa vlovitch 1-1 , Deceneu x G, Ba lsa n S: A vitamin v isualization of PV in ti ss ue sectio ns. T he published repo rts lo­ D-depcnd ent ca lcium-binding protein in rat sk in . FEBS Lett 111: 285-289, 1980 cali zing SCaBP to the epidermis utilized immuno flu o rescence of frozen sections [6]. It is therefore likely that the PV / SCaBP an­ 5. Rin aldi ML, Haiech J, Pav lovitch J, Ri zk M, Ferraz C, Derancourt J, Demaill e JG: Isolation and characte rization of a rat skin par­ tigen in the panniculus ca rnosus was no t detected at all. O ur fixed valbumin-like calcium-binding protein. Biochemistry 21:4805-4810, tissue immunohistochemistry dem o nstrates that PV / SCaBP an­ 1982 tigen is present in the panniculus ca rnosus of adult rats. 6. Sa urat Jl-1 , Didierjean L, Pavlovitch Jl-1 , Lao uari D. Balsan S: Ski n SCaBP antisera but not PV antisera recognize low-molecular ca lcium-binding protein is loca li zed in the cy toplasm of the basa l weig ht epidermal antigens in additio n to PV / SCaBP. By im­ layer of the epiderm is. J In vest Dermacol 76:221 -223, 198 1 munohistochemistry these antigens appear in all the living epi­ 7. Celio MR, Heizmann CW: Calcium-binding protein parvalbumin dermal cell Ia yers. In skin fractions, antigens are similarly locali zed is associated with f.1s t contracting muscle fibers. Nature 297:504--506, to the epidermis. PV /SCaBP is no t detectable in cultured m o use 1982 keratinocytes or in newborn rodent skin, but anti-SCaBP serum 8. Berchtold MW , Celi o MR, Heizmann CW: Parvalbumin in non­ recognizes the low-molecul ar weight anti gens in cul tured kera­ muscle tiss ues of th e rat. J Bioi Chcm 259:5 189-5196, 1984 tinocytes and in newborn as well as adult epidermis. In separate 9. Haw ley-Nelson P, Pav lovitch J, Yuspa SH: Regulation of skin ca l­ studies we have found that antigen synthesis is confined to pro­ cium-binding protein synthesis in normal and malignam epider­ liferating cultured keratinocytes in m edium containing 0.07 m M mal ce ll cultures. J In vest Dermatol 80:304--305 , 1983 calcium, and synthesis stops w hen differentiation is induced b y 10. Didi crj ea n L, Pav lovitch Jl-1 , Fusenig NE, Sa urat Jl-1 : Extracellul ar raising the calcium concentratio n to 1. 2 111M for 48-72 h (9]. The ca lcium concentration reg ulates the ex pression of skin calcium tumor pro m oter 12-0- tetradecanoyl phorbol-1 3-acctate, which binding protein (SCaBP) in mouse keratinocytc cultures. Arch also induces differentiati on in cultures [1 8], sup­ Derm:Itol Res 275:202-203, 1983 pressed synthesis of the antigens. These results arc consistent with II. Kawamura H, Strickl an d JE, Yu spa Sf-\: Association of resis tance the !ow-mo lecular weig ht antigens being exclusivel y synthesized to terminal diffe rentiation with initiation of carcinogenesis in adult in basal cells. Using immunofluorescence, Didierjean et al [10] mouse epidermal cel ls. Cancer Res . 45:2748-2752,1 985 showed regulated distribution of the "SCaBP" antigens in epi­ 12. Yuspa SH, Haw ley-Nelson P, Stanle y JR, Hennings H: Epiderm al dermal cell cultures. cell culture. Transplant Proc 12(s uppl 1): 11 4-122. '1980 162 HAWLEY-NE LSON ET AL T H E JOURNAL OF INVESTIG ATIVE DERMATOLOGY

13. Lac mmli UK: C lea vage of structural proteins during the assembly I fi. MacManus JP, Watson DC, Yaguchi M : Rat skin ca lcium-binding of th e head of bacteri ophage T4. Nature 227 :680-fi85, 1970 protei n is parvalbumin. Biochem J 229:39-45, 1985 14. Gcrshoni JM, Palade GE: Electrophoretic transfer of proteins from 17. Berchtold MW, Means AR: The Ca2+ binding protein parva lburnin: sodium dodecy l sul fa te-polyacryla mide gels to a positivel y charged Molecular cloning and developmental rcgubrion of mHNA abun­ membrane fil ter. Anal Biochem 124:396-405, 1982 dance. Proc Nat! Aca d Sci USA 82: 1414-141 8. 1985 15. Brandtzaeg P: Prolonged incubati on time in immunohistochemistry: effects on flu orescence staining of immunoglobulins and epithelial 18. Yu spa SH, 13en TB, Hennings H. Li chti U: Phorbol ester tumor components in ethanol- and fo rmald ehyde-fi xed paraffin-embed­ promoters induce epidermal nansglutaminasc activity. Biochem ded tiss ues. J Histochem Cytochcm 29:1302- 1315. 1981 Biophys Res Commun 97:700-708. 1980