peptidylarginine deiminase isoforms are differentially

Peptidylarginine Deiminase Isoforms Are Differentially Expressedin the Anagen Hair Follicles and Other Human Skin Appendages

Rachida Nachat,� Marie-Claire Mechin,� Marie Charveron,w Guy Serre,� Jacques Constans,� andMichel Simon��CNRS-University of Toulouse III UMR 5165 ‘‘Epidermis differentiation and rheumatoid autoimmunity’’, Institut Federatif de Recherche 30 (INSERM; CNRS; CentreHospitalier Universitaire de Toulouse; Universite Paul Sabatier), Toulouse Cedex, France; wCERPER/Institut de Recherche Pierre Fabre, Toulouse Cedex, France

Peptidylarginine deiminases (PAD) catalyze the conversion of arginine residues to citrullines. Five isoforms are

known that present distinct tissue locations. In the epidermis, like in the skin, only PAD1, 2, and 3 are expressed.

Their pattern of expression in skin appendages is not known. Here, confocal microscopy analysis using highly

specific antibodies demonstrated that PAD1 and 3 are expressed in human anagen hair follicles, PAD1 and 2, in

arrector pili muscles and sweat glands, whereas no PAD were detected in sebaceous glands. PAD1 was detected in

the cuticle and the Huxley layer of the inner root sheath (IRS), and in the companion layer. PAD3 was localized in

the medulla, and in the three layers of the IRS. Using anti-modified citrulline antibodies, we also showed that

deiminated proteins appeared in the lower part of the IRS, first in the Henle layer, then in the cuticle, and finally in

the Huxley layer. Our data demonstrate that PAD3 is the enzyme that deiminates trichohyalin in the medulla and the

Henle layer, indicate that PAD1 and 3 are involved in the hair follicle program of differentiation, and suggest a role

for PAD1 and 2 in the physiology of sweat glands and arrector pili muscles.

Key words: citrulline/confocal microscopy/post-translational modifications/skin/trichohyalinJ Invest Dermatol 125:34 –41, 2005

The peptidylarginine deiminase (PAD) enzymes (protein-arginine deiminase, protein-L-arginine iminohydrolase, EC3.5.3.15) are responsible for the formation of protein-boundcitrulline in the presence of calcium ions. First detected inthe hair follicles of the guinea-pig (Rogers and Taylor, 1977),PAD activity has been identified in various tissues of almostall vertebrates (for a review, see Vossenaar et al, 2003). Fivehuman PAD genes, clustered on chromosome 1p35–36,have been described and named PADI1, 2, 3, 4, and 6 onthe basis of their cDNA nucleotide sequence and of the invitro biochemical features of their products: the PAD1, 2, 3,4 (formerly named PAD type 5), and 6 (Nakashima et al,1999; Rus’d et al, 1999; Kanno et al, 2000; Ishigami et al,2002; Guerrin et al, 2003; Chavanas et al, 2004). PAD1 ismainly found in the epidermis and uterus (Terakawa et al,1991; Rus’d et al, 1999; Guerrin et al, 2003; Nachat et al,2005). PAD2 is widely expressed in various tissues includingskeletal muscle, brain, uterus, salivary glands, pancreas,and epidermis (Kubilus and Baden, 1985; Takahara et al,1989; Watanabe and Senshu, 1989; Akiyama et al, 1990;Terakawa et al, 1991; Ishigami et al, 2002; Nachat et al,2005). PAD3 is expressed in the epidermis and hair follicles(Terakawa et al, 1991; Rogers et al, 1997; Rus’d et al, 1999;Kanno et al, 2000; Nachat et al, 2005). PAD4 is found in

hematopoietic cells (Nakashima et al, 1999; Asaga et al,2001; Vossenaar et al, 2004). Human PAD6 mRNA weremainly detected in ovary, peripheral blood leukocytes, andtestis (Chavanas et al, 2004). The structure of PAD4 deter-mined after crystallization shows two elongated domains,each with at least two calcium-binding sites. Calcium-bind-ing induces conformational changes that generate the ac-tive site cleft, formed by five b-strands. Moreover, PAD4was shown to form a head-to-tail dimer (Arita et al, 2004).

Little is known about the exact functions and substratesof PAD. Deimination dramatically alters the charge of argi-nine residues from positive to neutral and results in the un-folding of target proteins. Recently, the deimination ofhistones by PAD4 was described as a novel mechanism forantagonizing the transcription induction mediated by argi-nine methylation (Cuthbert et al, 2004; Wang et al, 2004).

This post-translational modification has been implicatedin the pathogenesis of multiple sclerosis and rheumatoidarthritis. Indeed, increased deimination of myelin basic pro-tein and glial fibrillary acidic protein has been observed inthe brain of multiple sclerosis patients (Kim et al, 2003; Ni-cholas et al, 2004). In rheumatoid arthritis, deiminated formsof fibrin deposited in the rheumatoid synovial membranesare the major target of disease-specific autoantibodies(Masson-Bessiere et al, 2001). In a Japanese population,PADI4 gene polymorphisms were strongly linked to sus-ceptibility to the rheumatic disease (Suzuki et al, 2003).Moreover, a recent study revealed that PAD can act as anautoantigen in patients with various inflammatory rheumaticdiseases (Nissinen et al, 2003).

Abbreviations: CDSN, corneodesmosin; DSG, desmoglein; HF, hairfollicles; IF, immunofluorescence; IRS, inner root sheath; MoAb,monoclonal antibody; ORS, outer root sheath; PAD, peptidylargi-nine deiminase; THH, trichohyalin

Copyright r 2005 by The Society for Investigative Dermatology, Inc.

34

In normal epidermis, where keratin K1 was identified asthe major deiminated epidermal protein, K10 and filaggrinas the minor ones, the deiminated proteins are located inthe cornified layers, suggesting a role for PAD during theterminal stages of epidermal differentiation (Senshu et al,1995; Senshu et al, 1996). Deimination of filaggrin inducesits disassembly from keratin intermediate filaments, andprobably facilitates its proteolysis. This degradation gener-ates a pool of amino acids that contribute to the cutaneousnatural moisturizing factor (Rawlings and Harding, 2004).Alterations in the pattern of deiminated proteins in humanepidermis have been reported in some skin disorders,namely psoriasis and bullous congenital ichthyosiform er-ythroderma (Ishida-Yamamoto et al, 2000, 2002).

In the hair follicles (HF), trichohyalin (THH), a high mo-lecular weight structural a-helix-rich protein of the inner rootsheath (IRS) and the medulla, is deiminated by PAD (Rogersand Taylor, 1977). The following model of THH processing,including its deimination, has been proposed. First, THH issynthesized in the IRS keratinocytes as a large insolubleprotein-forming cytoplasmic granules. Then, deimination ofTHH at the periphery of the granules generates a more sol-uble form that is released and intermixed with keratin inter-mediate filaments. THH is subsequently cross-linked bytransglutaminase 3 to itself and to keratins (Tarcsa et al,1997; Steinert et al, 2003). In the medulla, which containsfew if any keratin intermediate filaments, the disperse THHseems to be cross-linked as vacuolated aggregates en-trapping air. But, the PAD isoforms used for THH deimina-tion are unknown. Until now, only PAD3 has been detectedin HF. In situ hybridization and double immunofluorescence(IF) staining revealed its co-expression with THH in woolfollicles of sheep and in human HF, respectively (Rogerset al, 1997; Kanno et al, 2000). By RT-PCR experiments, werecently demonstrated that three PADI genes are expressedin human skin, i.e., PADI1, 2, and 3. Accordingly, using an-tibodies highly specific for each PAD, we showed thatPAD1, 2, and 3 are actually expressed in the epidermis.These results suggested that PAD other than PAD3 may beexpressed in HF. Here, we investigated the pattern of ex-pression of these PAD in the human anagen HF and in otherskin appendages.

Results

By RT-PCR experiments, we previously demonstrated thatPADI1, 2, and 3 are the only PAD genes expressed in theskin. In this study, we analyzed the expression of thesethree PAD in human HF and skin appendages by indirectIF and confocal microscopy using specific affinity purifiedanti-peptide antibodies. Anagen HF were not stained withthe anti-PAD2(C2) antibodies (data not shown), but showedclear expression of PAD1 and 3.

Localization of PAD1 in anagen HF Several distinct con-centric compartments compose the HF: the outer rootsheath (ORS), which is the outermost layer, the IRS, and thecentral hair shaft itself. The ORS and IRS are separated bythe companion layer. Three subcompartments further com-posed the IRS: the Henle and Huxley layers, and the cuticle

in direct contact with the cuticle of the hair shaft. To preciselylocalize PAD1 in the different layers of the HF, we performeddouble-label IF staining using the anti-PAD1 antibodiesand two monoclonal antobodies (MoAb) directed to eitherthe desmosomal cadherins DSG1 and DSG2 (DG3.10) orto corneodesmosin (CDSN), a corneodesmosomal protein(G36-19). DG3.10 labeled the IRS from the stem cells tothe keratogenous zone, and part of the hair shaft (Fig 1A–C,see also Fig 2A–D). As described (Mils et al, 1992), G36-19

Figure1Expression of peptidylarginine deiminase type 1 (PAD1) in hair fol-licles. Cryosections of human scalp were analyzed by indirect immuno-fluorescence (double staining) and confocal observations with DG3-10,a monoclonal antibody directed against desmoglein 1 and 2 (DSG1-2),G36-19, a monoclonal antibody directed against corneodesmosin(CDSN), and with affinity-purified anti-PAD1 antibodies. (A) A longitu-dinal section of a hair follicle double stained with the anti-PAD1 andDG3.10 antibodies. (B, C) Higher magnification of parts of the sectionshown in (A). (D–F) A transverse section of a hair follicle stained withG36-19 and anti-PAD1 antibodies. (F) Image superimposition of CDSN/PAD1 labeling. The red arrow in (A) and the double-headed red arrowsin (A) and (B) denote the site of abrupt cornification of Henle and Huxleylayers, respectively. The white open arrowhead in (A) shows a stainedFlugelzellen cell. He, Henle layer; He�, cornified Henle layer; Hu, Huxleylayer; iCu, IRS cuticle; cl, companion layer; dp, dermal papilla. Scalebar¼150 mm (A), ¼75 mm (B), and ¼ 50 mm (C–F).

EXPRESSION OF PAD IN HUMAN SKIN APPENDAGES 35125 : 1 JULY 2005

also stained the IRS, but the labeling appeared first (from thebase of the HF) in the Henle layer, then in the IRS cuticle, andfinally in the Huxley layer. It persisted in the three layers up tothe isthmus (Fig 1D, see also Fig 3A).

PAD1 was detected first in the IRS cuticle at the height ofthe dermal papilla, then in the Huxley layer (Fig 1A and C),and finally, in the middle portion of the HF, in the companionlayer (Fig 1B). PAD1 persisted in the cornified Huxley layer,characterized by the abrupt disappearance of DG3.10staining (Fig 1A and B). Double-label IF staining using an-ti-PAD1 and G36-19 antibodies on transversal sections(Fig 1D–F) revealed that in the upper portion of the HF, PAD1was restricted to the companion layer. PAD1 expressionwas also observed in cells of the Huxley layer that devel-oped pseudopodal extensions passing through the corni-fied Henle layer (Fig 1A, triangle). These cells probablycorrespond to the specialized so-called Flugelzellen cells(Langbein et al, 2003). In every case, the labeling producedby the anti-PAD1 was cytoplasmic.

Localization of PAD3 in anagen HF We performed dou-ble-label IF staining using the anti-PAD3(B3) antibodies andthe MoAb DG3.10, G36-19, and AE16. AE16 is known tolabel the THH granules of the IRS and the medulla of the HFbut not to react with THH when it is associated with the IRSfilaments (O’Guin et al, 1992).

Double-label IF using the anti-PAD3(B3) and DG3.10 an-tibodies revealed cytoplasmic expression of PAD3 in the

Henle and Huxley layers and in the cuticle of the IRS, inthe bulb region (Fig 2A and B). Then, the labeling of theHenle layer abruptly disappeared, as observed on longitu-dinal (Fig 2A) and transversal (Fig 2C) sections of HF. Finally,PAD3 was no longer detected in the cornified Huxley lay-er characterized by the disappearance of DG3.10 staining(Fig 2A and D). A higher magnification showed that thelabeling in the Huxley layer was granular. In some cases,only the periphery of the granules seemed to be stained(Fig 2E, arrows). Fig 2A, G, and F show that PAD3 is alsoexpressed in the Flugelzellen cells and in the medulla,respectively.

Double-label IF staining on longitudinal and transversalsections of HF, using the anti-PAD3(B3) and G36-19, anti-bodies showed that the loss of PAD3 detection in the IRScoincided with the appearance of CDSN expression, first inthe Henle layer (Fig 3A and C), then in the cuticle, and finallyin the Huxley layer (Fig 3A and D). Therefore, disappearanceof anti-PAD3(B3) staining corresponded to the asynchro-nous onset of abrupt cornification of these three layers.

Double-label IF staining on tangential sections of HF,using the anti-PAD3(B3) and AE16 antibodies, showed thatthe onset of AE16 labeling nearly coincided with the disap-pearance of PAD3 detection (Fig 4A–C). AE16 labeling ap-peared abruptly in distinct cells, and then rapidly becamediffuse before its total disappearance (Fig 4B). This corre-sponds to the dispersion of THH granules and the associ-ation of THH with the IRS filaments (Rothnagel and Rogers,

Figure 2Expression of peptidylarginine deimi-nase type 3 (PAD3) and desmoglein 1and 2 (DSG1-2) in hair follicles. Cryo-sections of the human scalp were analy-zed by indirect immunofluorescence(double (A–D) or simple (E–G) staining)and confocal observations with DG3-10and/or affinity-purified anti-PAD3(B3) anti-bodies. Longitudinal (A) and cross (B–D)sections of hair follicles analyzed by dou-ble staining are shown. The position ofeach transverse section on the longitudi-nal section is indicated as follows: 1—B,2—C, 3—D. The red arrow and the dou-ble-headed red arrows in (A) denote thesite of abrupt cornification of Henle andHuxley layers, respectively. Note the con-comitant abrupt disappearance of PAD3detection. The arrows in E show the labe-ling at the periphery of granules. Thewhite open arrowhead in (A), (C), and (G)shows a PAD3-stained Flugelzellen cell.M, medulla; the other abbreviations arethe same as in Fig 1. Scale bar¼150 mm(A and F), ¼ 75 mm (B–D), ¼50 mm (E),and ¼ 10 mm (G).

36 NACHAT ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

1986; O’Guin et al, 1992). In some cells, the anti-PAD3(B3)and AE16 labelings were co-localized (Fig 4C).

Localization of PAD in other skin appendages: sweatglands, sebaceous glands, and hair arrector pili mus-cles Analysis of human scalp sections by indirect IF withthe anti-PAD antibodies showed the presence of PAD1 andPAD2 but not of PAD3 in sweat glands or in the arrector pilimuscles, whereas no immunoreactivities were detected insebaceous glands (data not shown). We then performeddouble-label IF staining using either the anti-PAD1 or anti-PAD2(C2) antibodies and a MoAb specific to smooth musclea-actin (Figs 5 and 6). Both PAD1 and PAD2 were observed

to co-localize with a-actin in the arrector pili muscles(Fig 5A–C and D–F, respectively) and in the myoepithelialcells at the periphery of the sweat glands (Fig 6A–C andD–F, respectively). Staining of the cytoplasm of the secre-tory cells of sweat glands was also observed with the anti-PAD1 and anti-PAD2(C2) antibodies (Fig 6A, C, D, and F).

Localization of deiminated proteins in anagen HF Todetermine whether the presence of PAD in skin appendageswas associated with deimination of proteins, we performedimmunohistochemistry analyses using AMC, purified anti-bodies specific to chemically modified citrullines, as previ-ously published (Senshu et al, 1995). On longitudinalsections of HF, AMC labeling appeared in the lower partof the IRS, first in the Henle layer (Fig 7A), then in the IRScuticle, and finally in the Huxley layer (Fig 7B and C). Thelabeling persisted in the IRS at the isthmus. The medullawas also strongly labeled (Fig 7C). The detection of dei-minated proteins coincided with the asynchronous onset ofabrupt cornification of the IRS Henle layer, cuticle, andHuxley layer. These results are consistent with the distribu-tion of citrulline in rat HF, as observed long ago usinga colorimetric reaction with Ehrlich reagent (Rogers, 1963).No staining was observed in the ORS, sweat glands, seba-ceous glands or arrector pili muscles (data not shown).

Discussion

PAD activity was first identified in the HF of the guinea-pig(Rogers and Taylor, 1977) where THH was described as asubstrate for deimination. Since then, the existence of fiveisoforms of PAD has been reported, but their tissue distri-bution has yet to be explored in detail. By RT-PCR exper-iments, we demonstrated that PADI1, 2, and 3 are the onlyPADI genes expressed in skin (Nachat et al, 2005). In orderto identify the different PAD isoforms expressed in humananagen HF and other skin appendages, we performed dou-ble-label IF staining on unfixed cryosections of human scalpepidermis using our previously described anti-PAD anti-bodies highly specific to each of these isoforms (Guerrinet al, 2003; Nachat et al, 2005)

In human anagen HF, we demonstrated the expression ofPAD1, in the Huxley layer and the cuticle of the IRS and inthe companion layer. We also confirmed the expression ofPAD3. Indeed, PAD3 was described as the hair follicle PAD,the expression of its mRNA being very similar to that of THHin the IRS and medulla of wool follicles of sheep, as shownby in situ hybridization (Rogers et al, 1997). In agreement,we showed that PAD3 expression, at the protein level, isrestricted to the IRS and to the medulla of human anagenHF. These results somehow showed discrepancy with thoseobtained by Kanno et al (2000), who reported expressionnot only in the IRS and medulla but also in the ORS. A likelyexplanation is that the treatment performed by Kanno andcoworkers with sodium citrate buffer to unmask antigenicsites on fixed sections might have produced artifacts. Al-ternatively, the anti-PAD3 antibodies they used could havecross-reacted with another epidermal protein.

The companion layer was initially defined as the inner-most layer of the ORS. Because the companion layer cells

Figure 3Expression of peptidylarginine deiminase type 3 (PAD3) and corn-eodesmosin (CDSN) in hair follicles. Cryosections of the humanscalp were analyzed by indirect immunofluorescence (double staining)and confocal observations with the anti-PAD3(B3) and G36-19 anti-bodies. Longitudinal (A) and transverse (B–D) sections are shown. Theposition of each transverse section on the longitudinal section is in-dicated as follows: 1—B, 2—C, 3—D. The red arrow and the double-headed red arrows in (A) denote the site of abrupt cornification of Henleand Huxley layers, respectively, accompanied by the abrupt disap-pearance of PAD3 detection. The white open arrowheads in (A) showstained Flugelzellen cells. The abbreviations are the same as in Fig 1.Scale bar¼ 150 mm (A) and ¼75 mm (B–D).


display properties different from those of the adjacent ORScells and because their origin in the hair bulb is unrelated tothat of the ORS cells, Schweizer’group proposed that thecompanion layer is an integral compartment of the IRS, andcontributes to the guidance of hair shaft growth (Winter et al,1998). Our findings add a new biochemical argument tosupport this view: IRS and companion layer cells but notORS cells express PAD.

The only PAD detected in the medulla and the IRS Henlelayer is PAD3. Therefore, it is this isoform that is responsiblefor the THH deimination in these parts of the HF. In the IRScuticle and Huxley layer, PAD1 and PAD3 are expressedsimultaneously, suggesting that PAD1 may also be involvedin the deimination of THH. Similarly, we previously demon-strated the co-location of PAD1, 3, and (pro)filaggrin in thegranular layer and lower stratum corneum of the epidermis,and suggested that only one of the two enzymes or a com-bination of the two might be involved in the deimination offilaggrin (Nachat et al, 2005). The presence of PAD1 in thecornified Huxley layer, however, suggests that deiminationof THH can be completed by this isoform after cornification.PAD1 expression was also described in the superficial stra-tum corneum, where we proposed that it is involved in thedeimination of keratin K1 (Nachat et al, 2005). Whether K1 isalso deiminated by PAD1 in the Huxley layer remains to beexplored.

To study the function of THH in the HF, three MoAb al-lowed three distinct stages of maturation of the protein tobe defined: the AE15-stained form, which is found through-out the THH granules; the AE16-stained form which is lo-cated on the surface of the granules; and the AE17-stainedform, which is associated with the keratin intermediate fil-aments (O’Guin et al, 1992). O’Guin et al proposed thatAE16 recognizes a modified form of THH in the process ofbeing released from the granules. Our results suggest that itis the deimination of THH by PAD3 that generates its labe-ling by AE16. In particular, detection of THH by AE16 co-incides with the appearance of deiminated proteins.Moreover, we were able to locate PAD3 at the peripheryof granules, probably THH granules, in the cytoplasm ofkeratinocytes in the IRS. This is in agreement with the modelpresented by Steinert’s group, in which insoluble cytoplas-mic droplets of THH that are located in the IRS and themedulla are initially modified by PAD, which converts THHto a more soluble form, and allows subsequent cross-linkingby transglutaminase 3 (Tarcsa et al, 1997).

A mouse type-I keratin, KRT1-c29, specifically ex-pressed in the IRS cells, was shown to be deiminated be-fore being cross-linked to THH and other cornified cellcomponents (Steinert et al, 2003). Our data suggest thatKRT1-c29 and its human ortholog, the IRS-specific keratin25Cirs3, are deiminated by PAD1 or 3.

Figure 4Expression of peptidylarginine deimi-nase type 3 (PAD3) and trichohyalin(THH) in hair follicles. Cryosections ofthe human scalp were analyzed by indi-rect immunofluorescence with the anti-PAD3(B3) and anti-THH AE-16 antibodies.Staining with anti-PAD3(B3) (A) and AE-16(B) of tangential sections of hair follicles.(C) Image superimposition of THH/PAD3labelings. The double-headed red arrowsin (A) denote the site of abrupt cornificat-ion of Huxley (Hu) layer. The white arrowsshow the co-location of PAD3 and THH.Scale bar¼150 mm.

Figure 5Expression of peptidylarginine deimi-nase type 1 (PAD1) and type 2 (PAD2) inarrector pili muscles. Cryosections ofhuman scalp were analyzed by indirectimmunofluorescence and confocal mi-croscopy with anti-PAD1 (A), anti-PAD2(C2) (D) and anti-a-actin (B and E)antibodies. (C and F) Image superimposi-tion of a-actin/PAD1 and a-actin/PAD2labeling, respectively. Scale bar¼ 100 mm(A–C) and ¼ 75 mm (D–E).


As observed in the human epidermis, by confocal mi-croscopy analysis (Nachat et al, 2005), the distribution ofPAD in the HF contrasts with the distribution of deiminatedproteins only detected in the cornified IRS and in the me-

dulla (Table I) (Steinert et al, 1969). The absence of immuno-detection of PAD in the cornified layers is probably due toepitope masking in these compact cell layers, which is wellknown to occur in the cornified layer of the epidermis.These data also suggest that PAD are activated during thefinal stages of terminal differentiation of the different layersof the IRS, e.g., by an increase of intracellular calcium con-centration as proposed for the epidermis. Several calcium-binding proteins expressed in human HF may be involved inthis control, such as THH itself (Fietz et al, 1993; Lee et al,1993), calcyclin (Wood et al, 1991), CLED (Sun et al, 2000),or proteins of the S100 family (Takizawa et al, 1999; Schmidtet al, 2001).

We also detected PAD1 and 2 in arrector pili musclesand sweat glands, both in secretory and myoepithelial cells.PAD2 localization in sweat glands was in agreement witha previous study on human skin using a serum directedagainst rat-PAD2, whereas its detection in arrector pili mus-cles was in apparent discrepancy (Urano et al, 1990). Thiscould be due to experimental differences, e.g., the fixationstep of cryosections with acetone used by Urano et al. De-spite the expression of PAD in sweat glands and arrector pilimuscles, no deiminated proteins were detected using anti-modified citrulline antibodies (Table I). Therefore, PAD maybe produced as inactive forms that could be activatedin particular events, like apoptosis (Asaga et al, 1998;Mizoguchi et al, 1998) or hormonal stimulation. WhetherPAD expression or activation is modulated during the var-ious phases of the HF cycle remains to be explored.

The production of anti-PAD specific antibodies allowedus to define the pattern of PAD expression in human skinappendages. The specific location of the enzymes and dei-minated proteins in the IRS suggests a crucial role for de-imination during terminal differentiation of HF. In particular,PAD1 and 3 may be essential for controlling mechanicalresistance of the sheath due to the peripheral cell envelopeand cytoplasmic keratin filament network, through THH de-imination and subsequent cross-linking.

Figure 6Expression of peptidylarginine deimi-nase type 1 (PAD1) and type 2 (PAD2) insweat glands. Cryosections of the hu-man scalp were analyzed by indirectimmunofluorescence and confocal mi-croscopy with anti-PAD1 (A), anti-PAD2(C2) (D), and anti-a-actin (B and E)antibodies. (C and F) Image superimposi-tion of a-actin/PAD1 and a-actin/PAD2labeling, respectively. Scale bar¼75 mm.

Figure 7Location of deiminated proteins in hair follicles (HF). Longitudinalcryosections of hair follicles were analyzed by immunoperoxidase withAMC antibodies specific to chemically modified citrullines. Note theappearance of deiminated proteins (red) first in the Henle layer (bluearrows in A and B), then in the cuticle (double-headed blue arrows in Band C) and finally in the Huxley layer (red arrows in B and C) of the innerroot sheath. Deiminated proteins were also localized in the medulla (M)of the HF. Note the black pigment deposited in the hair follicles (A andB). Scale bar¼ 100 mm.


Materials and Methods

Antibodies The MoAb G36-19 directed to CDSN was producedand characterized in our laboratory (Serre et al, 1991). DG3.10, aMoAb directed to desmoglein 1 and 2 (DSG1 and DSG2), waspurchased from Progen Biotechnik GmbH (Heidelberg, Germany).AE16, a MoAb directed to THH, was a kind gift from T. T. Sun(O’Guin et al, 1992). Affinity-purified polyclonal antibodies directedto PAD1, 2 and 3, anti-PAD1, anti-PAD2(C2), and anti-PAD3(B3)were elicited in rabbits, and previously characterized (Guerrin et al,2003; Nachat et al, 2005). AMC, a monospecific antibody tochemically modified citrullines, was kindly given by N. Blaes,T. Senshu and has already been described (Senshu et al, 1995).The MoAb 1A4 directed to the smooth muscle a-actin was pur-chased from Sigma Aldrich, St. Louis, Missouri.

Immunohistology All described studies concerning human tis-sues/subjects were conducted in adherence with the Declarationof Helsinki Principles, and in accordance with the ethical guidelinesof the University of Toulouse III and of the French Ministry of Re-search and Technology. Indirect IF was performed on unfixed 5–7mm thick cryosections of human scalp skin as previously described(Nachat et al, 2005). In the case of double staining, the sectionswere further incubated with MoAb AE16, DG3.10, 1A4, or G36-19

diluted to 2.5 mg per mL. Sections were observed under a confocallaser microscope (LSM 410, Carl Zeiss, Oberkochen, Germany),excited at 488 and 543 nm with Ar and HeNe lasers, respectively.For negative controls, sections were incubated without primaryantibodies. For analyses of deiminated proteins, cryosections wereincubated in 0.0125% FeCl3, 2.3 M H2SO4, 1.5 M H3PO4, 0.25%diacetyl monoxime, and 0.125% antipyrine (modification medium)at 371C for 3 h to modify citrulline residues chemically (Senshuet al, 1995). Immunoperoxidase staining of the modified proteinswas then performed with the AMC antibody diluted in PBS to 0.25mg per mL for 1 h at 371C, and the Histostain-Plus kit (Zymed, SanFrancisco, California) using aminoethyl carbazole as a chromo-genic substrate. Sections were counterstained with hematoxylin.

We wish to sincerely acknowledge N. Blaes, T. Senshu and T. T. Sun fortheir generous gift of antibodies. We thank Dr S. Chavanas for hiscritical reading of the manuscript, and I. Ceruti for her help with con-focal laser microscopy. This work was supported by grants from the‘‘Institut National de la Sante et de la Recherche Medicale’’ (INSERM),the ‘‘Universite Paul Sabatier-Toulouse III’’, the ‘‘Centre National de laRecherche Scientifique’’ (CNRS), and the ‘‘Centre Europeen de Re-cherche sur la Peau et les Epitheliums de Revetement’’ (CERPER, Un-iversite Paul Sabatier Toulouse III—CHU de Toulouse—Institut derecherche Pierre Fabre, France).

DOI: 10.1111/j.0022-202X.2005.23763.x

Manuscript received December 1, 2004; revised February 1, 2005;accepted for publication February 10, 2005

Address correspondence to: Dr Michel Simon, CNRS-UPS UMR 5165,‘‘Differenciation epidermique et autoimmunite rhumatoıde’’, Faculte deMedecine, 37 allees Jules Guesde, 31073 Toulouse Cedex, France.Email: [email protected]

References

Akiyama K, Inoue K, Senshu T: Immunocytochemical demonstration of skeletal

muscle type peptidylarginine deiminase in various rat tissues. Cell Biol Int

Rep 14:267–273, 1990

Arita K, Hashimoto H, Shimizu T, Nakashima K, Yamada M, Sato M: Structural

basis for Ca(2þ )-induced activation of human PAD4. Nat Struct Mol Biol

11:777–783, 2004

Asaga H, Nakashima K, Senshu T, Ishigami A, Yamada M: Immunocytochemical

localization of peptidylarginine deiminase in human eosinophils and ne-

utrophils. J Leukoc Biol 70:46–51, 2001

Asaga H, Yamada M, Senshu T: Selective deimination of vimentin in calcium

ionophore-induced apoptosis of mouse peritoneal macrophages. Bio-

chem Biophys Res Commun 243:641–646, 1998

Chavanas S, Mechin MC, Takahara H, Kawada A, Nachat R, Serre G, Simon M:

Comparative analysis of the mouse and human peptidylarginine deimi-

nase gene clusters reveals highly conserved non-coding segments and a

new human gene, PADI6. Gene 330:19–27, 2004

Cuthbert GL, Daujat S, Snowden AW, et al: Histone deimination antagonizes

arginine methylation. Cell 118:545–553, 2004

Fietz MJ, McLaughlan CJ, Campbell MT, Rogers GE: Analysis of the sheep

trichohyalin gene: Potential structural and calcium-binding roles of

trichohyalin in the hair follicle. J Cell Biol 121:855–865, 1993

Guerrin M, Ishigami A, Mechin MC, et al: cDNA cloning, gene organization and

expression analysis of human peptidylarginine deiminase type I. Biochem

J 370:167–174, 2003

Ishida-Yamamoto A, Senshu T, Eady RA, Takahashi H, Shimizu H, Akiyama M,

Iizuka H: Sequential reorganization of cornified cell keratin filaments in-

volving filaggrin-mediated compaction and keratin 1 deimination. J Invest

Dermatol 118:282–287, 2002

Ishida-Yamamoto A, Senshu T, Takahashi H, Akiyama K, Nomura K, Iizuka H:

Decreased deiminated keratin K1 in psoriatic hyperproliferative epider-

mis. J Invest Dermatol 114:701–705, 2000

Ishigami A, Ohsawa T, Asaga H, Akiyama K, Kuramoto M, Maruyama N: Human

peptidylarginine deiminase type II: Molecular cloning, gene organization,

and expression in human skin. Arch Biochem Biophys 407:25–31, 2002

Kanno T, Kawada A, Yamanouchi J, et al: Human peptidylarginine deiminase type

III: Molecular cloning and nucleotide sequence of the cDNA, properties of

Table I. Detection of PAD and deiminated proteins in human

skin appendages

PAD1 PAD2 PAD3Deiminatedproteins

IRS of anagen hairfollicles

Non-cornifiedHenle layer

� � þ þ þ �

Cornified Henle

layer

� � � þ þ þ

Non-cornified

Huxley layer

þ þ þ � þ þ þ �

Cornified Huxley

layer

þ þ þ � � þ þ þ

Non-cornified

IRS cuticle

þ þ þ � þ þ þ �

Cornified IRS

cuticle

� � � þ þ þ

Medulla � � þ þ þ þ þ þ

Companion layer þ þ þ � � �

ORS of anagen

hair follicles

� � � �

Arrector pili

muscles

þ þ þ þ þ þ � �

Sweat glands

Secretory cells þ þ þ þ þ þ � �

Myoepithelial cells þ þ þ þ þ þ � �

Sebaceous glands � � � �

PAD, peptidylarginine deiminase; IRS, inner root sheath; ORS, outerroot sheath.


the recombinant enzyme, and immunohistochemical localization in hu-

man skin. J Invest Dermatol 115:813–823, 2000

Kim JK, Mastronardi FG, Wood DD, Lubman DM, Zand R, Moscarello MA: Multiple

sclerosis: An important role for post-translational modifications of myelin

basic protein in pathogenesis. Mol Cell Proteomics 2:453–462, 2003

Kubilus J, Baden HP: The occurrence and immunology of peptidylarginine dei-

minase and its preparation from bovine epidermis by an improved meth-

od. J Invest Dermatol 85:232–234, 1985

Langbein L, Rogers MA, Praetzel S, Winter H, Schweizer J: K6irs1, K6irs2, K6irs3,

and K6irs4 represent the inner-root-sheath-specific type II epithelial

keratins of the human hair follicle. J Invest Dermatol 120:512–522, 2003

Lee SC, Kim IG, Marekov LN, O’Keefe EJ, Parry DA, Steinert PM: The structure of

human trichohyalin. Potential multiple roles as a functional EF-hand-like

calcium-binding protein, a cornified cell envelope precursor, and an

intermediate filament-associated (cross-linking) protein. J Biol Chem

268:12164–12176, 1993

Masson-Bessiere C, Sebbag M, Girbal-Neuhauser E, Nogueira L, Vincent C,

Senshu T, Serre G: The major synovial targets of the rheumatoid arthritis-

specific antifilaggrin autoantibodies are deiminated forms of the alpha-

and beta-chains of fibrin. J Immunol 166:4177–4184, 2001

Mils V, Vincent C, Croute F, Serre G: The expression of desmosomal and corn-

eodesmosomal antigens shows specific variations during the terminal

differentiation of epidermis and hair follicle epithelia. J Histochem Cytoc-

hem 40:1329–1337, 1992

Mizoguchi M, Manabe M, Kawamura Y, et al: Deimination of 70-kD nuclear pro-

tein during epidermal apoptotic events in vitro. J Histochem Cytochem

46:1303–1309, 1998

Nachat R, Mechin MC, Takahara H, Chavanas S, Charveron M, Serre G, Simon

M: Peptidylarginine deiminase isoforms 1–3 are expressed in the epider-

mis and involved in the deimination of k1 and filaggrin. J Invest Dermatol

124:384–393, 2005

Nakashima K, Hagiwara T, Ishigami A, et al: Molecular characterization of pep-

tidylarginine deiminase in HL-60 cells induced by retinoic acid and

1alpha,25-dihydroxyvitamin D(3). J Biol Chem 274:27786–27792, 1999

Nicholas AP, Sambandam T, Echols JD, Tourtellotte WW: Increased citrullinated

glial fibrillary acidic protein in secondary progressive multiple sclerosis.

J Comp Neurol 473:128–136, 2004

Nissinen R, Paimela L, Julkunen H, Tienari PJ, Leirisalo-Repo M, Palosuo T,

Vaarala O: Peptidylarginine deiminase, the arginine to citrulline converting

enzyme, is frequently recognized by sera of patients with rheumatoid

arthritis, systemic lupus erythematosus and primary Sjogren syndrome.

Scand J Rheumatol 32:337–342, 2003

O’Guin WM, Sun TT, Manabe M: Interaction of trichohyalin with intermediate

filaments: Three immunologically defined stages of trichohyalin matura-

tion. J Invest Dermatol 98:24–32, 1992

Rawlings AV, Harding CR: Moisturization and skin barrier function. Dermatol Ther

17 (Suppl. 1):43–48, 2004

Rogers G: The localization and significance of arginine and citrulline in proteins of

the hair follicle. J Histochem Cytochem 11:700–705, 1963

Rogers GE, Taylor LD: The enzymic derivation of citrulline residues from arginine

residues in situ during the biosynthesis of hair proteins that are cross-

linked by isopeptide bonds. Adv Exp Med Biol 86A:283–294, 1977

Rogers G, Winter B, McLaughlan C, Powell B, Nesci T: Peptidylarginine deimi-

nase of the hair follicle: Characterization, localization, and function in

keratinizing tissues. J Invest Dermatol 108:700–707, 1997

Rothnagel JA, Rogers GE: Trichohyalin, an intermediate filament-associated pro-

tein of the hair follicle. J Cell Biol 102:1419–1429, 1986

Rus’d AA, Ikejiri Y, Ono H, Yonekawa T, Shiraiwa M, Kawada A, Takahara H:

Molecular cloning of cDNAs of mouse peptidylarginine deiminase type I,

type III and type IV, and the expression pattern of type I in mouse. Eur J

Biochem 259:660–669, 1999

Schmidt M, Gillitzer R, Toksoy A, et al: Selective expression of calcium-binding

proteins S100a8 and S100a9 at distinct sites of hair follicles. J Invest

Dermatol 117:748–750, 2001

Senshu T, Akiyama K, Kan S, Asaga H, Ishigami A, Manabe M: Detection of

deiminated proteins in rat skin: Probing with a monospecific antibody

after modification of citrulline residues. J Invest Dermatol 105:163–169,

1995

Senshu T, Kan S, Ogawa H, Manabe M, Asaga H: Preferential deimination of

keratin K1 and filaggrin during the terminal differentiation of human ep-

idermis. Biochem Biophys Res Commun 225:712–719, 1996

Serre G, Mils V, Haftek M, et al: Identification of late differentiation antigens of

human cornified epithelia, expressed in re-organized desmosomes and

bound to cross-linked envelope. J Invest Dermatol 97:1061–1072, 1991

Steinert PM, Harding HW, Rogers GE: The characterisation of protein-bound

citrulline. Biochim Biophys Acta 175:1–9, 1969

Steinert PM, Parry DA, Marekov LN: Trichohyalin mechanically strengthens the

hair follicle: Multiple cross-bridging roles in the inner root shealth. J Biol

Chem 278:41409–41419, 2003

Sun L, Sun TT, Lavker RM: CLED: A calcium-linked protein associated with early

epithelial differentiation. Exp Cell Res 259:96–106, 2000

Suzuki K, Sawada T, Murakami A, et al: High diagnostic performance of ELISA

detection of antibodies to citrullinated antigens in rheumatoid arthritis.

Scand J Rheumatol 32:197–204, 2003

Takahara H, Tsuchida M, Kusubata M, Akutsu K, Tagami S, Sugawara K: Pep-

tidylarginine deiminase of the mouse. Distribution, properties, and

immunocytochemical localization. J Biol Chem 264:13361–13368, 1989

Takizawa T, Arai S, Kizawa K, Uchiwa H, Sasaki I, Inoue T: Ultrastructural local-

ization of S100A3, a cysteine-rich, calcium binding protein, in human

scalp hair shafts revealed by rapid-freezing immunocytochemistry. J His-

tochem Cytochem 47:525–532, 1999

Tarcsa E, Marekov LN, Andreoli J, Idler WW, Candi E, Chung SI, Steinert PM: The

fate of trichohyalin. Sequential post-translational modifications by pep-

tidyl-arginine deiminase and transglutaminases. J Biol Chem 272:27893–

27901, 1997

Terakawa H, Takahara H, Sugawara K: Three types of mouse peptidylarginine

deiminase: Characterization and tissue distribution. J Biochem (Tokyo)

110:661–666, 1991

Urano Y, Watanabe K, Sakaki A, et al: Immunohistochemical demonstration of

peptidylarginine deiminase in human sweat glands. Am J Dermatopathol

12:249–255, 1990

Vossenaar ER, Radstake TR, Van Der Heijden A, et al: Expression and activity of

citrullinating peptidylarginine deiminase enzymes in monocytes and ma-

crophages. Ann Rheum Dis 63:373–381, 2004

Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ: PAD, a growing family of

citrullinating enzymes: Genes, features and involvement in disease. Bio-

essays 25:1106–1118, 2003

Wang Y, Wysocka J, Sayegh J, et al: Human PAD4 regulates histone arginine

methylation levels via demethylimination. Science 306:179–283, 2004

Watanabe K, Senshu T: Isolation and characterization of cDNA clones encoding

rat skeletal muscle peptidylarginine deiminase. J Biol Chem 264:15255–

15260, 1989

Winter H, Langbein L, Praetzel S, et al: A novel human type II cytokeratin, K6hf,

specifically expressed in the companion layer of the hair follicle. J Invest

Dermatol 111:955–962, 1998

Wood L, Carter D, Mills M, Hatzenbuhler N, Vogeli G: Expression of calcyclin, a

calcium-binding protein, in the keratogenous region of growing hair fol-

licles. J Invest Dermatol 96:383–387, 1991


peptidylarginine deiminase isoforms are differentially

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