Lysyl Oxidase-Like and Lysyl Oxidase Are Present in the Dermisand Epidermis of a Skin Equivalent and in Human Skin and AreAssociated to Elastic Fibers

Emmanuelle Noblesse,� Valerie Cenizo,�w Charbel Bouez,�w Agnes Borel,w Claudine Gleyzal,wSimone Peyrol,z Marie-Paule Jacob,y Pascal Sommer,w and Odile Damour,�w�Laboratoire des Substituts Cutanes, Hopital E. Herriot, Lyon, France; wInstitut de Biologie et de Chimie des Proteines, UMR5086-IFR128, Centre National de laRecherche Scientifique, Universite Claude Bernard Lyon 1, France; zCentre de Microscopie Electronique, Faculte de Medecine RTH Laennec, Universite ClaudeBernard 1, Lyon, France; yUnite INSERM U460, Institut National de la Recherche Medicale, UFR de Medecine Bichat, Paris, France

Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-

linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have

been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human

foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model,

keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG

matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal

skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression

patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association

with elastin-positive materials within the SE and human skin, though interaction with collagen could not be

discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and

human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating

keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data

favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes

should play a role in post-translational modification of yet unknown substrates.

Key words: extracellular matrix/fibroblast/keratinocyte/protein cross-linkingJ Invest Dermatol 122:621 –630, 2004

The skin is a complex organ composed of two main tissues:epidermis and dermis. The epidermis, a pluri-stratified anddifferentiated epithelium, is responsible for the barrierfunction. The dermis, a connective tissue in which cellsare embedded in their own and abundant extracellularmatrix (ECM), is mainly composed of collagen and manymacromolecules such as elastin. Elastic fibers are respon-sible for skin elasticity (Gibson et al, 1989), and collagenfibers for its tensile strength. While collagen fibers can besynthesized throughout life, elastic fibers are synthesizedmainly during development and growth, by smooth musclecells in blood vessels and by fibroblasts in skin (Vrhovskiand Weiss, 1998). The progressive disorganization of elasticfibers and their replacement by collagen fibers are majormanifestations of skin aging. Otherwise, burns or deepwounds result in scarring with loss of elasticity.

Elastic fibers, in contrast to collagen fibers, do notspontaneously assemble. The elastic fiber consists of two

distinct components: elastin and fibrillin-rich microfibrils.Elastin is synthesized as a 70 kDa monomer precursor thatis secreted through a trans-Golgi pathway (Davis andMecham, 1998). Elastogenesis is controlled by the 67 kDaelastin binding protein that acts as a recycling chaperoneand may bring the tropoelastin molecule to the microfibrillarscaffold (Hinek and Rabinovitch, 1994; Hinek et al, 1995).Recently, fibulin-5 has been reported to act as a scaffoldprotein at the surface of elastic fibers and to organize andlink elastic fibers to cells (Nakamura et al, 2002; Yanagisawaet al, 2002). Interaction of tropoelastin with microfibrilsoccurs via the amino-terminal domains of fibrillins 1 and 2,and is mediated by ionic interactions involving non-oxidizedlysine side chains of tropoelastin (Trask et al, 2000; Jensenet al, 2001). The C-terminal domain of tropoelastin isnecessary for correct orientation and subsequent cross-linking (Vrhovski and Weiss, 1998; Robb et al, 1999), whilelocal exposure to matrix glycosaminoglycans may favortropoelastin deposition (Wu et al, 1999). Microfibril bindingis thought to contribute to the alignment of tropoelastinmolecules, thus bringing together each cluster of fourpositively charged lysine side chains necessary for cross-linking (Bedell-Hogan et al, 1993). The initial step of thecross-linking reaction, which renders tropoelastin insoluble,

Abbreviations: LO, lysyl oxidase; DEJ, dermo-epidermal junction;DMEM, Dulbecco’s modified Eagle’s medium; DS, dermal sub-strate; ECM, extracellular matrix; GAG, glycosaminoglycan; PCP,procollagen-C-terminal proteinase; SE, skin equivalent; TEM,transmission electron microscopy

Copyright r 2004 by The Society for Investigative Dermatology, Inc.

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is an oxidative deamination of lysine residues by lysyloxidase to produce allysine, also known as a-amino adipicd-semialdehyde. All subsequent reactions are spontaneousand involve the condensation of closely positioned lysineand allysine residues to produce a variety of intra- andintermolecular cross-links, some unique to elastin (Reiseret al, 1992; Vrhovski and Weiss, 1998).

Lysyl oxidases have been originally described ascatalysts of the oxidative deamination of the e-amino groupof lysyl and hydroxylysyl residues of collagen and of thelysyl residues of elastin (Kagan and Li, 2003). Over the lastfew years, five genes (LOX, LOXL, LOR/LOXL2, LOR2/LOXL3, and LOXC/LOXL4) encoding putative lysyl oxidaseisoforms have been identified (Csiszar, 2001). Their specificroles are yet unknown. LOX, LOXL, and LOXL4 have beenshown to display enzymatic activities in vitro, using bothcollagen and tropoelastin as substrates. The 50 kDaglycosylated LOX precursor requires a maturation by theprocollagen-C-terminal proteinase (PCP) to be processedas a 32 kDa active enzyme (Trackman et al, 1992; Cronshawet al, 1995). A 56 kDa LOXL precursor has been recentlyisolated from bovine aorta. It can be cleaved in vitro by PCP,leading to a fraction active on both tropoelastin andcollagen (Borel et al, 2001). The 82 kDa LOXL4 precursorhas been reported to display enzymatic activity withoutactivation (Ito et al, 2001). The relation at the tissue and celllevels between LOX, LOXL, collagens, and elastin hashardly been addressed however. The relationship betweenLOX and collagen cross-linking has been demonstrated infibroblasts overexpressing the recombinant LOX (Seve et al,2002). This observation has been confirmed in null homo-zygote LOX �/� mice that display less collagen cross-linking. These LOX �/� mice, however, also display a 60%decrease in elastin cross-linking, as judged by the desmo-sine residual level (Hornstra et al, 2003). This suggests bothan involvement of LOX in elastin cross-linking and theexistence of other enzymes responsible for the residualelastin cross-linking activity. As the LOXL gene productshave been reported to be associated with the ECM intissues, LOXL is one candidate for this elastin cross-linkingactivity (Decitre et al, 1998; Kim et al, 1999).

The aim of this study was therefore to determine whetherLOXL is expressed in a skin-equivalent (SE) model allowingthe neosynthesis and maturation of collagen and elasticfibers (Duplan-Perrat et al, 2000). This SE is composed of adermal equivalent made of fibroblasts seeded in a porousdermal substrate (collagen, glycosaminoglycans, and chit-osan; Collombel et al, 1989), on top of which are furtherseeded keratinocytes. The epidermization induces elastinexpression and the formation of typical structures of anelastic network in the dermis, with elastin deposition on themicrofibrillar scaffold after long-term culture (Duplan-Perratet al, 2000). By comparison however with other skin modelswhere amorphous elastin has not been detected (Fleisch-majer et al, 1993), it is known that the presence ofkeratinocytes is not sufficient to trigger elastin depositionon microfibrils. This suggests that other regulatory mecha-nisms should occur, such as the influence of the composi-tion of the three-dimensional scaffold and of other regulatorsof the elastic fibers synthesis. The present investigation wasthen performed to gain a better understanding of the key

lysyl oxidase isoform(s) that govern the synthesis andcorrect assembly of elastic fibers in the SE. This modelallows for continuous assessment of elastic tissue formationand deposition that is impossible in the human skin.

Results

Immunodetection of LOXL and LOX in the SE The initialdetection of LOXL was achieved using the anti-LOXL231–368

antibody, used to identify the precursor form of tissue LOXL(Decitre et al, 1998; Borel et al, 2001). LOXL could bedetected using this anti-LOXL antibody at various levels ofthe dermis and epidermis of SEs, at different times ofculture (Fig 1a, c, e). On day 16, 2 d after keratinocyteseeding, LOXL was detected in fibroblasts in the dermalpart of the SE and mostly just beneath the dermo-epidermaljunction (DEJ), where the expression pattern is concen-trated as local spots as exemplified in Fig 1a. On day 35 (Fig1c), LOXL was detected beneath the DEJ. Deeper in thedermis, LOXL was associated with ECM neosynthesiswhere the porous structure was in the process of beingfilled. LOXL staining was intense in all the dermis on day 45(Fig 1e). On day 35, LOXL immunolabelling was observedfor all epidermal layers, with a more intense stainingbeneath the DEJ. This staining expanded throughout theepidermis by day 45. LOXL detection was eliminated byincubation of the anti-LOXL antibody with the specific GST-LOXL231–368 peptide prior to the immunodetection (Fig 1g).

To confirm our data, the laboratory developed a newanti-LOXL antibody, against the residues 355–416, whichrecognizes the smallest maturation product obtained byPCP cleavage of the human LOXL precursor at the S337–D338 bond (equivalent to the S354–D355 bond described onbovine LOXL; Borel et al, 2001). As this region shares about56% of similarity with the corresponding human LOX region(in contrast to the 7% with the region 231–238), we checkedits specificity using recombinant human LOX and LOXLexpressed in HeLa cells. The anti-LOXL355–416 antibodyreacted strongly with the recombinant LOXL precursor ofabout 69 kDa, while it did not react with the recombinant56 kDa LOX precursor (Fig 2). In such conditions, the anti-LOX228–279 antibody reacted with the 56 kDa recombinantLOX precursor and gave no reaction with the 69 kDa LOXLprecursor (Fig 2). The pattern of immunostaining using thenew anti-LOXL antibody was similar, though lighter, to theone obtained using the anti-LOXL231–368 antibody, with alabelling of the dermis and the epidermis on day 45 (Fig 1i ).

As found with LOXL, LOX was detected both in theepidermis and in the dermis of SE and normal skin (Fig 1b,d, f ). In the SE dermis, on day 16, LOX was detected inassociation with ECM (Fig 1b), with a higher and continuousstaining in the upper part of the dermis and beneath theforming DEJ. On day 35, LOX was observed throughout thedermis, though labelling was weak beneath the DEJ (Fig 1d ).On day 45, the staining was intense in the deep region of theDS, while staining under the DEJ remained weak (Fig 1f ). Inthe epidermis, on day 16 the single layer of keratinocytesseeded on the dermal layer was unlabelled (Fig 1b ). Onday 35, LOX labelling of the SE mainly concernedthe suprabasal layers, especially the spinous layers, while the

622 NOBLESSE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

labelling of the basal layer remained weaker (Fig 1d). On day45, LOX epidermal staining was restricted to the basal andspinous layers (Fig 1f ), displaying a difference in expressionpattern with LOXL. LOX staining decorated the periphery ofkeratinocytes and formed a framework around juxtaposedcells. Labelling was eliminated by a competition with thecorresponding GST-LOX peptide (Fig 1h).

Immunodetection of LOXL and LOX in human skin Inorder to determine whether the positive expression of LOXL

and LOX in the SE could be correlated to human tissues,different samples of human skin were analysed. Here,observations made on the foreskin were shown (Fig 1). Theimmunodetection of LOXL within the epidermal layer of SEwas confirmed in human foreskin (Fig 1k). It was alsoobserved at the same location in skin samples from theneck or the breast (data not shown). The staining could bepericellular and cytoplasmic. LOXL staining was low in thedermis and stronger around blood vessels (Fig 1k). LOX wasweakly detected in the whole dermis of these differentskins, together with an intense detection within the basaland suprabasal layers of the epidermis (Fig 1l ).

LOXL and LOX gene expression in the SE and inkeratinocytes To further confirm LOX and LOXL expres-sion by keratinocytes, their gene expression was analyzedby RT-PCR. Preliminary data were obtained on cells fromthe separated dermis and epidermis of SE obtained after 45d of growth, once the basement membrane is built. Theamplification of LOXL and LOX PCR products indicated thatkeratinocytes could express both genes (data not shown). Alow amount of collagen mRNA was detected however,indicating a low amount of contamination by fibroblasts. Wetherefore performed RT-PCR on keratinocytes grown on amonolayer without a feeder layer. LOX expression was notsignificantly detected in undifferentiated keratinocytes (Fig3). Its detection increased in differentiated keratinocytespositive for Keratin K10. In contrast, LOXL was expressed inboth non-differentiated and differentiated keratinocytes.The slight amplification of collagen I should be due to theresidual fibroblasts from the original feeder layer. Thiscontamination was reduced after different cell passages indefined medium, as in differentiated keratinocytes whereLOX amplification increased.

Figure 1Immunohistological detection of LOXL and LOX in skin equivalents(SE) and human foreskin. LOXL immunodetection (a, c, e, g, i ) on day16 2 d after keratinocytes seeding (a), day 35 (c), and day 45(e, i ) using the anti-LOXL231–368 (a, c, e), the anti-LOXL231–368 antibodyadsorbed with the corresponding GST-LOXL231–368 peptide prior to theimmunodetection (g), and the new anti-LOXL355–416 (i ). LOX immuno-detection (b, d, f, h) on day16 (b), day 35 (d ), and day 45 (f ) using theanti-LOX228–279 (b, d, f ) or the anti-LOX228–279 antibody adsorbed withthe corresponding GST-LOX228–279 peptide prior the immunodetection(h). A control without the first antibody displayed no staining ( j ).Immunodetection in human foreskin using anti-LOXL231–368 (k) and anti-LOX228–279 (l ) antibodies. The immunolabelling is indicated by a browndeposition and the counterstaining gives the tissue its blue color. Thechitosan-cross-linked collagen-GAG matrix appears in blue. Scale bar:.50 mm.

Figure2Verification of antibody specificity against LOX and LOXL humanrecombinant proteins. Hela cells were transfected using pcLOXL-His(1, 3, 5) or pcLOX-His (2, 4, 6), and their cell layers were harvested 48 hpost-transfection. Cell layer proteins were analyzed on 10% SDS PAGEand immunodetected by western blotting using the new anti-LOXL355–

416 (lanes 1 and 2), anti-LOX228–279 (lanes 3 and 4) antibodies, and theanti-V5 antibody (lanes 5 and 6). The migration of the protein markers isindicated with their respective molecular masses (in kDa).

LYSYL OXIDASES IN A SKIN EQUIVALENT 623122 : 3 MARCH 2004

We performed in situ hybridization to confirm and locateLOXL or LOX gene expression on the SE (Fig 4). On day 16,LOX and COL1A1 mRNAs could be detected in the dermis(beneath the keratinocytes) by in situ hybridization, while atthe same time LOXL and tropoelastin (TE) mRNAs werealmost not detected (data not shown). On day 35,expression of both LOXL and LOX genes was observed(Fig 4a and b). The LOXL gene was expressed by fibroblastsin the dermis and by keratinocytes throughout the epidermis(Fig 4a). The LOX gene was intensively expressed byfibroblasts colonizing the DS (Fig 4b). Suprabasal keratino-cytes displayed strong labelling for LOX whereas basal cells

appeared not stained. Interestingly, the expression of theelastin gene, which was positive as expected in fibroblastsof the dermis, was also observed, though weakly, through-out the epidermis (Fig 4c). In contrast, the expression ofCOL1A1 gene (as a control) was restricted to the dermis ofthe SE (Fig 4d ).

These experiments demonstrated the expression of bothLOX and LOXL genes by keratinocytes and confirmed theirdifferential expression pattern. LOXL was expressed byproliferating and non-differentiated keratinocytes, both inmonolayer culture and in the basal layer of the epidermis ondays 35 or 45. LOXL was also expressed by differentiatedkeratinocytes and in the suprabasal cells of the epidermis.In contrast, the LOX gene was not or weakly expressedin proliferating keratinocytes (either grown in monolayer orin the basal layer of the epidermis). LOX required thedifferentiation of keratinocytes to be upregulated, eitherafter calcium induction of keratinocytes or within thesuprabasal layers of epidermis.

Immunoelectron microscopy To further localize LOXL andLOX antigens in the SE, we performed immunolabelling atthe electron microscopic level. Assays were performedon the SE on day 45, where elastin was detected asamorphous material, associated or not with microfibrils(Duplan-Perrat et al, 2000). Though the SE was fragile, theimmunodetection of LOXL allowed its detection throughoutthe dermis in relation to the same kind of materialsresponding positively to elastin immunolabelling (Fig 5a),using both anti-LOXL antibodies (Fig 5b and c). The goldparticles could also be localized, though more rarely, inregions rich in collagen fibers, using both anti-LOXL anti-bodies as exemplified with the anti-LOXL355–416 (Fig 5c).The immunolabelling of LOX allowed its detection in thedermis, close to collagen fibers and to microfibrils withoutsuggesting any preferences (Fig 5d ).

In order to confirm the relation of LOXL with elastin-positive materials, its detection was performed on normalhuman skin, with foreskin tissues, less fragile and therefore

Figure 3RT-PCR analysis of LOX and LOXL gene expression in fibroblastsand keratinocytes. RT-PCR was performed for LOXL, LOX, a1ColI,Keratin K10, and GAPDH on mRNA extracted from fibroblasts (F),undifferentiated keratinocytes (Ku), and differentiated keratinocytes(Kd).

Figure 4LOXL (a), LOX (b), elastin (c), and a1(I ) collagen mRNAs (d ) expression in SE on day 35. In situ hybridization was performed using Dig-labelleddouble-stranded DNA probes on paraffin-embedded sections. (a) LOXL mRNA expression is detected in the whole dermis and throughout theepidermis. (b) LOX mRNA expression is mainly observed in the deep dermis. In the epidermis, cells of the basal layer are negative for LOX geneexpression, while the spinous and granulous layer cells are strongly positive. (c) Elastin mRNA is found in association with dermal fibroblasts andwithin the epidermis. (d ) COL1A1 gene expression is detected in the dermis and not in the epidermis. (e) Control without probe. The DEJ position isindicated by arrows.

624 NOBLESSE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

more adequate to perform double immunostaining (Fig 6a).The anti-LOXL231–368 and anti-elastin antibodies were ableto recognize the amorphous materials, characteristic ofelastic fibers, and the microfibrils (Fig 6b). The labellingusing the anti-LOXL231–368 antibody was mainly observed atthe periphery of the elastic fibers. As for the SE, staining ofthe collagen fibers using the anti-LOXL231–368 antibodywas rare (Fig 6b). LOX was immunodetected in relationto microfibrils or to elastic fibers deposits (Fig 6c), but incontrast to LOXL, LOX could also be found in associationwith collagen fibers (Fig 6d ).

Discussion

This study was performed to examine one paradigm ofelastic and collagen fiber synthesis, i.e. the initiation of thecross-linking reaction by lysyl oxidases, in a SE model thatallows the synthesis of both kinds of fibers (Duplan-Perratet al, 2000). The SE thus provides a unique model to studythis elastogenesis process, in contrast to the skin. The lysyloxidases form a growing family of five enzymes (Csiszar,2001). The role of these enzymes in tissues is yet largelyunknown. Using enzymatic assays performed in vitro, therecombinant LOX, the 56 kDa LOXL precursor from bovineaorta activated by PCP, and the 82 kDa LOXL4 precursor

have been described to use collagen and tropoelastin assubstrates (Wang et al, 1996; Borel et al, 2001; Ito et al,2001). Recent studies suggested an involvement of LOX incollagen and elastin cross-linking in cells and tissues (Seveet al, 2002; Hornstra et al, 2003; Kozel et al, 2003). Here, thiswork describes the behavior of LOXL and LOX during thedevelopment of the SE in order to address the question oftheir respective role in collagen and elastin cross-linking.

In the dermis, immunohistological and in situ hybridiza-tion analyses show that both LOXL and LOX are expressed.LOX expression can be observed on day 16, together withcollagen I, 1 d after the addition of keratinocytes. It shouldbe noted that both proteins are already expressed prior tokeratinocytes seeding (data not shown). In contrast, on day16, LOXL and elastin are rarely observed. On day 35, LOXLis localized beneath the DEJ where LOX is nearly absent.On day 45, we still note an expression of LOXL, LOX,elastin, and collagen in the dermis. LOX is also found in thedeep dermis, a location that might correspond to thetransient expression at the front of ECM neosynthesisalready described for LOX (Sommer et al, 1993). Theultrastructural localization of LOXL within the dermis of theSE as well as the time course of its expression suggest itsproximity to elastin-positive dense deposits and to micro-fibrils and its functional relation to the elastic tissue,respectively. This is confirmed within human skin, where

Figure 5(a–d). Elastin, LOXL, and LOX immunodetection by electron microscopy in the dermal part of the SE on day 45. Elastin was detected inassociation with dense deposits or microfibrils (a). LOXL was also detected with such dense deposits or microfibrils positive for elastin, using anti-LOXL355–416 (b) and the anti-LOXL231–368 (c). LOXL could be, though rarely, associated with collagen fibers as exemplified here (c). LOX labellingcould be found with dense deposits, microfibrils and with collagen (d). Negative control without primary antibody in the dermis (e). e, elastin-positivematerial; m, microfibrils; c, collagen fibers. Scale bar: 200 nm.

LYSYL OXIDASES IN A SKIN EQUIVALENT 625122 : 3 MARCH 2004

LOXL antibody labels the periphery of elastic fibers, ratherthan collagen fibers. The ultrastructural immunodetection ofLOX is related both to collagen and elastic fibers of the SEand the human skin.

A putative privileged association of lysyl oxidase iso-forms toward collagen or elastic fibers should be viewed inthe light of divergent reports that have previously addressed

this question. Using antibodies against a 32 kDa lysyloxidase isolated from bovine aorta, which contains sepa-rable protein isoforms (Kagan et al, 1979; Williams andKagan, 1985), Kagan et al (1986) have been able to localizethe enzyme at the interface between amorphous elasticfibers and the surrounding microfibrils in aortic tissues,while no reaction was obtained on collagen fibers. Using

Figure 6Double immunodetection of LOXL, LOX and elastin by electron microscopy in the dermal part of human foreskin. (a, b) Double labelling withanti-LOXL231–368 antibody (10 nm) and anti-elastin antibody (20 nm). LOXL was detected in association with microfibrils and around elastic fibers,not in association with the amorphous elastin-positive material spreading on microfibrils but mainly at their periphery, and rarely with collagen fibers.(c) Double labelling with anti-LOX antibodies (10 nm) and anti-elastin antibodies (20 nm) allowed one to detect LOX with elastin-positive fibers (c).LOX could also be detected on collagen fibers, shown here below the DEJ (d, e). The labelling corresponds to gold particles which appear as blackdots of 10 and 20 nm. m, microfibrils; c, collagen fibers; e, amorphous elastin. Scale bar: 200 nm.

626 NOBLESSE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

antibodies against the 32 kDa protein isolated fromplacenta, Pasquali-Ronchetti and coworkers stained col-lagen fibers of human skin, but they did not find a specificlocalization on or adjacent to elastin and microfibrils(Baccarani-Contri et al, 1989). As discussed by these latterauthors, this discrepancy might reflect the different speci-ficity of the antibodies toward lysyl oxidase isoforms or thepresence of masking proteins, such as proteoglycans(Baccarani-Contri et al, 1989; Quaglino et al, 1993). Ourdata, using specific antibodies against LOX and LOXL, arein favor of a relation between LOX and both collagen andelastic fibers, and between LOXL and elastic fibers,respectively, while the relation between LOXL and collagencannot be discarded. The relation between LOX andcollagen has already been reported (Seve et al, 2002). Co-regulation of LOX and collagen I has been described in theschistosomiasis liver fibrosis model, and a DNA sequence,LOcol1, found in the promoters of the LOX, a1(I) and a2(I)collagen genes, regulates the LOX promoter (Sommer et al,1993; Jourdan-Le Saux et al, 1997). On the other hand,upregulation of LOXL expression precedes collagen I andLOX expression in the schistosomiasis and in the CCl4-induced hepatic fibrosis models (Decitre et al, 1998; Kimet al, 1999). In both cases, LOXL expression coincides withcollagen III expression, though the relation with elastin hasnot been addressed. Accordingly, if the relation betweenLOX and collagen I fibers has been conclusively establishedat the promoter, protein, cell, and tissue levels, the questionremained open for the relation of LOX, LOXL, and elasticfiber components. Our data suggest that both LOX andLOXL can be present on elastic fibers, though further workis required to study their interaction and their regulationduring the formation and aging of elastic fibers. On the otherhand, it also remains to clarify which lysyl oxidase isoformis responsible for collagen IV cross-linking, knowing thatinhibition of lysyl oxidase activity by b-aminoproprionitrile(b-APN), a specific inhibitor of lysyl oxidase activity (Smith-Mungo and Kagan, 1998), interferes with the formation ofthe basement membrane of the SE (Noblesse et al,unpublished data). The use of the SE described here couldbe useful to answer these questions.

One intriguing finding in this study is the observation ofLOX and LOXL within the epidermis. The expression of LOXby human keratinocytes has already been documented(Wakasaki and Ooshima, 1990; Kobayashi et al, 1994). Theexact location of LOX and LOXL within the epidermis couldnot be determined by immunoelectron microscopy, as adiffuse concentration of gold labels was observed in thecytoplasm of keratinocytes and between the keratinocytes,in association with amorphous structures (data not shown).Incidentally, we observed that neither LOX nor LOXL isassociated with desmosome junctions, which are essen-tially unlabelled. The substrates of LOX and LOXL could beone of the numerous proteins that fill the space betweenkeratinocytes, or intracellular proteins involved in cellsignalling. Collagens, the main classical substrates of LO,are not yet known to be present in the epidermis, excepttype XIII collagen (Peltonen et al, 1999). Although collagenXIII can be found between keratinocytes however, its mainlocalization is between the basal layer of keratinocytes andthe basement membrane, where neither LOX nor LOXL as

found in large amounts. Many other proteins of the cornifiedcell envelope, including involucrin, small proline-rich pro-teins, loricrin and filaggrin, are known to be substrates fortransglutaminases (Aeschlimann and Thomazy, 2000), butthe localization of LOX and LOXL suggests that some ofthem might also be cross-linked by lysyl oxidase isoforms.Another candidate substrate for LOX or LOXL betweenkeratinocytes might be elastin. Different reports havedescribed the expression of elastin and microfibril mRNAsin keratinocytes (Haynes et al, 1997; Kajiya et al, 1997).Interestingly, it has been reported that elastin mRNAexpression is increased after UV exposure in the epidermallayers (Seo et al, 2001). No data however have beenpresented on the role of elastin as an epidermal cellconstituent. The observation of elastin molecule, but notnecessarily elastic fibers, within the epidermis of the SE andother reports in the skin (Haynes et al, 1997; Kajiya et al,1997; Seo et al, 2001) lead to future studies where it will beinteresting to determine whether or not elastin can bedeposited in normal human epidermis. In this regard, it isinteresting to note that elastin peptides have been reportedto influence the keratinocyte migration and differentiation(Fujimoto et al, 2000a, b).

The putative existence of LOX substrates within oroutside the cell might be related to yet unexplained featuresof this enzyme, first discovered as a tumor suppressor geneand also using the basic fibroblast growth factor assubstrate (Contente et al, 1990; Kenyon et al, 1991; Li et al,2003). Deregulation of LOX, by expressing an antisenseLOX cDNA in fibroblasts, induces a loss of contact inhibitionand decreases the expression pattern of proteins involvedin cell–matrix interactions, resulting in a highly tumorigenicphenotype (Giampuzzi et al, 2001). As LOX can regulate theactivity of NF-kB in fibroblasts (Jeay et al, 2003) and isinduced during the first stages of keratinocyte differentiation(this study), it would be interesting to determine its roleduring the NF-kB-dependent differentiation of keratinocytes(Bell et al, 2002). The location of LOX (and LOXL) and theiryet unknown substrate(s), at the periphery of keratinocytesor fibroblasts, would make sense in the light of a role for thisenzyme in cell–matrix or cell–cell contacts. The SE providesa new cell model to study the mechanisms that target LOXand LOXL to unexpected locations and will help to identifynew functions for the LO family of proteins.

Materials and Methods

Human cell cultures Keratinocytes and fibroblasts were isolatedfrom human foreskin. Fibroblasts were grown in Dulbecco’smodified Eagle’s medium (DMEM with Glutamax-1, Life Technol-ogies, Cergy Pontoise, France) supplemented with 10% fetal calfserum (FCS) (HyClone, Logan, Utah), 20 mg per mL gentamicin(Panpharma, Fougeres, France), 100 IU per mL penicillin (Sarbach,Suresnes, France), and 1 mg per mL amphotericin B (Bristol MyersSquibb, Puteaux, France). For the SE, keratinocytes were grownin a 3:1 mixture of DMEM and Ham’s F12 (Life Technologies),respectively, supplemented with 10% FCS (HyClone), 10 ng permL epidermal growth factor (EGF) (Austral Biologic, San Ramon,California), 0.12 IU per mL insulin (Lilly, Saint-Cloud, France), 0.4 mgper mL hydrocortisone (UpJohn, St Quentin en Yvelines, France), 5mg per mL triiodo-L-thyronine (Sigma, St Quentin Fallavier, France),24.3 mg per mL adenine (Sigma), and antibiotics. For keratinocyte

LYSYL OXIDASES IN A SKIN EQUIVALENT 627122 : 3 MARCH 2004

monolayer culture, cells were grown on a feeder layer of irradiatedfibroblasts, or without a feeder layer in defined K-SFM medium(Life Technologies) supplemented or not during 5 d after con-fluence with 1.2 mM CaCl2.

Preparation of dermal equivalent Fibroblasts from the seventhpassage were seeded at a density of 250,000 cells per cm2 onto adermal substrate (DS) made of chitosan-cross-linked collagen-GAG matrix prepared as previously described (Collombel et al,1989). This dermal equivalent was grown for 14 d at 371C in a 5%CO2 atmosphere. The fibroblast medium was supplemented with50 mg per mL L-ascorbic acid (Sigma) and changed every day.

Preparation of SE Keratinocytes were seeded on the dermalequivalent on day 14, at a density of 250,000 cells per cm2

(Duplan-Perrat et al, 2000). Briefly, after 7 d of submerged culturein the keratinocyte medium, the SE was elevated at the air–liquidinterface and cultured in a simplified keratinocyte medium contain-ing DMEM supplemented with 10% FCS, 10 ng per mL EGF, 0.12IU per mL insulin, 0.4 mg per mL hydrocortisone, and antibiotics.The SE samples were harvested at 16, 35, and 45 d of culturefollowing fibroblasts seeding. Samples were harvested for histol-ogy, immunohistology studies, electron microscopy, and in situhybridization studies.

Production and characterization of anti-LOXL antibodies Thefirst polyclonal rabbit anti-LOX and anti-LOXL antibodies used inthis study have been described previously (Sommer et al, 1993;Borel et al, 2001). These rabbit anti-LOX and anti-LOXL antibodieshave been raised against the regions 222–356 of murine LOX and231–368 of human LOXL, respectively. One new anti-LOXL anti-body was raised against the region 355–416 of human LOXL, fusedin frame to the glutathion-S-transferase (GST). The chimerical genewas constructed by the in-frame insertion of human LOXL cDNA atthe end of the glutathion-S-transferase (GST) gene in the BamHI-XhoI sites of pGEX-4T-3 expression plasmid (Amersham, Orsay,France). The GST-LOXL355–416 fusion gene was constructed byintroducing the sequence of human LOXL cDNA, generated byPCR using the sense 50-TTGGATCCAGCGTAGGCAGCGTGTAC-30

primer, the antisense 50-AAACTCGAGCATCGTAGTCGGTGGC-30

primer, and bioTaq polymerase (Bioprobe). As control, a new anti-LOX antibody was raised in an equivalent region, against the region228–279 of human LOX. The GST-LOX228–279 fusion gene wasconstructed by introducing the human LOX sequence amplifiedusing the 50-TTGGATCCGTGCAGAAGATGTCCATGTAC-30 and 50-TTTCTCGAGGCTGGGTAAGAAATCTGATG-30 sense and antisenseprimers, respectively.

The antibodies were obtained in rabbits and purified asdescribed (Sommer et al, 1993; Borel et al, 2001). For controlexperiments, the antibodies were purified by affinity chromato-graphy on the corresponding peptide antigen as described (Borelet al, 2001). For the adsorption experiments, the antibodies wereincubated for 3 h at 201C with the respective GST-fusion proteinscoated on a nitrocellulose Hybond-ECL membrane (Amersham)prior to the immunodetection test.

To check the new antibodies, the full-length LOXL fragment wasamplified by PCR from cDNA of MRC5 fibroblasts using theExpand High Fidelity PCR System (Roche, Mey Pan, France). Thesense 50-CAGAGGATCACCATGGCTCTG-30 and antisense 50-CGAGACAGGAGTCGACAATTTTGCAG-30 primers were used.The amplified segment displayed one difference with the publishedsequence, with a guanine in position 421 instead of a thymine(Kenyon et al, 1993). This introduced a leucine141 to arginine141

transition, also found in all checked LOXL sequences from theNCBI library. We therefore used this LOXL coding sequence,introduced under the control of the CMV promoter and in framewith a V5-His C-terminal tail. Taking advantage of the restrictionsites introduced into the PCR primers, BamHI for the forward andHincII/SalI for the reverse (this primer eliminates also the stopcodon of LOXL), the 1744 bp PCR product was cloned into thepBluescript II SKþ vector (Ozyme, St. Quentin, France) and

sequenced (MWG Biotech, Ebersberg, Germany). The BamHI/SalIfragment was subcloned into the pcDNA4/V5-His type A (Invitro-gen) expression vector using its BamHI and XhoI sites. Theresulting vector was called pcLOXL-His and was used for transienttransfection experiments. The �33 to þ 1284 LOX sequences wasinserted into pcDNA4 (Invitrogen, Cergy-Pontoise, France) asdescribed (Seve et al, 2002), resulting in pcLOX-His. Transienttransfections of Hela cells were performed using the Lipofectaminereagent (Life Technologies) on pre-confluent cells. Protein expres-sion was detected from the cell layer using the anti-LO antibodiesor using an anti-V5 antibody (Invitrogen) at a dilution of 1:100, bywestern blotting as described (Decitre et al, 1998), using theAlkaline Phosphatase Conjugate Substrate Kit (Biorad, Marne laCoquette, France). Protein mass markers were purchased fromBiorad.

Histology and immunohistology procedures

Histology and immunohistochemistry studies The SE was fixed inBouin’s fixative or in 10% formalin solution (Sigma) and embeddedin paraffin. Sections (6 mm) were de-paraffinized and whitened inglycine-HCl (100 mmol per liter). Beside the anti-LOX and anti-LOXL antibodies, the anti-human elastin antibodies were obtainedfrom Sigma and Novotec (Lyon, France). The antibodies were usedat the following dilution: 1:1000 (anti-LOX222–356), 1:500 (anti-LOXL231–368, anti-elastinNovotec), 1:100 (anti-LOXL355–416), and 1:50(anti-elastin Sigma). The staining of elastin requires an unmaskingstep using 0.2% hyaluronidase (Sigma). Peroxidase-conjugatedgoat anti-rabbit IgG (DAKO, Trappes, France) was used to detectthe immunocomplexes, using diaminobenzidine as substrate(DAKO). Counterstaining was performed using Harris’ hematoxylin.For controls, the primary antibody was omitted. Multiple serialsections of each specimen were processed to ensure representa-tive samples.

Immunoelectron microscopy Tissues were fixed for 3 h at 41C with4% paraformaldehyde in PBS containing 0.1% glutaraldehyde,washed in phosphate buffer containing 0.4 M cacodylate-sacchar-ose and 0.2 M lysine, dehydrated in graded ethanol solutions, andembedded in LR White (Euromedex, Mundols, France). Post-embedding labelling was performed using the primary antibodies(anti-LOXL, anti-LOX, anti-elastin (Sigma)) diluted 1:50 in Tris–HClbuffer (pH 8.2), supplemented with 1% BSA, and then with 10 nmgold-conjugated anti-rabbit IgG or 20 nm gold-conjugated anti-mouse IgG (Biocell, Tebu, France) diluted 1:40, respectively.Samples were contrasted using aqueous 3% uranyl acetate andlead citrate buffer, and then examined on a JEOL 1200 EX electronmicroscope.

In situ hybridization and reverse transcriptase polymerasechain reaction (RT-PCR) Double-stranded DNA probes wereproduced using the polymerase chain reaction (PCR). Thefollowing sense and antisense primers were used: for humana1(I) collagen, primers 50-GTGGAGAGTACTGGATTG-30 and 50-TCGTGCAGCCATCGACAG-30 (allowing the amplification of themRNA region 3812–4249); for human tropoelastin, 50-GTATATACC-CAGGTGGCGTG-30 and 50-CGAACTTTGCTGCTGCTTTAG-30 (re-gion 443 of 799); for human LOX, 50-GGTGGCCGACCCCTACTACATCC-30 and 50-GCAAATCGCCTCTGGTAGCCATAGTC-30 (region645 of 1016); for human LOXL, 50-GACATAACCGACGTGCAGCC-30 and 50-ATCCACGTTCGCTCCCTGAG-30 (region 1564 of 1869).The DNA was amplified using Taq Polymerase (Promega, Char-bonnieres, France) and Dig-11-dUTP (Roche, Meylan, France)as labelled nucleotide. In situ hybridization was performed onparaffin-embedded sections, as previously described (Le Guellecet al, 1994), with modifications. Briefly, samples were de-paraffinized and treated using proteinase K (Roche) at 2 mg perml for 15 min at 201C. Endogenous peroxidases were inhibited asindicated in the TSAþ amplification kit (NEN, Boston, Massachus-sets). Prehybridization was performed for 2 h at 371C in 20 mMphosphate buffer (pH 7.4), supplemented with 50% deionized

628 NOBLESSE ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

formamide, 2 � SSC, 5 mM EDTA, 2.5 � Denhardt’s solution, 200mg per mL denaturated herring sperm DNA, 1 mg per ml salmonsperm DNA, and 10 mg per mL tRNA. Hybridization was performedfor 16 h at 371C in 20 mM phosphate buffer (pH 7.4), supplementedwith 50% deionized formamide, 2 � SSC, 5 mM EDTA,2.5 � Denhardt’s solution, 200 mg per mL denaturated herringsperm DNA, 10% dextran sulfate, with or without the probespreviously denaturated for 5 min in a boiling water bath. Followinghybridization, the slides were successively washed twice for 10min at 201C (or 371C for collagen) in 2 � SSC/50% formamide,1 � SSC/50% formamide, 1 � SSC, and 0.5 � SSC. After dehy-dration, the Dig-labelled hybrids were detected using an anti-DIGantibody conjugated to horseradish peroxidase (Roche). The finaldetection of complexes was carried out using the TSAþ

amplification kit (NEN) as described by the manufacturer. Positivesignals were shown to correspond to the alkaline phosphataseactivity linked to the TSA amplification procedure, carried out for2 h at room temperature, as judged by the precipitation oftetrazolium salts (using NBT/BCIP as substrates).

In addition to the LOX and LOXL primers, the following primerpairs were used for the detection of gene expression by RT-PCR onhuman fibroblast and keratinocyte mRNA: for glyceraldehyde 3-phospho-dehydrogenase (GAPDH), 50-ATCACTGCCACCCAGAA-GAC-30 and 50- ATGAGGTCCACCACCCTGTT-30; for KeratinK10, 50-ACCGGTGATGTGAATGTGG-30 and 50-CCTGAATCTGT-GAGAGCTGC-30 (amplifying the 878–1208 human K10 segment);for collagen a1(I), 50-GCCGTGACCTCAAGATGTGC-30 and 50-GGTTTGACTTGGGGGGGTTT-30.

We thank Dominique Le Guellec and Jean Farjanel (IBCP), Marie-LiseCouble (Universite Lyon 1), Eric Perrier and Valerie Andre (Coletica,Lyon), and Sylviane Guerret (Novotec) for their advice. This work wassupported by the Centre National de la Recherche Scientifique, theUniversite Lyon 1, the Hospices Civils de Lyon, the Region Rhone-Alpes, the French Association pour la Recherche sur le Cancer, theEuropean Community (QLK6-CT-2001-00332), and Coletica. E.N. wasa recipient of a studentship from the CNRS. Agnes Borel and CharbelBouez are recipients of studentships from the French government.Valerie Cenizo is a recipient of a studentship from the Region Rhone-Alpes.

DOI: 10.1111/j.0022-202X.2004.22330.x

Manuscript received July 10, 2003; revised October 22, 2003;accepted for publication October 23, 2003

Address correspondence to: Pascal Sommer, Institut de Biologie etChimie des Proteines, CNRS UMR 5086, 7 Passage du Vercors, 69367Lyon cedex 07, France. Email: p.sommer@ibcp.fr

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