Differentiation (1 992) 49 : 39-46

Differentiation Ontogeny, Neoplasia and Differentiation Therapy

0 Springer-Verlag 1992

Expression of loricrin is negatively controlled by retinoic acid in human epidermis reconstructed in vitro Thierry Magnaldo ', Frangoise Bernerd2, Daniel Asselineau ', and Michel Darmon I , * Cell Biology Department, Clinical Research Department, Centre International de Recherches Dermatologiques Galderma (CIRD Galderma), Sophia Antipolis, F-06565 Valbonne Cedex, France Accepted in revised form October 14, 1991

Abstract. In epidermis, the last steps of keratinocyte differentiation are characterized by the covalent cross-linking of cornified envelope precursors such as involucrin and loricrin, a hydrophobic protein recently described in mouse and human epidermis. In situ hybridization of normal human skin sections with a human loricrin cRNA probe and immunolabeling with an antiserum directed against a synthetic peptide corresponding to the carboxyterminus of human loricrin revealed the presence of loricrin transcripts and protein in the granular layers of epidermis. In human epidermis reconstructed in vitro by growing keratinocytes on dermal equivalents, loricrin and loricrin mRNAs were also restricted to granular cells, but their amounts seemed higher than in epidermis from skin biopsies. The reactivities for both loricrin and loricrin mRNAs were abolished by a treatment of the cultures with a retinoic acid concentration ( l o p 6 M) provoking a complete inhibition of terminal epidermal differentiation (parakeratosis). Thus, the regulation of loricrin synthesis is different from that of another envelope precursor, involucrin, which does not seem to be significantly modulated by retinoic acid. Together with the well-documented inhibition of epidermal transglutaminase by retinoic acid, our results provide a molecular basis for the inhibition of cornified envelope formation by retinoic acid.

Introduction Molecular studies have shown that the successive morphological steps of epidermal differentiation can be correlated to a program of sequential expression of major gene products of the keratinocyte such as keratins [16, 27, 29, 30, 33, 37, 44, 46, 551, involucrin [12, 19, 431 and Glaggrin [31, 341. Four stages of keratinocyte differentiation corresponding to the various layers of epider-

* To whom offprint requests should be sent

mis can be described : basal, spinous, granular and cornified [for review see 15, 591. Basal keratinocytes are actively proliferative and express mainly the K5-Kl4 keratin pair [61], although a subpopulation of these cells which starts to detach from the basement membrane already expresses the K1-K10 keratin pair [40, 46, 491. In the spinous layers, the K1-K10 keratin pair is fully expressed [16, 551 and the synthesis of K5-Kl4 keratins decreases [30, 531. It is also in the spinous layers that the last mitoses occur [40, 581. In the granular layers, membrane coating granules and keratohyaline granules appear [17, 311, and the process of cornified envelope formation is initiated [42, 43, 48, 54, 561. The crosslinking of envelope precursors is catalysed by the membranebound transglutaminase [7, 561, the nuclei disappear, and the corneocytes are assembled into the protective stratum corneum. Until recently, cornified envelopes were thought to be formed essentially by two epidermal-specific precursor proteins, involucrin [12, 19, 431 and keratolinin [62] and by other ubiquitous proteins [52]. However, the high percentage of Glu and Gln (Glx) residues (45%) found in involucrin [43] and deduced from the gene sequence [12] did not correlate with the amino acid composition of cornified envelopes purified either from skin (nearly 9% Glx) [35] or from cultured keratinocytes (nearly 16% Glx) [43]. This discrepancy was understood when Mehrel et al. [34] reported the isolation of a murine cDNA clone corresponding to a major keratinocyte envelope precursor that they called Ioricrin. This protein is a substrate for the membrane-bound transglutaminase and contains a high percentage of glycin (55.1%) and serin (22.3%) residues in good agreement with the ratios of these amino acids in cornified envelopes (nearly 44 and 23.6% respectively). We have previously reported the isolation from RNAs of human epidermis of a cDNA clone, A8 [32], closely homologous (about 80% in its 3' portion) to that isolated from murine epidermis by Nischt et al. [39] and Mehrel et al. [35]. We thus considered that this clone corresponded to human loricrin [32]. However, this as-

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sumption was only verified in the 3’ half of the A8 cDNA since its 5’ end differs completely from the human loricrin sequence published recently by Hohl et al. [25]. The present study (see Fig. 1) was thus performed using a subtotal A8 probe identical to the 3‘ end of the human loricrin cDNA [25]. In our previous work [32], we showed, using an A8 probe, that the amount of loricrin mRNA was reduced by retinoic acid. This was confirmed by Hohl et al. [24]. These observations are particularly interesting because retinoic acid plays important roles in the modulation of growth, differentiation, and morphogenesis of many tissues including epidermis (for review see 10, 13, 26, 45, 50, 601. In this paper, we report the localization by in situ hybridization and immunolabeling of loricrin mRNAs and protein in human skin and in cultured epidermis. Both loricrin mRNAs and protein were found to be dramatically reduced, in cultured epidermis, by concentrations of retinoic acid inhibiting stratum corneum formation and differentiation markers such as K10 keratin. Methods Construction of an epidermal c D N A library and differential hybridization. The construction of a human epidermal cDNA library and the cloning procedures of cDNAs corresponding to niRNAs regulated during epidermal differentiation have been described previously [S, 11, 18, 321. Among non-keratin “differentiation-spccific” cDNA clones, one of them (AS) was found to be homologous to a murine cDNA isolated by Nischt et al. [39], and to the 3‘ end of the mouse loricrin cDNA isolated later by Mehrel et al. [35]. We thus considered A8 to correspond to human loricrin [32]. Biological samples, cells and tissue culture. Normal human skin was obtained from either face lifting or plastic mammary reduclions. Samples were frozen in liquid nitrogen immediately after biopsy and stored at -70” C until use. Epidermal cell cultures on plastic: Human keratinocytes from normal skin samples were cultured according to the procedure of Rheinwald and Green [41] on a feeder layer of mitomycin-treated (2 h, 10 pg/ml) Swiss 3T3 fibroblasts. The growth medium was Eagle’s minimal essential medium supplemented with 10% total fetal calf serum (TFCS) (Seromed, FRG). Cultures were detached with dispase (grade 11, Boehringer, FRG) according to the technique of Green et al. [20] beforc freezing in tissue-Tek OTC (Miles, USA). Reconstructed epidermis: Dermal equivalcnts (lattices) were prepared as previously described in detail [ I , 51. Human keratinocytes were seeded into stainless steel rings on the lattice. Dermal equivalents were kept submerged for a week in culture medium until cells formed a confluent monolayer [2]. To induce stratification and differentiation, a second week of culture was performed at the air-liquid interface on stainless steel grids [3, 41. All-trans retinoic acid (CIRD Galderma) dissolved in diniethyl sulfoxide (DMSO) was added 24 11 bcfore emersion at a concentration of M (final DMSO concentration 0.1%) in media containing 10% TFCS. Control cultures contained 0.1 Yn DMSO. Tissue samples were prepared and stored as previously described [3]. Preparation of anti-loricrin antisera. A peptide (His-Gln-Thr-GlnGln-Lys-Gln-Ala-Pro-Thr-Trp-Pro-Ser-Lys) deduced from a unique coding sequencc immediately upstream of the TAG stop codon of A8 cDNA was synthesized (Neosystem, Strasbourg, France) and coupled to ovalbumin. This peptide was identical to the one

used by Mehrel et al. [35] for immunization, except for the presence of a Ser in the human sequence (aa: 480) instead of Cys in the murine sequence (aa: 314) [25, 321. Albino rabbits were immunized by injections in poplitheal lymph nodes [ 5 11. Immunoreactivities of sera were first assayed on samples of normal human breast skin according to the immunostaining procedures described below. One antiserum of good titer (AS-73) was selected for further studies. Immunoprecipitation with AS-73 antiserum : Cultures grown at the air-liquid interface were incubated overnight as previously described [2] except that 35S-methionine was replaced by 35S-cystein. Labeled proteins were extracted according to Mehrel et al. [35] in hot PBS-TDS (phosphate buffered saline containing 30% triton X100, 0.5% sodium deoxycholate, 0.1 % sodium dodecyl sulfate) except that lysed cells were sonicated for 10 min. Imniunoprecipitation was performed as described [21] using protein A-sepharose. Zmmunostainings. Mouse monoclonal antibody (Mab) to human K10 keratin (RKSE60) was purchased from Sanbio Laboratories (Netherlands). Mouse Mab to human collagen IV was purchased from Serotec (England). Mouse Mab to human K14 keratin (FBI) was a generous gift from Dr D. Parent (Belgium). Antiscra were diluted in phosphate buffered saline (PBS) as follows: 1/10 for anti-Kl0; ljl00 for anti-collagen IV; l/l000 for A8-73 anti-loricrin; mouse anti-rabbit IgG FITC conjugate (Dako, Denmark) was diluted 1/300. FB1 anti-K14 Mab was used undiluted. Indirect immunofluorescence on 5 pm frozen sections was performed as described [3]. After air-drying, the sections were rinsed in PBS, pH 7.2 (Biomkrieux Laboratorics, France) and immunolabeled at room temperature. Double staining with anti-KI0 and anti-collagen IV Mabs: Sections were incubated with both antibodies for 30 min, washed with PBS, incubated with rabbit anti-mouse IgG FITC conjugate (Dako, Denmark) for 30 min, washed again with PBS and mountcd. Immunoperoxidase staining with FBI anti-K14 MAb: All incubation steps were performed at room temperature and followed by washing in PBS. Tissue sections were incubated first with FBI Mab for 30 min, second with a biotinylated goat anti-rabbit IgG (1/200 in PBS) for 90 min, and third with an avidin-peroxidase complex (IjS0 in PBS) for 90 min (Vectastain Kit, Vector Laboratories, USA). Staining was revealed by incubation of samples in 33’diaminobenzidine (Sigma) for 15 min. In situ hybridization. Human loricin and K 5 and K10 keratin cRNA probes: The A8 cDNA used as a matrix for synthesis of loricrin riboprobes is a 432 bp fragment corresponding to the 3’ end, i.e. from nucleotides 803 to 1235 according to the numbering of Hohl et al. [25], of the previously reported AS sequence [32]. This is

467

-6

Fig. 1. Schematic representation of human loricrin cDNA. The coding sequence is shown as an open box; non-coding sequences are shown as solid lines. A : Full-length (1256 bp) human loricrin cDNA [25]. B: Region of human loricrin identical to AS cDNA [32] (from nucleotides 461 to 1235, according to the numbering published by Hohl et al. [25]).C: Human loricrin cDNA (432 bp) isolated from an epidermal pBR322 library [32] and used as a template to generate RNA probes for in situ hybridization

41 shown on Fig. I . Keratin K10 and K5 cDNAs were isolated previously [ I I , 181. The K10 fragment used as a probe corresponds to the first 230 nucleotides of the 3' noncoding region of exon 8 [ I I , 441. The K5 fragment corresponds to 500 nucleotides of the 3' end of the sequence [18]. All three cDNAs were subcloned in the same orientation into the Pstl restriction site of the pBluescript IT S K + vector (Stratagene, La Jolla, Calif., USA) and their sequence was checked by double-stranded DNA sequencing (Multiwell Sequencing Kit, Amersham) according to Sanger et ai. [47]. Loricrin, K5 and K10 keratin cRNA probes were generated by in vitro transcription from either the T3 (sense) or the T7 (antisense) promoters in the presence of 35S-UTP (1000 Ci/mMol, Amersham). After column purification and ethanol precipitation, 5 lo7 cpm/ml of RNA probes were added to a mixture of 50% deionized formamide, 50% 2 x hybridization buffer (40 m M piperazine-N, N-bis 2-ethanesulfonic acid (PIPES) pH 7.4, 1.2 M NaC1, 20% Dextran Sulfate, 200 mM dithiothreitol (DTT), 250 pg/ml of sheared salmon sperm DNA and 500 pg/ml yeast tRNA). In situ hybridization procedure: 6 pm vertical cryostat sections were put on 3-aminopropyltriethoxysilane pretreated glass slides (Digene Diagnostics Inc, USA), and fixed in 4% (w/v) paraformaldehyde in PBS, rinsed in PBS and stored in 70% ethanol at 4" C until use. Before hybridization, sections were rinsed in 2 x SSC (300 mM NaCI, 15 mM sodium citrate), acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine pH 8.0 for 15 min at room temperature, rinsed in 2 x SSC, dehydrated in 70% ethanol, then in 100% ethanol, and air dried. 20 pl of probe were added per section. Sections were covered with a slip and sealed with rubber cement. Hybridization was performed overnight a t 52" C in a water bath. After removing the rubber cement and excess hybridization mixture in a 2 x SSC wash, slides were rinsed in 4 x SSC 10 mM DDT for 1 h at room temperature, washed twice in 2 x SSC, 30 mM DDT, 50% formamide at 52" C for 30 min, and washed in 2 x SSC. Slides were immerged in RNAse A buffer, 10 niM TRIS HC1 pH 8.0, 1 mM EDTA, 0.3 M NaC1) and treated for 30 min at 37" C in RNAse A solution (40 mg RNase A/ml of buffer). Slides were washed in 2 x SSC, 50% formamide for 30 min, washed in 2xSSC, dehydrated in 70% ethanol, then in 100% ethanol, and air dried. Autoradiography was performed with Amersham LMI emulsion. Slides were revealed after 10 h, 3 days and 1 week of exposure and counterstained with Mayer's haemalum solution (Merck). Bright field micrographs were taken with a Zeiss Axiophot microscope.

Results Distribution of loricrin and loricrin mRNA in normal human epidermis

To study the distribution of loricrin mRNAs in human epidermis, skin sections were hybridized in situ with a specific human loricrin cRNA antisense probe corresponding to 432 bp located at the 3' end of A8 sequence (see Fig. 1, and Methods). In the section shown in Fig. 3A, a discrete labeling of the upper granular cells located just beneath the stratum corneum is apparent. This labeling is specific, as shown by the absence of signal in a section hybridized with the cRNA sense probe (Fig. 3B). For comparison, hybridizations were also performed with probes specific for K5 keratin (basal compartment) or K10 keratin (suprabasal compartment). With the K5 riboprobe, the labeling was seen predominantly in the basal layer although some grains were found in the first suprabasal layers (Fig. 3 C). The K10 riboprobe hybrid-

A

B

C

98 68

43

c 18

1

Fig. 2. Immunoprecipitation of keratinocyte extracts with the A873 anti-loricrin serum. Reconstructed epidermis was labeled with 35S-cystein.After immunoprecipitation, extracts were run in a 10% SDS-PAGE gel. A: total protein extract before immunoprecipitation ; B: immunoprecipitation with the preimmune serum corresponding to the A8-73 antiserum; C: immunoprecipitation with the A8-73 antiserum. The urrow indicates the putative loricrin band with the expected size of approximately 30 kD [24]. Heavy bands seen in all fractions correspond to keratins

ized preferentially to mRNAs present in several layers of cells of the spinous compartment [30, 531 and to a significant fraction of basal cells (Fig. 3 D), as previously reported by others [46]. Altogether, these results show that in normal human epidermis, loricrin transcripts appear in granular cells at a stage at which K10 mRNAs have already started to decrease. The reactivity of the A8-73 anti-loricrin serum was first checked by immunoprecipitation of 35S-cystein-labeled proteins extracted from cultured human epidermis. A protein of approximately 30 kD was specifically precipitated (Fig. 2). When used for immunofluorescence experiments, the A8-73 antiserum recognized antigens located not only in the upper granular cell layers (the site of loricrin transcripts), but also in the lower part of the stratum corneum (Fig. 3 E). Moreover, the staining predominated at the periphery of the granular keratinocytes where the cornified envelope is expected to be built (Fig. 3E). The pattern of loricrin expression was compared to the patterns of K14 (Fig. 3G) and K10 keratin expression (Fig. 3 H). As described in several studies, K14 keratin was detected in the basal layer and in a few parabasal layers [30, 531. K10 keratin was predominant in the spinous compartment of human epidermis but a few basal cells were also labeled [40, 46, 491. To confirm the specificity of the labeling obtained with the A8-73 antiserum, the corresponding rabbit preimmune serum was incubated with skin sections treated

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Fig. 3. Top: Distribution of loricrin, K5 and K10 keratin m R N A s in normal human epidermis. A (loricrin), C (K5) and E (K10) are in situ hybridizations with antisense probes. B: control performed with a sense loricrin probe. Note that in A, loricrin m R N A s are detected within the upper cells of the stratum granulosum (arrows) c: K5 transcripts were detected predominantly in the basal layer but also to a lesser extent in the first and second suprabasal rows of cells; D: K10 messages are detected in several suprabasal layers and in few basal cells ( u r r o ~ , ~Bottom: ). Distribution of loric-

rin, K5 and K10 keratins in normal human epidermis. E: indirect immunofluorescence labeling with A8-73 antiserum ; F: control staining with the preimniune serum corresponding to A8-73 ; C: staining of K14 keratin by FBI monoclonal antibody; H: staining of K10 keratin. Note that in E (loricrin antiserum), a specific staining was revealed a t the periphery of the uppermost granular cells. Triungles indicate the basement membrane (E, F, H) and u r r o ~ s point to the few K10-positive basal cells (H). Burs=25 pm

Fig. 4. Distribution of loricrin in keratinocytes grown on plastic. A : vertical section of the epithelium detached by dispase after two weeks of culture observed by phase contrast; B: indirect immunofluorescence with the A8-73 antiserum. Note that the staining is confined to the uppermost cells. Arrows indicate the bottom of the epithelium. Bars= 30 pm

subsequently with fluorescent conjugates. No specific labeling of granular cells was observed (Fig. 3 F). Moreover, preincubation of the A8-73 antiserum with the synthetic peptide used for immunization abolished the signal in granular cells (data nor shown). Expression of loricrin in keratinocytes cultured on plastic

When keratinocytes are cultured on plastic dishes, some differentiation and stratification occur, but the architecture of the epithelium remains rudimentary [4, 541. In the section shown on Fig. 4A, the nuclei are not com-

pletely digested in the upper layers, but the outmost cells are nevertheless flattened. Immunofluorescence staining of such sections with the anti-loricrin antiserum labeled specifically the superficial cells of the culture, which seemed ready to detach from the epithelium (Fig. 4B). Expression of loricrin and loricrin m R N A and of KIO keratin and K I O m R N A in human epidermis reconsiructed in absence and presence ofretinoic acid

Loricrin expression was studied in the reconstructed epidermis culture system developed by Asselineau et a].

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Fig. 5. Morphological aspect of reconstructed epidermis treated or not with retinoic acid and corresponding distribution of loricrin and loricrin mRNA and of KlO keratin and K10 mRNA. A, C, E, G , I: no retinoic acid; B, D, F, H, J: A4 retinoic acid. A, B: haemalum-eosin staining; C, D: in situ hybridization with the loricrin cRNA probe; E, F: immunostaining of loricrin; G , H: in situ hybridization with the K10 keratin cRNA probe; I, J: immunostaining of K10 keratin. Note that in cultures treated with retinoic acid, loricrin and loricrin mRNAs as well as K10 keratin and K10 keratin mRNAs, are not detected. Also note that in nontreated cultures, the loricrin transcripts were detected in the granular layers, whereas K 10 keratin transcripts were detected in the first suprabasal layers. In these cultures, loricrin was only found in granular cells, whereas K10 keratin was suprabasal. The staining of the nuclei seen in E and F was not specific since it was found with the preimmune serum (not shown). Triangles indicate the junction between the cultured epithelium and the dermal equivalent. Ban = 50 WM

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[3, 41. In a medium containing 10% TFCS, the overall architecture of the reconstructed epidermis (Fig. 5A) and the distribution of differentiation markers was closely similar to that of normal epidermis [4]. In cultures M retinoic acid (Fig. 5B), terminal treated with epidermal differentiation did not occur, as attested by the persistence of nuclei in the upper layers (parakeratosis), and the absence of most differentiation markers [4]. In situ hybridization of sections of reconstructed epidermis cultured in absence of retinoic acid were performed with either the loricrin probe or the K10 probe for comparison. Figure 5 C shows that loricrin transcripts are detected in the upper cells of the stratum granulosum. It is noteworthy that the loricrin messages appeared to be present in higher amounts of cultured epidermis than in skin biopsies (Fig. 3A). The A8-73 antiserum labeled as in skin biopsies the upper granular cells of the epithelium (Fig. 5E) and the lower part of the stratum corneum. Moreover, the periphery of the cytoplasm of the granular cells was preferentially stained (Fig. 5 E). The fluorescent staining observed in the spinous layers mainly in the nuclei was not specific, since it was also observed with the preimmune serum and was not observed in immunoperoxydase staining (data not shown). K10 mRNA (Fig. 5G) and K10 keratin (Fig. 51) were detected in the suprabasal layers of the cultured epidermis. In view of the density of silver grains, the amount of K10 messages was comparable to that detected in human epidermis (Fig. 3 D). We have previously shown that, in classical cultures of human keratinocytes, the amount of transcripts hybridizing with the human cDNA probe A8 [32] is significantly reduced by retinoic acid. The modulation by retinoic acid of human loricrin (and filaggrin) mRNA levels in cultured keratinocytes has also been reported by Hohl et al. [24]. To examine whether retinoic acid was also able to reduce loricrin expression in the reconstructed epidermis model, tissue sections of the parakeratotic epidermis obtained after culturing keratinocytes in the presence of M retinoic acid were hybridized in situ with either the loricrin or the K10 cRNA probe for comparison. In these culture conditions, neither loricrin nor K10 messages were detectable (Fig. 5D, H). Labeling with the A8-73 antiserum also showed a dramatic decrease if not a complete disappearance of loricrin (Fig. 5F). In fact, the residual staining obtained with the A8-73 antiserum was also seen with the preimmune serum and was probably non-specific (Fig. 3F). As previously reported [4, 281, K10 keratin was undetectable M retinoic acid in cultured epidermis treated with (Fig. 5 J). -

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Discussion

In this study we report the distribution of loricrin and loricrin transcripts in granular layers of human epidermis, both in vivo and in tissue culture. Moreover we show that retinoic acid provokes the disappearance of loricrin and loricrin transcripts from human epidermis reconstructed in vitro. These results are consistent with our previous data [32] and with data published recently

by Hohl et al. [24] obtained with classical cultures of keratinocytes. In newborn murine epidermis, Mehrel et al. [35], who also localized loricrin transcripts in granular cells, estimated that these transcripts were as abundant as those of K1 keratin. However, in human epidermis, loricrin transcripts seemed to us less abundant than those of K10 keratin (specific activities of the probes were similar). This point may also seem controversial to data obtained by Hohl et al. [25], showing that in human epidermis, loricrin transcripts are as abundant as the products of keratin and filaggrin genes. This discrepancy may be due to: 1) the different lengths of the riboprobes used by Hohl et al. [25] (approx. 200 bp) and by us (432 bp); 2) the fact that our probe contains approximately 180 bp of coding region, whereas the probe designed by Hohl et al. [25] corresponds only to the 3‘ non-coding region. Immunolabeling of loricrin was performed with a rabbit antiserum raised against a carboxyterminal synthetic peptide (14 mer). Both indirect immunofluorescence and immunoperoxydase staining (data not shown) localized the antigen in the upper granular cells. Moreover, the labeling was predominant a t the periphery of the cytoplasmic membranes. These results are in agreement with the one-sided internal pattern of loricrin distribution reported by Mehrel et al. [35]. In addition, the membranebound location of the envelope cross-linking enzyme transglutaminase [7], together with the peripheral detection of loricrin, supports the enzyme/substrate relationships of these proteins [35]. The isolation from cornified envelopes of peptides obviously derived from loricrin, but cross-linked by isodipeptide bonds [25] gives in addition a molecular basis for this relationship. A tissue culture model such as the “reconstructed epidermis” system [3, 4, 51 allowed us to study in situ, the regulation of loricrin expression, although loricrin transcripts seemed to be overexpressed in reconstructed epidermis compared to natural epidermis. Whereas in human skin, membrane-bound transglutaminase, involucrin and loricrin are coexpressed in granular cells, the regulations of these proteins by retinoic acid and/or stratification - which can be studied in vitro - appear to be different. Transglutaminase and involucrin are already detected in the spinous layers of non-treated cultured epidermis, i.e. more precociously than in skin [4, 561, but in contrast, loricrin occupies a correct location in the stratum granulosum of cultured epidermis. Moreover, when reconstructed epidermis cultures are treated with retinoic acid, both loricrin (this study and [22]) and transglutaminase [4] are repressed, while expression of involucrin, another substrate for epidermal transglutaminase, is not altered in our model [4], or slightly inhibited [22]. Thus, despite the lack of (or weak) sensitivity of involucrin to retinoic acid [4, 19, 221, the drastic repression of both loricrin [22, 24, 321 and transglutaminase [4, 14, 36, 571 are sufficient to explain the inhibitory effect of retinoic acid on cornified envelope assembly [9, 38, 571. Acknowledgements. We are indebted to Ms S. Robinson for helping us in immunization of rabbits and to Dr. B.A. Bernard for helping

45 us with immunoprecipitation experiments. We are grateful to Dr. D. Parent (Dermatology Department, H8pital Erasme, Brussels, Belgium) for the generous gift of FBI Mab.

References 1. Asselineau D, Prunieras M (1984) Reconstruction of simplified skin : control of fabrication. Br J Dermatol 111 , Supp 27 :21 9222 2. Asselineau D, Bernard BA, Bailly C, Darmon M (1985) Epidermal morphogenesis and induction of the 67 kD keratin polypeptide by culture at the liquid-air interface. Exp Cell Res 159:536-539 3. Asselineau D, Bernard BA, Bailly C, Darmon M, Prunieras M (1986) Human epidermis reconstructed by culture: is it normal? J Invest Dermatol 86:181-185 4. Asselineau D, Bernard BA, Bailly C, Darmon M (1989) Retinoic acid improves epidermal morphogenesis. Dev Biol 133 :322335 5. Bell E, Ivarsson B, Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 76: 12741278 6. Boukamp P, Rupniak HTR, Fusenig NE (1985) Environmental modulation of expression of differentiation and malignancy in six human squamous cell carcinoma cell lines. Cancer Res 45~5582-5592 7. Buxman MM, Wuepper K D (1978) Cellular localization of epidermal transglutaminase: A histochemical and immuno-chemical study. J Histochem Cytochem 26: 34Ck348 8. Chirgwin J, Przybyla A, MacDonald R, Rutter W (1979) Isolation of biologically active ribonucleic acids from sources enriched in rubonuclease. Biochemistry 18 : 5294-5299 9. Cline PR, Rice RH (1983) Modulation of involucrin and envelope competence in human keratinocytes by hydrocortisone, retinyl acetate, and growth arrest. Cancer Res 43 : 3203-3207 10. Darmon M (1991) Retinoic acid in skin and epithelia. In: Maden M, Tickle C (eds) Seminars in Developmental Biology. WB Saunders, London 2 :21 9-228 11. Darmon M, Semat A, Darmon MC, Vasseur M (1987) Sequence of a cDNA encoding human keratin number 10 selected according to structural homologies of keratins and their tissuespecific expression. Mol Biol Rep 12 :277-283 12. Eckert RL, Green H (1986) Structure and evolution of the human involucrin gene. Cell 46: 583-589 13. Eichele G (1989) Retinoids and vertebrate limb pattern formation. Trends Genet 5:24&251 14. Floyd EE, Jetten AM (1989) Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: Down regulation by retinoids. Mol Cell Biol9:4846-4851 15. Fuchs E (1990) Epidermal differentiation: the bare essentials. J Cell Biol 111 :2807-2814 16. Fuchs EV, Green H (1980) Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell 19:1033-1042 17. Fukuyama K, Epstein WL (1986) Keratohyalin. In: BereiterHahn J, Matoltsy AG, Richards KS (eds) The Biology of the Integument; Vertebrates, vol. 2. Springer Verlag, Berlin Heidelberg, pp 739-751 18. Galup C, Dannon M (1988) Isolation and characterization of a cDNA clone coding for human epidermal keratin K5. Sequence of the carboxyterminal half of this keratin. J Invest Dermatol91: 39-42 19. Green H, Watt FM (1982) Regulation by vitamin A of envelope cross-linking in cultured keratinocytes derived from different human epithelia. Mol Cell Biol2 : 1115-1 117 20. Green H, Kehinde 0, Thomas J (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 76: 5665-5668

21. Harper JR, Quaranta, V, Reisfeld RA (1986) Ammonium chloride interferes with a distinct step in the biosynthesis and cell surface expression of human melanoma-type condrohytin sulfate proteoglycan. J Biol Chem 261 :3600-3606 22. Hendricks L, Geesin JC, Roop DE, Gordon JS (1991) Retinoid suppression of loricrin expression in reconstituted human skin cultured at the liquid-air interface. J Invest Dermatol 96: 548A 23. Hennings H, Holbrook KA (1983) Calcium regulation of cellcell contact and differentiation of epidermal cells in culture. Exp Cell Res 143: 127-142 24. Hohl D, Lichti U, Breitkreutz Steinert PM, Roop DR (1991) Transcription of thc human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid. J Invest Dermatol 96:414-418 25. Hohl D, Lichti U, Turner ML, Roop DR, Steinert PM (1991) Characterization of human loricrin : structure and function of a new class of epidermal cell envelope proteins. J Biol Chem 266 :6626-6636 26. Jetten AM (1990) Multi-stage program of differentiation in human epidermal keratinocytes : regulation by retinoids. J Invest Dermatol 95 :44s-46s 27. Knapp B, Rentrop M, Schweizer J, Winter H (1987) The cDNA sequences of mouse type I keratins. Cellular localization of the mRNAs in normal and hyperproliferative tissues. J Biol Chem 262:938-945 28. Kopan R, Fuchs E (1989) The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells. J Cell Biol 109:295-307 29. Lehnert ME, Jorcano JL, Zentgraf H, Blessing M, Franz JK, Franke WW (1984) Characterization of bovine keratin genes : similarities of exon paterns in genes coding for different keratins. EMBO J 3 : 3279-3287 30. Lersch R, Fuchs E (1988) Sequence and expression of a type 11 keratin, K5, in human epidermal cells. Mol Cell Biol 8:486493 31. Lynley AM, Dale BA (1983) The characterization of human epidermal filaggrin : a histidine rich keratin filament aggregating protein. Biochem Biophys Acta 744: 28-35 32. Magnaldo M, Pommks L, Asselineau D, Darmon M (1990) Isolation of a GC-rich cDNA identifying mRNAs present in human epidermis and modulated by calcium and retinoic acid in cultured keratinocytes. Homology with murine loricrin mRNA. Mol Biol Rep 14:237-246 33. Marchuk D, McCrohon S, Fuchs E (1985) Complete sequence of a gene encoding a human type I keratin: sequences homolgous to enhancer elements in the regulatory region of the gene. Proc Natl Acad Sci USA 82: 1609-1613 34. McKinley-Grant LJ, Idler WW, Bernstein IA, Parry DAD, Cannizzaro L, Croce CM, Huebner K, Lessin SR, Steinert PM (1989) Characterization of a cDNA clone encoding human filaggrin and localization of the gene to chromosome region lq21. Proc Natl Acad Sci USA 86:4848-4852 35. Mehrel T, Hohl D, Rothnagel J, Longley M, Bundman D, Cheng C, Lichti U, Bisher M, Steven A, Steinert P, Yuspa S, Roop D (1990) Identification of a major keratinocyte cell envelope protein, loricrin. Cell 61 :1103-1112 36. Michel S, Reichert U, Isnard JL, Shroot B, Schmidt R (1988) Retinoic acid controls expression of epidermal transglutaminase at the pre-translational level. FEBS Lett 258:35-38 37. Moll R, Franke WW, Schiller DL, Geiger B, Knepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31 : 11-24 38. Nagae S, Lichti U, De Luca L, Yuspa SH (1987) Effect of retinoic acid on cornified envelope formation : difference between spontaneous envelope formation in vivo or in vitro and expression of envelope competence. J Invest Dermatol 89: 5158 39. Nischt R, Rentrop M, Winter H, Schweizer J (1987) Localization of a novel mRNA in keratinizing epithelia of the mouse:

46 evidcnce for the sequential activation of differentiation-specific genes. Epithelia 1 : 165-177 40. Regnier M, Vaigot P, Darmon M, Prunieras M (1986) Onset of epidcrmal differentiation in hyperproliferative basal keratinocytes. J Invest Dermatol87:472-476 41. Rheinwald JG, Green H (1975) Serial cultivation of strains of human keratinocytes : the formation of keratinizing colonies from single cells. Cell 6:331-343 42. Rice RH, Green H (1977) The cornified envelope of terminally differentiated human epidermal keratinocytes consists of crosslinked protein. Cell1 1 :417422 43. Rice R H , Green H (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of thc crosslinking by calcium ions. Cell 18 :681-694 44. Rieger M, Franke WW (1988) Identification of an orthologous mammalian cytokeratin gene. High degree of intron sequence conservation during evolution of human cytokeralin 10. J Mol Biol204:841-856 45. Roberts AB, Spron M B (1984) Cellular biology and biochemistry of the retinoids. In: Sporn MB, Roberts AB, Goodman DS (eds), The Retinoids, vol2. Academic Press, Fla, USA, pp 209-286 46. Roop DR, Krieg TM, Mehrel T, Cheng C K , Yuspa SH (1988) Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis. Cancer Res 4813245-3252 47. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 48. Schmidt R, Reichert U, Michel S, Shroot B, Bouclier M (1985) Plasma membrane transglutaminase and cornified envelope competence in cultured human keratinocytes. FEBS Lett 861201-204 49. Schweizer J, Kinjo M, Fiirstenberger G, Winter H (1984) Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis : basal cells with properties of terminally differentialing cells. Cell 37: 159-170 50. Sherman MI (1986) How do retinoids promote differentiation? In: Sherman MI (ed) Retinoids and cell differentiation. CRC Press, Boca Raton, Fla, USA, pp 161-186

51. Siege1 MB, Sinha YN, VanderLaan WP )1983) Production of antibodies by inoculation into lymph nodes. Methods Enzymol 93: 3-12 52. Simon M, Green H (1984) Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell 36: 827-834 53. Stoler A, Kopan R, Duvic M, Fuchs E (1988) Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation. J Cell Biol 107 :427-446 54. Sun TT, Green H (1976) Differentiation of the epidermal keratinocyte in cell culture: formation of cornified envelope. Cell 9 :511-521 55. Sun TT, Eichner R, Schermer A, Cooper D, Nelson WG, Weiss RA (1984) Classification, expression, and possible mechanisms of evolution of mammalian epithelial keratins: a unifying model. In: Levine AJ, Van de Woude GF, Topp WC, Watson J D (eds) Cancer Cells 1. The transformed phenotype. Cold Spring Harbor Laboratory, New York, pp 169-176 56. Thacher SM, Rice RH (1 985) Keratinocyte-specific transglutaminase of cultured human epidermal cells: relation to crosslinked envelope formation and terminal differentiation. Cell 40: 685-695 57. Thacher SM, Coe EL, Rice R H (1985) Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells. Differentiation 29 : 8287 58. Viac J , Reano A, Brochier J, Staquet MJ, Thivolet J (1983) Reactivity pattern of a monoclonal antibody (KLI). J Invest Dermatol 81 :351-354 59. Watt FM (1989) Terminal differentiation of epidermal keratinocytes. Curr Opin Cell Biol 1 :1107-1 115 60. Wolf G (1990) Recent progress in vitamin A research: nuclear retinoic acid receptors and their interaction with gene elements. J Nutr Biochem 1 :284289 61. Woodcock-Mitchell J , Eichner R, Nelson WG, Sun TT (1982) Immunoiocalization of keratin polypeptides in human epidermis, using monoclonal antibodies. J Cell Biol95 :580-588 62. Zettergren JG, Peterson LL, Wuepper K D (1984) Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue. Proc Natl Acad Sci USA 83 :238-242