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In vitro reconstructed normal human epidermis expresses differentiation-related proteoglycans
Journal of Dermatological Science (2008) 51, 135—138
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR In vitro reconstructed normal human epidermis expresses differentiation-related proteoglycans KEYWORDS In vitro reconstructed human epidermis; Proteoglycans; Immunohistochemistry
Intercellular spaces of human epidermis contain extracellular matrix elements, such as proteoglycans (PGs), which are susceptible of influencing the cell behaviour, e.g. regulate cell proliferation, migration or differentiation. We have previously described a new epidermal PG, desmosealin, present between keratinocytes and integrated into the extracellular parts of desmosomes [1,2]. Differentiation-related expression of desmosealin in normal human epidermis could be demonstrated with the post-embedding immunogold labelling [1]. Because the ‘‘outside-in’’ regulation of the keratinocyte behaviour may be mediated by extracellular matrix molecules [3,4], we have studied the expression of desmosealin and of some of the other epidermal PGs in two commercially available air-exposed keratinocyte culture systems, Episkin1 and SkinEthic1 (SkinEthic Laboratories, Nice, France). The differences between Episkin1 and SkinEthic1 lay essentially in the medium composition (SkinEthic medium is serum-free) and in the fact that the reconstructed human epidermis is grown either on a collagen substrate or a polycarbonate porous membrane, respectively. Ten, 13 and 17-day emerged cultures of keratinocytes from various donors of different ages (abdominal and foreskin) were compared. Fragments of normal human skin were used as the controls. The tissues were fixed and embedded in paraffin using standard procedures. Four-micrometer paraffin sections of all specimens were dewaxed, rehydrated, microwave pre-treated in the antigen retrieval citrate buffer (Dako ChemMate; Trappes, France; pH 6; 2 30 at 700 W) and used for immunohistochemical staining. In this study
we have concentrated on the family of syndecans, with its prominent differentiation-related member syndecan-1, and the hyaluronic acid receptor CD44, the proteoglycans with expression patterns similar to the two newly described molecules, desmosealin and 7C1. The following antibodies were employed: mouse monoclonals anti-CD44v3/epican (clone VFF-327V3; at 1:30; Abcys SA, Paris, France), anti-CD44 H-CAM (clone DF1485, recognizing all forms of CD44; 1:30; Santa Cruz Biotechnology, USA), anti-syndecan-4 (clone 5G9; 1:100; Santa Cruz Biotechnology), anti-desmosealin (KM48 hybridoma supernatant; Lyon [1]), 7C1 antibody to another chondroitin/ dermatan sulphate PG (a generous gift of Prof. H. Suzuki [2,5]), and rabbit polyclonal antibodies H174 anti-syndecan-1, M140 anti-syndecan-2 (both at 1:100; Santa Cruz Biotechnology). Localization of the respective antigens was revealed with the LSAB2 kit (Dako) and an AEC peroxidase substrate (Invitrogen). A biotinylated hyaluronic acid-binding protein, b-HABP (5 mg/ml; Seikagaku, Tokyo, Japan) was used with the same detection system to stain this free glycosaminoglycan. All sections were slightly counter-stained with haematoxylin. Both epidermal models showed normal-looking stratification and keratinisation upon emersion (Figs. 1 and 2). However, SkinEthic1 cultures preserved a higher number of viable cell layers for a longer time, whatever the source of keratinocytes–— from adults or children (data not shown). The hyaluronan receptor CD44 H-CAM was strongly expressed in all epidermal tissues studied. Its pericellular pattern of staining was observed from the basal layer on and persisted until the most superficial keratinocytes of the stratum spinosum. This distribution closely followed the presence of hyaluronic acid detected in the epidermal intercellular spaces with bHABP. The visible differences in the number of cell layers expressing CD44 were related mostly to the tissue architecture obtained in a particular experimental setting. The CD44 isoform recognized by an antiCD44v3 antibody showed an epidermal localization similar to H-CAM but was more restricted to the
0923-1811/$30.00 # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2008.03.007
136
Letter to the Editor
Fig. 1 Epidermal proteoglycan expression in the Episkin1 model of reconstructed human epidermis, compared to the staining patterns in normal human skin. Normal human skin (A—D) and Episkin1 at 10 days of air exposure (E—H) were immunohistochemically stained with chosen anti-proteoglycan antibodies (see the letter). The cultures showed a thick stratum spinosum, well visible granular layer, and an orthokeratotic horny layer. CD44 antibodies (A and E) stained the basal and spinous layers in all specimens. Syndecan-1 showed a gradient of pericellular expression increasing from the basal to the granular layer (B and F). In the reconstructed epidermis expressing a thick stratum granulosum, the gradient reached only the lower granular keratinocytes (F). The gradient of desmosealin (C and G) and 7C1 antigen (D and H) expression was more extended, comprising the entire granular layer (400).
deepest epithelial layers. Some differences in the expression of CD44 variants during keratinocyte proliferation and differentiation were previously described by Zhou et al., with a general down-regulation of the CD44 expression in the differentiated cells compared to the proliferating ones [6]. Also the polyclonal anti-syndecan-1 antibody detected the extracellular fragment of the protein in all studied tissues, with the pericellular staining
of keratinocytes increasing gradually from the basal to the lower granular layer. The upper granular keratinocytes showed considerably less labelling; the difference was particularly well visible in the cultures expressing a thick granular layer. Unusual for the syndecans cytoplasmic staining was observed with the anti-syndecan-2 antibody, showing predominant localization in the upper layers of the viable part of the epidermis. The nature and significance of
Fig. 2 Epidermal proteoglycan expression in the SkinEthic1 model of reconstructed human epidermis. At 10 days after emersion, SkinEthic1 model was characterized by well-developed viable epidermal layers, no matter the source of keratinocytes (A—D, child foreskin, E—H, adult abdominal skin). The tissues in E—H were fixed with FineFix instead of the buffered formaldehyde. Patterns of immunohistochemical staining were similar in all cultures and not different from the Episkin1, except of the desmosealin (C and G) which showed an incomplete and irregular gradient of distribution (400).
Letter to the Editor this labelling, observed also on the control skin sections, remains unexplained. Previously, syndecan-2 was scarcely observed in the normal human skin [7], sometimes on Western blot only [8]. The antibody to syndecan-4 showed no specific reactivity with the in vitro reconstructed tissues. This was not dependent on technical problems related to tissue fixation and/or embedding methods, since the antigen could be correctly detected, although with a variable staining intensity, on the control normal skin sections (at least in the epithelial structures of sweat glands; not shown). Syndecan-4 is an inducible molecule related to the keratinocyte activation during wound healing [8]. The lack of syndecan-4 induction in the tested cultures suggests that the in vitro reproduced Episkin1 and SkinEthic1 systems reach a relative homeostasis, similar to the situation in vivo, already at 10 days after emersion. 7C1 chondroitin/dermatan sulphate PG was strongly expressed in all viable epidermal layers, with a gradient increasing from the basal to the granular layer. The staining was stronger in the Episkin1 cultures, where its traces persisted in the horny layer, whereas immunoreactive deposits could be observed at the bottom of the SkinEthic1 cultures, in the polycarbonate membranes. Also desmosealin was stronger expressed in Episkin1 than in SkinEthic1 and the typical peripheral pattern of keratinocyte staining increasing gradually from the basal to granular layers could only be fully observed in the former. More acanthotic cultures showed a patchy pattern of desmosealin staining localized in the upper spinous layers, in the areas with no signs of modified expression of the other PGs. Poorer desmosealin visualization in these sections was not dependent on the tissue fixation since it was apparent both after FineFix (non-aldehydic fixative; Tech-Inter, Thoiry, France) and after standard buffered formalin treatment. Thus, desmosealin differs from the other epidermal proteoglycans by its expression closely dependent on the terminal differentiation of keratinocytes. The distinctive pattern of desmosealin expression indicates a more specific involvement of this PG, incorporated into desmosomes, in the process of epidermal keratinisation. Altogether, there were very few striking variations in the reactivity pattern of antibodies directed against PGs depending on the type of the reconstructed epidermal model employed. For most PGs, the existing differences in histochemical staining were mostly related to the epidermal morphology depending, in turn, on various durations of the emerged culture and on the culture conditions, e.g., medium composition. Our results underline the relationship between the PG expression and
137 regulation of keratinocyte differentiation. The in vitro epidermal culture systems may be useful for further studies on epidermal PG expression and function.
References [1] Haftek M, Viac J, Schmitt D, Gaucherand M, Thivolet J. Ultrastructural quantitation of desmosome and differentiation-related keratinocyte membrane antigen. Arch Dermatol Res 1986;278:283—92. [2] Duhieu S, Le Bitoux M-A, Schmitt D, Suzuki H, Ishikawa K, Haftek M. Proteoglycans in the intercellular cores of desmosomes and corneodesmosomes. J Invest Dermatol 2004;123: A4. [3] Duhieu S, Laperdrix C, Boukerche H, Ishikawa K, Sommer P, Schmitt D, et al. Epidermal extracellular matrix: new arguments for a new idea. J Invest Dermatol 2003;121: 1571. [4] Mu ¨ller EJ, Williamson L, Kolly C, Suter MM. Outside-in signalling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation? J Invest Dermatol 2008;128:501—16. [5] Suzuki H, Horii M, Miyamoto R, Ishikawa K, Inoue K, Tanaka S. Subcellular distribution of 220 kDa antigen in the intercellular spaces of normal human epidermis. Arch Dermatol Res 1997;289:360—6. [6] Zhou J, Haggerty JG, Milstone LM. Growth and differentiation regulate CD44 expression on human keratinocytes. In Vitro Cell Dev Biol Anim 1999;35:228—35. [7] Lundqvist K, Schmidtchen A. Immunohistochemical studies on proteoglycan expression in normal skin and chronic ulcers. Br J Dermatol 2001;144:254—9. [8] Gallo R, Kim C, Kokenyesi R, Adzick NS, Bernfield M. Syndecans-1 and -4 are induced during wound repair of neonatal but not fetal skin. J Invest Dermatol 1996;107:676—83.
Marek Haftek* Marie-Aude Le Bitoux1 Sylvie Callejon1 ´ Lyon 1, EA4169, ‘‘Normal and Universite Pathological Functions of the Skin Barrier’’, Laboratoire de Recherche Dermatologique, ˆpital E. Herriot, Pavillon R, Ho 69437 Lyon, France Alain Denis2 Ingrid Pernet2 Bioderma Laboratory, 75 Cours Albert Thomas, 69003 Lyon, France Carole Amsellem3 Episkin SNC, 4 Rue Alexander Fleming, 69366 Lyon, France Be ´atrice Bertino4 SkinEthic SA, 45 Rue St. Philippe, 06000 Nice, France
138
Letter to the Editor *Corresponding author. Tel.: +33 472 11 02 92; fax: +33 472 11 02 90 E-mail address: [email protected] (M. Haftek)
1
Tel.: Tel.: 3 Tel.: 4 Tel.: 2
+33 +33 +33 +33
472 442 437 493
11 60 28 97
02 73 72 77
93; 78; 08; 27;
fax: fax: fax: fax:
+33 +33 +33 +33
472 442 437 493
11 24 28 97
02 34 72 77
90. 54. 28. 28.
20 June 2007
Available online at www.sciencedirect.com
www.intl.elsevierhealth.com/journals/jods
LETTER TO THE EDITOR In vitro reconstructed normal human epidermis expresses differentiation-related proteoglycans KEYWORDS In vitro reconstructed human epidermis; Proteoglycans; Immunohistochemistry
Intercellular spaces of human epidermis contain extracellular matrix elements, such as proteoglycans (PGs), which are susceptible of influencing the cell behaviour, e.g. regulate cell proliferation, migration or differentiation. We have previously described a new epidermal PG, desmosealin, present between keratinocytes and integrated into the extracellular parts of desmosomes [1,2]. Differentiation-related expression of desmosealin in normal human epidermis could be demonstrated with the post-embedding immunogold labelling [1]. Because the ‘‘outside-in’’ regulation of the keratinocyte behaviour may be mediated by extracellular matrix molecules [3,4], we have studied the expression of desmosealin and of some of the other epidermal PGs in two commercially available air-exposed keratinocyte culture systems, Episkin1 and SkinEthic1 (SkinEthic Laboratories, Nice, France). The differences between Episkin1 and SkinEthic1 lay essentially in the medium composition (SkinEthic medium is serum-free) and in the fact that the reconstructed human epidermis is grown either on a collagen substrate or a polycarbonate porous membrane, respectively. Ten, 13 and 17-day emerged cultures of keratinocytes from various donors of different ages (abdominal and foreskin) were compared. Fragments of normal human skin were used as the controls. The tissues were fixed and embedded in paraffin using standard procedures. Four-micrometer paraffin sections of all specimens were dewaxed, rehydrated, microwave pre-treated in the antigen retrieval citrate buffer (Dako ChemMate; Trappes, France; pH 6; 2 30 at 700 W) and used for immunohistochemical staining. In this study
we have concentrated on the family of syndecans, with its prominent differentiation-related member syndecan-1, and the hyaluronic acid receptor CD44, the proteoglycans with expression patterns similar to the two newly described molecules, desmosealin and 7C1. The following antibodies were employed: mouse monoclonals anti-CD44v3/epican (clone VFF-327V3; at 1:30; Abcys SA, Paris, France), anti-CD44 H-CAM (clone DF1485, recognizing all forms of CD44; 1:30; Santa Cruz Biotechnology, USA), anti-syndecan-4 (clone 5G9; 1:100; Santa Cruz Biotechnology), anti-desmosealin (KM48 hybridoma supernatant; Lyon [1]), 7C1 antibody to another chondroitin/ dermatan sulphate PG (a generous gift of Prof. H. Suzuki [2,5]), and rabbit polyclonal antibodies H174 anti-syndecan-1, M140 anti-syndecan-2 (both at 1:100; Santa Cruz Biotechnology). Localization of the respective antigens was revealed with the LSAB2 kit (Dako) and an AEC peroxidase substrate (Invitrogen). A biotinylated hyaluronic acid-binding protein, b-HABP (5 mg/ml; Seikagaku, Tokyo, Japan) was used with the same detection system to stain this free glycosaminoglycan. All sections were slightly counter-stained with haematoxylin. Both epidermal models showed normal-looking stratification and keratinisation upon emersion (Figs. 1 and 2). However, SkinEthic1 cultures preserved a higher number of viable cell layers for a longer time, whatever the source of keratinocytes–— from adults or children (data not shown). The hyaluronan receptor CD44 H-CAM was strongly expressed in all epidermal tissues studied. Its pericellular pattern of staining was observed from the basal layer on and persisted until the most superficial keratinocytes of the stratum spinosum. This distribution closely followed the presence of hyaluronic acid detected in the epidermal intercellular spaces with bHABP. The visible differences in the number of cell layers expressing CD44 were related mostly to the tissue architecture obtained in a particular experimental setting. The CD44 isoform recognized by an antiCD44v3 antibody showed an epidermal localization similar to H-CAM but was more restricted to the
0923-1811/$30.00 # 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2008.03.007
136
Letter to the Editor
Fig. 1 Epidermal proteoglycan expression in the Episkin1 model of reconstructed human epidermis, compared to the staining patterns in normal human skin. Normal human skin (A—D) and Episkin1 at 10 days of air exposure (E—H) were immunohistochemically stained with chosen anti-proteoglycan antibodies (see the letter). The cultures showed a thick stratum spinosum, well visible granular layer, and an orthokeratotic horny layer. CD44 antibodies (A and E) stained the basal and spinous layers in all specimens. Syndecan-1 showed a gradient of pericellular expression increasing from the basal to the granular layer (B and F). In the reconstructed epidermis expressing a thick stratum granulosum, the gradient reached only the lower granular keratinocytes (F). The gradient of desmosealin (C and G) and 7C1 antigen (D and H) expression was more extended, comprising the entire granular layer (400).
deepest epithelial layers. Some differences in the expression of CD44 variants during keratinocyte proliferation and differentiation were previously described by Zhou et al., with a general down-regulation of the CD44 expression in the differentiated cells compared to the proliferating ones [6]. Also the polyclonal anti-syndecan-1 antibody detected the extracellular fragment of the protein in all studied tissues, with the pericellular staining
of keratinocytes increasing gradually from the basal to the lower granular layer. The upper granular keratinocytes showed considerably less labelling; the difference was particularly well visible in the cultures expressing a thick granular layer. Unusual for the syndecans cytoplasmic staining was observed with the anti-syndecan-2 antibody, showing predominant localization in the upper layers of the viable part of the epidermis. The nature and significance of
Fig. 2 Epidermal proteoglycan expression in the SkinEthic1 model of reconstructed human epidermis. At 10 days after emersion, SkinEthic1 model was characterized by well-developed viable epidermal layers, no matter the source of keratinocytes (A—D, child foreskin, E—H, adult abdominal skin). The tissues in E—H were fixed with FineFix instead of the buffered formaldehyde. Patterns of immunohistochemical staining were similar in all cultures and not different from the Episkin1, except of the desmosealin (C and G) which showed an incomplete and irregular gradient of distribution (400).
Letter to the Editor this labelling, observed also on the control skin sections, remains unexplained. Previously, syndecan-2 was scarcely observed in the normal human skin [7], sometimes on Western blot only [8]. The antibody to syndecan-4 showed no specific reactivity with the in vitro reconstructed tissues. This was not dependent on technical problems related to tissue fixation and/or embedding methods, since the antigen could be correctly detected, although with a variable staining intensity, on the control normal skin sections (at least in the epithelial structures of sweat glands; not shown). Syndecan-4 is an inducible molecule related to the keratinocyte activation during wound healing [8]. The lack of syndecan-4 induction in the tested cultures suggests that the in vitro reproduced Episkin1 and SkinEthic1 systems reach a relative homeostasis, similar to the situation in vivo, already at 10 days after emersion. 7C1 chondroitin/dermatan sulphate PG was strongly expressed in all viable epidermal layers, with a gradient increasing from the basal to the granular layer. The staining was stronger in the Episkin1 cultures, where its traces persisted in the horny layer, whereas immunoreactive deposits could be observed at the bottom of the SkinEthic1 cultures, in the polycarbonate membranes. Also desmosealin was stronger expressed in Episkin1 than in SkinEthic1 and the typical peripheral pattern of keratinocyte staining increasing gradually from the basal to granular layers could only be fully observed in the former. More acanthotic cultures showed a patchy pattern of desmosealin staining localized in the upper spinous layers, in the areas with no signs of modified expression of the other PGs. Poorer desmosealin visualization in these sections was not dependent on the tissue fixation since it was apparent both after FineFix (non-aldehydic fixative; Tech-Inter, Thoiry, France) and after standard buffered formalin treatment. Thus, desmosealin differs from the other epidermal proteoglycans by its expression closely dependent on the terminal differentiation of keratinocytes. The distinctive pattern of desmosealin expression indicates a more specific involvement of this PG, incorporated into desmosomes, in the process of epidermal keratinisation. Altogether, there were very few striking variations in the reactivity pattern of antibodies directed against PGs depending on the type of the reconstructed epidermal model employed. For most PGs, the existing differences in histochemical staining were mostly related to the epidermal morphology depending, in turn, on various durations of the emerged culture and on the culture conditions, e.g., medium composition. Our results underline the relationship between the PG expression and
137 regulation of keratinocyte differentiation. The in vitro epidermal culture systems may be useful for further studies on epidermal PG expression and function.
References [1] Haftek M, Viac J, Schmitt D, Gaucherand M, Thivolet J. Ultrastructural quantitation of desmosome and differentiation-related keratinocyte membrane antigen. Arch Dermatol Res 1986;278:283—92. [2] Duhieu S, Le Bitoux M-A, Schmitt D, Suzuki H, Ishikawa K, Haftek M. Proteoglycans in the intercellular cores of desmosomes and corneodesmosomes. J Invest Dermatol 2004;123: A4. [3] Duhieu S, Laperdrix C, Boukerche H, Ishikawa K, Sommer P, Schmitt D, et al. Epidermal extracellular matrix: new arguments for a new idea. J Invest Dermatol 2003;121: 1571. [4] Mu ¨ller EJ, Williamson L, Kolly C, Suter MM. Outside-in signalling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation? J Invest Dermatol 2008;128:501—16. [5] Suzuki H, Horii M, Miyamoto R, Ishikawa K, Inoue K, Tanaka S. Subcellular distribution of 220 kDa antigen in the intercellular spaces of normal human epidermis. Arch Dermatol Res 1997;289:360—6. [6] Zhou J, Haggerty JG, Milstone LM. Growth and differentiation regulate CD44 expression on human keratinocytes. In Vitro Cell Dev Biol Anim 1999;35:228—35. [7] Lundqvist K, Schmidtchen A. Immunohistochemical studies on proteoglycan expression in normal skin and chronic ulcers. Br J Dermatol 2001;144:254—9. [8] Gallo R, Kim C, Kokenyesi R, Adzick NS, Bernfield M. Syndecans-1 and -4 are induced during wound repair of neonatal but not fetal skin. J Invest Dermatol 1996;107:676—83.
Marek Haftek* Marie-Aude Le Bitoux1 Sylvie Callejon1 ´ Lyon 1, EA4169, ‘‘Normal and Universite Pathological Functions of the Skin Barrier’’, Laboratoire de Recherche Dermatologique, ˆpital E. Herriot, Pavillon R, Ho 69437 Lyon, France Alain Denis2 Ingrid Pernet2 Bioderma Laboratory, 75 Cours Albert Thomas, 69003 Lyon, France Carole Amsellem3 Episkin SNC, 4 Rue Alexander Fleming, 69366 Lyon, France Be ´atrice Bertino4 SkinEthic SA, 45 Rue St. Philippe, 06000 Nice, France
138
Letter to the Editor *Corresponding author. Tel.: +33 472 11 02 92; fax: +33 472 11 02 90 E-mail address: [email protected] (M. Haftek)
1
Tel.: Tel.: 3 Tel.: 4 Tel.: 2
+33 +33 +33 +33
472 442 437 493
11 60 28 97
02 73 72 77
93; 78; 08; 27;
fax: fax: fax: fax:
+33 +33 +33 +33
472 442 437 493
11 24 28 97
02 34 72 77
90. 54. 28. 28.
20 June 2007
Available online at www.sciencedirect.com