Regulation of Keratin 9 in Nonpalmoplantar Keratinocytes by Palmoplantar Fibroblasts Through Epithelial–Mesenchymal Interactions
1999; Elsevier BV; Volume: 112; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.1999.00544.x
ISSN1523-1747
AutoresYuji Yamaguchi, Satoshi Itami, Masahito Tarutani, Ko Hosokawa, Hiroyuki Miura, Kunihiko Yoshikawa,
Tópico(s)Plant Surface Properties and Treatments
ResumoPalms and soles differ from other body sites in terms of clinical and histologic appearance, response to mechanical stress, and the distribution of keratin 9. Because keratin 9 is exclusively expressed in the palmoplantar suprabasal keratinocyte layers, it is considered a differentiation marker of palms and soles. We studied palmoplantar mesenchymal influences on keratin 9 induction in nonpalmoplantar epidermis. Although palmoplantar keratinocytes when cultured alone continued to express keratin 9 mRNA in 12 (100%) of 12 cultures, nonpalmoplantar keratinocytes did not express it in 16 of 17 cultures. Although nonpalmoplantar keratinocytes did not express keratin 9 mRNA when cultured with nonpalmoplantar fibroblasts, they did express it within 2 h in cocultures with palmoplantar fibroblasts derived from papillary dermis. Grafting of these coculture sheets on severe combined immunodeficient mice resulted in an epidermis, which histologically showed hyperkeratosis and acanthosis and immunohistochemically expressed keratin 9. Furthermore, pure epidermal sheets from nonpalmoplantar skin grafted on the human sole wounds due to burn, injury, and the resection of acral lentiginous melanoma, demonstrated adoption of palmoplantar phenotype and expressed keratin 9. Our report indicates extrinsic keratin 9 regulation by signals from dermal fibroblasts. This is also the first to suggest the possibility of treating palmoplantar wounds with nonpalmoplantar epidermis, which is much easier to obtain and harvest. Palms and soles differ from other body sites in terms of clinical and histologic appearance, response to mechanical stress, and the distribution of keratin 9. Because keratin 9 is exclusively expressed in the palmoplantar suprabasal keratinocyte layers, it is considered a differentiation marker of palms and soles. We studied palmoplantar mesenchymal influences on keratin 9 induction in nonpalmoplantar epidermis. Although palmoplantar keratinocytes when cultured alone continued to express keratin 9 mRNA in 12 (100%) of 12 cultures, nonpalmoplantar keratinocytes did not express it in 16 of 17 cultures. Although nonpalmoplantar keratinocytes did not express keratin 9 mRNA when cultured with nonpalmoplantar fibroblasts, they did express it within 2 h in cocultures with palmoplantar fibroblasts derived from papillary dermis. Grafting of these coculture sheets on severe combined immunodeficient mice resulted in an epidermis, which histologically showed hyperkeratosis and acanthosis and immunohistochemically expressed keratin 9. Furthermore, pure epidermal sheets from nonpalmoplantar skin grafted on the human sole wounds due to burn, injury, and the resection of acral lentiginous melanoma, demonstrated adoption of palmoplantar phenotype and expressed keratin 9. Our report indicates extrinsic keratin 9 regulation by signals from dermal fibroblasts. This is also the first to suggest the possibility of treating palmoplantar wounds with nonpalmoplantar epidermis, which is much easier to obtain and harvest. keratin 9 keratinocyte growth medium Palmar and plantar epidermis histologically has an extremely thick, compact stratum corneum and a well-defined stratum lucidum, as compared with the thin and hairy epidermis of other body sites. Rete ridges in the palm are deep or shallow, whereas those of other body sites are relatively uniform in size. Palmoplantar stem cells are located at the tips of the deep rete ridges, whereas nonpalmoplantar stem cells are above the papillary dermis (Lavker and Sun, 1982Lavker R.M. Sun T.T. Heterogeneity in epidermal basal keratinocytes: morphological and functional correlations.Science. 1982; 215: 1239-1241Crossref PubMed Scopus (288) Google Scholar,Lavker and Sun, 1983Lavker R.M. Sun T.T. Epidermal stem cells.J Invest Dermatol. 1983; 81: 121s-127sAbstract Full Text PDF PubMed Scopus (227) Google Scholar;Jones et al., 1995Jones P.H. Harper S. Watt F.M. Stem cell patterning and fate in human epidermis.Cell. 1995; 80: 83-93Abstract Full Text PDF PubMed Scopus (698) Google Scholar). Palmoplantar epidermis expresses keratin 9 (K9), which is a large and highly differentiated acidic type 1 keratin (63K) in the suprabasal layers of epidermis (Knapp et al., 1986Knapp A.C. Franke W.W. Heid H. Hatzfeld M. Jorcano J.L. Moll R. Cytokeratin no. 9, an epidermal type 1 keratin characteristic of a special program of keratinocyte differentiation displaying body site specificity.J Cell Biol. 1986; 103: 657-667Crossref PubMed Scopus (91) Google Scholar;Moll et al., 1987Moll I. Heid H. Franke W.W. Moll R. Distribution of a special subset of keratinocytes characterized by the expression of cytokeratin 9 in adult and fetal human epidermis of various body sites.Differentiation. 1987; 33: 254-265Crossref PubMed Scopus (53) Google Scholar;Schweizer et al., 1989Schweizer J. Baust I. Winter H. Identification of murine type 1 keratin 9 (73 kDa) and its immunolocalization in neonatal and adult mouse foot sole epidermis.Exp Cell Res. 1989; 184: 193-206Crossref PubMed Scopus (16) Google Scholar;Langbein et al., 1993Langbein L. Heid H.W. Moll I. Franke W.W. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.Differentiation. 1993; 55: 57-71Crossref PubMed Scopus (102) Google Scholar). Although K9 expression in palmoplantar keratinocytes is considered to be endogenously rather than exogenously regulated (Limat et al., 1996Limat A. Stockhammer E. Breitkreutz D. et al.Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.Eur J Cell Biol. 1996; 69: 245-258PubMed Google Scholar), keratinocyte differentiation is also regulated by extrinsic factors, such as Ca2+ concentrations (Hennings et al., 1980Hennings H. Nfichael D. Cheng C. Steinert P. Holbrook K. Yuspa H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1450) Google Scholar), vitamin A (Fuchs and Green, 1981Fuchs E. Green H. Regulation of terminal differentiation of cultured human keratinocytes by vitamin A.Cell. 1981; 25: 617-625Abstract Full Text PDF PubMed Scopus (560) Google Scholar), mechanical stress (Gormar et al., 1990Gormar F.E. Bernd A. Bereiter-Hahn J. HoIzmann H. A new model of epidermal differentiation: induction by mechanical stimulation.Arch Dermatol Res. 1990; 282: 22-32Crossref PubMed Scopus (48) Google Scholar), and mesenchymal factors (Bohnert et al., 1986Bohnert A. Horming J. Mackenzie I.C. Fusenig N.E. Epithelial–mesenchymal interactions control basement membrane production and differentiation in cultured and transplanted mouse keratinocytes.Cell Tissue Res. 1986; 244: 413-429Crossref PubMed Scopus (104) Google Scholar). Using heterotypic recombinant experiments,Billingham and Silvers, 1966Billingham R.E. Silvers W.K. Studies on the conservation of epidermal specificities of skin and certain mucosas in adult mammals.J Exp Med. 1966; 125: 429-446Crossref Scopus (92) Google Scholar showed that the dermis determines the phenotypic expression of the transplanted epidermis and that sole dermis induces the sole type epidermis in ear and trunk epidermis. In this study, we report the induction of highly differentiated keratin synthesis by heterotypic fibroblasts; both in vitro and in vivo. Because there is increasing evidence that dermal fibroblasts are heterogeneous (Harper and Grove, 1979Harper R.A. Grove G. Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro.Science. 1979; 204: 526-527Crossref PubMed Scopus (203) Google Scholar;Schor and Schor, 1987Schor S.L. Schor A.M. Clonal heterogeneity in fibroblast phenotype: implications for the control of epithelial–mesenchymal interactions.Bioessays. 1987; 7: 200-204Crossref PubMed Scopus (75) Google Scholar;Goldring et al., 1990Goldring S.R. Stephenson M.L. Downie E. Krane S.M. Korn H. Heterogeneity in hormone responses and patterns of collagen synthesis in cloned dermal fibroblasts.J Clin Invest. 1990; 85: 798-803Crossref PubMed Scopus (58) Google Scholar;Falanga et al., 1995Falanga V. Zhou L.H. Takagi H. Murata H. Ochoa S. Martin T.A. Helfman T. Human dermal fibroblast clones derived from single cells are heterogeneous in the production of mRNAs for alpha 1 (I) procollagen and transforming growth factor-beta 1.J Invest Dermatol. 1995; 105: 27-31Crossref PubMed Scopus (18) Google Scholar), we also examined whether fibroblasts from papillary or reticular dermis could induce the same level of K9 expression in nonpalmoplantar keratinocytes. Considering the heterotypic mesenchymal–epithelial interaction, we also successfully treated with nonpalmoplantar epidermis plantar wounds due to burn, injury, and the resection of acral lentiginous melanoma, which is the most common melanoma in oriental races (Seiji et al., 1983Seiji M. Takematsu H. Hosokawa M. et al.Acral melanoma in Japan.J Invest Dermatol. 1983; 80: 56s-60sCrossref PubMed Scopus (70) Google Scholar). Skin specimens were obtained from patients during plastic surgery. Human keratinocytes were cultured by the modified method of explant culture (Halprin et al., 1979Halprin K.M. Lueder M. Fusenig N.E. Growth and differentiation of postembryonic mouse epidermal cells in explant cultures.J Invest Dermatol. 1979; 72: 88-98Crossref PubMed Scopus (31) Google Scholar;Itami et al., 1995Itami S. Kurata S. Takayasu S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-1 from dermal papilla cells.Biochem Biophys Res Commun. 1995; 212: 988-994Crossref PubMed Scopus (147) Google Scholar). The dermis were carefully scraped to produce a thin piece of skin under a stereoscopic microscope. The skin was cut into 0.2–0.3 mm squares and single pieces were placed in 3.5 cm collagen type 1 coated dishes (Iwaki Glass, Tokyo, Japan). They were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) with 10% fetal bovine serum (FBS; JRH Biosciences, KS). After epidermal outgrowth was observed, cultures were supplemented with ketatinocyte growth medium (KGM) (Boyce and Ham, 1983Boyce S.T. Ham R.G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.J Invest Dermatol. 1983; 81: 33s-40sAbstract Full Text PDF PubMed Scopus (917) Google Scholar). Keratinocytes from third passage were principally used in these experiments. Four different systems were used for the cocultures. In the first system, fibroblasts and keratinocytes were simply mixed 50:50 at a density of 3 × 104 cells per cm2 in KGM. Biocoat cell culture inserts (rat tail collagen type I, 6 well, 0.45 μm, Becton Dickinson, Bed Ford, MA) were used for the other three coculture systems. In the second system, keratinocytes were seeded onto the inserts in KGM. Fibroblasts were seeded onto the outer culture dish in 10% FBS/DMEM separately, and then the two were placed together after confluence in KGM. In the third system, the inserts were turned upside down and fibroblasts were seeded onto the bottom of the membranes. Two days later, the inserts were turned upright and keratinocytes were seeded onto the top of the membranes at a density of 5 × 104 cells per cm2 in KGM. Finally, in a fourth system, fibroblasts were embedded in 2 ml of collagen lattices (Cellmatrix type I, Nitta Gelatin, Osaka, Japan) at a density of 1.5 × 106 cells per ml into the outer culture dish, and then washed with KGM three times. The inserts filled with keratinocytes in KGM were placed on the collagen gel. In certain experiments, papillary and reticular dermal fibroblasts were cultured individually under a stereoscopic microscope after dispase treatment (Harper and Grove, 1979Harper R.A. Grove G. Human skin fibroblasts derived from papillary and reticular dermis: differences in growth potential in vitro.Science. 1979; 204: 526-527Crossref PubMed Scopus (203) Google Scholar). Reverse transcriptase-PCR analyses were performed as previously described (Itami et al., 1995Itami S. Kurata S. Takayasu S. Androgen induction of follicular epithelial cell growth is mediated via insulin-like growth factor-1 from dermal papilla cells.Biochem Biophys Res Commun. 1995; 212: 988-994Crossref PubMed Scopus (147) Google Scholar). The oligonucleotide primers for PCR were based on published mRNA sequences, which include the 1A rod domain of K9 (Langbein et al., 1993Langbein L. Heid H.W. Moll I. Franke W.W. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.Differentiation. 1993; 55: 57-71Crossref PubMed Scopus (102) Google Scholar), and were as follows: cytokeratin 9 (accession number: z29074), 5′-ATA GTA GTT CTG GGG GTT TT-3′ and 5′-GTG TCA TGC GAG TGT TGT CA-3′ or 5′-TGG GGG TGG TTT TAG TG-3′ and 5′-ACT GTC AGG TCC ACA ATC-3′: human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control (Tso et al., 1985Tso J.Y. Sun X.H. Kao T.H. Reece K.S. Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.Nucleic Acids Res. 1985; 13: 2485-2502Crossref PubMed Scopus (1744) Google Scholar), 5′-CCC ATC ACC ATC TTC CAG-3′ and 5′-CCT GCT TCA CCA CCT TCT-3′. PCR amplification times were 35 cycles (1 min at 94°C, 1 min at 62°C, 1 min at 72°C) for K9, and 20 cycles (1 min at 94°C, 1 min at 60°C, 1 min at 72°C) for GAPDH. The sizes of products were compared with 100 bp molecular weight markers (BRA Life Technology, Gaithersburg, MD). We confirmed the identity of the cDNA products by the digestion with Xbal, i.e., the specific restriction enzyme. The cDNA products were purified by electrophoresis on 2% agarose gels, extracted with Quiaex (Qiagen, Hilden, Germany), and cloned in Bluescript vector (Stratagene, La Jolla, CA). The cloned plasmid DNA were isolated and purified using the Quiagen Matrix Columns (Qiagen). The recombinant plasmids were digested and analyzed for sequencing by 373 DNA sequencer STRETCH (PE Applied Biosystems, Norwalk, CT). Frozen sections (5 μm) were fixed with cold acetone and incubated with the primary antibodies at 4°C overnight. The primary antibodies used were Serum HK9-AM2, which was developed against K9 specific aminoterminal sequence and stained the foot sole epidermis suprabasally and cells around acrosyringia of eccrine sweat gland ducts and Serum HM-TY1, which was also made against human K9 specific rod end sequence (Langbein et al., 1993Langbein L. Heid H.W. Moll I. Franke W.W. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.Differentiation. 1993; 55: 57-71Crossref PubMed Scopus (102) Google Scholar). These antibodies were very kind and generous gifts from Drs. W.W. Franke and L. Langbein. After washing with PBS containing 0.1% Tween 20, samples were incubated with fluorescein-5-isothiocyanate-conjugated goat affinity purified anti-guinea pig IgG antibody (Cappel, Westchester, PA) at room temperature for 60 min. The samples that were incubated with preimmune sera were used as negative control. Organotypic cocultures were grafted on the dorsal muscle fascia of SCID mice (Nomura et al., 1997Nomura T. Nakajima H. Hongyo T. et al.Induction of cancer, actinic keratosis, and specific p53 mutations by UVB light in human skin maintained in severe combined immunodeficient mice.Cancer Res. 1997; 57: 2081-2084PubMed Google Scholar). The transplants were excised 3 wk later and examined by anti-human HLA-Class I antigen antibody (DAKOPATTS, Glostrup, Denmark), hematoxylin and eosin, and anti-K9 antibodies as described above. All surgeries were done after written informed consent. Split-thickness skin was harvested from the anterior thigh of patients with plantar wounds due to burn, injury, and the resection of acral lentiginous melanoma. The skin was soaked into 500 U per ml of dispase (Godo Shusei, Tokyo, Japan) in DMEM at 37°C for 1 h to separate epidermis from dermis. The pure epidermal sheets were washed with DMEM three times and grafted on the plantar full-thickness skin defects. K9 mRNA expression in vitro We first determined whether keratinocytes from soles and palms continue to express K9 mRNA in vitro, and whether keratinocytes from nonpalmoplantar sites do not. Keratinocytes were cultured to confluence with serum free medium without 3T3 feeder layers to avoid undefined mesenchymal effects in these studies (Rheinwald and Green, 1975Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (3726) Google Scholar;Boyce and Ham, 1983Boyce S.T. Ham R.G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture.J Invest Dermatol. 1983; 81: 33s-40sAbstract Full Text PDF PubMed Scopus (917) Google Scholar). The explant culture technique instead of a single cell suspension method was used to culture the keratinocytes, because the former method results in better palmoplantar keratinocyte growth. Explant cultures may have improved keratinocyte growth because stem cells from palmoplantar skin are known to be located at the tips of the rete ridges (Jones et al., 1995Jones P.H. Harper S. Watt F.M. Stem cell patterning and fate in human epidermis.Cell. 1995; 80: 83-93Abstract Full Text PDF PubMed Scopus (698) Google Scholar). Furthermore, trypsinization is not needed in primary explant cultures, thereby preventing damage to the stem cells. Both palmoplantar and nonpalmoplantar keratinocytes looked microscopically similar during cell culture (data not shown). The expressions of K9-specific mRNA in keratinocytes from various body sites were examined by reverse transcriptase-PCR amplification. The positions of primer nucleotides were based on the published sequence and contained the 1A rod domain of K9, whose sequence is quite different from the other acidic type I keratins (Langbein et al., 1993Langbein L. Heid H.W. Moll I. Franke W.W. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.Differentiation. 1993; 55: 57-71Crossref PubMed Scopus (102) Google Scholar). Because of the homology among keratin genes, annealing temperature was set ≈7°C higher than the calculated Tm for stringent amplification. The cDNA products from cultured palmoplantar keratinocytes showed K9-specific bands in all of the specimens from 12 subjects (ages of the patients ranged from 12 to 47) from early to late passages (Figure 1a, lanes 1–4). Cultured nonpalmoplantar keratinocytes did not express K9 in 16 of 17 specimens (Figure 1a, lanes 5–8), except for one sample from thigh skin (data not shown). This expression of K9 in one sample may be attributed to the PCR amplification of K9 found in the acrosyringia (Moll et al., 1987Moll I. Heid H. Franke W.W. Moll R. Distribution of a special subset of keratinocytes characterized by the expression of cytokeratin 9 in adult and fetal human epidermis of various body sites.Differentiation. 1987; 33: 254-265Crossref PubMed Scopus (53) Google Scholar). Sequence analysis was performed to confirm the identity of the cDNA products (Figure 1b). No difference was observed in the 1A region between our sequence and the published sequence of K9 (Langbein et al., 1993Langbein L. Heid H.W. Moll I. Franke W.W. Molecular characterization of the body site-specific human epidermal cytokeratin 9: cDNA cloning, amino acid sequence, and tissue specificity of gene expression.Differentiation. 1993; 55: 57-71Crossref PubMed Scopus (102) Google Scholar), whereas 2 bp differences were observed in the variant subdomain V1 of the head region in all of our four samples. One was an A to T transition at position 468, which did not result in an amino acid change. The other was an A to C transition at position 495, which resulted in a leucine (L) to phenylalanine (F) substitution in codon 133 of K9. We next examined the induction of K9 expression in nonpalmoplantar keratinocytes cocultured with palmoplantar fibroblasts by four coculture systems (Figure 2a–e). Obvious K9 mRNA expression occurred when keratinocytes from hair-bearing skin were directly (Figure 2a e, lane 1) or closely (Figure 2c–e, lanes 3, 4) cocultured with palmoplantar fibroblasts, to mimic a juxtacrine or paracrine system. On the other hand, only slight K9 expression was observed in the coculture insert system, which separates keratinocytes from fibroblasts (Figure 2b, e, lane 2). The same cell strain of nonpalmoplantar keratinocytes did not express K9 mRNA when cocultured with nonpalmoplantar fibroblasts (Figure 2d, e lane 5). Moreover, palmoplantar fibroblasts did not express K9 mRNA when cultured alone or with either type of keratinocytes (data not shown). We next determined how long it takes to induce K9 mRNA in nonpalmoplantar keratinocytes by palmoplantar fibroblasts (Figure 2f). For this purpose, we used recombinant cocultures of collagen lattice with embedded fibroblasts and with the insert filled with confluent keratinocytes (Figure 2d). Induction of keratin 9 mRNA expression occurred at 30 min after the recombination, gradually increased in intensity from 2 h to 1 d, and continued for 3 d (Figure 2f). We next examined the difference in the ability to induce K9 between papillary and reticular dermal fibroblasts from sole skin. Figure 3 shows representative results obtained from nonpalmoplantar keratinocytes cocultured with either type of fibroblasts for 3 d (Figure 2d). Papillary palmoplantar fibroblasts were able to induce K9 mRNA expression in all seven cases (Figure 3, lanes 1, 2), while reticular fibroblasts could moderately induce the K9 expression in five of eight cases (Figure 3, lanes 3, 4). Nonpalmoplantar papillary fibroblasts could not induce K9 mRNA expression in nonpalmoplantar keratinocytes in all four cases (Figure 3, lanes 5, 6). Papillary palmoplantar fibroblasts had a higher ability to induce K9 in nonpalmoplantar keratinocytes. K9 protein, a highly differentiated keratin, was not observed in palmoplantar keratinocytes in submerged cultures, as previously reported (Limat et al., 1996Limat A. Stockhammer E. Breitkreutz D. et al.Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.Eur J Cell Biol. 1996; 69: 245-258PubMed Google Scholar). To solve this problem, the keratinocyte cultures were lifted to the air/liquid interface with collagen gels containing fibroblasts (Limat et al., 1996Limat A. Stockhammer E. Breitkreutz D. et al.Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.Eur J Cell Biol. 1996; 69: 245-258PubMed Google Scholar) using medium consisting of 50% KGM, 2.5% FBS, 7.5% delipidized FBS (Fuchs and Green, 1981Fuchs E. Green H. Regulation of terminal differentiation of cultured human keratinocytes by vitamin A.Cell. 1981; 25: 617-625Abstract Full Text PDF PubMed Scopus (560) Google Scholar), and 40% DMEM with 10–7 M vitamin D3. The organotypic cocultures were studied within 1 wk of preparations. We found that K9 mRNA expression occurred adequately in nonpalmoplantar keratinocytes within 3 d of coculture (Figure 2f), and that long-term cultures resulted in slight degeneration of the suprabasal layers. Immunohistochemical studies with the specific antibodies revealed K9 induction in nonpalmoplantar keratinocytes cocultured with palmoplantar fibroblasts as well as palmoplantar keratinocytes cocultured with palmoplantar fibroblasts (Figure 4). Because long-lasting differentiation could be achieved only by grafting the keratinocytes onto nude mice (Limat et al., 1996Limat A. Stockhammer E. Breitkreutz D. et al.Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.Eur J Cell Biol. 1996; 69: 245-258PubMed Google Scholar), we next transplanted 1 wk old organotypic cocultures onto the muscle fascia of the back of SCID mice (Nomura et al., 1997Nomura T. Nakajima H. Hongyo T. et al.Induction of cancer, actinic keratosis, and specific p53 mutations by UVB light in human skin maintained in severe combined immunodeficient mice.Cancer Res. 1997; 57: 2081-2084PubMed Google Scholar) to confirm the K9 induction (Figure 5). Organotypic cocultures of nonpalmoplantar keratinocytes and palmoplantar fibroblasts took well and were excised 3 wk later. The samples, which were shown to be of human origin by testing with anti-human HLA-class 1 antibody (Figure 5a), histologically showed hyperkeratosis and acanthosis-like palmoplantar-type skin (Figure 5b). Furthermore, they showed several K9-positive cells in suprabasal layers of the grafted epidermis (Figure 5c), which never appeared in the cocultures of nonpalmoplantar keratinocytes and fibroblasts (data not shown). The distribution of K9-positive cells in our heterogeneous grafts was comparable with the results reported byLimat et al., 1996Limat A. Stockhammer E. Breitkreutz D. et al.Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.Eur J Cell Biol. 1996; 69: 245-258PubMed Google Scholar in their composite grafts of palmoplantar keratinocytes and fibroblasts on nude mice. In this study and in previous work byLimat et al., 1996Limat A. Stockhammer E. Breitkreutz D. et al.Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.Eur J Cell Biol. 1996; 69: 245-258PubMed Google Scholar, the grafts were harvested shortly after the transplantation; long-term interactions may be necessary for the adequate K9 induction and/or contamination with mouse dermal fibroblasts might suppress K9 expression in human keratinocytes. To address these issues and to provide a clinical perspective, we grafted the nonpalmoplantar epidermis on five palmoplantar full-thickness skin wounds due to burn, acute injury, and the resection of acral lentiginous melanoma. We used dispase instead of trypsin in our cultures to minimize contamination with donor fibroblasts. The grafts took well and without contracture (Figure 6a) and, gradually, showed typical palmoplantar skin appearance (Figure 6b–d). Histologically, the grafted epidermis showed a thick and compact stratum corneum and a well-developed stratum lucidum at 5 wk post-grafting (data not shown). Immunohistochemical examinations also showed a sparse distribution of K9-positive keratinocytes at 3 wk post-grafting (Figure 6e) and expression increased at 5 wk (Figure 6f). These findings are comparable with those observed with normal palmoplantar epidermis (Moll et al., 1987Moll I. Heid H. Franke W.W. Moll R. Distribution of a special subset of keratinocytes characterized by the expression of cytokeratin 9 in adult and fetal human epidermis of various body sites.Differentiation. 1987; 33: 254-265Crossref PubMed Scopus (53) Google Scholar). Two years later, the grafted epidermis continued to show expression of K9-positive keratinocytes in the suprabasal layers (data not shown).Figure 5Transplantation of organotypic cocultures on SCID mice. Trunk keratinocytes were organotypically cultured with plantar fibroblasts for 1 wk and grafted onto the dorsal muscle fascia of SCID mice. The transplants were excised at 3 wk post-grafting and examined by anti-human HLA-class I antigen antibody (a), hematoxylin and eosin (b), and anti-K9 antibody (c). The left half of each figure shows mouse skin and the right half shows the grafted human culture. Hair follicles of mouse show nonspecific stains both in part (c) and in the negative control that was incubated with preimmune serum (d). Scale bars: 100 μm.View Large Image Figure ViewerDownload (PPT)Figure 6Transplantation of pure epidermal sheets derived from nonpalmoplantar skin on human palmoplantar wounds. The pure epidermis from trunk skin was grafted on the human plantar wound. The graft took well 1 wk later (a) and, gradually, showed typical palmoplantar skin appearance at 2 wk (b), 3 wk (c), and 5 wk (d) post-grafting. Immunohistochemical studies with anti-K9 antibodies were done at 3 wk (e) and 5 wk (f) post-grafting. These results show a representative of five independent studies. Scale bars: (a–d) 1 cm, (e, f) 50 μm.View Large Image Figure ViewerDownload (PPT) Once acquired, K9 expression in keratinocytes represents an intrinsic program, as shown by our results that palmoplantar keratinocytes continuously expressed the K9 mRNA during serial subcultivations and continued to express K9 when cocultured with nonpalmoplantar fibroblasts (data not shown). Our results are in agreement with the previous reports, mentioning that differentiation of epithelial cells is generally regulated intrinsically (Doran et al., 1980Doran T.I. Vidrich A. Sun T.T. Intrinsic and extrinsic regulation of the differentiation of skin, corneal and esophageal epithelial cells.Cell. 1980; 22: 17-25Abstract Full Text PDF PubMed Scopus (207) Google Scholar;Boukamp et al., 1990Boukamp P. Breitkreutz D. Stark H.J. Fusenig N.E. Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites.Differentiation. 1990; 44: 150-161Crossref PubMed Scopus (98) Google Scholar;Limat et al., 1996Limat A. Stockhammer E. Breitkreutz D. et al.Endogeneously regulated site-specific differentiation of human palmar skin keratinocytes in organotypic cocultures and in nude mouse transplants.Eur J Cell Biol. 1996; 69: 245-258PubMed Google Scholar;Miller et al., 1998Miller S.J. Burke E.M. Rader M.D. Coulombe P.A. Lavker R.M. Re-epithelialization of porcine skin by the sweat apparatus.J Invest Dermatol. 1998; 110: 13-19Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Epithelial–mesenchymal interactions are considered to play fundamental roles in the growth and differentiation of keratinocytes (Boukamp et al., 1990Boukamp P. Breitkreutz D. Stark H.J. Fusenig N.E. Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites.Differentiation. 1990; 44: 150-161Crossref PubMed Scopus (98) Google Scholar;Jahoda, 1992Jahoda C.A. Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds: vibrissa-type fibres are specified.Development. 1992; 115: 1103-1109PubMed Google Scholar;Smola et al., 1993Smola H. Thiekotter G. Fusenig N.E. Mutual induction of growth factor gene expression by epidermal–dermal cell interaction.J Cell Biol. 1993; 122: 417-429Crossref PubMed Scopus (318) Google Scholar;Byrne et al., 1994Byrne C. Tainsky M. Fuchs E. Programming gene expression in developing epidermis.Development. 1994; 120: 2369-2383Crossref PubMed Google Scholar). Extrinsic factors, such as mechanical stress, can induce the expression of a keratin probably corresponding to K9 in cultured keratinocytes (Gormar et al., 1990Gormar F.E. Bernd A. Bereiter-Hahn J. HoIzmann H. A new model of epidermal differentiation: induction by mechanical stimulation.Arch Dermatol Res. 1990; 282: 22-32Crossref PubMed Scopus (48) Google Scholar). Our report shows exogenous regulation of K9 expression in topologically different keratinocytes by signals from a subset of palmoplantar fibroblasts. Although the culture insert membrane prevented direct contact of the fibroblasts with the overlying keratinocytes, it remains possible that paracrine and/or juxtacrine interactions are involved in K9 induction. The fact that K9 expression could be induced weakly when palmoplantar fibroblasts were distant from keratinocytes is interesting, and suggests a secreted factor may be involved in the induction. The K9 sequence analysis revealed 2 bp differences between the previously published gene (Langbein et al., 1993Langbein L. Heid H.W. Moll I. Franke W.W. 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Human dermal fibroblast clones derived from single cells are heterogeneous in the production of mRNAs for alpha 1 (I) procollagen and transforming growth factor-beta 1.J Invest Dermatol. 1995; 105: 27-31Crossref PubMed Scopus (18) Google Scholar). Our results are in agreement with these previous reports. Fibroblasts derived from palms and soles were different from those from other body sites in terms of K9 induction in nonpalmoplantar keratinocytes. Moreover, papillary palmoplantar fibroblasts had a higher ability to induce K9 mRNA expression than those from reticular palmoplantar dermis. It is not easy to treat palmoplantar skin defects because palmoplantar skin is difficult to harvest from donor sites and differs from nonpalmoplantar skin in terms of its ability to support mechanical stress. In this report, we grafted epidermal sheets derived from nonpalmoplantar sites on sole wounds. These grafted keratinocytes took and grew well and assumed a palmoplantar phenotype with substantial expression of K9 at 5 wk after the transplantation. There are several possibilities to explain these findings. Because the epidermal sheets were grafted on the fatty layers of sole wounds, the remaining wound fibroblasts might have induced K9 in the grafted epidermis. Possibly, plantar fibroblasts from around the skin defect might have migrated into the wound. Alternatively, K9 expression might have occurred because of keratinocytes around sweat glands in the wound and which extended into the grafted epidermis. The latter possibility, however, is unlikely because re-epithelialized skin from the sweat apparatus in the deep wound looks different from normal skin and takes longer to be formed than our results indicate (Miller et al., 1998Miller S.J. Burke E.M. Rader M.D. Coulombe P.A. Lavker R.M. Re-epithelialization of porcine skin by the sweat apparatus.J Invest Dermatol. 1998; 110: 13-19Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In conclusion, K9 expression in human keratinocytes is controlled not only by intrinsic regulatory mechanism, but also by extrinsic signals from a subset of palmoplantar fibroblasts. Pure epidermal grafting may be effective in the treatment of palmoplantar skin defects as a result of heterotypic mesenchymal–epithelial interactions. We greatly appreciate Prof. Dr. Vincent Falanga, Boston University, for the critical review of this work. We also thank Mrs. Satomi Okamoto for her excellent immunohistochemical studies. Finally, we greatly appreciate Prof. Dr. Werner W. Franke and Dr. Lutz Langbein, German Cancer Research Center, for providing us with anti-human K9 antibodies.
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