Expression of the Keratinocyte Lipid Transporter ABCA12 in Developing and Reconstituted Human Epidermis
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.061207
ISSN1525-2191
AutoresYasuko Yamanaka, Masashi Akiyama, Yoriko Sugiyama‐Nakagiri, Kaori Sakai, Maki Goto, James R. McMillan, Mitsuhito Ota, Daisuke Sawamura, Hiroshi Shimizu,
Tópico(s)RNA Interference and Gene Delivery
ResumoSerious defects in the epidermal keratinocyte lipid transporter ABCA12 are known to result in a deficient skin lipid barrier, leading to harlequin ichthyosis (HI). HI is the most severe inherited keratinizing disorder and is frequently fatal in the perinatal period. To clarify the role of ABCA12, ABCA12 expression was studied in developing human skin and HI lesions artificially reconstituted in immunodeficient mice. By immunofluorescent study, ABCA12 was expressed in the periderm of the early stage two-layered human fetal epidermis. After formation of a three-layered epidermis, ABCA12 staining was seen throughout the entire epidermis. ABCA12 mRNA expression significantly increased during human skin development and reached 62% of the expression in normal adult skin, whereas the expression rate of transglutaminase 1, loricrin, and kallikrein 7 remained low. We transplanted keratinocytes from patients with HI and succeeded in reconstituting HI skin lesions in immunodeficient mice. The reconstituted lesions showed similar changes to those of patients with HI. Our findings demonstrate that ABCA12 is highly expressed in fetal skin and suggest that ABCA12 may play an essential role under both the wet and dry conditions, including the dramatic turning point from a wet environment of the amniotic fluid to a dry environment after birth. Serious defects in the epidermal keratinocyte lipid transporter ABCA12 are known to result in a deficient skin lipid barrier, leading to harlequin ichthyosis (HI). HI is the most severe inherited keratinizing disorder and is frequently fatal in the perinatal period. To clarify the role of ABCA12, ABCA12 expression was studied in developing human skin and HI lesions artificially reconstituted in immunodeficient mice. By immunofluorescent study, ABCA12 was expressed in the periderm of the early stage two-layered human fetal epidermis. After formation of a three-layered epidermis, ABCA12 staining was seen throughout the entire epidermis. ABCA12 mRNA expression significantly increased during human skin development and reached 62% of the expression in normal adult skin, whereas the expression rate of transglutaminase 1, loricrin, and kallikrein 7 remained low. We transplanted keratinocytes from patients with HI and succeeded in reconstituting HI skin lesions in immunodeficient mice. The reconstituted lesions showed similar changes to those of patients with HI. Our findings demonstrate that ABCA12 is highly expressed in fetal skin and suggest that ABCA12 may play an essential role under both the wet and dry conditions, including the dramatic turning point from a wet environment of the amniotic fluid to a dry environment after birth. One important event during terminal differentiation of stratified squamous epithelia such as the epidermis is the formation of intercellular lipid layers in the stratum corneum. Intercellular lipid layers in the stratum corneum are essential for epidermal barrier function. The lipid layers are formed from the extruded lipid contents secreted from lamellar granules within granular layer keratinocytes. 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67: 793-799Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar In 2005, ABCA12, a member of the ABCA subfamily, was reported to underlie harlequin ichthyosis (HI), one of the most devastating genodermatoses.18Akiyama M Sugiyama-Nakagiri Y Sakai K MacMillan JR Goto M Arita K Tsuji-Abe Y Tabata N Matsuoka K Sasaki R Sawamura D Shimizu H Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J Clin Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (312) Google Scholar, 19Kelsell DP Norgett EE Unsworth H Teh MT Cullup T Mein CA Dopping-Hepenstal PJ Dale BA Tadini G Fleckman P Stephens KG Sybert VP Mallory SB North BV Witt DR Sprecher E Taylor AE Ilchyshyn A Kennedy CT Goodyear H Moss C Paige D Harper JI Young BD Leigh IM Eady RA O'Toole EA Mutations in ABCA12 underlie the severe congenital skin disease harlequin ichthyosis.Am J Hum Genet. 2005; 76: 794-803Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar HI was known to show several morphological abnormalities reflecting defective lipid content: absent or abnormal lamellar granules in the granular keratinocytes, lipid droplets in the stratum corneum, and a lack of extracellular lipid lamellae.20Akiyama M Kim D-K Main DM Otto CE Holbrook KA Characteristic morphologic abnormality of harlequin ichthyosis detected in amniotic fluid cells.J Invest Dermatol. 1994; 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detection of severe genodermatoses expressed in the fetal period.J Invest Dermatol. 1994; 103: 674-677Abstract Full Text PDF PubMed Scopus (20) Google Scholar Affected neonates usually show the most severe, life-threatening symptoms such as large, thick, platelike scales over the whole body, ectropion, eclabium, and flattened ears from birth.32Williams ML Elias PM Genetically transmitted, generalized disorders of cornification; the ichthyoses.Dermatol Clin. 1987; 5: 155-178PubMed Google Scholar Patients with HI usually die during the first few weeks of life from secondary infection, severe anemia, dehydration, circulatory disturbance, or renal failure. However, once patients with HI have survived beyond the perinatal period, their skin symptoms tend to be less severe, and some long-term survivors even show clinical features of the milder nonbullous congenital ichthyosiform erythroderma.33Franco RC Successful treatment of harlequin ichthyosis with acitretin.Int J Dermatol. 2001; 40: 472-484Crossref PubMed Scopus (12) Google Scholar, 34Haftek M Cambazard F Dhouailly D Reano A Simon M Lachaux A Serre G Claudy A Schmitt D A longitudinal study of a harlequin infant presenting clinically as non-bullous congenital ichthyosiform erythroderma.Br J Dermatol. 1996; 135: 448-453Crossref PubMed Scopus (49) Google Scholar Some survivors can even stop taking oral retinoids. From these clinical findings, we hypothesized that ABCA12 deficiency is the most critical at around the time of birth and that the negative effects of ABCA12 deficiency might be reduced or compensated for after the baby grows beyond the perinatal period. In the present study, to clarify further the pathomechanisms of severe HI manifestations from birth and the critical role of ABCA12 in the neonatal period, ABCA12 expression was studied in detail in developing human skin and artificially reconstituted HI lesions grown on immunodeficient mice. This is the first report describing ABCA12 expression during embryonic and fetal skin ontogeny, and it demonstrates that ABCA12 is highly expressed in the upper epidermis from the second trimester. Furthermore, we succeeded in establishing a model system for regenerated HI lesions harboring ABCA12 mutations in adult skin with reduced ABCA12 expression in the reconstituted HI skin lesions. The skin lesions reconstituted in the dry environment were similar to the original lesions seen at birth. The present results suggest that ABCA12 may play an essential role both in the wet conditions during fetal development and in the dry conditions including the dramatic turning point from wet condition in the amniotic fluid to dry environment around the birth. Normal human fetal tissue was acquired (after informed consent was obtained) from Sapporo Maternity-Women's Hospital (Sapporo, Japan). Human embryonic and fetal skin specimens were obtained from abortuses of 7 to 22 weeks estimated gestational age (EGA). An HI fetal skin sample was obtained from an abortus at 21 weeks EGA that had been diagnosed with HI by prenatal skin biopsy.30Akiyama M Suzumori K Shimizu H Prenatal diagnosis of harlequin ichthyosis by the examinations of keratinized hair canals and amniotic fluid cells at 19 weeks' estimated gestational age.Prenat Diagn. 1999; 19: 167-171Crossref PubMed Scopus (28) Google Scholar Skin specimens were taken from the trunk, scalp, and fingers and processed for the present study. EGA was determined from maternal history, fetal measurements (crown, rump, and foot length), and comparative histological appearance of the epidermis. Immunofluorescence labeling was performed as described below. We used anti-ABCA12 antisera18Akiyama M Sugiyama-Nakagiri Y Sakai K MacMillan JR Goto M Arita K Tsuji-Abe Y Tabata N Matsuoka K Sasaki R Sawamura D Shimizu H Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J Clin Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (312) Google Scholar as a primary antibody. For control immunostaining, we also used mouse monoclonal anti-transglutaminase 1 (TGase1) antibody BT-621 (Biomedical Technologies, Inc., Stoughton, MA), because TGase1 is a major keratinization marker that is known to cross-link several precursor proteins in the formation of the cornified cell envelope during keratinocyte differentiation. Rabbit anti-human glucosylceramide antibody (Glycobiotech, Kükels, Germany) was used to clarify the expression sites of the lipid in the epidermis during development. Immunolabeling for keratin 10, a keratinization marker, and cathepsin D, a component of lamellar granules, was performed using mouse anti-human keratin 10 antibody (DAKO, Glostrup, Denmark) and rabbit anti-human cathepsin D antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in normal and HI fetal skin. Rat anti-human HLA class I antibody (Serotec Ltd., Oxford, UK) was also used to differentiate human keratinocyte-derived epidermis from host murine epidermis. Immunofluorescent labeling was performed as previously described.35Akiyama M Smith LT Yoneda K Holbrook KA Hohl D Shimizu H Periderm cells form cornified cell envelope in their regression process during human epidermal development.J Invest Dermatol. 1999; 112: 903-909Crossref PubMed Scopus (59) Google Scholar In brief, 6-μm-thick sections of fresh skin samples cut using a cryostat were prepared for immunolabeling. Sections were fixed in 4% paraformaldehyde for 15 minutes at 4°C for labeling with anti-human HLA class I antibody or in acetone for 10 minutes at room temperature for labeling with other antibodies (ABCA12, TGase1, keratin 10, and cathepsin D) except for anti-human glucosylceramide antibody. We performed glucosylceramide labeling without any fixation. The sections were incubated in primary antibody solution for 2 hours at room temperature. Primary antibodies and dilutions were as follows: rabbit polyclonal anti-human ABCA12 antibody,18Akiyama M Sugiyama-Nakagiri Y Sakai K MacMillan JR Goto M Arita K Tsuji-Abe Y Tabata N Matsuoka K Sasaki R Sawamura D Shimizu H Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J Clin Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (312) Google Scholar 1:800; mouse monoclonal anti-TGase1 antibody, BT-621, 1:100; rabbit polyclonal anti-human glucosylceramide antibody, 1:10; mouse polyclonal anti-human keratin 10 antibody, 1:100; rabbit polyclonal anti-human cathepsin D antibody, 1:10; and rat anti-human HLA class I antibody, 1:100. The sections were then incubated in each secondary antibody: fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin, anti-rabbit immunoglobulin, anti-rat immunoglobulin, or tetramethylrhodamine-5-(and -6)-isothiocyanate-conjugated donkey anti-rabbit immunoglobulin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:100 for 2 hours at room temperature, followed by 10 μg/ml TO-PRO-3 iodide (Molecular Probes, San Diego, CA) or propidium iodide (Dojindo Laboratories, Kumamoto, Japan) to counterstain nuclei for 10 minutes at 37°C. Sections were observed under an Olympus FluoView confocal laser scanning microscope (Olympus, Tokyo, Japan). To quantify the ABCA12 mRNA expression levels together with TGase1, loricrin, and kallikrein 7 (KLK7) in fetal skin, total RNA was extracted from fresh skin samples. Commercial epidermal mRNA obtained from Stratagene (La Jolla, CA) was used only for the specimen at 18 and 20 weeks EGA. In addition, total RNA was extracted from fresh skin samples obtained from a human adult (a generally healthy Japanese male without any skin disease) at a surgical operation of a benign subcutaneous tumor, and the RNA sample was used for the real-time polymerase chain reaction analysis. RNA samples were analyzed by the ABI prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Primers and probes specific for ABCA12, TGase1, loricrin, and KLK7 were obtained from the TaqMan gene expression assay (Applied Biosystems: Hs00292421_m1, Hs00165929_m1, Hs01894962_s1 and Hs00192503_m1). Differences between the mean CT values of ABCA12, TGase1, loricrin, and KLK7 and those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, or large ribosomal protein (Applied Biosystems) were calculated as ΔCTsample = CTABCA12 (or other keratinization markers) − CTGAPDH (or other housekeeping genes) and those of ΔCT for the normal adult skin as ΔCTcalibrator = CTABCA12 (or other keratinization markers) − CTGAPDH (or other housekeeping genes). Final results for fetal skin sample/adult skin (%) were determined by 2−(ΔCtsample − ΔCTcalibrator). Normal human fibroblasts and keratinocytes were purchased from Kurabo (Osaka, Japan). We established primary cultures of skin cells from two patients with HI. One patient harbored a homozygous splice site mutation c.3295-2A>G and the other harbored heterozygous mutations: p.Ser387Asn and c.4158_4160del (p.Thr1387del) as previously reported.36Akiyama M Sakai K Sugiyama-Nakagiri Y Yamanaka Y McMillan JR Sawamura D Niizeki H Miyagawa S Shimizu H Compound heterozygous mutations including a de novo missense mutation in ABCA12 led to a case of harlequin ichthyosis with moderate clinical severity.J Invest Dermatol. 2006; 126: 1518-1523Crossref PubMed Scopus (38) Google Scholar In detail, patients' keratinocytes were isolated from lesional epi-dermis after separation from the dermis by overnight treatment of dispase I (Godoshusei, Chiba, Japan). After 0.25% trypsin digestion for 5 minutes, epidermal cells were collected and cultured in defined keratinocyte serum-free medium (Invitrogen, San Diego, CA). Normal human keratinocytes were grown in the same culture medium. Normal human fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) and antibiotics. All of the cells were maintained at 37°C in 5% CO2 atmosphere. Engraftment was performed as previously described.37Sugiyama-Nakagiri Y Akiyama M Shimizu H Hair follicle stem cell-targeted gene transfer and reconstitution system.Gene Ther. 2006; 13: 732-737Crossref PubMed Scopus (25) Google Scholar Equal numbers of keratinocytes (normal human keratinocytes or keratinocytes from patients with HI) and normal human fibroblasts were combined at a final density of 6 to 8 × 106 cells, and the cells were thoroughly mixed. This cell slurry was engrafted into a silicon chamber attached to the back of an anesthetized severe combined immunodeficient mouse (Clea, Tokyo, Japan). After 1 week, the wounds had healed, and the chamber tops were removed. The skin reconstitution was completed 2 to 3 weeks thereafter. We succeeded in reconstituting HI skin using keratinocytes from a patient with HI who had a homozygous mutation, c.3295-2A>G,18Akiyama M Sugiyama-Nakagiri Y Sakai K MacMillan JR Goto M Arita K Tsuji-Abe Y Tabata N Matsuoka K Sasaki R Sawamura D Shimizu H Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J Clin Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (312) Google Scholar using the methods described above. Thus, with the same methods, we reconstituted HI lesions using keratinocytes from another patient with HI who had heterozygous mutations affecting both ABCA12 alleles, p.Ser387Asn and c.4158_4160del (p.Thr1387del) (see Ref. 37Sugiyama-Nakagiri Y Akiyama M Shimizu H Hair follicle stem cell-targeted gene transfer and reconstitution system.Gene Ther. 2006; 13: 732-737Crossref PubMed Scopus (25) Google Scholar for further detailed analysis of the reconstituted lesions). For transmission EM, fresh biopsies of fetal skin and reconstituted skin were fixed in 5% glutaraldehyde solution, postfixed in 1% OsO4, dehydrated, and embedded in Epon 812. All of the samples were ultrathin-sectioned at a thickness of 70 nm and stained with uranyl acetate and lead citrate. Photographs were taken using a Hitachi H-7100 transmission electron microscope. This study was approved by the medical ethical committees of Hokkaido University Graduate School of Medicine, Sapporo, Japan. The study was conducted according to the Declaration of Helsinki Principles. ABCA12 expression was seen in the periderm during the early period when the two-layered epidermis forms, about 6 to 9 weeks EGA (Figure 1B). In the two-layered epidermis, both ABCA12 and TGase1 were expressed only in periderm cells (Figure 1, A–D). In the three-layered epidermis (10 to 13 weeks EGA), ABCA12 staining was seen in the entire epidermis, including intense periderm staining, whereas TGase1 staining was restricted to the periderm (Figure 1, E–H). A similar pattern was observed in the period of four or more layered epidermis before keratinization (14 to 22 weeks EGA) (Figure 1, I–P). In the newborn skin, ABCA12 and TGase1 staining were restricted to upper layers of epidermis, mainly granular layers (Figure 1, Q–T). These staining patterns are similar to those in normal adult skin (Figure 1, U–X) as previously reported.18Akiyama M Sugiyama-Nakagiri Y Sakai K MacMillan JR Goto M Arita K Tsuji-Abe Y Tabata N Matsuoka K Sasaki R Sawamura D Shimizu H Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.J Clin Invest. 2005; 115: 1777-1784Crossref PubMed Scopus (312) Google Scholar We performed immunofluorescent staining of glucosylceramide, one of the most important precursors of ceramide, in normal and HI fetal skin. In normal fetal skin at 22 weeks EGA, the expression of glucosylceramide was observed in the upper epidermis, including periderm (Figure 2C). HI fetal skin at 21 weeks EGA shows marked hyperkeratosis by hematoxylin and eosin (H&E) staining (Figure 2B). In HI fetal skin, the expression of glucosylceramide was obviously reduced, and only weak expression was seen in the upper epidermis (Figure 2D), although the expression of keratin 10, a keratinization marker, and cathepsin D, a component of lamellar granules, in HI fetal skin (Figure 2, F and H) were similar to those in normal fetal skin (Figure 2, E and G). Thus, the reduced glucosylceramide expression was thought to be a specific change resulting from an abnormality in HI epidermis and was specifically caused by an ABCA12 deficiency in HI fetal skin. In two-layered epidermis (6 to 9 weeks EGA) (Figure 3, A and B), the periderm contained many vesicles (arrows). In the three-layered epidermal stage (10 to 13 weeks EGA) (Figure 3, C and D), vesicles were observed at the cell periphery of periderm cells. In four or more layered epidermis before keratinization (14 to 22 weeks EGA) (Figure 3, E and F), the number of vesicles close to the cell membrane significantly increased. Due to the intense ABCA12 staining in the cytoplasm of periderm cells, some of the vesicles were thought to be associated with ABCA12 staining seen in this period, although a large number of these vesicles are thought to be pinocytic vesicles. The thickening of the periderm cell membrane was markedly observed in the four-layered epidermis (Figure 3F). We examined the expression of ABCA12, TGase1, loricrin, and KLK7 mRNA by real-time reverse transcriptase-polymerase chain reaction. The results were normalized by expression of three housekeeping genes, GAPDH, β-actin, and large ribosomal protein, and the expression level of each mRNA was converted to a percentage rate compared with that of the normal adult skin. The expression level of ABCA12 mRNA normalized to GAPDH remarkably increased after 15 weeks EGA when compared with an earlier developmental stage (10 weeks EGA) (Figure 4). Expression rates of other keratinization-related molecules studied for controls, TGase1, loricrin, and KLK7, remained very low, whereas the expression rate of ABCA12 increased by up to 62% (at 18 and 20 weeks EGA) during development. The expression level of loricrin was very low (expression level at 18 and 20 weeks EGA/expression level in adult = 0.019%), probably due to the extremely high expression level of loricrin in adult samples. The results normalized to other housekeeping genes, β-actin, or large ribosomal protein, showed a similar pattern. ABCA12 mRNA expression during development became up-regulated to 86% (normalized to β-actin) or 62% (normalized to large ribosomal protein), whereas TGase1, loricrin, and KLK7 remained very low (data not shown). This increase in ABCA12 mRNA expression is consistent with ABCA12 immunofluorescence findings during human epidermal development. We regenerated normal human skin using cultured normal human keratinocytes and fibroblasts. Four weeks after transplantation, the grafts exhibited an ordinary skin appearance (Figure 5B). Immunohistochemical staining for anti-human HLA class I showed that the reconstituted epidermis and dermis had been organized by surviving human cells (data not shown). An epidermis with a morphology resembling that of normal epidermis was observed in reconstituted skin using normal control keratinocytes and normal human fibroblasts (Figure
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