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Premature Terminal Differentiation and a Reduction in Specific Proteases Associated with Loss of ABCA12 in Harlequin Ichthyosis

2009; Elsevier BV; Volume: 174; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2009.080860

1525-2191

Anna Thomas, Daniel Tattersall, Elizabeth E. Norgett, Edel A. O’Toole, David P. Kelsell,

Mast cells and histamine

One of the primary functions of skin is to form a defensive barrier against external infections and water loss. Disrupted barrier function underlies the most severe and often lethal form of recessive congenital ichthyosis, harlequin ichthyosis (HI). HI is associated with mutations in the gene that encodes the ABC transporter protein, ABCA12. We have investigated the morphological and biochemical alterations associated with abnormal epidermal differentiation and barrier formation in HI epidermis. An in vitro model of HI skin using human keratinocytes retrovirally transduced with shRNA targeting ABCA12 in a three-dimensional, organotypic co-culture (OTCC) system has also been developed. A robust reduction in ABCA12 expression had a dramatic effect on keratinocyte differentiation and morphology comparable with that observed in HI skin, including a thicker epidermis and abnormal lipid content with a reduction in nonpolar lipids. As seen in HI epidermis, proteins that are normally expressed in late differentiation were highly dysregulated in the ABCA12-ablated OTCC system. These proteins were expressed in the stratum basale and also in the stratum spinosum, indicative of a premature terminal differentiation phenotype. Expression of the proteases kallikrein 5 and cathepsin D was dramatically reduced in both HI epidermis and the OTCC model. These data suggest that ABCA12 is a key molecule in regulating keratinocyte differentiation and transporting specific proteases associated with desquamation. One of the primary functions of skin is to form a defensive barrier against external infections and water loss. Disrupted barrier function underlies the most severe and often lethal form of recessive congenital ichthyosis, harlequin ichthyosis (HI). HI is associated with mutations in the gene that encodes the ABC transporter protein, ABCA12. We have investigated the morphological and biochemical alterations associated with abnormal epidermal differentiation and barrier formation in HI epidermis. An in vitro model of HI skin using human keratinocytes retrovirally transduced with shRNA targeting ABCA12 in a three-dimensional, organotypic co-culture (OTCC) system has also been developed. A robust reduction in ABCA12 expression had a dramatic effect on keratinocyte differentiation and morphology comparable with that observed in HI skin, including a thicker epidermis and abnormal lipid content with a reduction in nonpolar lipids. As seen in HI epidermis, proteins that are normally expressed in late differentiation were highly dysregulated in the ABCA12-ablated OTCC system. These proteins were expressed in the stratum basale and also in the stratum spinosum, indicative of a premature terminal differentiation phenotype. Expression of the proteases kallikrein 5 and cathepsin D was dramatically reduced in both HI epidermis and the OTCC model. These data suggest that ABCA12 is a key molecule in regulating keratinocyte differentiation and transporting specific proteases associated with desquamation. Harlequin ichthyosis (HI; MIM 242500) is the most severe and often lethal form of recessive congenital ichthyosis.1Hsu WY Chen JY Lin WL Tsay CH [Harlequin fetus—a case report].Zhonghua Yi Xue Za Zhi (Taipei). 1989; 43: 63-66PubMed Google Scholar, 2Moreau S Salame E Goullet de Rugy M Delmas P Harlequin fetus: a case report.Surg Radiol Anat. 1999; 21: 215-216Crossref PubMed Scopus (12) Google Scholar, 3Sarkar R Sharma RC Sethi S Basu S Das R Mendiratta V Sardana K Kakar N Three unusual siblings with harlequin ichthyosis in an Indian family.J Dermatol. 2000; 27: 609-611Crossref PubMed Scopus (11) Google Scholar Infants born with HI have hard, thick skin covering most of their body. The skin forms large diamond-shaped plates resembling armor plating separated by deep red fissures, which restrict movement.4Buxman MM Goodkin PE Fahrenbach WH Dimond RL Harlequin ichthyosis with epidermal lipid abnormality.Arch Dermatol. 1979; 115: 189-193Crossref PubMed Scopus (67) Google Scholar These skin abnormalities also affect the shape of the eyelids and lips, causing ectropion and eclabion, respectively. HI sufferers can also have malformations or autoamputation of the fingers and toes because of constricting bands of skin in utero. Because of the impaired barrier function of the skin, neonates struggle to control water loss, regulate temperature, and are more susceptible to infection and have feeding difficulties.5Moskowitz DG Fowler AJ Heyman MB Cohen SP Crumrine D Elias PM Williams ML Pathophysiologic basis for growth failure in children with ichthyosis: an evaluation of cutaneous ultrastructure, epidermal permeability barrier function, and energy expenditure.J Pediatr. 2004; 145: 82-92Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar In addition, the tightened skin produced can cause breathing difficulties leading to respiratory failure. From our cohort data of more than 70 unrelated families, 45% of HI-affected neonates die soon after birth (if not still-born). Disease from ∼6 months onwards resembles a nonbullous congenital ichthyosiform erythroderma.6Haftek 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 (48) Google Scholar However, the skin barrier still remains severely compromised in HI patients, and they will always have problems with thermal regulation, have increased trans-epidermal water loss, and be at a greater risk of microbial infection.The gene defective in HI is ABCA12, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily of active transporters. ABCA12 mutations are responsible for all HI cases analyzed to date with the majority of mutations being either nonsense substitution or frameshift.7Kelsell 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 (259) Google Scholar, 8Akiyama M Sugiyama-Nakagiri Y Sakai K McMillan 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 (290) Google Scholar, 9Thomas AC Cullup T Norgett EE Hill T Barton S Dale BA Sprecher E Sheridan E Taylor AE Wilroy RS Delozier C Burrows N Goodyear H Fleckman P Stephens KG Mehta L Watson RM Graham R Wolf R Slavotinek A Martin M Bourn D Mein CA O'Toole EA Kelsell DP ABCA12 is the major harlequin ichthyosis gene.J Invest Dermatol. 2006; 126: 2408-2413Crossref PubMed Scopus (75) Google Scholar, 10Thomas AC Sinclair C Mahmud N Cullup T Mellerio JE Harper J Dale BA Turc-Carel C Hohl D McGrath JA Vahlquist A Hellstrom-Pigg M Ganemo A Metcalfe K Mein CA O'Toole EA Kelsell DP Novel and recurring ABCA12 mutations associated with harlequin ichthyosis: implications for prenatal diagnosis.Br J Dermatol. 2008; 158: 611-613Crossref PubMed Scopus (26) Google Scholar ABCA12 localizes to lamellar granules (LG) in normal epidermal keratinocytes.8Akiyama M Sugiyama-Nakagiri Y Sakai K McMillan 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 (290) Google Scholar The exact role of the ABCA12 protein in LG lipid transport is unknown, but it is hypothesized that ABCA12 transports glucosylceramide into the LGs and then the ABCA12-positive LGs secrete the lipid into the extracellular space to form the intercellular lipid layer.8Akiyama M Sugiyama-Nakagiri Y Sakai K McMillan 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 (290) Google Scholar Recent evidence has found that peroxisome proliferators-activated receptor (PPAR)-γ, PPAR-β, and PPAR-δ activators stimulate the expression of ABCA12 mRNA in cultured human keratinocytes.11Jiang YJ Lu B Kim P Paragh G Schmitz G Elias PM Feingold KR PPAR and LXR activators regulate ABCA12 expression in human keratinocytes.J Invest Dermatol. 2008; 128: 104-109Crossref PubMed Scopus (59) Google Scholar It was also found that liver X receptor (LXR) activators also increased ABCA12 mRNA expression but to a lesser extent than the PPAR activators. The PPAR and LXR activators have been shown to stimulate downstream effects in keratinocytes related to differentiation including increased LG secretion and lipid synthesis.12Schmuth M Jiang YJ Dubrac S Elias PM Feingold KR Thematic review series: skin lipids. Peroxisome proliferator-activated receptors and liver X receptors in epidermal biology.J Lipid Res. 2008; 49: 499-509Crossref PubMed Scopus (157) Google ScholarThe histology of HI skin is striking. The most obvious abnormality is the sheer thickness of the stratum corneum (SC) and indeed the entire epidermis.8Akiyama M Sugiyama-Nakagiri Y Sakai K McMillan 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 (290) Google Scholar, 13Milner ME O'Guin WM Holbrook KA Dale BA Abnormal lamellar granules in harlequin ichthyosis.J Invest Dermatol. 1992; 99: 824-829Abstract Full Text PDF PubMed Google Scholar With closer histological inspection, the nuclei become flattened early on in differentiation but are retained in the cornified layer (parakeratosis) and hyperkeratosis and hypergranulosis have also been noted as hallmarks of HI skin.4Buxman MM Goodkin PE Fahrenbach WH Dimond RL Harlequin ichthyosis with epidermal lipid abnormality.Arch Dermatol. 1979; 115: 189-193Crossref PubMed Scopus (67) Google Scholar In addition to the increased epidermal thickness, an abnormal localization of epidermal lipids has been described.14Dale BA Holbrook KA Fleckman P Kimball JR Brumbaugh S Sybert VP Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatol. 1990; 94: 6-18Abstract Full Text PDF PubMed Google Scholar, 15Akiyama M Shimizu H Yoneda K Nishikawa T Collodion baby: ultrastructure and distribution of cornified cell envelope proteins and keratins.Dermatology. 1997; 195: 164-168Crossref PubMed Scopus (15) Google ScholarA mouse knockout for ABCA12 has been described as having a postnatal lethal phenotype (line NIH-01279; neonatal lethal Mouse Genome Informatics). Recently, the Akiyama group16Yanagi T Akiyama M Nishihara H Sakai K Nishie W Tanaka S Shimizu H Harlequin ichthyosis model mouse reveals alveolar collapse and severe fetal skin barrier defects.Hum Mol Genet. 2008; 17: 3075-3083Crossref PubMed Scopus (57) Google Scholar has generated and characterized an ABCA12-null mouse. The phenotype resembles that of newborn HI skin plus reveals alveolar collapse as an additional phenotype linked to the nonviability in these mice. The expression of ABCA12 in normal fetal skin development and the grafting of HI keratinocytes on immunodeficient mice have also been described.17Yamanaka Y Akiyama M Sugiyama-Nakagiri Y Sakai K Goto M McMillan JR Ota M Sawamura D Shimizu H Expression of the keratinocyte lipid transporter ABCA12 in developing and reconstituted human epidermis.Am J Pathol. 2007; 171: 43-52Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar To complement these studies and because of the lethality of the ABCA12-null mice, we have generated and characterized an in vitro human model of HI skin using shRNA targeting ABCA12 in a normal keratinocyte cell line to study the role of ABCA12 in human epidermis.Materials and MethodsCell LinesThe immortalized keratinocyte cell line NEB118Morley SM Dundas S James J Brown RA Sexton C Navsaria HA Leigh IM Lane EB Temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from EBS patients.J Cell Sci. 1995; 108: 3463-3471Crossref PubMed Google Scholar was cultured in 3:1 Dulbecco's modified Eagle's medium-F12 medium, supplemented with 10% fetal calf serum, 2 mmol/L glutamine, 0.4 μg/ml hydrocortisone, 5 μg/ml insulin, 10 ng/ml epidermal growth factor, 10 × 10−10 mol/L cholera toxin, 5 μg/ml transferrin, 2 × 10−11 mol/L liothyronine, and 50 U/ml penicillin-G and 50 μg/ml streptomycin sulfate. Primary keratinocytes extracted from face lift skin were grown in co-culture with γ-irradiated mouse 3T3 fibroblast cells in the same media.Retroviral TransductionThe pSUPERIOR-retro-puro shRNA system (Oligoengine, Seattle, WA) was used to suppress the expression of ABCA12. Four pairs of oligos were designed as follows (sense sequence only shown): shRNA1: 5′-GATCCCGGAACTCCCAGGAAATAGCTTCAAGAGAGCTATTTCCTGGGAGTTCCTTTTTA-3′, shRNA2: 5′-GATCCCGGAGCACCTTCTCCTATATTTCAAGAGAATATAGGAGAAGGTGCTCCTTTTTA-3′, shRNA3: 5′-GATCCCGGACAGAGCTACCTCTATGTTCAAGAGACATAGAGGTAGCTCTGTCCTTTTTA-3′, shRNA4: 5′-GATCCCGCAAATGCATCTGCCCAGATTCAAGAGATCTGGGCAGATGCATTTGCTTTTTA-3′, scrambled control: 5′-GATCCCAAGCGCGGCAATAATCCTTTTCAAGAGAAAAGCGTAGGGTACTCTGTTTTTTA-3′. Subcloning of the oligos, transfection of the packaging cell line, production of retrovirus, and transduction of target cells were performed as per the manufacturer's instructions. shRNA1 was selected for subsequent experiments described in this article.Skin Biopsies and Organotypic Co-Culture (OTCC)Normal skin was obtained from redundant skin and a skin biopsy from a Bangladeshi male HI patient, age 14, homozygous for the ABCA12 mutation 6378delGC,7Kelsell 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 (259) Google Scholar was cryo-mounted before frozen sectioning. Consent and ethical approval was obtained for these studies. OTCC was performed as described previously.19Ojeh NO Frame JD Navsaria HA In vitro characterization of an artificial dermal scaffold.Tissue Eng. 2001; 7: 457-472Crossref PubMed Scopus (65) Google Scholar, 20Man YK Trolove C Tattersall D Thomas AC Papakonstantinopoulou A Patel D Scott C Chong J Jagger DJ O'Toole EA Navsaria H Curtis MA Kelsell DP A deafness-associated mutant human connexin 26 improves the epithelial barrier in vitro.J Membr Biol. 2007; 218: 29-37Crossref PubMed Scopus (48) Google ScholarTissue StainingEach tissue was cut in half such that one half was snap-frozen while embedded in Cryo-M-Bed (Bright) and was stored at −80°C. The other half was fixed in 4% paraformaldehyde and embedded in paraffin. Sections were cut at 5 to 6 μm in thickness. Hematoxylin and eosin (H&E) staining was performed on frozen sections following standard protocol.Epidermal Thickness MeasurementsNine frozen sections were cut from different positions of each OTCC and three measurements of each section were taken at random using the measuring tool with the MetaMorph software (Molecular Devices, Sunnyvale, CA) on an Eclipse microscope (Nikon, Tokyo, Japan). Statistical analysis was performed using an unpaired two-tailed t-test as described previously.20Man YK Trolove C Tattersall D Thomas AC Papakonstantinopoulou A Patel D Scott C Chong J Jagger DJ O'Toole EA Navsaria H Curtis MA Kelsell DP A deafness-associated mutant human connexin 26 improves the epithelial barrier in vitro.J Membr Biol. 2007; 218: 29-37Crossref PubMed Scopus (48) Google ScholarNile Red Lipid AnalysisA stock solution containing 0.05% Nile Red in acetone was diluted to 2.5 μg/ml with 75:25 glycerol:water, followed by brisk vortexing. A drop of the glycerol-dye solution was applied to each tissue section and immediately covered with a coverslip. Slides were then viewed using a Nikon Eclipse TE2000-S fluorescence microscope. Pictures were taken separately of the green and red fluorescence and then subsequently merged.AntibodiesThe ABCA12 antibody was generated by Harlen (Oxford, UK), using the 15-amino acid ABCA12 (NM_173076) motif present at residues 2504 to 2519 (QLHFPKTYLKDQHLS) plus a cysteine for conjugation. The rabbit polyclonal antibody obtained was then G-protein-purified before use, and used at a dilution of 1:500 for immunofluorescence. All other antibodies were used at 1:200 dilution. The LEKT1 antibody was a gift from Dr. W.L.Di (Institute of Child Health, UCL, London, UK). The involucrin (clone SY5) and K2e (clone LHK2E) antibodies were obtained from CRUK (Cancer Research UK, London, UK). The mouse anti-transglutaminase 1 (Biogenesis, Poole, UK), mouse anti-filaggrin (Biomeda, Foster City, CA), rabbit anti-KLK5 and goat anti-KLK7 (Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-cathepsin D (Abcam, Cambridge, UK) are available commercially.Immunohistochemical StainingFrozen sections were air-dried for 30 minutes and permeabilized in 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 15 minutes. Sections were washed in PBS, blocked in 0.2% gelatin from cold water fish skin for 15 minutes, and incubated with primary antibody for 1 hour. After three PBS washes, fluorescent secondary antibody (donkey anti-rabbit or donkey anti-mouse Alexa Fluor 488; Molecular Probes, Eugene, OR) was added at a 1/1000 dilution and incubated for 1 hour in the dark. Sections were washed three times in PBS, incubated with 4,6-diamidino-2-phenylindole (DAPI) (0.125 μg/ml) for 5 minutes, and washed three more times. Sections were mounted with immunomount reagent (Thermo Shandon, Waltham, MA) and viewed under a Nikon Eclipse TE2000-S microscope.Confocal ImmunofluorescenceImmunocytochemistry was performed using a standard protocol. Briefly, NEB1 cells that had been seeded onto glass coverslips were fixed in 4% paraformaldehyde for 30 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes. Nonspecific binding was blocked with 3% bovine serum albumin for 15 minutes before the cells were incubated in primary antibody solution for 1 hour. After washing, the cells were incubated with the relevant Alexa Fluor secondary antibodies (Molecular Probes) before being washed and mounted in Shandon Immu-mount (Thermo Electron Corporation, Waltham, MA) containing 10 μg/ml of DAPI. Cells were imaged using a Zeiss 510 confocal microscope (Zeiss, Thornwood, NY).ResultsABCA12 Expression Is Absent in HI SkinImmunofluorescence demonstrated ABCA12 was expressed throughout the normal interfollicular epidermis with prominent expression in the stratum granulosum (Figure 1A). Immunohistochemistry (IHC) with the ABCA12 antibody showed ABCA12 expression was significantly reduced/absent in the skin of this HI patient (Figure 1B).Abnormal Epidermal Differentiation and Protease Transport in HI SkinIHC analysis with antibodies raised against proteins expressed in late epidermal differentiation including involucrin, keratin 2e (K2e), transglutaminase 1 (TGase1), and filaggrin revealed an abnormal expression pattern in HI epidermis. Involucrin and K2e are primarily expressed in the granular suprabasal layers of normal control skin (Figure 2, A and B). In contrast, both K2e and involucrin were expressed throughout the entire suprabasal epidermis of the HI skin biopsy, with K2e expression also in the basal layer (Figure 2, E and F). The localization of both TGase1 and filaggrin was primarily in the SC in normal skin (Figure 2, C and D). This localization was consistent with the respective roles of these proteins in the epidermis. TGase1 is involved in the interprotein cross-linking of the cornified envelope.21Hennings H Steinert P Buxman MM Calcium induction of transglutaminase and the formation of epsilon(gamma-glutamyl) lysine cross-links in cultured mouse epidermal cells.Biochem Biophys Res Commun. 1981; 102: 739-745Crossref PubMed Scopus (79) Google Scholar Filaggrin is a product of proteolytic cleavage of profilaggrin and is thought to aggregate intermediate filaments during terminal differentiation.22Presland RB Kimball JR Kautsky MB Lewis SP Lo CY Dale BA Evidence for specific proteolytic cleavage of the N-terminal domain of human profilaggrin during epidermal differentiation.J Invest Dermatol. 1997; 108: 170-178Crossref PubMed Scopus (73) Google Scholar However, in HI skin TGase1 localization was membranous and was expressed throughout the entire epidermis including the basal layer (Figure 2G). The pattern of filaggrin expression was also abnormal in HI skin; it was absent from the SC but present throughout the upper spinous layer supporting a previously reported error in filaggrin processing in HI skin (Figure 2H).13Milner ME O'Guin WM Holbrook KA Dale BA Abnormal lamellar granules in harlequin ichthyosis.J Invest Dermatol. 1992; 99: 824-829Abstract Full Text PDF PubMed Google Scholar, 14Dale BA Holbrook KA Fleckman P Kimball JR Brumbaugh S Sybert VP Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: variable morphology and structural protein expression and a defect in lamellar granules.J Invest Dermatol. 1990; 94: 6-18Abstract Full Text PDF PubMed Google Scholar The filaggrin antibody also detects profilaggrin.Figure 2IHC of normal and HI patient skin biopsies with markers of late epidermal differentiation revealed an abnormal expression pattern in HI epidermis. In normal skin, the expression of involucrin (A) was observed primarily in the granular layer with keratin 2e (K2e, B) expressed variably throughout the suprabasal layers. In contrast, in HI skin both involucrin (E) and K2e (F) were expressed throughout the entire epidermis. The TGase 1 (C) and filaggrin (D) were expressed primarily in the SC in normal skin, however, in HI skin, TGase 1 (G) was expressed throughout the entire epidermis including the basal layer. H: Filaggrin was expressed throughout the upper spinous layer but was significantly reduced/absent from the SC. DAPI- or propidium iodide-stained nuclei are shown in blue and red, respectively, and the dermal-epidermal junctions are marked by a dotted white line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The expression of a number of LG-associated components was analyzed in HI and normal skin by immunofluorescence including lympho-epithelial Kazal-type-related inhibitor 1 (LEKT1), kallikrein (KLK) 7, KLK5, and cathepsin D (CTSD). LEKT1 was expressed throughout the epidermis but most strongly in the granular and upper spinous layers (Figure 3A) where it is associated with the LG network.23Ishida-Yamamoto A Deraison C Bonnart C Bitoun E Robinson R O'Brien TJ Wakamatsu K Ohtsubo S Takahashi H Hashimoto Y Dopping-Hepenstal PJ McGrath JA Iizuka H Richard G Hovnanian A LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.J Invest Dermatol. 2005; 124: 360-366Crossref PubMed Scopus (127) Google Scholar There was no obvious difference in the expression of LEKT1 between HI and control skin (Figure 3, A and E). In normal skin, KLK7 was identified in the stratum granulosum (Figure 3, B and F). However, some fainter expression of KLK7 was additionally identified in lower keratinocyte layers in HI skin. KLK5 expression in normal skin was observed in the upper epidermal layers but was significantly reduced/absent from the HI skin biopsy (Figure 3, C and G). CTSD was present in the stratum granulosum and upper spinous layers of normal skin but was also significantly reduced/absent from HI skin (Figure 3, D and H).Figure 3IHC of normal and HI patient skin biopsies with LG markers revealed KLK5 and CTSD (but not LEKT1 and KLK7) are significantly reduced/absent in HI skin. There was no obvious difference between the expression pattern of LEKT1 in normal (A) compared with HI skin (E) skin, being expressed in the granular and upper spinous layers. Kallikrein (KLK) 7 was localized to the stratum granulosum, in both normal skin (B) and HI skin (F), with some fainter expression in the lower keratinocyte layers in HI skin. In contrast, KLK5 (C) and cathepsin D (CTSD, D) were present in normal skin in the stratum granulosum and the SC, and the stratum granulosum and upper spinous layers, respectively, but both were significantly reduced/absent from HI skin (G and H). DAPI-stained nuclei are shown in blue, and the dermal-epidermal junctions are marked by a dotted white line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nonpolar Lipids Reduced in HI SkinNile Red analysis of lipids was performed to compare the lipid profile between the HI skin biopsy and normal control skin (Figure 4, A and B). Nile Red fluoresces green in the presence of nonpolar lipids (such as ceramides) and red in the presence of polar lipids (such as phospholipids and sphingomyelin). Pictures were taken to detect both red and green fluorescence separately and subsequently merged. In the SC of normal skin, a bright yellow-gold color is observed indicating the presence of both polar and nonpolar lipids. However, in HI skin the vast majority of lipid was polar because only red fluorescence was detected with no obvious yellow-gold color seen after the pictures were merged. The nonpolar ceramides comprise the bulk of the lipid contained within the lipid bilayers of the SC and are formed from the hydrolysis of glucosylceramide (GlcCer) transported via the LG system.24Hamanaka S Hara M Nishio H Otsuka F Suzuki A Uchida Y Human epidermal glucosylceramides are major precursors of stratum corneum ceramides.J Invest Dermatol. 2002; 119: 416-423Crossref PubMed Scopus (142) Google Scholar There is evidence to suggest ABCA12 is involved in the transport of GlcCer into the LG system. Human keratinocytes lacking functional ABCA12 show an abnormal intracellular localization of GlcCer that is resolved after functional gene transfer.8Akiyama M Sugiyama-Nakagiri Y Sakai K McMillan 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 (290) Google ScholarFigure 4Nile Red analysis reveals a disruption in the lipid profile in HI skin. A: The SC of normal skin contains both polar lipids (red) and nonpolar lipids (green). Expression of both lipid species is shown as yellow-gold in the merged image. B: In HI skin the vast majority of lipid is polar, with little green nonpolar lipid staining present.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Development of an in Vitro Skin Model for HITo ascertain if the abnormal epidermal differentiation and LG transport identified in HI skin was attributable specifically to lack of functional ABCA12, three-dimensional OTCCs using NEB1 cell line keratinocytes retrovirally transduced with a pSUPERIOR.retro.puro vector containing an ABCA12 shRNA construct. After shRNA retroviral transduction of NEB1 keratinocytes, cells were fixed onto coverslips for immunocytochemical analysis of ABCA12 expression. Robust ABCA12 knockdown was achieved (Figure 5A). OTCC using this shRNA construct was created on three separate occasions along with vector-only control (VOC) and a scrambled control (ScramC) (Figure 5, B and C). These cultures were allowed to grow at the air-liquid interface for 10 days.Figure 5H&E reveals a similar morphology in ABCA12 knockdown OTCCs than that seen in HI patients. Immunocytochemistry (A–C) demonstrating successful knockdown in NEB1 keratinocytes (A) compared with the VOC (B) and scrambled control (ScramC, C). IHC of OTCCs demonstrates that ABCA12 was strongly expressed throughout the epidermis in VOC (E) and ScramC (F) but was significantly reduced in the ABCA12 shRNA knockdown OTCC (D). H&E staining for the knockdown OTCC (G) showed a thicker and more disorganized epidermis compared with the control OTCCs (H and I). Dermal-epidermal junctions are marked by a dotted white line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)IHC with the ABCA12 antibody showed the protein was strongly expressed throughout the VOC (Figure 5E) and ScramC (Figure 5F) epidermis but was significantly reduced in the ABCA12 shRNA culture (Figure 5D). ABCA12 shRNA OTCCs (Figure 5G) formed a thicker and more disorganized epidermis compared with the VOC and ScramC sample (Figure 5, H and I).