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• W2005005120 abstract "CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an α/β-hydrolase fold and is also known as the α/β-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca2+ conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation. CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an α/β-hydrolase fold and is also known as the α/β-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca2+ conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation. CGI-58 (ABHD5) is one of the genes that has been identified using the comparative proteomic approaches between Caenorhabditis elegans and humans.1Lai CH Chou CY Ch'ang LY Liu CS Lin W Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics.Genome Res. 2000; 10: 703-713Crossref PubMed Scopus (314) Google Scholar CGI was named after comparative gene identification. CGI-58 (α/β-hydrolase domain-containing protein 5; ABHD5) is a member of α/β-hydrolase family and a putative esterase/lipase/thioesterase. The physiological roles of CGI-58 protein were reported to be lipolytic degradation of fat in the lipid storing cells.2Lass A Zimmermann R Haemmerle G Riederer M Schoiswohl G Schweiger M Kienesberger P Strauss JG Gorkiewicz G Zechner R Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar However, its roles in various tissues including the epidermis have not yet been clarified.1Lai CH Chou CY Ch'ang LY Liu CS Lin W Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics.Genome Res. 2000; 10: 703-713Crossref PubMed Scopus (314) Google Scholar In 2001, CGI-58 mutations were identified in Dorfman-Chanarin syndrome (DCS; MIM 275630) families from the Mediterranean region3Lefèvre C Jobard F Caux F Bouadjar B Karaduman A Heilig R Lakhdar H Wollenberg A Verret JL Weissenbach J Ozguc M Lathrop M Prud'homme JF Fischer J Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome.Am J Hum Genet. 2001; 69: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar and, later, in a DCS patient of Japanese origin.4Akiyama M Sawamura D Nomura Y Sugawara M Shimizu H Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome.J Invest Dermatol. 2003; 121: 1029-1034Crossref PubMed Scopus (80) Google Scholar Additional DCS cases with CGI-58 mutations have been reported.5Caux F Selma ZB Laroche L Prud'homme JF Fischer J CGI-58/ABHD5 gene is mutated in Dorfman-Chanarin syndrome.Am J Med Genet A. 2004; 129: 214Crossref Google Scholar, 6Schleinitz N Fischer J Sanchez A Veit V Harle JR Pelissier JF Two new mutations of the ABHD5 gene in a new adult case of Chanarin Dorfman syndrome: an uncommon lipid storage disease.Arch Dermatol. 2005; 141: 798-800Crossref PubMed Scopus (25) Google Scholar, 7Pujol RM Gilaberte M Toll A Florensa L Lloreta J Gonzalez-Ensenat MA Fischer J Azon A Erythrokeratoderma variabilis-like ichthyosis in Chanarin-Dorfman syndrome.Br J Dermatol. 2005; 153: 838-841Crossref PubMed Scopus (49) Google ScholarDCS is an autosomal recessively inherited neutral lipid storage disease and is characterized by ichthyosis.8Dorfman ML Hershko C Eisenberg S Sagher F Ichthyosiform dermatosis with systemic lipidosis.Arch Dermatol. 1974; 110: 261-266Crossref PubMed Scopus (169) Google Scholar, 9Chanarin I Patel A Slavin G Wills EJ Andrews TM Stewart G Neutral-lipid storage disease: a new disorder of lipid metabolism.Br Med J. 1975; 1: 553-555Crossref PubMed Scopus (222) Google Scholar This entity shows leukocyte lipid vacuoles and involvement of several internal organs, including liver dysfunction, myopathy, cataracts, and a variety of neurological symptoms. The most characteristic feature of DCS is ragged skin over the entire body, caused by thickening of the cornified layers of skin surface, termed ichthyosis. All DCS cases present with this skin manifestation, ie, moderate to severe non-bullous congenital ichthyosiform erythroderma.10Williams MLK Lynch PJ Generalized disorders of cornification: The ichthyoses.in: Sams Jr, WM Lynch PJ Principles and Practice of Dermatology. Second Edition. Churchill Livingstone, New York1996: 379-396Google Scholar, 11Pena-Penabad C Almagro M Martinez W Garcia-Silva J Del Pozo J Yebra MT Sanchez-Manzano C Fonseca E Dorfman-Chanarin syndrome (neutral lipid storage disease): new clinical features.Br J Dermatol. 2001; 144: 430-432Crossref PubMed Scopus (41) Google ScholarIt was reported that truncation of CGI-58 protein results in abnormal lamellar granule (LG) formation in DCS.4Akiyama M Sawamura D Nomura Y Sugawara M Shimizu H Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome.J Invest Dermatol. 2003; 121: 1029-1034Crossref PubMed Scopus (80) Google Scholar LGs have been shown to form a continuous network from the Golgi apparatus (trans-Golgi network; TGN). LGs are multifunctional and are involved in packaging and secretion of a variety of enzymes and structural proteins. LGs are known to be involved in lipid transport and secretion in keratinocytes and are thought to play a crucial role in epidermal lipid barrier formation.12Elias PM Stratum corneum defensive functions: an integrated view.J Invest Dermatol. 2005; 125: 183-200Abstract Full Text Full Text PDF PubMed Scopus (514) Google ScholarTo date, the function of CGI-58 in the skin, the liver and the brain has not been clarified and the exact mechanism of pathogenesis of ichthyosis, liver dysfunction and mental retardation in DCS is not well understood. In this study, to elucidate the function of CGI-58 in the skin and to better understand the pathogenic mechanisms underlying the DCS phenotype including the ichthyosis, the expression patterns of CGI-58 were examined in normal human skin, harlequin ichthyosis patients’ LG deficient skin, developing human fetal epidermis in vivo and cultured human keratinocytes in vitro. In addition, CGI-58 expression was studied in mouse organs including the skin. Our findings clearly demonstrated for the first time that CGI-58 is expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 is localized to the LGs, which are lipid transporting and secreting granules in keratinocytes. CGI-58 expression was up-regulated during keratinocyte differentiation (keratinization) and human skin development. Reduced expression of keratinocyte differentiation markers was seen in CGI-58 knockdown keratinocytes. These facts suggest that CGI-58 plays crucial role(s) in epidermal keratinocyte differentiation and keratinocyte LG lipid metabolism, contributing to the skin lipid barrier formation.Materials and MethodsProduction of Anti-CGI-58 AntibodyPolyclonal anti-CGI-58 antibody was raised in rabbits using a 14 amino acid sequence synthetic peptide (residues 181-194) derived from the CGI-58 sequence (AL606838) as the immunogen (Sigma Genosys, Hokkaido, Japan). Rabbits were immunized by antigen injection (every 2 weeks, total six times). During the immunization period, serum anti-CGI-58 antibody titers were checked by enzyme-linked immunosorbent assay three times to confirm antibody production. At 77 days after the first antigen injection, the rabbits were sacrificed and entire serum was obtained. Anti-CGI-58 antisera were subsequently purified using an antigen affinity column. Immunofluorescent and immunohistochemical staining confirmed that the anti-CGI-58 antisera worked on frozen tissue sections. However, we could not obtain consistent staining results with the anti-CGI-58 antibody on routine, formalin-fixed and paraffin-embedded tissue sections.Human Fetal and Adult Skin SpecimensNormal 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 79 to 135 days estimated gestational age (EGA). 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.Normal adult human skin samples were obtained at surgical operations of benign subcutaneous tumors under fully informed consent at the Department of Dermatology, Hokkaido University Graduate School of Medicine.Ichthyosis Patients with LG AbnormalitiesDCS Patient Harboring a CGI-58 Truncation MutationThe patient had presented with severe ichthyosis since birth. The detailed information on this patient was previously described.4Akiyama M Sawamura D Nomura Y Sugawara M Shimizu H Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome.J Invest Dermatol. 2003; 121: 1029-1034Crossref PubMed Scopus (80) Google Scholar He had demonstrated liver dysfunction since infancy and a liver biopsy showed cirrhosis with severe fatty degeneration. He also had slight mental retardation. White blood cells in the peripheral blood and the basal cells and the lowermost spinous cells of the epidermis demonstrated large cytoplasmic lipid vacuoles characteristic of DCS. The patient was a homozygote for a nonsense mutation 550C>T transition (c.550C>T) in exon 4 of CGI-58 [sequence according to Lefèvre et al3Lefèvre C Jobard F Caux F Bouadjar B Karaduman A Heilig R Lakhdar H Wollenberg A Verret JL Weissenbach J Ozguc M Lathrop M Prud'homme JF Fischer J Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome.Am J Hum Genet. 2001; 69: 1002-1012Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar] (GenBank accession No. AL606839) that changed an arginine residue to a stop codon (p.Arg184X).4Akiyama M Sawamura D Nomura Y Sugawara M Shimizu H Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome.J Invest Dermatol. 2003; 121: 1029-1034Crossref PubMed Scopus (80) Google ScholarHarlequin Ichthyosis Patients Harboring ABCA12 MutationsBoth harlequin ichthyosis patients showed typical clinical features including thick scales over their entire body surface, severe ectropion, eclabium, and malformed pinnae. Both patients were homozygotes for ABCA12 truncation mutations and these mutations were predicted to lead to severe defects in ABCA12 keratinocyte lipid transporter function. The details of the harlequin ichthyosis patients were reported previously.13Akiyama 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 ScholarHuman Liver and Brain SamplesFrozen tissue slides of human liver and brain were purchased from BioChain Institute Inc. (Hayward, CA). Normal human adult liver or brain tissue sections cut at 5 to 10 μm thickness were mounted on a positively charged glass slide. The slides were fixed and dehydrated with acetone.MiceC57BL/6J mice were purchased from Clea Japan Inc. (Tokyo, Japan). The mice were kept in isolator cages in a barrier facility under a 12 hours light cycle and maintained under specific pathogen-free conditions.Cell CultureNeonatal human keratinocytes were purchased from Cambrex BioScience Walkersville, Inc. (Walkersville, MD). Human keratinocytes were cultured in Defined keratinocyte serum-free medium (Invitrogen, San Diego, CA). The keratinocytes were cultured in low Ca2+ conditions (0.09 mmol/L) to maintain a basal cell-like population of undifferentiated cells. To induce terminal differentiation, CaCl2 was added directly to the culture media for a final 2 mmol/L calcium concentration. In addition, three-dimensional, organotypic cultured epidermal sheets cultured over a non-proliferative fibroblast feeder layer (Japan Tissue Engineering Co. J-TEC, Aichi, Japan) were used for the study.AntibodiesPrimary antibodies to keratinization-associated proteins used in the present study were rabbit polyclonal anti-involucrin antibody (Biomedical Technologies, Inc., Stoughton, MA), mouse monoclonal anti-loricrin antibody (Covance Lab., Richmond, CA), and mouse monoclonal anti-transglutaminase 1 antibody (Biomedical Technologies, Inc., Stoughton, MA). As markers for TGN and LG contents, a TGN-marker; sheep polyclonal anti-TGN-46 antibody (Serotec Inc., Oxford, UK), anti-LG content antibodies; rabbit anti-human cathepsin D antibody (Santa Cruz Biotechnology, Inc., CA), and rabbit polyclonal anti-human β defensin 3 antibody (Novus Biologicals, Littleton, CO). Human β defensin 3 was reported to be localized to LGs.14Sawamura D Goto M Shibaki A Akiyama M McMillan JR Abiko Y Shimizu H Beta defensin-3 engineered epidermis shows highly protective effect for bacterial infection.Gene Ther. 2005; 12: 857-861Crossref PubMed Scopus (46) Google ScholarIn addition, rabbit polyclonal anti-glucosylceramide antibody (Glycobiotech, Kuekel, Germany) was used in the present study.ImmunoblottingCell lysates were prepared by homogenization in lysis buffer (50 mmol/L Tris-HCl [pH 7.6], 150 mmol/L NaCl, 1% NP-40, 0.1% SDS, 0.25% sodium deoxycholate), followed by centrifugation at 15000 rpm for 5 minutes. In addition, normal mouse and human tissue extracts were purchased from BioChain Institute, Inc. (Hayward, CA). Cell lysates (100 μg of protein per lane) were resolved on 12.5% SDS-polyacrylamide gels that were then electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) using a wet transfer apparatus. Membranes were probed with the rabbit polyclonal anti-CGI-58 antiserum (final dilution 1:10). Proteins were detected with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratory, West Grove, PA), and specific bands were visualized by chemiluminescence.Real-Time Reverse Transcription-Polymerase Chain Reaction AnalysisTo quantify the CGI-58 mRNA expression levels, total RNA was extracted from human keratinocytes cultured in low Ca2+ or high Ca2+ condition, and extracted from human adult and fetal skin. RNA samples were analyzed by ABI prism 7000 sequence detection system (Applied Biosystems, Lincoln Centre Drive Foster City, CA). Probes for real-time RT-PCR were as follows: a probe for CGI-58, Applied Biosystems, ID Hs 00211205_m1; a probe for involucrin, Applied Biosystems, ID Hs 00846307_sl; a probe for loricrin, Applied Biosystems, ID Hs 01894962_sl; a probe for transglutaminase 1, Applied Biosystems, ID Hs 00165929_ml; a probe for filaggrin, Applied Biosystems, ID Hs 00863478_gl. Differences between the mean CT values of CGI-58 and that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were calculated as ΔCTsample = CTCGI-58 − CTGAPDH and those of CT for the normal adult skin as ΔCTcalibrator = CTCGI-58 − CTGAPDH.RNA Interference and Generation of LentivirusesCGI-58 expression was depleted using short interfering RNA duplexes. Target regions in CGI-58 were identified by the BLOCK-iT RNA interference (RNAi) Designer Program (Invitrogen, San Diego, CA). Two sequences were used to generate RNAi molecules that target either 498-518 coding region of CGI-58 gene (RNAi 498, sequence 5′-CAGTTTGTGGAATCCATTGAA-3′; CGI-58 #1) or to the 886-906 region (RNAi 886, sequence 5′-GTGAGACAGCTTTCAAGAATA-3′; CGI-58 #2). These sequences were synthesized as DNA oligonucleotides and in addition contained complementary sequences joined by a 5′-GTTTTGGCCACTGACTGAC-3′ loop. Synthetic duplexes were cloned into the pcDNA6.2GW/Em green fluorescent protein (GFP) vector in the middle of the micro RNA155 (miR155) sequence supplied as a part of the BLOCK-iT Lentiviral Pol II miR RNA expression system kit (Invitrogen, San Diego, CA). In the pcDNA6.2GW/EmGFP vector the chimeric miR155-CGI-58 sequence is located under the control of the cytomegalovirus promoter co-cis-tronically with EmGFP. On processing in the cells by the endogenous endonuclease machinery, the construct produces anti-CGI-58 RNA duplex (microRNA [miRNA]-CGI-58 #1 and #2). We used the control vector pcDNA6.2GW/EmGFP-miR-neg that encodes random mRNA that fails to target any known vertebrate gene (scrambled control miRNA, Invitrogen, San Diego, CA). The expression cassette was transferred to the lentiviral shuttle vector pLenti6/V5-DEST vector (Invitrogen, San Diego, CA) by Gateway recombination following the instructions. For the generation of infectious lentiviral particles pLenti6/V5-DEST vectors containing miRNA-CGI-58 or miR-neg cassettes were co-transfected with ViraPower packaging plasmid mixture: pLP1, pLP2, and pLP/VSV-G (Invitrogen) into 293FT cells using Lipofectamine 2000 (Invitrogen). Ten-cm tissue culture plates were used to produce each batch of lentivirus. Virus-containing media was collected 60 hours after transfection, centrifuged at 2000 rpm for 20 minutes and filtered through a 0.45 μm filter (Millipore). Transduction of miRNA viruses to normal human keratinocytes was conducted following the manufacture's protocol. Briefly, normal human keratinocytes in 6-well plates were infected by 2-ml supernatants in the presence of 6 μg/ml Polybrene (Invitrogen). Six hours later, we removed the medium containing virus and replaced it with KGM-2 culture medium. After switching to high Ca2+ culture conditions, expression of CGI-58 and keratinocyte differentiation markers including involucrin, loricrin, filaggrin, and transglutaminase 1 was studied by real-time PCR analysis.Immunofluorescent LabelingThe tissue samples or three-dimensional, organotypic cultured epidermal sheets were immediately frozen in optimal cutting temperature compound (Tissue-Tek; Sakura Finetechnical, Tokyo, Japan), and cut at a thickness of 6 μm. Immunofluorescent labeling was performed as previously described.15Akiyama 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 Briefly, 6-μm thick sections of fresh tissue samples cut using a cryostat or cultured cells were prepared for immunolabeling. Sections were fixed in acetone for 10 minutes at room temperature for labeling with anti-human CGI-58 antibody or other antibodies. 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 CGI-58 antibody, 1/10; mouse monoclonal anti-cathepsin D antibody, 1/10; rabbit polyclonal anti-human β-defensin 3 antibody, 1/250; sheep polyclonal anti-TGN-46 antibody, 1/4; rabbit polyclonal anti-involucrin antibody, neat; mouse monoclonal anti-loricrin antibody, 1/250; and mouse monoclonal anti-transglutaminase 1 antibody, 1/10. The sections were then incubated in fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA) or donkey anti-rabbit immunoglobulins (DAKO, Glostrup, Denmark) diluted 1:100 for 2 hours at room temperature, followed by 10 μg/ml TOPRO3 (Sigma Chemical Co., St. Louis, MO) to counterstain nuclei for 10 minutes at 37°C or 10 μg/ml propidium iodide for 10 seconds. Sections were observed under an Olympus Fluoview confocal laser scanning microscope (Olympus, Tokyo, Japan). Furthermore, cultured cells were stained with similar procedures.Immunohistochemistry on Mice TissuesUnder deep ether anesthesia, 1-month-old mice (C57Bl/6CrSlc) were fixed by transcardial perfusion with 4% paraformaldehyde in a phosphate buffer (0.1M, pH 7.4). The brains and livers were immersed in the same fixative overnight, cryoprotected with 30% sucrose in phosphate buffer for 2 days at 4°C, and then cut into coronal sections at a thickness of 20 μm with a cryostat. The sections, mounted on gelatin-coated glass slides were treated as follows; with methanol containing 0.3% H2O2 for 30 minutes, pepsin solution (1 μg/ml in 0.2N HCl) for 5 minutes at 37°C, phosphate buffer for 10 minutes, 3% normal goat serum in phosphate buffer for 1hour, and a rabbit anti-CGI-58 antibody (diluted 1:500) for 2days at 4°C. After rinsing three times with phosphate buffer for 15 minutes, sections were visualized using the avidin-biotin-peroxidase complex method. After finishing diaminobenzidine reaction, sections of brain and liver were stained with Toluidine blue and hematoxylin, respectively.Postembedding Immunoelectron MicroscopyNormal human skin samples were obtained from surgical operations of benign subcutaneous skin tumors under fully informed consent, and were processed for postembedding immunoelectron microscopy as previously described.13Akiyama 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 Cyrofixed cryosubstituted samples were embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with anti-CGI-58 antisera antibody, a secondary linker antibody, and a 5-nm gold-conjugated antibody for immunogold labeling.ResultsEstablishment of Anti-CGI-58 AntibodyImmunoreactivity of the antiserum to the synthetic peptide (residue 181-194 of CGI-58) was confirmed by enzyme-linked immunosorbent assay with the synthetic peptide (data not shown). By immunoblot analysis, anti-CGI-58 antiserum exhibited approximately 45-kDa bands both in the human skin and the mouse skin extracts (Figure 1).CGI-58 Expression in the Brain and the LiverDCS patients with CGI-58 deficiency show neurological and hepatic symptoms including mental retardation, liver dysfunction, and liver cirrhosis. Thus, using the anti-CGI-58 antisera that we have created CGI-58 localization in the brain and the liver was studied.By immunoblot analysis, approximately 45 kDa-bands representing the CGI-58 peptide were detected in extracts from mouse brain and liver (Figure 1).Strong CGI-58-immunolabeling was observed in the brain and liver of mice (Figure 2). In the brain, CGI-58-containing cells were confined within the hypothalamus (arrows in Figure 2A), and no labeled cells were detected in other areas, such as the cerebral cortex, hippocampus, thalamus, brain stem, cerebellum, and spinal cord. In the hypothalamus, CGI-58 was localized in medium-sized (diameter ∼15 to 30 μm) multipolar neurons. The stained neurons were scattered throughout the hypothalamus, but did not accumulate in specific nuclei, such as the dorsomedial hypothalamic nucleus, ventromedial hypothalamic nucleus complex, arcuate hypothalamic nucleus. In higher magnification pictures of the hypothalamus, CGI-58-immunolabeling exhibited quite a fine granular pattern in the perikarya, dendrites and axons of neurons, but glial cells and blood vessel endothelial cells were negative (Figure 2B). Furthermore, CGI-58-containing axons extended into the cerebral cortex (Figure 2, C and D) and hippocampus (Figure 2E).Figure 2CGI-58 Distribution in the Brain and the Liver. Immunohistochemical localization of CGI-58 in the mouse brain (A–E) and liver (F, G). A-D: Hypothalamus at lower magnification (A) and at higher magnification (B–E). A: CGI-58-positive neurons (arrows) scattered in the hypothalamus. B: CGI-58-immunolabeling exhibited quite fine granules in the dendrites (de) and perikarya, but nuclei (Nu) were negative. Small neurons and glial cells (gl) did not contain CGI-58. Axons (Ax) with varicosities were densely stained. C–E: CGI-58-positive axons (arrows) entered into the layer 2 (C) and layer 4 (D) of the somatosensory cortex, oriens layer (or) and stratum radiatum (rad) of hippocampal formation CA1 region (E). These axons were also detected within the pyramidal cell layer of hippocampus (Pyr). F, G: The liver at lower magnification (F) and at higher magnification (G). Hepatocytes abundantly expressed the CGI-58. The CGI-58-immunolabeling exhibited a fine granular pattern in the cytosol (arrows). H, I: CGI-58 distribution in the human brain (H) and liver (I). In the normal human brain, CGI-58-positive neurons (white arrows) were scattered (H). In the normal human liver, cytoplasmic CGI-58 staining (white arrows) was seen in the hepatocytes (I). Abbreviations: Arc; arcuate hypothalamic nucleus, DMH; dorsomedial hypothalamic nucleus, ec; endothelial cells, ia; interlobular artery, LH; lateral hypothalamic area, mt; mammillothalamic tract, opt; optic tract, sin; sinusoid, III; third ventricle, VMH; ventromedial hypothalamic nucleus. CGI-58, green (FITC); nuclear stain, red (propidium iodide). Scale bars = 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the liver, CGI-58 was abundantly expressed in hepatocytes, but the sinusoidal endothelial cells and blood cells were negative (Figure 2, F and G). In the hepatocytes, CGI-58-immunolabeling exhibited a fine granular pattern (arrows), occupying the cytosol.The corneal epithelium was only weakly labeled with anti-CGI-58 antisera (data not shown), and CGI-58 was not detected in the epithelia of the digestive organs or the urogenital organs.Immunofluorescent localization of CGI-58 revealed that human liver hepatocytes and brain neurons showed discrete cytoplasmic labeling (Figure 2, H and I).CGI-58 Distribution in the EpidermisCongenital ichthyosiform erythroderma on the whole body surface is one of the major symptoms of DCS caused by CGI-58 deficiency. Thus, we studied the distribution of CGI-58 in the epidermis to obtain clues to clarify the function of CGI-58 and the pathomechanisms of ichthyosis in DCS.In normal human skin, the anti-CGI-58 antiserum stained the upper epidermal layers, mainly the granular layers in addition to dermal fibroblasts (Figure 3, A and B), although no apparent CGI-58-positive neurons in dermal nerves were observed. In contrast, there was an absence of immunolabeling in the skin of our DCS patient, who harbors a homozygous truncation mutation p.Arg184X4Akiyama M Sawamura D Nomura Y Sugawara M Shimizu H Truncation of CGI-58 protein causes malformation of lamellar granules resulting in ichthyosis in Dorfman-Chanarin syndrome.J Invest Dermatol. 2003; 121: 1029-1034Crossref PubMed Scopus (80) Google Scholar (Figure 3, C and D). This mutatio" @default.
• W2005005120 created "2016-06-24" @default.
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• W2005005120 title "CGI-58 Is an α/β-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes" @default.
• W2005005120 cites W1504523524 @default.
• W2005005120 cites W1923838861 @default.
• W2005005120 cites W1967695632 @default.
• W2005005120 cites W1968658112 @default.
• W2005005120 cites W1976336093 @default.
• W2005005120 cites W1978921042 @default.
• W2005005120 cites W1981131466 @default.
• W2005005120 cites W1981454564 @default.
• W2005005120 cites W1996471792 @default.
• W2005005120 cites W1998505872 @default.
• W2005005120 cites W2001039269 @default.
• W2005005120 cites W2005565418 @default.
• W2005005120 cites W2008804318 @default.
• W2005005120 cites W2012478400 @default.
• W2005005120 cites W2018832136 @default.
• W2005005120 cites W2020573304 @default.
• W2005005120 cites W2027455626 @default.
• W2005005120 cites W2047084876 @default.
• W2005005120 cites W2047745952 @default.
• W2005005120 cites W2049358414 @default.
• W2005005120 cites W2059105163 @default.
• W2005005120 cites W2071656852 @default.
• W2005005120 cites W2073532048 @default.
• W2005005120 cites W2075220584 @default.
• W2005005120 cites W2080095703 @default.
• W2005005120 cites W2090946848 @default.
• W2005005120 cites W2094265952 @default.
• W2005005120 cites W2136056145 @default.
• W2005005120 cites W2139226646 @default.
• W2005005120 cites W2141691234 @default.
• W2005005120 cites W2145946855 @default.
• W2005005120 cites W2158531928 @default.
• W2005005120 cites W2159577930 @default.
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