Disruption of Aldh 3 a 2 gene 1 Disruption of the Sjögren-Larsson syndrome gene Aldh 3 a 2 in mice increases keratinocyte growth and retards skin barrier recovery

The fatty aldehyde dehydrogenase (FALDH) ALDH3A2 is the causative gene of Sjögren-Larrson syndrome (SLS). To date, the molecular mechanism underlying the symptoms characterizing SLS has been poorly understood. Using Aldh3a2 mice, we here found that Aldh3a2 was the major FALDH active in undifferentiated keratinocytes. Long-chain base metabolism was greatly impaired in Aldh3a2 keratinocytes. Phenotypically, the intercellular spaces were widened in the basal layer of the Aldh3a2 epidermis due to hyper-proliferation of keratinocytes. Furthermore, oxidative stress-induced genes were up-regulated in Aldh3a2 keratinocytes. Upon keratinocyte differentiation, the activity of another FALDH, Aldh3b2, surpassed that of Aldh3a2. As a result, Aldh3a2 mice were indistinguishable from wild-type mice in terms of their whole-epidermis FALDH activity, and their skin barrier function was uncompromised under normal conditions. However, perturbation of the stratum corneum caused increased transepidermal water loss and delayed barrier recovery in Aldh3a2 mice. In conclusion, Aldh3a2 mice replicated some aspects of SLS symptoms, especially at the basal layer of the epidermis. Our results suggest that hyper-proliferation of keratinocytes via oxidative stress responses may partly contribute to the ichthyosis symptoms of SLS. Sjögren-Larsson syndrome (SLS) is a hereditary neurocutaneous disorder caused by mutations in the fatty aldehyde dehydrogenase (FALDH) gene ALDH3A2. The major symptoms of SLS are mental retardation, spastic dior tetraplegia, and ichthyosis, with crystalline macular dystrophy sometimes comorbid (1). ALDH3A2 catalyzes conversion of fatty aldehydes with mediumto http://www.jbc.org/cgi/doi/10.1074/jbc.M116.714030 The latest version is at JBC Papers in Press. Published on April 6, 2016 as Manuscript M116.714030 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Disruption of Aldh3a2 gene 2 very-long-chain (medium-chain (MC), C5-10; long-chain (LC), C11-C20; and very-long-chain, ≥C21) to fatty acids (FAs), with the most preferred substrates being C16 and C18 aldehydes (2,3). To date, over 70 mutations have been found in the ALDH3A2 gene of SLS patients, and most of them cause >90 % reduction in enzyme activity (4). Since aldehyde molecules are reactive and toxic in general, it is considered that accumulated fatty aldehydes cause the SLS pathology by reacting with certain important proteins in the nervous system and epidermis and compromising their functions. Furthermore, the literature lacks a detailed description of the characteristics of Aldh3a2 knockout mice. The skin symptom ichthyosis is characterized by dry, thickened, and scaly skin, often likened to fish scales, and is caused by a skin permeability barrier defect related to multi-layered lipids (lipid lamellae) in the epidermis. The epidermis is composed of four cell layers—the stratum basale (SB), stratum spinosum, stratum granulosum, and the stratum corneum (SC)—the last of which is the site of these lipid lamellae, and accordingly has the most important role in skin barrier formation (5,6). Keratinocytes proliferate in the SB and migrate outward while differentiating into cell-layer-specific cell types. The major lipid components of lipid lamellae are ceramides, cholesterol, and FAs, with ceramides being the most abundant (5,6). A variety of ceramide species exist in the epidermis. Among them, acylceramides are a class of epidermis-specific ceramides and play an essential function in skin barrier formation (7,8). Ceramides are normally composed of a long-chain base (LCB) and a FA (9). However, acylceramides contain an additional hydrophobic chain (linoleic acid), which is esterified with the hydroxylated ω-carbon of the FA. In the epidermis of SLS patients, some ceramide species, including the acylceramide EOS (a combination of an esterified ω-hydroxy FA and the LCB sphingosine), are greatly reduced (10). Several metabolic pathways generate the substrates of ALDH3A2, fatty aldehydes. These include metabolic pathways of leukotriene B4, diet-derived phytol, plasmalogens, and fatty alcohols (11-15). Furthermore, we recently revealed that metabolism of LCBs also generates fatty aldehydes, i.e., hexadecanal (C16:0 aldehyde) from dihydrosphingosine (DHS) and trans-2-hexadecenal (16:1 aldehyde) from sphingosine (16-18). However, it is still unclear which fatty aldehyde or which metabolic pathway mainly contributes to the pathogenesis of SLS. ALDH3A2 belongs to the aldehyde dehydrogenase (ALDH) family, which is well represented in mammals (19 genes in human, and 21 in mouse) (19,20). The mammalian ALDH family is divided into 11 subfamilies (ALDH1-9, -16, and -18). Mouse Aldh3 in particular includes five subfamily members (20): Aldh3a2, Aldh3b1, Aldh3b2, and Aldh3b3, which exhibit high activities toward LC aldehydes, and Aldh3a1, which shows only weak activity toward them but high activity toward MC aldehydes (2). Human ALDH3, on the other hand, contains four ALDH3 subfamily members (ALDH3A1, ALDH3A2, ALDH3B1, and ALDH3B2, with no Aldh3b3 homolog). As their mouse homologs do, ALDH3A1, ALDH3A2, and ALDH3B1 exhibit high activity toward MC or LC fatty aldehydes (21-23). However, human ALDH3B2 lacks 94 N-terminal amino acids present in the corresponding mouse Aldh3b2, and has no enzyme activity, suggesting that human ALDH3B2 is a pseudogene (19). In the present study, to gain insight into the molecular mechanism behind SLS pathology, especially as it relates to ichthyosis symptoms, we by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Disruption of Aldh3a2 gene 3 analyzed Aldh3a2 KO mice. We revealed the contribution of Aldh3a2 to total fatty ALDH activities as well as mRNA expression profiles of Aldh3 subfamily members in various tissues. Although Aldh3a2 is the major FALDH in undifferentiated keratinocytes, Aldh3b2 mRNA levels began to surpass Aldh3a2 mRNA levels upon differentiation. Accordingly, gene disruption of Aldh3a2 had little effect on skin barrier formation and ceramide composition. However, Aldh3a2 KO mice did exhibit broadened intercellular spaces in the SB and delayed skin barrier recovery. Furthermore, proliferation and oxidative stress responses were enhanced in Aldh3a2 keratinocytes. Thus, our findings give a clue to understanding the molecular mechanism behind the early-stage pathogenesis of the SLS symptom ichthyosis. EXPERIMENTAL PROCEDURES Generation of Aldh3a2 Mice—Aldh3a2 gene trap mice (C57BL/6N background), in which exon 4 of the Aldh3a2 gene was flanked by two loxP sequences, were obtained from the European Mouse Mutant Archive (http://strains.emmanet.org/). Aldh3a2 heterozygous KO mice (Aldh3a2) were generated by crossing Aldh3a2 gene trap mice with CAG-Cre transgenic mice (24) obtained from the Riken BRC through the National Bio-Resource Project of the MEXT. The Aldh3a2 mice were maintained by repeated back-crossing with C57BL/6J mice. Aldh3a2 homozygous KO mice (Aldh3a2) were generated by intercrossing Aldh3a2 mice born from the second or higher generations of backcrossing. Genotyping was conducted by PCR using genomic DNAs and primers (p1 and p2 for detection of wild-type alleles; p3 and p2 for detection of KO alleles; Table 1). Mice were kept at 23 ± 1 °C in a 12-h light/dark cycle with a standard chow diet (PicoLab Rodent Diet 20, LabDiet, St. Louis, MO; or CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and water available ad libitum. Food intake of mice was measured from the ages of 16 to 23 weeks, every 24 h for 7 days. Statistical analysis was performed by one-way ANOVA using StatView (SAS Institute, Cary, NC). The animal experiments performed in this study were approved by the institutional animal care and use committees of Hokkaido University and Fujita Health University. Cell Culture—Mouse primary keratinocytes were isolated as described elsewhere (25). Briefly, skins prepared from P0 pups were treated with 3 ml of 5 mg/ml dispase (Thermo Fisher Scientific, Waltham, MA) for 24 h at 4 °C. Epidermis was then separated from dermis and washed with CnT-Prime, Epithelial Culture Medium (CELLnTEC Advanced Cell Systems AG, Bern, Switzerland). Keratinocytes were isolated by incubating with 500 μl of TrypLE Select (1x) (Thermo Fisher Scientific) for 15 min at room temperature, followed by rubbing the basal side of the epidermis. Keratinocytes were then washed with CnT-Prime, Epithelial Culture Medium, collected by centrifugation, suspended in the medium, and seeded at a cell density of 5x10 cells/cm. Differentiation was induced by replacing the medium with CnT-Prime, 3D Barrier Culture Medium (CELLnTEC Advanced Cell Systems AG) at subconfluency. Medium was freshly replaced every 3 days. RT-PCR—Total RNAs were isolated from various tissues and keratinocytes of mice using the NucleoSpin RNA II Kit (Machery-Nagel, Dueren, Germany), according to the manufacturer's instructions. RT-PCR was performed using the One-Step PrimeScript RT-PCR Kit II (Takara Bio, Shiga, Japan) and primers (for Aldh3a2, primers by gest on Sptem er 1, 2017 hp://w w w .jb.org/ D ow nladed from Disruption of Aldh3a2 gene 4 Aldh3a2-F and Aldh3a2-R; for Gapdh, primers Gapdh/GAPDH-F and Gapdh/GAPDH-R; Table 1). Real-time quantitative PCR was performed using the One-Step SYBR PrimeScript RT-PCR Kit II (Takara Bio) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA), according to the manufacturer’s manual. Forward (-F) and reverse (-R) primers for respective genes were used (Table 1). The gene amplification efficiencies of the used primers were all >90 %. The mRNA levels were normalized with those of Gapdh. The reaction was conducted by incubating the samples at 42 °C for 5 min and 95 °C for 10 sec, followed by 40 cycles of 95 °C for 5 sec, 63 °C for 30 sec, and 72 °C for 30 sec. Skin Permeability Assays—Transepidermal water loss (TEWL) was measured a