Expression of Acyl-Coa Synthetase 5 in Human Epidermis
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Histol Histopathol (2008) 23: 451-458 Histology and http://www.hh.um.es Histopathology Cellular and Molecular Biology Expression of acyl-CoA synthetase 5 in human epidermis N.T. Gaisa*, J. Köster*, A. Reinartz, K. Ertmer, J. Ehling, K. Raupach, A. Perez-Bouza, R. Knüchel and N. Gassler RWTH Aachen University, Institute of Pathology, Aachen, Germany *Contributed equally to this work Summary. The human epidermis is characterized by a melanocytes and Langerhans cells, whereas CD1a is constant renewal of keratinocytes embedded in a matrix exclusively found in Langerhans cells. Merkel cells are enriched with lipids. Numerous proteins involved in characterized by Cytokeratin 20 expression. lipid metabolism are found in human epidermis, Discrimination of different epidermal cell types is of especially in keratinocytes. Long-chain acyl-CoA relevance in order to understand complex intraepidermal derivatives, which are catalyzed by human ACSL5, are pathways. Stress response and protective properties of important metabolites in several biochemical pathways, human epidermis are due to a well-balanced homeostasis including ceramide de novo synthesis. The aim of the of different cell types and a permanent renewal of present study was to investigate expression of acyl-CoA keratinocytes, embedded in a matrix enriched with synthetase isoform 5 (ACSL5) in human epidermis by an lipids. It is suggested that intraepidermal lipid in situ, as well as a molecular approach. We show that metabolism is essential for extracellular matrix ACSL5 mRNA and protein are found in human composition and a key factor in stress response (de Jager epidermis, as well as in non-differentiated and et al., 2004; Jennemann et al., 2007). differentiated HaCaT cells. Keratinocytes of stratum Long-chain free fatty acids and their derivatives, like spinosum are the main source for ACSL5 expression in ceramide, are the main components of intercellular lipid both meshed facial or abdominal skin and ridged skin of matrix, as well as stratum corneum (Pyne and Pyne, upper or lower extremities including TUNEL-positive 2000; Ponec et al., 2003). This structural and cells in upper cellular layers. Single keratinocytes of biochemical feature is reflected by the expression of chronic solar-exposed meshed facial epidermis several enzymes and molecules which are involved in occasionally display a stronger ACSL5 immunostaining. lipid metabolism (Harris et al., 1998; Schmuth et al., In conclusion, our study indicates that epidermal ACSL5 2005). In detail, expression of fatty acid binding proteins expression might be involved in differentiation and the (FABP), fatty acid transport proteins (FATP), and acyl- stress response of keratinocytes. CoA synthetase isoforms (ACSL) has been described in human epidermis, as well as in cultured keratinocytes Key words: Acyl-CoA synthetase 5, Epidermis, Human, and melanocytes (Harris et al., 1998; Kucharekova et al., Keratinocyte 2003; Cario-Andre et al., 2005; Schmuth et al., 2005; Yen et al., 2005; Scott et al., 2006). ACSL proteins and other molecules, like FATP4, are able to catalyze acyl- Introduction CoA derivatives from long chain fatty acids and coenzym A (Herrmann et al., 2001; Lewin et al., 2001; The human epidermis, an epithelium with constant Mashek et al., 2004). Acyl-CoA derivatives represent the cellular turnover, consists of at least four different cell metabolic active form of fatty acids which are involved types, including keratinocytes, melanocytes, Langerhans in several biochemical pathways of lipid metabolism. In cells, and Merkel cells, which are distinguished by mammalians, synthesis of long-chain acyl-CoA- specific marker profiles. S100 protein is expressed by thioesters is the most important step in cellular long- chain fatty acid metabolism (Mashek et al., 2004). Offprint requests to: Prof. Dr. Nikolaus Gassler, RWTH Aachen Up to now, five different isoforms of human acyl- University, Institute of Pathology, Pauwelsstraße 30, 52074 Aachen, CoA synthetase have been characterized by molecular Germany. e-mail: [email protected] and biochemical techniques (Mashek et al., 2004). 452 ACSL5 expression in keratinocytes Biochemical parameters of ACSLs, as well as their and protein according to Chomczynski (1993). Briefly, tissue distribution, reflected by normalized abundance of phase separation was induced by application of 200 µl ACSL transcripts, vary (Lewin et al., 2001; chloroform per ml suspension and subsequent http://harvester.fzk.de). ACSL1 through ACSL4 centrifugation. Precipitated RNA was dissolved in transcripts, are found in epithelia of trachea, mouth, distilled water/RNasin (Promega, Mannheim, Germany). liver, and skin, whereas ACSL6 is found in nerval Spectrophotometric measurements were performed with tissues and leucocytes. Isoform 5 is abundantly a NanoDrop ND-1000 spectrophotometer (NanoDrop expressed in intestinal surface epithelia (Gassler et al., Technologies, Wilmington, USA). Protein 2006). The ACSL5 gen is located on chromosome concentrations were estimated using BioRad assay 10q25.1-q25.2, spans approximately 46 kb, and reagent (BioRad, München, Germany). Concentrated comprises 21 exons coding for the ACSL5 protein (fatty Laemmli’s buffer was added to preparation volumes in acid CoA ligases: AMP forming; E.C. 6.2.1.3.) order to obtain one fold final concentrations. (Yamashita et al., 2000). However, data concerning the synthesis of ACSL proteins and their functional Reverse transcription and polymerase chain reaction relevance in human epidermis are very rare up to now. (RT-PCR) An in-depth analysis of ACSL5 expression in human skin is not available. SuperScript amplification system (Invitrogen, The purpose of the present study was to analyze Karlsruhe, Germany) was used for oligo (dT) primed ACSL5 expression in human epidermis by in situ first-strand cDNA synthesis from DNase-treated total techniques and a molecular approach. RNA (5 µg). Reverse transcription was terminated with an incubation step at 70°C for 15 min, followed by Materials and methods digestion with RNase H (20 min at 37°C). In control experiments, transcription of a commercially provided Tissue and cell culture RNA or substitution of the enzyme reverse transcriptase by distilled water was performed. A segment of human Formalin-fixed paraffin-embedded (n=20), as well as ACSL5 sequence (accession no. NM_016234) was snap frozen (n=10) specimens, derived from surgical amplified by PCR using LightCycler equipment (Roche resections of meshed or ridged skin from lower Diagnostics, Mannheim, Germany) and a pair of extremities, abdominal as well as facial regions, and the corresponding primers: 5’-TTT TTG TAC ACG GGG back were used. The use of human tissues was approved AGA GC-3’, 5’-ACA GGC TGT CAA TTT GGG TC- by the local ethics committee at the RWTH Aachen 3’. Amplifications were performed in a total volume of University. For molecular analysis unfixed epidermal 20 µl (3 mM MgCl2; 0.5 µM each primer; 2 µl SYBR layers were mechanically dissected, immediately frozen green mix) under the following conditions [35 x (10 s, in liquid nitrogen, and stored at -80°C until use. 95°C; 15 s, 68-58°C; 10 s, 72°C)]. Amplicon integrity Dissection was controlled by H&E stainings. HaCaT was further evaluated by agarose gels (2%) stained with cells, a spontaneously immortalized keratinocyte cell ethidium bromide (Sigma). line displaying a non-tumorigenic phenotype (Fusenig and Boukamp, 1998), was used for all cell culture SDS-PAGE and Western blot analysis experiments. Cells were cultured under standard conditions with DMEM high glucose supplemented with Proteins diluted in Laemmli’s buffer were resolved 10 % FCS (both substances from Sigma, Deisenhofen, by one-dimensional SDS-PAGE (7.5% gels) and Germany) and harvested in TRI reagent (Sigma). transferred to PVDF Immobilon-P membranes Differentiation of HaCaT cells was induced in (Millipore Corp., Bedford, USA) by semidry blotting. organotypic co-cultures as described elsewhere (Mass- Transfer of proteins was controlled by Coomassie Szabowski et al., 2003). Briefly, HaCaT cells (stock staining of gels and membranes. For detection of ACSL5 passages 28-35) were grown in organotypic co-cultures protein the monoclonal rat anti-human ACSL5 antibody, with postmitotic fibroblasts in collagen type-1 gels in clone KD7, the secondary anti-rat horseradish DMEM (Sigma) with 2 ng/ml TGF-alpha and 100 ng/ml peroxidase-conjugated antibody (dilution 1:10.000; GM-CSF (both R&D Systems, Minneapolis, USA). Santa Cruz, Santa Cruz, USA), and ECL substrate Afterwards co-cultures were fixed in 10 % formalin, (Amersham Pharmacia Biotech, Little Chalfont, UK) embedded in paraffin, and used for immuno- were applied sequentially. Normal small intestinal histochemical approaches as described below. mucosa was used as a positive control, in negative controls the primary antibody KD7 was omitted. RNA and protein isolation Immunostainings Tissue specimens or cultured cells were homogenized in TRI reagent (Sigma) using an Ultra For immunohistochemistry, sections of formalin- Turrax homogenizer (IKA Labortechnik, Staufen, fixed paraffin-embedded tissues were used. Briefly, Germany) followed by simultaneous preparation of RNA sections were dewaxed and then incubated with 453 ACSL5 expression in keratinocytes monoclonal anti-ACSL5 antibody KD7 for 1 h in a corroborated by molecular analyses of mechanically moist chamber at room temperature. The ABC detection dissected epidermal layers derived from meshed or kit with DAB as chromogen was applied