Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis

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Research Article

Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis Maik Dahlhoffa, Thomas Fröhlichb, Georg J. Arnoldb, Udo Müllerc, Heinrich Leonhardtc, Christos C. Zouboulisd, Marlon R. Schneidera,n a

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Germany Laboratory for Functional Genome Analysis LAFUGA, Gene Center, LMU Munich, Germany c Human Biology and BioImaging, Department of Biology II, LMU Munich, Germany d Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germany b

article information

abstract

Article Chronology:

Lipid metabolism depends on lipid droplets (LD), cytoplasmic structures surrounded by a protein-

Received 12 October 2014

rich phospholipid monolayer. Although lipid synthesis is the hallmark of sebaceous gland cell

Received in revised form

differentiation, the LD-associated proteins of sebocytes have not been evaluated systematically.

3 December 2014 Accepted 5 December 2014

The LD fraction of SZ95 sebocytes was collected by density gradient centrifugation and associated proteins were analyzed by nanoliquid chromatography/tandem mass spectrometry. 54 proteins were significantly enriched in LD fractions, and 6 of them have not been detected

Keywords: Lipid droplets Proteomics Sebaceous glands Acne

previously in LDs. LD fractions contained high levels of typical LD-associated proteins as PLIN2/ PLIN3, and most proteins belonged to functional categories characteristic for LD-associated proteins, indicating a reliable dataset. After confirming expression of transcripts encoding the six previously unidentified proteins by qRT-PCR in SZ95 sebocytes and in another sebocyte line (SebE6E7), we focused on two of these proteins, ALDH1A3 and EPHX4. While EPHX4 was localized almost exclusively on the surface of LDs, ALDH1A3 showed a more widespread localization that included additional cytoplasmic structures. siRNA-mediated downregulation revealed that depletion of EPHX4 increases LD size and sebaceous lipogenesis. Further studies on the roles of these proteins in sebocyte physiology and sebaceous lipogenesis may indicate novel strategies for the therapy of sebaceous gland-associated diseases such as acne. & 2014 Published by Elsevier Inc.

Introduction Accumulation and storage of lipids in mammalian cells take place in lipid droplets (LDs), cytoplasmic organelles consisting of a

neutral lipid core surrounded by a phospholipid monolayer [1–4]. Traditionally viewed as rather inert fat repositories, LDs recently attracted the attention of researchers from different fields as these organelles became linked to diverse physiological processes

Abbreviations: LA, linoleic acid; LD, lipid droplets; SG, sebaceous gland n Corresponding author. Fax: þ49 89 2180 76849. E-mail address: [email protected] (M.R. Schneider). http://dx.doi.org/10.1016/j.yexcr.2014.12.004 0014-4827/& 2014 Published by Elsevier Inc.

Please cite this article as: M. Dahlhoff, et al., Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.12.004

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beyond lipid storage, as well as to a number of pathologies including cancer cachexia, hepatic steatosis, and cardiovascular disease [5–7]. Since the discovery of perilipin 1 (PLIN1) as a LD-associated protein in 1991 [8], a growing number of proteins were found to be localized to the periphery of LDs, including the remaining members of the PLIN family, PLIN2-5 [9]. The LD-associated proteins are essential for normal LD formation, fusion, and degradation, and, therefore, indispensable for the functions of these organelles. Importantly, progresses in the methods for the isolation of LDs and analysis of their proteins by mass spectrometry allowed the characterization of the LD proteome of a growing number of cell types [10,11]. So far, the LD proteome has been characterized in many cell lines, including fibroblasts [12,13], epithelial cells [13–19], adipocytes [20–23], hepatocytes [24,25], macrophages [26], myoblasts [27], and pancreatic β-cells [28], as well as in tissues, including the adipose tissue [29], mammary gland [30], and liver [30–32]. In contrast, except for the identification of histone H3 in cultured hamster sebocytes [33], the LD proteome of sebaceous glands (SGs) or cultured sebocytes has not been characterized so far. This is perplexing considering that the key feature of sebocyte differentiation is a progressive accumulation of intracellular lipids via expansion of LDs, culminating in cell rupture and release of lipids and cell debris into the hair follicle canal [34–37]. While numerous functions have been ascribed to the SGs and their product (sebum), including a role in epidermal barrier, hair follicle integrity, and antioxidant and antimicrobial functions, their precise raison d'être remains to be determined. From a medical point of view, SGs are appealing structures, since their deregulation is a hallmark of the most common skin disease, acne [38,39], and possibly of other skin diseases as cicatricial alopecia [40] and certain types of tumors [41,42]. Sebocytes have a different origin compared to hepatocytes, fibroblasts, or adipocytes, and synthesize lipids significantly different in their composition compared to that of adipocytes [43]. Furthermore, while the stored lipids are mobilized in response to metabolic stimuli in adipocytes (lipolysis), sebocytes release their lipids via cell disruption (holocrine secretion). Finally, sebocytes display a distinct array of lipogenic enzymes and a unique differentiation program as compared to keratinocytes. We therefore postulated that LD of sebocytes may have a unique composition of LD-associated proteins, whose identification may provide new insights into the biology of sebaceous lipogenesis and possibly indicate new targets for treating SG-associated diseases. Here, we employed density gradient centrifugation and LC-MS/MS to isolate and characterized the LD proteome of the immortalized human SG cell line SZ95.

Materials and methods Cell culture and induction of sebaceous lipogenesis SZ95 [44] and SebE6E7 [45] sebaceous gland cells were cultured in Sebomeds medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum and 5 mg/l epidermal growth factor (EGF, Biochrom). To increase the cellular lipid load, cells were cultured in the above indicated medium supplemented with

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linoleic acid (LA, 10  4 M, Merck, Darmstadt, Germany) for 48 h without EGF supplementation.

Analysis of the lipid accumulation To evaluate the efficacy of the employed lipid accumulation protocol, SZ95 sebocytes were washed with PBS, fixed in 2% paraformaldehyd for 10 min and washed again twice with PBS. After permeabilization for 10 min in 0.5% Triton X-100 in PBS the cells were stained for 10 min in DAPI (2.5 m/ml, Sigma, Taufkirchen, Germany), washed once with PBS and then stained with AdipoRed (Lonza, Walkersville, MD, USA). After 10 min, the released fluorescence was measured on a fluorimeter (Infinite M1000 microplate reader, Tecan Group, Männedorf, Switzerland) with excitation at 485 nm and emission at 572 nm. The readout is presented in relative fluorescence units (RFU). The Operetta high content screening system (Perkin Elmer, Waltham, MA) was used for detecting nuclei and quantifying lipid droplet size and numbers in DAPI and Nile red-stained cells (345 nm and 488 nm, respectively). Nonconfocal images of the cells were acquired on 96-well plates with a 40  objective.

Isolation and harvest of the LD fraction LDs were isolated by density gradient centrifugation according to a standard protocol [46], with minor modifications. In each round, twenty 10-cm plates were employed. After 48 h of lipid synthesis, cells were washed twice with and then harvested in ice-cold PBS using a cell scraper and centrifuged at 1100  g for 15 min at 4 1C in a 15 ml Falcon tube. The pellet was resuspended in 1 ml hypotonic lysis buffer (HLM; 20 mM Tris.Cl, 1 mM EDTA, and 10 mM sodium fluoride, supplemented with a protease inhibitor cocktail, Roche) and incubated on ice for 10 min. The resuspended cells were homogenized in an ice-cold Potter-Elvehjem tissue homogenizer with a loose-fitting Teflon pestle (Wheaton, Millville, USA) via 8–10 gentle strokes with the hand-driven pestle, transferred to a new Falcon tube and centrifuged again as above. The supernatant, containing the released LDs, was transferred to a polyallomer ultracentrifuge tube, while the cell pellet was frozen at  80 1C for posterior analysis. One-third volume of ice-cold HLM containing 60% sucrose was carefully mixed to the supernatant, resulting in a final concentration of 20% sucrose, and the mixture was transferred to an ultracentrifuge tube. Next, 4 ml icecold HLM containing 5% sucrose was gently layered over the sample, followed by 5.5 ml ice-cold HLM. Centrifugation took place in an ultracentrifuge (Beckman), using a SW41Ti swinging bucket rotor at 28,000  g for 30 min at 4 1C. Floating lipid droplets were harvested using a self-made tube slicer into a 1.5 ml microcentrifuge tube and centrifuged at 14,000  g for 10 min at 4 1C to remove any residual HLM solution. The LD proteins were isolated by solubilization in 30 ml Laemmli buffer and frozen at 20 1C. Prior to the proteome analysis, the whole LD protein sample was separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue.

Proteomic studies Gel lanes were cut into 10 sections. Each gel slice was transferred in a 1.5 ml reaction tube and equilibrated twice with 50 mM NH4HCO3 for 10 min. Cysteines were reduced with 45 mM

Please cite this article as: M. Dahlhoff, et al., Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.12.004

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dithiothreitol (30 min, 55 1C), and alkylated with 100 mM iodoacetamide (30 min at room temperature). Gel slices were minced and subjected to overnight digestion at 37 1C with 100 ng porcine trypsin (Promega, Madison, WI, USA). Supernatants were preserved and peptides were further extracted from the gel slices with 50 mM NH4HCO3 followed by a 80% acetonitrile (ACN) wash. Prior to mass spectrometry peptide samples were dried using a SpeedVac concentrator (Bachofer, Reutlingen, Germany). For nano-LC-ESI-MS/MS, samples were dissolved in 40 ml 0.1% formic acid. Nano-chromatography was performed on an Nano-LC system (Ettan, MDLC, GE Healthcare, Munich, Germany) using the following method: samples were loaded on a C18 trap column (flow rate 5 ml/min, C18 PepMap100, particle size: 5 mm, 100 Å, column size: 300 mm  5 mm, Dionex, Sunnyvale, California, USA) and separated using a nano-flow column (Reprosil-Pur C18 AQ, 3 mm; 150 mm  75 mm, Dr. Maisch, Ammerbuch-Entringen, Germany) at a flow rate of 280 nl/min. The gradients used were 1–30% B in 80 min, 30–60% B in 30 min, and 100% B for 10 min (A: 0.1% formic acid in water and B: 0.1% formic acid in 84% ACN). Mass spectrometry was performed on an Orbitrap instrument (LTQ Orbitrap XL, Thermo Fisher Scientific, San Jose, CA, USA). For electrospray ionization distal coated SilicaTip (FS-360-20-10-D20, New Objective, Woburn, MA, USA) and a needle voltage of 1.7 kV were used. Mass spectra were acquired in parallel mode performing precursor mass scanning in the Orbitrap (60000 FWHM resolution at m/z 400) and five data dependent CID MS/ MS scans in the LTQ ion trap.

Data processing and bioinformatics MS data were analyzed with MASCOT 2.4 (Matrix Science, London, UK) using the human subset of the SwissProt Database (Release 2013_07) and the following parameters: (a) “Fixed Q2 modifications”: Carbamidomethyl (C) (b) Variable modifications: Oxidation (M); (c) Decoy database: checked, (d) Peptide charge: 2þ and 3þ; (e) Peptide tol. 7: 10 ppm; (f) MS/MS tol. 7: 0.8 Da. MASCOT dat-files were imported in Scaffold 4.1.1 (Proteome Software, Portland, OR, USA) and filtered for a FDRo1%. Normalized spectral counts, termed as “Quantitative value” in Scaffold, were used for quantitative comparison between cell lysate and LD samples. Cluster analysis of proteins (right-sided hypergeometric with Bonferroni step down) and network visualization (kappascoreZ 0.3) was done with Cytoscape V3.0.2 [47] with the ClueGO v2.0.7 [48] and CluePedia V1.0.8 [49] plugins using the Reactome database [50].

Quantitative RT-PCR Total RNA was isolated with TRIZOL reagent (Invitrogen) and 1 mg of RNA samples were reverse-transcribed in a final volume of 20 ml using RevertAid Reverse Transcriptase (Thermo Scientific, Schwerte, Germany) according to the manufacturer's instructions. Quantitative RT-PCR was carried out in a LightCyclers480 (Roche, Mannheim, Germany) using the primers listed below (0.5 mM), 1 ml cDNA, 0.2 mM probe (Universal ProbeLibrary Set, Roche), and the LightCyclers 480 Probes Master (Roche) in a final volume of 10 ml. Cycle conditions were 95 1C for 5 min for the first cycle, followed by 45 cycles of 95 1C

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for 10 s, 60 1C for 15 s, and 72 1C for 1 s. Transcript copy numbers were normalized to peptidylprolyl isomerase A (PPIA) mRNA copies. The ΔCt value of the sample was determined by subtracting the average Ct value of the target gene from the average Ct value of the PPIA gene. For each primer pair we performed no-template control and no-RT control assays, which produced negligible signals that were usually greater than 40 in Ct value. Experiments were performed in duplicates for each sample. Following primers were used: ALDH1A3: Forward 50 -tggtggctttaaaatgtcagg-30 , Reverse 50 -tattcggccaaagcgtattc-30 and Universal probe #53 (Cat. no. 04688503001), CSE1L: Forward 50 -tgtaaacctaactgagttctttgtgaa-30 , Reverse 50 -ccgtcagctttaaggacagg-30 and Universal probe #88 (Cat. no. 04689135001), EPHX4: Forward 50 -tggcccaattaaccattacc-30 , Reverse 50 -gtggtcaccatgtgatgtttg-30 and Universal probe #27 (Cat. no. 04687582001), XPO1: Forward 50 -ccctaatcccccaacgag-30 , Reverse 50 -cataattgctggcatagattacca-30 and Universal probe #12 (Cat. no. 04685113001), ZW10: Forward 50 -tgttgtaccaacatatcacaagga-30 , Reverse 50 -catacagttgttgtgatgaatagcag-30 and Universal probe #60 (Cat. no. 04688589001), IQGAP1: Forward 50 -ctagaaacaccagccaccagt30 , Reverse 50 -tcacggatagcacgtctctg-30 and Universal probe #38 (Cat. no. 04687965001), and PPIA: Forward 50 -cctaaagcatacgggtcctg-30 , Reverse 50 -tttcactttgccaaacacca-30 and Universal probe #48 (Cat. no. 04688082001).

Immunofluorescence and immunohistochemistry Immunofluorescence analysis was carried out by growing SZ95 or SebE6E7 sebocytes on cover slips inserted into 6-well plates for the indicated time points. Cells were treated with 0.1 mg/ml BODIPYs 558/568C12 (ThermoFisher Scientific, Dreieich, Germany) for the last 24 h of LA treatment. Cover slips were fixed for 10 min in 2% paraformaldehyde and permeabilized for 10 min in 0.5% Triton X100 in PBS. The cells were blocked in 5% donkey serum and afterwards incubated with the primary antibodies rabbit antiALDH1A3 (1:500) (Novus, Littleton, USA), rabbit anti-EPHX4 (1:500) (Novus), or rabbit IgG (1:500) (DAKO, Glostrup, Denmark) diluted in PBS as negative control, for 2 h at room temperature. After washing in PBS, the cover slips were incubated with the secondary antibody ALEXAs488-conjugated donkey anti-rabbit (Dianova, Hamburg, Germany). Finally, the cover slips were mounted in DAPI-containing Vectashild (BIOZOL, Eching, Germany). Paraformaldehyde-fixed biopsy samples (normal neck skin from a male, caucasian, 36 years old patient) obtained after informed consent were kindly provided by C. Rose, M.D. (Department of Dermatology, University of Lübeck, Germany). For immunofluorescence staining, 5 mm thick sections were deparaffinized, rehydrated and boiled for antigen retrieval for 20 min in 10 mM citrate buffer pH 6.0. After blocking, the sections were incubated with primary antibodies rabbit anti-ALDH1A3 (1:500, as above) diluted in TBS, rabbit anti-EPHX4 (1:500, as above), or rabbit IgG (1:500) as negative control, overnight at 4 1C. Next day the slides were incubated with a secondary goat anti-rabbit biotin-conjugated antibody (1:200) (BIOZOL, Eching, Germany) for 1 h and then for 30 min with a streptavidin–biotin complex. 3,30 -diaminobenzidine (KEM-EN-TEC Diagnostics, Taastrup, Denmark) was used as chromogen and the sections were counterstained with hemalum. Images were acquired using the Leica DFC425C digital camera (Leica Mikrosystems, Wetzlar, Germany) with a 63  objective.

Please cite this article as: M. Dahlhoff, et al., Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.12.004

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siRNA-mediated downregulation Lipofectamine RNAiMAX (Invitrogen, Darmstadt, Germany) was used to transfect SZ95 sebocytes at  40% confluence in 6-well plates with siRNAs for ALDH1A3, EPHX4, or with a negative control siRNA (Silencer Select, Ambion, Austin, TX, USA). 24 h after transfection the medium was changed and cells were either induced to synthesize lipids by adding LA to the medium as described above or culture in vehicle-containing medium. The cells were allowed to differentiate for 48 h and either analyzed with the Operetta system or dissolved in protein lysis buffer for Western blot analysis.

Mean LD size (µm2)

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Western blot analysis Protein was extracted using Laemmli-extraction-buffer, and the protein concentration was estimated via bicinchoninic acid protein assay. 30 mg of total protein was separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Schwalbach, Germany) by semidry blotting. Membranes were blocked in 5% w/v fat-free milk powder (Roth, Karlsruhe, Germany) for 1 h at room temperature. After washing in Tris-buffered saline solution with 1% Tween20 (Sigma, Taufkirchen, Germany), membranes were incubated over night at 4 1C in 5% w/v BSA (Sigma) with the appropriated primary antibody. Primary antibodies were rabbit anti-ALDH1A3 (1:500, as above), rabbit anti-EPHX4 (1:500, Santa Cruz, Heidelberg, Germany), and rabbit anti-A-Tubulin (1:5000, Cell Signaling, Frankfurt, Germany, #2125). After washing, membranes were incubated in 5% w/v fat-free milk powder with horseradish peroxidase-labeled secondary antibody donkey antirabbit (1:2000; NA934V, GE Healthcare, Munich, Germany). Signals were detected using an enhanced chemiluminescence detection reagent (GE Healthcare) and appropriated X-ray films (GE Healthcare, Munich, Germany).

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Fig. 1 – Evaluation of sebaceous lipogenesis in SZ95 sebocytes treated with LA or vehicle for 48 h. The mean number of LDs/ cell (a) and the mean LD size (b) were evaluated in an Operetta high content screening system. The images of DAPI and Nile Red-stained sebocytes (c) and the measurement of the emitted fluorescence (d) confirmed the increase in the lipid content of LA-treated cells. N ¼12 replicates for all quantitative analyzes. Data were analyzed by Student's t-test and asterisks indicate statistically significant differences (nnpo0.01, nnnpo0.001). Scale bars in c represent 50 lm.

the released fluorescence in a fluorimeter revealed a 13-fold increase in the lipid content (Fig. 1d).

Statistical analysis Ct values of gene targets of qRT-PCR analysis are shown relative to Ct values of the PPIA cDNA in Fig. 3a–c. In Fig. 3b and c all values are related to the mean value of the undifferentiated cells. Groups were compared by Student's t-test (GraphPad Prism version 5.0 for Windows, GraphPad Software, San Diego, CA, USA). Data are presented as mean7SD or box-plots with median. Group differences were considered to be statistically significant if po0.05.

Results Evaluation of lipid accumulation Before isolating LD fractions from SZ95 sebocytes, cells were treated with linoleic acid (LA) or vehicle (ethanol) for 48 h and stained with DAPI and Nile Red to evaluate the extent of lipid accumulation. LA is an essential fatty acid (FA) frequently employed for inducing lipid synthesis in sebocytes [51–53]. Analysis of the cells with an automated imaging system (Operetta) revealed a 2.5-fold increase in the number of LDs/cell (Fig. 1a) and a 2-fold increase in the mean LD size (Fig. 1b) in LA-treated cells. The increase in the cellular lipid load was readily recognizable in microscopic images (Fig. 1c), and quantification of

Harvest of LDs and characterization of the SZ95 sebocyte LD proteome Having confirmed a significant increase in lipid load, we next carried out two fully independent rounds of LDs isolation from LA-treated SZ95 sebocytes by employing a standard method based on density gradient centrifugation followed by harvest of the LD fraction with a tube slicer [46]. The proteins in the LD fraction or in the cell pellet (corresponding to the remaining cellular components) were separated by SDS-PAGE, trypsinized, and analyzed by LC-MS/MS (Fig. 2a). The protein clusters identified in the LD fraction (874 proteins) and in the cell pellet (1510 proteins) of each round are listed separately in Supplementary Table 1. To generate a reliable dataset of proteins clearly enriched in the LD fraction, we applied a spectral counting approach [54] comparing the abundance of proteins between the cell pellet and the LD fractions. Proteins were considered as enriched in the LD fraction if they showed (1) at least 2-times more spectral counts compared to the cell pellet; (2) a combined spectral count of at least 10; and (3) were detectable in both runs. Application of these criteria resulted in the identification of 54 proteins (Table 1). These included several known LD-associated proteins, such as PLIN2 and PLIN3, proteins previously shown to be

Please cite this article as: M. Dahlhoff, et al., Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.12.004

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strongly expressed in SZ95 sebocytes [51,53]. Functionally clustering of the identified proteins using Cytoscape and the plug-ins Clue-GOþCluePedia in combination with the Reactome database revealed an enrichment for proteins related to the metabolism of lipids and lipoproteins (Fig. 2b), further supporting the relevance of the dataset.

LA-treated SZ95 cells

Cell lysis, centrifugation

Cell pellet

LD fraction

Pre-fractionation by SDS-PAGE

10 fractions

10 fractions

LC-MS/MS analysis on an Orbitrap

Protein IDs from Protein IDs from cell fractions LD fractions

Quantification by spectral counting

Proteins enriched in LD fractions

Bioinformatic analysis by ClueGO

Phospholipid metabolism

Metabolism of lipids and lipoproteins

Glycerophospholipid biosynthesis

Triglyceride Biosynthesis Metabolism

Fatty acid, triacylglycerol, and ketone body metabolism

Fig. 2 – (a) Workflow of the method employed for the isolation and analysis of the SZ95 sebocyte LD fraction and cell pellet proteome. (b) ClueGO/CluePedia analysis of 54 proteins showing higher abundance in LD fractions compared to cell pellets. Enriched terms from the Reactome database are displayed as red nodes. The node size corresponds to the statistical significance (adjusted p-value) for each term. Gene symbols of the corresponding proteins are shown as small yellow circles and are connected to related terms from the Reactome database.

Identification of previously uncharacterized LD-associated proteins By checking Table 1 against the so far published LD proteomic studies (see “Introduction”), we identified 6 proteins (11.1% of all identified LD-associated proteins) not reported before as LDassociated proteins: CSE1L, EPHX4, IQGAP1, XPO1, ZW10, and ALDH1A3 (see Supplementary Fig. 1 for the MS/MS spectra of these proteins). QRT-PCR (Fig. 3a) confirmed expression of the transcripts encoding these six proteins in SZ95 sebocytes and in a second SG cell line, SebE6E7 [45]. Interestingly, EPHX4 was the only transcript whose abundance was affected by LA, which was observed both in SZ95 (Fig. 3b) and in SebE6E7 sebocytes (Fig. 3c). We next focused on two of the identified proteins, ALDH1A3 (whose transcript showed the highest expression) and EPHX4 (whose transcript showed the lowest expression and was regulated by LA). Immunofluorescence staining for ALDH1A3 in SZ95 sebocytes showed a rather widespread cytoplasmic localization, suggesting that this protein is not exclusively LD-associated (Fig. 3d). EPHX4, in contrast was localized essentially to the periphery of SZ95 sebocyte LD (identified by staining with the lipophilic dye bodipy), indicating this protein to be a bona fide LDassociated protein (Fig. 3d). A similar staining pattern was observed for both proteins in SebE6E7 sebocytes (data not shown). Finally, immunohistochemistry using human skin confirmed a rather strong expression of ALDH1A3 in SG cells, while the expression of EPHX4 was considerably lower (Fig. 3e, upper panel). Higher magnification of selected regions revealed that while ALDH1A3 showed a widespread cytoplasmic localization, EPHX4 was localized around the LD, and was therefore compatible with the pattern expected for a LD-associated protein (Fig. 3e, lower panel). Thus, the immuhistochemical localization of ALDH1A3 and EPHX4 overall confirmed the expression data for these proteins obtained from SG cell lines.

Downregulation of EPHX4 increases LD size and lipid accumulation is sebocytes Next, to identify potential functions of these proteins in sebocytes, we employed siRNA to downregulate ALDH1A3 and EPHX4 and assessed the consequences of their reduction in SZ95 sebocytes. While an almost complete depletion of ALDH1A3 could be achieved (Fig. 4a), the levels of EPHX4 could only be reduced by approximately 50% in spite of several modifications in the transfection protocol (Fig. 4b). Analysis of the SZ95 sebocytes with the Operetta system revealed that downregulation of ALDH1A3 did not affect the parameters mean LD size and lipid accumulation in vehicle or LA-treated cells (Fig. 4c). Downregulation of EPHX4, in contrast, increased LD size and lipid accumulation in vehicletreated sebocytes, but not in LA-treated cells (Fig. 4c).

Please cite this article as: M. Dahlhoff, et al., Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.12.004

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Table 1 – List of the 54 LD-associated proteins identified in SZ95 sebaceous gland cells. The list includes proteins identified in both runs, enriched by factor 2 or higher in the LD fraction compared to the cell pellet, and with a spectral count of at least 10 (combined from the two LD isolation rounds). Gene symbol

PLIN3 ACSL3 PLIN2 CYB5R3 LPCAT2 AUP1 LPCAT1 DHRS1 SCCPDH PNPLA2 NSDHL DHRS3 FAF2 LSS LPCAT4 SDR16C5 RDH10 TPD52L2 RAB7A RAB5C MVP ACSL4 ABHD5 C2orf43 CSE1L HSD17B11 HSD17B7 METTL7A ELMOD2 RAB5A ATG2A NBAS DHRSX DHDDS SQLE PHGDH TPRG1L EPHX4 UBXN4 IQGAP1 ALG5 XPO1 ZW10 ALDH1A3

Protein name

Uniprot ID

Perilipin-3 Long-chain-fatty-acid–CoA ligase 3 Perilipin-2 NADH-cytochrome b5 reductase 3 Lysophosphatidylcholine acyltransferase 2 Ancient ubiquitous protein 1 Lysophosphatidylcholine acyltransferase 1 Dehydrogenase/reductase SDR family member 1 Saccharopine dehydrogenase-like oxidoreductase Patatin-like phospholipase domain-containing protein 2 Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating Short-chain dehydrogenase/reductase 3 FAS-associated factor 2 Lanosterol synthase Lysophospholipid acyltransferase Epidermal retinol dehydrogenase 2 Retinol dehydrogenase 10 Tumor protein D54 Ras-related protein Rab-7a Ras-related protein Rab-5C Major vault protein Long-chain-fatty-acid–CoA ligase 4 1-acylglycerol-3-phosphate O-acyltransferase UPF0554 protein Exportin-2 Estradiol 17-beta-dehydrogenase 11 3-keto-steroid reductase Methyltransferase-like protein 7A ELMO domain-containing protein 2 Ras-related protein Rab-5A Autophagy-related protein 2 homolog A Neuroblastoma-amplified sequence Dehydrogenase/reductase SDR family member X Dehydrodolichyl diphosphate synthase Squalene monooxygenase D-3-phosphoglycerate dehydrogenase Tumor protein p63-regulated gene 1-like protein Epoxide hydrolase 4 UBX domain-containing protein 4 Ras GTPase-activating-like protein Dolichyl-phosphate beta-glucosyltransferase Exportin-1 Centromere/kinetochore protein zw10 homolog Aldehyde dehydrogenase family 1 member A3

Discussion Although lipid synthesis and accumulation within cytoplasmic LDs are the hallmark of SG cell differentiation, the LD-associated proteins of sebocytes have been only incipiently characterized. Here, using a proteomic approach, we identified 54 proteins that were significantly enriched in the LD fraction of SZ95 sebocytes and thus represent potential LD-associated proteins. Inspection of this list revealed a number of typical, established LD-associated proteins [10,11]. These included proteins involved in membrane

O60664 O95573 Q99541 P00387 Q7L5N7 Q9Y679 Q8NF37 Q96LJ7 Q8NBX0 Q96AD5 Q15738 O75911 Q96CS3 P48449 Q643R3 Q8N3Y7 Q8IZV5 O43399 P51149 P51148 Q14764 O60488 Q8WTS1 Q9H6V9 P55060 Q8NBQ5 P56937 Q9H8H3 Q8IZ81 P20339 Q2TAZ0 A2RRP1 Q8N5I4 Q86SQ9 Q14534 O43175 Q5T0D9 Q8IUS5 Q92575 P46940 Q9Y673 O14980 O43264 P47895

Average spectral counts LD fraction

Cell pellet

322.50 252.00 174.50 108.50 95.50 89.00 88.00 85.50 76.00 65.00 63.50 52.50 46.50 40.00 39.00 39.00 36.00 35.50 35.00 32.00 30.50 30.00 30.00 28.50 22.00 20.50 19.50 17.00 15.00 13.50 13.50 12.50 12.50 12.00 10.50 10.00 10.00 9.00 8.50 8.50 8.00 7.50 7.50 7.50

29.00 33.00 19.50 30.00 8.00 2.50 13.50 13.50 9.50 2.00 9.00 4.50 12.00 2.00 7.00 1.00 2.00 11.00 16.50 17.00 5.50 12.00 0.00 5.00 6.00 1.00 0.00 3.00 5.00 6.00 0.00 0.00 0.00 0.00 3.00 8.00 3.00 0.00 3.00 2.50 3.00 2.00 1.00 3.00

trafficking such as Rab family members (RAB5A, RAB5C, RAB7A), in protein degradation (AUP1, UBXN4), in lipid metabolism (ACSL3, CYB5R3, LPCAT1, LPCAT2, LPCAT4 etc.), and perilipins (PLIN2, PLIN3). The abundance of previously known LD-associated proteins, and in particular the high abundance of PLIN2 and PLIN3, for which a strong expression in SZ95 sebocytes has already been shown [51,53], confirms the reliability of the LD preparation. For a more detailed analysis, we functionally clustered the identified proteins using Cytoscape and the plug-ins Clue-GOþCluePedia in combination with the Reactome database. Importantly, proteins related to the metabolism of lipids and

Please cite this article as: M. Dahlhoff, et al., Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.12.004

647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706

E X PE R IM EN TA L C ELL R E S EA RC H

lipoproteins are functionally enriched, again demonstrating the relevance of the dataset. Six out of the 54 identified proteins (11.1%) have not been reported before as LD-associated proteins: CSE1L, EPHX4, IQGAP1, XPO1, ZW10, and ALDH1A3. Notably, these proteins belonged to functional categories typical for LD-associated proteins such as retinol metabolism

10 Rel. expression

SZ95

Rel. expression (SZ95)

SebE6E7

1 0.1 0.01 0.001

ALDH3A1 CSE1L

EPHX4

XPO1

ZW10

IQGAP1

1.5

*

1.0

0.5 ALDH3A1

0.0

Rel. expression (SebE6E7)

707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766

-

LA

CSE1L

EPHX4

-

-

LA

LA

XPO1

-

LA

ZW10

IQGAP1

-

-

LA

LA

1.5

1.0

] (]]]]) ]]]–]]]

7

(ALDH1A3), epoxide-containing lipid metabolism (EPHX4), signal transduction (IQGAP1) and protein transport (XPO1, CSE1L and ZW10). After confirming expression of all six proteins in SZ95 and in a second sebocyte cell line (SebE6E7), we focused on two of the identified proteins, ALDH1A3 and EPHX4. ALDH1A3 is one of three retinaldehyde dehydrogenases involved in the conversion of retinalaldehyde to retinoic acid [55], while EPHX4 belongs to a large family of enzymes that transform epoxide-containing lipids into products with decreased chemical reactivity and increased water solubility [56]. In both sebocyte cell lines, EPHX4 was localized exclusively to the periphery of LDs, indicating that EPHX4 is a genuine LD-associated protein. ALDH1A3, in contrast, showed a more widespread localization that included, but was not limited to LDs. Downregulation of EPHX4 by siRNA transfection increased LD size and lipid accumulation in vehicle-treated SZ95 sebocytes, but not in LA-treated cells. At the moment, we can only speculate about EPHX4's functions during sebaceous lipogenesis. The fact that EPHX4 is downregulated by LA treatment suggests that this protein may be a component of nascent or small LD rather than of growing LDs. Such a role is supported by the increase in lipid synthesis followed by depletion of EPHX4 in vehicle-treated cells: removal of EPHX4 may represent a stimulus or even a necessary event for the progression of sebocyte LD growth. The fact that no changes in lipid synthesis are observed after downregulation of EPHX4 in LA-treated sebocytes is probably explained by the fact that LA treatment provides an extremely powerful stimulus that may not be influenced by loss of a single LD-associated protein.

* Conclusion

0.5 ALDH3A1

CSE1L

EPHX4

-

-

XPO1

ZW10

IQGAP1

-

-

0.0 -

LA

LA

LA

-

LA

LA

LA

DAPI

Bodipy

ALDH1A3

Merge

DAPI

Bodipy

EPHX4

Merge

Neg. control

ALDH1A3

EPHX4

In conclusion, by characterizing the LD proteome of the human SZ95 SG cells, we confirmed the presence of a number of wellknown LD-associated proteins and identified several proteins hitherto unknown to be associated with LDs. Importantly, we confirmed expression of these previously unidentified proteins in two SG cell lines and showed that reduction of at least one of them, EPHX4, can alter sebaceous lipogenesis. Future studies are likely to reveal important functions for these proteins during Fig. 3 – Expression of LD-associated proteins in sebocytes. Quantitative RT-PCR confirmed expression of the indicated transcripts in both SG cell lines (a). EPHX4 expression is reduced in SZ95 (b) and SebE6E7 sebocytes (c) after treatment with LA (npo0.05). The indicated expression level was adjusted to the peptidylprolyl isomerase A (PPIA) transcript levels. Immunofluorescence employing SZ95 sebocytes revealed a widespread cytoplasmic expression of ALDH1A3 (d, upper panel), while EPHX4 (d, lower panel) was localized essentially at the periphery of LDs (identified by bodipy staining). Immunohistochemistry (e) employing human skin demonstrates the expression pattern of ALDH1A3 and EPHX4 in sebaceous glands (the negative control represents omission of the primary antibody). In all immunofluorescence studies, no staining was observed when the first antibody was omitted (not shown). Scale bars in d represent 15 lm for ALDH1A3 and 7.5 lm for EPHX4; scale bars in e represent 50 lm in the upper panel and 25 lm in the lower panel.

Please cite this article as: M. Dahlhoff, et al., Characterization of the sebocyte lipid droplet proteome reveals novel potential regulators of sebaceous lipogenesis, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.12.004

767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826

EPHX4 siRNA

No treatment

ALDH1A3 siRNA

Control siRNA

No treatment

] (]]]]) ]]]–]]]

ALDH1A3

-55 kDa

EPHX4

-42 kDa

TUBA1A

-52 kDa

TUBA1A

-52 kDa

1.5

Mean LD size

1.5

Lipid accumulation (RFU)

Fold change

*** Fold change

827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886

E XP E RI ME N TAL CE L L R ES E ARC H

Control siRNA

8

1.0

Vehicle

** 1.0

Vehicle

LA

0.5

LA

0.5 Control ALDH1A3 EPHX4 Control ALDH1A3 EPHX4

Control ALDH1A3 EPHX4 Control ALDH1A3 EPHX4

Fig. 4 – Downregulation of EPHX4 stimulates sebaceous lipogenesis. Western blot analysis showing the downregulation of ALDH1A3 (a) or EPHX4 (b) protein following transfection of SZ95 sebocytes with specific siRNAs. Measurement of Nile red and DAPI-stained SZ95 sebocytes (c) revealed increased LD size (left graph) and lipid accumulation (right graph) after siRNA-mediated downregulation of EPHX4 after 48 h under basal conditions but not under LA treatment. The results (n ¼6 replicates/group) are Q 3 representative for three independent experiments and are expressed relative to the values obtained with the control siRNA. RFU, relative fluorescence units. nnpo0.01; nnnpo0.001. sebaceous lipogenesis, and may indicate novel strategies for modulating SG function.

Conflict of interest The authors have declared no conflicting interests.

Acknowledgments We thank Fiona Watt (Centre for Stem Cells and Regenerative Medicine, King's College London, UK) for providing the SebE6E7 sebaceous gland cell line. The authors also thank Miwako Kösters and Stefanie Riesemann for excellent technical support

Appendix A.

Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.12.004.

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E X PE R IM EN TA L C ELL R E S EA RC H

947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006

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