Sphingolipid metabolism in organotypic mouse keratinocyte cultures. Lipid molecules enriched both upon ELOVL1 or SLC27A1 knockdown and upon suspension-induced differentiation of primary human keratinocytes. • Multipotent epidermal stem cells are likely to be the main target cells for the various types of epidermal … In the principal component analysis of the lipidomic data the majority of the variance (component 1) separated cells that had been disaggregated and immediately lysed (time 0) from cells that had been maintained in suspension (Fig. When subculturing or seeding cells for an experiment, we filtered the disaggregated keratinocytes through a 40 µm nylon strainer to remove cell clumps and large differentiated keratinocytes. The environmentally responsive nature of plant development means that cell lineage does not play the same role in plant … For a summary of materials and resources used, see SI Appendix, Table S4. 1A and subsequently examined using sparse partial least squares regression (sPLS) analysis to maximize separation between the sample groups, coupled with discriminant analysis (DA) to pinpoint the main lipid species contributing to such separation (26). Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions, Diverse epigenetic strategies interact to control epidermal differentiation, Involucrin and other markers of keratinocyte terminal differentiation, RNAi transfection results in lipidome changes, Human epidermal glucosylceramides are major precursors of stratum corneum ceramides, ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis, Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain fatty acid production, Fatty acid transporters in skin development, function and disease, Differential expression of fatty acid transport proteins in epidermis and skin appendages, Fatty acid transport protein 1 can compensate for fatty acid transport protein 4 in the developing mouse epidermis, Exome sequencing identifies FATP1 mutation in Melkersson-Rosenthal syndrome, Activation of keratinization of keratinocytes from fetal rat skin with N-(O-linoleoyl) ω-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) as a marker of epidermis, Impaired epidermal ceramide synthesis causes autosomal recessive congenital ichthyosis and reveals the importance of ceramide acyl chain length, Stem cell signaling. Keywords: The predominant lipid species change across the different epidermal layers, with the basal and spinous layers enriched in more polar lipid classes such as phospholipids and sphingomyelins, the granular layers exhibiting higher levels of glycosylceramides and cholesterol sulfates, and the cornified layer mostly composed of cholesterol, fatty acids, and ceramides, particularly ω-acylceramides that are only found in the outermost epidermal layer (14). The bioactive lipid mediators we tested, ceramides and glucosylceramides, are known to be important in the biology of the upper epidermal layers (32). Conversely, the siSLC27A1 samples were never clearly distinguishable from the nontargeting control samples. Interestingly, CerS3 can influence the activity of ELOVL1 by enhancing further elongation of C24 fatty acyl-CoA to C26/C28 fatty acyl-CoA (34). The transfected cells were incubated under conditions (feeder-free keratinocyte serum-free medium [KSFM]) that would enrich for undifferentiated cells or were treated with medium supplemented with fetal bovine serum, which is known to stimulate accumulation of differentiated cells (28, 29). The overlapping lipid species were sphingolipids (12/42), more specifically ceramides (6/42) and hexosylceramides (6/42), and glycerophospholipids (30/42), with the most represented types being phosphatidylcholines (8/42) and phosphatidylserines (7/42). from the Wellcome Trust (Grant 110060/Z/15/Z), and from a grant to F.M.W. This potentially indicates the existence of compensatory mechanisms to maintain skin homeostasis in vivo. Single-cell suspension-induced differentiation of primary human keratinocytes can be blocked in the presence of a protein kinase C inhibitor (PKCi) (Fig. Enrichment was determined by comparing the median of each sample class. Analysis was performed in positive and negative ion modes (resolution = 280,000 at m/z = 200 for MS and resolution = 17,500 at m/z = 200 for tandem MS [MSMS], where m/z indicates the mass-to-charge ratio). Error bars indicate SDs; P values are calculated using Dunnet’s multiple comparison test (*P < 0.05, **P < 0.01). We transfected primary human keratinocytes with a custom siRNA library targeting 258 lipid biosynthetic enzymes purchased from Dharmacon (siGENOME pools) (23). Error bars indicate SDs, P values are calculated using one-way ANOVA with Dunnett’s multiple comparison test (colony number) or the Kruskal–Wallis test with Dunn’s multiple comparison test (colony size) (*P < 0.05, ***P < 0.001). For lipid-induced apoptosis induction, n ≥ 3 independent treatments. 1) with the ones that accumulated in ELOVL1 or SLC27A1 knocked-down keratinocytes to see if there were any common species. 1F and SI Appendix, Fig. 4C). 3B). S2A). Online ISSN 1091-6490. For colony formation assays, n ≥ 2 hexaplicates of independent transfections or n = 12 independent treatments. Cell 43, 387–401 (2017). Epigenetic factors are proteins that modify either DNA or histone. The outermost part of the skin, the epidermis, is a stratified squamous epithelium formed predominantly by keratinocytes (1, 2). Green). To examine how lipids were affected in suspension culture, we analyzed samples using a shotgun lipidomics approach (25), comparing control and PKCi-treated cells (Dataset S1). Recent studies have described a mutation in ELOVL1 in humans that causes similar alterations in the lipid profile and is associated with dry skin and ichthyotic keratoderma, pointing to abnormal differentiation and therefore supporting our findings (35, 36). While detailed transcriptomic and proteomic analysis of suspension-induced differentiation was carried out previously (9), little is known about changes in the lipid composition of keratinocytes as they exit the stem cell compartment. MS alone was used to monitor LPA, LPE, LPE O-, LPI, and LPS as deprotonated anions; Cer, HexCer, SM, LPC, and LPC O- as acetate adducts; and cholesterol as the ammonium adduct of an acetylated derivative. ELOVL1 catalyzes the elongation of saturated and monounsaturated C20 to C26 acyl-CoAs. Despite numerous advances, current methodologies do not allow direct and systematic manipulation of lipid molecular species (27). We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes. Analysis of control (R26 mT/mG) head skin epidermis showed that asymmetric divisions, once considered the predominant division type of epidermal progenitors, occurred in approximately 44% of cases, and that symmetric renewal was nearly perfectly balanced by symmetric differentiation … A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. The siRNA screen yielded reproducible results, as indicated by the good correlation observed between each replicate and the mean of the quadruplicates (Pearson’s r ∼ 0.8; SI Appendix, Fig. Upon treatment, the stock solutions were heated to 37 °C, diluted to the indicated concentrations in culture medium preheated to 37 °C, and finally added to the monolayers. This latter concept is consistent with the idea that lipids operate by affecting the functionality of whole assemblies of molecular partners within their localized environment, which makes their effects challenging to evaluate using the more linear approaches that are commonly employed when studying proteins (27). Functional blocking of this local feedback showed that differentiation continues to occur in the absence of cell division, resulting in a rapid depletion of the epidermal … Nature 551, 327–332 (2017). Enrichment and fold change were determined by comparing the means of the replicates of each sample. Representative images for each treatment are shown below the bar plot. None of the other lipid classes showed such a marked change with time in suspension, although it was notable that several families were affected by PKC inhibition (SI Appendix, Fig. Samples in the siRNA knockdown time course were analyzed by PCA using the mixOmics package (v6.10.9) (54) with parameter scale = T after removal of near-zero variance predictors. contributed new reagents/analytic tools; and M.V.R., U.S.E., and F.M.W. By integrating lipidomic datasets with an siRNA screen of lipid modifying enzymes, we identified ELOVL1 and SLC27A1 as genes that, when knocked down, caused keratinocytes to undergo differentiation. Author contributions: M.V.R., A.M., U.S.E., and F.M.W. 2018 Oct;92(1):97-105. doi: 10.1016/j.jdermsci.2018.07.002. Acad. 2014 Mar;1841(3):435-40. doi: 10.1016/j.bbalip.2013.08.009. Please enable it to take advantage of the complete set of features! Dermatologica. A common set of individual lipid molecules was enriched in cells stimulated to differentiate by suspension culture or by either ELOVL1 or SLC27A1 knockdown. Having identified a panel of lipids enriched during keratinocyte differentiation, we asked whether any of them regulated the onset of differentiation. A two-step chloroform/methanol procedure was used to extract the lipids (52), and samples were spiked with an internal lipid standard mixture. To investigate whether lipids can regulate cell fate decisions, we carried out a systematic lipidomic analysis and perturbation of lipid metabolism in cultured human epidermal keratinocytes, determining associations with the onset of differentiation. Keratinocyte lipid composition changes during…, Keratinocyte lipid composition changes during suspension-induced differentiation. Vehicle concentration was 1%, equal to the level present in the highest lipid concentration. 4A). For the knockdown time course lipidomics, n = 3 replicates (independent transfections). The lipids were dissolved in ethanol at a stock concentration of 10 mM and stored at −20 °C. This would be consistent with SLC27A1 knockdown affecting differentiation without strongly perturbating the overall lipid composition of keratinocytes. The suspended cells were subsequently incubated at 37 °C. To maximize separation between the samples at 48 and 72 h posttransfection and identify discriminating lipid species, we employed sPLS-DA using the mixOmics package with the following parameters: ncomp = 3, keepX = rep(250,3), near.zero.var = T. Samples in the suspension-induced differentiation time course were analyzed by PCA using the mixOmics package with parameters ncomp = 3, scale = T after removal of near-zero variance predictors. It also remains technically challenging to dissect the exact mechanisms through which the lipid subspecies identified are operating. 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