The establishment of an effective physical barrier protecting the living organism from its surrounding environment is the key function of mammalian skin (Hoffjan and Stemmler, 2007). The transport of most substances across the stratum corneum (SC) takes place through the lipid bilayer, suggesting its essential role for the epidermal barrier function (Jungersted et al., 2008). The major SC lipids, ceramides, cholesterol and fatty acids have distinct roles in maintaining normal barrier functions (Elias and Menon, 1991
; Ishikawa et al., 2010
). Ceramides as well as cholesterol were shown to have an influence on (and are influenced by) the integrity of the SC, while free fatty acids play a major role in the lipid bilayer formation and pH maintenance (Baroni et al., 2012). We have identified Ctip2 as a key regulator of skin lipid metabolism, which plays an important role in establishment of epidermal permeability barrier (EPB) during development. Ablation of Ctip2 leads to altered lipid composition in the developing mouse epidermis, such as reduced ceramide at E17.5-P0, as well as increased sphingomyelins at E16.5–E18.5. Ctip2 regulate sphingolipid homeostasis in mouse embryonic skin by modulating the expression of several key enzymes involved in lipid metabolism. Here we demonstrate that transcription factor Ctip2 is recruited to the promoter regions of a subset of genes (elox3, Lass2 and Gba2) involved in the sphingolipid biosynthesis pathways and could directly regulate their expression (Figure S5
Skin barrier defects are caused not only by altered levels of protein(s) involved in keratinocytes terminal differentiation, cross-linking of cornified envelops (CEs), formation of intercellular junctions, but also by impaired formation and maintenance of skin lipid barrier (Furuse et al., 2002; Kirschner and Brandner, 2012). Previous studies have shown that Ctip2 regulate keratinocyte proliferation and late terminal differentiation in a cell-autonomous manner; the ultrastructural analyses of the epidermis have revealed defects in lipid disc formation, loosely packed corneocytes, as well as disorganized intercellular lamellar body membranes in Ctip2 null mice (Golonzhka et al., 2008
and unpublished data). In this study, we have focused on the role of Ctip2 in controlling epidermal lipid barrier by modulating lipid metabolism. Herein, we have applied a mass spectrometry method to systemically detect and profile ceramides, sphingomyelins, sphingosines and sphinganines simultaneously in murine epidermis. As a crucial member of stratum corneum lipids, there are few reports on profiling of skin sphingomyelins. Our present data confirm deregulated levels of both long chain and very long chain saturated and unsaturated ceramides in the mutant epidermis at different stages of skin development. Along the same line, overall levels of long chain and very long chain saturated and unsaturated sphingomyelins were significantly enhanced in the mutants at all stages analyzed. In the present study, we have specifically looked at ceramide [NS] containing nonhydroxy fatty acids (N) and sphingosines (S), but were unable to perform mass-spectrometry studies on other subclasses containing (N) and phytosphingosines (P)-[NP]; (N) and 6-hydroxy sphingosines (H)-[NH]; hydroxy fatty acids (A) and sphingosines (S)-[AS]; [AP]; [AH]; ester-linked fatty acids (E), hydroxy fatty acids (O) and (S)-[EOS]; [EOP] and [EOH], due to the lack of available deuterated ceramide internal standards for each subclasses. Additional analyses for the different subclasses will be performed in future using specific internal standard for each subclass.
Our results indicate that altered expression(s) of ceramide synthases 1-6 (Lass 1-6), sphingomyelinases (Smpd1 and Smpd2), glucosylceramide synthase (Ugcg) and that of acid beta-glucosidase (Gba2) at different developmental stages of Ctip2 null skin contribute to an overall decrease in the epidermal ceramide levels in the mutant embryonic skin. It was also shown that several ceramidases involved in ceramide metabolism (Asah2 and Acer1) are reduced in Ctip2−/−
skin. As ceramide biosynthesis is a complex process regulated by multiple pathways, it is still unclear that which pathway contributes most to the reduction of epidermal ceramide content in ablation of Ctip2. Lass 1-6 have been reported to be involved in UVB-induced apoptosis and their expression was increased after UVB irradiation in normal human keratinocytes, but reduced in aged skin (Uchida et al., 2003; Jensen et al., 2005). A recent study has reported that Lass3 mutant mice die after birth due to increased skin barrier defects, characterized by increased transepidermal water loss (TEWL), loss of ultra-long chain ceramides as well as non-functional cornified envelopes (Jennemann et al., 2011). Upregulation of Sgms2 and reduced expression of Smpd1 and Smpd2 in Ctip2−/−
mice may partially account for increased amount of sphingomyelins observed at later stages in the mutant skin (). Furthermore, loss of Gba2, whose expression is altered in the mutant epidermis, has been reported to lead to barrier defects in Gaucher’s disease patients (Holleran et al., 1994). Similarly, eLox3 as well as its substrate 12R
-LOX) are involved in the formation of oxidized ceramides in mouse epidermis; 12R-LOX deficient mice die shortly after birth due to severe barrier abnormalities with absence of protein-bound EOS ceramide (Zheng et al., 2011). Interestingly, we have reported earlier that eLox3 expression is downregulated in the Ctip2−/−
embryonic skin (Golonzhka et al., 2008
). Here we have furthered our studies and have shown that Ctip2 could directly regulate the expression of eLox3, Gba2 and Lass2 in mouse embryonic skin by recruitment to their promoter regions. Also, the catabolism and metabolism of sphingosines were both downregulated in Ctip2−/−
epidermis, which may explain the consistency of its amount between wildtype and null mice epidermis. Altogether, we have identified Ctip2 as a key regulator of several lipid metabolizing genes and hence epidermal sphingolipid biosynthesis during skin development.
Sphingolipids are key components in skin barrier homeostasis, as well as in other cellular processes such as cell cycle arrest, apoptosis and stress responses (Hannun, 1996; Ogretmen and Hannun, 2004). Ceramide is a central metabolite, whose biosynthesis is regulated at multiple levels with spatial separation of enzymes involved in ceramide formation (Futerman and Riezman, 2005; Kitatani et al., 2008
). It remains unclear that if alteration of the enzymes in one of the pathways could have an effect on other pathways, and finally modulate the biological functions. It would be interesting to systemically compare the lipid composition in healthy subjects vs patients with skin disorders such as atopic dermatitis and psoriasis. It is possible that besides Ctip2, other regulatory pathways are also involved in controlling ceramide biosynthesis. B-cell lymphoma/leukemia 11A (Bcl11a/Ctip1) protein is a homolog of Ctip2, which was found to be upregulated in Ctip2 null mice at E18.5 (Golonzhka et al., 2008
). The role of Ctip1 in epidermal barrier development and maintenance is still unclear. Further investigation about the expression pattern and distribution of Ctip1, as well as its role in lipid barrier homeostasis in mouse epidermis will be performed utilizing Ctip1−/−