The role of Glucosylceramides in Keratinocyte Differentiation and Epidermal Barrier Function

Glycosphingolipids (GSLs) are amphiphatic molecules ubiquitiously expressed in eukaryotes and have been shown to play a crucial role in various (inherited) human disorders such as autoimmune diseases, ichthyoses and cancer. GSLs are one class of sphingolipids (SLs) and are constructed from ceramides (Cers), comprising a sphingoid base (typically sphingosine, a long-chain amino diol) which is amide-linked to a fatty acid (FA) of variable chain length. GSLs are formed by the addition of mono- or oligosaccharides via a glycosidic linkage to sphingosine C1. Glucosylceramides (GlcCers) are precursors for most of the higher GSLs and major compounds of the stratified squamous epithelia. In particular, GlcCers comprise ca. 4% of the total lipid mass in the epidermis and ca. 16% in the esophageal epithelium. The lipophilic anchor of epidermal GlcCers contains a unique ultra-long-chain amide-linked fatty acid (ULC-FA) with up to 36 carbon atoms. The ULC-FAs are typically ω-hydroxylated to give the so-called OS class of GlcCers, accounting for an essential part of the skin barrier. The ULC-GlcCers reside primarily in the outermost anucleated layers of the epidermis, i.e., the stratum corneum (SC), which is mainly composed of dead, flattened keratinocytes, the so-called corneocytes, embedded in a lipid-enriched extracellular matrix. This barrier protects the body from dehydration, mechanical insult and infection and derives from the interplay between proteins in the corneocyte membrane and the lipids. The lipid matrix surrounding the corneocytes is composed of Cers, cholesterol and free FAs in nearly equimolar ratios, which can be arranged into lamellar sheets. The Cers found in the SC are believed to be derived from GlcCers which have been synthesized at the trans-Golgi network by the enzyme UDP-glucose:ceramide glucosyltransferase (UGCG). Thus, it has been suggested that GlcCers function as intracellular carriers of the hydrophobic Cers and are transferred via vesicular transport within lamellar bodies (LBs) to the apical plasma membrane (PM) of SC keratinocytes. LBs fuse with the PM in the upper stratum granulosum (SG) and at the SG/SC interface, where they exocytose their lamellar lipid content, together with structural proteins, enzymes and antimicrobial peptides, into the extracellular space. By the action of enzymes such as glucosylceramidase and ceramidase, Cers, glucose, FAs and free sphingoid bases are released from GlcCer precursors. The ω-hydroxy group of ULC-(Glc)Cers enables their esterification to an additional FA, predominantly linoleic acid (18:2, ω–6), to give the EOS subclass of (Glc)Cers. Trans-esterification to proteins of the corneocyte membrane then leads to the formation of protein-bound Cers (POS) and the - 2 - cornified lipid envelope (CLE). EOS as well as POS species and their corresponding GlcCer-precursors establish the extremely hydrophobic extracellular lipid lamellae of the SC and thereby the skin barrier. An earlier study using mice with constitutive Ugcg deletion in the epidermis (Ugcgf/fK14Cre mice) highlighted the importance of Cer glucosylation. Mutant mice lost the water permeability barrier (WPB) and died postnatally at day P5. However, the exact molecular and cellular contribution of OS and specifically POS-Cers for the buildup and stability of the WPB could not be determined. In addition, the differentiation and function of UGCG-deficient epidermis and its function in clinically relevant pathophysiologic states, e.g. wound healing, could not be analyzed. Inducible Ugcgf/fK14CreERT2 mice were generated in our laboratory to circumvent the problem of early death experienced with Ugcgf/fK14Cre mice. Tamoxifen (TAM)-induced Ugcg gene deletion in keratin K14-positive basal cells resulted in a significant decrease in esophageal and epidermal GlcCers and, in contrast to newborn mice, to an almost complete loss of epidermal POS-Cers three weeks after initiation of TAM induction. Subsequent alterations in keratinocyte differentiation and epidermal barrier homeostasis were evidenced by an increase in transepidermal water loss (TEWL) and pH as well as by keratinocyte hyperproliferation to give a severe ichthyosiform skin phenotype. This skin phenotype was slightly reversed within three months following TAM induction, apparently due to the action of hair follicle stem cells. Furthermore, wound closure and reepithelialization was significantly delayed in mutant vs. control skin. Gene expression profiling of Ugcg mutant skin vs. control skin revealed a subset of differentially expressed genes which are involved in lipid signaling and epidermal differentiation/proliferation and which correlate with human skin diseases such as psoriasis and atopic dermatitis. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a Cer-sensitive transcription factor, was identified as a potential mediator of the altered gene sets. Thus, we conclude that the combined increase in free epidermal Cers and the expression of PPARβ/δ and target genes serves as the driving mechanism for impaired epidermal homeostasis and barrier loss, thereby emphasizing the critical role of GlcCer in epithelial differentiation.