Ceramide Synthase 3 and its Essential Role in Skin Barrier Function and Male Fertility

The functional specialization of sphingolipids (SLs) is determined by their structural diversity and their unique expression patterns according to cell type and degree of differentiation. Ultra long chain (ULC) SLs characterized by an N-acyl moiety longer than 26 carbon atoms in length are primarily expressed in mature male germ cells and epidermal keratinocytes. To unveil the functional role of these unconventional SLs, it is necessary to define their biosynthetic requirements at the molecular level. In mammals, fatty acids are incorporated into SLs at the stage of ceramide synthesis by a family of six homologue enzymes, the ceramide synthases (CerS1–6). By analyzing the expression levels of the CerS family in skin, as well as in juvenile and in “germ cell-free” testis, solely CerS3 mRNA distinctively correlated with the presence of ULC-SLs. As found in vitro, the synthesis of ceramides with cerotoyl- (26:0) and montanoyl-residues (28:0) required CerS3, as demonstrated by a non-radioactive and detergent-free enzymatic assay established in living human cells expressing CerS3. For this purpose, activated ULC-acyl-CoAs as assay substrates and ceramide internal standards for mass spectrometric quantifications were specifically synthesized. The crucial role of CerS3 in the synthesis of ULC-SLs could be further confirmed with CerS3 deficient mice. Mass spectrometric analysis of epidermal extracts established acyl-CoAs ranging from 24 to 36 carbon atoms in length as substrates of CerS3 in vivo. CerS3 deficiency resulted in a complete loss of ceramides with acyl-chains longer than 24 carbon atoms, including all ω-hydroxy-ULC-ceramides. Consequently, the total epidermal ceramides were drastically reduced by 90% leading to a severe impairment of the epidermal permeability barrier and ultimately to premature neonatal death. The metabolic defect of CerS3 mice gave rise to a multitude of alterations associated with the maturation and terminal cornification of keratinocytes. At the stratum granulosum (SG), diminished size in filaggrin-containing granules and reduced number of loricrin-containing granules were accompanied by aberrant processing of filaggrin and decreased levels of loricrin. At the stratum corneum (SC), remnants of glycogen, nuclear material and organelle structures were detected at the first corneocyte layers. CerS3d/d mice exhibited a markedly thickened and compact SC that could be associated with the persistence of corneodesmosomes, eventually resulting from the reduced expression of the protease cathepsin D at the SG and SC. Additionally, the defective degradation of corneodesmosomes led to the distinctive persistence of the embryonic peridermal layer in newborn mutant epidermis. Furthermore, CerS3 deficient mice exhibited disorganized lamellar sheets that yield the formation of non-lamellar lipid agglomerates, rather than building up evenly distributed lipid unit lamellar structures. The compromised skin integrity of mutant mice facilitated the severe invasion of pathogens evidenced by the increased microbial growth and the formation of pseudohyphae prior to colonization by Candida albicans on cultured skin biopsies. In summary, these results demonstrated the prerequisite of a functional CerS3 for the generation of ULC-SLs, which play an essential role in skin barrier function and male fertility. The phenotypic alterations derived from the in vivo depletion of CerS3 suggested in addition a crucial regulatory function that could be encoded within the homeobox domain. Taken all together, these findings established the basis for the identification and diagnosis of potential human skin disorders associated with a CerS3 dysfunction