Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis

The permeability barrier is required for terrestrial life and is localized to the stratum corneum, where extracellular lipid membranes inhibit water movement. The lipids that constitute the extracellular matrix have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids. Essential fatty acid deficiency results in abnormalities in stratum corneum structure function. The lipids are delivered to the extracellular space by the secretion of lamellar bodies, which contain phospholipids, glucosylceramides, sphingomyelin, cholesterol, and enzymes. In the extracellular space, the lamellar body lipids are metabolized by enzymes to the lipids that form the lamellar membranes. The lipids contained in the lamellar bodies are derived from both epidermal lipid synthesis and extracutaneous sources. Inhibition of cholesterol, fatty acid, ceramide, or glucosylceramide synthesis adversely affects lamellar body formation, thereby impairing barrier homeostasis. Studies have further shown that the elongation and desaturation of fatty acids is also required for barrier homeostasis. The mechanisms that mediate the uptake of extracutaneous lipids by the epidermis are unknown, but keratinocytes express LDL and scavenger receptor class B type 1, fatty acid transport proteins, and CD36. Topical application of physiologic lipids can improve permeability barrier homeostasis and has been useful in the treatment of cutaneous disorders.     

Epidermal sphingolipids: metabolism, function, and roles in skin disorders

Mammalian epidermis produces and delivers large quantities of glucosylceramide and sphingomyelin precursors to stratum corneum extracellular domains, where they are hydrolyzed to corresponding ceramide species. This cycle of lipid precursor formation and subsequent hydrolysis represents a mechanism that protects the epidermis against potentially harmful effects of ceramide accumulation within nucleated cell layers. Prominent skin disorders, such as psoriasis and atopic dermatitis, have diminished epidermal ceramide levels, reflecting altered sphingolipid metabolism, that may contribute to disease severity/progression. Enzymatic processes in the hydrolysis of glucosylceramide and sphingomyelin, and the roles of sphingolipids in skin diseases, are the focus of this review.     

Role of lipids in the formation and maintenance of the cutaneous permeability barrier

The major function of the skin is to form a barrier between the internal milieu and the hostile external environment. A permeability barrier that prevents the loss of water and electrolytes is essential for life on land. The permeability barrier is mediated primarily by lipid enriched lamellar membranes that are localized to the extracellular spaces of the stratum corneum. These lipid enriched membranes have a unique structure and contain approximately 50% ceramides, 25% cholesterol, and 15% free fatty acids with very little phospholipid. Lamellar bodies, which are formed during the differentiation of keratinocytes, play a key role in delivering the lipids from the stratum granulosum cells into the extracellular spaces of the stratum corneum. Lamellar bodies contain predominantly glucosylceramides, phospholipids, and cholesterol and following the exocytosis of lamellar lipids into the extracellular space of the stratum corneum these precursor lipids are converted by beta glucocerebrosidase and phospholipases into the ceramides and fatty acids, which comprise the lamellar membranes. The lipids required for lamellar body formation are derived from de novo synthesis by keratinocytes and from extra-cutaneous sources. The lipid synthetic pathways and the regulation of these pathways are described in this review. In addition, the pathways for the uptake of extra-cutaneous lipids into keratinocytes are discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice

The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope.     

Sphingolipid activator proteins are required for epidermal permeability barrier formation

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.     

The role of sphingolipid metabolism in cutaneous permeabilitybarrier formation

The epidermal permeability barrier of mammalian skin is localized in the stratum corneum. Corneocytes are embedded in an extracellular, highly ordered lipid matrix of hydrophobic lipids consisting of about 50% ceramides, 25% cholesterol and 15% long and very long chain fatty acids. The most important lipids for the epidermal barrier are ceramides. The scaffold of the lipid matrix is built of acylceramides, containing ω-hydroxylated very long chain fatty acids, acylated at the ω-position with linoleic acid. After glucosylation of the acylceramides at Golgi membranes and secretion, the linoleic acid residues are replaced by glutamate residues originating from proteins exposed on the surface of corneocytes. Removal of their glucosyl residues generates a hydrophobic surface on the corneocytes used as a template for the formation of extracellular lipid layers of the water permeability barrier. Misregulation or defects in the formation of extracellular ceramide structures disturb barrier function. Important anabolic steps are the synthesis of ultra long chain fatty acids, their ω-hydroxylation, and formation of ultra long chain ceramides and glucosylceramides. The main probarrier precursor lipids, glucosylceramides and sphingomyelins, are packed in lamellar bodies together with hydrolytic enzymes such as glucosylceramide-β-glucosidase and acid sphingomyelinase and secreted into the intercelullar space between the stratum corneum and stratum granulosum. Inherited defects in the extracellular hydrolytic processing of the probarrier acylglucosylceramides impair epidermal barrier formation and cause fatal diseases: such as prosaposin deficiency resulting in lack of lysosomal lipid binding and transfer proteins, or the symptomatic clinical picture of the “collodion baby” in the absence of glucocerebrosidase. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Sphingolipid metabolism during epidermal barrier development in mice

In rodents, a competent skin barrier to water loss is formed within 2 or 3 days prior to birth. Acquisition of barrier function during rat gestation correlates with the formation of a stratum corneum enriched in ceramides, cholesterol, and fatty acids (Aszterbaum, M., G. K. Menon, K. R. Feingold, and M. L. Williams. 1992. Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content. PEDIATR: Res. 31: 308-317). We analyzed the formation and epidermal localization of glucosylceramides during embryonic skin barrier development in Balb/c mice. Using immunohistochemistry, epidermal glucosylceramides were hardly detectable 3 days prior to birth. After further 24 h of gestation the level of glucosylceramides was maximal and decreased with increasing gestational age. In parallel, glucosylceramides were targeted to the apical side of the outermost granular keratinocyte layer. A spectrum of five distinct epidermal ceramides was present 2 days prior to birth. With ongoing gestation the composition of the ceramide fraction changed markedly. Most importantly, the level of omega-hydroxylated acylceramides decreased paralleled by the formation of the corneocyte lipid envelope. This structure consists of omega-hydroxylated ceramides and fatty acids bound to surface proteins of the corneocytes. The covalent attachment of ceramides or glucosylceramides correlated with the maturation of the stratum corneum and might contribute to its chemical and enzymatic resistance.     

Models of stratum corneum intercellular membranes: the sphingolipid headgroup is a determinant of phase behavior in mixed lipid dispersions.

During formation of the intercellular membranes of mammalian stratum corneum, sphingomyelin and glucosylceramide are converted enzymatically to ceramide. To model in isolation the possible effect of such a lipid modification on the phase behavior of the ensemble, we used proton and deuterium nuclear magnetic resonance to compare an equimolar dispersion of bovine brain sphingomyelin, cholesterol, and perdeuterated palmitic acid (at pH 6.2), with an equivalent dispersion in which bovine brain ceramide was substituted for sphingomyelin. While the sphingomyelin dispersions remain in a homogeneous fluid lamellar phase from 20-75 degrees C under these conditions, those containing ceramide display complex polymorphism.     

Sphingosylphosphorylcholine is upregulated in the stratum corneum of patients with atopic dermatitis

To clarify the functional relevance of sphingomyelin (SM) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [(14)C]acetic anhydride to yield [(14)C-C(2)]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase. These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD.     

Ceramide Stimulates ABCA12 Expression via Peroxisome Proliferator-activated Receptor �� in Human Keratinocytes

ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise ∼50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C₂-Cer and C₆-Cer), but not C₈-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C₆-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARδ expression (but not other PPARs or liver X receptors), whereas PPARδ knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARδ is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARδ-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter.The outermost layer of mammalian epidermis, the stratum corneum, is essential for permeability barrier function and critical for terrestrial life. The stratum corneum consists of terminally differentiated, anucleate keratinocytes, or corneocytes, surrounded by lipid-enriched lamellar membranes composed of three major lipids, ceramides, cholesterol, and free fatty acids (1). These lipids are delivered to the extracellular spaces of the stratum corneum through exocytosis of lamellar body contents from outermost stratum granulosum cells (2). Mature lamellar bodies contain primarily cholesterol, phospholipids, and glucosylceramides (3). Following lamellar body secretion, the secreted phospholipids and glucosylceramides are converted to free fatty acids and ceramides by phospholipases and β-glucocerebrosidase, respectively (1, 4). ABCA12 (ATP binding cassette transporter, family 12), a lipid transporter predominantly expressed in epidermis, has been shown to play a vital role in the formation of mature lamellar bodies (5, 6), although how this transporter is regulated remains unresolved.ABCA12 is a member of the ABCA subfamily of transporters, which are involved in the transport of a variety of lipids (7). Mutations in ABCA1 cause Tangier disease, which is due to a defect in transporting cholesterol and phospholipids from intracellular lipid stores to apolipoproteins, particularly apolipoprotein A-I (8–11). Mutations in ABCA3 cause neonatal respiratory failure due to a defect in surfactant transport from alveolar type II cells into the alveolar space (12). Mutations in ABCA4 cause Stargardt''s macular degeneration, with visual loss due to a defect in transporting phosphatidylethanolamine-retinylidene out of retinal pigment cells (13).Recently, mutations in ABCA12 have been shown to cause harlequin ichthyosis and a subgroup of lamellar ichthyosis, two disorders of keratinization (5, 14, 15). ABCA12 mutations lead to an abnormality in lamellar body formation, a decrease in lamellar membranes in the extracellular spaces of the stratum corneum, an accumulation of glucosylceramide in the epidermis with a reduction in ceramide (16), and ultimately loss of permeability barrier function (17), which in harlequin ichthyosis can result in neonatal lethality (5, 15). Strikingly, genetic correction of ABCA12 deficiency in patients'' keratinocytes by gene transfer normalized loading of glucosylceramides into lamellar bodies (5). These studies demonstrate a critical role for ABCA12 in epidermal physiology, specifically in the formation of mature lamellar bodies and subsequent permeability barrier homeostasis. Hence, it is crucial to understand how ABCA12 is regulated.Our laboratory recently demonstrated that activation of peroxisome proliferator-activated receptor (PPARδ and PPARγ) or liver X receptor (LXR) stimulates ABCA12 expression in cultured human keratinocytes (18). Both PPARs and LXR are important lipid sensors that stimulate keratinocyte differentiation and enhance permeability barrier function (19). Additionally, PPARα and -δ as well as LXR activators stimulate ceramide synthesis in keratinocytes (20, 21). Likewise, ceramide synthesis increases in keratinocytes during differentiation, foreshadowing the formation of lamellar bodies (22, 23).In addition to serving as structural membrane components, ceramides are also important signaling molecules that can induce growth arrest, differentiation, and apoptosis in various cells, including keratinocytes (24–26). Moreover, distal ceramide metabolites, sphingosine and sphingosine-1-phosphate (Fig. 1), are also important signaling molecules (27).Open in a separate windowFIGURE 1.The central role of ceramide in sphingolipid metabolism in keratinocytes. C1P, ceramide 1-phosphate; Sph, sphingosine; S1P, sphingosine-1-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin.It is well established that the expression of ABCA1 is regulated by cellular cholesterol levels in many cell types, including keratinocytes (28). Cholesterol, if metabolized to certain oxysterols, can activate LXR, which then stimulates ABCA1 expression and the transport of cholesterol out of cells (29). This example of feed-forward regulation leads us to hypothesize that either ceramide or a metabolite of ceramide might stimulate ABCA12 expression, thereby leading to an increase in the transport of glucosylceramides into maturing lamellar bodies. Here, we provide evidence that ceramide stimulates ABCA12 expression in keratinocytes via a mechanism involving PPARδ signaling.     

The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation

ATP-binding cassette (ABC) transporters form a large superfamily of transporters that bind and hydrolyze ATP to transport various molecules across limiting membranes or into vesicles. The ABCA subfamily members are thought to transport lipid materials. ABCA12 is a keratinocyte transmembrane lipid transporter protein associated with the transport of lipids via lamellar granules. ABCA12 is considered to transport lipids including ceramides to form extracellular lipid layers in the stratum corneum of the epidermis, which is essential for skin barrier function. ABCA12 mutations are known to underlie the three major types of autosomal recessive congenital ichthyoses: harlequin ichthyosis, lamellar ichthyosis and congenital ichthyosiform erythroderma. ABCA12 mutations result in defective lipid transport via lamellar granules in the keratinocytes, leading to ichthyosis phenotypes from malformation of the stratum corneum lipid barrier. Studies on ABCA12-deficient bioengineered models have revealed that lipid transport by ABCA12 is required for keratinocyte differentiation and epidermal morphogenesis. Defective lipid transport due to loss of ABCA12 function leads to the accumulation of intracellular lipids, including glucosylceramides and gangliosides, in the epidermal keratinocytes. The accumulation of gangliosides seems to result in the apoptosis of Abca12^−/− keratinocytes. It was reported that AKT activation occurs in Abca12^−/− granular-layer keratinocytes, which suggests that AKT activation serves to prevent the cell death of Abca12^−/− keratinocytes. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Human epidermis reconstructed on synthetic membrane: Influence of experimental conditions on terminal differentiation

Summary Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham’s F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham’s F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to thestratum spinosum, stratum granulosum, andstratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no properstratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham’s F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space. Conversely, differentiation appears to depend on elements of basal medium and/or components synthesized by keratinocytes under the influence of the culture medium.     

Impaired Epidermal Permeability Barrier in Mice Lacking Elovl1, the Gene Responsible for Very-Long-Chain Fatty Acid Production

The sphingolipid backbone ceramide (Cer) is a major component of lipid lamellae in the stratum corneum of epidermis and has a pivotal role in epidermal barrier formation. Unlike Cers in other tissues, Cers in epidermis contain extremely long fatty acids (FAs). Decreases in epidermal Cer levels, as well as changes in their FA chain lengths, cause several cutaneous disorders. However, the molecular mechanisms that produce such extremely long Cers and determine their chain lengths are poorly understood. We generated mice deficient in the Elovl1 gene, which encodes the FA elongase responsible for producing C₂₀ to C₂₈ FAs. Elovl1 knockout mice died shortly after birth due to epidermal barrier defects. The lipid lamellae in the stratum corneum were largely diminished in these mice. In the epidermis of the Elovl1-null mice, the levels of Cers with ≥C₂₆ FAs were decreased, while those of Cers with ≤C₂₄ FAs were increased. In contrast, the levels of C₂₄ sphingomyelin were reduced, accompanied by an increase in C₂₀ sphingomyelin levels. Two ceramide synthases, CerS2 and CerS3, expressed in an epidermal layer-specific manner, regulate Elovl1 to produce acyl coenzyme As with different chain lengths. Elovl1 is a key determinant of epidermal Cer chain length and is essential for permeability barrier formation.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Selectivity in cornified envelop binding of ceramides in human skin and the role of LXR inactivation on ceramide binding

The cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the unbound SC lipids. By profiling the bound CLE ceramides, a new subclass was discovered and identified as an omega-hydroxylated dihydrosphingosine (OdS) ceramide. Bound glucosylceramides were observed in superficial SC layers of healthy human skin. To investigate the relation between bound and unbound SC ceramides, the composition of both fractions was analyzed and compared. Selectivity in ceramide binding towards unsaturated ceramides and ceramides with a shorter chain length was observed. The selectivity in ceramide species bound to the cornified envelope is thought to have a physiological function in corneocyte flexibility. Next, it was examined if skin models exhibit an altered bound ceramide composition and if the composition was dependent on liver X-receptor (LXR) activation. The effects of an LXR agonist and antagonist on the bound ceramides composition of a full thickness model (FTM) were analyzed. In FTMs, a decreased amount of bound ceramides was observed compared to native human skin. Furthermore, FTMs had a bound ceramide fraction which consisted mostly of unsaturated and shorter ceramides. The LXR antagonist had a normalizing effect on the FTM bound ceramide composition. The agonist exhibited minimal effects. We show that ceramide binding is a selective process, yet, still is contingent on lipid synthesized.     

Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids.

We previously isolated a temperature-sensitive Chinese hamster ovary cell mutant (strain SPB-1) with thermolabile serine palmitoyltransferase, which is involved in the first step of sphingolipid synthesis (Hanada, K., Nishijima, M., and Akamatsu, Y. (1990) J. Biol. Chem. 265, 22137-22142). In this study, sphingolipid-deficient culture medium was used to examine the effect of exogenous sphingolipids on the cell growth of SPB-1. When cultivated in the sphingolipid-deficient medium, SPB-1 cells ceased growing at non-permissive temperatures. Under these conditions, de novo sphingolipid synthesis ceased in the SPB-1 cells, resulting in a decrease in levels of sphingomyelin and ganglioside sialyl lactosylceramide (GM3), whereas the parental CHO-K1 cells grew logarithmically with normal sphingolipid synthesis. Exogenous sphingosine restored the contents of both sphingomyelin and GM3 in the SPB-1 cells near to the parental levels through metabolic utilization and allowed the mutant cells to grow even at the non-permissive temperature. Similarly, exogenous sphingomyelin restored the sphingomyelin levels and only partly the GM3 levels and also suppressed the temperature-sensitivity of the SPB-1 cell growth. In contrast, exogenous glucosylceramide, which restored the GM3 levels but not the sphingomyelin levels, failed to suppress the temperature sensitivity of the SPB-1 cell growth. Combination of exogenous sphingomyelin with ceramide, glucosylceramide, GM3, or sphingoid bases did not show any synergistic or additive effect on the SPB-1 cell growth enhancement, compared with sphingomyelin alone. The results indicated that the temperature sensitivity of the SPB-1 cell growth was due to the lack of cellular sphingolipids, possibly that of sphingomyelin.     

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: Modulating effect of retinoic acid

Summary Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.