Growth Retardation, Impaired Triacylglycerol Catabolism, Hepatic Steatosis, and Lethal Skin Barrier Defect in Mice Lacking Comparative Gene Identification-58 (CGI-58)

Comparative gene identification-58 (CGI-58), also designated as α/β-hydrolase domain containing-5 (ABHD-5), is a lipid droplet-associated protein that activates adipose triglyceride lipase (ATGL) and acylates lysophosphatidic acid. Activation of ATGL initiates the hydrolytic catabolism of cellular triacylglycerol (TG) stores to glycerol and nonesterified fatty acids. Mutations in both ATGL and CGI-58 cause “neutral lipid storage disease” characterized by massive accumulation of TG in various tissues. The analysis of CGI-58-deficient (Cgi-58^−/−) mice, presented in this study, reveals a dual function of CGI-58 in lipid metabolism. First, systemic TG accumulation and severe hepatic steatosis in newborn Cgi-58^−/− mice establish a limiting role for CGI-58 in ATGL-mediated TG hydrolysis and supply of nonesterified fatty acids as energy substrate. Second, a severe skin permeability barrier defect uncovers an essential ATGL-independent role of CGI-58 in skin lipid metabolism. The neonatal lethal skin barrier defect is linked to an impaired hydrolysis of epidermal TG. As a consequence, sequestration of fatty acids in TG prevents the synthesis of acylceramides, which are essential lipid precursors for the formation of a functional skin permeability barrier. This mechanism may also underlie the pathogenesis of ichthyosis in neutral lipid storage disease patients lacking functional CGI-58.     

The role of lipoxygenases in epidermis

Lipoxygenases (LOX) are key enzymes in the biosynthesis of a variety of highly active oxylipins which act as signaling molecules involved in the regulation of many biological processes. LOX are also able to oxidize complex lipids and modify membrane structures leading to structural changes that play a role in the maturation and terminal differentiation of various cell types. The mammalian skin represents a tissue with highly abundant and diverse LOX metabolism. Individual LOX isozymes are thought to play a role in the modulation of epithelial proliferation and/or differentiation as well as in inflammation, wound healing, inflammatory skin diseases and cancer. Emerging evidence indicates a structural function of a particular LOX pathway in the maintenance of skin permeability barrier. Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 have been found to represent the second most common cause of autosomal recessive congenital ichthyosis and targeted disruption of the corresponding LOX genes in mice resulted in neonatal death due to a severely impaired permeability barrier function. Recent data indicate that LOX action in barrier function can be traced back to the oxygenation of linoleate-containing ceramides which constitutes an important step in the formation of the corneocyte lipid envelope. 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.     

Reassessing the Potential Activities of Plant CGI-58 Protein

Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.     

Formation and functions of the corneocyte lipid envelope (CLE)

Corneocytes in mammalian stratum corneum are surrounded by a monolayer of covalently bound ω-OH-ceramides that form the corneocyte (-bound) lipid envelope (CLE). We review here the structure, composition, and possible functions of this structure, with insights provided by inherited and acquired disorders of lipid metabolism. 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.     

Regulation of skeletal muscle lipolysis and oxidative metabolism by the co-lipase CGI-58

We investigated here the specific role of CGI-58 in the regulation of energy metabolism in skeletal muscle. We first examined CGI-58 protein expression in various muscle types in mice, and next modulated CGI-58 expression during overexpression and knockdown studies in human primary myotubes and evaluated the consequences on oxidative metabolism. We observed a preferential expression of CGI-58 in oxidative muscles in mice consistent with triacylglycerol hydrolase activity. We next showed by pulse-chase that CGI-58 overexpression increased by more than 2-fold the rate of triacylglycerol (TAG) hydrolysis, as well as TAG-derived fatty acid (FA) release and oxidation. Oppositely, CGI-58 silencing reduced TAG hydrolysis and TAG-derived FA release and oxidation (-77%, P < 0.001), whereas it increased glucose oxidation and glycogen synthesis. Interestingly, modulations of CGI-58 expression and FA release are reflected by changes in pyruvate dehydrogenase kinase 4 gene expression. This regulation involves the activation of the peroxisome proliferator activating receptor-δ (PPARδ) by lipolysis products. Altogether, these data reveal that CGI-58 plays a limiting role in the control of oxidative metabolism by modulating FA availability and the expression of PPARδ-target genes, and highlight an important metabolic function of CGI-58 in skeletal muscle.     

CGI-58在动物脂类代谢中的作用

细胞中脂滴(Lipid droplets, LDs)表面存在多个调控脂肪储存和分解的蛋白, 这些蛋白对机体的脂肪代谢起着很重要的调控作用。CGI-58(Comparative gene identification-58)分布在LDs表面, 属于α/β水解酶折叠家族, 是脂肪甘油三酯脂肪酶(Adipose triglyceride lipase, ATGL)和依赖酰基辅酶A溶血磷脂酸酰基转移酶(Lysophosphatidic acid acyltransferase, LPAAT)的激活剂。在脂肪分解过程中, CGI-58结合PAT蛋白家族成员之一的脂滴包被蛋白(Perlipin)和ATGL, 促进脂肪分解, 同时CGI-58对ATGL的激活功能受脂滴包被蛋白家族成员间蛋白质与蛋白质相互作用的影响。文章结合国内外研究热点, 针对CGI-58在动物脂类代谢中的作用进行了综述。     

Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice

Adipose triglyceride lipase (ATGL) was recently identified as a rate-limiting triglyceride (TG) lipase and its activity is stimulated by comparative gene identification-58 (CGI-58). Mutations in the ATGL or CGI-58 genes are associated with neutral lipid storage diseases characterized by the accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, is characterized by TG accumulation in coronary atherosclerotic lesions and in the myocardium. Recent reports showed that myocardial TG accumulation is significantly higher in patients with diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated the roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state. Histological examination with oil red O staining showed marked lipid deposition in the hearts of diabetic fatty db/db mice. Cardiac triglyceride and diglyceride contents were greater in db/db mice than in db/+ control mice. Next, we determined the expression of genes and proteins that affect lipid metabolism, and found that ATGL and CGI-58 expression levels were decreased in the hearts of db/db mice. We also found increased expression of genes regulating triglyceride synthesis (sterol regulatory element-binding protein 1c, monoacylglycerol acyltransferases, and diacylglycerol acyltransferases) in db/db mice. Regarding key modulators of apoptosis, PKC activity, and oxidative stress, we found that Bcl-2 levels were lower and that phosphorylated PKC and 8-hydroxy-2′-deoxyguanosine levels were higher in db/db hearts. These results suggest that reduced ATGL and CGI-58 expression and increased TG synthesis may exacerbate myocardial steatosis and oxidative stress, thereby promoting cardiac apoptosis in diabetic mice.     

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.     

CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80–95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.     

The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier

This review covers the background to discovery of the two key lipoxygenases (LOX) involved in epidermal barrier function, 12R-LOX and eLOX3, and our current views on their functioning. In the outer epidermis, their consecutive actions oxidize linoleic acid esterified in ω-hydroxy-ceramide to a hepoxilin-related derivative. The relevant background to hepoxilin and trioxilin biochemistry is briefly reviewed. We outline the evidence that linoleate in the ceramide is the natural substrate of the two LOX enzymes and our proposal for its importance in construction of the epidermal water barrier. Our hypothesis is that the oxidation promotes hydrolysis of the oxidized linoleate moiety from the ceramide. The resulting free ω-hydroxyl of the ω-hydroxyceramide is covalently bound to proteins on the surface of the corneocytes to form the corneocyte lipid envelope, a key barrier component. Understanding the role of the LOX enzymes and their hepoxilin products should provide rational approaches to ameliorative therapy for a number of the congenital ichthyoses involving compromised barrier function. 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.     

The important role of stratum corneum lipids for the cutaneous barrier function

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is 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. 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.     

Deficiency of liver Comparative Gene Identification-58 causes steatohepatitis and fibrosis in mice

Triglyceride (TG) accumulation in hepatocytes (hepatic steatosis) preludes the development of advanced nonalcoholic fatty liver diseases (NAFLDs) such as steatohepatitis, fibrosis, and cirrhosis. Mutations in human Comparative Gene Identification-58 (CGI-58) cause cytosolic TG-rich lipid droplets to accumulate in almost all cell types including hepatocytes. However, it is unclear if CGI-58 mutation causes hepatic steatosis locally or via altering lipid metabolism in other tissues. To directly address this question, we created liver-specific CGI-58 knockout (LivKO) mice. LivKO mice on standard chow diet displayed microvesicular and macrovesicular panlobular steatosis, and progressed to advanced NAFLD stages over time, including lobular inflammation and centrilobular fibrosis. Compared with CGI-58 floxed control littermates, LivKO mice showed 8-fold and 52-fold increases in hepatic TG content, which was associated with 40% and 58% decreases in hepatic TG hydrolase activity at 16 and 42 weeks, respectively. Hepatic cholesterol also increased significantly in LivKO mice. At 42 weeks, LivKO mice showed increased hepatic oxidative stress, plasma aminotransferases, and hepatic mRNAs for genes involved in fibrosis and inflammation, such as α-smooth muscle actin, collagen type 1 α1, tumor necrosis factor α, and interleukin-1β. In conclusion, CGI-58 deficiency in the liver directly causes not only hepatic steatosis but also steatohepatitis and fibrosis.     

Functional Cardiac Lipolysis in Mice Critically Depends on Comparative Gene Identification-58

Efficient catabolism of cellular triacylglycerol (TG) stores requires the TG hydrolytic activity of adipose triglyceride lipase (ATGL). The presence of comparative gene identification-58 (CGI-58) strongly increased ATGL-mediated TG catabolism in cell culture experiments. Mutations in the genes coding for ATGL or CGI-58 in humans cause neutral lipid storage disease characterized by TG accumulation in multiple tissues. ATGL gene mutations cause a severe phenotype especially in cardiac muscle leading to cardiomyopathy that can be lethal. In contrast, CGI-58 gene mutations provoke severe ichthyosis and hepatosteatosis in humans and mice, whereas the role of CGI-58 in muscle energy metabolism is less understood. Here we show that mice lacking CGI-58 exclusively in muscle (CGI-58KOM) developed severe cardiac steatosis and cardiomyopathy linked to impaired TG catabolism and mitochondrial fatty acid oxidation. The marked increase in ATGL protein levels in cardiac muscle of CGI-58KOM mice was unable to compensate the lack of CGI-58. The addition of recombinant CGI-58 to cardiac lysates of CGI-58KOM mice completely reconstituted TG hydrolytic activities. In skeletal muscle, the lack of CGI-58 similarly provoked TG accumulation. The addition of recombinant CGI-58 increased TG hydrolytic activities in control and CGI-58KOM tissue lysates, elucidating the limiting role of CGI-58 in skeletal muscle TG catabolism. Finally, muscle CGI-58 deficiency affected whole body energy homeostasis, which is caused by impaired muscle TG catabolism and increased cardiac glucose uptake. In summary, this study demonstrates that functional muscle lipolysis depends on both CGI-58 and ATGL.     

Chanarin–Dorfman syndrome: Deficiency in CGI-58, a lipid droplet-bound coactivator of lipase

Chanarin–Dorfman syndrome (CDS) is a rare autosomal recessive disease of lipid metabolism; it is associated with congenital ichthyosis typed as non-bullous congenital ichthyosiform erythroderma (NCIE). CDS is characterized by the presence of an abnormally large number of cytosolic lipid droplets containing triacylglycerol (TG) in various tissues such as the skin, liver, and leukocytes. Mutations in the CGI-58 (also called ABHD5) gene encoding a 39-kDa protein of the α/β hydrolase domain subfamily have been shown to be responsible for this disorder. In adipocytes, CGI-58 is involved in TG degradation on lipid droplets; in doing so, it coordinates with several lipolytic factors including perilipin, a member of the PAT protein family, and ATGL, a putative rate-limiting lipase in adipocytes. In quiescent adipocytes, CGI-58 interacts with perilipin on the surfaces of lipid droplets. Upon hormonal stimulation, CGI-58 facilitates massive lipolysis by activating ATGL. Some CGI-58 mutations found in CDS patients cancel the ability to interact with perilipin or activate ATGL, indicating that the loss of these interactions is physiologically important. However, based on the tissue distributions of these lipolytic factors, there are likely multiple molecular targets of CGI-58 actions. This in turn gives rise to the multiple phenotypes of CDS, such as ichthyosis, liver steatosis, or neurosensory diseases.     

Deletion of CGI-58 or adipose triglyceride lipase differently affects macrophage function and atherosclerosis

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58^−/− mice). CGI-58^−/− macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL^−/− macrophages. In contrast to ATGL^−/− macrophages, however, CGI-58^−/− macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58^−/− macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-58/ApoE-double KO (DKO) animals. In response to high-fat/high-cholesterol diet feeding, DKO animals showed comparable plaque formation as observed in ApoE^−/− mice. In agreement, antisense oligonucleotide-mediated knockdown of CGI-58 in LDL receptor^−/− mice did not alter atherosclerosis burden in the aortic root. These results suggest that macrophage function and atherosclerosis susceptibility differ fundamentally in these two animal models with disturbed TG catabolism, showing a more severe phenotype by ATGL deficiency.     

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.     

CGI-58/ABHD5 is phosphorylated on Ser239 by protein kinase A: control of subcellular localization

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS²³⁹S²⁴⁰, we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno­blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.     

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.     

The epidermal barrier function is dependent on the serine protease CAP1/Prss8

Serine proteases are proteolytic enzymes that are involved in the regulation of various physiological processes. We generated mice lacking the membrane-anchored channel-activating serine protease (CAP) 1 (also termed protease serine S1 family member 8 [Prss8] and prostasin) in skin, and these mice died within 60 h after birth. They presented a lower body weight and exhibited severe malformation of the stratum corneum (SC). This aberrant skin development was accompanied by an impaired skin barrier function, as evidenced by dehydration and skin permeability assay and transepidermal water loss measurements leading to rapid, fatal dehydration. Analysis of differentiation markers revealed no major alterations in CAP1/Prss8-deficient skin even though the epidermal deficiency of CAP1/Prss8 expression disturbs SC lipid composition, corneocyte morphogenesis, and the processing of profilaggrin. The examination of tight junction proteins revealed an absence of occludin, which did not prevent the diffusion of subcutaneously injected tracer (approximately 600 D) toward the skin surface. This study shows that CAP1/Prss8 expression in the epidermis is crucial for the epidermal permeability barrier and is, thereby, indispensable for postnatal survival.