UVB induces epidermal 11ß-hydroxysteroid dehydrogenase type 1 activity in vivo.

Detrimental consequences of ultraviolet radiation (UVR) in skin include photoageing, immunosuppression and photocarcinogenesis, processes also significantly regulated by local glucocorticoid (GC) availability. In man, the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) generates the active GC cortisol from cortisone (or corticosterone from 11-dehydrocorticosterone in rodents). 11ß-HSD1 oxo-reductase activity requires the cofactor NADPH, generated by hexose-6-phosphate dehydrogenase. We previously demonstrated increased 11ß-HSD1 levels in skin obtained from photoexposed versus photoprotected anatomical regions. However, the direct effect of UVR on 11ß-HSD1 expression remains to be elucidated. To investigate the cutaneous regulation of 11ß-HSD1 following UVR in vivo, the dorsal skin of female SKH1 mice was irradiated with 50, 100, 200 and 400 mJ/cm(2) UVB. Measurement of transepidermal water loss, 11ß-HSD1 activity, mRNA/protein expression and histological studies was taken at 1, 3 and 7 days postexposure. 11ß-HSD1 and hexose-6-phosphate dehydrogenase mRNA expression peaked 1 day postexposure to 400 mJ/cm(2) UVB before subsequently declining (days 3 and 7). Corresponding increases in 11ß-HSD1 protein and enzyme activity were observed 3 days postexposure coinciding with reduced GC receptor mRNA expression. Immunofluorescence studies revealed 11ß-HSD1 localization to hyperproliferative epidermal keratinocytes in UVB-exposed skin. 11ß-HSD1 expression and activity were also induced by 200 and 100 (but not 50) mJ/cm(2) UVB and correlated with increased transepidermal water loss (indicative of barrier disruption). UVB-induced 11ß-HSD1 activation represents a novel mechanism that may contribute to the regulation of cutaneous responses to UVR exposure.

A TALEN-based strategy for efficient bi-allelic miRNA ablation in human cells.

Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for bi-allelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells.

LncRNA: a new player in 1α, 25(OH)(2) vitamin D(3) /VDR protection against skin cancer formation.

Sunlight, vitamin D and skin cancer form a controversial brew. While too much sunlight exposure causes skin cancer, it is the major source of vitamin D from skin. We propose that these processes can be balanced. Vitamin D signalling (VDS) protects against skin cancer as demonstrated by the susceptibility of the skin to tumor formation in VDR null mice and protection from UVB-induced mutations when VDR agonists are administered. The question is how is protection afforded. Previously, we have focused on the Wnt/ß-catenin/hedgehog and DNA damage repair (DDR) pathways. As VDR regulates hundreds of genes with thousands of VDR response elements (VDRE) throughout the genome, and many VDREs are in non-coding regions, we decided to explore long non-coding RNAs (lncRNA). LncRNAs are mRNA-like transcripts ranging from 200 bases ~100 kb lacking significant open reading frames. They are aberrantly expressed in human cancers and involved in a spectrum of tumorigenic/metastatic processes (cell proliferation/apoptosis/angiogenesis). We discovered that VDS regulated the expression of certain lncRNAs in a manner consistent with VDS protection against skin cancer. Given the huge variation in genes actively regulated by 1,25(OH)2 D from different cell types, it is conceivable that our results could apply to personalized medicine based on the distinctive lncRNA profiles. These lncRNAs could also serve as skin cancer biomarkers secreted into the blood or urine via exosomes as demonstrated in other cancer types (breast, prostate). Modulation of lncRNA profile by VDS may also provide insight into regulating pathways such as Wnt/ß-catenin and hedgehog.

LncRNA profiling reveals new mechanism for VDR protection against skin cancer formation.

Accumulating evidence strongly suggests a protective role of vitamin D signaling against chemical and UVR-induced skin cancer formation. However, the mechanism remains largely unknown. Recently, the emerging role of long, non-coding RNA (lncRNA) as a hallmark of cancer has become better appreciated. LncRNAs are mRNA-like transcripts ranging in length from 200 bases to 100kb lacking significant open reading frames, which are involved in a broad spectrum of tumorigenic/metastatic processes. In this study we profiled 90 well-annotated mouse lncRNAs from cultured mouse keratinocytes after deleting the vitamin D receptor (VDR) (∼90%) vs. control cells using an lncRNA array analysis. We found that several well-known oncogenes, including H19, HOTTIP and Nespas, are significantly increased (6.3-1.8-fold), whereas tumor suppressors (Kcnq1ot1, lincRNA-p21) are decreased (up to 50-70%) in VDR deleted keratinocytes. A similar pattern of lncRNA profiling is observed in the epidermis of K14 driven, tamoxifen-regulated epidermal-specific VDR null vs. wild-type control mice. Additionally there is an increase in the expression levels of other oncogenes (mHOTAIR, Malat1 and SRA) and a decrease of other tumor suppressors (Foxn2-as, Gtl2-as, H19-as). The increased expression levels of HOTTIP and H19 were further confirmed by real-time PCR analysis with individually designed primer sets. The major finding of this study is a novel mechanism for protection by VDR against skin cancer formation by maintaining the balance of oncogenic to tumor suppressing lncRNAs. In keratinocytes lacking VDR this balance is disturbed with increased expression of oncogenes and decreased expression of tumor suppressors, a mechanism that predisposes the VDR deficient mice to skin cancer formation. This article is part of a Special Issue entitled "Vitamin D Workshop".

Ceramides stimulate caspase-14 expression in human keratinocytes.

Caspase-14 is an enzyme that is expressed predominantly in cornifying epithelia and catalyses the degradation of profilaggrin. Additionally, caspase-14 plays an important role in the terminal differentiation of keratinocytes. However, how caspase-14 expression is regulated remains largely unknown. Here we demonstrate that ceramides (C(2) -Cer and C(6) -Cer), but not other sphingolipids (C(8) -glucosylceramides, sphinganine, sphingosine-1-phosphate or ceramide-1-phosphate), increase caspase-14 expression (mRNA and protein) in cultured human keratinocytes in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase and ceramidase increase endogenous ceramide levels and also increase caspase-14 expression, indicating an important regulatory role for ceramides and suggesting that the conversion of ceramides to other metabolites is not required. The increase in caspase-14 expression induced by ceramides is first seen at 16 h and requires new protein synthesis, suggesting that the ceramide-induced increase is likely an indirect effect. Furthermore, ceramides increase caspase-14 gene expression primarily by increasing transcription. Blocking de novo synthesis of ceramides does not affect caspase-14 expression, suggesting that basal expression is not dependent on ceramide levels. These studies show that ceramides, an important structural lipid, stimulate caspase-14 expression providing a mechanism for coordinately regulating the formation of lipid lamellar membranes with the formation of corneocytes.

1α,25(OH)2-dihydroxyvitamin D3/VDR protects the skin from UVB-induced tumor formation by interacting with the ß-catenin pathway.

Ultra violet (UV) irradiation, in particular UVB, is the single most important carcinogen for skin tumor formation. UVB induces genetic mutations and immune suppression, which lead to abnormal cell proliferation and eventually tumor formation. Previously studies from our group and others demonstrated that both global and epidermal specific VDR knock out mice are predisposed to either chemical (DMBA)- or long-term UVB-induced skin tumor formation, paralleled by an increase in ß-catenin signaling. Using primary cultured human keratinocytes, we further demonstrated that 1,25(OH)2-dihydroxyvitamin D3 (1,25(OH)2D3) suppresses cyclin D1 and Gli1 which are regulated by ß-catenin/TCF signaling and have a critical role in epidermal carcinogenesis. Blockage of VDR by siRNA resulted in hyperproliferation of keratinocytes, and increased expression of cyclin D1 and Gli1. In addition, we also showed that 1,25(OH)2D3/VDR directly regulates transcriptional activity of ß-catenin/TCF signaling using the -catenin reporter TopGlow. Using K14 driven tamoxifen-induced cre recombinase to delete both VDR and ß-catenin in keratinocytes of mice following the first hair follicle cycle, we found that ablation of epidermal specific ß-catenin cannot rescue VDR null mice from UVB-induced skin tumor formation. Further study using VDR or ß-catenin single null mice is necessary to compare with the data from double null mice. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Tight junction properties change during epidermis development.

In terrestrial animals, the epidermal barrier transitions from covering an organism suspended in a liquid environment in utero, to protecting a terrestrial animal postnatally from air and environmental exposure. Tight junctions (TJ) are essential for establishing the epidermal permeability barrier during embryonic development and modulate normal epidermal development and barrier functions postnatally. We now report that TJ function, as well as claudin-1 and occludin expression, change in parallel during late epidermal development. Specifically, TJ block the paracellular movement of Lanthanum (La(3+)) early in rat in vivo prenatal epidermal development, at gestational days 18-19, with concurrent upregulation of claudin-1 and occludin. TJ then become more permeable to ions and water as the fetus approaches parturition, concomitant with development of the lipid epidermal permeability barrier, at days 20-21. This sequence is recapitulated in cultured human epidermal equivalents (HEE), as assessed both by ultrastructural studies comparing permeation of large and small molecules and by the standard electrophysiologic parameter of resistance (R), suggesting further that this pattern of development is intrinsic to mammalian epidermal development. These findings demonstrate that the role of TJ changes during epidermal development, and further suggest that the TJ-based and lipid-based epidermal permeability barriers are interdependent.

The expression and regulation of enzymes mediating the biosynthesis of triglycerides and phospholipids in keratinocytes/epidermis.

Triglycerides and phospholipids play an important role in epidermal permability barrier formation and function. They are synthesized de novo in the epidermis via the glycerol-3-phosphate pathway, catalyzed sequentially by a group of enzymes that have multiple isoforms including glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate acyltransferase (AGPAT), Lipin and diacylglycerol acyltransferase (DGAT). Here we review the current knowledge of GPAT, AGPAT, Lipin and DGAT enzymes in keratinocytes/epidermis focusing on the expression levels of the various isoforms and their localization in mouse epidermis. Additionally, the factors regulating their gene expression, including calcium induced differentiation, PPAR and LXR activators, and the effect of acute permeability barrier disruption will be discussed.

The mechanisms by which lipids coordinately regulate the formation of the protein and lipid domains of the stratum corneum: Role of fatty acids, oxysterols, cholesterol sulfate and ceramides as signaling molecules.

The formation of a permeability barrier between the external environment and the host is essential for survival. To provide this barrier keratinocytes undergo a complex pathway of differentiation, which culminates in keratinocyte cornification and the formation of extracellular lipid enriched lamellar membranes in the stratum corneum. The mechanisms that coordinately regulate the parallel formation of the corneocytes and lamellar membranes are unknown. The extracellular lamellar membranes are derived from the exocytosis of lamellar bodies and to synthesize lamellar bodies the keratinocyte must have abundant quantities of cholesterol, fatty acids and ceramides. These lipids could serve as signaling molecules and thereby coordinately regulate the formation of the stratum corneum. Fatty acids activate PPARs and studies have shown that PPAR activation stimulates keratinocyte differentiation. Cholesterol is converted to oxysterols that activate LXR and studies have shown that LXR activation also stimulates keratinocyte differentation. Additionally, PPAR and LXR activation also facilitates the formation of the lipid enriched lamellar membranes. Ceramides, via a number of mechanisms also stimulate keratinocyte differentiation. Recently, studies have shown that ceramides by increasing PPAR delta also increase the expression of ABCA12, which would facilitate the formation of lamellar bodies. Finally, keratinocytes accumulate a large quantity of cholesterol sulfate, which plays a key role in regulating desquamation. Cholesterol sulfate has also been shown to stimulate keratinocyte differentiation. Thus, cholesterol, cholesterol sulfate, fatty acids and ceramides all stimulate keratinocyte differentiation and thereby could coordinately regulate the formation of the stratum corneum.

PPARgamma activators stimulate aquaporin 3 expression in keratinocytes/epidermis.

Aquaporin 3 (AQP3), a member of the aquaglyceroporin family, which transports water and glycerol, is robustly expressed in epidermis and plays an important role in stratum corneum hydration, permeability barrier function and wound healing. PPAR and LXR activation regulates the expression of many proteins in the epidermis and thereby can affect epidermal function. Here, we report that PPARgamma activators markedly stimulate AQP3 mRNA expression in both undifferentiated and differentiated cultured human keratinocytes (CHKs). The increase in AQP3 mRNA by PPARgamma activator occurs in a dose- and time-dependent fashion. Increased AQP3 mRNA levels are accompanied by an increase in AQP3 protein in undifferentiated keratinocytes and a significant increase in glycerol uptake. Activation of LXR, RAR and RXR also increases AQP3 mRNA levels in undifferentiated and differentiated CHKs, but to a lesser extent. PPARdelta activation stimulates AQP3 expression in undifferentiated CHKs but decreases expression in differentiated CHKs. In contrast, PPARalpha activators do not alter AQP3 expression. AQP9 and AQP10, other members of aquaglyceroporin family, are less abundantly expressed in CHKs, and their expression levels are not significantly altered by treatment with LXR, PPAR, RAR or RXR activators. Finally, when topically applied, the PPARgamma activator, ciglitazone, induces AQP3 but not AQP9 gene expression in mouse epidermis. Our data demonstrate that PPAR and LXR activators stimulate AQP3 expression, providing an additional mechanism by which PPAR and LXR activators regulate epidermal function.

Regulation of ABCG1 expression in human keratinocytes and murine epidermis.

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3-6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.

Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis.

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

PPARδ activation promotes stratum corneum formation and epidermal permeability barrier development during late gestation.

The goal of epidermal ontogenesis is to form a stratum corneum (SC), which is required for post-natal permeability barrier function. The regulation of epidermal ontogenesis is poorly understood, but nuclear hormone receptors have been shown to have an important function. As peroxisome proliferator-activated receptor-delta (PPARdelta) is very abundant in fetal epidermis and PPARdelta activation stimulates differentiation and permeability barrier formation in adults, we hypothesized that PPARdelta might regulate epidermal ontogenesis. Treatment of fetal rat explants with the PPARdelta ligand, GW 610742X, accelerates permeability barrier development, evidenced by a decrease in transepidermal water loss and an enhanced outside-in barrier function, attributable to the presence of more mature lamellar membranes in the SC and enhanced expression of loricrin and involucrin. Similarly, the intra-amniotic administration of GW 610742X also accelerates the formation of the SC and permeability barrier development. Finally, in PPARdelta-deficient mice the formation of the SC and the expression of differentiation-related proteins were delayed on days 16.5 and 17.5 of gestation. However, at later stages (day 18.5 and after birth), there were no differences between wild-type- and PPARdelta-deficient mice, indicating only a transient delay in epidermal ontogenesis. These studies show that PPARdelta has a role in SC formation and permeability barrier development.

IL-6 stimulates but is not essential for stratum corneum formation and permeability barrier development during gestation.

The regulation of epidermal ontogenesis is a complex process. Previous studies have shown that cytokines (IL-1, TNFalpha and IL-6) regulate permeability barrier homeostasis in adult mice. Recently, we reported that IL-1 and TNFalpha accelerate stratum corneum (SC) formation and permeability barrier development in foetal rodents. Here, we determined whether IL-6 also regulates SC formation and permeability barrier development during late gestation. Using a rat skin explant model, we demonstrated that IL-6 accelerates permeability barrier formation in a time- and dose-dependent fashion. This acceleration of barrier formation is attributable to (a) accelerated lamellar membrane maturation, (b) formation of a multi-layer SC and (c) enhanced expression of epidermal differentiation markers. When comparing epidermis of IL-6-deficient (knockout mice) and wild-type foetal mice at days 16-18, we could not detect any abnormalities in either SC formation or the expression of differentiation markers in knockout (KO) mice. In parallel, the basal expression levels of IL-6 mRNA in epidermis and IL-6 protein in amniotic fluid were very low, with only a minimal change in IL-6 receptor mRNA levels in epidermis of days 16-22 foetal mice. These low IL-6 levels may account, at least in part, for the absence of epidermal abnormalities in IL-6 KO mice. In conclusion, exogenous IL-6 accelerates epidermal ontogenesis, but it is not essential for normal epidermal maturation.

Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated receptor {delta} 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 approximately 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 (C(2)-Cer and C(6)-Cer), but not C(8)-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 C(6)-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 PPARdelta expression (but not other PPARs or liver X receptors), whereas PPARdelta knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARdelta 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 PPARdelta-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter.

IL-1alpha accelerates stratum corneum formation and improves permeability barrier homeostasis during murine fetal development.

BACKGROUND: The ontogenesis of the epidermal permeability barrier is complex and incompletely understood. Previously we showed that IL-1 and TNFalpha regulate permeability barrier homeostasis in adult mice. OBJECT: We determined whether IL-1 and TNFalpha also regulate fetal barrier development. METHODS: Messenger RNA and protein levels in epidermis were determined by real-time PCR and immunohistochemistry, respectively. Epidermal ultra-structure was examined by electron microscopy. RESULTS: The protein expression of IL-1alpha/beta and TNFalpha peaked in fetal rat epidermis at gestational age d19-20, a time point that coincides with the formation of a competent barrier. Treatment of fetal rat explants with IL-1 or TNFalpha accelerates barrier formation in a time- and dose-related fashion, evidenced by a decrease in transepidermal water loss attributable to the presence of mature morphology and an increase in the expression of cornified envelope proteins. Using single receptor KO mice, we demonstrated a delay in both barrier formation and cornified envelope protein expression, paralleled with immature lamellar membranes in epidermis of IL-1R KO, but not TNFR KO vs. wild-type at day 17, differences that disappeared in later gestational stages and immediately after birth. Using TNF receptor and IL-1 receptor double knock out (D-KO) mice, we further demonstrated that a transient delay in barrier development consistently occurs in epidermis of D-KO mice. CONCLUSION: IL-1 plays a role in regulating the late stages of SC formation and permeability barrier ontogenesis.

Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism.

Many of the ichthyoses are associated with inherited disorders of lipid metabolism. These disorders have provided unique models to dissect physiologic processes in normal epidermis and the pathophysiology of more common scaling conditions. In most of these disorders, a permeability barrier abnormality "drives" pathophysiology through stimulation of epidermal hyperplasia. Among primary abnormalities of nonpolar lipid metabolism, triglyceride accumulation in neutral lipid storage disease as a result of a lipase mutation provokes a barrier abnormality via lamellar/nonlamellar phase separation within the extracellular matrix of the stratum corneum (SC). Similar mechanisms account for the barrier abnormalities (and subsequent ichthyosis) in inherited disorders of polar lipid metabolism. For example, in recessive X-linked ichthyosis (RXLI), cholesterol sulfate (CSO(4)) accumulation also produces a permeability barrier defect through lamellar/nonlamellar phase separation. However, in RXLI, the desquamation abnormality is in part attributable to the plurifunctional roles of CSO(4) as a regulator of both epidermal differentiation and corneodesmosome degradation. Phase separation also occurs in type II Gaucher disease (GD; from accumulation of glucosylceramides as a result of to beta-glucocerebrosidase deficiency). Finally, failure to assemble both lipids and desquamatory enzymes into nascent epidermal lamellar bodies (LBs) accounts for both the permeability barrier and desquamation abnormalities in Harlequin ichthyosis (HI). The barrier abnormality provokes the clinical phenotype in these disorders not only by stimulating epidermal proliferation, but also by inducing inflammation.

Thematic review series: skin lipids. Peroxisome proliferator-activated receptors and liver X receptors in epidermal biology.

The epidermis is a very active site of lipid metabolism, and all peroxisome proliferator-activated receptor (PPAR) and liver X receptor (LXR) isoforms are expressed in the epidermis. Activation of PPARalpha, -beta/delta, or -gamma or LXRs stimulates keratinocyte differentiation. Additionally, activation of these receptors also improves permeability barrier homeostasis by a number of mechanisms, including stimulating epidermal lipid synthesis, increasing lamellar body formation and secretion, and increasing the activity of enzymes required for the extracellular processing of lipids in the stratum corneum, leading to the formation of lamellar membranes that mediate permeability barrier function. The stimulation of keratinocyte differentiation and permeability barrier formation also occurs during fetal development, resulting in accelerated epidermal development. PPAR and LXR activation regulates keratinocyte proliferation and apoptosis, and studies have shown that these receptors play a role in cutaneous carcinogenesis. Lastly, PPAR and LXR activation is anti-inflammatory, reducing inflammation in animal models of allergic and irritant contact dermatitis. Because of their broad profile of beneficial effects on skin homeostasis, PPAR and LXR have great potential to serve as drug targets for common skin diseases such as psoriasis, atopic dermatitis, and skin cancer.

PPAR and LXR activators regulate ABCA12 expression in human keratinocytes.

ATP-binding cassette (ABC) transporter, family 12 (ABCA12), a member of the ABC superfamily, facilitates the delivery of lipids to lamellar bodies (LB) in keratinocytes, which is critical for permeability barrier function. Recently, gene mutations of ABCA12 were found to underlie Harlequin ichthyosis and lamellar ichthyosis, two devastating skin disorders. Previously we and others have demonstrated that peroxisome proliferators-activated receptors (PPARs) and liver X receptor (LXR) activation improved epidermal permeability barrier homeostasis by stimulating keratinocyte differentiation, lipid synthesis, and increasing LB formation/secretion. Here we report that both PPAR-gamma and -beta/delta activators markedly stimulate ABCA12 mRNA expression in cultured human keratinocyte (CHK) in a dose- and time-dependent manner. Increased ABCA12 mRNA levels are accompanied by an increase in ABCA12 protein, suggesting biological importance of this upregulation. LXR activators also increase ABCA12 mRNA levels in CHK, but to a lesser extent. In contrast, activators of PPAR-alpha, RAR, RXR, or vitamin D receptor did not alter ABCA12 expression. Two major ABCA12 alternative transcripts and their corresponding proteins are also expressed and upregulated by PPAR or LXR activator in both undifferentiated and differentiated CHK. Together, our data demonstrate that PPAR and LXR activators increase ABCA12 expression, providing an additional mechanism by which PPAR and LXR activators promote epidermal permeability barrier homeostasis.

Stratum corneum acidification is impaired in moderately aged human and murine skin.

Aged skin commonly is afflicted by inflammatory skin diseases or xerosis/eczema that could be triggered or exacerbated by impaired epidermal permeability barrier homeostasis. This defect is linked to reduced epidermal lipid synthesis in humans and in mice of advanced age (i.e., >75 years in human or >18-24 months in mice). We now report that barrier defects in moderately aged humans (50-80 years) or analogously aged mice (12-15 months) are linked instead to defective stratum corneum (SC) acidity. In moderately aged mouse epidermis, we find that abnormal acidification, in turn, is linked to decreased Na+/H+ antiporter (NHE1) expression. Decreased NHE1 levels lead to increased SC pH, which results in defective lipid processing and delayed maturation of lamellar membranes, due to suboptimal activation of the pH-sensitive essential, lipid-processing enzyme, beta-glucocerebrosidase. Conversely, impaired SC integrity in moderately aged mice is due to increased pH-dependent activation of serine proteases, leading to premature degradation of corneodesmosomes. These abnormalities were normalized by exogenously acidifying the SC, suggesting a basis for the well-known acidification therapies that are widely used to treat the pathologic xerosis/eczema seen in moderately aged humans.