Publications

Neutralization of the normally acidic stratum corneum (SC) has deleterious consequences for permeability barrier homeostasis and SC integrity/cohesion attributable to serine proteases (SPs) activation leading to deactivation/degradation of lipid-processing enzymes and corneodesmosomes (CD). As an elevated pH compromises SC structure and function, we asked here whether SC hyperacidification would improve the structure and function. We lowered the pH of mouse SC using two polyhydroxyl acids (PHA), lactobionic acid (LBA), or gluconolactone (GL). Applications of the PHA reduced the pH at all levels of SC of hairless mouse, with further selective acidification of SC membrane domains, as shown by fluorescence lifetime imaging. Hyperacidification improved permeability barrier homeostasis, attributable to increased activities of two key membrane-localized, ceramide-generating hydrolytic enzymes (beta-glucocerebrosidase and acidic sphingomyelinase), which correlated with accelerated extracellular maturation of SC lamellar membranes. Hyperacidification generated "supernormal" SC integrity/cohesion, attributable to an SP-dependent decreased degradation of desmoglein-1 (DSG1) and the induction of DSG3 expression in lower SC. As SC hyperacidification improves the structure and function, even of normal epidermis, these studies lay the groundwork for an assessment of the potential utility of SC acidification as a therapeutic strategy for inflammatory dermatoses, characterized by abnormalities in barrier function, cohesion, and surface pH.

BACKGROUND

Atopic dermatitis (AD) is a chronic inflammatory dermatosis now increasingly linked to mutations that alter the structure and function of the stratum corneum. Activators of peroxisome proliferator-activated receptors (PPARs) alpha, beta/delta, and gamma and liver X receptor (LXR) regulate epidermal protein and lipid production, leading to superior barrier function. Additionally, some of these activators exhibit potent antihyperplastic and anti-inflammatory activity in irritant contact dermatitis and acute allergic contact dermatitis murine models.

OBJECTIVE

We evaluated the efficacy of PPAR/LXR activation in a hapten (oxazolone [Ox])-induced AD-like model (Ox-AD) in hairless mice.

METHODS

Ox-AD was established with 10 Ox challenges (every other day) on the flank. After the establishment of Ox-AD, twice-daily topical application with individual PPAR/LXR activators was then performed for 4 days, with continued Ox challenges every other day. The efficacy of topical PPAR/LXR activators to reduce parameters of Ox-AD was assessed physiologically, morphologically, and immunologically.

RESULTS

Certain topical activators of PPARalpha, PPARbeta/delta, and LXR, but not activators of PPARgamma, reversed the clinical dermatosis, significantly improved barrier function, and increased stratum corneum hydration in Ox-AD mice. In addition, the same activators, but again not PPARgamma, largely reversed the immunologic abnormalities in Ox-AD mice, including the increased T(H)2 markers, such as tissue eosinophil/mast cell density, serum thymus and activation-related chemokine levels, the density of chemoattractant receptor-homologous molecule expressed on T(H)2-positive lymphocytes (but not serum IgE levels), and reduced IL-1alpha and TNF-alpha activation, despite ongoing hapten challenges.

CONCLUSION

These results suggest that topical applications of certain activators/ligands of PPARalpha, PPARbeta/delta, and LXR could be useful for the treatment of AD in human subjects.

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.

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.

Chinese herbal medicine (CHM) has been shown to have beneficial effects for both skin disorders with barrier abnormality and as skin care ingredients. Yet, how CHM exerts their benefits is unclear. As most, if not all, inflammatory dermatoses are accompanied by abnormal permeability barrier function, we assessed the effects of topical CHM extracts on epidermal permeability barrier function and their potential mechanisms. Topical CHM accelerated barrier recovery following acute barrier disruption. Epidermal lipid content and mRNA expression of fatty acid and ceramide synthetic enzymes increased following topical CHM treatment in addition to mRNA levels for the epidermal glucosylceramide transport protein, ATP-binding cassette A12. Likewise, CHM extract increased mRNA expression of antimicrobial peptides both in vivo and in vitro. These results demonstrate that the topical CHM extract enhances epidermal permeability barrier function, suggesting that topical CHM could provide an alternative regimen for the prevention/treatment of inflammatory dermatoses accompanied by barrier abnormalities.

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.

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.