Publications

Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur. This localized acidification correlated temporally with efficient processing of secreted lamellar body contents to mature extracellular lamellar bilayers. Since expression of the key acidifying mechanism NHE1 is maximal just prior to birth, and gradually declines over the first postnatal week, suboptimal SC acidification at birth cannot be attributed to insufficient NHE1 expression, but could instead reflect reduced NHE1 activity. Expression of the key lipid processing enzyme, beta-glucocerebrosidase (beta-GlcCer'ase), develops similar to NHE1, excluding a lack of beta-GlcCer'ase protein as rate limiting for efficient lipid processing. These results define a postnatal development consisting of initial acidification in the lower SC followed by outward progression, which is accompanied by formation of mature extracellular lamellar membranes. Thus, full barrier competence appears to require the extension of acidification in microdomains from the SC/SG interface outward toward the skin surface in the immediate postnatal period.

Both exposure of stratum corneum to neutral pH buffers and blockade of acidification mechanisms disturb cutaneous permeability barrier homeostasis and stratum corneum integrity/cohesion, but these approaches all introduce potentially confounding variables. To study the consequences of stratum corneum neutralization, independent of hydration, we applied two chemically unrelated superbases, 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo [5,4,0] undec-7-ene, in propylene glycol:ethanol (7:3) to hairless mouse skin and assessed whether discrete pH changes alone regulate cutaneous permeability barrier function and stratum corneum integrity/cohesion, as well as the responsible mechanisms. Both 1,1,3,3-tetramethylguanidine and 1,8-diazabicyclo [5,4,0] undec-7-ene applications increased skin surface pH in parallel with abnormalities in both barrier homeostasis and stratum corneum integrity/cohesion. The latter was attributable to rapid activation (<20 min) of serine proteases, assessed by in situ zymography, followed by serine-protease-mediated degradation of corneodesmosomes. Western blotting revealed degradation of desmoglein 1, a key corneodesmosome structural protein, in parallel with loss of corneodesmosomes. Coapplication of serine protease inhibitors with the superbase normalized stratum corneum integrity/cohesion. The superbases also delayed permeability barrier recovery, attributable to decreased beta-glucocerebrosidase activity, assessed zymographically, resulting in a lipid-processing defect on electron microscopy. These studies demonstrate unequivocally that stratum corneum neutralization alone provokes stratum corneum functional abnormalities, including aberrant permeability barrier homeostasis and decreased stratum corneum integrity/cohesion, as well as the mechanisms responsible for these abnormalities.

The synthesis of triglycerides is catalyzed by two known acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Although they catalyze the same biochemical reaction, these enzymes share no sequence homology, and their relative functions are poorly understood. Gene knockout studies in mice have revealed that DGAT1 contributes to triglyceride synthesis in tissues and plays an important role in regulating energy metabolism but is not essential for life. Here we show that DGAT2 plays a fundamental role in mammalian triglyceride synthesis and is required for survival. DGAT2-deficient (Dgat2(-/-)) mice are lipopenic and die soon after birth, apparently from profound reductions in substrates for energy metabolism and from impaired permeability barrier function in the skin. DGAT1 was unable to compensate for the absence of DGAT2, supporting the hypothesis that the two enzymes play fundamentally different roles in mammalian triglyceride metabolism.

At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.63) on postnatal days 0 to 1 to adult levels (pH 5.9) or even below over the subsequent 7 to 8 d. The postnatal decline in SC pH was paralleled by a progressive activation of a pH-dependent hydrolytic enzyme, beta-glucocerebrosidase. Because SC acidification could not be linked to commonly implicated exogenous factors, such as bacterial colonization, or the deposition of sebaceous gland products. We next assessed whether changes in one or more of three endogenous mechanisms demonstrate postnatal activity changes that contribute to the progressive development of an acidic SC pH. Although the histidine-to-urocanic acid pathway has been implicated in acidification of the adult SC, surface pH is completely normal in histidase-deficient (his/his, Peruvian) mice, ruling out a requirement for this mechanism. In contrast, when sodium/hydrogen antiporter-1 (NHE1), which predominantly acidifies membrane domains at the stratum granulosum-SC interface, is inhibited, postnatal acidification of the SC is partially blocked. Likewise, SC secretory phospholipase A2 (sPLA2) activity, measured with a fluorometric assay, is low at birth, but increases progressively (by 66%) over the first 5 d after birth, and inhibition of sPLA2 between days 0 to 1 and days 5 to 6 delays postnatal SC acidification. Together, these results describe a neonatal model, in which the development of an acidic surface pH can be ascribed, in part, to progressive SC acidification by two endogenous mechanisms, namely, sPLA2 and NHE1, which are known to be important for acidification of adult rodent SC. Conversely, the impaired acidification of neonatal SC, which has important functional and clinical consequences, can be explained by the relatively low activities of one or both of these mechanisms at birth.

Mutations in the gene for steroid sulfatase (SSase), are responsible for recessive x-linked ichthyosis (RXLI). As a consequence of SSase deficiency, its substrate, cholesterol sulfate (CSO4), accumulates in the epidermis. Accumulation of this amphipathic lipid in the outer epidermis provokes both a typical scaling phenotype and permeability barrier dysfunction. Research on RXLI has illuminated several, potentially overlapping pathogenic mechanisms and provided insights about the role of SSase and CSO4 in normal differentiation, barrier maintenance, and desquamation. We now show here that SSase is concentrated in lamellar bodies (LB), and secreted into the SC interstices, along with other LB-derived lipid hydrolases. There, it degrades CSO4, generating some cholesterol for the barrier, while the progressive decline in CSO4 (a serine protease (SP) inhibitor) permits corneodesmosome (CD) degradation leading to normal desquamation. Two molecular pathways contribute to disease pathogenesis in RXLI: 1) excess CSO4 produces nonlamellar phase separation in the stratum corneum (SC) interstices, explaining the barrier abnormality. 2) The increased CSO4 in the SC interstices inhibit activity sufficiently to delay CD degradation, leading to corneocyte retention. We also show here that increased Ca++ in the SC interstices in RXLI could contribute to corneocyte retention, by increasing CD and interlamellar cohesion. RXLI represents one of the best understood diseases in dermatology--from the gene to the SC interstices, its etiology and pathogenesis are becoming clear, and assessment of disease mechanisms in RXLI led to new insights about the role of SSase and CSO4 in epidermis terminal differentiation.

Although loricrin is the predominant protein of the cornified envelope (CE) in keratinocytes, loss or gain of loricrin function in mouse models produces only modest skin phenotypes. In contrast, insertional mutations resulting in a frameshift in the C-terminal domain of loricrin produce the characteristic ichthyosis of loricrin keratoderma in mouse and man. To ascertain the basis for the loricrin keratoderma phenotype, we assessed epidermal structure and stratum corneum (SC) function in a previously genotyped human loricrin keratoderma kindred. Our studies revealed abnormal corneocyte fragility and basal permeability barrier function, but accelerated repair kinetics. Despite fragility, increased water loss occurred predominantly via extracellular domains, which correlated with disorganized lamellar bilayers that were linked spatially to discontinuities of the CE. Accelerated barrier recovery was explicable by amplified lamellar body secretion, while partial normalization of the CE in the outer SC correlated with persistence of abundant calcium in the extracellular spaces (positioned to activate transglutaminase-1). These results show that the barrier abnormality in loricrin keratoderma is linked to a defective CE scaffold, resulting in increased extracellular permeability, as shown previously for another "scaffold disorder", lamellar ichthyosis. But in contrast to lamellar ichthyosis, the CE scaffold partially normalizes in the outer SC in loricrin keratoderma.

Peroxisome proliferator-activated receptor (PPAR) are nuclear hormone receptors that are activated by endogenous lipid metabolites. Previous studies have demonstrated that PPAR-alpha activation stimulates keratinocyte differentiation in vitro and in vivo, is anti-inflammatory, and improves barrier homeostasis. Recent studies have shown that PPAR-beta/delta activation induces keratinocyte differentiation in vitro. This study demonstrated that topical treatment of mice with a selective PPAR-beta/delta agonist (GW1514) in vivo had pro-differentiating effects, was anti-inflammatory, improved barrier homeostasis, and stimulated differentiation in a disease model of epidermal hyperproliferation [corrected]. In contrast to PPAR-alpha activation, PPAR-beta/deltain vivo did not display anti-proliferative or pro-apoptotic effects. The pro-differentiating effects persisted in mice lacking PPAR-alpha, but were decreased in mice deficient in retinoid X receptor-alpha, the major heterodimerization partner of PPAR. Furthermore, in vitro PPAR-beta/delta activation, aside from stimulating differentiation-related genes, additionally induced adipose differentiation-related protein (ADRP) and fasting induced adipose factor (FIAF) mRNA in cultures keratinocytes, which was paralleled by increased oil red O staining indicative of lipid accumulation, the bulk of which were triglycerides (TG). Comparison of differentially expressed genes between PPAR-beta/delta and PPAR-alpha activation revealed distinct profiles. Together, these studies indicate that PPAR-beta/delta activation stimulates keratinocyte differentiation, is anti-inflammatory, improves barrier homeostasis, and stimulates TG accumulation in keratinocytes.

Oxysterols, via activation of liver X receptor (LXR), regulate keratinocyte differentiation by stimulating transglutaminase cross-linking of several constituent proteins leading to the formation of the cornified envelope. We previously reported that oxysterols increase the expression of one of these cross-linked proteins, involucrin, and that this effect can be abolished by mutations of the distal activator protein (AP)-1 response element in the involucrin promoter. Furthermore, oxysterols increase AP-1 binding in an electrophoretic gel mobility shift assay and increase the expression of an AP-1 reporter. In this study, we describe the individual components of the AP-1 complex that are involved in the oxysterol-mediated AP-1 activation and stimulation of keratinocyte differentiation. We identified Fra-1 within the AP-1 DNA binding complex by supershift analysis of nuclear extracts from oxysterol-treated, cultured keratinocytes and confirmed that oxysterol treatment increased the levels of Fra-1 by western blot analysis. Additionally, on Western and Northern analysis, oxysterol treatment increased two other AP-1 proteins, Jun-D and c-Fos, whereas Fra-2, Jun-B, and c-Jun were not changed. Similar alterations in AP-1 proteins occurred when 25-OH-cholesterol or non-steroidal LXR agonists (GW3965, TO-901317) were used. These results indicate that oxysterols induce specific AP-1 proteins, thereby activating involucrin, one of the genes required for epidermal differentiation.

At birth, neonatal stratum corneum (SC) pH is close to neutral but acidifies with maturation, which can be ascribed, in part, to secretory phospholipase A(2) and sodium/hydrogen antiporter 1 (NHE1) activities. Here we assessed the functional consequences of a neutral SC pH in a newborn rat model. While basal transepidermal water loss rates are near normal, barrier recovery (BR) rates after acute barrier disruption were delayed in newborn animals. The abnormality in barrier homeostasis could be improved by topical applications of an acidic buffer, indicating that barrier abnormality is primarily due to high SC pH. The delay in BR correlated with incompletely processed lamellar membranes and decreased activity of beta-glucocerebrosidase. Inhibition of NHE1 delayed BR after acute barrier perturbation. SC integrity was abnormal in newborn animals. Electron microscopy demonstrated decreased corneodesmosomes (CD) in newborn animals with decreased expression of desmoglein 1 and corneodesmosin. Serine protease activation appears to be responsible for CD degradation in newborn animals, because serine protease activity is increased in the SC and it can be reduced by acidification of the SC. The delay in acidification of neonatal SC results in abnormalities in permeability barrier homeostasis and SC integrity and are likely due to pH-induced modulations in enzyme activity.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.