Differential expression of fatty acid transport proteins in epidermis and skin appendages.

Epidermis and sebocyte-derived lipids are derived both from de novo synthesis and through uptake of fatty acids from the circulation. Plasma membrane proteins can significantly contribute to the latter process. In particular, fatty acid transport proteins (FATP/solute carrier family 27) are integral transmembrane proteins that enhance the uptake of long-chain fatty acids into cells. Using specific antisera against all six mammalian FATP, we found that both human and mouse skin express FATP1, -3, -4, and -6. In adult skin, FATP1 and -3 are expressed predominantly by keratinocytes, whereas FATP4 is strongly expressed by sebaceous glands and FATP6 by hair follicle epithelia. Sustained barrier disruption leads to increases in FATP1 and -6 levels as well as a robust increase in CD36 protein. Notably, expression of FATP1 by embryonic keratinocytes at day 18.5 was lower, and FATP4 increased in comparison with adult epidermis. Together, these findings indicate that FATP are not only expressed by different cell types within the skin, but also that their localization is dynamically regulated during development.

Topical liver x receptor activators accelerate postnatal acidification of stratum corneum and improve function in the neonate.

In neonatal rat stratum corneum (SC), pH declines from pH 6.8 at birth to adult levels (pH 5.0-5.5) over 5-6 d. Liver X receptor (LXR) activators stimulate keratinocyte differentiation, improve permeability barrier homeostasis, and accelerate the in utero development of the SC. In this manuscript we determined the effect of LXR activators on SC acidification in the neonatal period and whether these activators correct the functional abnormalities in permeability barrier homeostasis and SC integrity/cohesion. Formation of the acid SC-buffer system was accelerated by topically applying the LXR activator, 22(R)-hydroxycholesterol, and non-oxysterol activators of LXR, TO-901317, and GW-3965. A sterol which does not activate LXR had no effect. LXR activation increased secretory phospholipase A(2) (sPLA(2)) activity and conversely, inhibition of sPLA(2) activity prevented the LXR induced increase in SC acidification, suggesting that increasing sPLA(2) accounts in part, for the LXR stimulation of acidification. LXR activation resulted in an improvement in permeability barrier homeostasis, associated with an increased maturation of lamellar membranes attributable to an increased beta-glucocerebrosidase activity. SC integrity cohesion also normalized in LXR-activator-treated animals and was associated with an increase in corneodesmosomes and in desmoglein 1 expression. These results demonstrate that LXR activators stimulate the formation of an acidic SC and improve both permeability barrier homeostasis and SC integrity/cohesion.

Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum.

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.

Glycerol regulates stratum corneum hydration in sebaceous gland deficient (asebia) mice.

The only known function of human sebaceous glands is the provocation of acne. We assessed here whether sebum influences stratum corneum hydration or permeability barrier function in asebia J1 and 2 J mice, with profound sebaceous gland hypoplasia. Asebia J1 mice showed normal permeability barrier homeostasis and extracellular lamellar membrane structures, but they displayed epidermal hyperplasia, inflammation, and decreased (>50%) stratum corneum hydration, associated with a reduction in sebaceous gland lipids (wax diesters/monoesters, sterol esters). The triglyceride content of both asebia and control stratum corneum was low, consistent with high rates of triglyceride hydrolysis within the normal pilosebaceous apparatus, despite high rates of triglyceride synthesis. Although a mixture of synthetic, sebum-like lipids (sterol/wax esters, triglycerides) did not restore normal stratum corneum hydration to asebia skin, topical glycerol, the putative product of triglyceride hydrolysis in sebaceous glands, normalized stratum corneum hydration, and the glycerol content of asebia stratum corneum was 85% lower than in normal stratum corneum. In contrast, another potent endogenous humectant (urea) did not correct the abnormality. The importance of glycerol generation from triglyceride in sebaceous glands for stratum corneum hydration was demonstrated further by (i) the absence of sebaceous-gland-associated lipase activity in asebia mice, whereas abundant enzyme activity was present in the glands of control mice; and (ii) the inability of high concentrations of topical triglyceride to correct the hydration abnormality, despite the presence of abundant lipase activity in asebia stratum corneum. These results show that sebaceous-gland-derived glycerol is a major contributor to stratum corneum hydration.

Functional consequences of a neutral pH in neonatal rat stratum corneum.

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.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

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.

Peroxisome proliferator-activated receptor (PPAR)-beta/delta stimulates differentiation and lipid accumulation in keratinocytes.

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.

Focal adhesion kinase controls pH-dependent epidermal barrier homeostasis by regulating actin-directed Na+/H+ exchanger 1 plasma membrane localization.

Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAK(K5 KO)), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAK(K5 KO) mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAK(K5 KO) mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAK(K5 KO) mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAK(K5 KO) and control littermates. We found that the critical SC acidification regulator Na(+)/H(+) exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na(+)/H(+) exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells.