Publications

Oxidized cholesterol is present in significant quantities in the typical Western diet. When ingested, oxidized cholesterol is absorbed by the small intestine and incorporated into both chylomicrons and LDL, resulting in LDL that is more susceptible to further oxidation. Feeding studies in animal models and epidemiological studies in humans have suggested that oxidized cholesterol in the diet increases the development of atherosclerosis. In this study, we determined the effect of ezetimibe, a drug that inhibits small intestinal absorption of cholesterol, on the levels of oxidized cholesterol in the serum after a test meal containing oxidized cholesterol. We demonstrate that ezetimibe, 10 mg per day for 1 month, markedly reduced the levels (50% decrease) of oxidized cholesterol in the serum after feeding a test meal containing either alpha-epoxy cholesterol or 7-keto cholesterol, two of the predominant oxidized cholesterols found in the diet. Moreover, the decrease in oxidized cholesterol in the serum was attributable to a decrease in the incorporation of dietary oxidized cholesterol into both chylomicrons and LDL. Because there was no decrease in postprandial triglyceride levels, we conclude that this decrease in oxidized cholesterol levels in the serum is attributable to decreased absorption and not to enhanced clearance. Whether this decrease in oxidized cholesterol absorption prevents or delays the development of atherosclerosis remains to be determined.

PLS3 (phospholipid scramblase-3) is a new member of the family of phospholipid scramblases and transports CL (cardiolipin) from the inner to the outer mitochondrial membrane. In the present paper we examined whether changing the levels of functional PLS3 in HeLa cells altered de novo CL biosynthesis and its resynthesis. HeLa cells overexpressing PLS3 or expressing a disrupted PLS3 (F258V) or control were incubated with [1,3-3H]glycerol and radioactivity incorporated into CL was determined. CL biosynthesis from [1,3-3H]glycerol was increased 1.8-fold in PLS3 cells and 2.1-fold in F258V cells compared with control. This was due to a 64% (P<0.05) and 2.6-fold (P<0.05) elevation in CL synthase activity in PLS3 and F258V cells respectively, compared with control, and not due to changes in phosphatidylglycerolphosphate synthase activity. The increase in CL synthase activity in these cells was due to an increase in its mRNA expression. In contrast, resynthesis of CL from [1-14C]linoleic acid was reduced 52% (P<0.05) in PLS3 and 45% (P<0.05) in F258V cells compared with control and this was due to a reduction in mitochondrial monolysocardiolipin acyltransferase activity. Although protein levels of mitochondrial monolysocardiolipin acyltransferase were unaltered, activity and mRNA expression of endoplasmic reticulum monolysocardiolipin acyltransferase was upregulated in PLS3 and F258V cells compared with controls. These data indicate that the CL resynthesis in HeLa cells is sensitive to the mitochondrial localization of CL and not the level of the reacylating enzymes. Alterations in functional PLS3 levels in PLS3 or F258V cells did not affect the mitochondrial decarboxylation of phosphatidylserine to phosphatidylethanolamine indicating that the biosynthetic changes to CL were specific for this mitochondrial phospholipid. We hypothesize that the cardiolipin resynthesis machinery in the cell 'senses' altered levels of CL on mitochondrial membranes and that de novo CL biosynthesis is up-regulated in HeLa cells as a compensatory mechanism in response to altered movement of mitochondrial CL. The results identify PLS3 as a novel regulator of CL de novo biosynthesis and its resynthesis.

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.

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.

Disruption of the permeability barrier stimulates a repair response that leads to the restoration of barrier function. Previous studies demonstrated that changes in ions, particularly calcium, and cytokines are positive signals, whereas serine protease activation of proteinase-activated receptor 2 is a negative signal regulating barrier recovery. Ikeyama and colleagues provide data that the nitric oxide signaling pathway also regulates barrier homeostasis.

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.

Atopic dermatitis (AD) is a chronic dermatosis bearing clinical, histological, and immunologic similarities to chronic allergic contact dermatitis (ACD). AD shows a Th2 cell-dominant inflammatory infiltrate, elevated serum IgE levels, a permeability barrier abnormality, and Staphylococcus aureus colonization. Repeated hapten challenges reportedly produce a Th2-like hypersensitivity reaction (Th2-like HR). Here, 9-10 challenges with oxazolone (Ox) to hairless mice also produced a chronic Th2-like HR. Permeability barrier function and expression of differentiation proteins, filaggrin, loricrin, and involucrin, became abnormal. CRTH-positive Th2-dominant inflammatory infiltrate, with increased IL-4 expression, and a large increase in serum IgE levels were observed. The barrier abnormality was associated with decreased stratum corneum (SC) ceramide content and impaired lamellar body secretion, resulting in abnormal lamellar membranes, as in human AD. Furthermore, as in human AD, epidermal serine protease activity in SC increased and expression of two lamellar body-derived antimicrobial peptides, CRAMP and mBD3, declined after Ox challenges, paralleling the decrease of their human homologues in AD. Thus, multiple Ox challenges to normal murine skin produce a chronic Th2-like HR, with multiple features of human AD. Because of its reproducibility, predictability, and low cost, this model could prove useful for evaluating both pathogenic mechanisms and potential therapies for AD.

In cultured human keratinocytes or murine epidermis, peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) (NR1C2) activators (1) stimulate keratinocyte differentiation; (2) decrease keratinocyte proliferation; (3) accelerate permeability barrier repair; (4) increase epidermal lipid synthesis; and (5) reduce cutaneous inflammation. Since these results suggest that PPARbeta/delta could play an important role in cutaneous homeostasis, we assessed here the skin phenotype of mice deficient in PPARbeta/delta. Gross cutaneous abnormalities were not evident, and both stratum corneum (SC) skin hydration and surface pH were normal. However, the epidermis was thickened and proliferating cell nuclear antigen (PCNA) staining was increased, indicating increased cell proliferation. No change in apoptosis was observed but the expression of differentiation markers, such as filaggrin, involucrin, and loricrin, was slightly increased in PPARbeta/delta(-/-) mice. Although basal permeability barrier function was normal, PPARbeta/delta knockout (KO) mice show a significant delay in barrier recovery rates following acute barrier disruption by either acetone treatment or tape-stripping. Delayed barrier recovery correlated with decreased production and secretion of lamellar bodies (LBs), and with reduced numbers of extracellular lamellar membranes in the SC. Finally, PPARbeta/delta KO mice displayed increased inflammation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Together, these results further demonstrate that PPARbeta/delta in the epidermis: (1) is required for permeability barrier homeostasis; (2) regulates keratinocyte proliferation; and (3) modulates cutaneous inflammation.

The permeability barrier is required for terrestrial life and is localized to the stratum corneum, where extracellular lipid membranes inhibit water movement. The lipids that constitute the extracellular matrix have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids. Essential fatty acid deficiency results in abnormalities in stratum corneum structure function. The lipids are delivered to the extracellular space by the secretion of lamellar bodies, which contain phospholipids, glucosylceramides, sphingomyelin, cholesterol, and enzymes. In the extracellular space, the lamellar body lipids are metabolized by enzymes to the lipids that form the lamellar membranes. The lipids contained in the lamellar bodies are derived from both epidermal lipid synthesis and extracutaneous sources. Inhibition of cholesterol, fatty acid, ceramide, or glucosylceramide synthesis adversely affects lamellar body formation, thereby impairing barrier homeostasis. Studies have further shown that the elongation and desaturation of fatty acids is also required for barrier homeostasis. The mechanisms that mediate the uptake of extracutaneous lipids by the epidermis are unknown, but keratinocytes express LDL and scavenger receptor class B type 1, fatty acid transport proteins, and CD36. Topical application of physiologic lipids can improve permeability barrier homeostasis and has been useful in the treatment of cutaneous disorders.

Human epidermis elaborates two small cationic, highly hydrophobic antimicrobial peptides (AMP), beta-defensin 2 (hBD2), and the carboxypeptide cleavage product of human cathelicidin (hCAP18), LL-37, which are co-packaged along with lipids within epidermal lamellar bodies (LBs) before their secretion. Because of their colocalization, we hypothesized that AMP and barrier lipid production could be coregulated by altered permeability barrier requirements. mRNA and immunostainable protein levels for mBD3 and cathelin-related antimicrobial peptide (CRAMP) (murine homologues of hBD2 and LL-37, respectively) increase 1-8 hours after acute permeability barrier disruption and normalize by 24 hours, kinetics that mirror the lipid metabolic response to permeability barrier disruption. Artificial permeability barrier restoration, which inhibits the lipid-synthetic response leading to barrier recovery, blocks the increase in AMP mRNA/protein expression, further evidence that AMP expression is linked to permeability barrier function. Conversely, LB-derived AMPs are also important for permeability barrier homeostasis. Despite an apparent increase in mBD3 protein, CRAMP-/- mice delayed permeability barrier recovery, attributable to defective LB contents and abnormalities in the structure of the lamellar membranes that regulate permeability barrier function. These studies demonstrate that (1) the permeability and antimicrobial barriers are coordinately regulated by permeability barrier requirements and (2) CRAMP is required for permeability barrier homeostasis.