Originally published In Press as doi:10.1194/jlr.R700013-JLR200 on September 13, 2007
Journal of Lipid Research, Vol. 48, 2531-2546, December 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology
Thematic review series: Skin Lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis
Kenneth R. Feingold1
Metabolism Section, Medical Service, Department of Veterans Affairs Medical Center, University of California San Francisco, San Francisco, CA 94121
Published, JLR Papers in Press, September 13, 2007.
1 To whom correspondence should be addressed. e-mail: kenneth.feingold{at}ucsf.edu
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ABSTRACT
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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.
Supplementary key words stratum corneum • lamellar body • cholesterol • fatty acids • ceramides
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JLR: DR. FEINGOLD, WHAT IS THE KEY FUNCTION OF THE SKIN?
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KRF: The chief function of the skin is to form a barrier between the external hostile environment and the internal milieu of the host (1). The skin must protect the host from mechanical insults, ultraviolet light, chemicals, pathogenic microorganisms, etc. Most importantly, to survive in a terrestrial environment without desiccating, the skin must provide a barrier to the loss of water and electrolytes (1). Without a permeability barrier, survival on land would be impossible. Severe burns abrogate these barrier properties and lead to an increased risk of infection and difficulties with maintaining fluid and electrolyte balance. Similarly, in premature infants, the skin is not fully developed and barrier function is impaired; therefore, they also have great difficulties in maintaining fluid and electrolyte balance (2, 3). More subtle functional abnormalities in skin barrier function occur in neonates, in the elderly, and in association with several cutaneous diseases, including psoriasis and atopic dermatitis (4–6).
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JLR: WHERE IN THE SKIN ARE THESE BARRIER PROPERTIES LOCALIZED?
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KRF: The permeability barrier properties are primarily localized to the outer epidermal layer, the stratum corneum (1). The stratum corneum consists of corneocytes, keratinocytes that have undergone terminal differentiation, surrounded by a neutral lipid-enriched extracellular matrix. The mechanical strength of the skin is provided by the corneocytes, which are encased by a cornified envelope consisting of extensively cross-linked proteins such as involucrin and loricrin. The hydrophobic extracellular lipid matrix provides the barrier to the movement of water and electrolytes (1).
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JLR: WHAT LIPIDS ARE IN THIS EXTRACELLULAR MATRIX?
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KRF: The lipids that constitute the extracellular matrix of the stratum corneum have a unique composition and are very different from the lipids that constitute most biological membranes. On a total lipid mass basis, human stratum corneum is 50% ceramides, 25% cholesterol, and 15% free fatty acids (7). Very little phospholipid is present in the stratum corneum, which is markedly different from what is observed in most other membranes. The specific ceramides present in the stratum corneum are unusual and very diverse. Walter Holleran and colleagues will discuss the origin and importance of this diversity in detail in a review in this series. However, it should be noted that linoleate is present in the acylceramides and that in essential fatty acid deficiency oleate replaces linoleate, resulting in marked abnormalities in cutaneous permeability barrier function associated with an abnormal appearance of the extracellular lipid membranes (8–11). These observations indicate that essential fatty acids are required for the normal structure and permeability barrier function of the stratum corneum. The free fatty acids in human stratum corneum are predominantly straight chained, with 22 and 24 carbon chain lengths being the most abundant (7). Although cholesterol is the major sterol in stratum corneum, cholesterol sulfate is a minor sterol metabolite that plays a key role in regulating desquamation (this will be discussed in detail by Peter Elias and colleagues in another review in this series) (12, 13). The synthesis of cholesterol sulfate in the epidermis is catalyzed by the enzyme cholesterol sulfotransferase. Cholesterol sulfotransferase activity increases with keratinocyte differentiation, and recent studies have shown that SULT2B1b is the isoform that accounts for the cholesterol sulfotransferase activity in the epidermis (14–16). For information on the organization of lipids in the stratum corneum, a recent review by Bouwstra and Ponec (17) provides a comprehensive state-of-the-art update.
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JLR: HOW ARE THE LIPIDS DELIVERED TO THE EXTRACELLULAR SPACES OF THE STRATUM CORNEUM?
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KRF: The lipid is secreted from keratinocytes in lamellar bodies, which are ovoid, 0.2 x 0.3 µm, membrane bilayer-encircled secretory organelles that are unique to the epidermis (18) (Fig. 1
). These lamellar bodies are not present in the undifferentiated basal layer of the epidermis, but they begin to appear as keratinocytes differentiate and are first observed in the upper stratum spinosum layer of the epidermis, with increasing numbers found in the stratum granulosum (18). These lamellar bodies contain phospholipids, glucosylceramides, sphingomyelin, and cholesterol (18). In addition, numerous enzymes, including lipid hydrolases such as ß glucocerebrosidase, acidic sphingomyelinase, secretory phospholipase A2, and neutral lipases, and proteases such as chemotryptic enzymes (kallikreins) and cathepsins, are localized to lamellar bodies (18). Moreover, recent studies have shown that antimicrobial peptides, such as human ß-defensin 2 and the cathelicidin LL-37, are also present in lamellar bodies (18).
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Fig. 1. Pathways for the formation of the extracellular lamellar lipid membranes that provide for the permeability barrier.
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JLR: DO THE LIPIDS IN THE EXTRACELLULAR LIPID MEMBRANES IN THE STRATUM CORNEUM DIFFER FROM THE LIPIDS PACKAGED INTO LAMELLAR BODIES?
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KRF: Yes. The lipids in the lamellar bodies are precursors of the stratum corneum extracellular lipids. After secretion, these lamellar body-derived lipids are further metabolized in the stratum corneum extracellular spaces by enzymes that are cosecreted in lamellar bodies (18–22). Specifically, ß-glucocerebrosidase converts glucosylceramides into ceramides (23, 24), acidic sphingomyelinase converts sphingomyelin into ceramides (25, 26), and phospholipases convert phospholipids into free fatty acids and glycerol (27, 28). Both Gaucher's disease, caused by a deficiency in ß-glucocerebrosidase, and Niemann-Pick disease, caused by a deficiency in acidic sphingomyelinase, lead to defects in the extracellular lipid membranes and abnormal permeability barrier function that result from the impaired conversion of lipid precursors into ceramides (23, 26). Walter Holleran and colleagues will discuss in greater detail the extracellular processing of sphingolipids in the stratum corneum in their review. Of note, disruption of the permeability barrier produces an increase in ß-glucocerebrosidase activity and mRNA levels in the epidermis (29). Similarly, disruption of the permeability barrier also increases acidic sphingomylinase activity in the epidermis (25). Thus, the activities of the two key enzymes that are required for the extracellular metabolism of lamellar body lipids to the lipid species that form the lamellar membranes are enhanced after permeability barrier disruption. Additionally, inhibition of phospholipase A2 activity, which blocks the conversion of phospholipids to free fatty acids, also leads to defects in the structure of the extracellular lipid membranes and permeability barrier homeostasis (27, 28). There are several different isoforms of phospholipase A2 expressed in the epidermis, and which specific isoforms are important for the extracellular catabolism of phospholipids to fatty acids in the stratum corneum remains to be determined (30, 31). Finally, the cholesterol sulfate in the stratum corneum is metabolized by the lamellar body-derived enzyme, steroid sulfatase, to cholesterol [see the review by Peter Elias and colleagues (32) for a detailed discussion of the important role of the steroid sulfatase-mediated breakdown of cholesterol sulfate in regulating corneocyte desquamation].
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JLR: DOES THIS EXTRACELLULAR PROCESSING OF LIPIDS HAVE OTHER IMPORTANT EFFECTS IN ADDITION TO PROVIDING THE LIPIDS REQUIRED FOR THE FORMATION OF THE EXTRACELLULAR LIPID MEMBRANES THAT MEDIATE PERMEABILITY BARRIER FUNCTION?
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KRF: Yes. In fact, a number of key stratum corneum functions are derived in part from this extracellular processing of lipids. The glycerol that is formed by the breakdown of phospholipids by phospholipases plays a role in the stratum corneum as a water-holding agent, which helps to keep the stratum corneum hydrated. Hydration is crucial for a smooth and flexible skin, and changes in hydration status signal several downstream responses, including epidermal DNA synthesis and catabolism of filaggrin into deiminated carboxylic acid metabolites (33–37).
The free fatty acids that are formed by phospholipid breakdown contribute to the acidification of the stratum corneum (38, 39). The pH of the outer stratum corneum and skin surface in humans and animals ranges from 5 to 5.5 (40). This acidic environment is very important, as it regulates the activity of many of the enzymes in the stratum corneum (40). For example, the activities of both ß-glucocerebrosidase and acidic sphingomyelinase are optimal at or below pH 5.5, which is very similar to the pH of the stratum corneum. Conversely, many of the proteases in the stratum corneum have a pH optimum of 7 or higher; therefore, their activities are decreased at the usual stratum corneum pH of 5.5. If the pH of the stratum corneum is increased, the activities of ß-glucocerebrosidase and acidic sphingomyelinase are reduced and the extracellular processing of glucosylceramides and sphingomyelins to ceramides is impaired, leading to abnormalities in the structure of the extracellular lipid membranes and decreased permeability barrier function (4, 41–43). Furthermore, increases in stratum corneum pH stimulate protease activity, resulting in increased corneocyte desquamation (4, 41, 42). In newborns, the pH of the stratum corneum is increased, which could explain the decreased permeability barrier homeostasis and epidermal fragility that is observed during the neonatal period (4). Similarly, many cutaneous inflammatory disorders also are associated with increases in stratum corneum pH, which could adversely affect enzyme activity in the stratum corneum, resulting in a decrease in permeability barrier function and stratum corneum integrity and cohesion (40). Finally, the breakdown of cholesterol sulfate to cholesterol by the enzyme steroid sulfatase plays an important role in regulating desquamation (12, 13, 32). Steroid sulfatase deficiency results in recessive X-linked ichthyosis, which will be discussed in detail in the review by Peter Elias and colleagues (12, 13, 32). Additionally, cholesterol sulfate stimulates keratinocyte differentiation, adversely affects permeability barrier function, and inhibits cholesterol synthesis and HMG-CoA reductase activity in keratinocytes (44–48).
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JLR: IS ANYTHING KNOWN ABOUT HOW LAMELLAR BODIES ARE FORMED?
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KRF: The structural proteins that constitute the lamellar bodies have not yet been identified, and the details of lamellar body formation are not well understood. The incorporation of the lipid hydrolases and proteases into lamellar bodies requires the prior or concurrent delivery of lipid to the lamellar bodies (49). If lipids are deficient, the enzymes that are characteristically found in lamellar bodies are not transported from the Golgi to the lamellar bodies (49). Recent studies have shown that ABCA12, a member of the ABC family of transporters, is required for lamellar body formation (50, 51). Mutations in ABCA12 result in the failure to form normal lamellar bodies and extracellular lipid membranes (50, 51). Severe mutations in ABCA12 are associated with harlequin ichthyosis, a disease that is often fatal in childhood, whereas milder partial loss-of-function mutations in ABCA12 are associated with a less severe phenotype of lamellar ichthyosis type 2 (these disorders will be discussed in greater detail in the review by Peter Elias and coworkers) (50–54).
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JLR: WHAT REGULATES LAMELLAR BODY SECRETION?
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KRF: Acute disruption of the permeability barrier by mechanical forces (i.e., sequential tape stripping), solvents (i.e., acetone), or detergents (i.e., SDS) initiates a homeostatic repair response that results in the rapid recovery of permeability barrier function (55, 56). The first step in this repair response is the rapid secretion (within minutes) of the contents of the lamellar bodies from the outer stratum granulosum cells, resulting in a marked decrease in the number of lamellar bodies in stratum granulosum cells (50–80% of preexisting lamellar bodies are secreted) (57). Newly formed lamellar bodies begin to reappear in the stratum granulosum cells, and accelerated secretion continues until permeability barrier function returns toward normal (57). If one artificially restores permeability barrier function to normal by application of an impermeable membrane, one can inhibit the further secretion of lamellar bodies (57).
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JLR: HOW DO THE STRATUM GRANULOSUM CELLS KNOW THAT THE PERMEABILITY BARRIER IS DISTURBED AND THAT IT IS TIME TO SECRETE LAMELLAR BODIES AND INITIATE THE HOMEOSTATIC REPAIR PROGRAM?
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KRF: Within the epidermis, there is a calcium gradient with high levels of extracellular calcium in the upper epidermis surrounding the stratum granulosum cells (58, 59). Immediately after barrier disruption, the increased water movement through the compromised stratum corneum carries calcium outward toward the skin surface, resulting in a reduction in the calcium concentration surrounding the stratum granulosum cells (60–62). This change in calcium concentration appears to be the primary signal inducing lamellar body secretion. If one prevents the reduction in calcium levels by providing exogenous calcium, lamellar body secretion does not occur and permeability barrier repair is not initiated (60–62). Conversely, if one decreases the calcium surrounding the stratum granulosum cells without disrupting the permeability barrier by either iontophoresis or sonophoresis, lamellar body secretion is stimulated (63, 64). It is likely that potassium and other ions also play a role in this signaling process (65–67). In addition, other nonionic signals generated in the stratum corneum and by keratinocytes may also influence the repair response (for review, see Ref. 68). For example, cytokines such as interleukin-1
(IL-1) are stored at high concentrations in the stratum corneum and are rapidly released after barrier disruption (69–71). Mice deficient in IL-1, IL-6, and tumor necrosis factor- signaling have a delay in permeability barrier repair after acute barrier disruption, indicating a role for these cytokines in regulating permeability barrier homeostasis (25, 72, 73).
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JLR: WHERE DO THE LIPIDS IN THE LAMELLAR BODIES COME FROM? FOR EXAMPLE, WHAT IS THE SOURCE OF LAMELLAR BODY CHOLESTEROL?
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KRF: The epidermis on a weight basis is a very active site of cholesterol synthesis (74). Moreover, after acute barrier disruption, there is a rapid and marked increase in epidermal cholesterol synthesis (75). The increase in cholesterol synthesis is associated with an increase in the activity, protein, and mRNA levels of HMG-CoA reductase, a key enzyme in the cholesterol biosynthetic pathway (76–78). Furthermore, after acute barrier disruption, a marked increase in the percentage of HMG-CoA reductase in the active dephosphorylated form is observed (77). Increased enzyme activation is observed as early as 15 min after acute permeability barrier disruption, and the degree of disruption required to activate the enzyme is less than that required to increase enzyme mass. The increase in HMG-CoA reductase activity occurs in both the upper and lower epidermis (79). Additionally, mRNA levels of other key enzymes in the cholesterol synthetic pathway, including HMG-CoA synthase, farnesyl diphosphate synthase, and squalene synthase, also increase after acute barrier disruption (80). Preliminary studies by our laboratory have suggested that the active forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 increase after barrier disruption, which could explain the concordant increase in the enzymes of the cholesterol synthetic pathway.
Evidence that disruption of the permeability barrier signals the increase in cholesterol synthesis is demonstrated by experiments in which an artificial permeability barrier is provided by occlusion with an impermeable membrane. Under these conditions, the increase in epidermal cholesterol synthesis and the increase in mRNA levels of the cholesterol synthetic enzymes are inhibited (75, 77, 80). Most importantly, if one inhibits the increase in epidermal cholesterol synthesis by topical application of statins, which inhibit HMG-CoA reductase activity and decrease cholesterol synthesis, the recovery of permeability barrier function is delayed (81). The initial wave of lamellar body secretion occurs, but the reappearance of lamellar bodies is delayed and those organelles that do appear have an abnormal internal structure. These abnormalities can be reversed by topical treatment with either cholesterol, the final product of the synthetic pathway, or mevalonate, the product formed by HMG-CoA reductase, indicating that these defects are not attributable to nonspecific effects of the topical application of statins (81). Of note, mice with a deficiency of 3ß-hydroxysterol-
24, the enzyme that catalyzes the conversion of desmosterol to cholesterol, have abundant desmosterol but no cholesterol in the epidermis. These animals die within a few hours after birth from an impaired cutaneous permeability, providing additional evidence for the importance of cholesterol for normal permeability barrier function (82). Together, these results demonstrate an important role for epidermal cholesterol synthesis in permeability barrier homeostasis.
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JLR: IS FATTY ACID SYNTHESIS IN THE EPIDERMIS ALSO IMPORTANT FOR BARRIER REPAIR?
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KRF: The epidermis is also a very active site of fatty acid synthesis, and disruption of the permeability barrier results in a rapid and marked increase in fatty acid synthesis (74, 83). Barrier disruption increases the activities and mRNA levels of both of the key enzymes required for de novo fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase (80, 84). The increase in acetyl-CoA carboxylase and fatty acid synthase induced by permeability barrier disruption is likely attributable to an increase in the activation of SREBPs. Once again, occlusion with an impermeable membrane that restores permeability barrier function prevents the increase in fatty acid synthesis and the increase in the expression of acetyl-CoA carboxylase and fatty acid synthase (80, 83, 84). Moreover, after acute barrier disruption, inhibition of fatty acid synthesis by the topical application of the acetyl-CoA carboxylase inhibitor, 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), delays the recovery of permeability barrier function (85). The initial wave of lamellar body secretion occurs normally, but the ability of the epidermis to synthesize new lamellar bodies is impaired and those lamellar bodies that are formed display abnormal lamellar membranes. These abnormalities in barrier repair and lamellar body formation can be reversed by topical treatment with free fatty acids, indicating that these defects are not the nonspecific effects of TOFA (85). These results demonstrate an important role for epidermal de novo fatty acid synthesis in permeability barrier homeostasis.
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JLR: IS THERE ANY EVIDENCE THAT THE ELONGATION OF FATTY ACIDS IS IMPORTANT FOR PERMEABILITY BARRIER HOMEOSTASIS?
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KRF: Relatively few studies have examined this issue, and the effect of permeability barrier disruption on the expression of the enzymes involved in the elongation of fatty acids has not yet been examined. Of note, animals deficient in ELOVL4 (for elongation of very long chain fatty acid-4) have a severely compromised permeability barrier and die soon after birth (86–89). These animals have deficient lamellar body contents and a paucity of lamellar membranes in the stratum corneum, which would account for the permeability barrier abnormality (89). Lipid analysis revealed a global deficiency of very long chain fatty acids in the epidermis and the absence of 
-O-acylceramides, which are key components of the extracellular lipid membranes of the stratum corneum (see the review by Walter Holleran and colleagues for additional information regarding the role of specific ceramides in permeability barrier homeostasis) (87–89). These observations demonstrate the importance of ELOVL4 in generating at least one of the lipids required for normal permeability barrier homeostasis. ELOV3 knockout mice also have a defective permeability barrier and abnormalities in stratum corneum structure, but because this enzyme is predominantly expressed in sebaceous glands and has only minimal expression in keratinocytes, it is currently hypothesized that the defects in stratum corneum structure and function are secondary effects (90).
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JLR: IS DESATURATION OF FATTY ACIDS IMPORTANT FOR PERMEABILITY BARRIER HOMEOSTASIS?
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KRF: The effects of permeability barrier disruption on the expression of enzymes that desaturate fatty acids have not been examined. Studies have shown that animals that are deficient in SCD2 (for stearoyl-CoA desaturase 2) have a defective permeability barrier, and many die soon after birth (91). The barrier defect is associated with a decrease in lamellar body contents and a decrease in lamellar membranes in the stratum corneum (91). In the SCD2-deficient mice, the content of linoleic acid in the acylceramide fraction was markedly reduced with increased linoleic acid in phospholipids, suggesting alterations in the partitioning of linoleic acid (91). Given the important role of acylceramides in permeability barrier function, the reduction of acylceramides containing linoleic acid could account for the observed barrier abnormalities. Of note is that 
30% of the animals survive, and in these animals SCD1 appears to compensate for the absence of SCD2 (91). However, in animals deficient in SCD1 (asebia mice), there are no abnormalities in permeability barrier homeostasis (SCD1-deficient mice have a sebaceous gland defect that will be discussed in Diane Thiboutot's review in this series) (92). The absence of defects in permeability barrier function in asebia mice that have marked abnormalities in sebaceous glands and the presence of normal permeability barrier function in areas of human skin with a paucity of sebaceous glands indicate that the lipids produced by sebaceous glands are not essential for permeability barrier homeostasis (92, 93). However, stratum corneum hydration is decreased in asebia mice that are deficient in sebaceous glands and in areas of human skin with a decreased number of sebaceous glands (92, 93). The triglycerides in sebaceous lipids are metabolized by lipases to free fatty acids and glycerol, and a decrease in glycerol in areas with reduced sebaceous gland activity leads to a decrease in stratum corneum hydration (92, 93).
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JLR: ARE THERE FATTY ACID BINDING PROTEINS IN KERATINOCYTES?
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KRF: Yes. Epidermal fatty acid binding protein (E-FABP) is expressed in keratinocytes (E-FABP has also been called C-FABP in rats, MAL 1 in mice, and PA-FABP in humans) (94–97). The amount of E-FABP increases with keratinocyte differentiation, and immunohistochemistry studies have demonstrated that the intensity of staining is greatest in the upper epidermis (96, 98, 99). The expression of brain, liver, and heart FABP is not usually detected in the epidermis (100, 101). Acute disruption of the permeability barrier induces E-FABP expression, and this increase can be prevented by covering with a vapor-permeable membrane (102). Additionally, inflammatory disorders, including psoriasis, are associated with increased E-FABP levels in the epidermis (95, 97, 98). In animals deficient in E-FABP, basal transepidermal water loss is lower than in wild-type animals, indicating better barrier function (100, 101). After acute barrier disruption, the return of barrier function to normal follows very similar kinetics to those observed in wild-type animals, indicating that a deficiency in E-FABP does not markedly impair normal permeability barrier homeostasis (100, 101). Of note, though, is that heart FABP is expressed in the epidermis of E-FABP knockout mice (usually not detectable in wild-type mice), and it is possible that this increase in heart FABP compensates for the absence of E-FABP (100, 101).
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JLR: THE FATTY ACIDS PRODUCED IN THE EPIDERMIS WILL SERVE AS PRECURSORS FOR BOTH PHOSPHOLIPIDS AND CERAMIDES. WHAT IS KNOWN ABOUT THE SYNTHESIS OF PHOSPHOLIPIDS IN THE EPIDERMIS?
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ABSTRACT
JLR: DR. FEINGOLD, WHAT...