Induction of Selected Lipid Metabolic Enzymes and Differentiation-Linked Structural Proteins by Air Exposure in Fetal Rat Skin Explants

Transcription

1 Induction of Selected Lipid Metabolic Enzymes and Differentiation-Linked Structural Proteins by Air Exposure in Fetal Rat Skin Explants László G. Kömüves,* Karen Hanley,*, Yan Jiang,* Chika Katagiri,* 1 Peter M. Elias,* Mary L. Williams,* and Kenneth R. Feingold* Departments of *Dermatology, Medicine, and Pediatrics, University of California, San Francisco, California, U.S.A.; Dermatology and Medical Services, Department of Veterans Affairs Medical Center, San Francisco, California, U.S.A. The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air medium interface, and (ii) occlusion with a waterimpermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, β-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mrna levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for airinduced acceleration of permeability barrier maturation in premature infants. Key words: filaggrin/involucrin/ loricrin/steroid sulfatase/stratum corneum/β-glucocerebrosidase. J Invest Dermatol 112: , 1999 The stratum corneum (SC) is a complex tissue, which provides both a permeability and a mechanical barrier between the outside environment and the internal milieu of the organism. It is composed of both corneocytes, which provide strength and rigidity, and an extracellular lipid-enriched matrix, which provides a barrier to water transit (Elias and Menon, 1991; Downing, 1992). Corneocytes are postapoptotic, anucleated cells that have a unique external envelope, the cornified envelope, which consists of proteins, such as involucrin and loricrin cross-linked by transglutaminase, and cytoplasmic keratin filaments aggregated into macrofibrils by Manuscript received September 2, 1998; revised November 3, 1998; accepted for publication November 6, Reprint requests to: Dr. László G. Kömüves, Dermatology Service (190), Department of Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA Abbreviations: CSTase, cholesterol sulfotransferase; GC synthase, UDPglucose:ceramide glucosyltransferase; SC, stratum corneum; SPT, serine palmitoyltransferase 1 Current address: Life Sciences Laboratories, Shiseido Research Center, Yokohama-shi, Japan. filaggrin (Fuchs, 1990). The extracellular lipids are enriched in ceramides, cholesterol, and fatty acids that are organized as multiple lamellar membranes (Elias and Menon, 1991). These lipids are delivered to the extracellular space as a mixture of polar precursors by the exocytosis of lamellar body contents. Lamellar bodies contain abundant quantities of glucosylceramides, whose catabolism to ceramides, by the enzyme β-glucocerebrosidase, is essential for normal permeability barrier function (Holleran et al, 1994, 1995). Requirements for permeability barrier function regulate epidermal lipid metabolism, with disruption of the permeability barrier stimulating epidermal lipid synthesis (Feingold, 1991; Proksch et al, 1993). Whereas the increase in cholesterol synthesis is due to an increase in the activity of HMG CoA reductase (Feingold et al, 1990; Proksch et al, 1993), the increase in sphingolipid synthesis is due to an increase in serine palmitoyl transferase (SPT) (Holleran et al, 1991a). Topical application of inhibitors of either of these enzymes delays the restoration of normal barrier function, indicating that these lipids are required for barrier homeostasis (Feingold et al, 1990; Holleran et al, 1991b). While the activity of glucosylceramide synthase (GC synthase) does not increase following barrier disruption, recent studies have shown that inhibition of this enzyme delays barrier repair (Chujor et al, 1998), indicating that GC synthase also is required for normal barrier homeostasis X/99/$10.50 Copyright 1999 by The Society for Investigative Dermatology, Inc. 303

2 304 KÖMÜVES ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY The signals that initiate the increase in lipid synthesis following barrier disruption are not fully understood. Transepidermal water loss must be an important initiating factor, however, because occlusion with a water vapor impermeable membrane blocks the increase in lipid synthesis and barrier recovery (Grubauer et al, 1989). Epidermal water loss also regulates permeability barrier development in premature infants following birth. Premature infants born before wk gestational age have an incompetent permeability barrier (Harpin and Rutter, 1983; Cartlidge and Rutter, 1998). Following birth and exposure to air, however, they develop a competent barrier (Evans and Rutter, 1986). Furthermore, fetal rat skin explants submerged in a serum-free medium develop a competent permeability barrier following a timetable identical to that in utero, while barrier formation accelerates when these explants are incubated at an air medium interface (Hanley et al, 1997a). This accelerated development again is inhibited by occlusion with a water vapor-impermeable membrane, but not by a vaporpermeable membrane, indicating that water movement regulates not only adult barrier repair, but also fetal epidermal permeability barrier development. We demonstrated previously in fetal epidermis that the activities of HMG CoA reductase and SPT peak prior to barrier formation and decrease as a competent barrier emerges (Hurt et al, 1995). In contrast, the activities of GC synthase and cholesterol sulfotransferase (CSTase), the enzymes responsible for the synthesis of glucosylceramide and cholesterol sulfate, respectively, peak somewhat later in gestation, but prior to the emergence of barrier competence (Hanley et al, 1997b, c). In contrast, the activity of β-glucocerebrosidase and steroid sulfatase rise coincident with the formation of a competent barrier (op. cit.). Thus, in fetal epidermis, the activities of the enzymes that synthesize lipids increase prior to barrier formation, while the activities of enzymes that catabolize lipid precursors in the SC increase progressively in the last stages of barrier formation. We and other laboratories have shown that gene expression of key structural proteins required for the formation of corneocytes (involucrin, profilaggrin/filaggrin, loricrin) is initiated prior to barrier development and increases progressively throughout barrier formation (Bickenbach et al, 1995; Kömüves et al, 1998). In this study, we determined the effect of air exposure on the activity of key enzymes of lipid synthesis (HMG CoA reductase, SPT, GC synthase, and CSTase) and catabolism (β-glucocerebrosidase and steroid sulfatase) in fetal epidermis. In addition, we examined air exposure-induced changes in the gene expression of key structural proteins of the SC (profilaggrin/filaggrin, loricrin, and involucrin). MATERIALS AND METHODS Organ culture Timed-pregnant Sprague Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). Samples were obtained from gestational day 17 fetal rats, and full-thickness flank skin was incubated dermis-side down on COL-well inserts (3 µm pore size, Costar, Cambridge, MA), either submerged in M199 medium (Life Technologies, Grand Isle, NY) or at the air medium interface, as described previously (Hanley et al, 1997a). In some experiments explants incubated at the air medium interface were covered with a water-impermeable plastic (Saran wrap) (occluded) as described previously (Hanley et al, 1997a). Tissue preparation and enzyme assays Following 0 4 d of incubation, skin explants were placed on ice and the appropriate homogenization buffer was added (10% wt/vol). Tissues were homogenized on ice using a ground glass homogenizer followed by sonication (3 10 s). SPT and SSase activity was measured in isolated microsomes as previously described (Hurt et al, 1995; Hanley et al, 1997c). HMG CoA Reductase, GC synthase, and β-glucocerebrosidase activity was measured in homogenates and CSTase in cytosol, as previously described (Hurt et al, 1995; Hanley et al, 1997b). In situ hybridization Explants were fixed in 4% paraformaldehyde and embedded in paraffin. As described previously (Kömüves et al, 1998), digoxigenin-labeled RNA probes to detect loricrin and profilaggrin mrna were made from linearized cdna sequences as templates (a gift from S. Yuspa, NIH), using reagent supplied by Boehringer (Indianapolis, IN). In situ hybridization was performed as described previously (Kömüves et al, 1998), with probes applied to the sections and hybridized at 45 C for loricrin and 40 C for filaggrin. The hybridization of DIG-labeled probes to the endogenous mrna was detected by anti-dig-alkaline phosphatase with BCIP/NTBT substrate. The sections were counterstained with methyl green. Probes of sense orientation served as controls to ensure the specificity of hybridization. Omitting the DIG-labeled probes resulted in no signal, indicating that only DIG-containing RNA hybrids were detected. The sense control probes resulted in no signal, indicating the specificity of hybridization with the anti-sense probe (not shown). Immunohistochemistry Affinity-purified rabbit antipeptide antibodies (BabCo, Berkeley, CA) specific for involucrin, loricrin, and filaggrin were used. These antibodies were found to recognize both mouse and the rat proteins following heat-induced antigen retrieval treatment (in 10 mm citrate buffer; ph 6.0, at 95 C, for 30 min). The primary antibodies were used at 4 µg per ml concentration. All the immunoreagents were diluted in 10 mm Tris buffer, ph 7.6, containing 4% bovine serum albumin, 1% teleostean skin gelatin, 0.1% Tween 20, and 500 mm NaCl. The binding of the primary antibodies to the sections was detected by affinity-purified, biotinylated goat anti-rabbit IgG, followed by ABC-peroxidase reagent, both purchased from Vector (Burlingame, CA). Peroxidase activity was revealed with DAB substrate (QualTek Laboratories, Santa Barbara, CA) followed by counterstaining with methyl green. Omitting the first antibodies resulted in no signal, and preabsorption of the antibodies with the immunizing peptides resulted in the abolition of staining (not shown). These experiments demonstrate that the antibodies used specifically recognize the antigens under the conditions used. Statistics Data are presented as mean SEM. Statistical significance was determined by a Student s t test. RESULTS HMG CoA reductase and SPT activity are not altered by air exposure We have previously reported that SC formation and epidermal barrier development in gestational day 17 fetal skin explants follows a timetable virtually identical to that which occurs in utero, such that a competent barrier forms after 3 4 d of incubation, corresponding to the formation of a competent barrier on gestational days in utero (Hanley et al, 1996). To determine whether the changes in activity of HMG CoA reductase and SPT during in vitro development also parallel those measured in utero, we measured their activities in fetal rat epidermis of gestational days and compared them to activity measured in explants taken from day 17 rats and incubated submerged for 0 4 d. The time course of changes in activity of HMG CoA reductase (Fig 1A) and SPT (Fig 1B) in vitro paralleled that measured in utero. For both enzymes, epidermal activity was highest on gestational day 17, and declined during gestation as a competent barrier formed. We showed previously that barrier formation accelerates in airexposed explants, such that structural and functional markers of barrier maturation are present after only 2 d of incubation (vs 4d in controls) (Hanley et al, 1997a). To determine whether increased HMG CoA reductase or SPT activity might contribute to this acceleration, we next measured the epidermal activities of these enzymes in explants incubated either submerged or at an air medium interface for 1 or 2 d (equivalent to days 18 or 19 of gestation). As shown in Fig 2(A, B), air exposure did not significantly alter the activity of either HMG CoA reductase or SPT compared with submerged explants. In several experiments, parallel samples were examined by light microscopy to verify the acceleration of epidermal maturation by air exposure (data not shown). These data indicate that both HMG CoA reductase and SPT, the key regulatory enzymes required for the de novo synthesis of cholesterol and sphingolipids, respectively, are not regulated by air exposure during epidermal ontogenesis. The peak in GC synthase and CSTase activity is accelerated by air exposure We recently demonstrated in cultured fetal skin explants that the activity of GC synthase and CSTase, required for the synthesis of glucosylceramides and cholesterol sulfate, respectively, closely parallel activities in utero. The activity of both

3 VOL. 112, NO. 3 MARCH 1999 AIR EXPOSURE INDUCES FETAL EPIDERMAL DEVELOPMENT IN VITRO 305 Figure 1. Epidermal HMG CoA reductase and SPT activity decreases during late development in utero and in vitro. Enzyme activity was measured in homogenate (HMG CoA reductase) or microsomal fractions (SPT) of epidermis isolated from fetal rats of gestational age days (in utero) or from skin explants taken from gestational day 17 rats and incubated 0 4 d (in vitro submerged) as described in Materials and Methods. Data are presented as mean SEM. Similar results were obtained in two separate experiments. enzymes rise sharply from gestational day 17 to peak on day 19 (or culture day 2), and decrease thereafter (Hanley et al, 1997b, c). To determine whether air exposure regulates the activity of these enzymes, we next measured these enzymes in submerged versus air-exposed explants after 1 or 2dofincubation. As shown in Fig 3(A), GC synthase activity increased significantly in explants incubated for 1 d at the air medium interface compared with submerged explants. GC synthase activity after 2 d of air exposure, however, was similar to that measured in submerged explants. Thus, GC synthase activity peaks earlier in air-exposed than in submerged explants. CSTase activity was also significantly higher in air-exposed explants after 1dofincubation (Fig 3B); however, CSTase activity decreased significantly in air-exposed explants incubated for 2 d. These data indicate that the normal time-activity profile for CSTase is accelerated by air exposure, so that the peak in activity, which normally is observed in submerged explants at culture day 2, occurs in air-exposed explants at day 1. These studies show that peak activity of the enzymes responsible for the synthesis of glucosylceramide and cholesterol sulfate accelerates with air exposure and occurs prior to the formation of a competent barrier. β-glucocerebrosidase and steroid sulfatase activity increase earlier with air exposure We next examined the activity of two enzymes, β-glucocerebrosidase and steroid sulfatase, associated Figure 2. Air exposure does not affect activity of HMG CoA reductase or SPT in fetal epidermis in vitro. Epidermis was isolated from full-thickness fetal skin explants incubated either submerged or at the air medium interface (air-exposed) for 1 or 2 d. Activity of HMG CoA reductase (A) or SPT (B) was measured as described in Materials and Methods. Data are expressed as mean SEM, n 4. with formation of two of the final lipid species of the SC. Previous studies have shown that the activity of both enzymes increases throughout epidermal maturation in utero, i.e., between gestational days 17 and 21, and that their activities during in vitro development parallel those in utero (Hanley et al, 1997b, c). As shown in Fig 4(A), the activity of steroid sulfatase was significantly increased after 1, 2, and3dofair-exposed incubation. No significant effect was observed after 4 d of incubation, when a competent barrier is present under both immersed and lifted conditions. Similarly, air exposure also increased β-glucocerebrosidase activity at culture days 1 3, with no significant difference observed at culture day 4 (Fig 4B). These data demonstrate that an increase in the activity of the lipid catabolic enzymes, β-glucocerebrosidase and steroid sulfatase, is stimulated by air exposure during fetal barrier development in vitro. mrna and protein levels of structural proteins increase with air exposure We next examined the effect of air exposure on the expression of three key epidermal structural proteins. Previous studies by our and other laboratories have shown that involucrin, profilaggrin/filaggrin, and loricrin are expressed in the upper epidermis during gestation just prior to SC formation (Bickenbach et al, 1995; Kömüves et al, 1998). As shown in Fig 5, using in situ hybridization, profilaggrin and loricrin mrna expression was not observed in epidermis from day 17 fetal rats or in submerged explants cultured for 1 or 2 d. In contrast, incubation at an air medium interface for 2 d resulted in enhanced profilaggrin and loricrin mrna expression in the upper epidermis. The degree

4 306 KÖMÜVES ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 3. GC synthase and CSTase activities are affected by air exposure. GC synthase activity (A) was measured in homogenate and CSTase (B) measured in cytosolic fractions of fetal epidermis isolated from explants incubated for 1 or 2 d either submerged or air-exposed. Average activity in day 17 fetal rat epidermis prior to incubation was 6.5 pmol per min per mg (GC synthase) and 46.6 pmol per h per mg (CSTase). Data are presented as mean SEM, *p 0.01, n 9. and location of expression in the air-exposed explants was similar to that in fully differentiated epidermis from 21 d fetal rats. Moreover, immunohistochemical localization of profilaggrin/ filaggrin, loricrin, and involucrin protein demonstrated that air exposure, in addition to increasing mrna expression, also increased protein levels (Fig 6). This indicates that exposure to air not only increases the activity of selected lipid enzymes, but also accelerates the expression of structural proteins required for the formation of normal corneocytes. Occlusion prevents the air induced increase in enzyme activity and structural protein expression To determine if increased water transit is an important stimulus for these changes in enzyme activity and expression of structural proteins, we next examined the effect of occlusion with a water-impermeable membrane. As shown in Fig 7(A, B), occlusion blocked most of the increase in β-glucocerebrosidase and steroid sulfatase activity produced by air exposure. Furthermore, the degree of the expression of loricrin and profilaggrin mrna, and the accumulation of involucrin, profilaggrin/filaggrin, or loricrin protein was markedly reduced in occluded epidermis (Figs 5 and 6). Thus, occlusion blocks the increase in both lipid catabolic enzymes and structural proteins induced by exposure to an air medium interface. DISCUSSION Our previous studies demonstrated that permeability barrier development accelerates in fetal rat skin explants incubated at the Figure 4. Induction of steroid sulfatase and β-glucocerebrosidase activity by air exposure. Enzyme activity in epidermis isolated from explants incubated either submerged or air-exposed for 1 4 d was measured as described in Materials and Methods. Average activity in day 17 fetal rat epidermis prior to incubation was 4.9 pmol per min per mg (steroid sulfatase) and 0.98 nmol per min per mg (β-glucocerebrosidase). Data are presented as mean SEM, *p 0.005, n 8. air medium interface (Hanley et al, 1997a). This change in the timetable of development is dependent on water movement, because occlusion with a water vapor impermeable, but not a water permeable membrane, blocks such acceleration. As with normal barrier ontogenesis, accelerated permeability barrier formation correlates with the formation of mature lamellar membranes in the SC and a multilayered SC. Thus, this in vitro model mimics observations in very premature infants where permeability barrier and SC development accelerate following birth and subsequent air exposure (Harpin et al, 1983; Evans et al, 1986). In this study, we examined the biochemical changes that account for accelerated SC and barrier formation, using the response of fetal rat skin explants to air exposure. We observed an accelerated appearance and an overall increase in the activity of key enzymes required for the formation of glucosylceramides and cholesterol sulfate, respectively; air exposure accelerated the developmental increase and earlier appearance of both GC synthase and CSTase activities, thereby providing increased quantities of glucosylceramides and cholesterol sulfate at an early stage of fetal epidermal development. As glucosylceramides are required for the formation and secretion of normal lamellar bodies (Chujor et al, 1998), while cholesterol sulfate is essential for normal SC cohesion (Epstein et al, 1981), the early increase in peak activity of these two enzymes would facilitate the accelerated formation of a functional SC. In addition to increasing the activity of GC synthase and CSTase,

5 VOL. 112, NO. 3 MARCH 1999 AIR EXPOSURE INDUCES FETAL EPIDERMAL DEVELOPMENT IN VITRO 307 Figure 5. Effects of air exposure and occlusion on the expression of profilaggrin and loricrin mrna in fetal rat skin in vitro. Skin explants from day 17 fetal rats were incubated submerged for 2 d (A, D, G), or incubated air-exposed for 2 d unoccluded (B, E, H) or occluded (C, F, I). Parts (A) (C) were stained with hematoxylin and eosin. Expression of profilaggrin (D F) and loricrin (G I) mrna was detected by in situ hybridization. The dotted line demarcates the basal lamina of the epidermis, whereas the arrows indicate the epidermal surface. Scale bar: 25 µm. Figure 6. Effects of air exposure and occlusion on the expression of involucrin, profilaggrin/filaggrin, and loricrin proteins in fetal rat skin in vitro. Fetal rat skin explants were incubated submerged for 2 d (A, D, G), or air-exposed for 2 d unoccluded (B, E, H) or occluded (C, F, I). Presence of involucrin (A C), profilaggrin/ filaggrin (D F), and loricrin (G I) proteins were detected by immunohistochemistry. Profilaggrin/filaggrin staining is seen both in the stratum spinosum and in the stratum granulosum, whereas loricrin is localized to the stratum granulosum. The dotted line demarcates the basal lamina of the epidermis, whereas the arrows indicate the epidermal surface. Scale bar: 25 µm. which are localized in the nucleated layers of the epidermis, air exposure also increases the activity of β-glucocerebrosidase and steroid sulfatase, enzymes which primarily function in the SC (Elias et al, 1984; Holleran et al, 1992). β-glucocerebrosidase catalyzes the conversion of glucosylceramides to ceramides that are crucial for the formation of mature extracellular lamellar membranes and a competent permeability barrier (Holleran et al, 1993). Steroid sulfatase in the SC catalyzes the conversion of cholesterol sulfate to cholesterol, an enzymatic step that is essential for both normal desquamation and barrier function (Epstein et al, 1981; Zettersten et al, 1998). A SC deficient in either of these enzymes does not function normally (op. cit.); and therefore, stimulation of these enzymes by air exposure allows for accelerated epidermal development and formation of a competent permeability barrier. In contrast to the stimulation of GC synthase, CSTase, β-glucocerebrosidase, and steroid sulfatase, air exposure does not increase the activity of either HMG CoA reductase or SPT. HMG CoA reductase is the rate-limiting enzyme in cholesterol synthesis, while SPT is the initial and rate-limiting enzyme in ceramide synthesis. The absence of an increase suggests that the rate of synthesis of both cholesterol and ceramides is sufficient in fetal skin to support the accelerated formation of the SC that is induced by air exposure. This observation agrees with our previous studies, which showed that the activity of these enzymes actually decreases during the later stages of epidermal ontogenesis in utero (Hurt et al, 1995). In contrast, in adult animals, disruption of the barrier results in a marked stimulation of HMG CoA reductase and SPT activity leading to an increase in cholesterol and ceramide synthesis (Feingold, 1991; Proksch et al, 1993). Moreover, the increase in either cholesterol or ceramide synthesis induced by barrier disruption is inhibited by topical application of compounds that inhibit HMG CoA reductase or SPT activity which delays SC formation and barrier recovery in adult animals (Feingold et al, 1990; Holleran et al, 1991b). These results indicate that while in adult epidermis

6 308 KÖMÜVES ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY not known. Water movement appears to play a part, however, as occlusion with a water vapor impermeable membrane blocks these responses. In adult epidermis the increased water movement associated with barrier disruption alters the epidermal calcium gradient thereby stimulating barrier recovery (Lee et al, 1992). Whether alterations in calcium or other ions play a part in the acceleration of stratum corneum and barrier development in airexposed fetal skin is unknown. We have recently shown that in fetal mice the epidermal calcium gradient appears as the SC barrier forms (Elias et al, 1998). Therefore, air exposure is not required for the establishment of the calcium gradient in the fetal epidermis. Whether it accelerates the formation of the epidermal calcium gradient remains to be determined. Finally, air exposure is a known stimulator of keratinocyte differentiation in vitro. Cultured keratinocytes incubated at an air medium interface (i.e., lifted) produce increased numbers of lamellar bodies, and exhibit a lipid composition and structural organization more closely resembling skin in vivo (Williams et al, 1988; Rosdy and Clauss, 1990; Fartasch and Ponec, 1994). In summary, this study demonstrates that air exposure of fetal skin stimulates the activity of epidermal GC synthase, CSTase, β-glucocerebrosidase, and steroid sulfatase, lipid enzymes, which are crucial for the formation and function of the SC. Additionally, air exposure stimulates the expression of structural proteins, such as involucrin, profilaggrin/filaggrin, and loricrin, required for the formation of the corneocyte. These changes form the biochemical basis for the acceleration of SC ontogenesis induced by air exposure. These results provide strong evidence that the developmental expression of these structural proteins and catabolic lipid enzymes is coordinately regulated. This study was supported by NIH grants HD 29706, AR 19098, PO 39448, and AR 39639, and the Medical Research Service, Department of Veterans Affairs Medical Center. Figure 7. Steroid sulfatase and β-glucocerebrosidase activity are not increased by air exposure in occluded explants. Explants were incubated for 2 d either submerged, or air exposed either unoccluded (air) or covered with a water-impermeable membrane (occluded) as described in Materials and Methods. Enzyme activity was measured in epidermal microsomal fractions (A, steroid sulfatase) or homogenates (B, β-glucocerebrosidase) as described in Materials and Methods. Data are expressed as mean SEM, n 6. cholesterol and ceramides may be in limited supply, in fetal epidermis the rates of cholesterol and ceramide synthesis are sufficient to sustain accelerated barrier development. A normal SC requires the formation of both mature extracellular lamellar membranes and corneocytes, anucleated cells with an outer cornified envelope linked to a dense network of intermediate filaments, which provides strength and rigidity. The present study shows that air exposure profoundly affects not only lipid metabolism in fetal epidermis, but also the expression of structural proteins required for keratinocyte differentiation. Using in situ hybridization the expression of profilaggrin and loricrin mrna increased in the upper epidermis of air-exposed fetal explants. Moreover, profilaggrin/filaggrin, loricrin, and involucrin protein levels increase in the upper epidermis with air exposure. The results indicate that air exposure not only increases the activity of specific lipid enzymes, but also accelerates the expression of the key structural proteins required for epidermal differentiation. 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