FEBS Letters 580 (2006) 54565466

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Epidermal sphingolipids: Metabolism, function, and roles in skin disorders

Walter M. Hollerana,b,c,*, Yutaka Takagid, Yoshikazu Uchidaa,b
c a Department of Dermatology, School of Medicine, University of California San Francisco, United States Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, United States Dermatology Service and Research Unit, Department of Veterans Aairs Medical Center, San Francisco, CA, United States d Biological Science Laboratories, Kao Corporation, Haga-gun, Tochigi, Japan b

Received 15 August 2006; revised 17 August 2006; accepted 17 August 2006 Available online 1 September 2006 Edited by Gerrit van Meer

Abstract Mammalian epidermis produces and delivers large quantities of glucosylceramide and sphingomyelin precursors to stratum corneum extracellular domains, where they are hydrolyzed to corresponding ceramide species. This cycle of lipid precursor formation and subsequent hydrolysis represents a mechanism that protects the epidermis against potentially harmful eects of ceramide accumulation within nucleated cell layers. Prominent skin disorders, such as psoriasis and atopic dermatitis, have diminished epidermal ceramide levels, reecting altered sphingolipid metabolism, that may contribute to disease severity/ progression. Enzymatic processes in the hydrolysis of glucosylceramide and sphingomyelin, and the roles of sphingolipids in skin diseases, are the focus of this review. 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: ABCA12; Barrier; Ceramide; Dermis; Epidermis; Gaucher; Glucocerebrosidase; Glucosylceramides; Ichthyosis; Niemann-Pick; Prosaposin; Saposins; Sphingolipid; Sphingomyelin; Sphingomyelinase; Stratum corneum

1. Sphingolipids in the structure and function of mammalian epidermis Mammalian skin is composed of two distinct layers divided by a basement membrane zone: (1) the underlying dermis containing primarily broblasts embedded in an acellular collagen/elastin matrix that accounts for the majority of skin thickness; and (2) the overlying epidermis, responsible for for-

* Corresponding author. Present address: Dermatology Service and Research Unit (190), UCSF & Veterans Aairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, United States. Fax: +1 415 751 3927. E-mail address: walt.holleran@ucsf.edu (W.M. Holleran).

Abbreviations: Cer, ceramide; CLE, corneocyte lipid envelope (covalently-bound omega-hydroxy ceramides); FA, fatty acid; GlcCer, glucosylceramide; GlcCerase, b-glucocerebrosidase; SC, stratum corneum; SM, sphingomyelin; SMase, sphingomyelinase; N, non-hydroxy fatty acid; A, alpha-hydroxy fatty acid; O, omega-hydroxy fatty acid; E, esteried (omega) fatty acid; S, sphingosine base; P, phytosphingosine base; H, 6-hydroxy-sphingosine base

mation and maintenance of the skin barrier to both desiccation and penetration of xenobiotics. The function of this barrier resides primarily in the extracellular lipid domains between the outermost, enucleated cells, called corneocytes, of the stratum corneum (SC). Mammalian SC contains extensive quantities of lipids, localized to these extracellular domains (interstices), comprised of nearly equimolar quantities of ceramides (Cer), cholesterol, and free fatty acids, a ratio that is imperative for normal organization into the lamellar membrane structures primarily responsible for epidermal barrier homeostasis [1,2]. Cer species comprise approximately half of the total intercellular lipid content by weight, and are critical for these lamellar membrane structures [3,4]. The specic roles for Cer in epidermal function have been revealed in studies with both normal and diseased human skin, as well as animal models and model membranes in vitro [5,6]. For example, the Cer content is altered in patients with both atopic dermatitis [79] and psoriasis [10,11] (discussed further below). Likewise, cholesterol as well as both essential and non-essential free fatty acids play separate, critical roles in epidermal barrier homeostasis. Given that human SC contains at least nine major Cer fractions (see Fig. 1), many of which are unique to the epidermis, including omega(x)-hydroxylated and x-acylated forms, as well as x-hydroxy-Cer species that are covalently attached via the x-hydroxyl of the N-acyl fatty acid to cornied envelope proteins of the enucleate stratum corneum cells (i.e., corneocytes), the metabolism of these unique sphingolipids is of signicant interest. It is intriguing that mammalian epidermis forms these and other barrier lipids as precursor species, storing them with other lipid precursors in membrane-limited organelles, called lamellar bodies (LBs), a process that requires at least one transport protein [12]. It is only following the fusion of these LBs with the apical plasma membrane of the outermost nucleated cell layer of the epidermis, the stratum granulosum, that their combined lipid and protein contents are secreted [13]. The subsequent hydrolysis of these lipid precursors constitutes the nal processing step that facilitates eective permeability barrier formation in the SC. Since the myriad Cer species in mammalian SC are generated nearly exclusively from glucosylceramide (GlcCer) and sphingomyelin (SM) precursors, this review focuses on the metabolism of these unique sphingolipids, as well as on the changes in the content of these lipids associated with common skin disorders such as atopic dermatitis and psoriasis.

0014-5793/$32.00 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2006.08.039

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Fig. 1. Structures of major human stratum corneum (SC) ceramide species. Sphingoid base structures include sphing-4-enine (sphingosine), 4hydroxysphinganine (phytosphingosine), and sphing-[6-hydroxy]-4-enine (6-OH-sphingosine) bases. Cer 1, Cer 4, and Cer 9 are commonly termed acyl-ceramides, as they contain linoleic acid esteried to the omega-hydroxy of the very long chain N-acyl fatty acid. Note that all nine ceramide species are generated via deglucosylation of the respective glucosylceramide species, while only Cer 2 [NS] and Cer 5 [AS] are generated via hydrolysis of corresponding sphingomyelin precursors. Abbreviations for Cer species (as per Motta S., et al. [11]): N, non-hydroxy-FA; A, a-hydroxy-FA; O, omega-hydroxy FA; E, esteried (omega)-FA; S, sphingosine base; P, phytosphingosine base; H, 6-hydroxy-sphingosine base.

The seemingly inecient cycle of lipid precursor formation and subsequent extracellular lipid hydrolysis noted above, appears to reect a mechanism by which the epidermis can produce and store very large quantities of Cer, while simultaneously protecting itself from potentially adverse intracellular events that could stem from an excessive accumulation of these bioactive lipids within the nucleated epidermal cell compartments. Specically, various Cer species and metabolites have well-recognized roles in signaling dierent cell fate outcomes, such as proliferation, dierentiation, and/or apoptosis depending on the sphingolipid species involved. In addition, inappropriate accumulation of Cer species leads to cell death in keratinocytes [1418]. Thus, the sequential formation and sequestration of Cer precursor lipids, followed by post-secre-

tion, extracellular hydrolysis to regenerate free Cer species, allows for the orderly accumulation of these sphingolipids required for the barrier, while simultaneously minimizing risks from adverse Cer signaling events.

2. Precursors of mammalian stratum corneum ceramides The synthesis of Cer species, although occurring throughout the epidermis [19], increases with epidermal dierentiation, consistent with elevated sphingolipid generation and content in more-dierentiated epidermal layers [2022]. During synthesis, the varied Cer species destined for the SC are modied at the 1-hydroxy position to either GlcCer species by GlcCer

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synthase (i.e., UDP-glucose:ceramide glucosyltransferase or GlcT1; EC 2.4.1.80) [23,24], or to sphingomyelin (SM) species by the transfer of phosphorylcholine from phospatidylcholine [25,26]. As noted above, although the majority of Cer species contained within epidermal lamellar bodies (LBs) are glucosylated [2731], sphingomyelin (SM) species are also present in substantial quantities [28,29]. Consistent with this, GlcCer synthase activity increases during epidermal dierentiation both in vitro [32,33] and in vivo [34], and as the permeability barrier is forming in utero [35]. Moreover, inhibitor studies revealed that GlcCer synthase activity and the generation of GlcCer species, are required for normal barrier homeostasis [34]. Biochemical studies show that the increase in Cer levels that occurs during the nal stages of mammalian epidermal dierentiation corresponds with a decrease in GlcCer and SM, following a peak of these latter sphingolipids in the uppermost nucleated cell layers, or stratum granulosum (SG) [2022,36]. These marked shifts in the content of sphingolipid precursors/products have been visualized in normal human epidermis by immunoelectron microscopy, using antibodies to both GlcCer and Cer [37]. The distinct molecular species of human SC Cer include three unique omega-hydroxylated Cer [3841], as noted above (see Fig. 1). The precursorproduct relationship for each of these SC Cer molecular species has recently been delineated [42,43]. Analysis of epidermal SM species revealed three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) composition; i.e., containing either long-chain FA (SM1; C(2226)), shorter-chain FA (SM-2; primarily C16), or short-chain alpha-hydroxy FA (SM-3; C1618) [42]. In addition, these three major epidermal SM species contain either sphingosine or sphinganine, but they lack phytosphingosine as the sphingoid base backbone. Thus, only two of the nine SC Cer species, Cer 2 (NS)1 and Cer 5 (AS) (see Fig. 1), derive from SM precursors [42]. A comparable analysis of epidermal glucosylceramides (GlcCer) indicates conversely that distinctive epidermal GlcCer precursors correspond to all Cer species in epidermis and SC [42,43]. Thus, all nine of the major human SC Cer species, including the omega(x)-hydroxy fatty-acid-containing Cer species; i.e., Cer 1 (EOS), Cer 4 (EOH), and the most-recently described Cer 9 (EOP) [41], derive from GlcCer, while only two Cer species (Cer 2 (NS) and Cer 5 (AS)), also derive in part from corresponding SM precursors (see Fig. 1). These results provide further details about sphingolipid processing, and suggest potential divergent roles for GlcCer- vs. SM-derived Cer species in epidermal structure and function.

3. Enzymatic processing of glucosylceramide and sphingomyelin is required for epidermal barrier function Although at least two enzymes can catalyze the hydrolysis of b-linked glucose-moieties, only b-glucocerebrosidase (GlcCerase; EC 3.2.1.45) specically hydrolyzes GlcCer-to-Cer. Early reports described b-glucosidase activity in both whole mam-

1 Abbreviations for Cer species to delineate structures (as per Ref. [11]), containing as follows: N, non-hydroxy-FA; A, a-hydroxy-FA; O, omega-hydroxy FA; E, esteried (omega)-FA; S, sphingosine base; P, phytosphingosine base; H, 6-hydroxy-sphingosine base.

malian skin as well as isolated epidermis (reviewed in [44]). In porcine epidermis, this b-glucosidase activity is active at acidic pH [45], and the activity in epidermis and palatal epithelium exceeded that in non-keratinized oral epithelium [46]. Thus, the relative inactivity of b-glucosidase in non-keratinized epithelium is consistent not only with the persistence of GlcCer species to the epithelial surface [47,48], but also with less-stringent barrier in non-keratinized oral epithelium [49]. The specicity of the epidermal activity toward GlcCer substrate(s) was later shown to be a consequence of specic GlcCerase activity, with higher activity in the outer, more differentiated epidermal cell layers, extending into the enucleate SC [44,50], with elevated activity evident in serial frozen sections at the region corresponding to the stratum granulosum-SC transition [51]. Immunohistochemical staining for GlcCerase protein also revealed enhanced uorescent signal in the outer nucleated layers of human epidermis, concentrated at the apex and margins of SG cells, as well as within extracellular membrane domains of the lower SC [37]. In situ enzyme zymography with both whole skin and tissue sections revealed that the highest levels of GlcCerase activity are present in the upper SG and the lower SC [50,52,53]. Finally, in extracts from individual epidermal layers, GlcCerase activity is present throughout murine epidermis, again with the highest activity in the SC, peaking in the lower-to-mid-SC [44,50]. Thus, the processing of GlcCer-to-Cer within the outer epidermis is attributable to retention of specic enzymatic activity within extracellular domains of the SC. The link between GlcCer-to-Cer conversion and normal epidermal permeability barrier homeostasis became evident when both mRNA and enzyme activity levels for GlcCerase were found to be increased during the recovery of the barrier following acute abrogation in a murine model [54]. In vivo inhibition of GlcCerase using topical application(s) of bromoconduritol B-epoxide (BrCBE), a potent and specic inhibitor for this enzyme [55], reduced murine epidermal GlcCerase activity by >90%, and signicantly diminished barrier function [54,56], demonstrating a requirement for this enzymatic processing in the maintenance of epidermal barrier homeostasis. This functional delay is due to replacement of the normal, highlyordered, SC lamellar membrane arrays with disordered linear arrays of incompletely-processed, immature-appearing membrane structures [56,57]. Additional evidence for the importance of GlcCer processing in epidermal function was obtained using a null-allele GlcCerase-decient mouse [58], a model that was developed as a murine analogue for Gaucher disease, and which retains less than 1% residual enzyme activity. The homozygous GlcCerase-decient animals displayed highly-disordered membrane arrays and a corresponding severely-compromised barrier [57]. The prole of SC and epidermal lipid contents revealed both increased GlcCer levels, and diminished Cer, consistent with a deciency of this enzyme. Interestingly, the epidermis of heterozygous animals, which retains approximately 50% of normal GlcCerase activity, revealed none of the lipid biochemical, ultrastructural, or functional abnormalities that were evident in the more-severely decient animals [57]. This knockout approach demonstrated not only the importance of GlcCer-to-Cer conversion for barrier homeostasis, but also that a single GlcCerase gene product solely accounts for this activity in mammalian epidermis. Additional murine models that more-closely mirror the molecular mutations in human Gaucher disease have revealed

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similar ndings. Specically, while animals homozygous for GlcCerase point mutations equivalent to the common mutations found in human Gaucher disease (e.g., L444P) revealed no neurologic pathology, severe ichthyotic (scaling) and histologic abnormalities were evident in the epidermis [59]. In fact, these animals do not survive beyond the initial 24 h postpartum, presumably because of abnormally high water loss resulting from the epidermal lipid processing defect [59]. Examination of SC ultrastructure (using ruthenium tetroxide-post-xed samples) from these animals revealed marked structural abnormalities in the SC membrane lipid domains, including incompletely processed lamellar body contents throughout the SC interstices (W. Holleran & R. Proia, unpublished observations), as observed in b-GlcCerase deciency of genetic or inhibitor-based origin. Skin samples from human Gaucher patients with severe GlcCerase deciency; i.e., neonatal Type 2 disease, demonstrated similar SC membrane structural abnormalities [57,60]. Interestingly, Gaucher patients with less-severe disease do not display signicant abnormalities in SC ultrastructure [60], consistent with the normal ndings in mice heterozygous for GlcCerase deciency described above. Together, these results suggest that an excess of this enzyme is available in normal epidermis under basal conditions; and conversely, that a minimal threshold of epidermal enzyme activity, that no longer can support normal SC lamellar membrane formation and barrier integrity, exists in severe Gaucher disease with skin involvement. Interestingly, epidermal ultrastructure abnormalities may help to predict the severity of Gaucher disease, and thereby assist with diagnostic assessment [61]. Although the studies summarized above were the rst to demonstrate the critical requirement for enzymatic hydrolysis of lipid precursors (GlcCer) to specic products (Cer) in mammalian epidermis, enzymatic processing of sphingomyelin (SM) to Cer by sphingomyelinase (SMase; EC 3.1.4.12) also is critical for SC structure and epidermal barrier homeostasis. At least two isoenzymes in human skin can hydrolyze SM to Cer, including a lysosomal-type acid-pH optimum sphingomyelinase (aSMase) and a non-lysosomal, magnesium-dependent neutral-pH optimum sphingomyelinase (nSMase). The epidermal aSMase has optimal activity at pH 4.55.0, and is activated by Triton X-100 (0.1% w/v) in vitro [62,63]. Although both the acidic- and neutral-SMase enzymes are involved in the recovery of the epidermal permeability barrier following an acute challenge [64], a number of ndings reveal that at an acid-pH, the lysosomal enzyme appears to be primarily responsible for the extracellular hydrolysis of SMto-Cer in mammalian SC. For example, aSMase activity in SC localizes primarily to intercellular membrane domains [13,65], making it ideally situated to catalyze this critical lipid processing step. In addition, aSMase activity increases, in parallel with accumulation of lipids, within lamellar bodies during the maturation of these critical epidermal organelles [13]. Furthermore, in normal mice aSMase activity increases two hours following acute barrier abrogation, an increase not observed in tumor necrosis factor-receptor (TNF-R)-decient mouse epidermis that display delayed barrier recovery [64]. Inhibition of this increase in aSMase activity by two independent approaches; i.e., a Cer-structural analog, PDHS, or intracellular inhibiton at the level of the lamellar body with either imipramine [64] or desipramine [63], signicantly delays epidermal barrier recovery following an acute challenge. This

functional delay is accompanied by an increase in SM content, as well as a reduction of normal extracellular lamellar membrane structures in the SC [63]. Consistent with these observations, patients with Niemann-Pick (type A) disease, characterized by a deciency of lysosomal aSMase, demonstrate abnormal permeability barrier homeostasis, including delayed recovery kinetics following an acute challenge [63]. Given that the inhibitor-induced delays in barrier recovery discussed above can be overridden by topical co-applications of Cer [63], alteration of the Cer-to-SM ratio, rather than SM accumulation itself, appears to be responsible for the diminished epidermal barrier function. This latter nding contrasts with the consequences of absent GlcCerase, where accumulation of enzyme substrates (i.e., GlcCers) in the SC, rather than reduction of enzyme products, is responsible for the resultant membrane alterations and diminished barrier function [56]. The contribution of specic SM precursors to SC Cer species was further addressed by structural analysis of human epidermal SM species (see above) [42]. Given that only two of the nine known human SC Cer species; i.e., Cer 2 (NS) and Cer 5 (AS) (see Fig. 1), are generated in part by SM hydrolysis, and that all nine major SC Cer species are derived from GlcCer precursors [42,43], the GlcCer-to-Cer pathway appears to have a more-critical role for generating the full spectrum of unique Cer species that comprise the epidermal barrier. Consistent with this interpretation is the more-signicant epidermal barrier dysfunction that is evident with GlcCerase deciency (i.e., in either severe Gaucher disease or GlcCerase knockout mice) [57,60] than has so far been observed with aSMase deciency (i.e., Niemann-Pick disease) [63,64]. The role of nSMase in epidermal structure and function is less-well resolved. Although aSMase activity appears primarily responsible for the majority of SM-to-Cer hydrolysis that occurs in the extracellular domains of the SC, nSMase activity also is involved in epidermal homeostasis. For example, delayed barrier recovery following an acute challenge to the skin of FAN-decient mice, with FAN being an adapter protein in TNFR55 signaling, suggests a role for nSMase activity in epidermal barrier recovery and homeostasis [66]. However, delayed barrier recovery without the normal increase in aSMase activity (or changes in nSMase activity), also was noted in TNF-R-decient mouse skin [64]. Although these studies do not distinguish between aSMase and nSMasedependent events, they are consistent with the SM-to-Cer conversion being critical for epidermal barrier homeostasis. The results summarized above represent strong evidence for the dynamic events occurring during the nal stage of epidermal/barrier dierentiation and development. As such, it should now be readily apparent that extracellular lipid processing of both GlcCer and SM precursors is critical for mammalian epidermal structure and function. Interestingly, the importance of the gradual decline in epidermal SMase activity [67] for the decline in barrier function in aging [68] has yet to be fully explored. Finally, given that SM-to-Cer turnover (and recycling) has been implicated in myriad cellular processes (reviewed in [69,70]), and that active sphingolipid remodeling is ongoing within proliferating and more-dierentiated keratinocytes (Uchida & Holleran; unpublished observations), it is highly likely that additional important roles will be revealed for both GlcCer and SM in cutaneous function and disease.

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4. Generation of covalently-attached omega-hydroxy ceramides requires GlcCer processing Mammalian SC contains a unique lipid layer that is covalently-attached to the external surface of cornied envelope proteins of fully-dierentiated corneocytes. This corneocyte lipid envelope (CLE) primarily consists of omega(x)-OH Cer covalently attached to highly-crosslinked, cornied envelope proteins [71] (see Fig. 2). The x-OH group of these Cer is required for CLE formation [72,73], as this moiety represents the attachment point between the Cer and specic amino acids of cornied envelope proteins, including involucrin and others [74]. Although puried transglutaminase 1 enzyme can perform this protein ceramidation reaction in vitro [75], whether this enzyme is solely responsible for this unique reaction in vivo remains unresolved, as transglutaminase 1-decient patient epidermis contains sporadic normal CLE structures [76], as well as near-normal quantities of covalently-attached Cer [77]. Similar to the formation of membrane bilayer structures in the SC described above, lipid processing also is required to generate the mature CLE structure (Fig. 2). A number of ndings support this conclusion. First, in epidermis lacking GlcCerase activity, the CLE contains only covalently-attached x-OH GlcCer species [78,79]. Second, CLE structures also are evident in Gaucher mouse SC by ultrastructural analysis (Y. Uchida & W. Holleran, unpublished observations), despite the parallel accumulation of covalently-attached x-OH GlcCer and an absence of the corresponding covalently-bound x-OH Cer species in b-GlcCerase-decient epidermis [78,79]. Thus,

deglucosylation, though clearly required for lamellar membrane formation/organization [54,56,57], is not required for initial CLE formation. However, since the covalent attachment of Cer to cornied envelope proteins involves the x-OH groups of Cer [73], the (bulky) hydrophilic glucose residue, facing toward the extracellular domains, could further interfere with organization of the compact lamellar membranes required for competent barrier function. Thus, mature CLE formation occurs in a sequential manner; i.e., attachment of x-OH GlcCer followed by subsequent enzymatic deglucosylation by GlcCerase.

5. Regulation of GlcCerase activity in the epidermis and stratum corneum As noted above, the post-secretory processing of GlcCer precursors into Cer within the SC interstices is catalyzed by GlcCerase, a lysosomal, acidic-pH optimum enzyme. The activity of epidermal GlcCerase has been shown to possess an acidic pH optimum, with peak in vitro activity at pH 5.2, and absent or signicantly reduced activity at neutral pH (7.4) [44,45,50]. Given that epidermal barrier homeostasis is perturbed when the normally-acidic SC is neutralized [52,80], SC acidication appears to have a signicant role in epidermal function(s), including lipid processing events. Specically, permeability barrier recovery proceeds normally when barrier-disrupted murine skin is exposed externally to solutions buered to an acidic pH [52]. In contrast, the initiation of barrier recovery slows when barrier-disrupted skin is exposed to a neutral

Fig. 2. Formation and secretion of LB at stratum granulosum stratum corneum interface. Precursor lipids, including glucosylceramide (GlcCer), sphingomyelin (SM), glycerophospholipids (PL), and cholesterol sulfate (CS), are packaged into lamellar bodies (LB) within the upper cell layers of the epidermis. Lipid packaging into the LB requires function of at least one ABC transporter protein, ABCA12, which appears to localize to the limiting membranes of the LB [12]. Fusion of the LB with the apical plasma membrane (PM) in the uppermost nucleated cell layer of the epidermis (stratum granulosum), allows extrusion of lipid precursors into the extracellular domain(s). Subsequent enzymatic processing of precursor lipids generates the major lipid classes required for epidermal barrier function; i.e., ceramides (Cer), cholesterol (Chol), and free fatty acids (FFA). The corneocyte lipid envelope (CLE) consists of x-OH-ceramides that are covalently attached to the highly-crosslinked proteins of the cornied envelope (CE), and is formed sequentially with the addition of omega-OH-GlcCer followed by deglucosylation.

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pH, independent of buer composition. Interestingly, both the formation and secretion of LB proceed comparably when skin is exposed to either acidic (pH 5.5) or neutral (pH 7.4) conditions. However, exposure to pH 7.4 (but not pH 5.5) results in both the persistence of immature, extracellular lamellar membrane structures accompanied by a marked decrease in the in situ activity of GlcCerase [52]. Indeed, despite an increased pH toward neutrality in the lower SC, extracellular microdomains remain acidic [81], strongly suggesting that GlcCerase could be activated by localized changes in SC pH. Consistent with the membrane localization of acidic microdomains in lower SC, increased GlcCerase activity is evident with increasing depth of tape stripping of mammalian skin [50,82]. Finally, while short-term changes in pH reversibly activate/inactivate GlcCerase [80], more prolonged elevations in pH ultimately result in an irreversible loss of enzyme content/activity due to a serine protease-mediated degradation of GlcCerase [83]. Thus, extracellular pH in the SC also has an important role in regulating GlcCer-to-Cer processing through enzyme-dependent hydrolysis. Additional mechanisms are likely to modulate the posttranslational changes in GlcCerase activity. For example, in extracutaneous tissues, GlcCerase is activated by small molecular weight glycoproteins, called saposins (SAPs) [84]. In particular, SAP-A and SAP-C can upregulate GlcCerase activity in reconstituted, in vitro enzyme systems [85,86]. Four of the SAP proteins; i.e., SAP-ASAP-D, each of which is expressed in human epidermis (Holleran et al., unpublished results), are derived by proteolytic processing of a common precursor protein, prosaposin [87]. Given the importance of GlcCerase activity in mammalian epidermis, it is not surprising that prosaposin-decient mice display GlcCer accumulation, subnormal levels of Cer, and immature SC lamellar membranes [78], as in severe Gaucher disease (see above). Furthermore, the decreased levels of epidermal prosaposin protein [88], also may explain, in part, the apparent decrease in SMase activities in patients with atopic dermatitis [89], contributing to the diminished SC Cer content of these patients (discussed further below). Finally, the striking abnormality in SC membrane domains in prosaposin-decient mice is accompanied by an accumulation of GlcCer species that are covalently-attached to cornied envelope proteins [78], again consistent with deglucosylation of x-OH-GlcCer occurring after covalent attachment to cornied envelope proteins. Together, these results suggest that extracellular SAP activation of GlcCerase activity is important for normal SC lipid processing.

6. Alterated ABCA12-dependent transport of glucosylceramide into lamellar bodies in harlequin ichthyosis and lamellar ichthyosis type 2 The role(s) of lipid transporter proteins in epidermal function and disease have only recently begun to be addressed. Recent discoveries regarding the genetic causes of specic congenital ichthyoses, severe skin disorders characterized by excessive and debilitating scaling of the SC, highlight the importance of lipid (and in particular, sphingolipid) transport in epidermal function. For example, the ABCA12 gene was initially mapped to a region of chromosome 2q34 that harbors the genes for lamellar ichthyosis [90]. Subsequently, nine families with the rare autosomal recessive skin disorder, lamellar

ichthyosis type 2, were shown to have mutations in the ABCA12 gene [91]. In addition, mutations in the ABCA12 gene also have been reported in patients with harlequin ichthyosis (HI) [12,92], the most severe form of congenital ichthosis that is often fatal in the neonatal period. A recent report conrms ABCA12 to be a major HI gene [93]. The ABCA12 protein belongs to the superfamily of ATP-binding cassette transporter proteins that require ATP for transport of molecules across cell membranes [94], and appears to be situated in the limiting membrane of the epidermal LBs [12]. Abnormal LB formation and secretion has long-been associated with HI pathogenesis [95,96]. Although the cytosol of stratum granulosum cells contains an extensive number of LBs [97], a paucity of these lipid-rich organelles are evident in HI skin [12,95,96]. Immunouorescence using an anti-GlcCer antibody [37] revealed diuse GlcCer distribution throughout the epidermis of HI patient skin, in contrast to the punctate pattern concentrated at the stratum granulosum level in normal skin [12], suggesting alterations in the transport of GlcCer species into the internal membrane structures of the LB. In addition, HI keratinocytes showed intense GlcCer staining around nuclei that failed to redistribute to the cell periphery during calcium-induced dierentiation in vitro, instead revealing a congested pattern of GlcCer distribution [12]. Importantly, genetic correction of HI keratinocytes with wild-type ABCA12 normalized the GlcCer distribution pattern in vitro [12]. Interestingly, HI corneocytes also contained abnormal lipid droplets that resembled immature LBs, suggestive of entombed lipids within SC cells. The apparent lack of GlcCer delivery to LB precursors in HI stratum granulosum cells impairs the formation and maturation of these membrane-bound organelles, and ultimately blocks the normal delivery to (and organization of) barrier lipids in the extracellular domains of the SC. The severe skin defects associated with ABCA12 mutations underscore the critical role(s) for epidermal lipid transport, especially that of GlcCer, in normal mammalian skin function. A parallel role has been reported for the structurally-related ABCA3 protein in the secretion of pulmonary surfactant, composed primarily of phosphatidylcholine, in alveolar type II cells [98]. The presence of ABCA3 in the limiting membrane of lamellar bodies in these lipid-secreting lung cells [98], reveals a common function for these related ABCA proteins in the protection from the challenges of survival in a dry (terrestrial) environment. Given the complex mixture of sphingolipid species that are formed and delivered to the apical plasma membrane of granular cells, additional as yet unresolved lipid transport and sorting proteins are likely required for normal epidermal function(s).

7. Lipid processing alterations in atopic dermatitis The contribution of SC lipid abnormalities to the pathogenesis of common cutaneous disorders continues to be an active area of research. Numerous studies have linked the distinctive barrier abnormality in patients with atopic dermatitis (AD) to altered SC lipid content, most-notably, diminished Cer levels [7,8,99]. Although dierences in sampling and analytical methods lead to variations in the absolute levels of these reported changes, all of these studies concur that total Cer levels are diminished in the SC of aected AD epidermis [79,99101].

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Fig. 3. Mechanisms for ceramide alterations in atopic dermatitis (AD). A number of possible mechanisms may account for the decreased ceramide content noted in the SC of AD patients, including: (1) a decrease in de novo ceramide synthesis; (2) deacylase activity to generate glucosylsphingosine and/or sphingosyl-phosphorylcholine; (3) increased ceramidase activity; and/or (4) decreased sphingomyelinase activity.

However, early studies also revealed no signicant dierences in either GlcCerase or alkaline-ceramidase (alk-CDase) activities in the SC from AD epidermis [102]. Subsequent studies by these same authors revealed not only high activity of a novel enzyme with properties of a sphingomyelin deacylase [103], but also elevated content of the unique end-product of this enzymatic activity, sphingosyl-phosphorylcholine (SPC or lyso-sphingomyelin) [104] (see Fig. 3). Additional studies later demonstrated a similar GlcCer deacylase activity in AD epidermis [105]. Since the responsible enzyme(s) has/have neither been cloned nor further identied, due in part to the low baseline expression in normal epidermis, it remains unclear whether this latter activity either represents a novel enzyme [105], a single enzyme with combined GlcCer and SM deacylase activities [106], or possibly is of bacterial origin. Despite this limitation, elevation of one-or-both of these novel enzyme activity(ies) could explain both the diminished Cer and sphingosine levels in the SC of AD patients [107] (Fig. 3, Step 2), and thus, contribute to the barrier abnormality, as well as other disease aspects. Indeed, sphingosyl-phosphorylcholine (SPC), coupled with decreased sphingosine could contribute to the increased proliferation, altered keratinization, inammation, and even increased tendency for secondary bacterial infections in AD [107109]. Interestingly, atopic SC was shown to be decient not only in free Cer species [7,8,99101], but also in covalently-bound x-hydroxyCer (corneocyte-bound lipid envelope) [9]. Signicant decreases in de novo synthesis of x-esteried Cer 4 (EOH), and Cer 3 (NP) also were noted in keratinocytes from lesional skin [9] (Fig. 3, Step 1). Thus, lesional AD epidermis contains signicant deciencies of key barrier Cer species that may contribute not only to permeability barrier alterations, but also to other functional defects (e.g., antimicrobial) in the skin of these patients. At least four ceramidase (CDase) activities are present in mammalian epidermis [110], with high alk-CDase activity reported in the SC [111]. Although no alteration in alk-CDase activity was noted in AD skin samples vs. age-matched controls [102,107], reduction of acid-CDase activity is evident [107]. Thus, the decreased sphingosine levels in involved AD skin [107] suggest that, in addition to elevations in the novel GlcCer/SM deacylase activities (Fig. 3, Step 2; see also above), reduced CDase activity(ies) also could underlie this abnormal-

ity (Fig. 3, Step 3). In addition, speculation continues regarding possible contributions of bacterial CDase both to normal epidermal function and to AD progression [112]. Although elevated bacterial colonization correlates with the severity of atopic skin lesions, an eect attributed to diminished content of the natural anti-bacterial sphingosine [107,113], the extent to which these changes in endogenous and/or exogenous CDase activity(ies) contribute to the pathogenesis of AD has yet to be resolved. Finally, contrary to the earlier studies that reported either no signicant alterations in SMase activity in AD skin [114], or increases in SMase protein levels by quantitative immunoblot analysis [115], reductions of both aSMase and nSMase activities have been noted in lesional AD skin [89] (Fig. 3, Step 4). In addition, the levels of prosaposin, the precursor for sphingolipid activator proteins, and/or saposin D itself, also appear to be diminished in psoriatic epidermis [88], suggesting yet another potential contributing factor to altered lipid processing in the epidermis of AD patients. Despite these contrasting ndings regarding enzyme activities that likely represent dierences in sampling and/or assay techniques, better understanding of the relative contributions of each of the above-mentioned alterations in lipid processing enzymes to the reduction in the content of specic epidermal Cer species, and the associated permeability barrier abnormality in AD [116,117], could reveal the mostappropriate therapeutic (non-immune-based) approaches to this debilitating dermatitis [118].

8. Altered lipid processing in psoriasis Not surprisingly, the severity of barrier defect in psoriatic skin is proportional to the disease phenotype. Although a number of studies suggest a role for altered lipid processing in psoriasis, whether these alterations are involved in the pathogenesis, or simply represent a consequence of this complex cutaneous disorder, remains unresolved. A number of reports cite altered Cer distribution and/or content in psoriatic epidermis. For example, Motta and colleagues reported decreased content of specic Cer species in psoriatic scale vs. normal SC; i.e., Cer 1 (EOS)1 (see structures in Fig. 1), as well as phytosphingosine-containing Cer species, Cer 3 (NP) and Cer 6 (AP), with simultaneous increases in other Cer species,

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5463 [2] Man, M.Q., Feingold, K.R. and Elias, P.M. (1993) Exogenous lipids inuence permeability barrier recovery in acetone-treated murine skin. Arch. Dermatol. 129, 728738. [3] Swartzendruber, D.C., Wertz, P.W., Kitko, D.J., Madison, K.C. and Downing, D.T. (1989) Molecular models of the intercellular lipid lamellae in mammalian stratum corneum. J. Invest. Dermatol. 92, 251257. [4] Elias, P.M. and Menon, G.K. (1991) Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv. Lipid Res. 24, 126. [5] Bouwstra, J.A., Cheng, K., Gooris, G.S., Weerheim, A. and Ponec, M. (1996) The role of ceramides 1 and 2 in the stratum corneum lipid organisation. Biochim. Biophys. Acta 1300, 177 186. [6] de Jager, M.W., Gooris, G.S., Ponec, M. and Bouwstra, J.A. (2005) Lipid mixtures prepared with well-dened synthetic ceramides closely mimic the unique stratum corneum lipid phase behavior. J. Lipid Res. 46, 26492656. [7] Imokawa, G., Abe, A., Jin, K., Higaki, Y., Kawashima, M. and Hidano, A. (1991) Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J. Invest. Dermatol. 96, 523526. [8] Melnik, B., Hollmann, J. and Plewig, G. (1988) Decreased stratum corneum ceramides in atopic individuals a pathobiochemical factor in xerosis? [letter]. Br. J. Dermatol. 119, 547549. [9] Macheleidt, O., Kaiser, H.W. and Sandho, K. (2002) Deciency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis. J. Invest. Dermatol. 119, 166173. [10] Motta, S., Sesana, S., Ghidoni, R. and Monti, M. (1995) Content of the dierent lipid classes in psoriatic scale. Arch. Dermatol. Res. 287, 691694. [11] Motta, S., Monti, M., Sesana, S., Caputo, R., Carelli, S. and Ghidoni, R. (1993) Ceramide composition of the psoriatic scale. Biochim. Biophys. Acta 1182, 147151. [12] Akiyama, M., Sugiyama-Nakagiri, Y., Sakai, K., McMillan, J.R., Goto, M., Arita, K., Tsuji-Abe, Y., Tabata, N., Matsuoka, K., Sasaki, R., Sawamura, D. and Shimizu, H. (2005) Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer. J. Clin. Invest. 115, 17771784. [13] Rassner, U., Feingold, K.R., Crumrine, D.A. and Elias, P.M. (1999) Coordinate assembly of lipids and enzyme proteins into epidermal lamellar bodies. Tissue Cell 31, 489498. [14] Wakita, H., Tokura, Y., Yagi, H., Nishimura, K., Furukawa, F. and Takigawa, M. (1994) Keratinocyte dierentiation is induced by cell-permeant ceramides and its proliferation is promoted by sphingosine. Arch. Dermatol. Res. 286, 350354. [15] Geilen, C.C., Wieder, T. and Orfanos, C.E. (1997) Ceramide signalling: regulatory role in cell proliferation, dierentiation and apoptosis in human epidermis. Arch. Dermatol. Res. 289, 559566. [16] Bektas, M., Dullin, Y., Wieder, T., Kolter, T., Sandho, K., Brossmer, R., Ihrig, P., Orfanos, C.E. and Geilen, C.C. (1998) Induction of apoptosis by synthetic ceramide analogues in the human keratinocyte cell line HaCaT. Exp. Dermatol. 7, 342 349. [17] Takami, Y., Abe, A., Matsuda, T., Shayman, J.A., Radin, N.S. and Walter, R.J. (1998) Eect of an inhibitor of glucosylceramide synthesis on cultured human keratinocytes. J. Dermatol. 25, 7377. [18] Uchida, Y., Murata, S., Schmuth, M., Behne, M.J., Lee, J.D., Ichikawa, S., Elias, P.M., Hirabayashi, Y. and Holleran, W.M. (2002) Glucosylceramide synthesis and synthase expression protect against ceramide-induced stress. J. Lipid Res. 43, 1293 1302. [19] Holleran, W.M., Gao, W.N., Feingold, K.R. and Elias, P.M. (1995) Localization of epidermal sphingolipid synthesis and serine palmitoyl transferase activity: alterations imposed by permeability barrier requirements. Arch. Dermatol. Res. 287, 254258. [20] Gray, G.M. and Yardley, H.J. (1975) Dierent populations of pig epidermal cells: isolation and lipid composition. J. Lipid Res. 16, 441447. [21] Elias, P.M., Brown, B.E., Fritsch, P., Goerke, J., Gray, G.M. and White, R.J. (1979) Localization and composition of lipids in

such that the total Cer content remained unchanged [11,119]. The contribution of altered lipid processing to these changes in lipid content has been explored, yielding contrasting results. An early report compared the in vitro activities of fourteen distinct acid hydrolases in epidermal keratome samples from eight psoriatic vs. 36 normal (non-diseased) patients [120, 121]. Signicant elevations in total b-glucosidase activity (i.e., 11-fold over mean control values) were noted [120]. Conversely, diminished GlcCerase mRNA has been reported in whole skin punch biopsies from lesional sites in ve patients with psoriasis vulgaris, when compared with skin from healthy, non-aected controls [122]. Immunohistochemical localization (using an antibody produced against a GST-fusion protein containing GlcCerase cloned from human skin) also revealed a corresponding decrease in GlcCerase protein in lesional psoriatic epidermis [122]. Interestingly, non-lesional skin also showed a signicant decrease in epidermal mRNA level and immunostaining for GlcCerase vs. normal control skin, with both of these parameters lower than in lesional skin. Decreased immunostaining for acid-SMase in SC from lesional vs. non-lesional psoriatic skin also has been reported [123]. These results are consistent with decreases in prosaposin mRNA and protein levels in psoriatic lesional skin [123]. Given that individual saposin proteins are co-activator proteins for both GlcCerase and SMase enzymes [84,124,125], as well as acid-CDase activity [126], and that prosaposin-decient animals retain GlcCer species in the SC associated with diminished permeability barrier function [78], altered GlcCer (and SM) lipid processing could contribute to disease expression in psoriasis.

9. Summary Studies into the metabolic activities localized within the mammalian SC are promoting continued reevaluation of the concept that this epidermal compartment is an inert accumulation of dead (enucleate) cells surrounded by static lipid-enriched domains. We now appreciate that the early bricks and mortar representation of the SC does not do full justice to the myriad biochemical and structural alterations required to subserve its numerous critical functions. The studies reviewed above reveal some of the biochemical complexities required for the normal formation and maturation of these protective lipid domains. However, the role that these key lipid changes play not only in orchestrating the normal corneocyte transition between the lower and outer layers, but also in disease pathogenesis and/or progression, remain highly fertile areas for future study. Additionally, dening the role(s) of lipid transport proteins, such as ABCA12, as determinants of epidermal lipid topology in both normal and diseased skin, represents an exciting new research area. Thus, the study of epidermal lipidomics, in conjunction with transgenic/knock-out approaches, will likely drive a continuing transformation of our concepts of the SC as a dynamic lipid and protein environs. References
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