J. Lipid Res. Acyl Labeled PIP's available August 1, 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Uchida, Y.
Right arrow Articles by Hamanaka, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Uchida, Y.
Right arrow Articles by Hamanaka, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Journal of Lipid Research, Vol. 41, 2071-2082, December 2000
Copyright © 2000 by Lipid Research, Inc.

Original Article

Epidermal sphingomyelins are precursors for selected stratum corneum ceramides

Yoshikazu Uchidaa,c, Mariko Harac, Hiroyuki Nishiod, Ellen Sidranskye, Shintaro Inouec, Fujio Otsukaf, Akemi Suzukig, Peter M. Eliasa, Walter M. Hollerana,b, and Sumiko Hamanakag
a Departments of Dermatology, University of California, San Francisco, CA 94143 and Dermatology Service and Research Unit, Department of Veterans Affairs Medical Center, San Francisco, CA 94121
b Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 and Dermatology Service and Research Unit, Department of Veterans Affairs Medical Center, San Francisco, CA 94121
c Basic Research Laboratory, Kanebo Ltd., Odawara, 250-0002 Japan
d Cosmetics Laboratory, Kanebo Ltd., Odawara, 250-0002 Japan
e Section on Molecular Neurogenetics, National Institute of Mental Health, Bethesda, MD 20892
f Department of Dermatology, University of Tsukuba, Tsukuba, 305-0006 Japan
g RIKEN Frontier Research System, Suprabiomolecular System Research Group, Sphingolipid Expression Laboratory, Wako 351-0198, Japan

Correspondence to: Walter M. Holleran


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Epidermal ceramides (Cer) comprise a heterogeneous family of seven species, including two unique {omega}-hydroxylated Cer, that are key components of the stratum corneum (SC) intercellular lamellar membranes responsible for the epidermal permeability barrier. Although both glucosylceramide (GlcCer) and the phospho-sphingolipid sphingomyelin (SM) are potential precursors of SC Cer, based on reported chemical structures of epidermal GlcCer and SC Cer, it is assumed that all major subfractions of SC Cer are generated from lamellar body-derived GlcCer. Yet, we and others have shown that SM-derived Cer are required for normal barrier homeostasis. Moreover, two pools of SM, one from plasma membrane, the other from lamellar body-derived contents, are potentially available for Cer production. To clarify the role of SM as a potential precursor of bulk or specific SC Cer, we compared Cer moieties in epidermal SM, Cer generated from epidermal SM by sphingomyelinase treatment, Cer within SC, and Cer that persist in Gaucher SC, where GlcCer cannot generate Cer due to an absence of ß-glucocerebrosidase. Using gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance for Cer characterization, epidermal SM comprise three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) compositions: that is, either long-chain FA (SM-1; C22;–26), short-chain FA (SM-2; primarily C16), and short-chain {alpha}-hydroxy FA (SM-3; C16;–18). In contrast, only trace quantities of {omega}-hydroxy FA were present. For each SM subfraction, the sphingoid base was either sphingosine or sphinganine, but phytosphingosine was not detected. Comparison of these SM with corresponding sphingomyelinase-generated epidermal Cer and SC Cer revealed that the Cer moieties of SM-1 and SM-3 are equivalent to Cer 2 (NS) and Cer 5 (AS), respectively. Moreover, both Cer 2 and Cer 5 occurred in Gaucher SC, whereas other Cer subfractions did not occur.

These results indicate that two epidermal SM, that is, SM-1 and SM-3, are important precursors of two corresponding Cer in mammalian SC, that is, Cer 2 and Cer 5, but other Cer species, including the {omega}-hydroxy Cer species, do not derive from SM. — Uchida, Y., M. Hara, H. Nishio, E. Sidransky, S. Inoue, F. Otsuka, A. Suzuki, P. M. Elias, W. M. Holleran, and S. Hamanaka. Epidermal sphingomyelins are precursors for selected stratum corneum ceramides. J. Lipid Res. 2000. 41: 2071;–2082.

Supplementary key words: glucosylceramide, epidermis, Gaucher disease


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

Ceramides (Cer) are biologically active lipid molecules associated with inhibition of cellular proliferation and induction of cellular differentiation and programmed cell death (1) (2). In the epidermis, a large, additional pool of secreted Cer, in combination with cholesterol and free fatty acids (FA), form extracellular lamellar membrane structures within the stratum corneum (SC) that subserve the permeability barrier to water loss, necessary for the survival of mammals in a terrestrial environment (3). These extracellular Cer comprise ~50% of the SC lipids (4) (5), and represent a heterogeneous family of at least seven molecules, with variations in the long-chain sphingoid base structure, as well as in the chain length and {alpha}-hydroxylation of constituent amide-linked FA (6) (7) (8). In addition, two of these seven Cer species, Cer 1 (EOS)2 and Cer 4 (EOH), also contain N-acylated {omega}-hydroxy FA ({omega}-OH Cer) that are unique to epidermis (6) (9) (10). These {omega}-OH Cer are not only key constituents of the extracellular lamellae, but also major components of the corneocyte lipid envelope (CLE), a covalently attached bilayer, bound to the external aspect of the cornified envelope (11) (12) (13) (13a).

Both non-OH FA-containing Cer (non-OH Cer) and {alpha}/{omega}-OH FA-containing Cer can be generated by ß-glucocerebrosidase-dependent hydrolysis of epidermal glucosylceramides (GlcCer) (14). Studies of patients with type 2 Gaucher disease, in which severe ß-glucocerebrosidase deficiency is a hallmark, as well as of ß-glucocerebrosidase-deficient null allele mice, demonstrate that conversion of GlcCer to Cer by ß-glucocerebrosidase is required for the formation of the highly organized SC membrane structures essential for normal barrier function (15) (16). Moreover, because of the similarity of the chemical structures of epidermal GlcCer, and the major SC Cer subfractions, it is generally assumed that Cer are generated from their corresponding, glucosylated lamellar body precursors (6) (8) (17) (18) (19) (20). Deglucosylation of the entire family of GlcCer occurs after the extrusion of lamellar body contents at the interface of the stratum granulosum (SG) and SC (3) (15).

However, some or all of the SC Cer could also originate from hydrolysis of the epidermal sphingophospholipid sphingomyelin (SM). SM is present in large quantities both in the plasma membrane of SG cells (21) (22), where it is degraded along with other phospholipids during terminal differentiation, and in the lipid-rich epidermal lamellar bodies (23) (24). Moreover, epidermis contains abundant sphingomyelinase, which is localized in the outer epidermis (25) (26) (27) (28) (29). Two studies demonstrate that normal barrier function requires sphingomyelinase activity either to generate a pool of SC Cer (30), and/or to remove the hydrophilic phosphorylcholine residue (31). Furthermore, the decreased Cer levels reported in SC of atopic dermatitis patients, which demonstrate a defective barrier (32) (33) (34), have been attributed to enhanced Cer degradation within epidermal SM due to increased activity of epidermal SM deacylase (35). Together, these studies suggest that SM-derived Cer are important for permeability barrier homeostasis.

Yet, whether SM is a precursor of bulk or specific Cer subfractions remains unresolved. Human, murine, and porcine epidermal SM contain Cer moieties with either hydroxylated or nonhydroxylated N-acyl FA in the SC, with a carbon chain length of C14;–C28 (36) (37). However, whether epidermal SM species also contain {omega}-OH-containing amide-linked FA [i.e., N-({omega}-OH)-acyl-sphingosylphosphorylcholine, or acylsphingomyelin (N-{omega}-O-linoleoyl)-acyl-sphingosyl-phosphorylcholine)] is not known. Moreover, the sphingoid base composition of epidermal SM has not been analyzed or compared with SC Cer. We first analyzed the Cer moieties in epidermal SM isolated from both hairless mouse and human epidermis. The resultant Cer structures generated by sphingomyelinase treatment of epidermal SM were then compared with those of Cer within the SC. Second, we assessed the composition of Cer in Gaucher epidermis. Our results indicate, first, that the Cer moieties of epidermal SM correspond to only two of the seven Cer in SC, suggesting a precursor-product relationship. In contrast, we show that epidermal SM do not contain {omega}-OH Cer moieties, indicating that these unique species are produced solely from GlcCer precursors.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Preparation of epidermal fractions and lipid extraction
Hairless male mice (Skh: hr-1) were purchased from Japan SLC (Hamamatsu, Japan). Six- to-20-week-old animals were used in this study. Gaucher mice were generated by targeted disruption of the murine ß-glucocerebrosidase gene, as previously described (38). Both neonatal ß-glucocerebrosidase null allele mice and normal wild-type littermates were used. Human skin was obtained after informed consent from the amputated tissues of 17- to-86 year-old male and female patients, with no prior history of cutaneous disorders. Human and murine epidermal sheets were obtained by incubation in a phosphate-buffered saline (PBS) solution containing 10 mM ethylenediaminetetraacetic acid or 0.1% trypsin (37°C for 30 min). SC was then isolated from the underlying nucleated layers of the epidermis by incubation with 0.5% trypsin in PBS, as previously described (39). Alternatively, the outer epidermal layers (i.e., rich in SC and stratum granulosum) were obtained after incubation with staphylococcal epidermolytic toxin (Toxin Technology, Sarasota, FL), as previously described (39). The remaining inner epidermal layers (i.e., rich in stratum spinosum and stratum basale) were then separated from the underlying dermis after incubation in PBS at 60°C for 30 sec.

Total lipids were isolated from epidermal or SC samples by the method of Bligh and Dyer (40), as previously described (41).

Thin-layer chromatography
The major epidermal and SC lipid species were separated first by high performance thin-layer chromatography (HPTLC; Merck, Darmstadt, Germany), as previously described (42). SM were separated from other polar lipids with chloroform;–methanol;–acetic acid;–water 50:30:8:4 (v/v) or chloroform;–methanol;–28% NH4OH 65:35:4 (v/v). GlcCer and Cer species were separated with chloroform;–methanol;–water 40:10:1 (v/v) to 2.0 cm, and then to 5.0 cm; chloroform;–methanol;–acetic acid 94:4:1.5 (v/v) to the top of the plate; and finally, n-hexane;–diethyl ether;–acetic acid 65:35:1 (v/v) to the top of the plate. Individual Cer species were separated by HPTLC with the following solvent sequence: 1) chloroform to 1.5 cm; 2) chloroform;–methanol;–acetone 76:16:8 (v/v) to 1.0 cm; 3) chloroform;–methanol;–hexyl acetate;–acetone 86:4:1:10 (v/v) to 7.0 cm; 4) chloroform;–methanol;–acetone 76:20:4 (v/v) to 2.0 cm; 5) chloroform;–methanol –diethyl ether;–ethylacetate;–hexyl acetate;–acetone 72:4:4:1:4:16 (v/v) to 7.5 cm; 6) n-hexane;–diethyl ether;–ethyl acetate 80:16:4 (v/v) to the top of the plate (43) (44); and finally, 7) n-hexane;–diethyl ether;–acetic acid 65:35:1 (v/v) to the top of the plate. Lipids were visualized after treatment with cupric acetate-phosphoric acid, and heating to 160°C for 15 min. The quantity of each lipid separated by TLC was determined by spectrodensitometry (CS-9000; Shimadzu, Kyoto, Japan), as previously described (8) (45).

Fractionation of sphingolipids by HPLC
Total lipid extracts were first applied to an aminopropyl silica gel column (Varian, Harbor City, CA) equilibrated with n-hexane. After washing the column with six volumes of n-hexane, the fraction containing Cer was eluted with chloroform;–isopropanol 2:1 (v/v). The SM-containing phospholipid fraction was then eluted with chloroform;–methanol 1:1 (v/v) and 1:2 (v/v), after washing of the column with diethyl ether containing 2% acetic acid. Fractions were monitored for individual SM or Cer by HPTLC, as described above. The combined SM fractions were further separated by high performance liquid chromatography (HPLC) by gradient elution, using an Iatrobeads column (6RSP-8010; 10.0 x 250 mm; Iatron Laboratories, Tokyo, Japan); that is, chloroform;–methanol;–water 83:16:0.5 (v/v) to 20:80:8 (v/v) over 210 min. Ceramides were further separated by HPLC by gradient elution, using combined Aquasil SS-1251 ((60); 4.6 x 250 mm; Senshu, Tokyo, Japan) and Develosil (100 Diol-5; 4.6 x 250 mm; Nomura Chemical, Seto, Japan) columns with n-hexane;–isopropanol 97:3 (v/v) to 80:20 (v/v) over 55 min. The lipid content of eluate samples was monitored by HPTLC, as described above, and samples were combined, as appropriate, for further analysis (see below).

Preparation of ceramides from SM by sphingomyelinase treatment
To further determine the Cer structures derived from both human and murine epidermal SM, the combined SM eluate fraction(s), obtained by HPLC separation, were treated with recombinant Bacillus cereus sphingomyelinase (Funakoshi, Tokyo, Japan) to remove the phosphorylcholine group, as described previously (46). Briefly, the total phospholipid fraction was suspended into a small volume of diethyl ether;–ethanol 98:2 (v/v) and incubated in 0.1 M Tris-HCl buffer (pH 7.2), containing 0.02 mM CaCl2 and sphingomyelinase (4.5 U/ml), for 3 h (30°C). Lipids were then extracted from the reaction mixture, as described above (40), and fractionated by HPTLC, as described above. Using standard amounts of SM (Sigma, St. Louis, MO), we determined that more than 90% of extracted SM was converted to free Cer species under these experimental conditions.

GC-MS analysis of amide-linked FA methyl esters and long-chain bases of ceramides
Fractions of both Cer isolated from SC, and Cer resulting from epidermal SM hydrolysis, were further methanolyzed to their corresponding FA methyl esters with aqueous methanolic HCl (70°C for 20 h) (47). The FA methyl esters were then extracted with n-hexane and silaylated for gas chromatography-mass spectrometry (GC-MS); that is, incubated with a mixture of 1,1,1,3,3,3-hexamethyldisilazane and trimethylchlorosilane in pyridine (3:1, v/v; GL Sciences, Tokyo, Japan). The long-chain bases also were recovered from the methanol phase after methanolysis, and likewise were converted to their corresponding O-trimethylsilyl derivatives. The derivatives of both FA methyl esters and long-chain bases were then analyzed by GC-MS (HP 5972; Hewlett-Packard, Palo Alto, CA) on a 30 cm x 0.32 µm DB-1 capillary column (J&W Scientific, Folsom, CA). For amide-linked FA analyses, the initial temperature was 100°C (5 min), with 5°C/min increases to 300°C; for long-chain base analyses, the starting temperature was 180°C (5 min), increasing at 5°C intervals per minute to 230°C.

1H NMR spectrometry analysis
1H NMR spectra were recorded in chloroform;–DL-methanol-d4 1:1 (v/v), using a 400-MHz nuclear magnetic resonance (NMR) spectrometer (JEOL-LA 400; JEOL, Tokyo, Japan).

Positive-ion fast atom bombardment-mass spectrometry
SM were further analyzed by fast atom bombardment (FAB)-MS (JMS-MS 303 mass spectrometer; JEOL), using the positive ion-mode with 2-nitrobenzylalcohol as the matrix.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

Epidermal SM of hairless mice
We first characterized the epidermal phospholipids, including SM species, obtained from isolated mouse epidermis. Epidermal phospholipids contained three HPTLC bands; that is, with Rf values of 0.19 (SM-3), 0.23 (SM-2), and 0.27 (SM-1) ( Fig 1A, lane 2) that correspond to authentic SM (from bovine brain; Fig 1A, lane 7). To confirm further that these three species were SMs, the whole phospholipid fraction was treated with bacterial sphingomyelinase to selectively remove the phosphorylcholine residue from all SM species, leaving other phospholipid fractions intact. Whereas the three SM components in the phospholipid fraction were hydrolyzed to three Cer species (i.e., Cer-A, Cer-B, and Cer-C; Fig 1B, lane 3), other phospholipid components [e.g., phosphatidylethanolamine (PE) and phosphatidylcholine (PC)] remained unaltered by sphingomyelinase treatment (Fig 1A, lane 3). The relative amounts of each fraction, as determined by spectrodensitometry, were as follows: murine SM-1 (mSM-1) and mCer-A: 25.7% and 27.1%, respectively; mSM-2 and mCer-B: 30.1% and 27.8%, respectively; and mSM-3 and mCer-C: 44.2% and 45.1%, respectively. These results show that murine epidermis contains three distinct SM species, with variations in HPTLC mobility due to variations in their respective Cer structures.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 1. Murine epidermal SM and Cer fractions. HPTLC separation of phospholipids (A) and ceramides (B) contained in lipid extracts from murine epidermis (see Materials and Methods for details). Lane 1 (A and B): standard GlcCer, {alpha}-OH ceramide ({alpha}-OH Cer), and non-OH ceramide (non-OH Cer) as indicated by arrows; lane 2 (A and B): epidermal phospholipid fraction, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and three major sphingomyelin species (i.e., SM-1 through SM-3), as indicated; note that the most polar band (i.e., below SM-3) is unknown, and is unaffected by sphingomyelinase or saponification treatments. Lane 3, lipid fractions remaining/obtained after sphingomyelinase: (A) shows loss of SM species, while PE and PC are unaffected by sphingomyelinase; (B) shows the corresponding resultant Cer species (Cer-A, Cer-B, and Cer-C). Lane 4 (A): saponified phospholipids, showing SM species to be resistant, while both PE and PC species are sensitive to saponification; lane 4 (B): resultant ceramide bands after treatment of standard SM (Sigma) with sphingomyelinase. Lane 5 (A): saponified phospholipid after sphingomyelinase treatment; lane 5 (B): standard SM. Lane 6 (A): lipid extract of the sphingomyelinase enzyme preparation. Lane 7 (A): standard SM.

To further define the Cer structures contained within murine epidermal SM, we next saponified these lipid species, and analyzed the amide-linked FA and long-chain bases by GC-MS. Isolated SMs were converted to their respective Cer species by sphingomyelinase (see Materials and Methods), and again fractionated into mCer-A, mCer-B, and mCer-C, corresponding to mSM-1, -2, and -3, respectively (Fig 1B, lane 3). The composition of the amide-linked FA revealed that both mCer-A (primarily C22 to C26) and mCer-B (primarily C16) contained non-OH FA as their amide-linked acyl residues ( Table 1). In contrast, a large proportion of the major N-acyl species of mCer-C (primarily C16) contained {alpha}-OH FA. In all three murine SM species, d18-sphingosine was the predominant long-chain base in the Cer moiety (i.e., 80;–90%), while phytosphingosine base was not detected ( Table 2). Because Cer 1 (acylCer) is the most hydrophobic epidermal Cer (6), acyl-SM, if present, also would be expected to be a highly hydrophobic SM species. However, neither more hydrophobic mSM species, nor significant quantities of {omega}-OH FA-containing molecules, were detected; that is, only a negligible amount (i.e., <0.4%) of the amide FA of SM-derived Cer-A was {omega}-hydroxylated (Table 1). These studies show that the Cer moieties in murine epidermal SMs are composed primarily of both non-OH- and {alpha}-OH-containing FA moieties that are amide linked to a sphingosine base.


 
View this table:
[in this window]
[in a new window]
  		Table 1. Amide-linked fatty acid composition of murine ceramide moieties of corresponding epidermal sphingomyelin


 
View this table:
[in this window]
[in a new window]
  		Table 2. Long-chain sphingoid base composition of the ceramide moieties of murine epidermal sphingomyelin

Human epidermal SM
We next examined the structures of SM species isolated from normal human epidermis. Similar to murine epidermis, three distinct SM bands, denoted hSM-1, hSM-2, and hSM-3, with Rf values (HPTLC) identical to those of murine sphingomyelins; that is, mSM-1, mSM-2, and mSM-3, respectively, were identified ( Fig 2). However, hSM-2, rather than hSM-3, was the predominant component of epidermal hSM (i.e., 45%) (Fig 2 and Table 3).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 2. Densitometric scan of both human (h) and murine (m) epidermal SM. Lipids were separated by HPTLC, visualized with cupric acetate reagent, and scanned by spectrodensitometry (see Materials and Methods for details). The respective species are labeled for both human (hSM) and murine (mSM) sphingomyelin, and demonstrate nearly identical Rf values for the three respective SM species from each.


 
View this table:
[in this window]
[in a new window]
  		Table 3. Composition of epidermal sphingomyelin of human and hairless mouse

To further determine the structures of the major human SM species, the three hSM bands were separated by HPLC, and analyzed separately both by 1H NMR ( Fig 3 and Table 4) and by FAB-MS ( Fig 4). As in mouse epidermis, hSM-1 and hSM-2 contained only non-OH FA (C16;–26), amide linked to sphingosine base. Moreover, the most hydrophobic species, hSM-3, again was an {alpha}-OH-containing, but not an {omega}-OH-containing, SM species. The predicted structures of the major murine and human epidermal SM are presented in Fig 5. These results show that the three major Cer moieties of human epidermal SMs are nearly identical to those in murine epidermis.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. 1H NMR spectra of human epidermal sphingomyelin. Major 1H NMR chemical shift peaks are shown and labeled (i.e., a;–n for hSM-1 and hSM-2; and a' ;– l' for hSM-3). Details for each corresponding peak are summarized in Table 4, using the identical nomenclature. (A) 1H NMR spectrum for hSM-1; (B) 1H NMR spectrum for hSM-2; (C) 1H NMR spectrum for hSM-3. Peak labeled with an asterisk (*) corresponds to the chemical shift for the solvent (CD3OH).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Positive-ion FAB mass spectra of human sphingomyelin (hSM) species. Mass specta for each of the three hSM species are shown. Individual ions with the largest relative abundance, with their respective N-acyl FA and sphingoid base composition (i.e., 24:0/18:1 for N-tetracosanoic acid with sphingosylphosphorylcholine, respectively), are delineated (arrows). (A) hSM-1; (B) hSM-2; (C) hSM-3.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Deduced structures of most abundant SM species from both murine and human epidermis. Shown are SM-1 (24:0/18:1), SM-2 (16:0/18:1), and SM-3 ({alpha}-OH 16:0/18:1) species.


 
View this table:
[in this window]
[in a new window]
  		Table 4. Summary of 1H NMR analysis of human epidermal sphingomyelin

Identity of epidermal SM and SC ceramides
A comparison of the Cer species in the SC with those released by sphingomyelinase from murine epidermal SM revealed that mCer-A and mCer-C (i.e., from murine SM-1 and SM-3) displayed the same HPTLC mobilities as mCer 2 and mCer 5, respectively, isolated from murine SC ( Fig 6). In contrast, no band corresponding to mCer-B (i.e., from epidermal mSM-2) was detected in the Cer isolated from murine SC. To further confirm that SM-1 and SM-3 are hydrolyzed to Cer 2 and Cer 5, respectively, the FA composition of both the amide-linked FA and the long-chain bases of these two mCer species were compared with these moieties in their respective epidermal mSMs (Table 5 and Table 6). mCer 2 contained long chain (>=C18), non-OH FA(s) amide linked with either d18 sphingosine or d18 sphinganine (Table 6), while {alpha}-OH palmitic acid (C16:0) was the major amide-linked FA of Cer 5 (Table 5 and Table 6). Thus, the FA and base composition of mCer 2 and 5 are similar to the FA composition of mSM-1 and mSM-3 (cf. Table 1 and Table 2), respectively, providing futher support for a precursor-product relationship.



View larger version (64K):
[in this window]
[in a new window]
  		Figure 6. Comparison of ceramides prepared from murine epidermal sphingomyelin with complete SC Cer species. Representative HPTLC of standard GlcCer, {alpha}-OH ceramide ({alpha}-OH Cer), and non-OH ceramide (non-OH Cer) is shown in lane 1. Ceramides obtained after treatment of murine epidermal phospholipid-enriched, Cer-free fraction with sphingomyelinase (see Materials and Methods) (lane 2); Cer from lipid extracts of hairless mouse SC (lane 3), showing the spectrum of Cer species present in vivo, including Cer-1 through Cer-7. Note the absence of Cer-B band; that is, corresponding to SM-2 in SC Cer fraction (lane 3).


 
View this table:
[in this window]
[in a new window]
  		Table 5. Composition of amide-linked fatty acid in ceramides 2 and 5 in murine stratum corneum


 
View this table:
[in this window]
[in a new window]
  		Table 6. Long-chain sphingoid base composition of ceramide generated from human epidermal sphingomyelin

Distinct distribution of SM-1, SM-2, and SM-3 generation within murine epidermis
As described above, only two of the three epidermal mSM species are reflected in corresponding Cer species in murine SC. During keratinocyte differentiation both GlcCer production (48) and heterogeneity increase; that is, at least seven distinct species are present in the mammalian SC (6) (7) (10) (49) (50). Therefore, we next examined whether the distinctive fate of SM-2 versus SM-1 and -3 is linked to epidermal differentiation. As shown in Table 7, the percentage of mSM-3, versus other sphingolipid species, increases in the outer (i.e., SG/SC) more than in the inner (i.e., stratum basale and stratum spinosum) epidermal fraction, consistent with increased production of SM-3 during keratinocyte differentiation. In contrast, the quantities of both SM-1 and SM-2, which are commonly encountered in the plasma membranes of most cells (51) (52), did not change with differentiation. As noted above, only SM-1- and SM-3-derived Cer are retained as SC Cer.


 
View this table:
[in this window]
[in a new window]
  		Table 7. Distribution of epidermal sphingomyelin in hairless mouse epidermis

Cer species formed in the SC of ß-glucocerebrosidase-deficient epidermis
GlcCer are considered the predominant precursor pool for the generation of SC Cer. To determine further whether GlcCer and SM are precursors of specific Cer species in the SC, we next examined the profile of epidermal Cer in Gaucher mouse epidermis, which accumulates GlcCer species in the SC because of a deficiency of ß-glucocerebrosidase (14) (15). As described previously (15) (53) (54), GlcCer, including acylGlcCer, again increase in Gaucher mouse SC, while each Cer species, excluding Cer 5, is decreased ( Fig 7, lane 2). Pertinently, the major Cer components present in Gaucher mouse epidermis were identical to those prepared by sphingomyelinase treatment of both Gaucher and normal mouse epidermal phospholipids (Fig 7, lane 2 vs. lanes 3 and 4). Again, two of these species corresponded with Cer 2 and Cer 5 of normal SC (Fig 7, lane 5). Because Gaucher mouse epidermis cannot generate Cer from GlcCer, these studies provide further evidence that Cer 2 and 5 in the SC are generated from SM precursors, by sphingomyelinase-dependent hydrolysis of SM-1 and SM-3, respectively.



View larger version (78K):
[in this window]
[in a new window]
  		Figure 7. Ceramides generated by sphingomyelinase treatment of ß-glucocerebrosidase-deficient (Gaucher) epidermal lipid extracts. Lane 2 contains Gaucher mouse epidermal lipid. Lanes 3 and 4 contains Cer obtained by sphingomyelinase treatment of either Gaucher (lane 3) or hairless (lane 4) mouse epidermal phospholipid-enriched, Cer-free fraction. Lane 1 contains standard GlcCer, {alpha}-OH Cer, and non-OH Cer, while lane 5 shows the spectrum of Cer species obtained from hairless mouse SC.


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

Cer are key components of the extracellular lamellar membrane structures of the SC, which provide the epidermal permeability barrier (3). Not only their bulk content, but also the molecular heterogeneity of these Cer (i.e., representing at least seven subfractions) (6) (7) (10) (49), appear to be critical for the formation of membrane bilayer structures, which subserve the barrier (55). Although these Cer appear to be generated primarily from GlcCer precursors by the activity of ß-glucocerebrosidase (14) (15), the contribution of one or more epidermal SM as precursors is not known. Studies from this (30) and other laboratories (31) demonstrate a requirement for bulk Cer generation from SM for normal permeability barrier homeostasis. However, the relative contribution of GlcCer and SMs to both total and individual SC Cer species remains unresolved. Specifically, which subfraction(s) of Cer is (are) generated from sphingomyelinase-mediated hydrolysis during terminal differentiation is not known. To address this question, we compared the chemical structures of Cer moieties in epidermal SMs (36) (37), isolated both from hairless mouse and human epidermis, with individual Cer species in SC, and with Cer generated by sphingomyelinase treatment of epidermal SM. In both human and hairless murine epidermis, SMs comprise three major subfractions, containing long-chain FA (SM-1), short-chain FA (SM-2), and short-chain {alpha}-OH FA (SM-3) in amide linkage(s) with either a sphingosine or sphinganine base. These results are consistent with a previous study of Cer in whole SENCAR mouse epidermis, in which a similar SM distribution was reported (37). A further comparison of the Cer moieties in individual epidermal SMs with SC Cer species described here revealed that two of the SM Cer moieties correlated structurally with Cer 2 (NS)1 and Cer 5 (AS) [using the current nomenclature (10) (49)]. These results suggest that only two of the three epidermal SM, that is, SM-1 and SM-3, are likely precursors of SC Cer; that is, Cer 2 and 5. The third epidermal SM, SM-2, although present in all epidermal nucleated cell layers, does not appear to generate Cer species in either mouse or human SC.

To further confirm the role of specific SM as precursors of SC Cer species, we also assessed Cer profiles in ß-glucocerebrosidase-deficient Gaucher mice (15) (38). Epidermal Cer in the Gaucher mouse again matched the pattern of the Cer moieties generated by sphingomyelinase hydrolysis of SM in normal mouse epidermis. This finding provides further evidence of the generation of non-OH Cer (i.e., Cer 2) and {alpha}-OH palmitic acid-containing Cer (Cer 5), from SM ( Fig 8). Similar Cer species have been noted in the epidermis of another Gaucher mouse model (54), and in the epidermis of the prosaposin (precursor of sphingolipid activator protein)-deficient mouse, in which the enzymatic hydrolysis of sphingolipids, particularly GlcCer, is decreased (56). In fact, the content of both the {alpha}-OH palmitic acid-containing Cer (i.e., corresponding to Cer 5) and epidermal SM actually increase in this latter model, suggesting that SM represents an obligate precursor and/or is increased as a possible compensatory response by this pathway. However, the fact that Cer-2 content decreases in both Gaucher and prosaposin-deficient mice suggests that not only SM, but also GlcCer, serve as precursors for this SC Cer species. Together, these results are consistent with SM-3 as a significant precursor for Cer 5, while either or both SM-1 and GlcCer contribute to the Cer 2 pool in normal SC (Fig 8).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 8. Proposed pathway for production of SC ceramides. Scheme for the production of the seven SC Cer species (Cer 1;–Cer 7) distinguishes between GlcCer- and sphingomyelin-dependent pathways, and is based on the results presented in this article. Abbreviations: PC, phosphatidylcholine; DG, diacylglycerol; UDP, uridine diphosphate.

SM is a common membrane component of all mammalian cells, where it is concentrated primarily in the outer leaflet of the plasma membrane (57) (58). SMs generally contain non-OH FA with a carbon chain length of C16;–24 (21) (37) (51), with palmitic acid (C16) as the predominant N-acyl FA component. Conversely, although SM containing {alpha}-OH FA have been detected in bovine stomach (59), guinea pig harderian gland (60), mammalian testes/spermatozoa (61), and murine epidermis (37), this relatively hydrophilic SM is not widely distributed in mammalian tissues. The present study now demonstrates the presence of significant quantities of C16 {alpha}-OH N-acyl FA-containing SM in both hairless mouse and human epidermis. In contrast to epidermal SM species, both epidermal GlcCer and SC Cer also contain very long-chain {alpha}-OH N-acyl FA (C>=24) (19) (62), further supporting divergent metabolic pathways for the generation of specific Cer from epidermal SM versus GlcCer precursors.

It is interesting to speculate that specific Cer species, destined for different functions within the epidermis, may be directed toward either SM or GlcCer precursor pools. Whereas {alpha}-hydroxylation of N-acyl FA in brain galactosylceramide(s) occurs prior to Cer synthesis (63), {alpha}-hydroxylation of (mannosylinositolphosphoryl)-Cer in Saccharomyces cerevisiae occurs after Cer synthesis (64). Thus, if {alpha}-OH Cer are synthesized after {alpha}-hydroxylation of the FA has occurred in mammalian epidermis, then the substrate preferences for SM synthase and/or GlcCer synthase could be restricted solely by selection for short- versus long-chain amide-linked FAs, respectively. Divergence of the pathways of SM and GlcCer synthesis is also implicit from their differing subcellular sites of synthesis: Cer glucosylation occurs on the cytosolic aspect of the ER and proximal Golgi, while SM synthesis is localized to the lumenal side (65), while trafficking of the precursor Cer for GlcCer versus SM appears distinct (66). Together, these studies suggest that the transport pathways for de novo-synthesized GlcCer versus SM may be dependent on their molecular structures; that is, the pathway for SM synthesis may select for short-chain, {alpha}-OH Cer with a sphingosine base, while that for GlcCer may select for long-chain, {alpha}-OH-containing or {omega}-OH-containing Cer, independent of base composition. Further studies will be required to clarify the structural determinants for de novo generation of SM and GlcCer within mammalian epidermis.

We also demonstrated that the ratio of individual SM to total SM in the outer versus inner epidermal layers is altered, and that production of SM-3 is enhanced during keratinocyte differentiation. The likely source of this SM-3 precursor pool is the contents of epidermal lamellar bodies, which are known to contain not only GlcCer, but also SM (23) (24). Lamellar bodies first appear in the spinous cell layer, and increase in number from the spinous layer through the SG. The contents of lamellar bodies are then extruded at the interface of the SG and SC, eventually forming the extracellular lamellar membrane system. Thus, it appears likely that SM-3 (but not SM-2) may be stored in lamellar bodies, rather than in plasma membranes, and that it is delivered to the extracellular spaces of SC concurrent with lamellar body secretion. Conversely, Cer 2 may derive both from lamellar body GlcCer precursors, and/or plasma membrane SM-1. In either case, the results of Jensen and colleagues (31) demonstrate a critical role for SM-derived Cer-2 in murine permeability barrier function, as Cer 2 may account for as much as 40% to 50% of murine SC Cer. Whereas the fate of SM-2 remains unresolved, these shorter N-acyl FA-containing Cer (i.e., C16:0) do not contribute significantly to SC Cer composition. Perhaps these short-chain Cer species are involved in as yet unresolved signaling pathways, or they could be degraded during the latter stages of epidermal differentiation.

Although the functional role of each SC Cer species has not yet been resolved, Cer species appear to be critical for SC membrane structure and function. For example, only {omega}-OH-containing Cer species are covalently attached to cornified envelope proteins, underscoring their putative role in lamellar membrane organization. In addition, one of the {omega}-acylated Cer species (Cer 1) has been shown to be an essential component in the formation of the broad-narrow-broad sequence of the lamellar membrane unit structure (67). The present study supports the hypothesis that the majority of SC Cer 1 is derived from acylGlcCer hydrolysis, rather than from SM precursors (15) (68) (69). This finding also may explain the fact that significant permeability barrier dysfunction and abnormal SC lamellar structures occur in Gaucher type 2 patients (16), while in contrast, patients with Niemann-Pick disease display lesser barrier abnormalities (30). Because critical SC Cer, such as Cer 1, appear only to be generated from GlcCer precursors, alteration of this pathway may have more severe cutaneous consequences. Moreover, the {alpha}-OH-containing Cer (Cer 5), derived all, or at least in part, from the SM-3 precursor pool, is predicted to be localized to the narrow, electron-dense region in the lamellar phase (67), suggesting a role in membrane organization. Interestingly, although the Cer composition of human and mouse SC lipids exhibits certain differences [e.g., Cer 5 comprises approximately 20% of the total Cer in the hairless mouse SC, but only 5.7% of the total Cer in human SC (data not shown)], their lamellar membrane structures are strikingly similar. In addition, because Cer 5 can be partially replaced by cholesterol in lamellar structures in vitro (70), this Cer species may lack an obligate role in cutaneous function. Finally, as SM-2 consists primarily of a sphingosine base with a non-OH C16:0 N-acyl substituent, the structure resembles "normal" SM observed in the majority of mammalian cells, often localized to the plasma membrane (21) (37) (51) (57) (58). We would propose that this SM species is involved with cell membrane functions that are not unique to the epidermis. For example, it is possible that SM-2 is involved in signal transduction events induced by the activation of SMase activity. In this regard, one study demonstrated that C16:0 Cer is specifically increased during Fas, or radiation-induced apoptosis in Jurkat cells (71). Conversely, Jensen and colleagues (31) found no change in C16 Cer during the early phase of epidermal barrier repair. Because limited content of shorter chain, saturated, non-{alpha}-OH N-acyl Cer species (i.e., C16:0) is present in mammalian SC [ (6) (7) (8) (9) (10) (49) (50), and the present study], only the longer chain, non-OH N-acyl-containing SM species are utilized for generating SC Cer species that appear to be important for permeability barrier function. Together, these results suggest that certain Cer species, including those derived from SM hydrolysis, have specific structural roles within mammalian SC.

In summary, both SM and GlcCer play roles in the generation of Cer that are critical for epidermal barrier function. The present results indicate, first, that two epidermal SM (i.e., SM-1 and SM-3) are important precursors of Cer 2 and Cer 5, respectively, in the SC. However, the precise contribution of SM versus GlcCer to the content of these two Cer species in normal and diseased SC will require further investigation. Second, our results demonstrate that {omega}-OH Cer species in mammalian SC do not derive from SM, indicating that these unique species derive solely from GlcCer precursors.


  FOOTNOTES

2 Abbreviations for Cer structures are according to Motta et al. (50) and Robson et al. (10): EOS (Cer 1), esterified {omega}-hydroxy (OH) FA with sphingosine base; NA (Cer 2), non-OH FA, sphingosine; NP (Cer 3), non-OH FA, phytosphingosine; EOH (Cer 4), esterified {omega}-OH FA, 6-OH-sphingosine; AS (Cer 5), {alpha}-OH FA, sphingosine; AP (Cer 6), {alpha}-OH FA, phytosphingosine; AH (Cer 7), {alpha}-OH FA, 6-hydroxysphingosine. Back


  ACKNOWLEDGMENTS

We thank Mr. Takeshi Ikemoto (Cosmetics Laboratory, Kanebo) for technical advice in gas chromatography-mass spectrometry analysis, and Ms. Sue Allen for assistance with manuscript preparation. This work was supported by National Institutes of Health grants AR 39448 and AR19098.

Manuscript received April 7, 2000; and in revised form July 24, 2000

Abbreviations: Cer, ceramide; FA, fatty acid; GlcCer, glucosylceramide; HPLC, high performance liquid chromatography; HPTLC, high performance thin-layer chromatography; non-OH, nonhydroxy; {alpha}-OH, {alpha}-hydroxy; {omega}-OH, {omega}-hydroxy; SC, stratum corneum; SM, sphingomyelin


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

  1. Kolesnick, R. N., Krönke, M. 1998. Regulation of ceramide production and apoptosis. Annu. Rev. Physiol. 60:643-665[Medline].

  2. Perry, S. W., Hamilton, J. A., Tjoelker, L. W., Dbaibo, G., Dzenko, K. A., Epstein, L. G., Hannun, Y., Whittaker, J. S., Dewhurst, S., Gelbard, H. A. 1998. Platelet-activating factor receptor activation. An initiator step in HIV-1 neuropathogenesis. J. Biol. Chem. 273:17660-17664[Abstract/Free Full Text].

  3. Elias, P. M., Menon, G. K. 1991. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv. Lipid Res. 24:1-26[Medline].

  4. Gray, G. M., Yardley, H. J. 1975. Different populations of pig epidermal cells: isolation and lipid composition. J. Lipid Res. 16:441-447[Abstract].

  5. Schoephoerster, R. T., Wertz, P. W., Madison, K. C., Downing, D. T. 1985. A survey of polar and non-polar lipids of mouse organs. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 82:229-232.

  6. Wertz, P. W., Downing, D. T. 1983. Ceramides of pig epidermis: structure determination. J. Lipid Res. 24:759-765[Abstract].

  7. Wertz, P. W., Miethke, M. C., Long, S. A., Strauss, J. S., Downing, D. T. 1985. The composition of the ceramides from human stratum corneum and from comedones. J. Invest. Dermatol. 84:410-412[Medline].

  8. Uchida, Y., Iwamori, M., Nagai, Y. 1988. Distinct differences in lipid composition between epidermis and dermis from footpad and dorsal skin of guinea pigs. Jpn. J. Exp. Med. 58:153-161[Medline].

  9. Bowser, P. A., Nugteren, D. H., White, R. J., Houtsmuller, U. M., Prottey, C. 1985. Identification, isolation and characterization of epidermal lipids containing linoleic acid. Biochim. Biophys. Acta. 834:419-428[Medline].

  10. Robson, K. J., Stewart, M. E., Michelsen, S., Lazo, N. D., Downing, D. T. 1994. 6-Hydroxy-4-sphingenine in human epidermal ceramides. J. Lipid Res. 35:2060-2068[Abstract].

  11. Wertz, P. W., Downing, D. T. 1987. Covalently bound omega-hydroxyacylsphingosine in the stratum corneum. Biochim. Biophys. Acta. 917:108-111[Medline].

  12. Wertz, P. W., Madison, K. C., Downing, D. T. 1989. Covalently bound lipids of human stratum corneum. J. Invest. Dermatol. 92:109-111[Medline].

  13. Marekov, L. N., Steinert, P. M. 1998. Ceramides are bound to structural proteins of the human foreskin epidermal cornified cell envelope. J. Biol. Chem. 273:17763-17770[Abstract/Free Full Text].

  14. Elias, P. M., Fartasch, M., Crumrine, D., Behne, M., Uchida, Y., Holleran, W. M. 2000. Origin of the corneocyte lipid envelope (CLE): observations in Harlequin Ichthyosis and cultured human keratinocytes. J. Invest. Dermatol. 115:765-769[Medline].

  15. Holleran, W. M., Takagi, Y., Menon, G. K., Legler, G., Feingold, K. R., Elias, P. M. 1993. Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function. J. Clin. Invest. 91:1656-1664.

  16. Holleran, W. M., Ginns, E. I., Menon, G. K., Grundmann, J. U., Fartasch, M., McKinney, C. E., Elias, P. M., Sidransky, E. 1994. Consequences of beta-glucocerebrosidase deficiency in epidermis. Ultrastructure and permeability barrier alterations in Gaucher disease. J. Clin. Invest. 93:1756-1764.

  17. Sidransky, E., Fartasch, M., Lee, R. E., Metlay, L. A., Abella, S., Zimran, A., Gao, W., Elias, P. M., Ginns, E. I., Holleran, W. M. 1996. Epidermal abnormalities may distinguish type 2 from type 1 and type 3 of Gaucher disease. Pediatr. Res. 39:134-141[Medline].

  18. Wertz, P. W., Downing, D. T. 1983. Acylglucosylceramides of pig epidermis: structure determination. J. Lipid Res. 24:753-758[Abstract].

  19. Wertz, P. W., Downing, D. T. 1982. Glycolipids in mammalian epidermis: structure and function in the water barrier. Science. 217:1261-1262[Abstract/Free Full Text].

  20. Hamanaka, S., Takemoto, T., Hamanaka, Y., Asagami, C., Suzuki, M., Suzuki, A., Otsuka, F. 1993. Structure determination of glycosphingolipids of cultured human keratinocytes. Biochim. Biophys. Acta. 1167:1-8[Medline].

  21. Hamanaka, S., Asagami, C., Suzuki, M., Inagaki, F., Suzuki, A. 1989. Structure determination of glucosyl beta 1-N-(omega-O-linoleoyl)-acylsphingosines of human epidermis. J. Biochem. (Tokyo). 105:684-690[Abstract/Free Full Text].

  22. Gray, G. M., King, I. A., Yardley, H. J. 1978. The plasma membrane of granular cells from pig epidermis: isolation and lipid and protein composition. J. Invest. Dermatol. 71:131-135[Medline].

  23. Gray, G. M., King, I. A., Yardley, H. J. 1980. The plasma membrane of Malpighian cells from pig epidermis: isolation and lipid and protein composition. Br. J. Dermatol. 103:505-515[Medline].

  24. Grayson, S., Johnson-Winegar, A. G., Wintroub, B. U., Isseroff, R. R., Epstein, E. H., Jr., Elias, P. M. 1985. Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization. J. Invest. Dermatol. 85:289-294[Medline].

  25. Freinkel, R. K., Traczyk, T. N. 1985. Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis. J. Invest. Dermatol. 85:295-298[Medline].

  26. Bowser, P. A., Gray, G. M. 1978. Sphingomyelinase in pig and human epidermis. J. Invest. Dermatol. 70:331-335[Medline].

  27. Menon, G. K., Grayson, S., Elias, P. M. 1986. Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis. J. Invest. Dermatol. 86:591-597[Medline].

  28. Yamamura, T., Tezuka, T. 1990. Change in sphingomyelinase activity in human epidermis during aging. J. Dermatol. Sci. 1:79-83[Medline].

  29. Kusuda, S., Chang-Yi, C., Takahashi, M., Tezuka, T. 1998. Localization of sphingomyelinase in lesional skin of atopic dermatitis patients. J. Invest. Dermatol. 111:733-738[Medline].

  30. Rassner, U., Feingold, K. R., Crumrine, D. A., Elias, P. M. 1999. Coordinate assembly of lipids and enzyme proteins into epidermal lamellar bodies. Tissue Cell. 31:489-498[Medline].

  31. Schmuth, M., M-Q. Man, F., Weber, W., Gao, K., Feingold, P., Fritsch, P. M., Elias,, Holleran, W. M. 2000. Permeability barrier disorder in Niemann-Pick disease: sphingomyelin-ceramide processing required for normal barrier homeostasis. J. Invest. Dermatol. 115:459-466[Medline].

  32. Jensen, J. M., Schütze, S., Förl, M., Krönke, M., Proksch, E. 1999. Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier. J. Clin. Invest. 104:1761-1770[Medline].

  33. Melnik, B., Hollmann, J., Plewig, G. 1988. Decreased stratum corneum ceramides in atopic individuals — a pathobiochemical factor in xerosis? Br. J. Dermatol. 119:547-549. [letter][Medline].

  34. Yamamoto, A., Serizawa, S., Ito, M., Sato, Y. 1991. Stratum corneum lipid abnormalities in atopic dermatitis. Arch. Dermatol. Res. 283:219-223[Medline].

  35. Imokawa, G., Abe, A., Jin, K., Higaki, Y., Kawashima, M., Hidano, A. 1991. Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J. Invest. Dermatol. 96:523-526[Medline].

  36. Murata, Y., Ogata, J., Higaki, Y., Kawashima, M., Yada, Y., Higuchi, K., Tsuchiya, T., Kawainami, S., Imokawa, G. 1996. Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency? J. Invest. Dermatol. 106:1242-1249[Medline].

  37. Gray, G. M., Yardley, H. J. 1975. Lipid compositions of cells isolated from pig, human, and rat epidermis. J. Lipid Res. 16:434-440[Abstract].

  38. Kitano, Y., Iwamori, Y., Kiguchi, K., DiGiovanni, J., Takahashi, T., Kasama, K., Niwa, T., Harii, K., Iwamori, M. 1996. Selective reduction in alpha-hydroxypalmitic acid-containing sphingomyelin and concurrent increase in hydroxylated ceramides in murine skin tumors induced by an initiation-promotion regimen. Jpn. J. Cancer Res. 87:437-441[Medline].

  39. Tybulewicz, V. L., Tremblay, M. L., LaMarca, M. E., Willemsen, R., Stubblefield, B. K., Winfield, S., Zablocka, B., Sidransky, E., Martin, B. M., Huang, S. P., Mintzer, S. P., Westphal, K. A., Mulligan, H., Ginns, R. C. 1992. Animal model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase gene. Nature. 357:407-410[Medline].

  40. Hara, M., Uchida, Y., Haratake, A., Mimura, K., Hamanaka, S. 1998. Galactocerebroside and not glucocerebroside or ceramide stimulate epidermal beta-glucocerebrosidase activity. J. Dermatol. Sci. 16:111-119[Medline].

  41. Bligh, E. G., Dyer, W. J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.

  42. Holleran, W. M., Man, M. Q., Gao, W. N., Menon, G. K., Elias, P. M., Feingold, K. R. 1991. Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation. J. Clin. Invest. 88:1338-1345.

  43. Holleran, W. M., Uchida, Y., Halkier-Sorensen, L., Haratake, A., Hara, M., Epstein, J. H., Elias, P. M. 1997. Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function. Photodermatol. Photoimmunol. Photomed. 13:117-128[Medline].

  44. Ponec, M., Weerheim, A. 1990. Retinoids and lipid changes in keratinocytes. Methods Enzymol. 190:30-41[Medline].

  45. Ponec, M., Weerheim, A., Kempenaar, J., Mulder, A., Gooris, G. S., Bouwstra, J., Mommaas, A. M. 1997. The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C. J. Invest. Dermatol. 109:348-355[Medline].

  46. Uchida, Y., Iwamori, M., Nagai, Y. 1990. Activation of keratinization of keratinocytes from fetal rat skin with N-(O-linoleoyl) omega-hydroxy fatty acyl sphingosyl glucose (lipokeratinogenoside) as a marker of epidermis. Biochem. Biophys. Res. Commun. 170:162-168[Medline].

  47. Stahl, W. L. 1973. Phospholipase C purification and specificity with respect to individual phospholipids and brain microsomal membrane phospholipids. Arch. Biochem. Biophys. 154:47-55[Medline].

  48. Karlsson, K. A., Mårtensson, E. 1968. Studies on sphingosines. XIV. On the phytosphingosine content of the major human kidney glycolipids. Biochim. Biophys. Acta. 152:230-233[Medline].

  49. Sando, G. N., Howard, E. J., Madison, K. C. 1996. Induction of ceramide glucosyltransferase activity in cultured human keratinocytes. Correlation with culture differentiation. J. Biol. Chem. 271:22044-22051[Abstract/Free Full Text].

  50. Motta, S., Sesana, S., Ghidoni, R., Monti, M. 1995. Content of the different lipid classes in psoriatic scale. Arch. Dermatol. Res. 287:691-694[Medline].

  51. Motta, S., Monti, M., Sesana, S., Caputo, R., Carelli, S., Ghidoni, R. 1993. Ceramide composition of the psoriatic scale. Biochim. Biophys. Acta. 1182:147-151[Medline].

  52. Barenholz, Y., Thompson, T. E. 1980. Sphingomyelins in bilayers and biological membranes. Biochim. Biophys. Acta. 604:129-158[Medline].

  53. Barenholz, Y., and S. Gatt. 1982. Sphingomyelin: metabolism, chemical synthesis, chemical and physical properties. In Phospholipids. J. N. Hawthorne and G. B. Ansell, editors. Elsevier Biomedical, Amsterdam. 129–177.

  54. Uchida, Y., Sidransky, E., Ginns, E. I., Elias, P. M., Holleran, W. M. 1999. Formation of the lipid-bound envelope (LBE): insights from glucocerebrosidase-deficient Gaucher mouse epidermis. J. Invest. Dermatol. 112:543a.

  55. Doering, T., Proia, R. L., Sandhoff, K. 1999. Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice. FEBS Lett. 447:167-170[Medline].

  56. Bouwstra, J. A., Cheng, K., Gooris, G. S., Weerheim, A., Ponec, M. 1996. The role of ceramides 1 and 2 in the stratum corneum lipid organisation. Biochim. Biophys. Acta. 1300:177-186[Medline].

  57. Doering, T., Holleran, W. M., Potratz, A., Vielhaber, G., Elias, P. M., Suzuki, K., Sandhoff, K. 1999. Sphingolipid activator proteins are required for epidermal permeability barrier formation. J. Biol. Chem. 274:11038-11045[Abstract/Free Full Text].

  58. Allan, D., Quinn, P. 1988. Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with. Staphylococcus aureus sphingomyelinase.(Biochem. J. 254):765-771.

  59. Slotte, J. P., Bierman, E. L. 1988. Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts. Biochem. J. 250:653-658[Medline].

  60. Breimer, M. E., Karlsson, K. A., Samuelsson, B. E. 1974. The distribution of molecular species of monoglycosylceramides (cerebrosides) in different parts of bovine digestive tract. Biochim. Biophys. Acta. 348:232-240[Medline].

  61. Yasugi, E., Kasama, T., Shibahara, M., Seyama, Y. 1990. Composition of long-chain bases in sphingomyelin of the guinea pig Harderian gland. Biochem. Cell Biol. 68:154-160[Medline].

  62. Robinson, B. S., Johnson, D. W., Poulos, A. 1992. Novel molecular species of sphingomyelin containing 2-hydroxylated polyenoic very-long-chain fatty acids in mammalian testes and spermatozoa. J. Biol. Chem. 267:1746-1751[Abstract/Free Full Text].

  63. Wertz, P. W., Downing, D. T. 1983. Glucosylceramides of pig epidermis: structure determination. J. Lipid Res. 24:1135-1139[Abstract].

  64. Hoshi, M., Kishimoto, Y. 1973. Synthesis of cerebronic acid from lignoceric acid by rat brain preparation. Some properties and distribution of the {alpha}-hydroxylation system. J. Biol. Chem. 248:4123-4130[Abstract/Free Full Text].

  65. Haak, D., Gable, K., Beeler, T., Dunn, T. 1997. Hydroxylation of. Saccharomyces cerevisiae ceramides(requires Sur2p and Scs7p. J. Biol. Chem. 272):29704-29710.

  66. Ichikawa, S., Hirabayashi, Y. 1998. Glucosylceramide synthase and glycosphingolipid synthesis. Trends Cell Biol. 8:198-202[Medline].

  67. Fukasawa, M., Nishijima, M., Hanada, K. 1999. Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells. J. Cell Biol. 144:673-685[Abstract/Free Full Text].

  68. Bouwstra, J. A., Gooris, G. S., Dubbelaar, F. E., Weerheim, A. M., Ijzerman, A. P., Ponec, M. 1998. Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J. Lipid Res. 39:186-196[Abstract/Free Full Text].

  69. Wertz, P. W., Downing, D. T., Freinkel, R. K., Traczyk, T. N. 1984. Sphingolipids of the stratum corneum and lamellar granules of fetal rat epidermis. J. Invest. Dermatol. 83:193-195[Medline].

  70. Wertz, P. W., Downing, D. T. 1989. Beta-glucosidase activity in porcine epidermis. Biochim. Biophys. Acta. 1001:115-119[Medline].

  71. Bouwstra, J. A., Gooris, G. S., Cheng, K., Weerheim, A., Bras, W., Ponec, M. 1996. Phase behavior of isolated skin lipids. J. Lipid Res. 37:999-1011[Abstract].

  72. Thomas, R. L., Jr., Matsko, C. M., Lotze, M. T., Amoscato, A. A. 1999. Mass spectrometric identification of increased C16 ceramide levels during apoptosis. J. Biol. Chem. 274:30580-30588[Abstract/Free Full Text].


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
P. M. Elias, M. L. Williams, W. M. Holleran, Y. J. Jian