Integrity and Barrier Function of the Epidermis Critically Depend on Glucosylceramide Synthesis
2006; Elsevier BV; Volume: 282; Issue: 5 Linguagem: Inglês
10.1074/jbc.m610304200
ISSN1083-351X
AutoresRichard Jennemann, Roger Sandhoff, Lutz Langbein, Sylvia Kaden, Ulrike Rothermel, Hichem Gallala, Konrad Sandhoff, Herbert Wiegandt, Hermann-Josef Gröne,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoCeramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains ω-hydroxy very long chain fatty acids (C30–C36). These ω-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular ω-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free ω-hydroxy ceramides increased 4-fold and ω-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier. Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains ω-hydroxy very long chain fatty acids (C30–C36). These ω-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular ω-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free ω-hydroxy ceramides increased 4-fold and ω-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier. Glucosylceramide synthase (Ugcg), catalyzing the initial step of glycosphingolipid synthesis (see Fig. 1A), is vital during embryogenesis as revealed by systemic deletion in mice (1Yamashita T. Wada R. Sasaki T. Deng C. Bierfreund U. Sandhoff K. Proia R.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9142-9147Crossref PubMed Scopus (399) Google Scholar). To investigate the different functions of glucosylceramide (GlcCer) 4The abbreviations used are: GlcCer, glucosylceramide; AS, sphingolipids containing α-hydroxy fatty acids (A) and sphingosine (S); EOS, linoleyl-ω-esters (E) of sphingolipids containing ω-hydroxy very long chain fatty acids (O) and sphingosine (S); ESI-MS/MS, nanoelectrospray ionization tandem mass spectrometry; LB, lamellar body; NS, sphingolipids containing non-hydroxy fatty acids (N) and sphingosine (S); OS, sphingolipids containing ω-hydroxy long chain fatty acids (O) and sphingosine (S); POS, sphingolipids containing protein-bound (P) ω-hydroxy long chain fatty acids (O) and sphingosine (S); SM, sphingomyelin. 4The abbreviations used are: GlcCer, glucosylceramide; AS, sphingolipids containing α-hydroxy fatty acids (A) and sphingosine (S); EOS, linoleyl-ω-esters (E) of sphingolipids containing ω-hydroxy very long chain fatty acids (O) and sphingosine (S); ESI-MS/MS, nanoelectrospray ionization tandem mass spectrometry; LB, lamellar body; NS, sphingolipids containing non-hydroxy fatty acids (N) and sphingosine (S); OS, sphingolipids containing ω-hydroxy long chain fatty acids (O) and sphingosine (S); POS, sphingolipids containing protein-bound (P) ω-hydroxy long chain fatty acids (O) and sphingosine (S); SM, sphingomyelin.-based glycolipids in vivo, cell-specific deletions of this enzyme are indispensable. Successful deletion of glucosylceramide synthase in neural cells led to loss of brain gangliosides. Nevertheless, mice were born developing severe dysfunctions postnatally (2Jennemann R. Sandhoff R. Wang S. Kiss E. Gretz N. Zuliani C. Martin-Villalba A. Jager R. Schorle H. Kenzelmann M. Bonrouhi M. Wiegandt H. Grone H.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12459-12464Crossref PubMed Scopus (149) Google Scholar, 3Yamashita T. Allende M.L. Kalkofen D.N. Werth N. Sandhoff K. Proia R.L. Genesis. 2005; 43: 175-180Crossref PubMed Scopus (35) Google Scholar). In this report we focus on the function of GlcCer in skin.Land dwelling animals have a water and electrolyte barrier in the skin to prevent dehydration and electrolyte disturbances. Both membrane proteins of keratinocytes and lipids generated in the epidermis are important for maintenance of the skin barrier and, therefore, for the impermeability of the skin to water. Ceramides (Cers) constitute a major component of the lipid barrier. They are synthesized by amidation of sphingoid bases with long chain fatty acids at the endoplasmic reticulum. Conversion into GlcCer by Ugcg takes place at the cytoplasmic surface of the Golgi apparatus (4Futerman A.H. Pagano R.E. Biochem. J. 1991; 280: 295-302Crossref PubMed Scopus (242) Google Scholar, 5Jeckel D. Karrenbauer A. Burger K.N. van Meer G. Wieland F. J. Cell Biol. 1992; 117: 259-267Crossref PubMed Scopus (255) Google Scholar).GlcCers are the dominant glycosphingolipids of the epidermis and constitute ∼4% of the total epidermal lipid mass (6Madison K.C. Wertz P.W. Strauss J.S. Downing D.T. J. Investig. Dermatol. 1986; 87: 253-259Abstract Full Text PDF PubMed Scopus (34) Google Scholar). They are thought to act as an intracellular carrier for secreted ceramides and constitute the main components of lamellar bodies (LBs) (7Doering T. Proia R.L. Sandhoff K. FEBS Lett. 1999; 447: 167-170Crossref PubMed Scopus (88) Google Scholar, 8Grayson S. Johnson-Winegar A.G. Wintroub B.U. Isseroff R.R. Epstein Jr., E.H. Elias P.M. J. Investig. Dermatol. 1985; 85: 289-294Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 9Holleran W.M. Takagi Y. Menon G.K. Legler G. Feingold K.R. Elias P.M. J. Clin. Investig. 1993; 91: 1656-1664Crossref PubMed Scopus (244) Google Scholar). LBs are found in cells of the upper stratum granulosum and are extruded into the extracellular cleft of the stratum corneum interstices by exocytosis (10Bouwstra J.A. Honeywell-Nguyen P.L. Gooris G.S. Ponec M. Prog. Lipid Res. 2003; 42: 1-36Crossref PubMed Scopus (471) Google Scholar, 11Madison K.C. J. Investig. Dermatol. 2003; 121: 231-241Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar) together with enzymes such as β-glucocerebrosidase. In the presence of its activator protein saposin C, β-glucocerebrosidase catalyzes the release of ceramides from GlcCer in the extracellular space (9Holleran W.M. Takagi Y. Menon G.K. Legler G. Feingold K.R. Elias P.M. J. Clin. Investig. 1993; 91: 1656-1664Crossref PubMed Scopus (244) Google Scholar, 12Doering T. Holleran W.M. Potratz A. Vielhaber G. Elias P.M. Suzuki K. Sandhoff K. J. Biol. Chem. 1999; 274: 11038-11045Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar).Besides Cers and GlcCers with a fatty acid chain length of 16–26 carbon atoms common to many cells, Cers and GlcCers with very long chain ω-hydroxy fatty acids containing up to 36 carbon atoms (OS-Cer and -GlcCer) form a major portion of Cer compounds in the epidermis. At the ω-position, many of the latter are additionally esterified with linoleic acid (EOS-Cer and -GlcCer) (6Madison K.C. Wertz P.W. Strauss J.S. Downing D.T. J. Investig. Dermatol. 1986; 87: 253-259Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 12Doering T. Holleran W.M. Potratz A. Vielhaber G. Elias P.M. Suzuki K. Sandhoff K. J. Biol. Chem. 1999; 274: 11038-11045Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar); these OS-GlcCers can be covalently linked to proteins in the cornified layer (POS-Cer and in trace amounts, POS-GlcCer) obviously by transesterification of EOS-GlcCer (12Doering T. Holleran W.M. Potratz A. Vielhaber G. Elias P.M. Suzuki K. Sandhoff K. J. Biol. Chem. 1999; 274: 11038-11045Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 13Schuette C.G. Doering T. Kolter T. Sandhoff K. Biol. Chem. 1999; 380: 759-766Crossref PubMed Scopus (27) Google Scholar) to glutamine-glutamate rich regions of involucrin, envoplakin, and periplakin (14Marekov L.N. Steinert P.M. J. Biol. Chem. 1998; 273: 17763-17770Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). EOS- and POS-Cers as well as their corresponding GlcCers seem to be important contributors to the extremely hydrophobic extracellular lipid lamellae of the stratum corneum (15Candi E. Schmidt R. Melino G. Nat. Rev. Mol. Cell Biol. 2005; 6: 328-340Crossref PubMed Scopus (1252) Google Scholar). It has not been conclusively shown whether indeed all sphingolipid components (Cers, GlcCers, and sphingomyelins) or merely one or two of them are needed for maintenance of the epidermal water barrier. A deficiency of β-glucocerebrosidase or a deficiency of its sphingolipid activator protein saposin C in epidermis led to strong reduction of ceramides concomitant with an accumulation of GlcCers including the protein-bound fraction, suggesting GlcCer as a precursor of Cer, impairing the skin barrier function (7Doering T. Proia R.L. Sandhoff K. FEBS Lett. 1999; 447: 167-170Crossref PubMed Scopus (88) Google Scholar, 12Doering T. Holleran W.M. Potratz A. Vielhaber G. Elias P.M. Suzuki K. Sandhoff K. J. Biol. Chem. 1999; 274: 11038-11045Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Whether glucosylceramide synthesis is obligatory for the lipid part of the skin barrier is not known. Therefore, a keratinocyte-specific deficiency of glucosylceramide synthase was generated.In the epidermis the genes expressing keratins K5 and K14 are expressed in basal cells (16Byrne C. Tainsky M. Fuchs E. Development. 1994; 120: 2369-2383Crossref PubMed Google Scholar, 17McGowan K.M. Coulombe P.A. J. Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (251) Google Scholar). Because of its early expression in the basal layer, the keratin K14 promoter seems optimal to express Cre recombinase to achieve epidermis-specific gene deficiencies (18Huelsken J. Vogel R. Erdmann B. Cotsarelis G. Birchmeier W. Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar, 19Indra A.K. Li M. Brocard J. Warot X. Bornert J.M. Gerard C. Messaddeq N. Chambon P. Metzger D. Horm. Res. (Basel). 2000; 54: 296-300Crossref PubMed Scopus (47) Google Scholar, 20Li M. Indra A.K. Warot X. Brocard J. Messaddeq N. Kato S. Metzger D. Chambon P. Nature. 2000; 407: 633-636Crossref PubMed Scopus (273) Google Scholar). K14-promoted Cre-mice were bred with mice with a loxP flanked glucosylceramide synthase gene (2Jennemann R. Sandhoff R. Wang S. Kiss E. Gretz N. Zuliani C. Martin-Villalba A. Jager R. Schorle H. Kenzelmann M. Bonrouhi M. Wiegandt H. Grone H.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12459-12464Crossref PubMed Scopus (149) Google Scholar) to obtain a blockade of the initial step of the GlcCer biosynthesis in epidermal keratinocytes.It is now shown that lack of GlcCer in the epidermis led to an irregular arrangement of the lamellar bodies in stratum granulosum cells and of extracellular lipids in the stratum corneum. Protein-bound ceramides were not reduced, and free and esterified ω-hydroxy sphingomyelins were synthesized as functionally insufficient substitutes of GlcCer. Our data document for the first time that an intact stratum corneum with an ordered lipid arrangement depending on GlcCer-formation is vital for skin barrier function.EXPERIMENTAL PROCEDURESTransgenic Animals—Mice with loxP-flanked exons 6–8 of the Ugcg gene locus were generated as described (2Jennemann R. Sandhoff R. Wang S. Kiss E. Gretz N. Zuliani C. Martin-Villalba A. Jager R. Schorle H. Kenzelmann M. Bonrouhi M. Wiegandt H. Grone H.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12459-12464Crossref PubMed Scopus (149) Google Scholar) (see Fig. 1B). Generation of mutant animals and experiments were performed according to federal laws for animal experiments and approved (Regierungspräsidium Karlsruhe, Germany).For the generation of mice with the Ugcg gene deletion in the epidermis, homozygous floxed mice were crossed with K14-constitutive Cre animals (18Huelsken J. Vogel R. Erdmann B. Cotsarelis G. Birchmeier W. Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar). In a second mating step, heterozygous floxed mice with the respective K14 promoted Cre transgene were mated again with homozygous floxed Ugcgflox/flox mice, resulting in epidermal cell-specific glycosylceramide synthase-deficient Ugcgflox/flox//K14Cre mice.Genotyping of Mutant Mice—PCR and Southern blot analysis of tail biopsies were performed for confirmation of the wild type, floxed, and null allele (see Fig. 1C). For PCR, the following primers were used: Ugcg wild type forward, 5′-GATCTA AGAGGGTGAAGGCGCA-3′; wild type/Ugcg-flox reversed, 5′-AAGCCAGTCCAGTCAAACCGAG-3′; Ugcg-null reversed, 5′-CTGCCTTGCAATCCTGTCTGTC-3′. PCR products of 259, 383, and 458 bp indicated the wild type, floxed, and null alleles (see Fig. 1C, upper).The constitutive K14Cre transgene was determined using the primers K14Cre forward, 5′-ATTGCCAGGATCAGGGTTAAAGA-3′, and K14Cre, reverse, 5′-CCCGGCAAAACAGGTAGTTATTC-3′. A 200-bp PCR product was indicative for the K14Cre transgene (see Fig. 1C, lower image).The data obtained from PCR were further confirmed by Southern blot analysis after BglII restriction digestion using a digoxigenin-labeled 3′ “outside” southern probe amplified according to the protocol of the DIG Probe Synthesis Kit and DIG Luminescent detection kit (Roche Applied Science).mRNA Isolation, Analysis, Profile—Skins were prepared from decapitated animals at different time points after birth. Adhering adipose tissue was removed. The epidermis was separated from the dermis after incubation with 0.5 m ammonium thiocyanate in 0.1 m potassium sodium hydrogen phosphate, pH 6.8, for 30 min on ice according to Diaz et al. (21Diaz L.A. Heaphy M.R. Calvanico N.J. Tomasi T.B. Jordon R.E. J. Investig. Dermatol. 1977; 68: 36-38Abstract Full Text PDF PubMed Scopus (26) Google Scholar).Total mRNA was extracted using the RNA easy kit (Qiagen GmbH, Hilden, Germany) as described by the manufacturer. Quantitative real-time reverse transcription-PCR was done using the LC-fast DNA Master SYBR Green I kit PCR for the LightCycler (Roche Applied Science) as described, and glyceraldehyde-3-phosphate dehydrogenase was used as reference gene (2Jennemann R. Sandhoff R. Wang S. Kiss E. Gretz N. Zuliani C. Martin-Villalba A. Jager R. Schorle H. Kenzelmann M. Bonrouhi M. Wiegandt H. Grone H.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12459-12464Crossref PubMed Scopus (149) Google Scholar).Lipid Extraction—Skins were trypsinized in 0.25% trypsin for 16 h at 4 °C. Epidermis was separated from dermis (22Mertens A.E. Rygiel T.P. Olivo C. van der Kammen R. Collard J.G. J. Cell Biol. 2005; 170: 1029-1037Crossref PubMed Scopus (196) Google Scholar). Tissue was lyophilized, and glycosphingolipids were extracted from epidermis essentially according to Doering et al. (12Doering T. Holleran W.M. Potratz A. Vielhaber G. Elias P.M. Suzuki K. Sandhoff K. J. Biol. Chem. 1999; 274: 11038-11045Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar).In brief, ∼5–10 mg of epidermis was extracted with 2 ml of chloroform/methanol/distilled water 30:60:8 by vol. The samples were treated under sonification at 50 °C for 15 min and subsequently centrifuged at 3000 rpm for 10 min. Supernatant was taken, and pellets were extracted two additional times as described using the solvent mixtures chloroform/methanol/distilled water, 10:10:1, and chloroform/methanol, 2:1 by vol. Supernatants were combined with the previous fractions. For further purification, an aliquot of the extract pool was further purified by saponification under mild alkaline conditions followed by desalting using reversed phase RP-18 columns of 200 μl of volume as described (23Jennemann R. Mennel H.D. Bauer B.L. Wiegandt H. Acta Neurochir. 1994; 126: 170-178Crossref PubMed Scopus (19) Google Scholar, 24Jennemann R. Rodden A. Bauer B.L. Mennel H.D. Wiegandt H. Cancer Res. 1990; 50: 7444-7449PubMed Google Scholar).For the extraction of protein-bound sphingolipids, pellets, which were obtained after extraction above, were treated 3 times with 2 ml of 100% methanol at room temperature for 10 min followed by two additional extraction steps with 2 ml of 95% methanol in H20 at 60 °C for 2 h to remove residual free lipids. The respective supernatants were taken after centrifugation as described, and the absence of free ceramides in the final supernatant was confirmed by nano-ESI-MS/MS analyses.From the residual pellet, protein-bound sphingolipids were cleaved after treatment with 1 ml of 1 m KOH in 95% methanol at 60 °C for 2 h. Supernatants were taken as described, neutralized with 1 m acetic acid, and dried and desalted using reversed phase RP-18 columns of 100 μl of volume as described (23Jennemann R. Mennel H.D. Bauer B.L. Wiegandt H. Acta Neurochir. 1994; 126: 170-178Crossref PubMed Scopus (19) Google Scholar).TLC running solvents used were chloroform/methanol/water 65:25:4 (see Fig. 2, A and B) and chloroform/methanol/glacial acetic acid 190:9:1 (used twice; see Fig. 2, C and E). The amounts spotted corresponded to extracts from 1 mg of dry epidermis. Visualization was performed with 0.2% orcinol in 10% sulfuric acid, 120 °C, 10 min (TLC, see Fig. 2A) and 10% CuSO4 in 8% H3PO4 at 180 °C for 10 min (TLC, see Fig. 2, B, C, and E).FIGURE 2Lipid analysis by TLC and ESI-MS/MS. A–C, TLC of crude epidermal extracts. GlcCer is almost completely depleted (A), and SM (B) is slightly altered already at the age P2 due to a shift to SM with very long chain fatty acids. Epidermal ceramides showed no significant alterations in TLC analysis (C). Chol, cholesterol. DAG, diacylglycerol. St., standards. D, quantification of total GlcCer in epidermal extracts at different time points by densitometry. Partial, however significant deletion of GlcCer could be seen at P0 (p < 0.01). Approximately 80% was deleted at P2 (p < 0.0009). Animals reached the highest degree of GlcCer deletion shortly before death (95%, p < 0.0002), n = 4). f, flox; +, wild type. E, no significant changes could be seen in the protein-bound lipids. PS, phosphocholine; FFA, free fatty acid. F–H, quantification of sphingolipids from epidermal extracts at P4 by nano-ESI-MS/MS. GlcCer, as already shown by TLC (A), was drastically reduced in mutant mice (p < 0.0009 for NS/AS and p < 0.03 for EOS; F). ω-Hydroxy long chain fatty acid ceramides (OS) accumulated significantly due to Ugcg-deficiency in the epidermis (p < 0.002; G), but no differences were observed for esterified (EOS) and protein-bound (POS). Lack of GlcCer was partly substituted by sphingomyelin for ω-hydroxy and linoleic acid-esterified very long chain fatty acids (SM-OS/EOS; p < 0.02 and p < 0.05; H). Values shown are the means of two different control and mutant animals (±S.E.).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantification of sphingolipids was performed by nano-ESI-MS/MS using a precursor ion scan of m/z +184 for sphingomyelin (collision energy, 35 eV) (25Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2339-2344Crossref PubMed Scopus (726) Google Scholar) and of m/z +264 for ceramide and GlcCer detection (collision energy, 50 eV) as described by Sandhoff (26Sandhoff R. Hepbildikler S.T. Jennemann R. Geyer R. Gieselmann V. Proia R.L. Wiegandt H. Grone H.J. J. Biol. Chem. 2002; 277: 20386-20398Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) with slight modifications: Starting with 35 V at m/z = 500, the cone voltage was increased in a linear way up to 65 V at m/z = 1300 during the measurement. For quantification of GlcCers, sphingomyelins (SMs), and Cers, the following internal standards were synthesized and mixed in an equimolar ratio according to Sandhoff et al. (26Sandhoff R. Hepbildikler S.T. Jennemann R. Geyer R. Gieselmann V. Proia R.L. Wiegandt H. Grone H.J. J. Biol. Chem. 2002; 277: 20386-20398Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar): Cer(d18:1,14:0), Cer(d18:1,19:0), Cer(d18:1,25:0), Cer(d18:1,31:0), and Cer(t18:0,31:0), GlcCer(d18:1,14:0), GlcCer(d18:1,19:0), GlcCer(d18:1,25:0), and Cer(d18:1,31:0), and SM(d18:1,14:0), SM(d18:1,25:0), and SM(d18:1,31:0).Linoleic acid-esterified sphingolipids were determined by subtraction of the amount of OS sphingolipids of the non-treated epidermal extracts from the respective amounts of OS sphingolipids evaluated in the saponified extracts (linoleic acid esterified-sphingolipid standards were not available). For determination of total GlcCer, mildly alkaline-treated epidermal extracts were quantified densitometrically using a Shimadzu CS-9301PC TLC-scanner (Shimadzu Europe, Duisburg, Germany).Skin Permeability Assay—Skin permeability was tested by Lucifer yellow diffusion essentially as described (27Herrmann T. van der Hoeven F. Grone H.J. Stewart A.F. Langbein L. Kaiser I. Liebisch G. Gosch I. Buchkremer F. Drobnik W. Schmitz G. Stremmel W. J. Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (158) Google Scholar, 28Matsuki M. Yamashita F. Ishida-Yamamoto A. Yamada K. Kinoshita C. Fushiki S. Ueda E. Morishima Y. Tabata K. Yasuno H. Hashida M. Iizuka H. Ikawa M. Okabe M. Kondoh G. Kinoshita T. Takeda J. Yamanishi K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1044-1049Crossref PubMed Scopus (248) Google Scholar). In brief, animals at P4 were sacrificed with CO2, briefly washed with 70% ethanol, and incubated in 1 mm Lucifer yellow (Sigma) dissolved in isotonic NaCl at 37 °C for 1 h. The solution was removed, and bodies were incubated for one additional hour at 37 °C in the dark. Subsequently, skin sections were mounted, and slices with a thickness of 5 μm were prepared. Nuclear counterstaining was performed using DRAQ5 (Alexis, San Diego, CA), and slices were investigated by confocal laser microscopy (Leica, Wetzlar, Germany).Determination of Transepidermal Water Loss—Transepidermal water loss was measured using a Tewameter® TM 300 (Courage-Khazaka Electronics, Cologne, Germany) as described (27Herrmann T. van der Hoeven F. Grone H.J. Stewart A.F. Langbein L. Kaiser I. Liebisch G. Gosch I. Buchkremer F. Drobnik W. Schmitz G. Stremmel W. J. Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (158) Google Scholar).Light Microscopy and Immunohistochemistry—Skin of snout, ear, and midline abdomen (two pieces of each tissue type) of Ugcgflox/flox//K14Cre mice and their respective controls were immersion-fixed at several time points with 4% phosphate-buffered formaldehyde or zinc buffer. Tissue was embedded in paraffin wax. Sections of 3-μm thickness were stained by hematoxylin (HE), periodic acid-Schiff (PAS), and Goldner trichrome. Tissues from same locations were immediately frozen in isopentane precooled with liquid nitrogen. Cryo-conserved skin sections (5 μm) were used for indirect immunofluorescence microscopy for membrane-associated proteins and cytoskeleton components (27Herrmann T. van der Hoeven F. Grone H.J. Stewart A.F. Langbein L. Kaiser I. Liebisch G. Gosch I. Buchkremer F. Drobnik W. Schmitz G. Stremmel W. J. Cell Biol. 2003; 161: 1105-1115Crossref PubMed Scopus (158) Google Scholar, 29Langbein L. Rogers M.A. Praetzel S. Winter H. Schweizer J. J. Investig. Dermatol. 2003; 120: 512-522Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).Primary antibodies were as follows: 1) guinea pig antibodies against keratins K5 (1:2000), K14 (1:3000), K17 (1:2000), K2e (1:2000), Ha5 (1:2000), Hb5 (1:1000), and K6hf (1:2000) as well as cingulin (1:5000) (all from PROGEN Biotechnik, Heidelberg, Germany) and desmoplakin (Dp495rb, 1:4000, German Cancer Research Center, Dept. of Cell Biology Heidelberg, Germany), 2) mouse antibodies against transglutaminase 1 (1:20, Cell Systems, St. Katharinen, Germany), occludin (1:300, Zytomed, Berlin, Germany), and keratin K16 (1:30, NatuTec, Frankfurt, Germany), and 3) rabbit antibodies against keratins K10 (1:500) and K6 (1:500, both from Covance, Berkeley, CA), K6 (1:1000, a gift of P. Coulombe, Dept. of Biological Chemistry, The Johns Hopkins University, Baltimore, MD), mK6irs1 (1:500, a gift of A. Aoki, Dept. of Dermatology, Niigata University, School of Medicine, Niigata, Japan), filaggrin (1:500, Covance, Berkeley, CA), loricrin (1:500, Covance, Berkeley, CA), claudin-1 (1:100, Neo Markers Labvision, Fremont, CA), ZO-1 (1:250, Zytomed, Berlin, Germany), and occludin (1:100, Zytomed, Berlin, Germany). The secondary antibodies used for indirect immunofluorescence were goat anti-guinea pig, anti-rabbit, or anti-mouse immunoglobulins coupled to Cy3 (1:300, Dianova, Hamburg, Germany) or Alexa 568 (1:200; Invitrogen).Furthermore, rat anti-mouse Ki67, (1:200, DAKO, Hamburg, Germany), rat anti-mouse HR3, (1:50, Acris, Hiddenhausen, Germany), and rat anti-mouse F4/80, (1:2000, Serotech, Düsseldorf, Germany) as well as a biotinylated secondary rabbit anti-rat antibodies (1:200) and alkaline phosphatase-labeled streptavidin, (1:200, Vector BA-4001, Vector Laboratories, Burlington, CA) were used for immunohistochemistry on paraffin-embedded tissue sections fixed either in paraformaldehyde or in zinc buffer. Terminal dUTP nick-end labeling was performed according to the instructions of the in situ cell death detection kit (Roche Applied Science).Electron Microscopy—Freshly dissected skin sections were fixed in 0.5% ruthenium tetroxide, 2.5% glutaraldehyde, 0.2 m cacodylate buffer pH 7.2, 1:1:1 v/v, and embedded in araldite (Serva, Heidelberg, Germany). For detection of lamellar bodies, tissues were fixed with 2.5% glutaraldehyde for 30 min followed by 0.5% ruthenium tetroxide (Polysciences, Eppelheim, Germany) for 1 h. Ultrathin sections were stained with lead citrate and uranyl acetate. Photographs were taken with a digital camera mounted on an electron microscope (Zeiss EM 9000, Carl Zeiss Inc. Germany).Statistics—All experiments were done at least twice, and statistics were calculated using Student's unpaired t test.RESULTSSkin-specific Glycosphingolipid Deletion in Mice—Offspring with the deleted Ugcg gene in the epidermis, Ugcgflox/flox//K14Cre (mutant), were born according to Mendelian inheritance constituting ∼25% of the litter, indicating no prenatal death.Southern blot analysis showed an approximately 70% deletion of the Ugcg gene locus in the epidermis of newborn mice at P0 (Fig. 1D). An almost complete deletion of the gene was observed at P4 (Fig. 1D), and minimal mRNA expression could be demonstrated using real time reverse transcription-PCR (Fig. 1E). Residual Ugcg DNA and mRNA might be explained by expression in non-keratinocyte cells in which the K14 promoter is not active, e.g. Langerhans cells.Glucosylceramide Was Deleted in the Epidermis of Ugcg-deficient Mice—DNA and mRNA levels of the glucosylceramide synthase were negligibly low in epidermis of mutant mice at P4 (Fig. 1, D and E). The enzyme product, GlcCer, was determined at different days after birth. The total GlcCer content in epidermis of control animals decreased approximately by 40% within the first 4 days after birth (Fig. 2, A and D, controls).Compared with controls, a significant decrease (42%) of GlcCer could be seen in the epidermis of animals with Ugcg gene deletion at P0, p < 0.01 (Fig. 2, A and D, P0). Two days after birth, epidermal GlcCer was reduced by 80% in Ugcg-deficient mice compared with their respective controls, p < 0.0009 (Fig. 2, A and D, P2). The highest degree of GlcCer reduction of ∼95%, which correlated well with very low DNA and mRNA levels of Ugcg, could be measured shortly before most animals died at P4 and P5, p < 0.0002 (Fig. 2, A and D, P4).Free (Fig. 2, B and C) and protein-bound epidermal sphingolipids (Fig. 2E) were analyzed by TLC. In the SM pattern a slight decrease of the slow migrating SM band could be seen in Ugcg-deficient epidermis at P2/P4 (Fig. 2B) that correlates with a 50% decrease of α-hydroxy palmitoyl containing sphingomyelin measured by mass spectrometry. In the ceramide pattern slight quantitative differences occurred (Fig. 2C). A TLC of the protein-bound epidermal lipids reflected no striking changes in lipid compositions between wild type and mutant mice (Fig. 2E).To analyze changes of sphingolipid subpopulations grouped according to their fa
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