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TNF receptor p55 signaling and ceramides generated by sphingomyelinases in cutaneous barrier repair
ESDR I JSID I SID Abstracts
s34
0199
0202
CHARACTERIZATION OF THE PROTEIN STRUCTURE OF SCIELLIN, A UNIQUE PRECURSOR OF THE CORNIFIBD ENVELOPE OF KERATINIZING TISSUES.
GLUCOSYLCERAMIDE FORMATION IS CRITICAL FOF EPIDERMAL PERMEABILITY BARRIER HOMEOSTASIS Cbuior S.N. Chum. Taisuke Sek &mneth R. F&g&i. Peter M. &a & Walter M Holleran Dept of Dermatology. University of California, 8 Dermatol Service, Veterans A& Medical C;r., San Francisco, CA.
Harvard Medical School, Boston, MA Sciellin, a putative protein precursor of the corn&d envelope of mammalian keratinizing tissues, has unique features which are only partially characterized. To determine its structure the cDNA was cloned from an expression library derived from cultured human keratinccytes using a monoclonal antibody to xi&n. A polyckmal rabbit antibody (ScPab) was made to the expressed protein and tested by Western blotting and by indiiect immunofluorescence (IIF) on tissue.sections. A composite EDNA was isolated containing 2.35 kb and having an open reading frame of 1.93 kb which encoded au unique r&n of 72 kD with a calculated PI of 10.16. The translated sequence showed a 2,able hydrophilic protein which contained 16 repeats of 20 amino acids each and was rich in Glu and Lys residues, potential transglutamiwe substrates. The gene was mapped to chromosome 13q22 by FISH, while radiation hybrid mapping showed linkage to the STS marker W&457. The ScPab identified sciellin on Western blots of a lysis buffer extracts of human cultured keratinwytes, human epidermis and bovine tongue epithelium. The ScPab showed peripheral staining by IIF of the upper cell layers of epidermis and stratified squamous but not simple epithetium, with the exception of the amnion which reacted strongly. This was confirmed by Western and Nor&m blots of extracts of amnion. The ScPab also reacted with the hair follicle as previously reported. Sciellin was present by IIF in the developing mouse fetus at 17.5 days, when late markers of keratinocyte differentiation are detected. These data f&y establish that sciellin is a unique protein impatant in the terminal differentiation of keratinocyte and detail its structw. Its presence in amnion suggesrs a potential additional role in development.
Glucosylceramide synthase (GCS; EC 2.4.1.80) catalyzes the glucosylation of ceramide to form glucosylceramide (GlcCer). a key step in the generation of lamellar body-derived ceramides critical for epidermal permeability barrier homeostasis. Although we previously showed that acute barrier disruption does not upregulate epidermal GCS activity, the current studies were undertaken to further delineate the role of GlcCer and GCS in permeability barrier function. A single topical application of PPPP (P4). a specific GCS inhibitor, immediately after acute barrier disruption by tapestripping of hairless mice, significantly inhibited both in vitro GCS activity (i.e., 70% inhibition vs. vehicle controls; p
0200
0203
COMPLEX CERAMIDES ARE BOUND TO INVOLUCRIN, ENVOPLAKIN, DESMOPLAKIN PERIPLAKIN AND PERHAPS OTHER PROTEINS OF HUMAN FORESKIN EPIDERMAL CORNIFIED CELL ENVELOPES. Lwben N. Marekov. Peter M. Steinert. Laboratory of Skin Biology, NIAMS, NM, Bethesda, MD, USA. An important component of banier function in mammalian epidermis is contributed by axamides that are bound by ester linkages to unknown proteins of the corn&d celI envelope (CE). In this paper, we have examined the protein substrates for ceramide attachment in human foreskin eoidenuin. Bv oartial sauonification of isolated CEO followed by limited proteolysia, we have m&reh a series’of highly hydrophobic lipopeptides by chromatography. Biochemical and mass spectroscopic characterization &Aed that all contained near stoichiometric amounts of a complex series of ceramides of masses raugmg between 690-890 atomic mess umts (amu). Many of these differed in we from each other by I4 emu, suggesting an array of ceramides differing by an average of one metbylene on their fatty alcohol and sphingosine moieties. Of these about six quantitatively major classesof ceramides of masses of 736: 750, 776, 778 and 804 emu commonly occurred in several of the lipo-peptides A simdar army of ceramides of the same masses was obtained from pig skin, the composition of which is well known. These data thereby afford information on the fatty acid and sphingonine composition of the major human ceramide species. By amino acid sequeuang, we demonstrated that 30% of the lipo-peptides were derived from several ancestral glutamine-glutamate rich regions of involucrin, an important CE protein constituent. Other peptides were derived from the rod domain regions of envoplakin. Several others were too short for unequivocal identlficatlon, but some were most likely derived from glutamine-glutamate rich regions on the rod domains of dcsmoplakm, envoplakin or periplakiu? which are also known or suspectedCE protems. These protans occupy the mner portlo” of the CE lmmedmtcly adjacent to the cell penphely corresponding to the mmtial stages of its assembly. The attachment of ceramides which occurs from the exfenor of the comeocyte to these early CE scaffold proteins IS thus consistentwith current models of CE structure.
DIRECT EVIDENCE TEAT OMEGA-HYDROXY CERAMIDES ARE IMPORTANT FOR EPIDERMAL BARRIER FUNCTION. Martin Beh Y o s’lukazu Uchida. ws. & Welter M. Holleran, Dept. of Dermatology, UCSF, & Dermatology Serv., V.A. Medical Center, San Francisco, CA, In the epidermis, omega-hydroxy ceramides (c&OH Cer) are the predominant lipid species of the covalently-attached lipid-bound envelope (LBE), and their o-acylatedderivatives (acylC!er) are major components of the stratum corneum (SC) extracellular lamellae responsible for barrier function. Since epiderrnal o-OH Cer formation appears to be a cytochrome P450-dependent process (Uchida, et al. JID 108:606a. 1997), we determined the effects of a mechanism-based inhibitor of P450 enzymes, aminobenzoltriazol (ABT), on epidennal o-OH Cer formation and barrier function. ABT (IO&) inhibited the incorporation of [?I-acetate into the o-OH-containing Cer species in cultured human keratinocytes (CHK; 68.1 f6.9% inhibition vs. vebicletreated controls; p
0204
0201 TNF RECEPTOR P55 SIGNALING AND CERAMIDES GENERATED BY SPHINGOMYELINASES IN CUTANEOUS BARRIER REPAIR E. Proksch*, J.-M. Jensen*, M. Kmnke’, S. Schiitze’ Dept. of ‘Dermatol. and *Immunol. Univ. of Kiel, Germany The multiple biological activities of TNF are mediatedby two receptors TNF-R ~55 and TNFR ~75. Central to the TNF-R ~55 signaling is the second messengerceramide, which is generatedby sphingomyelin(SM) breakdown catalyzedby acidic and neutral sphingomyelinases (A-SMase and N-SMase). We recently showed that during permeability barrier repair A-SMx%? generate ceramides for the physical harrier in the stratum corneum, whereas N-SMase is probably related to signal transductionin permeability barrier repair. We now asked, a) which of the known TNF receptors is responsible for barrier repair in viw? b) if the increase in SMase activity induced by barrier disruption leads to a decreasein SM content and c) which ceramides are generatedin TNF signaling by SMases? We used TNF-R ~55 or ~75 deficient mice generated by gene targeting (TNF-R ~75 knockout mice were kindly provided by T.S. Kupper, Boston (USA)), and determined the time curve of skin barrier repair by measurementl of transepidermal water loss (TEWL) at 0, 3, 5, 7, and 24 hours after tape stripping. In normal hairless mouseskin we isolated epidermul samplesat 2 and 4h after barrier disruption and analyzed SM and ceramide content by high performance thin layer chromatography (HPTLC). In TNF-R ~55 and ~75 deficient mice baseline TEWL was unchanged.However, in TNF-R ~55 deficient mice we found after experimentalbarrier disruptiona significantly delayed barrier repair at all points oftime from I to 24 h (lh: -3O%, 24h: -14%; n=7, p
I,
A NOVEL WEEAT GLIADIN
CAUSES EXERCISFJNDUCED
ANAPHYLAXIS
&a,& Departmentof Dermatology, Institute ofOccupatmnal Health and B,otechnology, Helsmla, Fmland. Food-deoendent, exercwe-Induced anauhvlaxls or urhcana (EIA) 1s a special form of allergy where netther exercisenor the &e&d food alone elicus the react& Wlthm two years we found 18 Pabents (age 24-78 years) wtb wheat-dependent EIA. Twelve patients had exptienced anaphylaxls and 6 generalized urt~caria with collapse dunng e~ercw after having eaten wheat bread, pizza, hamburgers etc. All patients had a posttwe skin pnck test and RAST to wheat flour. Immunoblomng showed strong IgE-bmding to 65 kD and 40 kD wheat proteins. These protans were purified by HPLC gel tiltratmn and reversed-phase chromatography and submitted to N-terminal ammo amd sequencmg. The 65 kD protem (30 amino ac& analyzed) showed a 52% sequence homology to w-&&n and the 40 kD protem (22 ammo amds analyzed) a 100% homology to a@-glladin. Tbc purltied protems (2 &ml) were used as solid-phase antigens m IgE-ELISA. The novel wehadm bound IaE from the sera of 17118 and the a@-gliadin from the sera of &en& wth EIA whereas all control sera from 55 paueng (16 Baker’s asthma, I5 wheat allergy and atopy, 12 urbcarw 12 coeliac disease) were negative Skm pnck tests were pormve wuh the novel ghadm (0.05-50 &ml) m 1915 and wth the a/!%ghadm (500 ,&,I) m 5115 pauents wth EIA. The present study shows that wheat IS a frequent cause of food-dependent EIA. The in wrro and in viva results suggest that the major allergen m this hfe-threatening dwxder 1s a novel wheat ghadm which may have cross-reactwe epltopeswth c+ghadm.
I l/l8
s34
0199
0202
CHARACTERIZATION OF THE PROTEIN STRUCTURE OF SCIELLIN, A UNIQUE PRECURSOR OF THE CORNIFIBD ENVELOPE OF KERATINIZING TISSUES.
GLUCOSYLCERAMIDE FORMATION IS CRITICAL FOF EPIDERMAL PERMEABILITY BARRIER HOMEOSTASIS Cbuior S.N. Chum. Taisuke Sek &mneth R. F&g&i. Peter M. &a & Walter M Holleran Dept of Dermatology. University of California, 8 Dermatol Service, Veterans A& Medical C;r., San Francisco, CA.
Harvard Medical School, Boston, MA Sciellin, a putative protein precursor of the corn&d envelope of mammalian keratinizing tissues, has unique features which are only partially characterized. To determine its structure the cDNA was cloned from an expression library derived from cultured human keratinccytes using a monoclonal antibody to xi&n. A polyckmal rabbit antibody (ScPab) was made to the expressed protein and tested by Western blotting and by indiiect immunofluorescence (IIF) on tissue.sections. A composite EDNA was isolated containing 2.35 kb and having an open reading frame of 1.93 kb which encoded au unique r&n of 72 kD with a calculated PI of 10.16. The translated sequence showed a 2,able hydrophilic protein which contained 16 repeats of 20 amino acids each and was rich in Glu and Lys residues, potential transglutamiwe substrates. The gene was mapped to chromosome 13q22 by FISH, while radiation hybrid mapping showed linkage to the STS marker W&457. The ScPab identified sciellin on Western blots of a lysis buffer extracts of human cultured keratinwytes, human epidermis and bovine tongue epithelium. The ScPab showed peripheral staining by IIF of the upper cell layers of epidermis and stratified squamous but not simple epithetium, with the exception of the amnion which reacted strongly. This was confirmed by Western and Nor&m blots of extracts of amnion. The ScPab also reacted with the hair follicle as previously reported. Sciellin was present by IIF in the developing mouse fetus at 17.5 days, when late markers of keratinocyte differentiation are detected. These data f&y establish that sciellin is a unique protein impatant in the terminal differentiation of keratinocyte and detail its structw. Its presence in amnion suggesrs a potential additional role in development.
Glucosylceramide synthase (GCS; EC 2.4.1.80) catalyzes the glucosylation of ceramide to form glucosylceramide (GlcCer). a key step in the generation of lamellar body-derived ceramides critical for epidermal permeability barrier homeostasis. Although we previously showed that acute barrier disruption does not upregulate epidermal GCS activity, the current studies were undertaken to further delineate the role of GlcCer and GCS in permeability barrier function. A single topical application of PPPP (P4). a specific GCS inhibitor, immediately after acute barrier disruption by tapestripping of hairless mice, significantly inhibited both in vitro GCS activity (i.e., 70% inhibition vs. vehicle controls; p
0200
0203
COMPLEX CERAMIDES ARE BOUND TO INVOLUCRIN, ENVOPLAKIN, DESMOPLAKIN PERIPLAKIN AND PERHAPS OTHER PROTEINS OF HUMAN FORESKIN EPIDERMAL CORNIFIED CELL ENVELOPES. Lwben N. Marekov. Peter M. Steinert. Laboratory of Skin Biology, NIAMS, NM, Bethesda, MD, USA. An important component of banier function in mammalian epidermis is contributed by axamides that are bound by ester linkages to unknown proteins of the corn&d celI envelope (CE). In this paper, we have examined the protein substrates for ceramide attachment in human foreskin eoidenuin. Bv oartial sauonification of isolated CEO followed by limited proteolysia, we have m&reh a series’of highly hydrophobic lipopeptides by chromatography. Biochemical and mass spectroscopic characterization &Aed that all contained near stoichiometric amounts of a complex series of ceramides of masses raugmg between 690-890 atomic mess umts (amu). Many of these differed in we from each other by I4 emu, suggesting an array of ceramides differing by an average of one metbylene on their fatty alcohol and sphingosine moieties. Of these about six quantitatively major classesof ceramides of masses of 736: 750, 776, 778 and 804 emu commonly occurred in several of the lipo-peptides A simdar army of ceramides of the same masses was obtained from pig skin, the composition of which is well known. These data thereby afford information on the fatty acid and sphingonine composition of the major human ceramide species. By amino acid sequeuang, we demonstrated that 30% of the lipo-peptides were derived from several ancestral glutamine-glutamate rich regions of involucrin, an important CE protein constituent. Other peptides were derived from the rod domain regions of envoplakin. Several others were too short for unequivocal identlficatlon, but some were most likely derived from glutamine-glutamate rich regions on the rod domains of dcsmoplakm, envoplakin or periplakiu? which are also known or suspectedCE protems. These protans occupy the mner portlo” of the CE lmmedmtcly adjacent to the cell penphely corresponding to the mmtial stages of its assembly. The attachment of ceramides which occurs from the exfenor of the comeocyte to these early CE scaffold proteins IS thus consistentwith current models of CE structure.
DIRECT EVIDENCE TEAT OMEGA-HYDROXY CERAMIDES ARE IMPORTANT FOR EPIDERMAL BARRIER FUNCTION. Martin Beh Y o s’lukazu Uchida. ws. & Welter M. Holleran, Dept. of Dermatology, UCSF, & Dermatology Serv., V.A. Medical Center, San Francisco, CA, In the epidermis, omega-hydroxy ceramides (c&OH Cer) are the predominant lipid species of the covalently-attached lipid-bound envelope (LBE), and their o-acylatedderivatives (acylC!er) are major components of the stratum corneum (SC) extracellular lamellae responsible for barrier function. Since epiderrnal o-OH Cer formation appears to be a cytochrome P450-dependent process (Uchida, et al. JID 108:606a. 1997), we determined the effects of a mechanism-based inhibitor of P450 enzymes, aminobenzoltriazol (ABT), on epidennal o-OH Cer formation and barrier function. ABT (IO&) inhibited the incorporation of [?I-acetate into the o-OH-containing Cer species in cultured human keratinocytes (CHK; 68.1 f6.9% inhibition vs. vebicletreated controls; p
0204
0201 TNF RECEPTOR P55 SIGNALING AND CERAMIDES GENERATED BY SPHINGOMYELINASES IN CUTANEOUS BARRIER REPAIR E. Proksch*, J.-M. Jensen*, M. Kmnke’, S. Schiitze’ Dept. of ‘Dermatol. and *Immunol. Univ. of Kiel, Germany The multiple biological activities of TNF are mediatedby two receptors TNF-R ~55 and TNFR ~75. Central to the TNF-R ~55 signaling is the second messengerceramide, which is generatedby sphingomyelin(SM) breakdown catalyzedby acidic and neutral sphingomyelinases (A-SMase and N-SMase). We recently showed that during permeability barrier repair A-SMx%? generate ceramides for the physical harrier in the stratum corneum, whereas N-SMase is probably related to signal transductionin permeability barrier repair. We now asked, a) which of the known TNF receptors is responsible for barrier repair in viw? b) if the increase in SMase activity induced by barrier disruption leads to a decreasein SM content and c) which ceramides are generatedin TNF signaling by SMases? We used TNF-R ~55 or ~75 deficient mice generated by gene targeting (TNF-R ~75 knockout mice were kindly provided by T.S. Kupper, Boston (USA)), and determined the time curve of skin barrier repair by measurementl of transepidermal water loss (TEWL) at 0, 3, 5, 7, and 24 hours after tape stripping. In normal hairless mouseskin we isolated epidermul samplesat 2 and 4h after barrier disruption and analyzed SM and ceramide content by high performance thin layer chromatography (HPTLC). In TNF-R ~55 and ~75 deficient mice baseline TEWL was unchanged.However, in TNF-R ~55 deficient mice we found after experimentalbarrier disruptiona significantly delayed barrier repair at all points oftime from I to 24 h (lh: -3O%, 24h: -14%; n=7, p
I,
A NOVEL WEEAT GLIADIN
CAUSES EXERCISFJNDUCED
ANAPHYLAXIS
&a,& Departmentof Dermatology, Institute ofOccupatmnal Health and B,otechnology, Helsmla, Fmland. Food-deoendent, exercwe-Induced anauhvlaxls or urhcana (EIA) 1s a special form of allergy where netther exercisenor the &e&d food alone elicus the react& Wlthm two years we found 18 Pabents (age 24-78 years) wtb wheat-dependent EIA. Twelve patients had exptienced anaphylaxls and 6 generalized urt~caria with collapse dunng e~ercw after having eaten wheat bread, pizza, hamburgers etc. All patients had a posttwe skin pnck test and RAST to wheat flour. Immunoblomng showed strong IgE-bmding to 65 kD and 40 kD wheat proteins. These protans were purified by HPLC gel tiltratmn and reversed-phase chromatography and submitted to N-terminal ammo amd sequencmg. The 65 kD protem (30 amino ac& analyzed) showed a 52% sequence homology to w-&&n and the 40 kD protem (22 ammo amds analyzed) a 100% homology to a@-glladin. Tbc purltied protems (2 &ml) were used as solid-phase antigens m IgE-ELISA. The novel wehadm bound IaE from the sera of 17118 and the a@-gliadin from the sera of &en& wth EIA whereas all control sera from 55 paueng (16 Baker’s asthma, I5 wheat allergy and atopy, 12 urbcarw 12 coeliac disease) were negative Skm pnck tests were pormve wuh the novel ghadm (0.05-50 &ml) m 1915 and wth the a/!%ghadm (500 ,&,I) m 5115 pauents wth EIA. The present study shows that wheat IS a frequent cause of food-dependent EIA. The in wrro and in viva results suggest that the major allergen m this hfe-threatening dwxder 1s a novel wheat ghadm which may have cross-reactwe epltopeswth c+ghadm.
I l/l8