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Role of glucosylation of ceramide in epidermal barrier formation
14
Oct. 28/Concurrent
Session 5 - Keratinocyte Biology and Biochemistr_
059
062
ROLE OF GLUCOSYLATION OF CERAMIDE IN EPIDERMAL BARRIER FORMATION t&&t, Minors Tw Genii hnokawa. Kao Biological Science Laboratories, Tochigi. Japan We have investigated the physiological slgnlficance of glucosylation 01 ceramide in the synthetic pafhway 01 acylceramide. an important ceramide species lor epidermal barrier, by examining the metabolic pathway of [‘4C]-labelled serine in mouse epidermal tissue cullwe system and by determining the substrafe specilicity of glucosyftransferase (UDP-glucose:ceramlde glucosylfransferase) in wlro. Cultured new born mouse (Balbic) epldermal tissue synthesized several types of [14C]-labelled ceramides including ceramide type 1 or acylceramlde as analyzed by TLC followed by auforadiography. When Br-CBE (bromocondritol-B-epoxide). an inhibifor of glucocerebrosldase. was added in fhe culfure medium. synlhesis of acylceramide was inhibited compfefefy while other types 01 ceramides remained unchanged. Furthermore. addition of PDMP (D-three-I-phenyl-2.decanoylamino3.morphofino.l-propanol). an inhibitor of glucosyltransferase. also specifically abolished the synthesis of acykeramide, indicating that glucosylalion of ceramides was an essential requrement for acylceramide synthesis. We further determined if the phystological subshale of glucosyltransferase was omega.hydroxylceramide (C30) or acylceramide (linoleoyl C30 ceramide). Neither of fhem was good subsfrafe for glucosyllransferase as measured in vifro. suggesting that glucosylafion occurs before the formation of omega-hydoroxylceramide although the mechanism of omega-hydroxylceramide synthesis remanned 10 be clarified. These findings demonstrate for the first tlrne that acylceramide found in stratum corneum is synthesized through deftmte successive processes conststtng of glucosylalion of cerarmde. acylatlon of glucosylceramide and de-glucosylafion 01 acylglucosylceramlde.
CHARACTERIZATION OF THE S-1MLIKE CALCIUM BINDING DOMAIN OF HUMAN PROFILAGGRIN. R.B.. J. R. Ku&all and B.A. Dale Dept. of Oral Biology, University of Washington, Seattle, WA, U.S.A. Filaggrin (FG) is the intemxdiate-filament associated protein which functions to aggregate keratin IFS in the stratum corneum of epidermis. It is synthesized as a large precursor protein, protilaggrin @roFG), that consists of multiple FG repeats and is localized in keratohyalin (KH). We recently reported the genomic organization of the human proFG gene and identitied an S-1OCMike EF hand domain in the first 81 amino acids of the predicted protein sequence (JBC 267, 23772.81, 1992). To study its possible role in the processing of proFG to FG, a peptide antibody (EF-Ab) directed against part of the first EF hand (aa 16-30) was prepared and wed for immunohistwhemical and Western analysis of human and mouse epidermis. The EF-Ab reacts strongly with cells in the upper granular and transition layers where proFG is being processed, but only weakly with KH. There war no detectable staining of other epidermal cell layers or the corniced cell envelope. The EF-AC reacts weakly with proFG, strongly with polypeptides of 50-55 and 16.5 kD, and less intensely with bands of 90 kD and -150 kD. They may represent processing intermediates of proFG or non-specific pmtwlytic products. To study calcium binding, the 293 aa N-terminal domain was expressed in E. coli and calcium binding was analyzed by the 4sCaz+ overlay technique. Results obtained with whole cell bacterial er.tracts suggests that the 36 kD recombinant protein hinds calcium, although the binding is weak compared to yeast calmodulin which contains three functional EF hands. We hypothesize that the N-terminal EF hand domain of proFG is critical for proFG expression or processing to FG.
060
063
STRUCTURE DETERMINATION OF ACYLOLUCOSYLCERAMIDES OF HUMAN CULTURED FERATINOCYTES. Sumiko Hamanaka’ Chidori Asagami’, FuJia otsu!a3 ,lkpartments of Dermatology. 1Yamaguchi Rosai Hosplral, onoria. Yamaauchi. ZYamarruchi llniversitv School or Medicine. me.
EXPRESSION OF CORNIFIN IN SQUAMOUS DIFFERENTIATED EPITHELIAL TISSUES. W 1. t ru Fu.1 Department of Dermatology. Okayama University Medical School, Okayama, Japan. *Cell Biology Section, Laboratory of Pulmonary Pathobrology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, U.S.A. Comifin is a putative cross-linked envelope precursor in keratinocytes. In order to examine the expression and regulation of cornifin m viva, we investigated several squamous differentiated tissues by in situ hybridization and immunohistochemlcal analysis. Cornlfin mRNA and protein, which are absent in the normal mucocihary eprthelium, are induced in the suprabasal layers of the squamous metaplastic tracheal epithelium of vitamin-A deficient hamsters. Sxnilar to the induction of squamous metaplasia in viva culture of rabbit tracheal cells in the absence of rewords results m squamous differentratron and expression of cornitin. This induction of cornitin expression is suppressed by retinoic acid and several of its analogs. Cornltin mRNA and protein are also detected rn the suprabasal layers of the squamous epithehum of rabbit esophagus and tongue. The dismbuhon of cornifin m human epidermis was compared with that of two other cross-linked envelope precursor proreins. mvolucrin and loricrin. Comifin and involucrin are induced in the spinous layer and appear at an earlier stage during epidemxd differentiation than loncnn. The expression of comrfin is greatly increased in psoriatic skin. Comitin mRNA is barely detectable m normal epidermis whereas it is present at relatively high levels in the suprabasal layers of psoriatrc epidermis. Topical treatment with RA results in thickenmg of the skin and increases the level of corrofm mRNA and protein in the upper spinous layers of mouse skin. In conclusion. cormtin expression correlates generally with squamous differentiation in a variety of tissues and is abnomully regulated m psonahc skm and in skin treated topically wth retinoic acid.
Yamaguchi, ‘Tsukuba University, Acylgl”cosylCeramide9 ,ACCl
Tsukubi; Ibaragi, are
mportant
Japan. glycospningolipids
or tne
061 EXPRESSION AND FUNCTION OF NERVE GROWTH FACTOR AND NERVE GROWTH FACTOR RECEPTORS ON CULTURED NORMAL HUMAN KERATINOCYTES. Carlo Sevienani, Luisa Benassi. Fabrizio Fantini. Luiei Aloe. Sereio Ferrari and Alberta Institute of Dermatology and Hematology, University of Modena and Institute of Neurobiology, C.N.R., Rome, Italy. Nerve growth factor (NGF), in addition to its neurotrophic properties, possesses a number of biological effects on several cell types. NGF receptor (NGF-R) has been recently detected in normal human skin by immunohistochemistry. In the present study we evaluated function and expression of NGF and NGF-R on normal human keratinocytes (NHK) cultivated on 3T3 cells. Using the reverse-transcription polymerase chain reaction (RT-PCR), both the low- and the high-affinity (trk) NGF-R mRNA were detected on NHK Addition of either NGFbeta or NGF 2.55 (10, 100, 500 “g/ml) to the medium significantly stimulated the proliferation of NHK in a dose dependent manner (NGFbeta = 25, 55,75 % cell number increase respectively, p< 0.01; NGF 2.55 = 10, 35, 60 C cell number increase respectively, p-z 0.05). The NGF-induced keratinocyte proliferation was significantly blocked by anti-NGF neutralizing mAb. Furthermore, NGF mRNA was detected in NHK by RT-PCR and NGF was shown to be released by NHK, as measured by ELISA. In particular, NGF levels (pg/ml) were secreted in increasing amounts during proliferation (day 1=96+33; day 5=140+31), whereas they began to decrease after confluence (day 7=64.4%33.6; day 8=45.1+7.6; day 13=21.4*4.4). These results indicate that NGF might have an important function in human skin and suggest the hypothesis that NGF plays an autocrine role in the proliferation of NHK.
064 EPIDBRMAL EXPRESSION OF PROFILAGGRIN PHOSPHATASE, A PROTEIN PHOSPHATASE TYPE 2A. B.A. Dale. E. Kam. A. Glass. R., Depts. of Oral Biology, Periodontics, and Medicine/Dermatology, Univ. of Washington, Seattle, WA, USA. Epidermal granular cells undergo an abrupt transition to fully differentiated comitied cells lacking organelles and tilled with densely packed keratin proteins. During this transition the keratoh alin protein, protilaggrin, is dephosphorylated and pmteolytically processed to fy.. ~laggnn which aggre $. ates keratin filaments. In order to better understand this process, a pro daggnn phosphatasc was characterized from rat epidermis. This enzyme is a phospbatase type 2A @‘&?_A) not dependent on divalent cations and inhibited by okadaic acid. PP2A is a highly conserved serine/threonine protein phosphatax made up of one catalytic subunit and two regulatory subunits. We studied the expression of both the protein and mRNA of the PP2A catalytic subunit. Using an antibody (a gift of M. Mumby), we identified a band of the expected size (36 kDa) by Western analysis of human epidermal extracts. Immunostaining gave a strong reaction m the granular layer where protilaggrin is first expressed and then processed. Probes specific for the alpha and beta isoforms of the PP2A catalytic subunit were prepared and used for Northern analysis and for in siru hybridization. Both &forms are expressed in epidermis and cultured keratinocytes and are strongly expressed in the granular cell layer. The PP7.A catalytic subunit beta was cloned from an epidermal cDNA library and cloned into the PET-15b vector for expression in E. co/i. The fusion protein is being purified and tested for phosphatase activity with phosphotilaggrin. Optimization of expression will enable detailed studies of the regulation of this enzyme and its role in profilaggrin processing and keratinization.
Oct. 28/Concurrent
Session 5 - Keratinocyte Biology and Biochemistr_
059
062
ROLE OF GLUCOSYLATION OF CERAMIDE IN EPIDERMAL BARRIER FORMATION t&&t, Minors Tw Genii hnokawa. Kao Biological Science Laboratories, Tochigi. Japan We have investigated the physiological slgnlficance of glucosylation 01 ceramide in the synthetic pafhway 01 acylceramide. an important ceramide species lor epidermal barrier, by examining the metabolic pathway of [‘4C]-labelled serine in mouse epidermal tissue cullwe system and by determining the substrafe specilicity of glucosyftransferase (UDP-glucose:ceramlde glucosylfransferase) in wlro. Cultured new born mouse (Balbic) epldermal tissue synthesized several types of [14C]-labelled ceramides including ceramide type 1 or acylceramlde as analyzed by TLC followed by auforadiography. When Br-CBE (bromocondritol-B-epoxide). an inhibifor of glucocerebrosldase. was added in fhe culfure medium. synlhesis of acylceramide was inhibited compfefefy while other types 01 ceramides remained unchanged. Furthermore. addition of PDMP (D-three-I-phenyl-2.decanoylamino3.morphofino.l-propanol). an inhibitor of glucosyltransferase. also specifically abolished the synthesis of acykeramide, indicating that glucosylalion of ceramides was an essential requrement for acylceramide synthesis. We further determined if the phystological subshale of glucosyltransferase was omega.hydroxylceramide (C30) or acylceramide (linoleoyl C30 ceramide). Neither of fhem was good subsfrafe for glucosyllransferase as measured in vifro. suggesting that glucosylafion occurs before the formation of omega-hydoroxylceramide although the mechanism of omega-hydroxylceramide synthesis remanned 10 be clarified. These findings demonstrate for the first tlrne that acylceramide found in stratum corneum is synthesized through deftmte successive processes conststtng of glucosylalion of cerarmde. acylatlon of glucosylceramide and de-glucosylafion 01 acylglucosylceramlde.
CHARACTERIZATION OF THE S-1MLIKE CALCIUM BINDING DOMAIN OF HUMAN PROFILAGGRIN. R.B.. J. R. Ku&all and B.A. Dale Dept. of Oral Biology, University of Washington, Seattle, WA, U.S.A. Filaggrin (FG) is the intemxdiate-filament associated protein which functions to aggregate keratin IFS in the stratum corneum of epidermis. It is synthesized as a large precursor protein, protilaggrin @roFG), that consists of multiple FG repeats and is localized in keratohyalin (KH). We recently reported the genomic organization of the human proFG gene and identitied an S-1OCMike EF hand domain in the first 81 amino acids of the predicted protein sequence (JBC 267, 23772.81, 1992). To study its possible role in the processing of proFG to FG, a peptide antibody (EF-Ab) directed against part of the first EF hand (aa 16-30) was prepared and wed for immunohistwhemical and Western analysis of human and mouse epidermis. The EF-Ab reacts strongly with cells in the upper granular and transition layers where proFG is being processed, but only weakly with KH. There war no detectable staining of other epidermal cell layers or the corniced cell envelope. The EF-AC reacts weakly with proFG, strongly with polypeptides of 50-55 and 16.5 kD, and less intensely with bands of 90 kD and -150 kD. They may represent processing intermediates of proFG or non-specific pmtwlytic products. To study calcium binding, the 293 aa N-terminal domain was expressed in E. coli and calcium binding was analyzed by the 4sCaz+ overlay technique. Results obtained with whole cell bacterial er.tracts suggests that the 36 kD recombinant protein hinds calcium, although the binding is weak compared to yeast calmodulin which contains three functional EF hands. We hypothesize that the N-terminal EF hand domain of proFG is critical for proFG expression or processing to FG.
060
063
STRUCTURE DETERMINATION OF ACYLOLUCOSYLCERAMIDES OF HUMAN CULTURED FERATINOCYTES. Sumiko Hamanaka’ Chidori Asagami’, FuJia otsu!a3 ,lkpartments of Dermatology. 1Yamaguchi Rosai Hosplral, onoria. Yamaauchi. ZYamarruchi llniversitv School or Medicine. me.
EXPRESSION OF CORNIFIN IN SQUAMOUS DIFFERENTIATED EPITHELIAL TISSUES. W 1. t ru Fu.1 Department of Dermatology. Okayama University Medical School, Okayama, Japan. *Cell Biology Section, Laboratory of Pulmonary Pathobrology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, U.S.A. Comifin is a putative cross-linked envelope precursor in keratinocytes. In order to examine the expression and regulation of cornifin m viva, we investigated several squamous differentiated tissues by in situ hybridization and immunohistochemlcal analysis. Cornlfin mRNA and protein, which are absent in the normal mucocihary eprthelium, are induced in the suprabasal layers of the squamous metaplastic tracheal epithelium of vitamin-A deficient hamsters. Sxnilar to the induction of squamous metaplasia in viva culture of rabbit tracheal cells in the absence of rewords results m squamous differentratron and expression of cornitin. This induction of cornitin expression is suppressed by retinoic acid and several of its analogs. Cornltin mRNA and protein are also detected rn the suprabasal layers of the squamous epithehum of rabbit esophagus and tongue. The dismbuhon of cornifin m human epidermis was compared with that of two other cross-linked envelope precursor proreins. mvolucrin and loricrin. Comifin and involucrin are induced in the spinous layer and appear at an earlier stage during epidemxd differentiation than loncnn. The expression of comrfin is greatly increased in psoriatic skin. Comitin mRNA is barely detectable m normal epidermis whereas it is present at relatively high levels in the suprabasal layers of psoriatrc epidermis. Topical treatment with RA results in thickenmg of the skin and increases the level of corrofm mRNA and protein in the upper spinous layers of mouse skin. In conclusion. cormtin expression correlates generally with squamous differentiation in a variety of tissues and is abnomully regulated m psonahc skm and in skin treated topically wth retinoic acid.
Yamaguchi, ‘Tsukuba University, Acylgl”cosylCeramide9 ,ACCl
Tsukubi; Ibaragi, are
mportant
Japan. glycospningolipids
or tne
061 EXPRESSION AND FUNCTION OF NERVE GROWTH FACTOR AND NERVE GROWTH FACTOR RECEPTORS ON CULTURED NORMAL HUMAN KERATINOCYTES. Carlo Sevienani, Luisa Benassi. Fabrizio Fantini. Luiei Aloe. Sereio Ferrari and Alberta Institute of Dermatology and Hematology, University of Modena and Institute of Neurobiology, C.N.R., Rome, Italy. Nerve growth factor (NGF), in addition to its neurotrophic properties, possesses a number of biological effects on several cell types. NGF receptor (NGF-R) has been recently detected in normal human skin by immunohistochemistry. In the present study we evaluated function and expression of NGF and NGF-R on normal human keratinocytes (NHK) cultivated on 3T3 cells. Using the reverse-transcription polymerase chain reaction (RT-PCR), both the low- and the high-affinity (trk) NGF-R mRNA were detected on NHK Addition of either NGFbeta or NGF 2.55 (10, 100, 500 “g/ml) to the medium significantly stimulated the proliferation of NHK in a dose dependent manner (NGFbeta = 25, 55,75 % cell number increase respectively, p< 0.01; NGF 2.55 = 10, 35, 60 C cell number increase respectively, p-z 0.05). The NGF-induced keratinocyte proliferation was significantly blocked by anti-NGF neutralizing mAb. Furthermore, NGF mRNA was detected in NHK by RT-PCR and NGF was shown to be released by NHK, as measured by ELISA. In particular, NGF levels (pg/ml) were secreted in increasing amounts during proliferation (day 1=96+33; day 5=140+31), whereas they began to decrease after confluence (day 7=64.4%33.6; day 8=45.1+7.6; day 13=21.4*4.4). These results indicate that NGF might have an important function in human skin and suggest the hypothesis that NGF plays an autocrine role in the proliferation of NHK.
064 EPIDBRMAL EXPRESSION OF PROFILAGGRIN PHOSPHATASE, A PROTEIN PHOSPHATASE TYPE 2A. B.A. Dale. E. Kam. A. Glass. R., Depts. of Oral Biology, Periodontics, and Medicine/Dermatology, Univ. of Washington, Seattle, WA, USA. Epidermal granular cells undergo an abrupt transition to fully differentiated comitied cells lacking organelles and tilled with densely packed keratin proteins. During this transition the keratoh alin protein, protilaggrin, is dephosphorylated and pmteolytically processed to fy.. ~laggnn which aggre $. ates keratin filaments. In order to better understand this process, a pro daggnn phosphatasc was characterized from rat epidermis. This enzyme is a phospbatase type 2A @‘&?_A) not dependent on divalent cations and inhibited by okadaic acid. PP2A is a highly conserved serine/threonine protein phosphatax made up of one catalytic subunit and two regulatory subunits. We studied the expression of both the protein and mRNA of the PP2A catalytic subunit. Using an antibody (a gift of M. Mumby), we identified a band of the expected size (36 kDa) by Western analysis of human epidermal extracts. Immunostaining gave a strong reaction m the granular layer where protilaggrin is first expressed and then processed. Probes specific for the alpha and beta isoforms of the PP2A catalytic subunit were prepared and used for Northern analysis and for in siru hybridization. Both &forms are expressed in epidermis and cultured keratinocytes and are strongly expressed in the granular cell layer. The PP7.A catalytic subunit beta was cloned from an epidermal cDNA library and cloned into the PET-15b vector for expression in E. co/i. The fusion protein is being purified and tested for phosphatase activity with phosphotilaggrin. Optimization of expression will enable detailed studies of the regulation of this enzyme and its role in profilaggrin processing and keratinization.