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Analysis of the control of expression and tissue specificity of the human trichohyalin gene
ESDR f JSID f SID Abstracts
s133
0793
0796
TRANSCRIPTIONAL REGULATION OF COSTlMUL4TORY MOLECULE, B7-I, EXPRESSION IN HUMAN KERATINOCYTES TREATED WIT? ALLERGENS AND IRRITANTS. P W&m. F. Ramirez. R. Bums. A. C&pau DqmQnent of Dermatology, University of Rochester School of Medicine end Dentistry: Rahester, NY. ~ttr lab has previously reported that human kemtinccytes (KC) express a low basal level of ~7-1 (CDSO)on their cell surface. which is greatly increased both in viva, on KC at sites of allergic contact dermatitis, and k vitro. on cultured KC treated with the allergen mckel chloride (Ni) or the irritant sodium lamyl sulfate (SLS). lo addition, B7-1 expression is tmnscriptically upregulated in cultured KC treated with Ni or SLS (increased levels of B7-1 RNA transcripts in nuclear mnoff experiments). We are developing an in virro assay to detect allergens and irritanrs using primary ~ultore human KC, transiently tmnsfected with lncifemse reporter vectors containing httmao B7-1 promoter segments. Transfected KC are treated with an allergen or irritant, then assayed for B7- I promoter expression, meawed as luciferase activity. An 84 bp 87-l promoter fragment expresses a basal level of luciferrtse which is not upregulated by Vestment with allergens or irritants. In contrast, a 230 bp B7- 1 promoter fragment expresses a high level of luciferase activity (-30,ooO rlu) which is upregulated (2-5 fold) in KC treated with ceti_ainallergens or irritants. Using this construct in our in vitro assay, we have tested 14 allergens and 7 irritants for their ability to upregulate B7-1 promoter fragment expression. This upmgolruion of B7-1 expression is donor-specific, i.e. KC from different donors upmgulate promoter activity in response to different chemicals. In assays of KC derived from different donors, Ni produces a positive response in over 75% of the aSsays (N=9). In addition, some allergens (mercaptobeozothiamle, cinnamic tidehyde) and irtitanls (phenol) prodwe frequent positive responses (>60%). while other allergens (trinitmchlomhenzene) and irritants (SLS) am less frequently positive (~30%). We conclude: (1) allergens and irritants have direct effects on KC B7-1 expression, (2) this assay may be the basis of a predictive test for chemicals that cause contact dermatitis.
INTERFERON ENHANCES TNF -INDUCED VCAM-I (CDl06) EXPRESSION IN HUMAN ENDOTHELIAL CELLS BY AN IRF- I DEPENDENT PATHWAY. Saia * lens Gille**. and Peter Paz&aaeQepartm~f Lechletto~~F?x!x Dermatology, Dzision of General Dermatology. University of Vienna .Medical School. Vienna, A-1090 Austria; *Boyer Center of Molecular Bmlogy, Yale University, CT, USA; **Department of Dermatology. Goethe Umversity, Frankfurt, Germany TNF and IL-1 are known to initiate endothelial VCAM-I transcnption primarily by activating N&B, which translccates to the nucleus. In addition to two NF-& elements found wthin the minimal cytokine-inducible VCAM-1 promoter, an IRF-1 element has been identified close to the transcription initiauon site, suggesting that cytokines which induce IRF-I might affect VCAM-1 expression levels. We therefore investigated the effects of interferons. which strongly induce IRF-1, on VCAM-1 transcriotion and exoression. We show that IFN-a and IF& enhance TNF-induced VCAM-I mRNA ani protein expression in human endothelial’cells. IFN enhancement of TNF-induced expression is also seen using CAT repmer genes linked to the minimal cvtokine inducible VCAM-I oromoter. Nuclear IRF-1 is the molecular basis of IFN enhancement, because I) IFN’increased nuclear IRF-1. whereas nuclear NF-& levels remained unchanged m cells treated with tFN plus TNF as compared with cells treated wth TNF alone; 2) kineucs of nuclear m-1 levels correlated with VCAM-1 mRNA levels; 3) transfection with an IRF-I construct substituted for IFN treatment; and 4) transfectton with an expression construct encoding IRF-2, a competitive inhtbitor of IRF-I. reduced TNF -Induced VCAM-I exoression. Our exoer~mentsshow that levels of VCAM-I transcribed and expressed on the cell surface a& critically correlated with IRF- I levels, thus the IFN / IRFI pathway represents a dlstmct modulator of endothelial VCAM-I expressaon.
0794
0797 ANALYSIS OF THE CONTROL OF EXPRESSION AND TISSUE SPECIFICITY OF THE HUMAN TRICHOHYALIN GENE. Kenneth Wttl, Jeanne M. Andreoli’, ChnsancM.Coticchia’, &&&a G Markov$, &er M. Steiner+. Shvh-lee Jaq’. ‘Laboratory of Skin Biology, NIAMS, NIH, Bethesda. MD, and &pamnent of Oral Biology, SUNY Stony Brook, NY, USA. Trichohyalin (‘IHH) is an intermediate filament-associated protein which is expressed primarily in the inner nmt sheath and mdulla cells of haii follicle, and also in eoidermis. hard oalate. and fdiform ridees of the toneue. Previonslv we demonstrated &at as few as 1% bp Gf sequences f&the imnsuipptin strut site &capable of driving expression of the p-galactosidase gene, and four fnoctional elements, NFkB (-86), APl (-95), et.+like (-IOO), and Spl (-122). were involved. ln the present study, we have further analyzed the consul of the u-anscri*onal activity and calcium responsiveness of the THH promoter in cultnred mouse hair follicles (mHF) or e@rmal keradoocytes (mEK). In transfected mHF, mutations of either one of these elements reduced the levels of expression by at least 50%; when combinations of two were mutated, the activity fell to near basal levels. In contrast in t&K, expression of the wildtypc C~“S~IC~was at the basal level. Fwthermore, activity levels of the wildtype construct transfected into mHF cells grown in high calcium media was IO-fold higher than in low calcium media. Whereas mutations of the Spl or APl sites had no effect mutations of either the ets-like or NFkB motifs abolished the calcium responsiveness to the basal level. Together, these data show that a synergistic interplay between Spl, a-like, APl and NFkB elements is required for the cell-type specific control of the THH promoter activity in mHP cells, and that both as-like and NFkB elements are necessay for the calcium responsiveness. Other elements located outside this proximal promoter region must be required for specific expression in mEK cells.
0798
0795 Efficient transduction influenza
at mature virus as e vector
human
dandritic
cells
by using
an avia”
I. StrobeI’. R. Grassmaw?. E. Hofmann’, G. Hoborn’, U. Schulze’. E. Niedobilek’, E Wagner’, S. Fleckenstein’ end G. Schuler’
Dendrlbc cells (DC) are anbgen pmsenbng cells with the highest capeclty to initmte primary end secondary T-cell mediated immune respansee. Most importanw, several reports indite that DC also play a crucial role rn tumor mmwne response. Therefore DC are a promising target for immune- and gene therapy of tumors. The aim of our study is the tmnsfer of different genes emx&g tomOr antigens and cytokines into DC to enhance and-lumw response. FW this purlaxe DC ware generated in viva from adherent CD14+ bkcd monocytss by a z&step method (pdming phase with GM-CSF and IL-4 followed by e cytokine-induced rnetwetion phase). Mature DC we identified by their morphology. phenotypic markers (eg. CD33+, CD86+. fesan+) and potent T-0811sensitlring capacify In a fint approach we tested different gene transfer methods using green flwrescent protein (GFP) as marker protein which can be detected in living c& by conventional fluorescence techniques. Low expressjon of GFP (~5 % Of DC) Was Obtained by receptor-mediated bansfeclion, by lipid es well es non-lipid c&ionic transfecllon techniques. Intermediate expression lewls (~20 % of DC) resulted from eledroporalion High exppssion of GFP wee found in DC after infection v.ith an avian Influenzavirus Vectorharbouring a @p repcrter g%ne.Acmrdig to FACS-aoa,ys,s al least 70 % of DC express GFP Thee-e results indicate that the cave1 influenze virus-based vector is one of the most effident transfection methods for mature human DC desaibed SO feat. lt should prove useful for in !afm studies (e.g. reverse immunology) end after some modifications for Clmlcal use as we,,
EXPRESSION OF COXSACKIE / ADENOVIRUS RECEPTOR (CAR) PREDICTS SUSCEPTABILITY TO ADENOVIYL GENE T,wNSFER IN HUMAN M~~NOMA CELL CULTURES R.1. s. HanIll F.-Y. Yue I.2 lRD&; ‘Departmen of Dermatology, University Hospllal, IGrich, Switzerland and 21nstltole 01 Molecular Biology I, Universe of Zorich. Mtich. Switzedand. Adt%wirus m&&d gene expression of immune stimulators represents a valuable in viva approach for gene therepy of human center. The expresSiOnlevel of the therapeutic gene is of cnw;Y ~rnportence for the efficacy ol this type 01 treatma EnbY of subgroup C adenovirus (AdV) ir dependent of the primary adenovws receptor CAR (Sergslson et ti., Science 1997 275:1320-1323) and the secondary AdV receptor identified eadiir to be a member of the mtegrin fami!y of surface molecules. il has been disputed whether additional pimary AdV receptors exist. We have analyzed 14 different human melanoma cell cultures from dlfferent stages end one cell line for their adenowrus-medmted transduction and expressloo effiilency. Recombinant viruses were used for expression of 87.1 costimulabxy molecule (COSO) or IL-12 under the oontrol of eaherlhe RSV. CMV or EF-1 promoter. The expression levels of 87.1 were analyzed by ROWcytometry end compared lo the expression levels of CAR and the wb and avRr, integrins. The APAAP method was used to at?&ze in bilu the expression-pattern of CAR on cryosections 01biopllc male&l from the seme melanoma patients. Culture supernetants were measured for levels of IL-12 by ELISA. In 4/14 cell cultures tested, presence of CAR correlated with high 87.1 and IL-12 expression levels et a relatively low multiplicity of infecliosity (moi) of SO. With immmohistochemistry cryosections iron tha sema patients yie&,d a strong ?@-a, specilii for CAR. Additionally. transduced melarane cells expressing both 87.1 and IL-12 strongly increased proliferation of allogeneic and autologous PSMC in cocultivatlon experiments. In contrast, cell cuilures ex&xessing very low or undetectable levels of CAR needed al least a 40. told higher nxi to show compareb!e expression of 07.1 / IL-12. Expression levels 01 the trensgenes varied only lhtle Hith difterenl promoters and no correlabon was observed with presence 01 lntegdns or HLA-I. We comlude from these date, that CAR is the relevant maker for transduction effkciency using adenowrue, et least for human melammacel~s. Preanalysis of CAR expression mey allow to adjust AdV concenlretions intended to use for treatment modalnies
s133
0793
0796
TRANSCRIPTIONAL REGULATION OF COSTlMUL4TORY MOLECULE, B7-I, EXPRESSION IN HUMAN KERATINOCYTES TREATED WIT? ALLERGENS AND IRRITANTS. P W&m. F. Ramirez. R. Bums. A. C&pau DqmQnent of Dermatology, University of Rochester School of Medicine end Dentistry: Rahester, NY. ~ttr lab has previously reported that human kemtinccytes (KC) express a low basal level of ~7-1 (CDSO)on their cell surface. which is greatly increased both in viva, on KC at sites of allergic contact dermatitis, and k vitro. on cultured KC treated with the allergen mckel chloride (Ni) or the irritant sodium lamyl sulfate (SLS). lo addition, B7-1 expression is tmnscriptically upregulated in cultured KC treated with Ni or SLS (increased levels of B7-1 RNA transcripts in nuclear mnoff experiments). We are developing an in virro assay to detect allergens and irritanrs using primary ~ultore human KC, transiently tmnsfected with lncifemse reporter vectors containing httmao B7-1 promoter segments. Transfected KC are treated with an allergen or irritant, then assayed for B7- I promoter expression, meawed as luciferase activity. An 84 bp 87-l promoter fragment expresses a basal level of luciferrtse which is not upregulated by Vestment with allergens or irritants. In contrast, a 230 bp B7- 1 promoter fragment expresses a high level of luciferase activity (-30,ooO rlu) which is upregulated (2-5 fold) in KC treated with ceti_ainallergens or irritants. Using this construct in our in vitro assay, we have tested 14 allergens and 7 irritants for their ability to upregulate B7-1 promoter fragment expression. This upmgolruion of B7-1 expression is donor-specific, i.e. KC from different donors upmgulate promoter activity in response to different chemicals. In assays of KC derived from different donors, Ni produces a positive response in over 75% of the aSsays (N=9). In addition, some allergens (mercaptobeozothiamle, cinnamic tidehyde) and irtitanls (phenol) prodwe frequent positive responses (>60%). while other allergens (trinitmchlomhenzene) and irritants (SLS) am less frequently positive (~30%). We conclude: (1) allergens and irritants have direct effects on KC B7-1 expression, (2) this assay may be the basis of a predictive test for chemicals that cause contact dermatitis.
INTERFERON ENHANCES TNF -INDUCED VCAM-I (CDl06) EXPRESSION IN HUMAN ENDOTHELIAL CELLS BY AN IRF- I DEPENDENT PATHWAY. Saia * lens Gille**. and Peter Paz&aaeQepartm~f Lechletto~~F?x!x Dermatology, Dzision of General Dermatology. University of Vienna .Medical School. Vienna, A-1090 Austria; *Boyer Center of Molecular Bmlogy, Yale University, CT, USA; **Department of Dermatology. Goethe Umversity, Frankfurt, Germany TNF and IL-1 are known to initiate endothelial VCAM-I transcnption primarily by activating N&B, which translccates to the nucleus. In addition to two NF-& elements found wthin the minimal cytokine-inducible VCAM-1 promoter, an IRF-1 element has been identified close to the transcription initiauon site, suggesting that cytokines which induce IRF-I might affect VCAM-1 expression levels. We therefore investigated the effects of interferons. which strongly induce IRF-1, on VCAM-1 transcriotion and exoression. We show that IFN-a and IF& enhance TNF-induced VCAM-I mRNA ani protein expression in human endothelial’cells. IFN enhancement of TNF-induced expression is also seen using CAT repmer genes linked to the minimal cvtokine inducible VCAM-I oromoter. Nuclear IRF-1 is the molecular basis of IFN enhancement, because I) IFN’increased nuclear IRF-1. whereas nuclear NF-& levels remained unchanged m cells treated with tFN plus TNF as compared with cells treated wth TNF alone; 2) kineucs of nuclear m-1 levels correlated with VCAM-1 mRNA levels; 3) transfection with an IRF-I construct substituted for IFN treatment; and 4) transfectton with an expression construct encoding IRF-2, a competitive inhtbitor of IRF-I. reduced TNF -Induced VCAM-I exoression. Our exoer~mentsshow that levels of VCAM-I transcribed and expressed on the cell surface a& critically correlated with IRF- I levels, thus the IFN / IRFI pathway represents a dlstmct modulator of endothelial VCAM-I expressaon.
0794
0797 ANALYSIS OF THE CONTROL OF EXPRESSION AND TISSUE SPECIFICITY OF THE HUMAN TRICHOHYALIN GENE. Kenneth Wttl, Jeanne M. Andreoli’, ChnsancM.Coticchia’, &&&a G Markov$, &er M. Steiner+. Shvh-lee Jaq’. ‘Laboratory of Skin Biology, NIAMS, NIH, Bethesda. MD, and &pamnent of Oral Biology, SUNY Stony Brook, NY, USA. Trichohyalin (‘IHH) is an intermediate filament-associated protein which is expressed primarily in the inner nmt sheath and mdulla cells of haii follicle, and also in eoidermis. hard oalate. and fdiform ridees of the toneue. Previonslv we demonstrated &at as few as 1% bp Gf sequences f&the imnsuipptin strut site &capable of driving expression of the p-galactosidase gene, and four fnoctional elements, NFkB (-86), APl (-95), et.+like (-IOO), and Spl (-122). were involved. ln the present study, we have further analyzed the consul of the u-anscri*onal activity and calcium responsiveness of the THH promoter in cultnred mouse hair follicles (mHF) or e@rmal keradoocytes (mEK). In transfected mHF, mutations of either one of these elements reduced the levels of expression by at least 50%; when combinations of two were mutated, the activity fell to near basal levels. In contrast in t&K, expression of the wildtypc C~“S~IC~was at the basal level. Fwthermore, activity levels of the wildtype construct transfected into mHF cells grown in high calcium media was IO-fold higher than in low calcium media. Whereas mutations of the Spl or APl sites had no effect mutations of either the ets-like or NFkB motifs abolished the calcium responsiveness to the basal level. Together, these data show that a synergistic interplay between Spl, a-like, APl and NFkB elements is required for the cell-type specific control of the THH promoter activity in mHP cells, and that both as-like and NFkB elements are necessay for the calcium responsiveness. Other elements located outside this proximal promoter region must be required for specific expression in mEK cells.
0798
0795 Efficient transduction influenza
at mature virus as e vector
human
dandritic
cells
by using
an avia”
I. StrobeI’. R. Grassmaw?. E. Hofmann’, G. Hoborn’, U. Schulze’. E. Niedobilek’, E Wagner’, S. Fleckenstein’ end G. Schuler’
Dendrlbc cells (DC) are anbgen pmsenbng cells with the highest capeclty to initmte primary end secondary T-cell mediated immune respansee. Most importanw, several reports indite that DC also play a crucial role rn tumor mmwne response. Therefore DC are a promising target for immune- and gene therapy of tumors. The aim of our study is the tmnsfer of different genes emx&g tomOr antigens and cytokines into DC to enhance and-lumw response. FW this purlaxe DC ware generated in viva from adherent CD14+ bkcd monocytss by a z&step method (pdming phase with GM-CSF and IL-4 followed by e cytokine-induced rnetwetion phase). Mature DC we identified by their morphology. phenotypic markers (eg. CD33+, CD86+. fesan+) and potent T-0811sensitlring capacify In a fint approach we tested different gene transfer methods using green flwrescent protein (GFP) as marker protein which can be detected in living c& by conventional fluorescence techniques. Low expressjon of GFP (~5 % Of DC) Was Obtained by receptor-mediated bansfeclion, by lipid es well es non-lipid c&ionic transfecllon techniques. Intermediate expression lewls (~20 % of DC) resulted from eledroporalion High exppssion of GFP wee found in DC after infection v.ith an avian Influenzavirus Vectorharbouring a @p repcrter g%ne.Acmrdig to FACS-aoa,ys,s al least 70 % of DC express GFP Thee-e results indicate that the cave1 influenze virus-based vector is one of the most effident transfection methods for mature human DC desaibed SO feat. lt should prove useful for in !afm studies (e.g. reverse immunology) end after some modifications for Clmlcal use as we,,
EXPRESSION OF COXSACKIE / ADENOVIRUS RECEPTOR (CAR) PREDICTS SUSCEPTABILITY TO ADENOVIYL GENE T,wNSFER IN HUMAN M~~NOMA CELL CULTURES R.1. s. HanIll F.-Y. Yue I.2 lRD&; ‘Departmen of Dermatology, University Hospllal, IGrich, Switzerland and 21nstltole 01 Molecular Biology I, Universe of Zorich. Mtich. Switzedand. Adt%wirus m&&d gene expression of immune stimulators represents a valuable in viva approach for gene therepy of human center. The expresSiOnlevel of the therapeutic gene is of cnw;Y ~rnportence for the efficacy ol this type 01 treatma EnbY of subgroup C adenovirus (AdV) ir dependent of the primary adenovws receptor CAR (Sergslson et ti., Science 1997 275:1320-1323) and the secondary AdV receptor identified eadiir to be a member of the mtegrin fami!y of surface molecules. il has been disputed whether additional pimary AdV receptors exist. We have analyzed 14 different human melanoma cell cultures from dlfferent stages end one cell line for their adenowrus-medmted transduction and expressloo effiilency. Recombinant viruses were used for expression of 87.1 costimulabxy molecule (COSO) or IL-12 under the oontrol of eaherlhe RSV. CMV or EF-1 promoter. The expression levels of 87.1 were analyzed by ROWcytometry end compared lo the expression levels of CAR and the wb and avRr, integrins. The APAAP method was used to at?&ze in bilu the expression-pattern of CAR on cryosections 01biopllc male&l from the seme melanoma patients. Culture supernetants were measured for levels of IL-12 by ELISA. In 4/14 cell cultures tested, presence of CAR correlated with high 87.1 and IL-12 expression levels et a relatively low multiplicity of infecliosity (moi) of SO. With immmohistochemistry cryosections iron tha sema patients yie&,d a strong ?@-a, specilii for CAR. Additionally. transduced melarane cells expressing both 87.1 and IL-12 strongly increased proliferation of allogeneic and autologous PSMC in cocultivatlon experiments. In contrast, cell cuilures ex&xessing very low or undetectable levels of CAR needed al least a 40. told higher nxi to show compareb!e expression of 07.1 / IL-12. Expression levels 01 the trensgenes varied only lhtle Hith difterenl promoters and no correlabon was observed with presence 01 lntegdns or HLA-I. We comlude from these date, that CAR is the relevant maker for transduction effkciency using adenowrue, et least for human melammacel~s. Preanalysis of CAR expression mey allow to adjust AdV concenlretions intended to use for treatment modalnies