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DNA synthesis in cultured human keratinocytes and HaCaT keratinocytes is reduced by specific inhibition of dipeptidylpeptidase IV (CD26) activity
ESDR I JSID I SID Abstracts
0496 DEFINED ORGANOTYPIC COCULTURES OF NORMAL HUMAN KERATINOCYTES AND FIBROBLASTS EXERTING REGULAR EPIDERMAL MORPHOGENESIS. m Baud. D. Breilkreutz. N_Mirancea”. N F [email protected]_Dlvlslon 0240. Germen Cancer Rese&h Center. Heidelberg, Germany. ‘NE&% Research Center. Leusenne. Switzerland. “Romanian Academy. Institute of Biology, Bucharest, Romania. Skin equivalents formed by keretinwytes grow air exposed on lop of cdlegen gels with embedded fibroblasls represent promising twls for phammmlogical testing and experimental analysis. ln this context defined media would help to avoid Interferences with ill aefined factors of common cultire supplements such es serum. Herein we have studied Ifs defined medium (SKDM) could sup+mrl equally well epidermal marphogenesis es serum ccnteining medium (FAD) in wganotyplc wcullures. For this purpose we examined hallmarks of the epidermal phenotype by immunoRuorescence and electmn microscopy. Bolh the MCDB-IS3 based defined SKDM and FAD with 10% serum odginelly developed for keratiwxytes allowed also flbmblest growth e1 1.3 or 1.8 mM Ca” respectiv~y. In both cases regularly struclwed. orthokeretlnlred epithelis evolved with similar kinetics, while in FAD the mwpholcgy was sllghlly hypwplastic. Furthermore. in FAD the sywfhesis of bratins Ki and KIO wes less coordinated and delayed in contrast to dearly supra-basal localizeSon in SKDM. The late differentiation markers Baggrin. involuain. loriain K2e and lrensgluteminase corresponded in meir regular dislribution in upper suprebasal layers. K16 persisted under both conditions resembling the activated stale in epidermsl regeneration. At the ullreslmdurel lwel reconstltullon of basement membrane and archlfecfwel feelures ofepidermal tiiwtiation became evident in bofh media. Hmvevsrimmature lamellete bodies and cytoplasmic vesides probably ldglyceride droplels - indicated impaired lipid metabolism in SKDM. Nevertheless also in SKDM abundanl stellate keretohyslin granules were observed together with ccmifled envelopes and en electron dense stnetum comeurn. One possible ress~n for me more nomml status in lhe serum free svstem mav be lhe better ccmlrol of flbroblsst ordifersoon. Ths mav omvide e better basis for hbmeostsds and prolonged lifespan of organ&ypic wcultures. This: the direct inRuence on fibmblest growth end activity might be en addtiional promising appmach to modulate epidermel development end e starting point to optimize tie system further.
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0494
0497
NITRIC OXIDE PRODUCTION AND DIFF-EP.ENTIAL CELLULAR LOCALIZATION OF NITRIC OXIDE SYNTHASES IN HUMAN KERATINOCYTES. Peter K. PhiIio B. A. Our&i. &aH I&m aan A. Lerne?&nco”s B-Center. Dcpart~cnt Ef Dermatology. Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA. Nitric oxide (NO) is an important molecule in many biological systems. However, its role in cutaneous biology is relatively unknown. We studled the production of NO and the enzymes involved using BEKOOl, a human kcradnocyte line transformed with an HPV16 E6/E7 plasmid. Human keratinocytcs produce both constituitive brain nitric oxide syntbase (bNOS) and the inducible form (iNOS). As measured by a Griess colorime.tric assay, NO level was increased when HEKOLll cells were stimulated bv both LPS and interferon-gamma. NOS enzymatic assay examining 3H-Lag&e conversion to citmlliic showed increased NOS activitv in stimulated cells Induced NO synthesis was secondary to increased iradscription and translation of iNOS and not bNOS BS determined by RT-PCR and immunostaining. Stimulated HEKOOl cells labeled with anti-iNOS-FlTC monoclonal antibody showed a shift in fluorescence by FACS analysis compared with unstimulated cells. Examination by confocal laser microscopy showed that bNOS was constituitively present and located in the nucleus or nuclear membrane. In contrast, iNOS was expressed in the cytosol or perinuclear region of stimulated cells. These findings suggest that bNOS and iNOS have variable nuclear and cytoplasmic functions.
ANALYSIS OF INTEGRlN AND BASEMENT MEMBRANE COMPONENT EXPRESSION IN THE UVING SKIN EQUIVALENT. R. O’Learv. M. Arrowsmith. E.I. Wood. School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK and 3M Health Care Ltd. Morley House, Morley Street, Loughbomugh, UK. Organotypic systems such as the living skin equivalent &SE) have been used both for tissue engineered therapy and basic research into fields such ss wound healing and keratinocyte differentiation. The lSE is formed by culturing, at the airliquid Interface, keratinocytes seeded onto a Rbmblast populated type I collagen lattice. This creates an epidermis with morphology very close to that of skin in viva. However, the morphological characteristics of such models do not provide sufficient detail with which to decide how faithfully the LSE repmduces keratlnocvte behaviour in normal skin. In order to beoin to dissect the molecular architect&a of the LSE compared to skin, we have &alysed the lntegrln profile and basement membrane composition of the LSE durlnq development of the epidermis. Skin equivalents were maintained at the air liquid interface for 10 days, and hlgh levels of expression of integrins a&, a&, a$?,, @+ a& and a& were observed throughout epidermal development. After 10 days of exposure to air all integrins apD@ared onlv in the basal laver of the eoidermis and were sharolv locaiised to’tire basal pile. These results 6re in contra& to normal skin where i$;, a& and a& are expressed at high levels whereas ay& and a& are present at very low levels, although as in thi LSE all intagrln expression &wrs dnly in the basal layer. Additionally keratinocytas were shown to express laminin-1 and type VI collagen, although at levels significantly below those observed in viva These results indicate that whilst the LSE does share a number of characteristics with normal skin, there an important differences which may indicate similwities to hyperpmliferative or ‘activated” keratinocytes such as those in monolayer culture and disease.
0495
0498
METHOTREXATE (MTX) INDUCES APOPTOSIS IN KERATINOCYTES. &gi.@ Barrv. Steohan Niland. Ania Cremer and Gustav Mabrle, Department of Dermatology. University of Cologne, Cologne, Germany. Recently we reported on the expression of ICAM-I on the surface of keratinocytes by cytostatic agents including MTX, which may aid Tlymphocytes in rapidly detecting damaged keratinocytes (JID 104:660,1995). The present study shows fhat MTX may also induce apoptosis in keratinocytes. HaCaT keratinocytes were studled by the cell death detection (CDD) ELISA. which detects monoand oligonucleosomes with antibodies aeainst nucelosomal DNA and histones. MTX induced awxtosis is concentration (0.001-0.1 mM/l) and time-dependent (lo-24 h). In this concentration range, there was at leasf a twofold steeper increase of apoptotic DNA damage compared to the plasma membrane damage as revealed by the LDH release assay. Apoptotic cells were stained by the TUNEL techniaue. The aearosc eel electrouhoresis of MTX treated keratinocytes revealed the typical ladder-iike DNA fragmentation. Cycloheximide (2100 ng/ml) and EGF (~0.01 “g/ml) inhibited MTX induced apoptosis concentration dependently. IFN-gamma (~500 U/ml, 24 h) stimulated MTX induced apoptosis, but the addition of the antioxidant Trolox (2 0.5 mM) inhibited this effect. Thus MTX may have various effects on keratinocytes, besides the inhibition of cell growth, the support of the interaction with lymphocytes and the induction of apoptosis.
DNA SYNTHESIS IN CULTURED HUMAN KERATIh’OCYTES AN’D HACAT KERATINOCYTES IS REDUCED BY SPECIFIC INHIBITION OF DIP!*‘TIDFEPT*IDASE IV ‘$:26’ ACTIVITY. & Vetter*. 9 d F. B~*.*Depwtment of
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Dermatolo~v and Venereolo~~. **Institute of Eaoerimental Internal Medicine,&&-wn-Guericke-U&emity, Magdebwg, G&many. Dipqtidylpeptidsse IV (DP IV, CD26, E.C.3.4.14.6) is a txansmembrane exopeptidase that is present on most mammalian cells in&ding keratinocytes. DP IV catalyses the hydrolytic cleavage of N-terminal XaaPro- and Xaa-Ala- dipeptides from peptides. Using specific inhibitors of DP IV, it has been demonstrated that DP IV is involved inthe r&ationof DNA synthesis and in pmdwtionof various cytokines in lymphocytes. Aim of our project was to investigate how and to what extent DNA synthesis of human keratinocytes is intluencedby the enzymatic activity of DP IV. CD26 was detected using flow cytometry, RT-PCR and immunoblotting (SDS-PAGE and westernblot), specific enzymatic activity calorimetric assays and fluorescence microscopy. Expression of DP IV is found on all primary keratinocytes and HaCaT keratinocytes. The synthetic DP IV inhibitors Lys[Z(NOB)] -thiamlidide and -pyrmlidide directly suppress DNA synthesis of these keratimxytes in a dose-dependent manner. These data demonstrate that CD26 is also involved in regulation of DNA synthesis of keratinocytes and that the enzymatic activity is requiredfor mediating these effects. Thus, DP IV of keratinocytes could play a role in hyperproliferative skin diseases. Moreover, HaCaT cells, representing a wellatablished hyperpmliferative keratinocyte cell line, could serve as a promising model for further inwsti@tion.
0496 DEFINED ORGANOTYPIC COCULTURES OF NORMAL HUMAN KERATINOCYTES AND FIBROBLASTS EXERTING REGULAR EPIDERMAL MORPHOGENESIS. m Baud. D. Breilkreutz. N_Mirancea”. N F [email protected]_Dlvlslon 0240. Germen Cancer Rese&h Center. Heidelberg, Germany. ‘NE&% Research Center. Leusenne. Switzerland. “Romanian Academy. Institute of Biology, Bucharest, Romania. Skin equivalents formed by keretinwytes grow air exposed on lop of cdlegen gels with embedded fibroblasls represent promising twls for phammmlogical testing and experimental analysis. ln this context defined media would help to avoid Interferences with ill aefined factors of common cultire supplements such es serum. Herein we have studied Ifs defined medium (SKDM) could sup+mrl equally well epidermal marphogenesis es serum ccnteining medium (FAD) in wganotyplc wcullures. For this purpose we examined hallmarks of the epidermal phenotype by immunoRuorescence and electmn microscopy. Bolh the MCDB-IS3 based defined SKDM and FAD with 10% serum odginelly developed for keratiwxytes allowed also flbmblest growth e1 1.3 or 1.8 mM Ca” respectiv~y. In both cases regularly struclwed. orthokeretlnlred epithelis evolved with similar kinetics, while in FAD the mwpholcgy was sllghlly hypwplastic. Furthermore. in FAD the sywfhesis of bratins Ki and KIO wes less coordinated and delayed in contrast to dearly supra-basal localizeSon in SKDM. The late differentiation markers Baggrin. involuain. loriain K2e and lrensgluteminase corresponded in meir regular dislribution in upper suprebasal layers. K16 persisted under both conditions resembling the activated stale in epidermsl regeneration. At the ullreslmdurel lwel reconstltullon of basement membrane and archlfecfwel feelures ofepidermal tiiwtiation became evident in bofh media. Hmvevsrimmature lamellete bodies and cytoplasmic vesides probably ldglyceride droplels - indicated impaired lipid metabolism in SKDM. Nevertheless also in SKDM abundanl stellate keretohyslin granules were observed together with ccmifled envelopes and en electron dense stnetum comeurn. One possible ress~n for me more nomml status in lhe serum free svstem mav be lhe better ccmlrol of flbroblsst ordifersoon. Ths mav omvide e better basis for hbmeostsds and prolonged lifespan of organ&ypic wcultures. This: the direct inRuence on fibmblest growth end activity might be en addtiional promising appmach to modulate epidermel development end e starting point to optimize tie system further.
I
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0494
0497
NITRIC OXIDE PRODUCTION AND DIFF-EP.ENTIAL CELLULAR LOCALIZATION OF NITRIC OXIDE SYNTHASES IN HUMAN KERATINOCYTES. Peter K. PhiIio B. A. Our&i. &aH I&m aan A. Lerne?&nco”s B-Center. Dcpart~cnt Ef Dermatology. Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA. Nitric oxide (NO) is an important molecule in many biological systems. However, its role in cutaneous biology is relatively unknown. We studled the production of NO and the enzymes involved using BEKOOl, a human kcradnocyte line transformed with an HPV16 E6/E7 plasmid. Human keratinocytcs produce both constituitive brain nitric oxide syntbase (bNOS) and the inducible form (iNOS). As measured by a Griess colorime.tric assay, NO level was increased when HEKOLll cells were stimulated bv both LPS and interferon-gamma. NOS enzymatic assay examining 3H-Lag&e conversion to citmlliic showed increased NOS activitv in stimulated cells Induced NO synthesis was secondary to increased iradscription and translation of iNOS and not bNOS BS determined by RT-PCR and immunostaining. Stimulated HEKOOl cells labeled with anti-iNOS-FlTC monoclonal antibody showed a shift in fluorescence by FACS analysis compared with unstimulated cells. Examination by confocal laser microscopy showed that bNOS was constituitively present and located in the nucleus or nuclear membrane. In contrast, iNOS was expressed in the cytosol or perinuclear region of stimulated cells. These findings suggest that bNOS and iNOS have variable nuclear and cytoplasmic functions.
ANALYSIS OF INTEGRlN AND BASEMENT MEMBRANE COMPONENT EXPRESSION IN THE UVING SKIN EQUIVALENT. R. O’Learv. M. Arrowsmith. E.I. Wood. School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK and 3M Health Care Ltd. Morley House, Morley Street, Loughbomugh, UK. Organotypic systems such as the living skin equivalent &SE) have been used both for tissue engineered therapy and basic research into fields such ss wound healing and keratinocyte differentiation. The lSE is formed by culturing, at the airliquid Interface, keratinocytes seeded onto a Rbmblast populated type I collagen lattice. This creates an epidermis with morphology very close to that of skin in viva. However, the morphological characteristics of such models do not provide sufficient detail with which to decide how faithfully the LSE repmduces keratlnocvte behaviour in normal skin. In order to beoin to dissect the molecular architect&a of the LSE compared to skin, we have &alysed the lntegrln profile and basement membrane composition of the LSE durlnq development of the epidermis. Skin equivalents were maintained at the air liquid interface for 10 days, and hlgh levels of expression of integrins a&, a&, a$?,, @+ a& and a& were observed throughout epidermal development. After 10 days of exposure to air all integrins apD@ared onlv in the basal laver of the eoidermis and were sharolv locaiised to’tire basal pile. These results 6re in contra& to normal skin where i$;, a& and a& are expressed at high levels whereas ay& and a& are present at very low levels, although as in thi LSE all intagrln expression &wrs dnly in the basal layer. Additionally keratinocytas were shown to express laminin-1 and type VI collagen, although at levels significantly below those observed in viva These results indicate that whilst the LSE does share a number of characteristics with normal skin, there an important differences which may indicate similwities to hyperpmliferative or ‘activated” keratinocytes such as those in monolayer culture and disease.
0495
0498
METHOTREXATE (MTX) INDUCES APOPTOSIS IN KERATINOCYTES. &gi.@ Barrv. Steohan Niland. Ania Cremer and Gustav Mabrle, Department of Dermatology. University of Cologne, Cologne, Germany. Recently we reported on the expression of ICAM-I on the surface of keratinocytes by cytostatic agents including MTX, which may aid Tlymphocytes in rapidly detecting damaged keratinocytes (JID 104:660,1995). The present study shows fhat MTX may also induce apoptosis in keratinocytes. HaCaT keratinocytes were studled by the cell death detection (CDD) ELISA. which detects monoand oligonucleosomes with antibodies aeainst nucelosomal DNA and histones. MTX induced awxtosis is concentration (0.001-0.1 mM/l) and time-dependent (lo-24 h). In this concentration range, there was at leasf a twofold steeper increase of apoptotic DNA damage compared to the plasma membrane damage as revealed by the LDH release assay. Apoptotic cells were stained by the TUNEL techniaue. The aearosc eel electrouhoresis of MTX treated keratinocytes revealed the typical ladder-iike DNA fragmentation. Cycloheximide (2100 ng/ml) and EGF (~0.01 “g/ml) inhibited MTX induced apoptosis concentration dependently. IFN-gamma (~500 U/ml, 24 h) stimulated MTX induced apoptosis, but the addition of the antioxidant Trolox (2 0.5 mM) inhibited this effect. Thus MTX may have various effects on keratinocytes, besides the inhibition of cell growth, the support of the interaction with lymphocytes and the induction of apoptosis.
DNA SYNTHESIS IN CULTURED HUMAN KERATIh’OCYTES AN’D HACAT KERATINOCYTES IS REDUCED BY SPECIFIC INHIBITION OF DIP!*‘TIDFEPT*IDASE IV ‘$:26’ ACTIVITY. & Vetter*. 9 d F. B~*.*Depwtment of
. .
_
_
.
Dermatolo~v and Venereolo~~. **Institute of Eaoerimental Internal Medicine,&&-wn-Guericke-U&emity, Magdebwg, G&many. Dipqtidylpeptidsse IV (DP IV, CD26, E.C.3.4.14.6) is a txansmembrane exopeptidase that is present on most mammalian cells in&ding keratinocytes. DP IV catalyses the hydrolytic cleavage of N-terminal XaaPro- and Xaa-Ala- dipeptides from peptides. Using specific inhibitors of DP IV, it has been demonstrated that DP IV is involved inthe r&ationof DNA synthesis and in pmdwtionof various cytokines in lymphocytes. Aim of our project was to investigate how and to what extent DNA synthesis of human keratinocytes is intluencedby the enzymatic activity of DP IV. CD26 was detected using flow cytometry, RT-PCR and immunoblotting (SDS-PAGE and westernblot), specific enzymatic activity calorimetric assays and fluorescence microscopy. Expression of DP IV is found on all primary keratinocytes and HaCaT keratinocytes. The synthetic DP IV inhibitors Lys[Z(NOB)] -thiamlidide and -pyrmlidide directly suppress DNA synthesis of these keratimxytes in a dose-dependent manner. These data demonstrate that CD26 is also involved in regulation of DNA synthesis of keratinocytes and that the enzymatic activity is requiredfor mediating these effects. Thus, DP IV of keratinocytes could play a role in hyperproliferative skin diseases. Moreover, HaCaT cells, representing a wellatablished hyperpmliferative keratinocyte cell line, could serve as a promising model for further inwsti@tion.