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elafin is related to the degree of differentiation of squamous cell carcinoma
30
Oct. ZB/Concarrent Session 10 - Keratinocyte Biology and Biochemistry
126
129
EXPRESSION OF THE SERINE PROTEINASE INHIBITOR SKALPIELAFIN IS RELATED TO THE DEGREE OF DIFFERENTIATION OF SQUAMOUS CELL CARCINOMA. Hans A.C. Alkemade. Hem! O.F. Molhuiren, lvonne M.J.J. van Vlllmen-Willems. Urban J.G.M. van Ha&t’ and Joosi Schalkwilk. Depts. of Dermatology and ‘Pathology, University Hospital Nijmegen, The Netherlands. Skin-derived antlleukoproteinase (SKALP)/Elafin is a wine proteinase inhibitor that specifically inhlblts elastase end proteinase 3 with high affinity. SKALP/elafin IS expressed in lesional psoriatic epidermis and in epidermis following injury. but not in normal epidermis. Recently. we demonstrated a differential expression of SKALP/elafin in several types of human epidermai tumors. Since a relation between SKALP/elafin expression and extent of differentiation of the tumor cells was suggested we westigated its expressloo in a large number of squemoue cell carcinomas (SCC) using immunohistology and in situ hybridization. 1. At the protein level. well-differentiated SCC showed generally the highest expression; moderately-differentiated SCC showed SKALP/elafin expression in pans of the tumor fields that showed keratinization, and in the cells showing individual keratinization es well. In poody-differenbated (parts of the) SCC. no SKALP/elafin was demonstrated using immunohistology. 2. At the mRNA level these flndings were confirmed by non-radIoactive in situ hybridization. SKALP/Elalin may interfere with proteolytic activity of tumor cells or infiltrating InflemmetOry cells, and we hypothesize that within the spectrum of SCC wth varying degrees of differentlatlon. a progressive loss of SKALP/elafin expressjon could facil#.ete tumor invasion.
AND MOLECULAR CHARACTERIZATION OF PURIFICATION SKALP/ELAf=lN. AN ELASTASE INHIBITOR WITH TRANSGLUTAMINASE SUBSTRATE MOTIFS Hcnn 0.F Molhuizen. Hens A.C. Alkemade. Patnck L.J.M. Zeeuwen. Gil!, J. de Jongh, Be W~ennra’, and Jowl Schalkwik, Department of Dermatology, Acadcmrc Hospital. and the *Department of Cell Biology and Histology, Faculty of Medune. University of NiJmegen. Nljmegen. The Netherlands. SKALP (skin-derived antileukopmteinase), also termed &fin. 8s a proteinase inhibitor found in psoriadc epidemxs as P shon polypeptide of 6 kDa. A luger molecule with the same biochemical ch;tiacteristics as SKALPielafin was Shown to be present m cultured human keratmocytes. Purification and N-terminal sequencing of this molecule and the cloning of it> cDNA revealed the exlstrnce of a 117 xnmo acid precursor molecule Cleavage of an hydrophobic signal requence of 22 amino acids resuhs in a 9.9 kDa mature protein (95 ammo acids). Tlus mature protein appeared to contain a domain with putative Vansglutaminase substmte motifs in addnion to the known proieinase inhlbitmg domain of SKALP/elafin. Three different stratagies were followed to show that this domain can act as a functional tmosglutammase substrate. The results clearly show that SKALP can Indeed become crosslinked by transglutaminase to proteins extracted from psoriatic scales. Based on the\e results we speculate that SKALP/elafin. secreted by epidermal keratinocyw in psoriatic rkm. can ~~1st both 3s a free 6 kDa form. lnrinly conswmg 01. the proteinax mhlbmng doman. and 8% a 9.9 kDa form. covalently attached 10 dw coin,f,rd an”elopc\ by tr~nsgluiamma\r cios\hnkmg
127
130 CELL:MTRIX INTERACTlONS DIRECT CoLLAGENA% AND STROMELYSIN EXPRESSION
PROCESSING OF CATHEPSINS B AND L BY ASPARTIC PROTEINASES FROM RAT EPIDERMIS.
A Kawada’, K.Hara”.
T.Matsuyana”‘,
Francisco,CA. ‘National “‘Tokyo “nlverslty. Cathepslns precursor
Oefenee Medical College,
B end L. lysosomal cystelne ot- qudermal
aspartic
on the
Proteus
extracted
rat
in 20 mMsodium phosphate buffer,
epldermls
ulth emmo”~“msulfate peaks of epldermal
and applied
aspartlc
hemoglobin hydrolyzmg wth
“nlverslty,
(cathepslns
vhlch was lnhlblted
ZmPhemArg-MCAhydrolytx
contatu,ed
pH 7.0,
actlvlty
eluted
profarms uhlch was uu,,“nablotted
L I@
The two fractions
lyzlng
actlvlty)
as
protelnases.
processing
oC
from Z-day-old
were fractionated
to an SXepharose Fast Flaw column. Three
protelnaeee
activity,
and
are sy&hesxed
protelnases
B and L was uwestigated.
telne
“Nag&l
San
forlns end processed to the mature forms by aspartx
The actloo
N&l
of California.
protex,ases.
procathepslns
BY KERATINOCYTESiiCT1”El.Y lNYOL”ED 1N WOUNDHEALING. H.G. We&w, “. Saarialho-Kere, 5. Kovacs, A. Pentland, and W.C. Parks, Dermatology Di”S., Jewish Hosp. an* Washingron ““i”. school of Med., st. LO”&., MO
K.Fu!ayema, and
A.Ishlbashl’.Dapartmenent of Dermatology. Unlverslty
E, Di.
0,)
eluted
by pepstatln
had
A. Pro-
>n 0.18 M and 0.38 M
by antrcathepsu,s
B and
uere incubated with each (9.2 mUof hemoglobin hydra-
of 3 espartlc
protelnases
L was processed to mature form by cathepsln
at ZO’C for D,,
12 h. Procathepsu,
but not by D, or E, while
procathepeln B wee converted to mature form by cathepsin D,. These flndxngs lndlcate that different processed procathepslns
cathepsin D wxxymes in rat B and L to mature form.
epldermls
~nltlally
128 ANTILEUKOPROTEASE IN HUMAN KERATINOCYTE DERIVED CELL LINES. Oliver Wuedow. Reinhard Kulke. and Enno Christoohers, Department of Dermatology, University of Kiel. K!el. Germany Potent inhlbltors of human leukocyte elastase such es elafin end antdeukoprotease can be extracted from psormtic scale material. Since keratmocytes are known producers of &fin, we were interested whether they may also be the source of antdeukoprotease in human skin. We teeted the squamous carcinoma cell lhne A431 and the basal cell carcinoma cell hne KB for their capabilitY to produce proteaee inhibltors. The A431 cell line was grown ln Dulbecco’s modlfled Eagle medium and the KB cell lme in Minimum Essential Medium, both supplemented with 10% FCS. Subconfluent cultures were washed three times with PBS and further cultured m essentially protease Inhibitor free medium for 24 h under standard culture condmons. Culture supernetants contaned inhibitors for human leukocyte elastase end cathewn G. whereas the lysed cells did only contain HLE inhibitory actiwty, but no cathepsln G inhlbltory activity. The identity of the inhibitor released by the A431 and KB cell lanes with antileukoprotease could be further substantmted by the use of en antdeukoprotease ELISA. It could be shown that A431 and KB cells contam and secrete antileukoprotease immunoreectrwty. Thts rmunoreactivtty coelutes wth the HLE plus cathepsin G inhibttory actwtY m reversed phase HPLC. Therefore, we conclude that antdeukoprotease is present m keratinocyte derived cell lanes supporting the essumpt~on that antlleukoprotease I” human skin is dewed from keratinocytes.
131
Oct. ZB/Concarrent Session 10 - Keratinocyte Biology and Biochemistry
126
129
EXPRESSION OF THE SERINE PROTEINASE INHIBITOR SKALPIELAFIN IS RELATED TO THE DEGREE OF DIFFERENTIATION OF SQUAMOUS CELL CARCINOMA. Hans A.C. Alkemade. Hem! O.F. Molhuiren, lvonne M.J.J. van Vlllmen-Willems. Urban J.G.M. van Ha&t’ and Joosi Schalkwilk. Depts. of Dermatology and ‘Pathology, University Hospital Nijmegen, The Netherlands. Skin-derived antlleukoproteinase (SKALP)/Elafin is a wine proteinase inhibitor that specifically inhlblts elastase end proteinase 3 with high affinity. SKALP/elafin IS expressed in lesional psoriatic epidermis and in epidermis following injury. but not in normal epidermis. Recently. we demonstrated a differential expression of SKALP/elafin in several types of human epidermai tumors. Since a relation between SKALP/elafin expression and extent of differentiation of the tumor cells was suggested we westigated its expressloo in a large number of squemoue cell carcinomas (SCC) using immunohistology and in situ hybridization. 1. At the protein level. well-differentiated SCC showed generally the highest expression; moderately-differentiated SCC showed SKALP/elafin expression in pans of the tumor fields that showed keratinization, and in the cells showing individual keratinization es well. In poody-differenbated (parts of the) SCC. no SKALP/elafin was demonstrated using immunohistology. 2. At the mRNA level these flndings were confirmed by non-radIoactive in situ hybridization. SKALP/Elalin may interfere with proteolytic activity of tumor cells or infiltrating InflemmetOry cells, and we hypothesize that within the spectrum of SCC wth varying degrees of differentlatlon. a progressive loss of SKALP/elafin expressjon could facil#.ete tumor invasion.
AND MOLECULAR CHARACTERIZATION OF PURIFICATION SKALP/ELAf=lN. AN ELASTASE INHIBITOR WITH TRANSGLUTAMINASE SUBSTRATE MOTIFS Hcnn 0.F Molhuizen. Hens A.C. Alkemade. Patnck L.J.M. Zeeuwen. Gil!, J. de Jongh, Be W~ennra’, and Jowl Schalkwik, Department of Dermatology, Acadcmrc Hospital. and the *Department of Cell Biology and Histology, Faculty of Medune. University of NiJmegen. Nljmegen. The Netherlands. SKALP (skin-derived antileukopmteinase), also termed &fin. 8s a proteinase inhibitor found in psoriadc epidemxs as P shon polypeptide of 6 kDa. A luger molecule with the same biochemical ch;tiacteristics as SKALPielafin was Shown to be present m cultured human keratmocytes. Purification and N-terminal sequencing of this molecule and the cloning of it> cDNA revealed the exlstrnce of a 117 xnmo acid precursor molecule Cleavage of an hydrophobic signal requence of 22 amino acids resuhs in a 9.9 kDa mature protein (95 ammo acids). Tlus mature protein appeared to contain a domain with putative Vansglutaminase substmte motifs in addnion to the known proieinase inhlbitmg domain of SKALP/elafin. Three different stratagies were followed to show that this domain can act as a functional tmosglutammase substrate. The results clearly show that SKALP can Indeed become crosslinked by transglutaminase to proteins extracted from psoriatic scales. Based on the\e results we speculate that SKALP/elafin. secreted by epidermal keratinocyw in psoriatic rkm. can ~~1st both 3s a free 6 kDa form. lnrinly conswmg 01. the proteinax mhlbmng doman. and 8% a 9.9 kDa form. covalently attached 10 dw coin,f,rd an”elopc\ by tr~nsgluiamma\r cios\hnkmg
127
130 CELL:MTRIX INTERACTlONS DIRECT CoLLAGENA% AND STROMELYSIN EXPRESSION
PROCESSING OF CATHEPSINS B AND L BY ASPARTIC PROTEINASES FROM RAT EPIDERMIS.
A Kawada’, K.Hara”.
T.Matsuyana”‘,
Francisco,CA. ‘National “‘Tokyo “nlverslty. Cathepslns precursor
Oefenee Medical College,
B end L. lysosomal cystelne ot- qudermal
aspartic
on the
Proteus
extracted
rat
in 20 mMsodium phosphate buffer,
epldermls
ulth emmo”~“msulfate peaks of epldermal
and applied
aspartlc
hemoglobin hydrolyzmg wth
“nlverslty,
(cathepslns
vhlch was lnhlblted
ZmPhemArg-MCAhydrolytx
contatu,ed
pH 7.0,
actlvlty
eluted
profarms uhlch was uu,,“nablotted
L I@
The two fractions
lyzlng
actlvlty)
as
protelnases.
processing
oC
from Z-day-old
were fractionated
to an SXepharose Fast Flaw column. Three
protelnaeee
activity,
and
are sy&hesxed
protelnases
B and L was uwestigated.
telne
“Nag&l
San
forlns end processed to the mature forms by aspartx
The actloo
N&l
of California.
protex,ases.
procathepslns
BY KERATINOCYTESiiCT1”El.Y lNYOL”ED 1N WOUNDHEALING. H.G. We&w, “. Saarialho-Kere, 5. Kovacs, A. Pentland, and W.C. Parks, Dermatology Di”S., Jewish Hosp. an* Washingron ““i”. school of Med., st. LO”&., MO
K.Fu!ayema, and
A.Ishlbashl’.Dapartmenent of Dermatology. Unlverslty
E, Di.
0,)
eluted
by pepstatln
had
A. Pro-
>n 0.18 M and 0.38 M
by antrcathepsu,s
B and
uere incubated with each (9.2 mUof hemoglobin hydra-
of 3 espartlc
protelnases
L was processed to mature form by cathepsln
at ZO’C for D,,
12 h. Procathepsu,
but not by D, or E, while
procathepeln B wee converted to mature form by cathepsin D,. These flndxngs lndlcate that different processed procathepslns
cathepsin D wxxymes in rat B and L to mature form.
epldermls
~nltlally
128 ANTILEUKOPROTEASE IN HUMAN KERATINOCYTE DERIVED CELL LINES. Oliver Wuedow. Reinhard Kulke. and Enno Christoohers, Department of Dermatology, University of Kiel. K!el. Germany Potent inhlbltors of human leukocyte elastase such es elafin end antdeukoprotease can be extracted from psormtic scale material. Since keratmocytes are known producers of &fin, we were interested whether they may also be the source of antdeukoprotease in human skin. We teeted the squamous carcinoma cell lhne A431 and the basal cell carcinoma cell hne KB for their capabilitY to produce proteaee inhibltors. The A431 cell line was grown ln Dulbecco’s modlfled Eagle medium and the KB cell lme in Minimum Essential Medium, both supplemented with 10% FCS. Subconfluent cultures were washed three times with PBS and further cultured m essentially protease Inhibitor free medium for 24 h under standard culture condmons. Culture supernetants contaned inhibitors for human leukocyte elastase end cathewn G. whereas the lysed cells did only contain HLE inhibitory actiwty, but no cathepsln G inhlbltory activity. The identity of the inhibitor released by the A431 and KB cell lanes with antileukoprotease could be further substantmted by the use of en antdeukoprotease ELISA. It could be shown that A431 and KB cells contam and secrete antileukoprotease immunoreectrwty. Thts rmunoreactivtty coelutes wth the HLE plus cathepsin G inhibttory actwtY m reversed phase HPLC. Therefore, we conclude that antdeukoprotease is present m keratinocyte derived cell lanes supporting the essumpt~on that antlleukoprotease I” human skin is dewed from keratinocytes.
131