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Desmoplakin is expressed in dermal microvascular endothelial cells and is coupled to VE-cadherin by plakoglobin but not β-catenin
ESDR I JSID I SID Abstracts
0139
0142
ECTOPIC WHN EXPRESSION IN TRANSGENIC MICE CAUSES DEVELOPMENTAL DEFECTS AND EPIDERMAL HYPERPLASIA. David Prowse. David Lee. Cvnthia Magm. Howard Baden and Janice Brissette. Cutaneous Biology Research Center, Massachusetts General wand Harvard Medical School, Charlestown, MA, USA. The nude mouse phenotype is characterized by the abnormal development of the epidermis, hair follicles, and thymus, and results from loss-of-function mutations in the Whn transcription factor. In previous studies, we found that the cutaneous nude phenotype is due to an intrinsic defect in the keratinocytes. To better understand Whn function in the skin, we have generated transgenic mice which strongly express the murine Whn protein from the promoter of the involucrin gene. Two classes of transgenic animals were obtained. One class exhibited a severe uhenotvoe that resulted in death shortlv after birth. while the other catkgory &played milder effects and sur;ived to adulthood. The most severely affected animals were born with open eyes and tight, shiny skin that appeared defective in its barrier function. At the histological level skins from both classes of transgenics exhibited prominent basalar hyperplasia, with architectural disarray, dysmaturation and hyperkeratosis. Collectively, these results demonstrate that suprabasal Whn expression can perturb the normal keratinocyte growth controls, and suggest a role for Whn in the etiology of hyperproliferative skin diseases.
FIBRIN ENHANCES MICROVASCULAR CELL a~433 INTBGRIN GENE EXPRESSION, MEDIATES CELL SPREADING THROUGH THE RGDS CBLL BINDING DGMAIN. AND STIMULATES CAPILLARY TUBE FORMATION Clark. and Departments of Damatology and Medicine and the Blood Center, School of Medicine. SUNY at Stony Brook, NY and Dermatology Section, VAMC. Nor&port, NY. During cutaneous wound repair the tips of angiogenic capillary sprouts express ~43 integri” receptor as they invade the provisional maaix of the clot, rich in tibrin(oge”) and tibmnectin. We have previously shown that fibm”ectin upregulates a~/733mRNA expression in human damal miaovascolar endahelial cells (BDMBC). ‘I&se studies have now been extended to address the i”ter&tion of HDMEC with the fibrin clot. To simulate the early wound environment, HDMEC gmwn on collagen were overlaid with a fibrin gel. Marked upregulation of w/p3 mRNA was dewted compared to levels in cells overlaid by a collagen gel. Fibrin gel also enhanced both bFGF and VBGF induction of p3 mRNA expression. Ovalaid fibrin gel caused HDMEC to form capiuay tube-like st~“ctwes which were enhanced by the addition of VEGF. ox!33 mediates attachment of HDMEC to substrates of fibrin en or its degradation fragments; however, these cells do not spread. In con~st, HDh2 C attached and spread on intact tibri” gels. Spreading was enhanced by the additio” of fibronectin. The RGDS cell biiding domain in the wboxyl end of the a chain of fibrinogen appearsto be criticalfor HDMEC to spread on a fibrin gel, since cells attached but did not spread on gels formed by I-9 fibrinogen, a degradation product lacking RGDS. The addition of fibmnectin to I-9 fibrinogen resulted i” CeIIattachment and partial sprc&i”g. Thus the fibri” clot may actively participate in wound repair by upregulating HDMEC a-433 mRNA expression and by facilitating cell spreading and formation of capiUay tubes. HDMEC behavior may therefore be regulated in pan by the fibrin clot during the early phase of cofaneoos wound repair.
0143 CDNA CLONING
AND PEPTIDE
FACTOR(COLFI)
WHICH
Atsushi
SEQUENCE
ANALYSIS
OF A NOVEL
BlNDS TO THE a Z(1) COLLAGEN PROMOTER. Department of Dermatology
and *Center for Biomedical Science, Chiba University School of Medicine, Chiha, Japan. Collagen type I, a most abundant protein in the dermis, consists af twwo(I l(1) chain and one a 2(I) chain which are eoerdhmtely expressed. Excessive deposition of type I collagen is characteristic of many fibrotic disorders e.g. scleroderma end most likely results from transcriptional activation of collagen genes in response to eytokincs and other factors. ColFl, a DNA binding protein which speeitically binds to a segment of the a L(I)collagen promoter at -4ClObpupstream of the start of transcription, activates transcription
of the LIZ(I)collagcn gene in vitro. We report here on the partial sequences
of pelypeptide and the cDNA cloning of this transcriptional factor of theu 2(l)eollagen gene. A nuclear extract was prepared fmm lZ6g of NM-3T3 cellr: and purified by sequence-specific DNA affinity chromatography. The purified protein were found to consist of 42kDa and 40.5 kDa polypeptides. The pmtein were digested hy lysylendopeptidase and each peptide fragment W&Ffractionated by rewrse phase HPLC, and then sequenced. Sequences of peptide fragments from 42kDa polypptide were identical to Pur (I, protein which has been reported to binds to e upstream twgion of human c-myc gene. Some of those from 40kDa pelypepttde were identical to Pur 8, has been partially sequenced and has repions of strong homoloav to Pura Full
isPnuclear
_
-
length of Pur P cDNA were sequenced. The deductive amino acid sequences of Pura and Pur 0 showed 61.4% homology. Since the novel factor specifically enhances transcription of collagen in vitro, further studies concentrate on effect of co-expression of polypeptides on collagen transcription in viva and on measurement of expression levels of the factor in tihrotic dborden.
0141
0144
EXPRESSEDIN DBRMALMICROVASCULARBNDOTJXELIAL CELLSAND IS COUPLEDTO VEXADH+BJNBY PL$xoGLOBIN BUT ‘YOT p-CATJWIN.Andrew P. Kowalczvk*. Pdar Navarro . Ehsabelta Dwna . Kathleen J. Green*. Daniel S. Kooo*. and Jeffrev E. Borawardt*. *Depts. of$ermatolagy and Pathology, Northwestern Univ. Med School, Chicago, IL and Laboratory of Vascular Biology, Mtio Negri Institute for Pharmacologxal Research, Mlla~$ Italy Vascular endothehal cells assemble adheswe intercellular Junctions comprising a unique cadherin, VEcadhetin, which is coupled to the actin cytoskeleton through cytoplasmic interactions with plakoglobin (Pg), p-catenin and a-catenin. However, the mechanisms bv which VE-cadherin is hnked to the vim&in intermediate filament cytoskelet& are not known. Recent evidence indicates that lymphatic and vascular endothelial cells express desmoplakm (DP), a desmosomal component that attaches mtemxdiate filaments to the plasma membrane. To deternune if VEcadherin can associate with DP, we co-expressed VE-cadhexin and a DP aminoterminal polypeptide @P-NT?) in Lrcell tibroblasts. In the presence of VEcadherin, both Pg and DP-NTP were recruited to cell-cell borders. However, DPNTP was not recruited to borders by a deletion mutant of VE-cadherin lacking the cytoplasmic catenin binding domain. In addition, p-catenin could not substitute for Pg in the recruitment of DP-NTP to cell borders. Furthermore, DP-NTP bound to Pg but not p-wdenin in the yeast two hybnd system. DP-NTP co-localized at cellcell borders with a-catenin in the Gcell lines, and endogenous DP and a-catenin co-localized in cultured dermal microvascular endothehal cells. This is in striking contxwt to epithelial cells, where DP and a-catenin are restricted to desmosomes and adherens junctions, respectively. These results suggest endothelial cell! assemble unique junctional complexes in which VE-cadherin-Pg complexes recnnt both actin and intermediate filament binding proteins to mtercellular junctiocs. These cytoskele~al n&motions may play Important roles m anglogene% inflammation and endothelial barrier function.
NC1 DOMAIN OF TYPE VII (ANCHORING FIBRIL) COLLAGEN BINDS TO THR 03 CHAIN OF LAMININ 5 VIA A UNIQUE SUBDOMAIN WlTHIN THE FIBRONECITN-LIKE REPEATS. M. Chea. M. P. Marlnkovich’, Jonathan CR. Jones E. A O’Toole. Y.-Y, Li, and David T Woodley Depts fD 1 Cell an d I cul Biology, Nortbwestem Univ. dhi IL and S&&d U%?%%y&ti CA $ze & (anchoring Bbdl) collagen medi%.?epi&rmal-dexmal adherence and ‘con&s of a cellfral collagenow triple-helix flanked by non-collagenous &mah~~. NC1 and NC2. NC1 contains subdomalns with homology to flhronectln-llke repeats (FIN) and the A domain of van Wlllebmnd factor (VWP-A). These subdomains may be responsible for interactions with other extracellular matrix mot&s. Uslnn full-lenntb nxombinant NC1 produced from stably aansfected human 293 cells, we p&iously dgmonsaated that NC1 binds to a complex of lamloin 5 and laminin 6 (JBC 27214516. 1997). In this study, we examined the lntemctlon of the NC1 domain wkb pure isolated leminln 5 (a component of anchoring filaments). Authentic NC1 end purified rwombinam NC1 bound to human and rat lambda 5 as measured by enzyme-linked immunosorbant assay and by binding of ‘%radtolal-&?d NC1 to laminin 5.coated wells, but not to leminln albumin. NC1 bound predominantly to the 83 chain of lamioln 5 in a protein overlay assay wing electmpboretically separated laminin 5 chains. The binding of ‘%NCl to laminin 5 yllg inbiblted by a SO-fold excess of unlabeled NCl, a polyclonal antibody to landnin 5 and a monoclonal antibody to the 83 chain. In contrast, the NCI-lambdn 5 affinity war not at&ted by a monoclonal antibody to the a3 chain or a IOK-fold excess of albumin or laminin 1. Epidermolysis bullosa acquisita (EBA) autoantibodies to NC1 also abolished ule NCl-laminin 5 interaction. To further identify ECM protein biting sites within NC1. we generated a peoel of NC1 deletion mutant proteins in 293 cells. Using these mutant proteins, the 285 AAs (residues 760.1045) subdomain in the FIN of NC1 WBSidentlfled BS the binding site for laminin 5. In contrast, the 208 AAs (residues 1045-1253) subdomaln at the carboxyl-terminal of NC1 containing the VWKA motif wes the binding stte for collagen I and collagen N. We conclude that the aftinity of NC1 and lamioln 5 is mediated vie a FlN repeat submodule and the 83 chain, respenively, and pertubatlons of the NCl-lamloin 5 interaction may contribute to the patbogenesis of EBA.
DESMOPLAKlN IS
IM
0139
0142
ECTOPIC WHN EXPRESSION IN TRANSGENIC MICE CAUSES DEVELOPMENTAL DEFECTS AND EPIDERMAL HYPERPLASIA. David Prowse. David Lee. Cvnthia Magm. Howard Baden and Janice Brissette. Cutaneous Biology Research Center, Massachusetts General wand Harvard Medical School, Charlestown, MA, USA. The nude mouse phenotype is characterized by the abnormal development of the epidermis, hair follicles, and thymus, and results from loss-of-function mutations in the Whn transcription factor. In previous studies, we found that the cutaneous nude phenotype is due to an intrinsic defect in the keratinocytes. To better understand Whn function in the skin, we have generated transgenic mice which strongly express the murine Whn protein from the promoter of the involucrin gene. Two classes of transgenic animals were obtained. One class exhibited a severe uhenotvoe that resulted in death shortlv after birth. while the other catkgory &played milder effects and sur;ived to adulthood. The most severely affected animals were born with open eyes and tight, shiny skin that appeared defective in its barrier function. At the histological level skins from both classes of transgenics exhibited prominent basalar hyperplasia, with architectural disarray, dysmaturation and hyperkeratosis. Collectively, these results demonstrate that suprabasal Whn expression can perturb the normal keratinocyte growth controls, and suggest a role for Whn in the etiology of hyperproliferative skin diseases.
FIBRIN ENHANCES MICROVASCULAR CELL a~433 INTBGRIN GENE EXPRESSION, MEDIATES CELL SPREADING THROUGH THE RGDS CBLL BINDING DGMAIN. AND STIMULATES CAPILLARY TUBE FORMATION Clark. and Departments of Damatology and Medicine and the Blood Center, School of Medicine. SUNY at Stony Brook, NY and Dermatology Section, VAMC. Nor&port, NY. During cutaneous wound repair the tips of angiogenic capillary sprouts express ~43 integri” receptor as they invade the provisional maaix of the clot, rich in tibrin(oge”) and tibmnectin. We have previously shown that fibm”ectin upregulates a~/733mRNA expression in human damal miaovascolar endahelial cells (BDMBC). ‘I&se studies have now been extended to address the i”ter&tion of HDMEC with the fibrin clot. To simulate the early wound environment, HDMEC gmwn on collagen were overlaid with a fibrin gel. Marked upregulation of w/p3 mRNA was dewted compared to levels in cells overlaid by a collagen gel. Fibrin gel also enhanced both bFGF and VBGF induction of p3 mRNA expression. Ovalaid fibrin gel caused HDMEC to form capiuay tube-like st~“ctwes which were enhanced by the addition of VEGF. ox!33 mediates attachment of HDMEC to substrates of fibrin en or its degradation fragments; however, these cells do not spread. In con~st, HDh2 C attached and spread on intact tibri” gels. Spreading was enhanced by the additio” of fibronectin. The RGDS cell biiding domain in the wboxyl end of the a chain of fibrinogen appearsto be criticalfor HDMEC to spread on a fibrin gel, since cells attached but did not spread on gels formed by I-9 fibrinogen, a degradation product lacking RGDS. The addition of fibmnectin to I-9 fibrinogen resulted i” CeIIattachment and partial sprc&i”g. Thus the fibri” clot may actively participate in wound repair by upregulating HDMEC a-433 mRNA expression and by facilitating cell spreading and formation of capiUay tubes. HDMEC behavior may therefore be regulated in pan by the fibrin clot during the early phase of cofaneoos wound repair.
0143 CDNA CLONING
AND PEPTIDE
FACTOR(COLFI)
WHICH
Atsushi
SEQUENCE
ANALYSIS
OF A NOVEL
BlNDS TO THE a Z(1) COLLAGEN PROMOTER. Department of Dermatology
and *Center for Biomedical Science, Chiba University School of Medicine, Chiha, Japan. Collagen type I, a most abundant protein in the dermis, consists af twwo(I l(1) chain and one a 2(I) chain which are eoerdhmtely expressed. Excessive deposition of type I collagen is characteristic of many fibrotic disorders e.g. scleroderma end most likely results from transcriptional activation of collagen genes in response to eytokincs and other factors. ColFl, a DNA binding protein which speeitically binds to a segment of the a L(I)collagen promoter at -4ClObpupstream of the start of transcription, activates transcription
of the LIZ(I)collagcn gene in vitro. We report here on the partial sequences
of pelypeptide and the cDNA cloning of this transcriptional factor of theu 2(l)eollagen gene. A nuclear extract was prepared fmm lZ6g of NM-3T3 cellr: and purified by sequence-specific DNA affinity chromatography. The purified protein were found to consist of 42kDa and 40.5 kDa polypeptides. The pmtein were digested hy lysylendopeptidase and each peptide fragment W&Ffractionated by rewrse phase HPLC, and then sequenced. Sequences of peptide fragments from 42kDa polypptide were identical to Pur (I, protein which has been reported to binds to e upstream twgion of human c-myc gene. Some of those from 40kDa pelypepttde were identical to Pur 8, has been partially sequenced and has repions of strong homoloav to Pura Full
isPnuclear
_
-
length of Pur P cDNA were sequenced. The deductive amino acid sequences of Pura and Pur 0 showed 61.4% homology. Since the novel factor specifically enhances transcription of collagen in vitro, further studies concentrate on effect of co-expression of polypeptides on collagen transcription in viva and on measurement of expression levels of the factor in tihrotic dborden.
0141
0144
EXPRESSEDIN DBRMALMICROVASCULARBNDOTJXELIAL CELLSAND IS COUPLEDTO VEXADH+BJNBY PL$xoGLOBIN BUT ‘YOT p-CATJWIN.Andrew P. Kowalczvk*. Pdar Navarro . Ehsabelta Dwna . Kathleen J. Green*. Daniel S. Kooo*. and Jeffrev E. Borawardt*. *Depts. of$ermatolagy and Pathology, Northwestern Univ. Med School, Chicago, IL and Laboratory of Vascular Biology, Mtio Negri Institute for Pharmacologxal Research, Mlla~$ Italy Vascular endothehal cells assemble adheswe intercellular Junctions comprising a unique cadherin, VEcadhetin, which is coupled to the actin cytoskeleton through cytoplasmic interactions with plakoglobin (Pg), p-catenin and a-catenin. However, the mechanisms bv which VE-cadherin is hnked to the vim&in intermediate filament cytoskelet& are not known. Recent evidence indicates that lymphatic and vascular endothelial cells express desmoplakm (DP), a desmosomal component that attaches mtemxdiate filaments to the plasma membrane. To deternune if VEcadherin can associate with DP, we co-expressed VE-cadhexin and a DP aminoterminal polypeptide @P-NT?) in Lrcell tibroblasts. In the presence of VEcadherin, both Pg and DP-NTP were recruited to cell-cell borders. However, DPNTP was not recruited to borders by a deletion mutant of VE-cadherin lacking the cytoplasmic catenin binding domain. In addition, p-catenin could not substitute for Pg in the recruitment of DP-NTP to cell borders. Furthermore, DP-NTP bound to Pg but not p-wdenin in the yeast two hybnd system. DP-NTP co-localized at cellcell borders with a-catenin in the Gcell lines, and endogenous DP and a-catenin co-localized in cultured dermal microvascular endothehal cells. This is in striking contxwt to epithelial cells, where DP and a-catenin are restricted to desmosomes and adherens junctions, respectively. These results suggest endothelial cell! assemble unique junctional complexes in which VE-cadherin-Pg complexes recnnt both actin and intermediate filament binding proteins to mtercellular junctiocs. These cytoskele~al n&motions may play Important roles m anglogene% inflammation and endothelial barrier function.
NC1 DOMAIN OF TYPE VII (ANCHORING FIBRIL) COLLAGEN BINDS TO THR 03 CHAIN OF LAMININ 5 VIA A UNIQUE SUBDOMAIN WlTHIN THE FIBRONECITN-LIKE REPEATS. M. Chea. M. P. Marlnkovich’, Jonathan CR. Jones E. A O’Toole. Y.-Y, Li, and David T Woodley Depts fD 1 Cell an d I cul Biology, Nortbwestem Univ. dhi IL and S&&d U%?%%y&ti CA $ze & (anchoring Bbdl) collagen medi%.?epi&rmal-dexmal adherence and ‘con&s of a cellfral collagenow triple-helix flanked by non-collagenous &mah~~. NC1 and NC2. NC1 contains subdomalns with homology to flhronectln-llke repeats (FIN) and the A domain of van Wlllebmnd factor (VWP-A). These subdomains may be responsible for interactions with other extracellular matrix mot&s. Uslnn full-lenntb nxombinant NC1 produced from stably aansfected human 293 cells, we p&iously dgmonsaated that NC1 binds to a complex of lamloin 5 and laminin 6 (JBC 27214516. 1997). In this study, we examined the lntemctlon of the NC1 domain wkb pure isolated leminln 5 (a component of anchoring filaments). Authentic NC1 end purified rwombinam NC1 bound to human and rat lambda 5 as measured by enzyme-linked immunosorbant assay and by binding of ‘%radtolal-&?d NC1 to laminin 5.coated wells, but not to leminln albumin. NC1 bound predominantly to the 83 chain of lamioln 5 in a protein overlay assay wing electmpboretically separated laminin 5 chains. The binding of ‘%NCl to laminin 5 yllg inbiblted by a SO-fold excess of unlabeled NCl, a polyclonal antibody to landnin 5 and a monoclonal antibody to the 83 chain. In contrast, the NCI-lambdn 5 affinity war not at&ted by a monoclonal antibody to the a3 chain or a IOK-fold excess of albumin or laminin 1. Epidermolysis bullosa acquisita (EBA) autoantibodies to NC1 also abolished ule NCl-laminin 5 interaction. To further identify ECM protein biting sites within NC1. we generated a peoel of NC1 deletion mutant proteins in 293 cells. Using these mutant proteins, the 285 AAs (residues 760.1045) subdomain in the FIN of NC1 WBSidentlfled BS the binding site for laminin 5. In contrast, the 208 AAs (residues 1045-1253) subdomaln at the carboxyl-terminal of NC1 containing the VWKA motif wes the binding stte for collagen I and collagen N. We conclude that the aftinity of NC1 and lamioln 5 is mediated vie a FlN repeat submodule and the 83 chain, respenively, and pertubatlons of the NCl-lamloin 5 interaction may contribute to the patbogenesis of EBA.
DESMOPLAKlN IS
IM