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Integrins, cell matrix interactions and cell migration strategies: Fundamental differences in leukocytes and tumor cells
ESDR I JSID I SID Abstracts
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CONTRIBUTION OF DERMAL COMPARTMENT TO SYNTHESIS AND ASSEMBLY OF BASEMENTMEMBRANE PROTEOGLYCANS INWVO. YasuhiroYamane ,1”31, .John R Couchman’“, Hidao Yamta “) Department of Dermatology, Higashi-Saitama Hospfal, Satama,Japan”‘. Department of Cell Biology, University of Alabama at Birmmgham Aluhama.U S.A 121Department of Dermatology. J&x Medical School, Tachlgl .Japan (” Prevmuslv we demonstrated that basemsnt membrane moteoelvcans (BMPGs) ban&an and perleeanare of epidemxd or&in in de~~al.e~~dermal~unctian (DEJ) of the slim However. r&s of dermal conmartment on avnthesis and assemblv of BM-PGs are not fully understmd Therefore, m thi study, we i&st@ed whether ti dermal compartment could dxectly contnbute the syntheslv and assemblyof Bhl.PGsm DEJ Tw ce” hne cells, DJM., cells and SCC-1 cells (both were dewed from human squamous cell carcmoma of skin) were xenografted in nude rats and nude mice by mjeclmg the cell ruspensmn mtracutaneously After 7.12 days, sahd tumors of 0 5 to 1 cm m diameter were obtamed and the xenografis were waned wth specms.specitic monoclonal antlbodxq reaogmzing rat barnscan, mouse perlecan mouse laminm.1 and mouse entactin The immunatluorescence staming of xenografts m nude rats with anti-rat bamacan revealed that in ncnograftr ofDJM.1 cells, m, bamacan was prosent in the basement membrane surrounding tumor&l nests (BM.0 in acontinuous andhnear fashmn BM-tcontainedrat bamacan in xenografts of SCC-1 cells NS well, but discontinuously and less in intensxy. In xenografts of tumor cells in nude mice, moune perlecan ,vas deposIted m the interstrtml stmma mtensoly and, m parts. in a fibrillarpattern and was absent or ,at best, sparse m BM.1 In co,,trast, mouse lamuun-l and mouse entadm were present manly m BM.t These resulta suggest 1) Dermnlcells in tumor stroms could synthesize bamacan invwo and bamacan produced by mesenchymal cells could be assembled m basement membrane (BM) and, therefore, dermal compartment may contribute directly tc the synthew and assembly of bamacan of BM of DEJ m normal skm 2) The capacity for the assembly of bamacan d&red among tumor cells 3) Perlecan 1s synthesized by mesonchymal cells but IS not assembled in BM.t resultmg m mtense depasltmn in stroma
REGULATIONOF COLLAGENAND COLLAGENASEGENE EXPRESSION IN HUMAN FIBROBLASTS BY MONOCYTE CHEMOAlTRA~ANT PROTEIN-1 (MCP-1). Toshivuki Yamamoto. Karin Hartmann. Bests Eekea and Thomas
0740 ,N,WXD,S, CELL MATRlX
Krieg, Department of Dermatology, University of Cologne, Cologne, Germany. Monocyte chemoattractant protein-l (MCP-1) has recently been suggeested to play a role in experimental murine pulmonary fibrosis or murine interstitial kidney fibrosis by inducing collagen synthesis. Excessive deposition of connective tissue, however, is the result of an imbalance between synthesis and degradation. We therefore intended ta characterize the influence of MCP-1 sy&natieally at both levels in the human system, and examined the effect of MCP-1 on collagen and collagenase mFtNA expression in primary human dermal fibroblasts and in the human tibroblast cell line Wi-26/SV-40 (derived from human lung). Confluent cultures of tibroblasts were incubated with different concentrations of MCP-1 (l-50 n&n11 for 6-48 hours, and total RNA was isolated. Northern blot hvbridization revealed that not onlv collawn. but also matrix metalloprotein&l (MMP-1) mRNA was u&gul~ted’ by stmmlation with MCP-1 in a dose-dependent manner. Expression of collagen gene was upregulated at 48 hours in both tibroblasts. In contrast, MMP-1 expression was markedly enhanced by MCP-1 at 24 hours in dermal fibrohlasts only. In addition, ILla gene expression was also upregulated at 24 hours by Incubation with MCP-1 (10 “g/ml). Our results suggest that MCP-1 also affects metalloproteinase expression and can be involved in human fibrotic processes. In addition, the data indicate that the effect of MCP-1 on fibroblasts may be mediated by &I.
0743
WTERACTlONS AND CELL MIGRATION STRATEGIES: FUNDAMENTAL DIFFERENCES M LEUKOCYTES ANDTUMORCELLS.PeterFriedl, Eva-B.Briicker. and K. S. Zjinker’. Deparbnent ofDermatology, University of WUrzburg, and the ‘Institute of hnmunology, University of Wiierdecke, Germany The principles determining the migration of different cell types may result from their differences in origin, size and shape, function of adhesion receptors, and envimnmental factors, including the extracellular matrix. Polarizal leukocytes (T lymphocytes and dendritic cells) migrating in three-dimensional collagen lattices are small developing a highly dynamic leading edge and a trailing umpcd, whereas invasive melanoma cells are larger, highly polarized and less dynamic. In contmst to leukocytes, tumor cells may additionally develop migrating cell clusters maintaining intense cell-cell-interaction and cluster polarity. Leukocytes show a speed-oriented, oscillating and directionally unpredictable path profile strongly guided by matrix fibers, while melanoma cells and migrating cell clusters exhibit slow yet highly directional migration. Whereas leukccytes form short-lived interactions with collagen fibers in complete absence of tissue remodelin& melanoma cells and neoplastic cell clusters reorganize the matrix via profound pulling at attachment sites, limited fiber disruption upon detachment, and the shedding of cell surface determinants. Using blacking anti-integrin antibodies, tumor cell migration and migration-associated matrix reorganization were shown to be dependent on 01 integrinmediated adhesion, whereas migrating T cells cannot be inhibited by a panel of anti-fll-, R2-, 03-, and BYintegrin antibodies, either alone or in combination. Consequently, migrating melanoma cells use focal adhesions of integrins ccclustered with cytoskeletal components at contacts with collagen fibers. T cells, however, lack typical focal adhesions, redistribute !31 integrins to the uropod and the focal adhesion kinase to the leading edge. In conclusion, an adhesion- and integrin-dependent and reorganizing migration type employed by melanoma cells is based on strength and steadiness to overcome biophysical matrix resistance. In contrast, the dynamic migration type employed by leukocytes may occur largely i&grin-independent and in the absence of matrix remodeling.
THE EFFECT OF TOPOISOMERASE I INHIBITOR - CAMPTOTHECIN ON EXPRESSION OF ~53 IN SCLERODERMA FIBROBLASTS Joanna Czuwara. Urszula Nowicka. lzabela Uhrvnowska Zbianiew Gacionq, MatoorzatsOlszewska,Stawomir Maiewski.Stefania Jablofiska. Maria Blaszczvk,
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CROSS-TALK BETWEEN LAMININ-BINDING INTEGRINS. #~W.W&L Emmanuel Laolantine. Holoer Sondermann. Daamar Doalc. Patricia -y. lnsitut for Biochemistry II, Cologne, Germany, and IBCP, Lyon, France. Laminin 1 and 5 are ligands for a6Pl integrin, while a3Pl integrin has affinity for laminin 5 only. Both isoforms induced adhesion of normal human skin fibroblasts but promoted different overall cell shapes. On laminin 1 the cells formed filopodias, while on laminin 5 they developed lamellipodias. In addition, integrins and several cytoskeletal linker proteins, including vinculin, talin, and paxillin. showed a laminin isoform-specific clustering. To test whether this was due to a specific involvement of the a3Pl integrin, different strategies were used, including antibody occupancy of the integrin and composite ligands. The results indicated that there is a cross-talk between a3p1 and a6bl integrins and that the a3pl integrinlrans-dominantly regulated the clustering of cr6pl integrins and of focal adhesion components. To elucidate the underlying mechanisms of these effects the yeast two-hybrid system was used to search the intracellular ligands of the integrin a3 chain. Several clones were identified and shown to have specific potential interactions with either several integrin a subuntis or the a3 subunit only.
Lidla Rudnicka. Deoattment of DermatolowWarsaw Medical School, Poland Sclemdena (systemic sclerosis, SSc) is a chronic autoImmune disease characterized by progressive fibrosis A highly specific feature of SSc is the presence of circulating anti-topoisomerase antibodies. Our prewous studies have shown that the expression of topoisomerase I is significantly increased in fibmblasts of patients with SSc Camptothecin, a topoisomerase inhibitor, induces apoptosis in fibroblasts of SSc patients and decreases type I collagen production in these cells The aim of the present study was lo assess the effect of CamptotheCin on p53 expression in fibroblasts of patients with SSc. The study was performed with tne use of first passage SSc fibroblasts isolated from affected skin. The expression of p53 was evaluated by enzyme-linked immunosorbent assay, immunohistochemistry and immunofluorescence Our results show that camptothecin at the concentration of IO-7 and @M significantly increases the expression of p53 in fibroblasts The effect is dose and time dependent and is significantly more pronounced in SSc fibroblasts as compared to healthy Controls. These results indicate that one of the modes of action by which camptothecin shows it’s cytostatic activity on SSc cells is via ~53. The increased susceptibility of SSc cells to camptothecin might be due to increased topoisomerase I expression in these cells and confins the potentI. indication of camptothecins in SSc management.
I
I
, Iti
REGULATION
OF MMP
EXPRESSlON
COLLAGEN MATRICBSK
BY 3-DIMENSIONAL
tdach_ lZwartmentof Dermatolcw, Unlwxsityof Cologne,Germany
FIBRIN
. .
AND
Exp&on of matrixmetaUop%einases@iWs) is &btly regul&d and restricted to processes requiting matrix degradation such as wound healing. Intetwlngly, evidence is accumulating that cell-matrix interactions can also regulate MMP expression. To determine the potential role of early and late wound healing environmentson MMP expression. we investigatedthe effect of fibrin (early) and collagen (late) m&ices on fibroblast MMP expression. As previously reported, tibmblasts culhlredin 3-D colIsgen gels increasedW-1 gene expression,while concurrenttydecreasingtype I collagensynthesis.Additionstly, we detecteds strong increasein IvlTl-hiMPmRNA. Over tbe same periodof time,fibmblass in 3-D fibrin gels showed no expressionof MMP-1and MTl-MMP mRNA. but did down-regulate type I cottagengeneexpression.However.over longerperiods, fibringel culturesdid increaseW-1 andMTl-hiMP mRNA BecauseMTl-MMF’can activatepro-MMP2, we also analyzedMMP-2activationin bathgel systems.In collagengel cultures,we detectedlatentand activatedMMP-2,butin fibringel cultureswe observed only latent MMP-2, suggesting that MTl-MMP expression alone.was not sufficient for the activationof MMPIZ.One possible explanationwas immunodetwtablepresence of TIM&2. a ootentinhibitorof MTI-MMF’activitv.in tibrin4s. These t&i6 suggest thatmatrixcomponentst&y exert a-stronginfluenceon MMP expressionand activityduringwound healing.
0739
0742
CONTRIBUTION OF DERMAL COMPARTMENT TO SYNTHESIS AND ASSEMBLY OF BASEMENTMEMBRANE PROTEOGLYCANS INWVO. YasuhiroYamane ,1”31, .John R Couchman’“, Hidao Yamta “) Department of Dermatology, Higashi-Saitama Hospfal, Satama,Japan”‘. Department of Cell Biology, University of Alabama at Birmmgham Aluhama.U S.A 121Department of Dermatology. J&x Medical School, Tachlgl .Japan (” Prevmuslv we demonstrated that basemsnt membrane moteoelvcans (BMPGs) ban&an and perleeanare of epidemxd or&in in de~~al.e~~dermal~unctian (DEJ) of the slim However. r&s of dermal conmartment on avnthesis and assemblv of BM-PGs are not fully understmd Therefore, m thi study, we i&st@ed whether ti dermal compartment could dxectly contnbute the syntheslv and assemblyof Bhl.PGsm DEJ Tw ce” hne cells, DJM., cells and SCC-1 cells (both were dewed from human squamous cell carcmoma of skin) were xenografted in nude rats and nude mice by mjeclmg the cell ruspensmn mtracutaneously After 7.12 days, sahd tumors of 0 5 to 1 cm m diameter were obtamed and the xenografis were waned wth specms.specitic monoclonal antlbodxq reaogmzing rat barnscan, mouse perlecan mouse laminm.1 and mouse entactin The immunatluorescence staming of xenografts m nude rats with anti-rat bamacan revealed that in ncnograftr ofDJM.1 cells, m, bamacan was prosent in the basement membrane surrounding tumor&l nests (BM.0 in acontinuous andhnear fashmn BM-tcontainedrat bamacan in xenografts of SCC-1 cells NS well, but discontinuously and less in intensxy. In xenografts of tumor cells in nude mice, moune perlecan ,vas deposIted m the interstrtml stmma mtensoly and, m parts. in a fibrillarpattern and was absent or ,at best, sparse m BM.1 In co,,trast, mouse lamuun-l and mouse entadm were present manly m BM.t These resulta suggest 1) Dermnlcells in tumor stroms could synthesize bamacan invwo and bamacan produced by mesenchymal cells could be assembled m basement membrane (BM) and, therefore, dermal compartment may contribute directly tc the synthew and assembly of bamacan of BM of DEJ m normal skm 2) The capacity for the assembly of bamacan d&red among tumor cells 3) Perlecan 1s synthesized by mesonchymal cells but IS not assembled in BM.t resultmg m mtense depasltmn in stroma
REGULATIONOF COLLAGENAND COLLAGENASEGENE EXPRESSION IN HUMAN FIBROBLASTS BY MONOCYTE CHEMOAlTRA~ANT PROTEIN-1 (MCP-1). Toshivuki Yamamoto. Karin Hartmann. Bests Eekea and Thomas
0740 ,N,WXD,S, CELL MATRlX
Krieg, Department of Dermatology, University of Cologne, Cologne, Germany. Monocyte chemoattractant protein-l (MCP-1) has recently been suggeested to play a role in experimental murine pulmonary fibrosis or murine interstitial kidney fibrosis by inducing collagen synthesis. Excessive deposition of connective tissue, however, is the result of an imbalance between synthesis and degradation. We therefore intended ta characterize the influence of MCP-1 sy&natieally at both levels in the human system, and examined the effect of MCP-1 on collagen and collagenase mFtNA expression in primary human dermal fibroblasts and in the human tibroblast cell line Wi-26/SV-40 (derived from human lung). Confluent cultures of tibroblasts were incubated with different concentrations of MCP-1 (l-50 n&n11 for 6-48 hours, and total RNA was isolated. Northern blot hvbridization revealed that not onlv collawn. but also matrix metalloprotein&l (MMP-1) mRNA was u&gul~ted’ by stmmlation with MCP-1 in a dose-dependent manner. Expression of collagen gene was upregulated at 48 hours in both tibroblasts. In contrast, MMP-1 expression was markedly enhanced by MCP-1 at 24 hours in dermal fibrohlasts only. In addition, ILla gene expression was also upregulated at 24 hours by Incubation with MCP-1 (10 “g/ml). Our results suggest that MCP-1 also affects metalloproteinase expression and can be involved in human fibrotic processes. In addition, the data indicate that the effect of MCP-1 on fibroblasts may be mediated by &I.
0743
WTERACTlONS AND CELL MIGRATION STRATEGIES: FUNDAMENTAL DIFFERENCES M LEUKOCYTES ANDTUMORCELLS.PeterFriedl, Eva-B.Briicker. and K. S. Zjinker’. Deparbnent ofDermatology, University of WUrzburg, and the ‘Institute of hnmunology, University of Wiierdecke, Germany The principles determining the migration of different cell types may result from their differences in origin, size and shape, function of adhesion receptors, and envimnmental factors, including the extracellular matrix. Polarizal leukocytes (T lymphocytes and dendritic cells) migrating in three-dimensional collagen lattices are small developing a highly dynamic leading edge and a trailing umpcd, whereas invasive melanoma cells are larger, highly polarized and less dynamic. In contmst to leukocytes, tumor cells may additionally develop migrating cell clusters maintaining intense cell-cell-interaction and cluster polarity. Leukocytes show a speed-oriented, oscillating and directionally unpredictable path profile strongly guided by matrix fibers, while melanoma cells and migrating cell clusters exhibit slow yet highly directional migration. Whereas leukccytes form short-lived interactions with collagen fibers in complete absence of tissue remodelin& melanoma cells and neoplastic cell clusters reorganize the matrix via profound pulling at attachment sites, limited fiber disruption upon detachment, and the shedding of cell surface determinants. Using blacking anti-integrin antibodies, tumor cell migration and migration-associated matrix reorganization were shown to be dependent on 01 integrinmediated adhesion, whereas migrating T cells cannot be inhibited by a panel of anti-fll-, R2-, 03-, and BYintegrin antibodies, either alone or in combination. Consequently, migrating melanoma cells use focal adhesions of integrins ccclustered with cytoskeletal components at contacts with collagen fibers. T cells, however, lack typical focal adhesions, redistribute !31 integrins to the uropod and the focal adhesion kinase to the leading edge. In conclusion, an adhesion- and integrin-dependent and reorganizing migration type employed by melanoma cells is based on strength and steadiness to overcome biophysical matrix resistance. In contrast, the dynamic migration type employed by leukocytes may occur largely i&grin-independent and in the absence of matrix remodeling.
THE EFFECT OF TOPOISOMERASE I INHIBITOR - CAMPTOTHECIN ON EXPRESSION OF ~53 IN SCLERODERMA FIBROBLASTS Joanna Czuwara. Urszula Nowicka. lzabela Uhrvnowska Zbianiew Gacionq, MatoorzatsOlszewska,Stawomir Maiewski.Stefania Jablofiska. Maria Blaszczvk,
0741
0744
CROSS-TALK BETWEEN LAMININ-BINDING INTEGRINS. #~W.W&L Emmanuel Laolantine. Holoer Sondermann. Daamar Doalc. Patricia -y. lnsitut for Biochemistry II, Cologne, Germany, and IBCP, Lyon, France. Laminin 1 and 5 are ligands for a6Pl integrin, while a3Pl integrin has affinity for laminin 5 only. Both isoforms induced adhesion of normal human skin fibroblasts but promoted different overall cell shapes. On laminin 1 the cells formed filopodias, while on laminin 5 they developed lamellipodias. In addition, integrins and several cytoskeletal linker proteins, including vinculin, talin, and paxillin. showed a laminin isoform-specific clustering. To test whether this was due to a specific involvement of the a3Pl integrin, different strategies were used, including antibody occupancy of the integrin and composite ligands. The results indicated that there is a cross-talk between a3p1 and a6bl integrins and that the a3pl integrinlrans-dominantly regulated the clustering of cr6pl integrins and of focal adhesion components. To elucidate the underlying mechanisms of these effects the yeast two-hybrid system was used to search the intracellular ligands of the integrin a3 chain. Several clones were identified and shown to have specific potential interactions with either several integrin a subuntis or the a3 subunit only.
Lidla Rudnicka. Deoattment of DermatolowWarsaw Medical School, Poland Sclemdena (systemic sclerosis, SSc) is a chronic autoImmune disease characterized by progressive fibrosis A highly specific feature of SSc is the presence of circulating anti-topoisomerase antibodies. Our prewous studies have shown that the expression of topoisomerase I is significantly increased in fibmblasts of patients with SSc Camptothecin, a topoisomerase inhibitor, induces apoptosis in fibroblasts of SSc patients and decreases type I collagen production in these cells The aim of the present study was lo assess the effect of CamptotheCin on p53 expression in fibroblasts of patients with SSc. The study was performed with tne use of first passage SSc fibroblasts isolated from affected skin. The expression of p53 was evaluated by enzyme-linked immunosorbent assay, immunohistochemistry and immunofluorescence Our results show that camptothecin at the concentration of IO-7 and @M significantly increases the expression of p53 in fibroblasts The effect is dose and time dependent and is significantly more pronounced in SSc fibroblasts as compared to healthy Controls. These results indicate that one of the modes of action by which camptothecin shows it’s cytostatic activity on SSc cells is via ~53. The increased susceptibility of SSc cells to camptothecin might be due to increased topoisomerase I expression in these cells and confins the potentI. indication of camptothecins in SSc management.
I
I
, Iti
REGULATION
OF MMP
EXPRESSlON
COLLAGEN MATRICBSK
BY 3-DIMENSIONAL
tdach_ lZwartmentof Dermatolcw, Unlwxsityof Cologne,Germany
FIBRIN
. .
AND
Exp&on of matrixmetaUop%einases@iWs) is &btly regul&d and restricted to processes requiting matrix degradation such as wound healing. Intetwlngly, evidence is accumulating that cell-matrix interactions can also regulate MMP expression. To determine the potential role of early and late wound healing environmentson MMP expression. we investigatedthe effect of fibrin (early) and collagen (late) m&ices on fibroblast MMP expression. As previously reported, tibmblasts culhlredin 3-D colIsgen gels increasedW-1 gene expression,while concurrenttydecreasingtype I collagensynthesis.Additionstly, we detecteds strong increasein IvlTl-hiMPmRNA. Over tbe same periodof time,fibmblass in 3-D fibrin gels showed no expressionof MMP-1and MTl-MMP mRNA. but did down-regulate type I cottagengeneexpression.However.over longerperiods, fibringel culturesdid increaseW-1 andMTl-hiMP mRNA BecauseMTl-MMF’can activatepro-MMP2, we also analyzedMMP-2activationin bathgel systems.In collagengel cultures,we detectedlatentand activatedMMP-2,butin fibringel cultureswe observed only latent MMP-2, suggesting that MTl-MMP expression alone.was not sufficient for the activationof MMPIZ.One possible explanationwas immunodetwtablepresence of TIM&2. a ootentinhibitorof MTI-MMF’activitv.in tibrin4s. These t&i6 suggest thatmatrixcomponentst&y exert a-stronginfluenceon MMP expressionand activityduringwound healing.