- Email: [email protected]
tPA-mediated plasminogen activation at melanoma cell surface
BINDING OF FIBRINOLYTIC FACTORS TO HUMAN ENDOTHELIAL CELLS Anil K. Dudani’, Douglas S. Palmer’ and Peter R. Ganz*
tPA-MEDIATED PLASMINOGEN ACTIVATION AT MELANOMA CELL SURFACE Jozef Bizik and Antti Vaheri* Department
of
Tumor
Institute,
Bratislava,
Virology,
Haartman
Cell
Biology,
Slovakia Institute,
and
Cancer
*Department
University
of
‘Ottawn Blood Centre, Canadian Red Cross Sociev and 2Healih Canada, Ottawa, Ontario, Canada
Research of
mechanism by which endothelial cells (ECs) regulate tibrinolysis is through the regulated assembly of fibrinolytic
One
Helsinki,
Helsinki, Finland
proteins on their membrane surface. Studies done in our laboratory show that ECs isolated from veins, arteries & capillaries exhibit differential binding of plasminogen (plg). Further characterization of this interaction on venous ECs identified a 45 kDa cell surface component that interacted specifically with plg. Using 2-D
Tissue-type plasminogen activator (tPA), an essential component of the plasminogen activation cascade, is the key enzyme in fibrinolysis. However, in recent years it was also observed that tPA can mediate plasminogen activation at the cell surface. We have demonstrated previously that tPA secreted by melanoma cells is bound to the cell surfaces by a process dependent on a lysine-binding mechanism where it effectively mediates generation of cell bound plasmin. Recently we have undertaken the study to identify the domains on the tPA molecule that interact with its binding site on human melanoma cells. The consistent results obtained by the three different experimental approaches provide evidence that tPA binds to melanoma cells via its kringle-2 domain but binding sites within kringle-1 domain and protease domain may support the interaction. The finger domain did not contribute to the binding. These type of data may be helpful for better understanding of the precise function of tPA in the process of cell surface plasminogen activation and should assist in the design of further studies to isorate and identify the putative tPA cell surface receptor.
electrophoresis followed by ligand blotting, the 45kDa component has been resolved into two plg binding proteins. One protein (pI=5.1) has been identified as the cytoskeletal protein, actin. The presence of actin on tt.e extracellular surface of cultured, resting ECs was confirmed by immunofluorescence and flow cytometric analyses. This observation is consistent with a number of reports of actin localized to the outside of other cell types. In vitro studies using purified actin demonstrate that plg binds to actin saturably and with relatively high affinity via its kringles. Tissue plasminogen activator (tPA) and the plg homologue lipoprotein(a) [Lp(a)] also bound to actin. Addition of plg and low density lipoproteins inhibited Lp(a) binding to actin. Moreover, partial inhibition of plg binding to actin was also observed in competition with tPA. Using peptide array technology, we identified domains/sequences within the actin molecule that interact specifically with plg and tPA. At the cellular level, binding of plg, tPA & Lp(a) to ECs was significantly inhibited, but not eliminated, by anti-a&n antibodies confirming actin as a binding site for these ligands while attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing the binding of plg, tPA and Lp(a) to EC surfaces.
REGULATION BY CELLULAR Vincent Ellis
STROMAL CELL INVOLVEMENT AND FUNCTIONAL OVERLAP IN EXTRACXLLULAR PROTEOLYSIS IN CANCER INVASION AND NON-NEOPLASTIC TISSUE REMODELING Keld Da&, Leif R. Lund’, Thomas H. Buggel**, Boye Schnack Nielsen’, Lone Rmmov-Jessen’, Chung-Huyn Lee-Segaard’ and Helene Solberg’
Thrombosis
OF PLASMINOGEN RECEPTORS
Research
Institute, London,
ACTIVATION
UK
The generation of the broad specificity protease plasmin catalysed by the plasminogen activators uPA (urokinasetype plasminogen activator) and tPA (tissue-type plasminogen activator) is implicated in a variety of pathophysiological processes, including vascular fibrin dissolution, extracellular matrix degradation and remodelling, and cell migration. Due to the potency of this proteolytic system, an array of mechanisms exist to regulate its function, including those to control the initiation, progression, localisation and termination of its activity. A general mechanism for the regulation of plasmin generation is the interaction of the plasminogen activators with cellular binding sites; uPAR (the specific cellular receptor for uPA) being the established prototype for this type of regulation. The binding of pro-uPA to uPAR on many cell-types leads to a large increase in plasmin generation due to the assembly of an efficient system of reciprocal zymogen activation, dependent on the concurrent cellular binding of plasminogen. The molecular interactions involved in the formation of these specific plasminogen activation complexes are not fully elucidated. Recently we have observed that vascular smooth muscle cells bind tPA specifically, with relatively high-affmity and by a novel mechanism. As with the uPA/uPAR system, this binding leads to efficient plasminogen activation with similar kinetic characteristics. However the two systems also have significant differences, particularly in the regulation of their activity by protease inhibitors. The characteristics of the functional regulation of these two systems suggest that they may have somewhat different roles in the pericellular environment.
‘Finsen Laboratory, Rigshospitalet, DK-2100 Copenhagen 0., Denmark. ‘Childrens Hospital Research Foundation, Cincinatti, Ohio, USA
Cancer invasion can be considered as a tissue remodeling process in which normal tissue is substituted by cancer tissue. Expression studies suggest that generation and regulation of extracellular proteolysis in cancer invasion, like is remodeling processes, non-neoplastic in many accomplished by an interaction between several cell types which each produce one or more proteases, protease inhibitors or protease receptors. The pattern of expression of these proteins appears to be unique for each type of cancer and often to mimic certain non-neoplastic tissue remodeling Several examples will be discussed. The processes. similarity between cancer invasion and other tissue remodeling processes is also supported by studies in plasminogen-deficient transgenic mice, showing that although cancer metastasis and non-neoplastic tissue remodeling (mammary gland involution, wound healing) are impaired, these processes nevertheless can proceed at decreased speed. We have found that in these processes there is both in wild-type and plasminogen-deficient mice a coexpression of urokinase plasminogen activator and several matrix metalloproteases (MMP’s), such as gelatinase B, stromelysin- 1, stromelysin-3 and interstitial collagenase. We hypothesize that there is a functional overlap between uPAgenerated plasmin and one or more of these MMP’s. We are currently testing this assumption. Preliminary results will be discussed at the meeting.
139
tPA-MEDIATED PLASMINOGEN ACTIVATION AT MELANOMA CELL SURFACE Jozef Bizik and Antti Vaheri* Department
of
Tumor
Institute,
Bratislava,
Virology,
Haartman
Cell
Biology,
Slovakia Institute,
and
Cancer
*Department
University
of
‘Ottawn Blood Centre, Canadian Red Cross Sociev and 2Healih Canada, Ottawa, Ontario, Canada
Research of
mechanism by which endothelial cells (ECs) regulate tibrinolysis is through the regulated assembly of fibrinolytic
One
Helsinki,
Helsinki, Finland
proteins on their membrane surface. Studies done in our laboratory show that ECs isolated from veins, arteries & capillaries exhibit differential binding of plasminogen (plg). Further characterization of this interaction on venous ECs identified a 45 kDa cell surface component that interacted specifically with plg. Using 2-D
Tissue-type plasminogen activator (tPA), an essential component of the plasminogen activation cascade, is the key enzyme in fibrinolysis. However, in recent years it was also observed that tPA can mediate plasminogen activation at the cell surface. We have demonstrated previously that tPA secreted by melanoma cells is bound to the cell surfaces by a process dependent on a lysine-binding mechanism where it effectively mediates generation of cell bound plasmin. Recently we have undertaken the study to identify the domains on the tPA molecule that interact with its binding site on human melanoma cells. The consistent results obtained by the three different experimental approaches provide evidence that tPA binds to melanoma cells via its kringle-2 domain but binding sites within kringle-1 domain and protease domain may support the interaction. The finger domain did not contribute to the binding. These type of data may be helpful for better understanding of the precise function of tPA in the process of cell surface plasminogen activation and should assist in the design of further studies to isorate and identify the putative tPA cell surface receptor.
electrophoresis followed by ligand blotting, the 45kDa component has been resolved into two plg binding proteins. One protein (pI=5.1) has been identified as the cytoskeletal protein, actin. The presence of actin on tt.e extracellular surface of cultured, resting ECs was confirmed by immunofluorescence and flow cytometric analyses. This observation is consistent with a number of reports of actin localized to the outside of other cell types. In vitro studies using purified actin demonstrate that plg binds to actin saturably and with relatively high affinity via its kringles. Tissue plasminogen activator (tPA) and the plg homologue lipoprotein(a) [Lp(a)] also bound to actin. Addition of plg and low density lipoproteins inhibited Lp(a) binding to actin. Moreover, partial inhibition of plg binding to actin was also observed in competition with tPA. Using peptide array technology, we identified domains/sequences within the actin molecule that interact specifically with plg and tPA. At the cellular level, binding of plg, tPA & Lp(a) to ECs was significantly inhibited, but not eliminated, by anti-a&n antibodies confirming actin as a binding site for these ligands while attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing the binding of plg, tPA and Lp(a) to EC surfaces.
REGULATION BY CELLULAR Vincent Ellis
STROMAL CELL INVOLVEMENT AND FUNCTIONAL OVERLAP IN EXTRACXLLULAR PROTEOLYSIS IN CANCER INVASION AND NON-NEOPLASTIC TISSUE REMODELING Keld Da&, Leif R. Lund’, Thomas H. Buggel**, Boye Schnack Nielsen’, Lone Rmmov-Jessen’, Chung-Huyn Lee-Segaard’ and Helene Solberg’
Thrombosis
OF PLASMINOGEN RECEPTORS
Research
Institute, London,
ACTIVATION
UK
The generation of the broad specificity protease plasmin catalysed by the plasminogen activators uPA (urokinasetype plasminogen activator) and tPA (tissue-type plasminogen activator) is implicated in a variety of pathophysiological processes, including vascular fibrin dissolution, extracellular matrix degradation and remodelling, and cell migration. Due to the potency of this proteolytic system, an array of mechanisms exist to regulate its function, including those to control the initiation, progression, localisation and termination of its activity. A general mechanism for the regulation of plasmin generation is the interaction of the plasminogen activators with cellular binding sites; uPAR (the specific cellular receptor for uPA) being the established prototype for this type of regulation. The binding of pro-uPA to uPAR on many cell-types leads to a large increase in plasmin generation due to the assembly of an efficient system of reciprocal zymogen activation, dependent on the concurrent cellular binding of plasminogen. The molecular interactions involved in the formation of these specific plasminogen activation complexes are not fully elucidated. Recently we have observed that vascular smooth muscle cells bind tPA specifically, with relatively high-affmity and by a novel mechanism. As with the uPA/uPAR system, this binding leads to efficient plasminogen activation with similar kinetic characteristics. However the two systems also have significant differences, particularly in the regulation of their activity by protease inhibitors. The characteristics of the functional regulation of these two systems suggest that they may have somewhat different roles in the pericellular environment.
‘Finsen Laboratory, Rigshospitalet, DK-2100 Copenhagen 0., Denmark. ‘Childrens Hospital Research Foundation, Cincinatti, Ohio, USA
Cancer invasion can be considered as a tissue remodeling process in which normal tissue is substituted by cancer tissue. Expression studies suggest that generation and regulation of extracellular proteolysis in cancer invasion, like is remodeling processes, non-neoplastic in many accomplished by an interaction between several cell types which each produce one or more proteases, protease inhibitors or protease receptors. The pattern of expression of these proteins appears to be unique for each type of cancer and often to mimic certain non-neoplastic tissue remodeling Several examples will be discussed. The processes. similarity between cancer invasion and other tissue remodeling processes is also supported by studies in plasminogen-deficient transgenic mice, showing that although cancer metastasis and non-neoplastic tissue remodeling (mammary gland involution, wound healing) are impaired, these processes nevertheless can proceed at decreased speed. We have found that in these processes there is both in wild-type and plasminogen-deficient mice a coexpression of urokinase plasminogen activator and several matrix metalloproteases (MMP’s), such as gelatinase B, stromelysin- 1, stromelysin-3 and interstitial collagenase. We hypothesize that there is a functional overlap between uPAgenerated plasmin and one or more of these MMP’s. We are currently testing this assumption. Preliminary results will be discussed at the meeting.
139