Cytokeratin 8 ectoplasmic domain binds urokinase-type plasminogen activator to breast tumor cells and modulates their adhesion, growth and invasiveness.
BACKGROUND
Generation of plasmin is a characteristic of tumor cells, promoting the degradation of extracellular matrix, tumor progression and metastasis. The process is accelerated if plasminogen and plasminogen activator are bound to their cell surface receptors.


RESULTS
In this study we show that the monoclonal antibody that recognizes an epitope on the cytokeratin 8 (CK8) ectoplasmic domain (anti-CK MAb) inhibits plasminogen activation mediated by urokinase-type plasminogen activator (uPA) in MCF-7 and MCF-10A neoT cells. The ectoplasmic domain of CK8 acts as a binding site for plasminogen, however, by using confocal microscopy, we demonstrated that it is also co-localized with uPA. CK8, therefore, function also as a receptor for uPA on the cell surface, and the presence of anti-CK MAb may prevent the binding of uPA to a designated CK8 motif. The consequent inhibition of plasmin generation resulted in changed cell morphology, enhanced cell adhesion to fibronectin, reduced invasion potential, and an enhanced G1/S transition. Moreover, surface plasmon resonance analysis showed that the synthetic dodecapeptide corresponding to the epitope sequence (VKIALEVEIATY), binds uPA in the nanomolar range.


CONCLUSIONS
These novel findings suggest a model in which CK8, together with uPA, plasminogen and fibronectin, constitutes a signaling platform capable of modulating cell adhesion/growth-dependent signal transduction in breast tumor cells. Anti-CK MAb, which competes for the binding site for uPA, could be used as an agent to reduce the invasive potential of breast tumor cells.
Jozef Stefan Institute, Department of Biotechnology, Jamova 39, 1000 Ljubljana, Slovenia.
Natasa
N
Obermajer
Bojan
B
Doljak
Janko
J
Kos
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D005260:Female; D005353:Fibronectins / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D053533:Keratin-8 / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D010455:Peptides / Q000737:chemistry; D010958:Plasminogen / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D016196:S Phase / Q000187:drug effects; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D053533:Keratin-8; D010455:Peptides; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator