Plasminogen is shown to be expressed endogenously in these cells and localized intracellularly as well as on the plasma membrane

Plasminogen is shown to be expressed endogenously in these cells and localized intracellularly as well as on the plasma membrane. and metastasis. The process is accelerated if plasminogen and plasminogen activator are bound to their cell surface receptors. Results In this study we show Amyloid b-Protein (1-15) 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 Amyloid b-Protein (1-15) 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. Conclusion 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. Background Proteases are essential for invasion by tumor cells. For this, they need to be activated, either on the tumor cell surface or in the pericellular space. In breast cancer, urokinase type plasminogen activator (uPA), a serine proteinase, has been associated with an aggressive malignant phenotype [1,2]. Cell surface uPA, bound to uPA receptor (uPAR), activates plasminogen Amyloid b-Protein (1-15) to plasmin, a central player in breast cancer progression and metastasis. Plasmin activates growth factors and protease cascades that lead to the disruption of cell-cell and cell-extracellular matrix adhesion via pericellular proteolysis of glycoproteins [3,4]. Plasmin can also affect the cell phenotype by activating or inactivating growth factors, and by modifying growth factor receptors and adhesion receptors [5-7]. Plasminogen is activated on the cell surface much faster than in solution, due to the presence of proteins that promote a plasminogen conformation more open to proteolytic attack [8]. In addition to uPAR, cytokeratin 8 (CK8) is an important plasminogen-binding protein in the membrane of breast cancer cells. CK8 is an intermediate filament protein and associates with CK18 to form an insoluble matrix within the cell. However, the C-terminal end of CK8 also penetrates the cellular membrane, as shown for hepatocellular and breast carcinoma cells [9,10]. CK8 is unique among cytokeratins in that it contains a carboxyl-terminal lysine that can interact with the lysine binding sites of plasminogen, although it is able to bind plasminogen even when the C-terminal lysine is mutated [11]. Plasminogen activation is promoted by tissue type plasminogen activator (tPA), which can bind to both components of the cytokeratin heterodimer, CK8/CK18. Plasminogen and tPA cannot bind to CK8 at the same time, but the binding of tPA to CK18 enhances the binding of plasminogen to CK8 and its activation [11]. The CK8/CK18 complex modulates the signaling pathways intracellularly by binding kinases involved in signal transduction. It integrates signals generated by stimulated surface membrane receptors, such as insulin receptor and integrin beta 1 receptor, and modulates their signal transduction in appropriate reaction sequences. The absence of CK8/CK18 in hepatocytes results in a phenotype that attaches more rapidly to fibronectin and undergoes an enhanced G1/S transition compared with wild-type hepatocytes [12]. On the other hand, the cytoskeleton itself can undergo rearrangements as a result of outside-in signals triggered by fibronectin binding to cell surface receptors, the integrins. The aim of this study was to investigate the involvement of CK8 in plasminogen activation, and consequently in cell adhesion, invasion and signaling of breast tumor cells. An anti-cytokeratin MAb directed against an epitope on the ectoplasmic tail of CK8 was used to determine whether it prevents uPA NBN mediated activation of plasminogen, resulting in enhanced adhesion of.