Cytokeratin 8 ectoplasmic domain binds urokinase-type plasminogen activator to breast tumor cells and modulates their adhesion, growth and invasiveness

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Inspection at State and Municipal Level and the Contribution Thereof to Security in Municipalities

Namen prispevka: Namen prispevka je prikaz pravne ureditve inšpekcijskega nadzora na državni in lokalni ravni in umestitev inšpekcijskega nadzora v proces modernizacije in razvoja slovenske javne uprave. Prispevek je namenjen tudi razumevanju pravnega položaja inšpektorjev in strank ter inšpekcijskih postopkov z vidika državne in občinske regulative ter z vidika prispevka k varnosti v občinah. Metode: Raziskovalni pristop je kombinacija pravnih metod deskriptivne, primerjalne (komparativne) jezikovne in teleološke razlage s študijami šestih primerov ureditve v treh večjih in treh manjših oz. srednje velikih občinah. S teleološko razlago avtorji prikažejo ratio legis zakonodajalca v zakonski ureditvi pravnega položaja inšpektorjev, njihovih pooblastil ter pravnega položaja strank v teh postopkih. Ugotovitve: Inšpekcijski nadzor prispeva k večji varnosti v občinah, če so s predpisi ustrezno zajete in urejene situacije, ki predstavljajo varnostno tveganje, in če je učinkovit. Učinkovitost inšpekcijskega nadzora se praviloma obravnava parcialno, in sicer kvantitativno, kar ni skladno s kompleksnostjo upravnega, političnega in družbenega sistema. Učinkovitost inšpekcijskega nadzora in večja varnost v občinah torej nista odvisna samo od inšpekcij, ampak tudi od številnih drugih dejavnikov. Dosedanji razvoj na tem področju v Sloveniji kaže na težavnost uvajanja sistemskih sprememb na državni ravni, zlasti na organizacijskem področju, na občinski ravni pa na uspešnost v obliki ustanavljanja medobčinskih inšpektoratov in redarstev. Izvirnost/pomembnost prispevka: Članek bralcu predstavi veljavno pravno ureditev inšpekcijskega nadzora v Republiki Sloveniji in njegovo umestitev v kontekst modernizacije in reforme slovenske javne uprave. Izpostavlja dobre in slabe strani dosedanjega razvoja in predlaga rešitve. Na primeru izbranih občinskih aktov in sodne prakse prikaže prispevek inšpekcijskega nadzora k varnosti v občinah.Purpose: The article aims to provide an overview of the statutory regulation of inspection at the national and local levels and its placement in the process of modernization of Slovenian public administration. The article is intended to deepen the understanding of the legal position of inspectors and parties to inspection procedures, as well as the inspection procedure itself from the perspective of national and local regulations and the legal regulation of certain security aspects in local communities. Design/Methods/Approach: The applied research approach is a combination of the legal methods of descriptive, comparative linguistic, and teleological interpretations, combined with an analysis of six cases involving the application of the relevant regulation in three larger and three smaller or medium sized municipalities. By means of teleological interpretation, the authors demonstrate the ratio legis of the legislature in the statutory regulation of the state and local legal positions of inspectors, their powers, and the legal position of parties to these procedures. Findings: Inspections contributes to security in municipalities, if situations that pose a security risk are regulated properly and if it is effective. The effectiveness of inspections is normally considered partially, especially quantitative, which is not consistent with the complexity of administrative, political and social system. The effectiveness of inspection and higher security in municipalities therefore depend not only on inspections, but also on many other factors. The current development in this field in Slovenia shows the difficulties of introducing systemic changes at the national level, especially in the field of organization, and success at the municipal level in the form of the establishment of inter-municipal inspectorates and local police. Originality/Value: The article is a summary presentation that provides readers with an overview of the legal regulation of the institute of inspection in the Republic of Slovenia and its placement in the context of modernization and reform of Slovenian public administration. It highlights the strengths and weaknesses of current development and proposes solutions. By using selected municipal acts and the case law it presents the contribution of inspections to the security in the municipalities

Profilin 1 as a Target for Cathepsin X Activity in Tumor Cells

<div><p>Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.</p> </div

Cathepsin X increases migration, adhesion and invasion of cancer cells.

<p>Migration (A, B), adhesion (C) and invasion (D) assays were done using xCELLigence System. (<b>A and B</b>) Diagrams show a slope (cell index vs. time) of cells that migrated from the upper to the lower chamber. Cells migrated in the presence of DMSO (red line on graph) or 10 µM inhibitor of cathepsin X (green line on graph) (A) or cells, transfected with control (red line on graph) or cathepsin X specific siRNA (green line on graph) were used (B). (<b>C</b>) Diagram shows a slope for cells transfected with a control (red line on graph) or cathepsin X specific siRNA (green line on graph), that adhered to fibronectin (10 µg/ml). (<b>D</b>) Diagram shows a slope for cells transfected with a control (red line on graph) or cathepsin X specific siRNA (green line on graph) that invaded through Matrigel from the upper to the lower chamber. ***P≤0.01; ***P<0,001. Graphs show real-time curves of cell index (CI) as a function of time. Vertical lines represent the start and end of time intervals within which corresponding diagrams are calculated. Four (A), eight (B), four (C) and five (D) biological repeats were performed.</p

Cathepsin X modulates profilin 1 function by affecting the binding of poly-L-proline ligands.

<p>(<b>A and B</b>) Profilin 1 forms a stable complex with actin and clathrin. Representative co-immunoprecipitations of profilin 1 with clathrin (A-inset) and actin (B-inset) in PC-3 cells, treated with DMSO or cathepsin X specific inhibitor. (<b>A and B insets</b>) Cell lysates were treated with anti-clathrin or anti β-actin antibody and immunoprecipitated on Protein A Sepharose beads. Profilin 1 was detected by Western blot in total cell lysates and in immunoprecipitated pellets. β-actin was used as a loading control. (<b>A and B</b>) Quantification of data from the insets. The graphs represent densitometric analysis of bands using Sygene's GeneTools Software (Sygene, U.K.). Three or two biological experiments indicate the normalized amount of profilin 1 that is in complex with clathrin (A) or actin (B), respectively. **P≤0.01. (<b>C</b>) Cathepsin X regulates actin polymerization. Flow cytometric analysis of permeabilized PC-3 cells is shown. Filamentous actin was stained with phalloidin conjugate. Cells were treated with DMSO or AMS-36 or were transfected with control or cathepsin X specific siRNA. Increase in actin polymerization is shown with values of both control experiments set to 0% increase in actin polymerization. Values are representative of four independent experiments. *P≤0.05; **P<0.01.</p

Co-localization of cathepsin X and profilin 1 (A) with clathrin (B) and actin (C).

<p>All proteins were visualized by immunofluorescence staining using primary antibodies to cathepsin X, profilin 1 and clathrin, followed by Alexa Fluor conjugated secondary antibodies Alexa Fluor 488, 555 and 633 or phalloidin conjugate for actin. (<b>A</b>) Cathepsin X is shown in green, profilin 1 in red and co-localization in yellow. For co-localization, also Zen 2011 Software (Carl Zeiss) option for improved visibility of co-localized pixels was used and co-localization is shown in white with corresponding scatter diagram. (<b>B</b>) Clathrin is shown in red, profilin 1 and cathepsin X are both in green due to clearer merged image. (<b>C</b>) Profilin 1 is shown in green, cathepsin X in blue and actin in red. Zen 2011 Software option for improved visibility of co-localization is used. Bars, 10 µm.</p

Cathepsin X in MCF-7, MDA-MB-231 and PC-3 cells.

<p>(<b>A</b>) Activity was measured in cell lysates using cathepsin X specific substrate Abz-FEK(Dnp)-OH. Mean values of 5 independent experiments are shown. ***P<0,001 (<b>B</b>) The amount of cathepsin X (ng/ml) in different cell lines was determined with ELISA. Mean values of 2 (MCF-7 and MDA-MB-231) or 4 (PC-3) independent experiments are shown.</p