Author: Menakongka ; Sripa ; Parichut ; Rutaiwan ; Apaporn ; Tohtong ; Thummarati ; Tuangporn ; Suthiphongchai ; Banchob ; Sitsom ; Prasopthum ; Wijitburaphat ; Aruna

AIMTo investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters.METHODSuPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay.RESULTSThe immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA.CONCLUSIONuPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.

01 Oct 2009·Investigative Opthalmology & Visual ScienceQ2 · MEDICINE

Novel Laminin 5 γ2-chain Fragments Potentiating the Limbal Epithelial Cell Outgrowth on Amniotic Membrane

Q2 · MEDICINE

ArticleOA

Author: Cheng, Ching-Yi ; Hsieh, Hsi-Lung ; Yang, Chuen-Mao ; Ma, David Hui-Kang ; Pang, Jong-Hwei Su ; Yu, Wei-Hsuan ; Sun, Chi-Chin

PURPOSEMatrix metalloproteases (MMPs)-mediated extracellular matrix (ECM) degradation potentially releases cryptic motility factors involved in somatic stem cell migration and epithelial outgrowth. The authors previously demonstrated that MMP-9 is upregulated in limbal epithelial cells cultivated on amniotic membrane (AM). Here, the authors further investigated whether plasminogen activator (PA)/plasmin regulates MMP-9 activity in this model implicated in the processing of laminin 5 (Ln5), a component of amniotic basement membrane.METHODSLimbal epithelial cells migrated from limbal explants were expanded on intact AM. The activities and proteins of uPA and MMP-9 in limbal epithelial cells were determined by fibrin and gelatin zymography, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Specific pharmacological inhibitors including MMPs inhibitor GM6001, MMP-2/-9 inhibitor, and uPA inhibitor B428 were used to determine whether the PA/plasmin/MMP-9 axis induces cell growth via Ln5 in this model.RESULTSThese data showed that MMP-9 activity was attenuated by a selective uPA inhibitor, B428. Furthermore, MMP-9 activity was enhanced by exogenous addition or pre-incubation with plasmin. These results demonstrated that PA/plasmin regulates MMP-9 expression. An interesting proteolytic fragment of Ln5 gamma 2-chain was suppressed by pretreatment with GM6001, B428, or neutralizing antibodies of MMP-9 and uPA, indicating that Ln5 gamma 2-chain is processed by uPA/MMP-9. Moreover, the extent of limbal outgrowth was also retarded by B428.CONCLUSIONSThis study suggested that MMP-9 activity was upregulated by PA/plasmin, which in turn processed Ln5 gamma 2-chain to facilitate limbal outgrowth on intact AM.

01 Aug 2008·Behavioural Brain ResearchQ3 · PSYCHOLOGY

Effects of urokinase-type plasminogen activator in the acquisition, expression and reinstatement of cocaine-induced conditioned-place preference

Q3 · PSYCHOLOGY

Article

Author: Jean Luc Dreyer ; Amine Bahi ; Alexander W. Kusnecov

Cocaine and many other psychostimulants strongly induce urokinase-type plasminogen activator (uPA) expression in the mesolimbic dopaminergic pathway, which plays a major role in drug-mediated behavioral plasticity [Bahi A, Boyer F, Gumy C, Kafri T, Dreyer JL. In vivo gene delivery of urokinase-type plasminogen activator with regulatable lentivirus induces behavioral changes in chronic cocaine administration. Eur J Neurosci 2004;20:3473-88; Bahi A, Boyer F, Kafri T, Dreyer JL. Silencing urokinase in the ventral tegmental area in vivo induces changes in cocaine-induced hyperlocomotion. J Neurochem 2006;98:1619-31; Bahi A, Dreyer JL. Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine- and morphine-induced reward and behavioral sensitization. Genes Brain Behav 2007]. In this study, the role of mesolimbic dopamine (DA) pathways in the development of cocaine reward was examined by conditioned-place preference in rats with bilateral intra-accumbens injections of uPA-expressing lentiviral vectors. We show that overexpression of uPA in the Nac significantly augments cocaine-induced place preference. Furthermore, while this did not affect the ability of preference to be extinguished, reinstatement with a low dose of cocaine produced significantly greater preference to the cocaine-associated context. Once CPP had been established, and the preference extinguished, reinstatement induced by a priming dose of cocaine was facilitated by uPA. Inhibition of this serine protease expression using doxycycline abolished the augmented acquisition produced by overexpression of uPA but not the expression of the cocaine-induced CPP. When uPA is inhibited during the acquisition phase, animals no longer demonstrate place preference for the environment previously paired with cocaine. B428, a specific uPA inhibitor does not affect drug reinstatement after extinction if uPA has been activated during acquisition, a clear indication that uPA is involved in the acquisition phase of conditioned-place preference. Our results suggest that that increased uPA expression with repeated drug exposure produces conditions for enhanced acquisition of cocaine-induced CPP, indicating that cocaine-induced CPP and reinstatement may be dependent on active extracellular uPA.

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