Régulation protéolytique du récepteur de l'urokinase et inflammation pulmonaire

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Rôle de la protéine "Disc Large" dans les cellules de la paroi vasculaire humaine

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Disruption of the endothelial barrier by proteases from the bacterial pathogen Pseudomonas aeruginosa: implication of matrilysis and receptor cleavage

International audienceWithin the vasculature, uncontrolled pericellular proteolysis can lead to disruption of cell-to-cell and cell-to-matrix interactions and subsequent detachment-induced cell apoptosis, or anoikis, contributing to inflammatory vascular diseases, with the endothelium as the major target. Most studies so far have focused on endogenous proteinases. However, during bloodstream infections, bacterial proteinases may also trigger endothelial anoikis. We thus investigated the potential apoptotic activity of the proteinases secreted by the haematotropic opportunistic pathogen, Pseudomonas aeruginosa, and particularly its predominant metalloproteinase, LasB. For this, we used the secretome of the LasB-expressing pseudomonal strain, PAO1, and compared it with that from the isogenic, LasB-deficient strain (PAO1?lasB), as well as with purified LasB. Secretomes were tested for apoptotic activity on cultured human endothelial cells derived from the umbilical vein or from the cerebral microvasculature. We found that the PAO1 secretome readily induced endothelial cell anoikis, as did secretomes of LasB-positive clinical pseudomonal isolates, while the PAO1?lasB secretome had only a limited impact on endothelial adherence and viability. Notably, purified LasB reproduced most of the effects of the LasB-containing secretomes, and these were drastically reduced in the presence of the LasB-selective inhibitor, phosphoramidon. A precocious and extensive LasB-dependent degradation of several proteins associated with the endothelial extracellular matrix, fibronectin and von Willebrand factor, was observed by immunofluorescence and/or immunoblotting analysis of cell cultures. Moreover, the PAO1 secretome, but not that from PAO1?lasB, specifically induced rapid endoproteolysis of two major interendothelial junction components, VE-cadherin and occludin, as well as of the anti-anoikis, integrin-associated urokinase receptor, uPAR. Taken as a prototype for exogenous haemorrhagic proteinases, pseudomonal LasB thus appears to induce endothelial anoikis not only via matrilysis, as observed for many pro-apoptotic proteinases, but also via cleavage of some essential cell-to-cell and cell-to-matrix adhesion receptors implicated in the maintenance of the endothelial barrier

The Pseudomonas aeruginosa LasB Metalloproteinase Regulates the Human Urokinase-Type Plasminogen Activator Receptor through Domain-Specific Endoproteolysis▿

Pseudomonas aeruginosa is an opportunistic pathogen in human lungs, where its secretable LasB metalloproteinase can be a virulence factor. The urokinase-type plasminogen activator receptor (uPAR) participates in pericellular proteolysis and the adherence/migration of epithelial cells and leukocytes recruited during infection and shows functional regulation by various proteinases via limited endoproteolysis occurring within its three domains (D1 to D3). We thus examined the proteolytic activity of LasB on uPAR by using recombinant uPAR as well as uPAR-expressing, human monocytic, and bronchial epithelial cell lines. Protein immunoblotting and flow immunocytometry using a panel of domain-specific anti-uPAR antibodies showed that LasB is able to cleave uPAR both within the sequence linking D1 to D2 and at the carboxy terminus of D3. Comparison of LasB-producing and LasB-deficient bacterial strains indicated that LasB is entirely responsible for the uPAR cleavage ability of P. aeruginosa. Based on amino-terminal protein microsequencing and mass spectrometry analysis of the cleavage of peptides mimicking the uPAR sequences targeted by LasB, cleavage sites were determined to be Ala84-Val85 and Thr86-Tyr87 (D1-D2) and Gln279-Tyr280 (D3). Such a dual cleavage of uPAR led to the removal of amino-terminal D1, the generation of a truncated D2D3 species, and the shedding of D2D3 from cells. This proteolytic processing of uPAR was found to (i) drastically reduce the capacity of cells to bind urokinase and (ii) abrogate the interaction between uPAR and the matrix adhesive protein vitronectin. The LasB proteinase is thus endowed with a high potential for the alteration of uPAR expression and functioning on inflammatory cells during infections by P. aeruginosa

The LasB-containing secretome induces cleavage of the urokinase receptor.

<p>Confluent cultures of hCMEC/D3 cells were exposed for 1 to 24 h to low FCS culture medium alone or with 10% of either LB, PAO1-Sec, or PAO1∆<i>lasB</i>-Sec (left-hand panels in A and B), or for 24 h to culture medium with LB (20%) or with the bacterial secretomes in the range of 1 to 20% (right-hand panels in A and B). (<i>A</i>) Total proteins extracted from residual adherent cells were analyzed by SDS-PAGE/IB (5 μg <i>per</i> well) for detection of cell-associated uPAR using an anti-uPAR mAb directed against uPAR domain D2 (1/250 dilution of hybridoma cell culture medium; upper panels), then reprobed with the anti-GAPDH mAb (lower panels). Located on the left-hand side are full-length three-domain (D1D2D3) uPAR and its truncated two-domain D2D3 derivative (large black arrows), and GAPDH (small black arrows). Results illustrated are representative of four (left-hand panels) to five (right-hand panels) independent experiments performed on HUVECs or hCMEC/D3 cells. (<i>B</i>) Densitometric analysis of bands corresponding to the full-length uPAR species as illustrated in (A) is reported as the mean percentage + or - SEM of the value measured for control cell extracts exposed to LB.</p

Residual adherent endothelial cells in the presence of pseudomonal secretomes are not apoptotic.

<p>Confluent hCMEC/D3 cell cultures were exposed for up to 24 h in low FCS culture medium with 10% of either LB, PAO1-Sec, or PAO1∆<i>lasB</i>-Sec, or to staurosporine. Residual adherent cells were evaluated for apoptotic nuclei by staining with DAPI (upper panels) or the TUNEL assay (lower panels). Representative areas (100x magnification) are shown for one of three similar experiments performed on either hCMEC/D3 cell or HUVEC cultures. Cultures with LB or PAO1∆<i>lasB</i>-Sec are depicted at 24 h.</p