Enhanced Activity of Meprin-α, a Pro-Migratory and Pro-Angiogenic Protease, in Colorectal Cancer

Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF)- induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer) stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer

The Metalloprotease Meprinβ Processes E-Cadherin and Weakens Intercellular Adhesion

BACKGROUND: Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used MDCK and Caco-2 cells stably transfected with meprin alpha and or meprin beta to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. CONCLUSIONS/SIGNIFICANCE: By identifying E-cadherin as a substrate for meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression

Metalloprotease Meprinβ in Rat Kidney: Glomerular Localization and Differential Expression in Glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprinβ in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprinβ in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprinβ expression. The glomerular meprinβ expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprinβ staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprinβ is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprinβ in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprinβ in this form of glomerulonephritis

A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively

The direct binding partners of E-cadherin are processed in MDCK and Caco-2 cells expressing meprinβ.

<p>β-catenin and plakoglobin were analyzed in protein extracts of MDCKwt, MDCKαβ, MDCKα and MDCKβ cells (A), as well as in Caco-2-TC7 and Caco-2-β21 cells (B). Two cleaved fragments were detected in lysates of cells expressing meprinβ. α-catenin remained intact (C).</p

<i>In vitro</i> cleavage of E-cadherin by purified recombinant active meprinβ.

<p>(A) MDCKwt cell lysates were incubated with different concentrations of meprinβ for the indicated times. Cleavage products were analyzed on Western blots using an anti-E-cadherin antibody (DECMA). Processed E-cadherin in MDCKαβ cells are shown in comparison. (B) E-cadherin was first immunoprecipitated from MDCKwt cell lysates and subsequently incubated on the beads with recombinant meprinβ at the indicated concentration for 40 minutes. E-cadherin cleavage fragments in eluates from the beads were analyzed on immunoblots using the monoclonal N-terminal antibody (DECMA). Samples of MDCKwt and MDCKαβ cell lysates were loaded as reference. (C,D) Specific generation of the 97-kDa E-cadherin fragment by meprinβ, but not MMP-7 and ADAM-10. MDCKwt cell lysates (C) or immunoprecipitated E-cadherin on beads (D) were incubated for 1 hour or overnight with increasing concentrations of recombinant active meprinβ, MMP-7 and ADAM-10 (0.0125 nM, 0.125 nM and 1.25 nM).</p

Meprinβ affects intercellular adhesion.

<p>(A) Cell-cell contact strength was measured using the dispase assay (described in experimental procedures). The graph shows the mean values +/− SD from 3 independent experiments. * = p<0.05. (B) Cell aggregation assay with MDCK cell lines (<i>a–h</i>) and Caco-2 cell lines (<i>i–l</i>). Hanging drops of cell suspensions were incubated overnight. Representative pictures of three independent experiments with each condition in 6 replicates are shown. Cells expressing meprinβ form smaller aggregates (<i>b</i>, <i>d</i>, <i>j</i> compared to <i>a</i>, <i>c</i>, <i>i</i>). The presence of actinonin (right panels) in the resuspension medium reverted the phenotype of meprinβ expressing cells (<i>f</i>, <i>h</i>, <i>l</i>) without having an effect in the other cell lines (<i>e</i>, <i>g</i>, <i>k</i>).</p