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

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

E-cadherin is truncated in MDCK and Caco-2 cells expressing meprinβ.

<p>(A,B) Wild-type and meprin-expressing cells were grown on transwell filter inserts, <i>in situ</i> treated with trypsin or actinonin (meprin activator and inhibitor, respectively), or left untreated. Immunoblot analysis of indicated cell lysates was done with a monoclonal antibody directed against the EC1 domain at the N-terminus of E-cadherin (DECMA). An additional 97-kDa fragment of E-cadherin was detected in meprinβ-expressing cells. Blots were stripped and reprobed for meprin and tubulin. (C) Protein levels of meprinβ were similar in meprinβ-expressing MDCK cells and homogenates of whole mouse kidneys (C57Bl/6 strain). 15 µg of total protein extracts were analyzed on immunoblots. (D) E-cadherin is processed in meprinβ-expressing Caco-2 cells (β21) but not in parental TC7 Caco-2 cells.</p

Molecular mapping of the cleavage site of E-cadherin by meprinβ.

<p>(A) Schematic representation of E-cadherin and antibody epitopes. <i>a</i>: DECMA, <i>b</i>: SC7870, <i>c</i>: BDclone 36. S: Signal peptide, Pro: Propeptide, EC: Cadherin-domain, TM: transmembrane domain. (B) Protein extracts from MDCKwt and MDCKαβ cell lines were resolved by SDS-PAGE and analyzed by immunoblot using antibodies <i>a–c</i>. The same membrane was successively stripped and reprobed.</p

Meprinβ is partially colocalized with E-cadherin in MDCKαβ cells.

<p>The distribution of meprinβ and E-cadherin was analyzed by double immunostaining and CLSM in preconfluent (A) and confluent (B) monolayers. Pictures shown are single optical sections in the x–y axis. Corresponding orthogonal sections in the x–z and y–z axis are shown beside. Arrows in the merged picture indicate were meprinβ and E-cadherin colocalize (evidenced by the yellow color). Bar = 10 µm.</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

Meprinβ distribution in polarized and depolarized MDCKαβ cells.

<p>MDCKαβ cells were grown to confluence on filter supports in complete medium and subjected to a Ca²⁺-switch (described in experimental procedures). After fixation and immunostaining, meprinβ localization was analyzed by CLSM. Pictures (<i>a–c</i>) are single optical sections (x–y). Corresponding orthogonal sections in the x–z plane are shown below (<i>d–f</i>). Picture <i>a</i> shows meprinβ localization in confluent polarized MDCKαβ cells. Pictures <i>b</i>+<i>e</i> and <i>c</i>+<i>f</i> show meprinβ relocalization 30 minutes and 120 minutes after removal of extracellular Ca²⁺. Bar = 10 µm.</p