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

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

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

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

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

Proteolytic activity of meprins α and β on AIEC LF82 outer membrane proteins and flagellin.

<p>Total protein extracts from untreated or meprin-treated (100 µg/mL) whole bacteria were immunoblotted for OmpA and OmpC/F (A) or Flagellin (B). The inner membrane protein Lep was used as internal control. Amounts of proteins were quantified by using <i>Image J</i> software. Results are expressed as protein amount relative to Lep. Data are mean ± SEM for at least three independent experiments. Student's <i>t</i>-test, * <i>P</i><0.05.</p

Intestinal meprin α and meprin β mRNA.

<p>A and B, Mep1A (A) and Mep1B (B) mRNA levels were determined by TaqMan quantitative real time PCR and are displayed as amounts relative to the intestinal epithelial marker villin-1. Healthy controls (hc) were compared with ulcerative colitis (UC) and Crohn's disease (CD) patients. CD patient biopsies were separated into groups with normal appearance or with macroscopic inflammation, as determined by an experienced endoscopist. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the non-parametric Mann-Whitney test. C and D, Mep1A (C) and Mep1B (D) mRNA levels in C57Bl/6J mouse ileum and colon uninfected or infected with AIEC LF82 bacteria. The effect of AIEC LF82 infection on meprin expression was determined in mouse ileum and colon by quantitative real time PCR. Data are displayed as meprin amounts relative to the housekeeping TATA box binding protein (TBP) gene. Statistics were performed using GraphPad Prism 5.0 Software, and <i>P</i> values were calculated with the one-way ANOVA test. Dot plots show individual samples with relative mRNA (cDNA) amounts on a linear scale. Horizontal bars represent the median.</p

Meprin treatment affect mannose residue recognition by AIEC and AIEC-induced IL-8 secretion by T84 cells.

<p>A, ability of type 1 pili to bind D-mannose residues as determined by a yeast aggregation test. AIEC LF82 bacteria were treated with 100 µg/ml of meprin α or β at 37°C for 120 min. A fixed amount of inactivated yeast cells (<i>Saccharomyces cerevisiae</i>) suspension and decreasing concentrations of treated and untreated bacteria were mixed, and the loss of the ability to form homogenous aggregation was used as the read-out for impaired type 1 pili-yeast interaction. B, Amount of IL-8 secreted by uninfected or AIEC LF82- or type 1 pili negative mutant LF82-Δ<i>fimA</i>-infected T84 cells, at 24 h post-infection. AIEC LF82 and LF82-Δ<i>fimA</i> bacteria were treated with 100 µg/ml of meprins. Il-8 secretion was determined by ELISA. Data are expressed as fold increase in the amount of secreted IL-8 ± SEM by T84 cells infected with untreated or treated bacteria relative to non infected cells. Student's <i>t</i>-test, * <i>P</i><0.05 for comparison between IL-8 secretion induced by untreated versus meprin-treated AIEC LF82 or LF82-Δ<i>fimA</i> bacteria. C, LF82-Δ<i>fimA</i> bacteria were pretreated with exogenous meprin α or meprin β at 100 µg/ml and undifferentiated T84 cells were infected at a MOI of 10. The number of associated bacteria was determined. Results are expressed as the percentage of cell-associated bacteria pretreated with exogenous meprins relative to untreated bacteria, defined as 100%.D, effect of meprins on recombinant human IL-8. Recombinant human IL-8 (110 ng/ml) was treated with meprin α or β (100 µg/ml), electroblotted and detected with mouse anti-human IL-8.</p