Proteolytic Processing of the Epithelial Adherens Junction Molecule E-Cadherin by Neutrophil Elastase Generates Short Peptides With Novel Wound-Healing Bioactivity

Abstract

We report the novel observation that the inflammatory protease, neutrophil elastase (NE), present in high abundance in inflamed tissue in inflammatory bowel disease (IBD) patients, is capable of cleaving the cellular adherens junction protein, E-cadherin. Proteolysis of E-cadherin by NE generates a variety of short peptides, several of which were observed in patient tissue samples, showing biological activity to promote wound closure in an in vitro model system. This effect is independent of proliferation either in a wounded monolayer or under subconfluent conditions, suggesting a primarily migratory activity upon colonic epithelial monolayers. We report the novel observation that inflammatory proteases post-translationally modify cellular junction proteins to create signaling peptides that contribute to the wound healing response and identifies a new mechanism of mucosal healing to be examined further in the context of chronic inflammatory diseases. IBDs, including ulcerative colitis and Crohn’s disease, comprise a spectrum of chronic inflammatory gastrointestinal diseases of complex etiology. Although there is no one defined cause or trigger for IBD, the unifying feature across the spectrum of IBD is the concept of chronic relapsing and remitting inflammatory disease, primarily in the colon (although in Crohn’s disease inflammation may occur anywhere along the gastrointestinal tract). 1 Mucosal healing now is considered the current gold standard in assessing IBD therapeutic remission, 2 however, our understanding of how and why many patients fail to achieve healing remains poorly elucidated. Upon an inflammatory stimulus, the intestinal epithelial monolayer is compromised by bacterial insult at the luminal surface as collateral damage from degranulation and an oxidative burst from lamina propria granulocytes, primarily neutrophils, and from cytokines released from leukocytes. 3 Neutrophils are the first immune cells recruited to areas of inflammation in IBD and sustained high infiltration of activated neutrophils in inflamed tissue is a hallmark of disease. 4 However, neutrophils now also are considered to be important players in the resolution phase of the inflammatory response.5, 6 Neutrophils can interact directly with epithelial cells by transmigrating through epithelia and interacting with apical intercellular adhesion molecule 1 to enhance wound healing through activation of the Akt and β-catenin pathways. 7 Damage to the intestinal epithelium causes a shift from a tight barrier to a migratory/repair phenotype, a process that involves the proteolytic cleavage of junctional proteins such as E-cadherin. Proteases can cleave epithelial junctional proteins to generate peptides that have biological activity that can affect the intestinal mucosa, 8 and recently NE was shown to cleave E-cadherin in bronchial epithelial cells, 9 although the potential effects of E-cadherin degradation peptides was not assessed in that study. Of particular interest is the recent observation that NE can be internalized by cells and is thus capable of processing both intracellular and extracellular substrates.10, 11 In this context, we examined whether the inflammatory protease NE could proteolytically cleave the adherens junction protein E-cadherin and, specifically, whether the peptides resulting from this cleavage event could affect epithelial wound healing. We first used a cell-free system to digest human recombinant E-cadherin with purified human NE and used liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify the resulting peptides and cleavage events that occurred. We found that NE was capable of cleaving E-cadherin efficiently, with 48 peptides identified with high confidence using mass spectrometry. Of these 48 peptides, we focused on 24 of these based on their frequency of occurrence, P value scoring, and accessibility of peptide cleavage site to proteases (Supplementary Table 1 and Supplementary Figure 1 A and B). To further focus on the most physiologically relevant peptides, we sought to confirm the presence of these peptides in patient tissues. By using a modified extraction protocol, we obtained protein/peptide fractions from banked formalin-fixed, paraffin-embedded IBD and control samples, and identified peptides originating from E-cadherin using mass spectrometry enriched in IBD samples. Six of these peptides showed substantial overlap with 6 peptides identified from our cell-free digest, and we chose these 6 peptides for biological activity screening (Table 1). First, we tested our peptides for effects on wound healing capacity using a scratch assay, under both 10% serum and serum-free conditions, over 48 hours at concentrations of 1, 10, and 100 μg/mL using a high-throughput protocol we designed for the Incucyte live cell imaging system (EssenBiosciences Inc, Ann Arbor, MI) (see the Supplementary Methods section for detailed methodologies). Three peptides, designated E-cadherin peptide (EP)-15, EP-17, and EP-22, at concentrations of 100, 1, and 10 μg/mL, respectively, showed increased wound closure compared with untreated and vehicle controls, in both 10% serum (Figure 1) and serum-free (Supplementary Figure 1 C) conditions. The peptides appeared to have a synergistic effect with 10% serum. Table 1 Alignment of E-Cadherin Peptides Generated by Neutrophil Elastase In Vitro With Peptide Fragments Isolated From IBD Patient Tissue Peptide Position Overlap TAYFSLDTR 66-74 None VTEPLDR 216-222 None NTGVISVVTTGLDR 332-335 EP-22 KNMFTINR NTGVISVVTTGLDR ESFPTYTL GQVPENEANVVITTLK 382-397 EP-13, partially EP-23 IFNPTTYK GQVPENEANVVITTLK VTDADAPN TVTDTNDNPPIFNPTTYK GQVPENEANVVITTLK VTDADAPN DTANWLEINPDTGA 528-541 None ISTRAELDR 542-550 None TIFFCER 559-565 None MALEVGDYK 656-664 None EPLLPPEDDTR 739-749 EP-17 LRRRAVVK EPLLPPEDDTR DNVYYYDE GLDARPEVTR 775-784 Partially EP-14 FDLSQLHR GLDARPEVTR NDVAPTLM NDVAPTLMSVPR 785-796 EP-14 DARPEVTR NDVAPTLMSVPR YLPRPANP PANPDEIGNFIDENLK 801-816 Partially EP-15 SVPRYLPR PANPDEIGNFIDENLK AADTDPTAPPYD NOTE. The left column indicates the peptides identified in patient tissue, the second column shows its position in the full E-cadherin protein, and the right columns indicate overlap with in vitro neutrophil elastase digestion of E-cadherin. Highlighted text indicates peptides identified in patient samples, and underlined text indicates peptides before trypsin treatment. Boxed text indicates E-cadherin peptides generated by NE cleavage. Figure 1 E-cadherin peptides enhance wound healing in Caco-2 cells. E-cadherin peptides (designated EP-15, EP-17, and EP-22) significantly and synergistically enhanced healing in scratch-wounded Caco-2 monolayers over 48 hours in the presence of 10% serum. *P < .05, **P < .01, and ***P < .001 compared with vehicle and untreated controls (using a 2-way analysis of variance with the Bonferroni post-test). N = 5 independent experiments, each with 3 or more technical replicates. Tx, treatment. The pro-healing effect was replicated by NE in a concentration-dependent manner, with 1 ng/mL having a small but significant effect in the presence of serum (Supplementary Figure 1 D). The effect of NE was dependent on its catalytic activity because the effect on wound healing was blocked with heat denaturation, which was confirmed to significantly reduce NE activity (Supplementary Figure 1 E). To determine whether this effect was due to an increase in the proliferation of cells in response to the presence of E-cadherin peptides, we used the 5-Ethynyl-2′-deoxyuridine (EdU) system to identify actively dividing cells and found that there was no significant difference in proliferation under serum-free or serum conditions. Concurrently, we used the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling enzyme to identify cell death to determine any cytotoxicity of our peptides at their effective doses and found no cytotoxicity of these peptides on Caco-2 monolayers (Supplementary Figure 2). To examine whether these peptides may have a mitogenic effect under nonwounding conditions, peptides also were tested for biological activity under subconfluent conditions. Caco-2 cells were transfected with a green fluorescent protein construct, seeded at medium density and exposed to the 6 peptides at the concentrations described earlier. Cell number and morphology were tracked over 48 hours. No significant changes in cell number or cell spreading were seen in response to any of the 6 peptides in either serum-free or 10% serum conditions (data not shown), suggesting that these peptides do not have mitogenic properties, and that the biological activity of the E-cadherin peptides is primarily to increase the migratory capacity under wound-healing conditions. To assess whether E-cadherin peptides could enter Caco-2 cells to potentially evoke intracellular signaling pathways, 7-amino-4-methylcoumarin (AMC) fluorescently tagged versions of EP-15 and EP-17 were synthesized and shown to transmigrate across the plasma membrane and into the cytosol (Supplementary Figure 3). In this research letter, we show the ability of an inflammatory protease, neutrophil elastase, to process the adherens junction protein E-cadherin to generate short peptide fragments with effects on epithelial function, and a novel role for low levels of NE being pro-resolution. These peptide fragments are present in IBD patient tissues and appear to enhance the wound-healing response of intestinal epithelial cell monolayers independently of cellular proliferation. Our study raises important questions about the cellular mechanism whereby NE-derived peptide fragments of E-cadherin stimulate an epithelial wound-healing response. Is the site of action of E-cadherin peptides on epithelial cells extracellular or intracellular, and what is the mechanism of transport across the cell membrane? What intracellular pathway(s) are altered by these peptides to modify epithelial cell behavior? Are other bioactive peptides proteolytically produced by NE or other inflammatory proteases as a resolution response, and what are the cellular targets of these peptides? Thus, our work provides the impetus for further research that will determine the signaling mechanisms underlying this phenomenon, identify potential new peptide biomarkers of inflammatory diseases, and develop new therapeutic targets. Overall, our discovery adds a new layer of complexity to our understanding of the signaling mechanisms underlying mucosal repair after inflammatory insult and suggests a new potential arm of repair signaling that may be dysregulated during IBD and other chronic inflammatory diseases.