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

Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1β- Dependent Stabilization of Snail in Tumor Cells

We recently reported that colon tumor cells stimulate macrophages to release IL-1beta, which in turn inactivates GSK3beta and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1beta by neutralizing IL-1beta antibody, or silencing of IL-1beta in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1beta was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Delta psi) and activation of caspases were prevented by macrophages or by recombinant IL-1beta. Pharmacological inhibition of IL-1beta release from macrophages by vitamin D(3), a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1beta failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIkappaB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1beta stabilized Snail in tumor cells in an NF-kappaB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1beta, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1beta, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D(3) halts this amplifying loop by interfering with the release of IL-1beta from macrophages. Accordingly, vitamin D(3) sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent

Inflammation and breast cancer. Metalloproteinases as common effectors of inflammation and extracellular matrix breakdown in breast cancer

Two rapidly evolving fields are converging to impact breast cancer: one has identified novel substrates of metalloproteinases that alter immune cell function, and the other has revealed a role for inflammation in human cancers. Evidence now shows that the mechanisms underlying these two fields interact in the context of breast cancer, providing new opportunities to understand this disease and uncover novel therapeutic strategies. The metalloproteinase class of enzymes is well studied in mammary gland development and physiology, but mostly in the context of extracellular matrix modification. Aberrant metalloproteinase expression has also been implicated in breast cancer progression, where these genes act as tumor modifiers. Here, we review how the metalloproteinase axis impacts mammary physiology and tumorigenesis and is associated with inflammatory cell influx in human breast cancer, and evaluate its potential as a regulator of inflammation in the mammary gland

Apoptosis-induced cleavage of keratin 15 and keratin 17 in a human breast epithelial cell line

Keratin 15 (K15) and keratin 17 (K17) are intermediate filament (IF) type I proteins that are responsible for the mechanical integrity of epithelial cells. By analyzing the human breast epithelial cell line H184A1 before and after induction of apoptosis by high-resolution two-dimensional gel electrophoresis (2-DE) we identified the caspase-mediated cleavage of keratins 15 and 17. After induction of apoptosis three fragments of both K15 and K17 could be observed by 2 -DE. K15 and K17 proteolysis was observed during staurosporine-induced apoptosis and anoikis (anchorage-dependent apoptosis) as well and was shown to be caspase-dependent. By using mass spectrometry we could determine the caspase cleavage sites, one in K15 and two in K17. The sequence VEMD/A at the cleavage site located in the conserved linker region was found in K15 and K17. A further cleavage site was identified in the tail region of K17 with the recognition motif EVQD/G

Cleavage and shedding of E-cadherin after induction of apoptosis

Apoptotic cell death induces dramatic molecular changes in cells, becoming apparent on the structural level as membrane blebbing, condensation of the cytoplasm and nucleus, and loss of cell-cell contacts. The activation of caspases is one of the fundamental steps during programmed cell death. Here we report a detailed analysis of the fate of the Ca2+-dependent cell adhesion molecule E-cadherin in apoptotic epithelial cells and show that during apoptosis fragments of E-cadherin with apparent molecular masses of 24, 29, and 84 kDa are generated by two distinct proteolytic activities. In addition to a caspase-3-mediated cleavage releasing the cytoplasmic domain of E-cadherin, a metalloproteinase sheds the extracellular domain from the cell surface during apoptosis. Immunofluorescence analysis confirmed that concomitant with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulates in the cytosol. In the presence of inhibitors of caspase-3 and/or metalloproteinases, cleavage of E-cadherin was almost completely blocked. The simultaneous cleavage of the intracellular and extracellular domains of E-cadherin may provide a highly efficient mechanism to disrupt cadherin-mediated cell-cell contacts in apoptotic cells, a prerequisite for cell rounding and exit from the epithelium

Apoptosis-induced cleavage of beta-catenin by caspase-3 results in proteolytic fragments with reduced transactivation potential

{Beta}-Catenin is a member of the Armadillo repeat protein family with a dual cellular function as a component of both the adherens junction complex and the Wnt/wingless signaling pathway. Here we show that {beta}-catenin is proteolytically cleaved during anoikis and staurosporine-induced apoptosis. Cleavage of {beta}-catenin was found to be caspase-dependent. Five cleavage products of {beta}-catenin were identified in vivo and after in vitro cleavage by caspase-3. Amino acid sequencing and mass spectrometry analysis indicated two caspase-3 cleavage sites at the C terminus and three further sites at the N terminus, whereas the central Armadillo repeat region remained unaffected. All {beta}-catenin cleavage products were still able to associate with E-cadherin and {alpha}-catenin and were found to be enriched in the cytoplasm. Functional analysis revealed that {beta}-catenin deletion constructs resembling the observed proteolytic fragments show a strongly reduced transcription activation potential when analyzed in gene reporter assays. We therefore conclude that an important role of the {beta}-catenin cleavage during apoptosis is the removal of its transcription activation domains to prevent its transcription activation potential

Cleavage of Armadillo/beta-catenin by the caspase DrICE in Drosophila apoptotic epithelial cells

<p>Abstract</p> <p>Background</p> <p>During apoptosis cells become profoundly restructured through concerted cleavage of cellular proteins by caspases. In epithelial tissues, apoptotic cells loose their apical/basal polarity and are extruded from the epithelium. We used the <it>Drosophila </it>embryo as a system to investigate the regulation of components of the zonula adherens during apoptosis. Since Armadillo/beta-catenin (Arm) is a major regulator of cadherin-mediated adhesion, we analyzed the mechanisms of Arm proteolysis in apoptosis.</p> <p>Results</p> <p>We define early and late apoptotic stages and find that early in apoptosis <it>D</it>α-catenin remains relatively stable, while Arm and <it>D</it>E-cadherin protein levels are strongly reduced. Arm is cleaved by caspases in embryo extracts and we provide evidence that the caspase-3 homolog drICE cleaves Arm in vitro and in vivo. Cleavage by drICE creates a stable protein fragment that remains associated with the plasma membrane early in apoptosis. To further understand the role of caspase-mediated cleavage of Arm, we examined potential caspase cleavage sites and found that drICE cleaves Arm at a unique DQVD motif in the N-terminal domain of the protein. Mutation of the drICE cleavage site in Arm results in a protein that is not cleaved in vitro and in vivo. Furthermore we provide evidence that cleavage of Arm plays a role in the removal of <it>D</it>E-cadherin from the plasma membrane during apoptosis.</p> <p>Conclusion</p> <p>This study defines the specificity of caspase cleavage of Arm in <it>Drosophila </it>apoptotic cells. Our data suggest that N-terminal truncation of Arm by caspases is evolutionarily conserved and thus might provide a principal mechanism involved in the disassembly of adherens junctions during apoptosis.</p