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Jean-michel Sallenave - One of the best experts on this subject based on the ideXlab platform.

  • sheep lung segmental delivery strategy demonstrates adenovirus priming of local lung responses to bacterial lps and the role of Elafin as a response modulator
    PLOS ONE, 2014
    Co-Authors: Thomas I. Brown, David Collie, Darren J Shaw, Nina Marie Rzechorzek, Jean-michel Sallenave
    Abstract:

    Viral lung infections increase susceptibility to subsequent bacterial infection. We questioned whether local lung administration of recombinant adenoviral vectors in the sheep would alter the susceptibility of the lung to subsequent challenge with bacterial lipopolysaccharide (LPS). We further questioned whether local lung expression of Elafin, a locally produced alarm anti-LPS/anti-bacterial molecule, would modulate the challenge response. We established that adenoviral vector treatment primed the lung for an enhanced response to bacterial LPS. Whereas this local effect appeared to be independent of the transgene used (Ad-o-Elafin or Ad-GFP), Ad-o-Elafin treated sheep demonstrated a more profound lymphopenia in response to local lung administration of LPS. The local influence of Elafin in modulating the response to LPS was restricted to maintaining neutrophil myeloperoxidase activity, and levels of alveolar macrophage and neutrophil phagocytosis at higher levels post-LPS. Adenoviral vector-bacterial synergism exists in the ovine lung and Elafin expression modulates such synergism both locally and systemically.

  • The WAP protein Trappin-2/Elafin: A handyman in the regulation of inflammatory and immune responses
    The International Journal of Biochemistry & Cell Biology, 2012
    Co-Authors: Thomas Verrier, Jean-michel Sallenave, Brigitte Solhonne, Ignacio Garcia-verdugo
    Abstract:

    Abstract Trappin-2/Elafin is a potent serine protease inhibitor which prevents excessive damage under inflammatory status. This “alarm-antiprotease” is locally expressed by epithelial cells and immune cells such as macrophages and γδ T cells. It has also been proven to modulate a wide range of parameters that are critical for the inflammation process like modulating the NFκB pathway, cytokine secretion and cell recruitment. In addition, Trappin-2/Elafin was shown to possess anti-microbial properties against different classes of pathogens including viruses, fungi and bacteria. Studies also linked Trappin-2/Elafin to either susceptibility or protection against inflammatory disease and infections, even though the mechanisms remains poorly understood. This review will discuss some of the pleiotropic effects displayed by Trappin-2/Elafin, and the properties that could be used to prevent infection or to protect against inflammation.

  • the antimicrobial elastase inhibitor Elafin regulates lung dendritic cells and adaptive immunity
    American Journal of Respiratory Cell and Molecular Biology, 2006
    Co-Authors: Ali Roghanian, Steven Williams, Tara A. Sheldrake, Thomas I. Brown, Karen Oberheim, Zhou Xing, Sarah E. M. Howie, Jean-michel Sallenave
    Abstract:

    Infections with bacteria and viruses such as adenovirus are a feature of chronic lung diseases such as chronic obstructive pulmonary diseases (COPD), and may be instrumental in the generation of disease exacerbations. We have previously shown in acute models that Elafin (a lung natural chemotactic molecule for macrophages and neutrophils, with potent antimicrobial and neutrophil elastase inhibitor activity) is upregulated in infection and modulates innate immunity. Here we present data using two independent systems of Elafin overexpression in vivo (recombinant adenovirus [Ad-Elafin] and an Elafin transgenic mouse line) to examine the function of Elafin in adaptive immunity. We show that Elafin increases the number (immunofluorescence) and activation status (flow cytometric measurement) of CD11c+/MHCII+ lung dendritic cells in vivo. Analysis of cytokines produced by spleen and lung cells, and of antibodies measured in serum and bronchoalveolar lavage fluid, shows that the immunity induced is biased toward a type 1 response (production of IL-12, IFN-gamma, and IgG2a). Furthermore, Elafin overexpression protected mice against further challenge with Ad-LacZ, as assessed by antibody levels and neutralization titer, as well as LacZ expression in lung tissue. Thus, the pleiotropic molecule Elafin has significant potential in modulating antigen-presenting cell numbers and activity, and could be beneficial in mucosal protective strategies.

  • SLPI and Elafin: one glove, many fingers
    Clinical Science, 2006
    Co-Authors: Steven e. Williams, Thomas i. Brown, Ali Roghanian, Jean-michel Sallenave
    Abstract:

    Elafin and SLPI (secretory leucocyte protease inhibitor) have multiple important roles both in normal homoeostasis and at sites of inflammation. These include antiprotease and antimicrobial activity as well as modulation of the response to LPS (lipopolysaccharide) stimulation. Elafin and SLPI are members of larger families of proteins secreted predominantly at mucosal sites, and have been shown to be modulated in multiple pathological conditions. We believe that Elafin and SLPI are important molecules in the controlled functioning of the innate immune system, and may have further importance in the integration of this system with the adaptive immune response. Recent interest has focused on the influence of inflamed tissues on the recruitment and phenotypic modulation of cells of the adaptive immune system and, indeed, the local production of Elafin and SLPI indicate that they are ideally placed in this regard. Functionally related proteins, such as the defensins and cathelicidins, have been shown to have direct effects upon dendritic cells with potential alteration of their phenotype towards type I or II immune responses. This review addresses the multiple functions of Elafin and SLPI in the inflammatory response and discusses further their roles in the development of the adaptive immune response.

  • The antimicrobial antiproteinase Elafin binds to lipopolysaccharide and modulates macrophage responses
    American Journal of Respiratory Cell and Molecular Biology, 2005
    Co-Authors: Jonathan W. Mcmichael, Ali Roghanian, Lu Jiang, Robert Ramage, Jean-michel Sallenave
    Abstract:

    Lipopolysaccharides (LPS) of the outer membrane of Gram-negative bacteria represent a primary target for innate immune responses. We demonstrate here that the antimicrobial/anti-neutrophil elastase full-length Elafin (FL-EL) is able to bind both smooth and rough forms of LPS. The N-terminus was shown to bind both forms of LPS more avidly. We demonstrate that the lipid A core-binding proteins polymyxin B (PB) and LPS-binding protein (LBP) compete with Elafin for binding, and that LBP is able to displace prebound Elafin from LPS. When PB, FL-EL, N-EL, and C-EL were pre-incubated with LPS before addition to immobilized LBP, PB was the most potent inhibitor of LPS transfer to LBP. These data prompted us to examine the biological consequences of Elafin binding to LPS, using tumor necrosis factor (TNF)-alpha release by murine macrophages. In serum-containing conditions, N-EL had no effect, whereas both C-EL and FL-EL inhibited TNF-alpha production. In serum-free conditions, however, all moieties had a stimulatory activity on TNF-alpha release, with C-EL being the most potent at the highest concentration. The differential biological activity of Elafin in different conditions suggests a role for this molecule in either LPS detoxification or activation of innate immune responses, depending on the external cellular environment.

Khandan Keyomarsi - One of the best experts on this subject based on the ideXlab platform.

  • Elafin is downregulated during breast and ovarian tumorigenesis but its residual expression predicts recurrence
    Breast Cancer Research, 2014
    Co-Authors: Joseph A. Caruso, Cansu Karakas, Jing Zhang, Constance Albarracin, Aysegul A. Sahin, Melissa L. Bondy, Jinsong Liu, Kelly K. Hunt, Khandan Keyomarsi
    Abstract:

    Elafin is an endogenous serine protease inhibitor. The majority of breast cancer cell lines lack Elafin expression compared to human mammary epithelial cells. In this study, we hypothesized that Elafin is downregulated during breast and ovarian tumorigenesis. We examined Elafin expression by immunohistochemistry (IHC) in specimens of normal breast tissue (n = 24), ductal carcinoma in situ (DCIS) (n = 54), and invasive breast cancer (n = 793). IHC analysis of Elafin expression was also performed in normal fallopian tube tissue (n = 20), ovarian cystadenomas (n = 9), borderline ovarian tumors (n = 21), and invasive ovarian carcinomas (n = 216). To understand the significance of Elafin in luminal breast cancer cell lines, wild-type or M25G Elafin (lacking the protease inhibitory function) were exogenously expressed in MCF-7 and T47D cells. Elafin expression was downregulated in 24% of DCIS and 83% of invasive breast tumors when compared to Elafin expression in the normal mammary epithelium. However, the presence of Elafin-positive cells in invasive breast tumors, even at low frequency, correlated with poor recurrence-free survival (RFS), reduced overall survival (OS), and clinicopathological markers of aggressive tumor behavior. Elafin-positive cells were an especially strong and independent prognostic marker of reduced RFS in IHC-defined luminal A-like tumors. Elafin was also downregulated in 33% of ovarian cystadenomas, 43% of borderline ovarian tumors, and 86% of invasive ovarian carcinomas when compared to Elafin expression in the normal fallopian tube. In ovarian tumors, Elafin-positive cells were correlated with reduced RFS, OS and disease-specific survival (DSS) only in stage I/II patients and not in stage III/IV patients. Notably, exogenous expression of Elafin or Elafin M25G in the luminal breast cancer cell lines MCF-7 and T47D significantly decreased cell proliferation in a protease inhibitory domain-independent manner. Elafin predicts poor outcome in breast and ovarian cancer patients and delineates a subset of endocrine receptor-positive breast cancer patients susceptible to recurrence who could benefit from more aggressive intervention. Our in vitro results suggest that Elafin arrests luminal breast cancer cells, perhaps suggesting a role in tumor dormancy.

  • The serine protease inhibitor Elafin maintains normal growth control by opposing the mitogenic effects of neutrophil elastase
    Oncogene, 2014
    Co-Authors: Joseph A. Caruso, Kelly K. Hunt, Said Akli, Laura R. Pageon, Khandan Keyomarsi
    Abstract:

    The serine protease inhibitor, Elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared with normal human mammary epithelial cells (HMECs). Here, we evaluated the role of Elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor-deprived HMECs as they enter a quiescent (G0) state, suggesting that Elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of Elafin compromises the ability of HMECs to maintain G0 arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type Elafin but not Elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of Elafin to arrest cells in G0. In fact when purified NE was added to Elafin-knocked down HMECs, these cells demonstrated greater sensitivity to the growth-promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples. Immunohistochemical analysis of normal breast tissue revealed robust Elafin expression in the mammary epithelium; however, Elafin expression was dramatically downregulated in a significant proportion of human breast tumor specimens. The loss of Elafin expression during breast cancer progression may promote tumor growth as a consequence of increased NE activity. To address the role of NE in mammary tumorigenesis, we next examined whether deregulated NE activity enhances mammary tumor growth. NE knockout in the C3(1)TAg mouse model of mammary tumorigenesis suppressed proliferation and reduced the kinetics of tumor growth. Overall, the imbalance between NE and its inhibitors, such as Elafin, presents an important therapeutic target in breast cancer.

  • Elafin an inhibitor of elastase is a prognostic indicator in breast cancer
    Breast Cancer Research, 2013
    Co-Authors: Kelly K. Hunt, Ming Zhang, Tomoya Yokota, Yanna Liu, Hannah Wingate, Gordon B Mills, Fan Zhang, Bingliang Fang, Khandan Keyomarsi
    Abstract:

    Elafin is an elastase-specific inhibitor with increased transcription in normal mammary epithelial cells compared to mammary carcinoma cells. In this report, we test the hypothesis that inhibition of elastase, through induction of Elafin, leads to inhibition of human breast cancer cell viability and, therefore, predicts survival in breast cancer patients. Panels of normal and immortalized breast epithelial cells, along with breast carcinoma cells, were used to examine the impact of adenoviral-mediated Elafin expression or shRNA-mediated inhibition of elastase on the growth of cells and xenografts in nude mice. To determine the prognostic significance of decreased Elafin in patients with invasive breast cancer, previously published gene array datasets were interrogated. Elafin expression had no effect on non-tumorigenic cells but resulted in marked inhibition of cell growth in breast cancer cell lines. Control-treated xenografts generated a tumor burden that necessitated sacrifice within one month of initial treatment, whereas xenograft-bearing mice treated with Ad-Elafin were alive at eight months with marked reduction in tumor growth. Elastase inhibition mimicked these results, showing decreased tumor cell growth in vitro and in vivo. Low expression of Elafin gene correlated with significantly reduced time to relapse, and when combined with high expression of elastase gene was associated with decreased survival in breast cancer patients. Our data suggest that Elafin plays a direct role in the suppression of tumors through inhibition of elastase and thus serves as a prognostic indicator for breast cancer patients.

  • Abstract 2466: Elafin, a serine protease inhibitor, is deregulated during breast cancer progression
    Tumor Biology, 2012
    Co-Authors: Joseph A. Caruso, Cansu Karakas, Kelly K. Hunt, Khandan Keyomarsi
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Deregulated protease activity promotes diverse aspects of tumorigenesis, including tumor growth, invasion, inflammation, and angiogenesis. Elafin is an endogenous inhibitor of the serine protease, Neutrophil Elastase (NE). Imbalance between NE and its inhibitors is implicated in the pathogenesis of many chronic inflammatory diseases. We hypothesize that Elafin is deregulated during breast cancer progression, resulting in increased NE activity, accelerated tumor progression, and overall poor patient outcome. To test this hypothesis, we examined Elafin expression by immunohistochemistry (IHC) in normal breast tissue from reduction mammoplasty, pre-malignant Ductal Carcinoma In Situ (DCIS), and invasive breast carcinoma (n>1000, using Tissue Microarray [TMA]). We observed that Elafin is intracellularly (predominate nuclear localization) expressed in normal breast tissue. In the majority (>80%) of DCIS cases, intracellular Elafin is expressed in a manner consistent with normal breast tissue. However, in cases of invasive breast cancer, intracellular Elafin is largely downregulated (>80% of cases) suggesting Elafin loss may have a role in progression to invasive carcinoma. Interestingly, examination of Elafin expression in invasive breast cancer also revealed a subset (10-20%) of tumors with robust cytoplasmic overexpression of Elafin, correlating with poor outcome. Similar results were seen in TMAs of high-grade ovarian carcinoma (n>200). Examination of publically available microarray data (TCGA) demonstrates that Elafin measured at the mRNA level is correlates with poor outcome in ovarian cancer, consistent with our IHC results, and good outcome in breast cancer, incompatible with our IHC results. To further investigate the expression of Elafin in breast cancer we examined by IHC a patient cohort (>200 patients) for whom full sections (not TMA) were available. Using these patient samples we found a unique subset of breast cancer patients that demonstrate high-level secretion of Elafin into the tumor stroma, significantly correlating with increased survival especially in ER negative patients. Collectively, these data suggest that the prognostic role of extracellular Elafin is related to the well-characterized ability of Elafin to modulate inflammation and immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2466. doi:1538-7445.AM2012-2466

  • The Neutrophil Elastase Inhibitor Elafin Triggers Rb-Mediated Growth Arrest and Caspase-Dependent Apoptosis in Breast Cancer
    Cancer Research, 2010
    Co-Authors: Joseph A. Caruso, Kelly K. Hunt, Khandan Keyomarsi
    Abstract:

    Elafin, an endogenous inhibitor of neutrophil elastase, is expressed in human mammary epithelial cells but is transcriptionally downregulated in breast cancer cells. We hypothesized that Elafin may exert a tumor-suppressive activity in the context of breast cancer. In this study, we show that the retinoblastoma (Rb) pathway governs the antitumor properties of Elafin. In breast cancer cells with functional Rb, the expression of Elafin triggered Rb-dependent cell cycle arrest. Elafin also exhibited suppressive activity in breast cancer cell lines lacking Rb, but this was associated with an induction of caspase-3–dependent, p53-independent apoptotic cell death. Normal mammary epithelial cells were not affected by Elafin. Collectively, these results argue that Elafin mediates tumor-suppressive effects that are cytostatic or cytotoxic depending on the Rb status. Our findings suggest that Elafin could be engineered as a therapeutic modality to treat breast cancer without toxicity to normal proliferating cells. Cancer Res; 70(18); 7125–36. ©2010 AACR.

Thierry Moreau - One of the best experts on this subject based on the ideXlab platform.

  • A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation
    Molecular Therapy, 2015
    Co-Authors: Donna M. Small, Marie Louise Zani, Derek J. Quinn, Sandrine Dallet-choisy, Arlene Glasgow, Cecilia O'kane, Daniel F. Mcauley, Paul Mcnally, Sinéad Weldon, Thierry Moreau
    Abstract:

    Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of Elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of Elafin (GG- and QQ-Elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) Elafin. Similar to WT-Elafin, GG- and QQ-Elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-Elafin, GG- and QQ-Elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-Elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-Elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-Elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-Elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-Elafin showed increased functionality when compared to WT-Elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden.

  • Secretory Leukocyte Protease Inhibitor (SLPI) Is, like Its Homologue Trappin-2 (Pre-Elafin), a Transglutaminase Substrate
    PLoS ONE, 2011
    Co-Authors: Kevin Baranger, Marie Louise Zani, Sandrine Dallet-choisy, Valérie Labas, Thierry Moreau
    Abstract:

    Human lungs contain secretory leukocyte protease inhibitor (SLPI), Elafin and its biologically active precursor trappin-2 (pre-Elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, Elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to Elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and Elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, Elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal Elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the Elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.

  • multifaceted roles of human Elafin and secretory leukocyte proteinase inhibitor slpi two serine protease inhibitors of the chelonianin family
    Biochimie, 2008
    Co-Authors: Thierry Moreau, Nicolas Guyot, Kevin Baranger, Sebastien Dade, Sandrine Dalletchoisy, Marie Louise Zani
    Abstract:

    Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-Elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and Elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and Elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.

  • Elafin and its precursor trappin 2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase
    Biochemistry, 2005
    Co-Authors: Nicolas Guyot, Marie Louise Zani, Sandrine Dalletchoisy, Mariechristine Maurel, Thierry Moreau
    Abstract:

    Elafin and its precursor trappin-2 (also called pre-Elafin) are potent protein inhibitors of neutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conserved sequence motifs rich in Gin and Lys residues. These motifs are substrates for transglutaminases that may enable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its site of action. We have used Western blotting and ELISA-based assays to demonstrate that both Elafin and trappin-2 can be conjugated to various extracellular matrix proteins in vitro by a type 2 transglutaminase. Cross-linked Elafin and trappin-2 still inhibited their target proteases. Surface plasmon resonance studies allowed the determination of the kinetic constants governing the interaction of fibronectin-bound Elafin and trappin-2 with neutrophil elastase and proteinase 3. Both inhibitors were potent inhibitors when cross-linked to fibronectin by transglutamination, with equilibrium dissociation constants K i for their interaction with target proteases of 0.3 nM (elastase-Elafin), 20 nM (proteinase 3-Elafin), 0.3 nM (elastase-trappin-2), and 12 nM (proteinase 3-trappin-2). The conjugated inhibitors reacted more slowly with their target enzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constants of 2-7 x 10 5 M - 1 s - 1 for elastase and 1-4 x 10 4 M - 1 s - 1 for proteinase 3. We believe this is the first demonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preserves their inhibitory capacities.

  • Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-Elafin): evidence for tryptase-mediated generation of Elafin.
    Biological Chemistry, 2005
    Co-Authors: Nicolas Guyot, Marie Louise Zani, Sandrine Dallet-choisy, Patrick Berger, Thierry Moreau
    Abstract:

    A number of serine, cysteine, metallo- and acid proteases were evaluated for their ability to proteolytically cleave the serine protease inhibitor trappin-2, also known as pre-Elafin, and to release Elafin from its precursor. None of the metalloproteases or acid proteases examined cleaved trappin-2, while serine and cysteine proteases preferentially cleaved trappin-2 within its non-inhibitory N-terminal moiety. Cathepsin L, cathepsin K, plasmin, trypsin and tryptase were able to release Elafin by cleaving the Lys 3 8 -Ala 3 9 peptide bond in trappin-2. However, purified tryptase appeared to be efficient at releasing Elafin. Incubation of trappin-2 with purified mast cells first challenged with anti-immunoglobulin E or calcium ionophore A23187 resulted in the rapid generation of Elafin. This proteolytic release of Elafin from trappin-2 was inhibited in the presence of a tryptase inhibitor, suggesting that this mast cell enzyme was involved in the process. Finally, ex vivo incubation of trappin-2 with sputum from cystic fibrosis patients indicated the production of a proteolytic immunoreactive fragment with the same mass as that of native Elafin. This cleavage did not occur when preincubating the sputum with polyclonal antibodies directed against tryptase. Taken together, these findings indicate that tryptase could likely be involved in the maturation of trappin-2 into Elafin under physiological conditions.

Kelly K. Hunt - One of the best experts on this subject based on the ideXlab platform.

  • The Serine Protease Inhibitor Elafin Maintains Normal Growth Control by Opposing the Mitogenic Effects of Neutrophil
    2016
    Co-Authors: Joseph A. Caruso, Said Akli, Kelly K. Hunt
    Abstract:

    The serine protease inhibitor, Elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared to normal human mammary epithelial cells (HMECs). Here, we evaluated the role of Elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor deprived HMECs as they enter a quiescent (G0) state, suggesting that Elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of Elafin compromises the ability of HMECs to maintain G0-arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type Elafin, but not Elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of Elafin to arrest cells in G0. In fact when purified NE was added to Elafin knockdown HMECs, these cells demonstrated greater sensitivity to the growth promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples, and immunohistochemical analysis of norma

  • Elafin is downregulated during breast and ovarian tumorigenesis but its residual expression predicts recurrence
    Breast Cancer Research, 2014
    Co-Authors: Joseph A. Caruso, Cansu Karakas, Jing Zhang, Constance Albarracin, Aysegul A. Sahin, Melissa L. Bondy, Jinsong Liu, Kelly K. Hunt, Khandan Keyomarsi
    Abstract:

    Elafin is an endogenous serine protease inhibitor. The majority of breast cancer cell lines lack Elafin expression compared to human mammary epithelial cells. In this study, we hypothesized that Elafin is downregulated during breast and ovarian tumorigenesis. We examined Elafin expression by immunohistochemistry (IHC) in specimens of normal breast tissue (n = 24), ductal carcinoma in situ (DCIS) (n = 54), and invasive breast cancer (n = 793). IHC analysis of Elafin expression was also performed in normal fallopian tube tissue (n = 20), ovarian cystadenomas (n = 9), borderline ovarian tumors (n = 21), and invasive ovarian carcinomas (n = 216). To understand the significance of Elafin in luminal breast cancer cell lines, wild-type or M25G Elafin (lacking the protease inhibitory function) were exogenously expressed in MCF-7 and T47D cells. Elafin expression was downregulated in 24% of DCIS and 83% of invasive breast tumors when compared to Elafin expression in the normal mammary epithelium. However, the presence of Elafin-positive cells in invasive breast tumors, even at low frequency, correlated with poor recurrence-free survival (RFS), reduced overall survival (OS), and clinicopathological markers of aggressive tumor behavior. Elafin-positive cells were an especially strong and independent prognostic marker of reduced RFS in IHC-defined luminal A-like tumors. Elafin was also downregulated in 33% of ovarian cystadenomas, 43% of borderline ovarian tumors, and 86% of invasive ovarian carcinomas when compared to Elafin expression in the normal fallopian tube. In ovarian tumors, Elafin-positive cells were correlated with reduced RFS, OS and disease-specific survival (DSS) only in stage I/II patients and not in stage III/IV patients. Notably, exogenous expression of Elafin or Elafin M25G in the luminal breast cancer cell lines MCF-7 and T47D significantly decreased cell proliferation in a protease inhibitory domain-independent manner. Elafin predicts poor outcome in breast and ovarian cancer patients and delineates a subset of endocrine receptor-positive breast cancer patients susceptible to recurrence who could benefit from more aggressive intervention. Our in vitro results suggest that Elafin arrests luminal breast cancer cells, perhaps suggesting a role in tumor dormancy.

  • The serine protease inhibitor Elafin maintains normal growth control by opposing the mitogenic effects of neutrophil elastase
    Oncogene, 2014
    Co-Authors: Joseph A. Caruso, Kelly K. Hunt, Said Akli, Laura R. Pageon, Khandan Keyomarsi
    Abstract:

    The serine protease inhibitor, Elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared with normal human mammary epithelial cells (HMECs). Here, we evaluated the role of Elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor-deprived HMECs as they enter a quiescent (G0) state, suggesting that Elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of Elafin compromises the ability of HMECs to maintain G0 arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type Elafin but not Elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of Elafin to arrest cells in G0. In fact when purified NE was added to Elafin-knocked down HMECs, these cells demonstrated greater sensitivity to the growth-promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples. Immunohistochemical analysis of normal breast tissue revealed robust Elafin expression in the mammary epithelium; however, Elafin expression was dramatically downregulated in a significant proportion of human breast tumor specimens. The loss of Elafin expression during breast cancer progression may promote tumor growth as a consequence of increased NE activity. To address the role of NE in mammary tumorigenesis, we next examined whether deregulated NE activity enhances mammary tumor growth. NE knockout in the C3(1)TAg mouse model of mammary tumorigenesis suppressed proliferation and reduced the kinetics of tumor growth. Overall, the imbalance between NE and its inhibitors, such as Elafin, presents an important therapeutic target in breast cancer.

  • Elafin an inhibitor of elastase is a prognostic indicator in breast cancer
    Breast Cancer Research, 2013
    Co-Authors: Kelly K. Hunt, Ming Zhang, Tomoya Yokota, Yanna Liu, Hannah Wingate, Gordon B Mills, Fan Zhang, Bingliang Fang, Khandan Keyomarsi
    Abstract:

    Elafin is an elastase-specific inhibitor with increased transcription in normal mammary epithelial cells compared to mammary carcinoma cells. In this report, we test the hypothesis that inhibition of elastase, through induction of Elafin, leads to inhibition of human breast cancer cell viability and, therefore, predicts survival in breast cancer patients. Panels of normal and immortalized breast epithelial cells, along with breast carcinoma cells, were used to examine the impact of adenoviral-mediated Elafin expression or shRNA-mediated inhibition of elastase on the growth of cells and xenografts in nude mice. To determine the prognostic significance of decreased Elafin in patients with invasive breast cancer, previously published gene array datasets were interrogated. Elafin expression had no effect on non-tumorigenic cells but resulted in marked inhibition of cell growth in breast cancer cell lines. Control-treated xenografts generated a tumor burden that necessitated sacrifice within one month of initial treatment, whereas xenograft-bearing mice treated with Ad-Elafin were alive at eight months with marked reduction in tumor growth. Elastase inhibition mimicked these results, showing decreased tumor cell growth in vitro and in vivo. Low expression of Elafin gene correlated with significantly reduced time to relapse, and when combined with high expression of elastase gene was associated with decreased survival in breast cancer patients. Our data suggest that Elafin plays a direct role in the suppression of tumors through inhibition of elastase and thus serves as a prognostic indicator for breast cancer patients.

  • Elafin, an inhibitor of elastase, is a prognostic indicator in breast cancer.
    Breast Cancer Research, 2013
    Co-Authors: Kelly K. Hunt, Tomoya Yokota, Yanna Liu, Hannah Wingate, Gordon B Mills, Fan Zhang, Bingliang Fang, Ming Zhang
    Abstract:

    Introduction Elafin is an elastase-specific inhibitor with increased transcription in normal mammary epithelial cells compared to mammary carcinoma cells. In this report, we test the hypothesis that inhibition of elastase, through induction of Elafin, leads to inhibition of human breast cancer cell viability and, therefore, predicts survival in breast cancer patients.

Marie Louise Zani - One of the best experts on this subject based on the ideXlab platform.

  • A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation
    Molecular Therapy, 2015
    Co-Authors: Donna M. Small, Marie Louise Zani, Derek J. Quinn, Sandrine Dallet-choisy, Arlene Glasgow, Cecilia O'kane, Daniel F. Mcauley, Paul Mcnally, Sinéad Weldon, Thierry Moreau
    Abstract:

    Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of Elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of Elafin (GG- and QQ-Elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) Elafin. Similar to WT-Elafin, GG- and QQ-Elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-Elafin, GG- and QQ-Elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-Elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-Elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-Elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-Elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-Elafin showed increased functionality when compared to WT-Elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden.

  • Secretory Leukocyte Protease Inhibitor (SLPI) Is, like Its Homologue Trappin-2 (Pre-Elafin), a Transglutaminase Substrate
    PLoS ONE, 2011
    Co-Authors: Kevin Baranger, Marie Louise Zani, Sandrine Dallet-choisy, Valérie Labas, Thierry Moreau
    Abstract:

    Human lungs contain secretory leukocyte protease inhibitor (SLPI), Elafin and its biologically active precursor trappin-2 (pre-Elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, Elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to Elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and Elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, Elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal Elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the Elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.

  • multifaceted roles of human Elafin and secretory leukocyte proteinase inhibitor slpi two serine protease inhibitors of the chelonianin family
    Biochimie, 2008
    Co-Authors: Thierry Moreau, Nicolas Guyot, Kevin Baranger, Sebastien Dade, Sandrine Dalletchoisy, Marie Louise Zani
    Abstract:

    Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-Elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and Elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and Elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.

  • Elafin and its precursor trappin 2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase
    Biochemistry, 2005
    Co-Authors: Nicolas Guyot, Marie Louise Zani, Sandrine Dalletchoisy, Mariechristine Maurel, Thierry Moreau
    Abstract:

    Elafin and its precursor trappin-2 (also called pre-Elafin) are potent protein inhibitors of neutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conserved sequence motifs rich in Gin and Lys residues. These motifs are substrates for transglutaminases that may enable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its site of action. We have used Western blotting and ELISA-based assays to demonstrate that both Elafin and trappin-2 can be conjugated to various extracellular matrix proteins in vitro by a type 2 transglutaminase. Cross-linked Elafin and trappin-2 still inhibited their target proteases. Surface plasmon resonance studies allowed the determination of the kinetic constants governing the interaction of fibronectin-bound Elafin and trappin-2 with neutrophil elastase and proteinase 3. Both inhibitors were potent inhibitors when cross-linked to fibronectin by transglutamination, with equilibrium dissociation constants K i for their interaction with target proteases of 0.3 nM (elastase-Elafin), 20 nM (proteinase 3-Elafin), 0.3 nM (elastase-trappin-2), and 12 nM (proteinase 3-trappin-2). The conjugated inhibitors reacted more slowly with their target enzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constants of 2-7 x 10 5 M - 1 s - 1 for elastase and 1-4 x 10 4 M - 1 s - 1 for proteinase 3. We believe this is the first demonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preserves their inhibitory capacities.

  • Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-Elafin): evidence for tryptase-mediated generation of Elafin.
    Biological Chemistry, 2005
    Co-Authors: Nicolas Guyot, Marie Louise Zani, Sandrine Dallet-choisy, Patrick Berger, Thierry Moreau
    Abstract:

    A number of serine, cysteine, metallo- and acid proteases were evaluated for their ability to proteolytically cleave the serine protease inhibitor trappin-2, also known as pre-Elafin, and to release Elafin from its precursor. None of the metalloproteases or acid proteases examined cleaved trappin-2, while serine and cysteine proteases preferentially cleaved trappin-2 within its non-inhibitory N-terminal moiety. Cathepsin L, cathepsin K, plasmin, trypsin and tryptase were able to release Elafin by cleaving the Lys 3 8 -Ala 3 9 peptide bond in trappin-2. However, purified tryptase appeared to be efficient at releasing Elafin. Incubation of trappin-2 with purified mast cells first challenged with anti-immunoglobulin E or calcium ionophore A23187 resulted in the rapid generation of Elafin. This proteolytic release of Elafin from trappin-2 was inhibited in the presence of a tryptase inhibitor, suggesting that this mast cell enzyme was involved in the process. Finally, ex vivo incubation of trappin-2 with sputum from cystic fibrosis patients indicated the production of a proteolytic immunoreactive fragment with the same mass as that of native Elafin. This cleavage did not occur when preincubating the sputum with polyclonal antibodies directed against tryptase. Taken together, these findings indicate that tryptase could likely be involved in the maturation of trappin-2 into Elafin under physiological conditions.