Project description:The balance between protecting tissue integrity and efficient immune response is critical for host survival. Here we investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection using a combined genomic and proteomic approach. We followed the transcriptional dynamics and ECM- related responses in a mouse model of influenza virus infection, integrated with whole tissue imaging and functional assays. Our study identifies MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza virus infection. We show that selective inhibition of MT1-MMP-driven ECM proteolysis protects the tissue from infection-related structural and compositional damage. Inhibition of MT1-MMP did not significantly alter the immune response or cytokine expression, indicating its dominant role in ECM remodeling. We demonstrate that the available treatment for influenza virus (Tamiflu/ Oseltamivir) does not prevent lung ECM damage and is less effective than anti-MT1-MMP treatment in influenza virus and Streptococcus pneumoniae coinfection paradigms. Importantly, combination therapy of Tamiflu with anti-MT1-MMP shows a strong synergistic effect and results in complete recovery in mice. This study highlights the importance of tissue tolerance agents for surviving infectious diseases, and the potential of such host-pathogen therapy combination for respiratory infections.
Project description:This a model from the article:
A theoretical model of type I collagen proteolysis by matrix metalloproteinase
(MMP) 2 and membrane type 1 MMP in the presence of tissue inhibitor of
metalloproteinase 2.
Karagiannis ED, Popel AS. J Biol Chem
2004 Sep 10;279(37):39105-14 15252025
,
Abstract:
One well documented family of enzymes responsible for the proteolytic processes
that occur in the extracellular matrix is the soluble and membrane-associated
matrix metalloproteinases. Here we present the first theoretical model of the
biochemical network describing the proteolysis of collagen I by matrix
metalloproteinases 2 (MMP2) and membrane type 1 matrix metalloproteinases
(MT1-MMP) in the presence of the tissue inhibitor of metalloproteinases 2
(TIMP2) in a bulk, cell-free, well stirred environment. The model can serve as a
tool for describing quantitatively the activation of the MMP2 proenzyme
(pro-MMP2), the ectodomain shedding of MT1-MMP, and the collagenolysis arising
from both of the enzymes. We show that pro-MMP2 activation, a process that
involves a trimer formation of the proenzyme with TIMP2 and MT1-MMP, is
suppressed at high inhibitor levels and paradoxically attains maximum only at
intermediate TIMP2 concentrations. We also calculate the conditions for which
pro-MMP2 activation is maximal. Furthermore we demonstrate that the ectodomain
shedding of MT1-MMP can serve as a mechanism controlling the MT1-MMP
availability and therefore the pro-MMP2 activation. Finally the proteolytic
synergism of MMP2 and MT1-MMP is introduced and described quantitatively. The
model provides us a tool to determine the conditions under which the synergism
is optimized. Our approach is the first step toward a more complete description
of the proteolytic processes that occur in the extracellular matrix and include
a wider spectrum of enzymes and substrates as well as naturally occurring or
artificial inhibitors.
This model was taken from the CellML repository
and automatically converted to SBML.
The original model was:
Karagiannis ED, Popel AS. (2004) - version01
This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2011 The BioModels.net Team.
To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication
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To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.
Project description:Invasive cancers employ pericellular proteolysis to breach the extracellular matrix and basement membrane barriers and invade the surrounding tissue. Pro-invasive, pro-tumorigenic MT1-MMP is the primary mediator of proteolytic events on the cancer cell surface. Cellular MT1-MMP is synthesized as a latent zymogen. The latency of MT1-MMP is maintained by its N-terminal inhibitory prodomain. Our study reveals a critical mechanism underlying the activation pathway and subsequent execution of the tumor-promoting function of MT1-MMP. Evidence suggests that the prodomain undergoes intradomain cleavage at the PGD↓L50 cleavage site followed by the release of the degraded prodomain by furin cleavage of the R108RKR111↓Y112 site. These events, only if combined, cause the activation of MT1-MMP. The significance of these molecular events to the pro-tumorigenic function of MT1-MMP in malignancy remained, however, unidentified. To identify the functional importance of the PGD↓L50 intradomain cleavage in the activation and tumorigenic program of MT1-MMP, our current studies employed the cells which expressed the wild-type prodomain-based fluorescent biosensor and the mutant biosensor with the inactivated PGD↓L50 cleavage site (L50D mutant) and also the cells with the enforced expression the wild-type and mutant MT1-MMP. Using cell-based tests and orthotopic breast cancer xenografts in mice, we demonstrated that the intradomain cleavage of the PGD↓L50 sequence of the prodomain is essential for the pro-tumorigenic function of MT1-MMP. Our study contributes to the growing consensus for the design of selective, precisely focused MT1-MMP inhibitors in cancer. Analysis of global gene expression in orthotopic tumor and cultured breast cancer cells expressing wild-type and mutant Mt1-MMP forms
Project description:Mammary gland branching morphogenesis is thought to depend on the mobilization of the membrane-anchored matrix metalloproteinases, MT1-MMP and MT2-MMP, that drive epithelial cell invasion by remodeling the extracellular matrix and triggering associated signaling cascades. However, the roles that these proteinases play during mammary gland development in vivo remains undefined. A mammary gland branching program that occurs during the first 10 days of early postnatal development was used to characterize the impact of global Mt1-mmp or Mt2-mmp targeting on mammary gland morphogenesis. Transcriptome profiling of ductal networks and associated stroma was used to investigate the functional roles of MT1-MMP in the early postnatal mammary gland in an unbiased fashion.
Project description:The balance between tissue integrity and efficient immune response is critical for host survival. Here we investigate the role of extra cellular matrix (ECM)-remodeling events in the lung during influenza infection. Using an unbiased genomic and proteomic approach we follow the dynamics of gene and ECM responses in an influenza-infected mouse model, integrated with whole tissue as well as ECM imaging and functional assays. Our study identifies membrane type 1 matrix metalloproteinase (MT1-MMP) as a major host-regulated ECM remodeling collagenase in influenza infection. We show that inhibition of MT1-MMP-driven proteolytic activity protected tissue structural and compositional features. We demonstrate that available influenza treatment (Oseltamivir) is ineffective in preventing the lung ECM damage and is therefore less effective than anti-MT1-MMP treatment in influenza and Streptococcus pneumoniae co-infections. Importantly, the combination between the two treatments resulted in 100% survival. This study highlights the importance of ECM protection for survival during infectious diseases, and paves the way for combined host-pathogen theraphy to fight emerging pathogens.
Project description:Invasive cancers employ pericellular proteolysis to breach the extracellular matrix and basement membrane barriers and invade the surrounding tissue. Pro-invasive, pro-tumorigenic MT1-MMP is the primary mediator of proteolytic events on the cancer cell surface. Cellular MT1-MMP is synthesized as a latent zymogen. The latency of MT1-MMP is maintained by its N-terminal inhibitory prodomain. Our study reveals a critical mechanism underlying the activation pathway and subsequent execution of the tumor-promoting function of MT1-MMP. Evidence suggests that the prodomain undergoes intradomain cleavage at the PGD↓L50 cleavage site followed by the release of the degraded prodomain by furin cleavage of the R108RKR111↓Y112 site. These events, only if combined, cause the activation of MT1-MMP. The significance of these molecular events to the pro-tumorigenic function of MT1-MMP in malignancy remained, however, unidentified. To identify the functional importance of the PGD↓L50 intradomain cleavage in the activation and tumorigenic program of MT1-MMP, our current studies employed the cells which expressed the wild-type prodomain-based fluorescent biosensor and the mutant biosensor with the inactivated PGD↓L50 cleavage site (L50D mutant) and also the cells with the enforced expression the wild-type and mutant MT1-MMP. Using cell-based tests and orthotopic breast cancer xenografts in mice, we demonstrated that the intradomain cleavage of the PGD↓L50 sequence of the prodomain is essential for the pro-tumorigenic function of MT1-MMP. Our study contributes to the growing consensus for the design of selective, precisely focused MT1-MMP inhibitors in cancer.
Project description:Mammary gland branching morphogenesis is thought to depend on the mobilization of the membrane-anchored matrix metalloproteinases, MT1-MMP and MT2-MMP, that drive epithelial cell invasion by remodeling the extracellular matrix and triggering associated signaling cascades. However, the roles that these proteinases play during mammary gland development in vivo remains undefined. A mammary gland branching program that occurs during the first 10 days of early postnatal development was used to characterize the impact of global Mt1-mmp or Mt2-mmp targeting on mammary gland morphogenesis. Transcriptome profiling of ductal networks and associated stroma was used to investigate the functional roles of MT2-MMP in the early postnatal mammary gland in an unbiased fashion.
Project description:Macrophages dominate inflammatory environments where they modify the extracellular matrix by mobilizing complex repertoires of proteolytic enzymes. Nevertheless, the dominant proteinases used by macrophage as they confront physiologic tissue barriers remain undefined. Herein, we have characterized the molecular mechanisms that define human macrophage-extracellular matrix interactions ex vivo. Resting and immune-polarized macrophages are shown to proteolytically remodel basement membranes while infiltrating the underlying interstitial matrix. In an unbiased screen to identify key proteases, we find that the macrophage metalloproteinase, MT1-MMP, is the dominant effector of basement membrane degradation and invasion. These studies not only identify MT1-MMP as a key proteolytic effector of extracellular matrix remodeling by human macrophages, but also define the invasive strategies used by macrophages to traverse physiologic tissue barriers.
Project description:Full-length membrane PTK7 and its N-terminal and C-terminal proteolytic fragments induced differential transcriptional profiles in HT1080 cells. Pseudokinase PTK7 is an essential regulator of planar cell polarity (PCP) and directional cell motility in the course of vertebrate development and embryogenesis, and regulates both the non-canonical Wnt/PCP and canonical Wnt pathways. In contrast to its well-appreciated, crucial role in embryo development, the functional importance of the intact full-length PTK7 in malignancy is still a matter of debate. In humans, the full-length membrane PTK7 consists of seven extracellular immunoglobulin-like (Ig) domains, a transmembrane region, a juxtamembrane region and a catalytically inert cytoplasmic tyrosine kinase (PTK) domain. Because pericellular proteolysis plays a primary role in cell migration, especially in the directional locomotion of tumor cells, it is likely that proteolysis and PCP converge to promote efficient directed cancer cell migration. In agreement, the functionality of PTK7 is directly regulated by proteolysis. Ubiquitous membrane type-1 matrix metalloproteinase (MT1-MMP), arguably the primary enzyme in pericellular proteolysis and cancer cell migration, cleaves the PKP621M-bM-^FM-^SLI sequence in the seventh Ig-like domain of membrane PTK7 and this cleavage results in the liberation of the N-terminal soluble PTK7 fragment (sPTK7). MT1-MMP proteolysis is followed by the cleavage of the C-terminal residual portion of PTK7 by ADAMs, including ADAM17. The ectodomain shedding is a prerequisite for the intramembrane cleavage of PTK7 by M-NM-3-secretase. This cleavage releases the C-terminal cytoplasmic tail fragment of PTK7, which is then either degraded by the proteasome or transported to the nucleus.The limited pre-existing data suggest that the full-length membrane PTK7 and its proteolytic products cause an opposing effect on the efficiency of cell migration. Thus, the continuing presence of the full-length membrane PTK7 on the plasma membrane down-regulated the myosin light chain (MLC) phosphorylation (a downstream event of the Wnt/PCP pathway) and, in agreement, reduced migration efficiency of fibrosarcoma HT1080 cells. MT1-MMP proteolysis of PTK7 reversed the inhibitory effect of the full-length membrane PTK7, resulted in the accumulation of the stable N-terminal sPTK7 fragment in the extracellular milieu and promoted cell invasion of HT1080 cells. Expression of the Chz PTK7 mutant that exhibited an additional PEKM-bM-^FM-^SLK503 MT1-MMP cleavage site in the junction region between the fifth and the sixth Ig-like domains stimulated cell migration even further. These effects suggest the existence of the intriguing and specific downstream mechanisms by which the intact PTK7, its digest fragments and the homo- and heterodimeric complexes between the PTK7 membrane, soluble and intracellular portions control cell function. These mechanisms, however, have not been precisely investigated in the earlier works by us and others. Understanding of these mechanisms will shed additional light on the role that PTK7 alone as well as in its combination with MT1-MMP, ADAMs and M-NM-3-secretase plays in cancer cell migration. To evaluate in detail the effects of PTK7 and its proteolytic fragments on genome-wide transcriptional regulation, we specifically employed fibrosarcoma HT1080 cells. These highly invasive cells express low endogenous levels of PTK7 but high levels of active MT1-MMP and ADAMs. Because of these parameters, we could manipulate this preudokinase functionality using HT1080 cells transfected with the recombinant PTK7 constructs. The cells we employed included the PTK7 knock-out cells (shPTK7 cells), cells with the enforced overexpression of the original membrane PTK7 (PTK7 cells) and its Chuzhoi (Chz) mutant that exhibited an additional PEKM-bM-^FM-^SLK503 MT1-MMP cleavage site (Chz cells), and the cells, which overexpresssed multiple deletion species of PTK7. These species represented the soluble (sPTK7), membrane and cytoplasmic C-terminal digest fragments that resulted because of PTK7 cleavage by MT1-MMP (cPTK7/622-1070) and ADAM/gammaM-bM-^@M-^Ssecretase (cPTK7/726-1070). Expression of these constructs allowed us to specifically determine the downstream effect of PTK7 proteolysis on gene expression. HT1080 cells were stably transfected with the PTK7 constructs. Cells (1 x 10^4/ml) were plated in DMEM- 10% FBS in a 100-mm dish and grown for 72 h to produce a subconfluent culture. Total cellular RNA was extracted using a Direct-zol RNA MiniPrep kit (Zymo Research). Biotin-labeled cRNA samples were prepared using the Illumina RNA Amplification Kit (Ambion). The labeled cRNA (750 ng) was hybridized for 18 h at 58M-BM-0C to the HumanHT-12 v4 Expression BeadChip (Illumina). BeadChips were then developed using fluorolink streptavidin-Cy3 (GE Healthcare). Array chips were scanned using an Illumina BeadArray Reader. The initial data extraction and normalization were performed using the BeadArray Reader and GeneSpring GX software (Agilent).
Project description:Macrophages dominate inflammatory environments where they modify the extracellular matrix by mobilizing complex repertoires of proteolytic enzymes. Nevertheless, the dominant proteinases used by macrophage as they confront physiologic tissue barriers remain undefined. Herein, we have characterized the molecular mechanisms that define human macrophage-extracellular matrix interactions ex vivo. Resting and immune-polarized macrophages are shown to proteolytically remodel basement membranes while infiltrating the underlying interstitial matrix. In an unbiased screen to identify key proteases, we find that the macrophage metalloproteinase, MT1-MMP, is the dominant effector of basement membrane degradation and invasion. Unexpectedly, macrophages can alternatively use actomyosin-dependent forces to transmigrate native basement membrane pores that provide cells with proteinase-independent access to the interstitial matrix. These studies not only identify MT1-MMP as a key proteolytic effector of extracellular matrix remodeling by human macrophages, but also define the invasive strategies used by macrophages to traverse physiologic tissue barriers.