The mouse matrix metalloproteinase, epilysin (MMP-28), is alternatively spliced and processed by a furin-like proprotein convertase

Epilysin (MMP-28) is a recently identified member of the matrix metalloproteinase (MMP) family. To explore the expression of epilysin in vivo and to gain insight into its biological functions, we have cloned the mouse epilysin cDNA and determined its expression. The amino acid sequence of the mouse protein is 85% identical with the human sequence and contains conserved features such as an RKKR furin-activation sequence following the prodomain. Unexpectedly, we found two alternatively spliced forms of the epilysin mRNA lacking 30 and 72 nt at the beginning of the seventh exon coding for part of the haemopexin domain. Expression of recombinant epilysin in HT-1080 fibrosarcoma cells indicated that epilysin was secreted as a major 48 kDa form and a minor 58 kDa form. Expression of the 58 kDa form was increased by a synthetic furin inhibitor at the expense of the 48 kDa form, suggesting that furin cleaves and activates epilysin. Epilysin mRNA was detected in a number of mouse tissues, with the highest expression in the lung, placenta, heart and uterus, and lower levels in the testis and gastrointestinal tract. The wide expression of epilysin in intact, healthy tissues suggests that this MMP functions in physiological tissue homoeostasis and turnover.

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Fluorescence imaging to assess the matrix metalloproteinase activity and its inhibitor in vivo

Journal of Biomedical Optics ◽

10.1117/1.2830659 ◽

2008 ◽

Vol 13 (1) ◽

pp. 011006 ◽

Cited By ~ 12

Author(s):

Zhihong Zhang ◽

Jie Yang ◽

Jinling Lu ◽

Juqiang Lin ◽

Shaoqun Zeng ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Fluorescence Imaging ◽

The Matrix ◽

Metalloproteinase Activity

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Overexpression of the Matrix Metalloproteinase Matrilysin Results in Premature Mammary Gland Differentiation and Male Infertility

Molecular Biology of the Cell ◽

10.1091/mbc.9.2.421 ◽

1998 ◽

Vol 9 (2) ◽

pp. 421-435 ◽

Cited By ~ 36

Author(s):

Laura A. Rudolph-Owen ◽

Paul Cannon ◽

Lynn M. Matrisian

Keyword(s):

Matrix Metalloproteinase ◽

Transgenic Animals ◽

Reproductive Organs ◽

Mammary Development ◽

Seminiferous Tubules ◽

Interstitial Tissue ◽

Wild Type ◽

The Matrix

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce β-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner.

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Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo

Blood ◽

10.1182/blood.v100.4.1160.h81602001160_1160_1167 ◽

2002 ◽

Vol 100 (4) ◽

pp. 1160-1167 ◽

Cited By ~ 392

Author(s):

G. Angus McQuibban ◽

Jiang-Hong Gong ◽

Julie P. Wong ◽

John L. Wallace ◽

Ian Clark-Lewis ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Chemokine Receptor ◽

Chemokine Receptors ◽

Leukemic Cell ◽

Proteolytic Processing ◽

Cc Chemokine ◽

Monocyte Chemoattractant ◽

The Matrix

Monocyte chemoattractant protein (MCP)–3 is inactivated upon cleavage by the matrix metalloproteinase (MMP) gelatinase A (MMP-2). We investigated the susceptibility to proteolytic processing of the 4 human MCPs by 8 recombinant MMPs to determine whether MCP-3 is an isolated example or represents a general susceptibility of chemokines to proteolytic inactivation by these important inflammatory proteases. In addition to MMP-2, MCP-3 is efficiently cleaved by membrane type 1 (MT1)–MMP, the cellular activator of MMP-2, and by collagenase-1 and collagenase-3 (MMP-1, MMP-13) and stromelysin-1 (MMP-3). Specificity was shown by absence of cleavage by matrilysin (MMP-7) and the leukocytic MMPs neutrophil collagenase (MMP-8) and gelatinase B (MMP-9). The closely related chemokines MCP-1, MCP-2, and MCP-4 were not cleaved by MMP-2 or MT1-MMP, but were cleaved by MMP-1 and MMP-3 with varying efficiency. MCPs were typically cleaved between residues 4 and 5, but MCP-4 was further processed at Val7-Pro8. Synthetic MCP analogs corresponding to the MMP-cleaved forms bound CC chemokine receptor (CCR)–2 and CCR-3, but lacked chemoattractant activity in pre-B cells transfected with CCR-2 and CCR-3 or in THP-1 monocytic cells, a transformed leukemic cell line. Moreover, the truncated products of MCP-2 and MCP-4, like MCP-3, were potent antagonists of their cognate CC chemokine receptors in transwell cell migration assays in vitro. When they were injected 24 hours after the initiation of carrageenan-induced inflammation in rat paws, their in vivo antagonist activities were revealed by a greater than 66% reduction in inflammatory edema progression after 12 hours. We propose that MMPs have an important role in modulating inflammatory and immune responses by processing chemokines in wound healing and in disease.

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Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9

Biochemical Society Transactions ◽

10.1042/bst0300120 ◽

2002 ◽

Vol 30 (2) ◽

pp. 120-126 ◽

Cited By ~ 30

Author(s):

K. E. Porter ◽

N. A. Turner

Keyword(s):

Matrix Metalloproteinase ◽

Intimal Hyperplasia ◽

Therapeutic Potential ◽

Matrix Metalloproteinase 2 ◽

Neointima Formation ◽

Graft Stenosis ◽

The Matrix ◽

And Migration ◽

Proliferation And Migration

Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.

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Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo

Journal of Cellular Biochemistry ◽

10.1002/jcb.20878 ◽

2006 ◽

Vol 99 (2) ◽

pp. 545-557 ◽

Cited By ~ 50

Author(s):

Nagarajan Selvamurugan ◽

Stephen C. Jefcoat ◽

Sukyee Kwok ◽

Rodney Kowalewski ◽

Joseph A. Tamasi ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Bone Remodeling ◽

Matrix Metalloproteinase 13 ◽

The Matrix

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Tissue Specific Expression of Alternatively Spliced Forms of Tissue Factor Pathway Inhibitor (TFPI) in Mice

Blood ◽

10.1182/blood.v112.11.1026.1026 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1026-1026

Author(s):

Susan A. Maroney ◽

Josephine P. Ferrel ◽

Shuchong Pan ◽

Thomas A. White ◽

Robert D. Simari ◽

...

Keyword(s):

Western Blot ◽

Tissue Factor ◽

Terminal Region ◽

Heparin Infusion ◽

Mouse Plasma ◽

Mouse Placenta ◽

Mouse Tissues ◽

Kunitz Domain ◽

Alternatively Spliced

Abstract TFPI is an endothelium associated anticoagulant protein that directly inhibits tissue factor (TF) pro-coagulant activity and prevents disease produced by intravascular thrombosis. TFPI is produced in three alternatively spliced isoforms in mice, α, β and γ, that differ in domain structure and mechanism for association with endothelium. TFPIα has three Kunitz-type serine protease inhibitory domains and a basic C-terminal region. It produces anticoagulant activity by direct inhibition of factor Xa (fXa) via the second Kunitz domain (K2) and, in a fXa dependent manner, inhibition of TF-fVIIa via the first Kunitz domain (K1). The third Kunitz domain (K3) and C-terminal region do not directly inhibit proteolysis. However, alignment of sequences from various vertebrate species demonstrates that K3 has maintained a high degree of sequence conservation for over 430 million years suggesting that K3 maintains a physiological function. TFPIα indirectly associates with endothelium by binding to a glycosylphosphatidyl inositol (GPI) anchored protein and binds non-specifically to glycosaminoglycans. TFPIβ has K1 and K2 but lacks K3 and has a distinct C-terminal region that encodes a GPI-anchor attachment sequence. TFPIγ has K1 and K2 but lacks K3 and has a distinct C-terminal region that results in production of a secreted protein. It is not known if these structurally diverse forms of TFPI with different mechanisms for cell surface association have variable efficacy in their ability to inhibit TF activity in vivo. We characterized TFPI isoform expression in mouse tissues in order to define specific physiological functions for alternative splicing of TFPI and how it may hinder diseases mediated by intravascular TF activity. Message for TFPIα is ~16-fold more abundant than message for TFPIβ or TFPIγ in mouse tissues. In situ hybridization studies of mouse heart and lung found that the isoforms are expressed in the same pattern, primarily in endothelial cells. No cell or vascular bed exclusively producing a single isoform was identified. Western blot analyses revealed the somewhat surprising finding that TFPIβ is the predominant isoform produced by adult mouse tissues. Thus, protein synthesis of TFPI isoforms in mice is regulated at least in part at the level of protein translation. Mouse plasma TFPI levels increase only 10–20% following heparin infusion. These data contrast with the 2- to 4-fold increase in plasma TFPI observed following heparin infusion in humans. The presence of only small amounts of heparin releasable TFPI in mouse plasma is consistent with TFPIβ as the major isoform because TFPIβ is directly GPI-anchored to endothelium and lacks the highly basic C-terminal region that allows TFPIα to associate non-specifically with endothelium glycosaminoglycans. To our knowledge, these are the first data demonstrating TFPIβ production in vivo. In contrast to the adult tissues, western blot analysis of mouse placenta revealed that it produces primarily TFPIα. This finding led us to examine mouse fetal tissue to determine if there is developmental regulation of the production of different TFPI isoforms. These studies demonstrated that TFPIα and TFPIβ are produced in approximately equal amounts in E14.5 embryos. The relative proportion of TFPIα in mouse tissues then decreases during development and very little is produced at 12 weeks of age. The evolutionary conservation of TFPIα and its production in mouse placenta and embryonic tissues suggests that unique features of TFPIα, such as K3, the basic C-terminal region and/or its binding to the endothelial surface through a GPI-anchored co-receptor, perform specific functions in the regulation of intravascular TF activity during vasculogenesis/angiogenesis that are not performed by TFPIβ or TFPIγ.

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TGF-β Plays a Key Role in Morphogenesis of the Pancreatic Islets of Langerhans by Controlling the Activity of the Matrix Metalloproteinase MMP-2

The Journal of Cell Biology ◽

10.1083/jcb.143.3.827 ◽

1998 ◽

Vol 143 (3) ◽

pp. 827-836 ◽

Cited By ~ 126

Author(s):

Francisco Miralles ◽

Tadej Battelino ◽

Paul Czernichow ◽

Raphael Scharfmann

Keyword(s):

Extracellular Matrix ◽

Matrix Metalloproteinase ◽

Islets Of Langerhans ◽

Neutralizing Antibody ◽

Matrix Metalloproteinase 2 ◽

Pancreatic Islets Of Langerhans ◽

The Matrix ◽

Vitro Model

Islets of Langerhans are microorgans scattered throughout the pancreas, and are responsible for synthesizing and secreting pancreatic hormones. While progress has recently been made concerning cell differentiation of the islets of Langerhans, the mechanism controlling islet morphogenesis is not known. It is thought that these islets are formed by mature cell association, first differentiating in the primitive pancreatic epithelium, then migrating in the extracellular matrix, and finally associating into islets of Langerhans. This mechanism suggests that the extracellular matrix has to be degraded for proper islet morphogenesis. We demonstrated in the present study that during rat pancreatic development, matrix metalloproteinase 2 (MMP-2) is activated in vivo between E17 and E19 when islet morphogenesis occurs. We next demonstrated that when E12.5 pancreatic epithelia develop in vitro, MMP-2 is activated in an in vitro model that recapitulates endocrine pancreas development (Miralles, F., P. Czernichow, and R. Scharfmann. 1998. Development. 125: 1017–1024). On the other hand, islet morphogenesis was impaired when MMP-2 activity was inhibited. We next demonstrated that exogenous TGF-β1 positively controls both islet morphogenesis and MMP-2 activity. Finally, we demonstrated that both islet morphogenesis and MMP-2 activation were abolished in the presence of a pan-specific TGF-β neutralizing antibody. Taken together, these observations demonstrate that in vitro, TGF-β is a key activator of pancreatic MMP-2, and that MMP-2 activity is necessary for islet morphogenesis.

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Specific Deletion of the FHA Domain Containing SLMAP3 Isoform in Postnatal Myocardium Has No Impact on Structure or Function

Cardiogenetics ◽

10.3390/cardiogenetics11040018 ◽

2021 ◽

Vol 11 (4) ◽

pp. 164-184

Author(s):

Taha Rehmani ◽

Jana Mlynarova ◽

Joseph Byers ◽

Maysoon Salih ◽

Balwant S. Tuana

Keyword(s):

Sodium Channel ◽

Protein Trafficking ◽

Cardiac Electrophysiology ◽

Hippo Signaling ◽

Fha Domain ◽

Hypertrophic Response ◽

Associated Proteins ◽

Alternatively Spliced ◽

A Minor

Sarcolemmal membrane-associated proteins (SLMAPs) belong to the superfamily of tail-anchored membrane proteins known to regulate diverse biological processes, including protein trafficking and signal transduction. Mutations in SLMAP have been linked to Brugada and defective sodium channel Nav1.5 shuttling. The SLMAP gene is alternatively spliced to generate numerous isoforms, broadly defined as SLMAP1 (~35 kDa), SLMAP2 (~45 kDa) and SLMAP3 (~80–95 kDa), which are highly expressed in the myocardium. The SLMAP3 isoform exhibits ubiquitous expression carrying an FHA domain and is believed to negatively regulate Hippo signaling to dictate cell growth/death and differentiation. Using the αMHC-MerCreMer-flox system to target the SLMAP gene, we specifically deleted the SLMAP3 isoform in postnatal mouse hearts without any changes in the expression of SLMAP1/SLMAP2 isoforms. The in vivo analysis of mice with SLMAP3 cardiac deficiency revealed no significant changes to heart structure or function in young or aged mice without or with isoproterenol-induced stress. SLMAP3-deficient hearts revealed no obvious differences in cardiac size, function or hypertrophic response. Further, the molecular analysis indicated that SLMAP3 loss had a minor impact on sodium channel (Nav1.5) expression without affecting cardiac electrophysiology in postnatal myocardium. Surprisingly, the loss of SLMAP3 did not impact Hippo signaling in postnatal myocardium. We conclude that the FHA domain-containing SLMAP3 isoform has no impact on Hippo signaling or sodium channels in postnatal myocardium, which is able to function and respond normally to stress in its absence. Whether SLMAP1/SMAP2 isoforms can compensate for the loss of SLMAP3 in the affairs of the postnatal heart remains to be determined.

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Quantifying the Matrix Metalloproteinase 2 (MMP2) Spatially in Tissues by Probe via MALDI Imaging Mass Spectrometry

Frontiers in Chemistry ◽

10.3389/fchem.2021.786283 ◽

2021 ◽

Vol 9 ◽

Author(s):

Daojiang Yu ◽

Peng Lai ◽

Tao Yan ◽

Kai Fang ◽

Lei Chen ◽

...

Keyword(s):

Mass Spectrometry ◽

Matrix Metalloproteinase ◽

Cancer Progression ◽

Imaging Mass Spectrometry ◽

Matrix Metalloproteinase 2 ◽

Maldi Tof Mass Spectrometry ◽

Maldi Tof ◽

The Matrix ◽

Important Approach

As a matrix metalloproteinase, the abnormal expression of MMP2 is associated with multiple biological processes, including tissue remodeling and cancer progression. Therefore, spatial analysis of MMP2 protein in tissues can be used as an important approach to evaluate the expression distribution of MMP2 in complex tissue environments, which will help the diagnosis and treatment of various diseases, including tissue or organ injuries. Moreover, this analysis will also help the evaluation of prognoses. However, MMP2 is difficult to be spatially determined by MALDI TOF mass spectrometry due to its large molecular weight (over 72 KD) and low content. Therefore, a new method should be developed to help this detection. Here, we have designed a specific MMP2 probe that closely binds to MMP2 protein in tissue. This probe has a Cl on Tyr at the terminal, which can provide two isotope peaks to help the accuracy quantitative of MMP2 protein. Based on this, we used the probe to determine the spatial expression of MMP2 in the tissues based on MALDI TOF mass spectrometry. This approach may help to study the influence of multifunctional proteases on the degree of malignancy in vivo.

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