Extracellular Matrix Proteolysis Research Articles

Genotype and Injury Effect on the Expression of a Novel Hypothalamic Protein Sushi Repeat-Containing Protein X-Linked 2 (SRPX2)

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel hypothalamic protein and a ligand of the urokinase-type plasminogen activator receptor (uPAR), which is essential for proteolysis of extracellular matrix and tissue remodeling after an insult to the brain. However, little is known about regulation of SRPX2. Our objective was to investigate if SRPX2 expression is altered by (i) the deficiency of uPAR or uPA (urokinase-type plasminogen activator), and (ii) traumatic brain injury (TBI). SRPX2 expression was assessed in wild type (Wt), Plaur−/− (uPAR-deficient), and Plau−/− (uPA-deficient) mice, with and without controlled cortical impact injury (CCI). The number of SRPX2+ neurons in hypothalamus was comparable to that in Wt littermates in Plaur−/− (2985 ± 138 vs. 2890 ± 92, p > 0.05) and Plau−/− mice (2180 ± 232 vs. 2027 ± 77, p > 0.05). The number of hypothalamic SRPX2+ neurons in the Wt-CCI group was comparable to that in controls (3645 ± 288 vs. 3385 ± 192, p > 0.05). Hypothalamic, hippocampal and thalamic Srpx2 gene expression was unaltered after TBI. However, at 4 days post-TBI Srpx2 gene expression was upregulated in the perilesional cortex of Plau−/−-CCI mice up to 123% of that in the sham group (p < 0.05). Unsupervised hierarchical clustering using SRPX2 expression did not identify genotype or injury-specific clusters. Our data demonstrate that SRPX2 expression in the hypothalamus is resistant to genetic deficiencies in the urokinase-system or to the hypothalamus-affecting TBI. The contribution of elevated Srpx2 gene expression in perilesional cortex to post-TBI recovery process, however, requires further exploration.

Collagen‐derived matricryptin reduces cardiac dysfunction post‐MI via integrin alpha 4

Adverse cardiac remodeling post myocardial infarction (MI) triggers a compensatory maladaptive cardiac response that contributes to heart failure. The infarcted area is replaced by a fibrotic scar that favors collagen deposition instead of its degradation. The evolution of the scar formation depends on the interaction between cardiac cells, particularly fibroblasts, and the extracellular matrix (ECM). Matricryptins are biologically active peptides, generated from ECM proteolysis, able to regulate cell function and survival. We recently identified a collagen‐derived matricryptin (p1158/59) that gradually forms post‐MI. p1158/59 plasma levels negatively correlate with left ventricle (LV) filling pressure, suggesting a beneficial effect for this matricryptin. p1158/59 stimulates LV fibroblast migration in vitro, and mice treated with p1158/59 for 7 days post‐MI displayed less collagen deposition and reduced LV dilation. Accordingly, our goal was to identify the fibroblast receptor(s) involved in p1158/59 actions on cell migration and ECM secretion responsible for the beneficial effects observed on LV remodeling. Additionally, we aim to establish the therapeutic potential of p1158/59 in long term cardiac remodeling.MethodsUsing LV and NIH 3T3 fibroblasts, we performed automatic migration assays with increasing doses of p1158/59 to evaluate changes in receptor gene expression. Blocking antibodies against integrins were used to validate the receptor's findings. We used a murine permanent occlusion MI model (male and female C57Bl/6 mice 3–5 months old, n=4–6/group) to assess effects of p1158/59 treatment (950 μg/day for 14 days), and cardiac function was assessed by serial echocardiography (D0, D7, D14).ResultsCardiac fibroblasts incubated for 24h with 100nM p1158/59 displayed &gt;2‐fold increase of several integrins, with the highest increase on integrin a4 (Itga4), suggesting it as a receptor for p1158/59. Using a migration assay, fibroblasts were incubated with p1158/59 100nM ± Itga4 blocking antibody (15μg/mL) for 2h before wound. The antibody abolished the pro‐migratory effects of the matricryptin, indicating Itga4 as the receptor for p1158/59. Echocardiography analysis demonstrated that mice treated with p1158/59 post‐MI had an increase of 42% in ejection fraction, compared to controls. Additionally, cardiac output exceeded 50% when compared to vehicle controls. These results indicate that p1158/59 attenuated cardiac dysfunction post‐MI.ConclusionOur data suggest that matricryptin p11/58/59 treatment reduces maladaptive LV remodeling post‐MI. Noteworthy, p1158/59 beneficial effects occur through activation of signaling pathways in cardiac fibroblasts via the receptor Itga4.Support or Funding InformationWe acknowledge support from the American Heart Association (18AIREA33960311 and 19PRE34450066) and the Research and Graduate School at East Carolina University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interactions of Omentin and Lactotransferrin in the Progression of Metastatic Ovarian Cancer

Advanced stage serous ovarian cancer metastasizes preferentially to the omentum, which is a major site of intra‐abdominal fat accumulation and is covered by a layer of mesothelial cells (mesothelium). Ovarian cancer cells first adhere to and then penetrate through the mesothelium. It was previously shown the expression of adipokine omentin (ITLN1) in mesothelial cells was down‐regulated when mesothelial cells were co‐cultured with ovarian cancer cells. Survival correlation studies demonstrated that patients with serum ITLN1 levels of &gt;350 ng/mL at the time of first treatment experienced longer survival times than those with lower levels of ITLN1. In addition, wound‐healing assay and invasion assay demonstrated that ovarian cancer cells (SKOV3 and A224) treated with physiological levels of ITLN1 showed a significant decrease in motility and invasion potential. However, the molecular mechanism by which ITLN1 modulates these malignant phenotypes remains unknown. Cancer cells migrate using cell‐matrix mechanocoupling mechanisms through paths generated by the degradation and remodeling of the extracellular matrix (ECM) and by force‐mediated deformation (powered by cell traction force from cancer cells). To explore whether omentin suppresses ovarian cancer invasion potential via inhibiting ECM proteolysis, RNA sequencing and pathway analyses were performed on ITLN1 treated cancer cells. Significantly lower levels of MMP1 (matrix metalloproteinase‐1) was found in cancer cells treated with ITLN1 than untreated cells, which was confirmed by qRT‐PCR analysis. Since ITLN1 has been shown to interact with LTF (lactotransferrin), and binding of LTF to its receptors LRP1 (low‐intensity lipoprotein‐receptor‐related protein 1) can transcriptionally upregulate MMP1, an in vitro pull‐down assay on purified LTF and recombinant ITLN1 in serum‐free media was first performed to confirm that ITLN1 interacts and binds with LTF. To determine whether ITLN1 can interfere with the binding of LTF to LRP1 on ovarian cancer cells, a Duolink proximity ligation assay was performed using both anti‐LTF and anti‐LRP1 antibodies. There was a significant decrease in fluorescent signals in cells that were treated with ITLN1 compared to the control. The results suggested that ITLN1 interacts with LTF and prevents LTF from binding to its receptor LRP1 on ovarian cancer cells. Furthermore, ITLN1 abrogated the effects of LTF on MMP1 expression in a dose‐dependent manner in ovarian cancer cells. LTF not only induced MMP1 expression, but also triggered an increase of cytosolic Ca2+ and cell traction force in ovarian cancer cells, which were attenuated by pre‐incubation with ITLN1. Taken together, these data suggest that ITLN1 suppressed LTF's effect on the motility and invasion potential of ovarian cancer cells by decreasing MMP1 expression and cell traction force generation through modulating a calcium dependent LTF‐LRP1 signaling pathway.Support or Funding InformationDepartment of Defense: grant W81XWH‐17‐1‐0146This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aging and metalloproteinases expression in mussels extracellular matrix

Abstract Emerging traits of cachexia in the elderly are usually attributed to disease, reduced physical activity, reduced appetite, reduced metabolic rate, and reduced energy demand. Lower physical activity and malnutrition lead to sarcopenia, understood as age-related skeletal muscle loss, often associated with reductions in strength, speed of contraction, and muscle power. In addition, the loss of muscle mass observed in old age may be due to changes in the nervous and endocrine systems, changes in muscle metabolism, decreased cell proliferation capacity, chronic age-related inflammation and oxidative stress, chronic disease conditions, and use of pharmaceuticals. Many diseases of muscle are accompanied by increased expression and activity of matrix metalloproteinases (MMP) from the zinc-dependent endopeptidase family, which are involved in the matrix remodeling process that accompanies muscle regeneration. Disorders of collagen accumulation resulting from changes in MMP expression may lead to pathological processes of muscle fibrosis. With the exception of the membrane-type matrix metalloproteinases (MT-MMP), MMP are synthesized in cells and secreted into the extracellular matrix (ECM). Secretory MMP can aggregate on cell surfaces by binding to MT-MMP, along with molecules responsible for cell adhesion, integrins or proteoglycans. To date, over 20 subtypes of metalloproteinases have been classified, each coded by different genes. MMP–induced degradation of structural matrix components (such as collagen, laminin, fibronectin) creates space for suitable cells and facilitates cell migration through the matrix. These enzymes are also involved in the regulation of cell activ ity and take part in inflammatory reactions. Many of the peptides released during partial ECM proteolysis may also act as signaling molecules with autocrine or paracrine action. MMPs also participate in cell-cell communication. Under physiological conditions, MMP are expressed at a very low level in tissues. Induction of expression occurs only in times of ECM reconstruction. Due to their protease activity, these enzymes remain under strict multi-level regulation, i.e. at the level of transcription, post-translational processing, secretion, activation, and degradation. Active MMPs are regulated by tissue inhibitors of metalloproteinases and by numerous cytokines and growth factors.

Dual functions of ARP101 in targeting membrane type-1 matrix metalloproteinase: Impact on U87 glioblastoma cell invasion and autophagy signaling.

Membrane type-1 matrix metalloproteinase (MT1-MMP) possesses both extracellular proteolytic and intracellular signal-transducing functions in tumorigenesis. An imbalance in MT1-MMP expression and/or function triggers a metastatic, invasive, and therapy resistance phenotype. MT1-MMP is involved in extracellular matrix (ECM) proteolysis, activation of latent MMPs, as well as in autophagy signaling in human hepatoma and glioblastoma cells. A low autophagy index in tumorigenesis has been inferred by recent studies where autophagic capacity was decreased during tumor progression. Here, we establish ARP101 as a dual-function small-molecule inhibitor against MT1-MMP ECM hydrolysis and autophagy signal-transducing functions in a model of grade IV glioblastoma cells. ARP101 inhibited concanavalin-A-mediated proMMP-2 activation into MMP-2, as well as MT1-MMP auto-proteolytic processing. When overexpressing recombinant Wt MT1-MMP, ARP101 inhibited proMMP-2 activation and triggered the formation of MT1-MMP oligomers that required trafficking to the plasma membrane. ARP101 further induced cell autophagy as reflected by increased formation of acidic vacuole organelles, LC3 puncta, and autophagy-related protein ATG9 transcription. These were all significantly reversed upon siRNA-mediated gene silencing of MT1-MMP. ARP101 can thus concomitantly inhibit MT1-MMP extracellular catalytic function and exploit its intracellular transducing signal function to trigger autophagy-mediated cell death in U87 glioblastoma cancer cells.

Expression of human papillomavirus oncoproteins E6 and E7 inhibits invadopodia activity but promotes cell migration in HPV-positive head and neck squamous cell carcinoma cells.

BackgroundThe rapid increase in the incidence of head and neck squamous cell carcinoma (HNSCC) is caused by high‐risk human papillomavirus (HPV) infections. The HPV oncogenes E6 and E7 promote carcinogenesis by disrupting signaling pathways that control survival and proliferation. Although these cancers are often diagnosed with metastases, the mechanisms that regulate their dissemination are unknown.AimsThe aim of this study was to determine whether the HPV‐16 E6 and E7 oncogenes affected the invasive and migratory properties of HNSCC cells which promote their spread and metastasis.Methods and resultsInvasiveness was determined using invadopodia assays which allow for quantitation of extracellular matrix (ECM) degradation by invadopodia which are proteolytic membrane protrusions that facilitate invasion. Using cell lines and genetic manipulations, we found that HPV inhibited invadopodia activity in aggressive cell lines which was mediated by the E6 and E7 oncogenes. Given these findings, we also tested whether HPV caused differences in the migratory ability of HNSCC cells using Transwell assays. In contrast to our invadopodia results, we found no correlation between HPV status and cell migration; however, blocking the expression of the E6 and E7 oncoproteins in a HPV‐positive (HPV+) HNSCC cell line resulted in decreased migration.ConclusionsOur data suggest that the E6 and E7 oncoproteins are negative regulators of invadopodia activity but may promote migration in HPV+ HNSCC cells. Despite the need for ECM proteolysis to penetrate most tissues, the unique structure of the head and neck tissues in which these cancers arise may facilitate the spread of migratory cancer cells without significant proteolytic ability.

Hypoxia promotes breast cancer cell invasion through HIF-1\u03b1-mediated up-regulation of the invadopodial actin bundling protein CSRP2

Hypoxia is a common feature of solid tumours that promotes invasion and metastatic dissemination. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix proteolysis and facilitate tumour cell invasion. Here, we show that CSRP2, an invadopodial actin bundling protein, is upregulated by hypoxia in various breast cancer cell lines, as well as in pre-clinical and clinical breast tumour specimens. We functionally characterized two hypoxia responsive elements within the proximal promoter of CSRP2 gene which are targeted by hypoxia-inducible factor-1 (HIF-1) and required for promoter transactivation in response to hypoxia. Remarkably, CSRP2 knockdown significantly inhibits hypoxia-stimulated invadopodium formation, ECM degradation and invasion in MDA-MB-231 cells, while CSRP2 forced expression was sufficient to enhance the invasive capacity of HIF-1α-depleted cells under hypoxia. In MCF-7 cells, CSRP2 upregulation was required for hypoxia-induced formation of invadopodium precursors that were unable to promote ECM degradation. Collectively, our data support that CSRP2 is a novel and direct cytoskeletal target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion by promoting invadopodia formation.

Abstract SY38-01: Inflammation-induced neutrophil extracellular traps (NETs) awaken dormant cancer in the lung microenvironment

Abstract To elucidate mechanisms of cancer metastasis and recurrence from minimal residual disease, we use intravital imaging allowing for long-term visualization of cancer cells and surrounding host cells in living mice. Using intravital imaging, we found that neutrophils, the most abundant type of leukocytes in blood, form neutrophil extracellular traps (NETs) near disseminated cancer cells in the lung of mice during experimental inflammation. NETs are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes in response to, e.g., infections. However, we found that metastatic breast cancer cells themselves can induce neutrophils to form metastasis-supporting NETs, in the absence of infection. We also found NETs in clinical samples of metastatic, triple-negative human breast cancer. Using mouse models, we determined that NETs promote metastasis by facilitating proteolytic extracellular matrix remodeling. Pharmacologic inhibition of NET formation or digestion of NETs with DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs is an important host-mediated mechanism of promoting metastasis and a potential therapeutic target. Citation Format: Mikala Egeblad. Inflammation-induced neutrophil extracellular traps (NETs) awaken dormant cancer in the lung microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY38-01.

DOWN REGULATION OF u-PA BY A NUTRIENT MIXTURE IN HEMANGIOMA (EOMA) CELLS BY INDUCING CASPASE-DEPENDENT APOPTOSIS

To examine, whether the antitumor effects of a specific nutrient mixture are due to induction of apoptosis by inhibition of u-PA. A nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract which has showed anticancer activity against a number of cancer cell lines was used as an experimental composition. EOMA cells were grown in appropriate media with antibiotics in 24-well tissue culture plates. At near confluence, the cells were treated with nutrition mixture at 10, 100, 1000µg/ml in triplicate. Analysis of u-PA activity was carried out by fibrin zymography. Morphological changes and caspase activation associated with apoptosis induction was checked by H&amp;E staining and Live Green caspase assay, respectively. Apoptosis inducing anticancer drug camptothecin (10µM) was used as positive control. The nutrition mixture exhibited dose response toxicity with maximum toxicity 55% (p &lt; 0.001) at 1000µg/ml. EOMA cells expressed u-PA, which was inhibited by nutrition mixture in a dose-dependent manner. The caspase analysis revealed a dose dependent increase in apoptosis of EOMA hemangioma cells, with an increasing apoptosis observed at 100μg/ml, and maximum at 1000μg/ml. Cells treated with nutrition mixture showed significantly more apoptotic changes than the control or camptothecin-treated cells. These results suggest that NM may induce apoptosis of hemangioma cells in vitro thus warranting further investigation.

Studies on sporadic non-syndromic thoracic aortic aneurysms: II. Alterations of extra-cellular matrix components and focal adhesion proteins.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. Here, we focused on morphologic and molecular changes of the extracellular matrix of the tunica media of SNSTAAs. Design Single centre design. Methods Surgical media samples from seven SNSTAAs and seven controls underwent quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, histology and immunohistochemistry analysis. Results A down-regulation of Decorin mRNA with unchanged protein levels associated with a remarkable increase of collagen fibres. A reduced and distorted network of elastic fibres partnered with an attenuated expression of microfibril-associated glycoprotein1 despite the rise of MFAP2 gene-encoded mRNA levels. An increasingly proteolysed paxillin (55 kDa PXN), a focal adhesion protein, combined with an upregulated 62 kDa PXN holoprotein, without changes in amount and phosphorylation of focal adhesion kinase (pp125FAK). The upregulation of SPOCK2-encoded Testican2 proteoglycan and of ectodysplasin (EDA) protein was coupled with a down-regulation of EDA2 receptor (EDA2R). Conclusions Several tunica media extracellular matrix-related changes favour SNSTAA development. A steady level of decorin and a microfibril-associated glycoprotein1 protein shortage cause the assembly of structurally defective collagen and elastic fibres. Up-regulation of PXN holoproteins perturbs PXN/pp125FAK interaction and focal adhesion functioning. Testican2 up-regulation suppresses the membrane-type matrix metalloproteinase inhibiting activities of other SPOCK family members thus enhancing extracellular matrix proteolysis. Finally, the altered EDA•EDA2R signalling would impact on the remodelling of SNSTAA tunica media. Altogether, our results pave the way to a deeper molecular understanding of SNSTAAs necessary to identify their early diagnostic biochemical markers.

Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis.

Acute lung injury is characterized by excessive extracellular matrix proteolysis and neutrophilic inflammation. A major risk factor for lung injury is bacterial pneumonia. However, host factors that protect against pathogen-induced and host-sustained proteolytic injury following infection are poorly understood. Pseudomonas aeruginosa (PA) is a major cause of nosocomial pneumonia and secretes proteases to amplify tissue injury. We show that thrombospondin-1 (TSP-1), a matricellular glycoprotein released during inflammation, dose-dependently inhibits PA metalloendoprotease LasB, a virulence factor. TSP-1-deficient (Thbs1-/-) mice show reduced survival, impaired host defense, and increased lung permeability with exaggerated neutrophil activation following acute intrapulmonary PA infection. Administration of TSP-1 from platelets corrects the impaired host defense and aberrant injury in Thbs1-/- mice. Although TSP-1 is cleaved into 2 fragments by PA, TSP-1 substantially inhibits Pseudomonas elastolytic activity. Administration of LasB inhibitor, genetic disabling of the PA type II secretion system, or functional deletion of LasB improves host defense and neutrophilic inflammation in mice. Moreover, TSP-1 provides an additional line of defense by directly subduing host-derived proteolysis, with dose-dependent inhibition of neutrophil elastase from airway neutrophils of mechanically ventilated critically ill patients. Thus, a host matricellular protein provides dual levels of protection against pathogen-initiated and host-sustained proteolytic injury following microbial trigger.

Biophysical evidence for differential gallated green tea catechins binding to membrane type-1 matrix metalloproteinase and its interactors

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP which triggers intracellular signaling and regulates extracellular matrix proteolysis, two functions that are critical for tumor-associated angiogenesis and inflammation. While green tea catechins, particularly epigallocatechin gallate (EGCG), are considered very effective in preventing MT1-MMP-mediated functions, lack of structure-function studies and evidence regarding their direct interaction with MT1-MMP-mediated biological activities remain. Here, we assessed the impact in both cellular and biophysical assays of four ungallated catechins along with their gallated counterparts on MT1-MMP-mediated functions and molecular binding partners. Concanavalin-A (ConA) was used to trigger MT1-MMP-mediated proMMP-2 activation, expression of MT1-MMP and of endoplasmic reticulum stress biomarker GRP78 in U87 glioblastoma cells. We found that ConA-mediated MT1-MMP induction was inhibited by EGCG and catechin gallate (CG), that GRP78 induction was inhibited by EGCG, CG, and gallocatechin gallate (GCG), whereas proMMP-2 activation was inhibited by EGCG and GCG. Surface plasmon resonance was used to assess direct interaction between catechins and MT1-MMP interactors. We found that gallated catechins interacted better than their ungallated analogs with MT1-MMP as well as with MT1-MMP binding partners MMP-2, TIMP-2, MTCBP-1 and LRP1-clusterIV. Overall, current structure-function evidence supports a role for the galloyl moiety in both direct and indirect interactions of green tea catechins with MT1-MMP-mediated oncogenic processes.

From genetics to response to injury: vascular smooth muscle cells in aneurysms and dissections of the ascending aorta.

Vascular smooth muscle cells (vSMCs) play a crucial role in both the pathogenesis of Aneurysms and Dissections of the ascending thoracic aorta (TAAD) in humans and in the associated adaptive compensatory responses, since thrombosis and inflammatory processes are absent in the majority of cases. Aneurysms and dissections share numerous characteristics, including aetiologies and histopathological alterations: vSMC disappearance, medial areas of mucoid degeneration, and extracellular matrix (ECM) breakdown. Three aetiologies predominate in TAAD in humans: (i) genetic causes in heritable familial forms, (ii) an association with bicuspid aortic valves, and (iii) a sporadic degenerative form linked to the aortic aging process. Genetic forms include mutations in vSMC genes encoding for molecules of the ECM or the TGF-β pathways, or participating in vSMC tone. On the other hand, aneurysms and dissections, whatever their aetiologies, are characterized by an increase in wall permeability leading to transmural advection of plasma proteins which could interact with vSMCs and ECM components. In this context, blood-borne plasminogen appears to play an important role, because its outward convection through the wall is increased in TAAD, and it could be converted to active plasmin at the vSMC membrane. Active plasmin can induce vSMC disappearance, proteolysis of adhesive proteins, activation of MMPs and release of TGF-β from its ECM storage sites. Conversely, vSMCs could respond to aneurysmal biomechanical and proteolytic injury by an epigenetic phenotypic switch, including constitutional overexpression and nuclear translocation of Smad2 and an increase in antiprotease and ECM protein synthesis. In contrast, such an epigenetic phenomenon is not observed in dissections. In this context, dysfunction of proteins involved in vSMC tone are interesting to study, particularly in interaction with plasma protein transport through the wall and TGF-β activation, to establish the relationship between these dysfunctions and ECM proteolysis.

Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix.

Dendritic cells (DCs) infected by Toxoplasma gondii rapidly acquire a hypermigratory phenotype that promotes systemic parasite dissemination by a "Trojan horse" mechanism in mice. Recent paradigms of leukocyte migration have identified the amoeboid migration mode of DCs as particularly suited for rapid locomotion in extracellular matrix and tissues. Here, we have developed a microscopy-based high-throughput approach to assess motility and matrix degradation by Toxoplasma-challenged murine and human DCs. DCs challenged with T.gondii exhibited dependency on metalloproteinase activity for hypermotility and transmigration but, strikingly, also dramatically reduced pericellular proteolysis. Toxoplasma-challenged DCs up-regulated expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) and their supernatants impaired matrix degradation by naïve DCs and by-stander DCs dose dependently. Gene silencing of TIMP-1 by short hairpin RNA restored matrix degradation activity in Toxoplasma-infected DCs. Additionally, dissolution of podosome structures in parasitised DCs coincided with abrogated matrix degradation. Toxoplasma lysates inhibited pericellular proteolysis in a MyD88-dependent fashion whereas abrogated proteolysis persevered in Toxoplasma-infected MyD88-deficient DCs. This indicated that both TLR/MyD88-dependent and TLR/MyD88-independent signalling pathways mediated podosome dissolution and the abrogated matrix degradation. We report that increased TIMP-1 secretion and cytoskeletal rearrangements encompassing podosome dissolution are features of Toxoplasma-induced hypermigration of DCs with an impact on matrix degradation. Jointly, the data highlight how an obligate intracellular parasite orchestrates key regulatory cellular processes consistent with non-proteolytic amoeboid migration of the vehicle cells that facilitate its dissemination.

Mechanisms of plasminogen activator inhibitor 1 action in stromal remodeling and related diseases.

Plasminogen activator inhibitor type 1 (PAI-1) is the main physiologic inhibitor of fibrinolysis. However, it is also involved in many physiological processes such as extracellular matrix (ECM) proteolysis and remodeling, cell adhesion, motility, and apoptosis, angiogenesis, etc. The aim of the study was to summarize current knowledge and gain insights into the mechanisms of PAI-1 action in the processes of stromal remodeling and diseases with considerable matrix pathologies (atherosclerosis, tissue fibrosis, cancer metastasis, pregnancy related complications, etc). As a component of an early cellular response to injury, PAI-1 reacts with membrane surface proteins and participates in the initiation of intracellular signaling, specifically cytoskeletal reorganization and motility. Complexity of ECM homeostasis resides in varying relation of the plasminogen system components and other matrix constituents. Inflammatory mediators (transforming growth factor-β and interferon-γ) and hormones (angiotensin II) are in the close interdependent relation with PAI-1. Also, special attention is devoted to the role of increased PAI-1 concentrations due to the common 4G/5G polymorphism. Some of the novel mechanisms of ECM modification consider PAI-1 dependent stabilization of urokinase mediated cell adhesion, control of the vascular endothelial cadherin trafficking and interaction with endothelial cells proteasome, its relation to matrix metalloproteinase 2 and osteopontin, and oxidative inhibition by myeloperoxidase. Targeting and/or alteration of PAI-1 functions might bring benefit to the future therapeutic approaches in diseases where ECM undergoes substantial remodeling.

Cysteine Protease Cathepsins in Atherosclerotic Cardiovascular Diseases.

Atherosclerotic cardiovascular disease (ASCVD) is an inflammatory disease characterized by extensive arterial wall matrix protein degradation. Cysteine protease cathepsins play a pivotal role in extracellular matrix (ECM) remodeling and have been implicated in the development and progression of atherosclerosis-based cardiovascular diseases. An imbalance in expression between cathepsins (such as cathepsins S, K, L, C) and their inhibitor cystatin C may favor proteolysis of ECM in the pathogenesis of cardiovascular disease such as atherosclerosis, aneurysm formation, restenosis, and neovascularization. New insights into cathepsin functions have been made possible by the generation of knock-out mice and by the application of specific inhibitors. Inflammatory cytokines regulate the expression and activities of cathepsins in cultured vascular cells and macrophages. In addition, evaluations of the possibility of cathepsins as a diagnostic tool revealed that the circulating levels of cathepsin S, K, and L, and their endogenous inhibitor cystatin C could be promising biomarkers in the diagnosis of coronary artery disease, aneurysm, adiposity, peripheral arterial disease, and coronary artery calcification. In this review, we summarize the available information regarding the mechanistic contributions of cathepsins to ASCVD.

Nck deficiency is associated with delayed breast carcinoma progression and reduced metastasis.

Although it is known that noncatalytic region of tyrosine kinase (Nck) regulates cell adhesion and migration by bridging tyrosine phosphorylation with cytoskeletal remodeling, the role of Nck in tumorigenesis and metastasis has remained undetermined. Here we report that Nck is required for the growth and vascularization of primary tumors and lung metastases in a breast cancer xenograft model as well as extravasation following injection of carcinoma cells into the tail vein. We provide evidence that Nck directs the polarization of cell-matrix interactions for efficient migration in three-dimensional microenvironments. We show that Nck advances breast carcinoma cell invasion by regulating actin dynamics at invadopodia and enhancing focalized extracellular matrix proteolysis by directing the delivery and accumulation of MMP14 at the cell surface. We find that Nck-dependent cytoskeletal changes are mechanistically linked to enhanced RhoA but restricted spatiotemporal activation of Cdc42. Using a combination of protein silencing and forced expression of wild-type/constitutively active variants, we provide evidence that Nck is an upstream regulator of RhoA-dependent, MMP14-mediated breast carcinoma cell invasion. By identifying Nck as an important driver of breast carcinoma progression and metastasis, these results lay the groundwork for future studies assessing the therapeutic potential of targeting Nck in aggressive cancers.

Multifaceted Roles of Metzincins in CNS Physiology and Pathology: From Synaptic Plasticity and Cognition to Neurodegenerative Disorders.

The extracellular matrix (ECM) and membrane proteolysis play a key role in structural and functional synaptic plasticity associated with development and learning. A growing body of evidence underscores the multifaceted role of members of the metzincin superfamily, including metalloproteinases (MMPs), A Disintegrin and Metalloproteinases (ADAMs), A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTSs) and astacins in physiological and pathological processes in the central nervous system (CNS). The expression and activity of metzincins are strictly controlled at different levels (e.g., through the regulation of translation, limited activation in the extracellular space, the binding of endogenous inhibitors and interactions with other proteins). Thus, unsurprising is that the dysregulation of proteolytic activity, especially the greater expression and activation of metzincins, is associated with neurodegenerative disorders that are considered synaptopathies, especially Alzheimer’s disease (AD). We review current knowledge of the functions of metzincins in the development of AD, mainly the proteolytic processing of amyloid precursor protein, the degradation of amyloid β (Aβ) peptide and several pathways for Aβ clearance across brain barriers (i.e., blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB)) that contain specific receptors that mediate the uptake of Aβ peptide. Controlling the proteolytic activity of metzincins in Aβ-induced pathological changes in AD patients’ brains may be a promising therapeutic strategy.

A Glitch in the Matrix: Aberrant Extracellular Matrix Proteolysis Contributes to Alcohol Seeking

A Glitch in the Matrix: Aberrant Extracellular Matrix Proteolysis Contributes to Alcohol Seeking

High TMPRSS11D protein expression predicts poor overall survival in non-small cell lung cancer.

TMPRSS11D (HAT) belongs to the large type II transmembrane serine protease (TTSP) family, participating in various biological and physiological processes. TMPRSS11D expression has been reported during squamous cell carcinogenesis, however, its expression during non-small cell lung cancer (NSCLC) development has not been studied. In this study, we determined the mRNA and protein expression of TMPRSS11D in NSCLC tumorous and matched adjacent normal tissues by quantitative reverse transcription PCR (qRT-PCR) and tissue microarray immunohistochemistry analysis (TMA-IHC) respectively. TMPRSS11D protein expression in tumorous tissues were correlated with NSCLC patients’ clinical characteristics and overall survival. Both TMPRSS11D mRNA and protein expression levels were significantly higher in NSCLC tumorous tissues than in adjacent normal tissues. High TMPRSS11D protein expression was associated with high TNM stages, and high TMPRSS11D protein expression is an independent prognostic marker in NSCLC. Based on our results, we conclude that TMPRSS11D could play a role in NSCLC development and progression. Because of its role in proteolysis of extracellular matrix, targeting TMPRSS11D may prevent the development of metastasis in NSCLC.