Extracellular Matrix Proteolysis Research Articles

Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

Repair of airway epithelium after injury requires migration of neighboring epithelial cells to injured areas. However, the molecular mechanisms regulating airway epithelial cell migration is not well defined. We have previously shown that XB130, a scaffold protein, is required for airway epithelial repair and regeneration in vivo, and interaction between XB130 and another scaffold protein, Tks5, regulates cell proliferation and survival in human bronchial epithelial cells. The objective of the present study was to determine the role of XB130 and Tks5 interaction in airway epithelial cell migration. Interestingly, we found that XB130 only promotes lateral cell migration, whereas, Tks5 promotes cell migration/invasion via proteolysis of extracellular matrix. Upon stimulation with EGF, PKC activator phorbol 12, 13-dibutyrate or a nicotinic acetylcholine receptor ligand, XB130 and Tks5 translocated to the cell membrane in a stimulus-dependent manner. The translocation and distribution of XB130 is similar to lamellipodial marker, WAVE2; whereas Tks5 is similar to podosome marker, N-WASP. Over-expression of XB130 or Tks5 alone enhances cell migration, whereas co-expression of both XB130 and Tks5 inhibits cell migration processes and signaling. Furthermore, XB130 interacts with Rac1 whereas Tks5 interacts with Cdc42 to promote Rho GTPase activity. Our results suggest that dissociation between XB130 and Tks5 may facilitate lateral cell migration via XB130/Rac1, and vertical cell migration via Tks5/Cdc42. These molecular mechanisms will help our understanding of airway epithelial repair and regeneration.

Comparison of cell migration mechanical strategies in three-dimensional matrices: a computational study.

Cell migration on a two-dimensional flat surface has been extensively studied and is generally characterized by a front-protrusion-rear-contraction process. In a three-dimensional (3D) environment, on the other hand, cells adopt multiple migration strategies depending on the cell type and the properties of the extracellular matrix (ECM). By using computer simulations, we find that these migration strategies can be classified by various spatial-temporal dynamics of actin protrusion, actin-myosin contraction and actin-ECM adhesion. We demonstrate that if we include or exclude proteolysis of ECM, and vary adhesion dynamics and spatial distributions of protrusion, contraction and adhesion, our model can reproduce six experimentally observed motility modes: mesenchymal, chimneying, amoeboid, blebbing, finger-like protrusion and rear-squeezing cell locomotory behaviours. We further find that the mode of the cell motility evolves in response to the ECM density and adhesion detachment rate. The model makes non-trivial predictions about cell speed as a function of the adhesion strength, and ECM elasticity and mesh size.

Extracellular Matrix Proteolysis by MT1-MMP Contributes to Influenza-Related Tissue Damage and Mortality.

Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival.We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.

Distinct biological events generated by ECM proteolysis by two homologous collagenases

It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.

5'-Inositol phosphatase SHIP2 recruits Mena to stabilize invadopodia for cancer cell invasion.

Invadopodia are specialized membrane protrusions that support degradation of extracellular matrix (ECM) by cancer cells, allowing invasion and metastatic spread. Although early stages of invadopodia assembly have been elucidated, little is known about maturation of invadopodia into structures competent for ECM proteolysis. The localized conversion of phosphatidylinositol(3,4,5)-triphosphate and accumulation of phosphatidylinositol(3,4)-bisphosphate at invadopodia is a key determinant for invadopodia maturation. Here we investigate the role of the 5'-inositol phosphatase, SHIP2, and reveal an unexpected scaffold function of SHIP2 as a prerequisite for invadopodia-mediated ECM degradation. Through biochemical and structure-function analyses, we identify specific interactions between SHIP2 and Mena, an Ena/VASP-family actin regulatory protein. We demonstrate that SHIP2 recruits Mena, but not VASP, to invadopodia and that disruption of SHIP2-Mena interaction in cancer cells leads to attenuated capacity for ECM degradation and invasion in vitro, as well as reduced metastasis in vivo. Together, these findings identify SHIP2 as a key modulator of carcinoma invasiveness and a target for metastatic disease.

Abstract 5063: The receptor for urokinase-plasminogen activator controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style

Abstract Introduction. The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility. Materials and methods. To evaluate the mesenchymal ameboid transition we performed RhoA and Rac1 activation assay, immunofluorescence analysis of protein involved in cytoskeleton organization, and collagen degradation assay. For a clear visualization of collagen fiber breakdown in the process of proteolytic migration we used reconstruction by time-lapse video microscopy. Cell invasion was studied in Boyden chambers using filters coated with Matrigel. uPAR gene expression was inhibited using a 18-mer phosphorothioate aODN, and as a negative control we used a degenerated oligodeoxynucleotide, which is a mixture of all possible combinations of bases that compose the aODN. Results and discussion. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the “path generating” mesenchymal to the “path finding” amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one. Conclusion. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly. Citation Format: Anastasia Chillà, Francesca Margheri, Anna Laurenzana, Cristina Luciani, Alessio Biagioni, Gabriella Fibbi, Mario Del Rosso. The receptor for urokinase-plasminogen activator controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5063.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis.

BackgroundThrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis.MethodsTo assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM.ResultsInhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa.ConclusionsTaken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells.

Epicatechin-3-gallate reverses TGF-β1-induced epithelial-to-mesenchymal transition and inhibits cell invasion and protease activities in human lung cancer cells

Epithelial-to-mesenchymal transition (EMT) and invasion potential have been considered as essential factors in cancer metastasis, which is the major cause of cancer death. EMT is a multi-step process that involves gain invasion, cytoskeleton change, cell adhesion, and proteolytic extracellular matrix degradation. Epicatechin-3-gallate (ECG), which is a natural polyphenolic component of green tea, elicits several antioxidant and anti-inflammatory effects. However, the effects of ECG on cancer invasion and EMT of human lung carcinoma remain unknown. We provided molecular evidence supporting the anti-metastatic effect of ECG. This compound suppressed the invasion (P < 0.001) of highly metastatic A549 cells by reducing the activities of matrix metalloproteinase-2 (P < 0.001) and urokinasetype plasminogen activator (P < 0.001). ECG also reversed the transforming growth factor (TGF)-β1-induced EMT and upregulated epithelial markers, such as E-cadherin. Conversely, ECG inhibited mesenchymal markers, such as fibronectin and p-FAK. The subcutaneous inoculation of this compound also inhibited the tumor growth of the A549 cells in vivo. Therefore, ECG may be used as an anti-cancer and anti-invasion agent for the adjuvant treatment and metastasis control of human lung cancer cells. ECG may also be administered as an effective chemopreventive agent against TGF-β1-induced EMT.

Membrane-anchored proteases in endothelial cell biology.

The endothelial cell plasma membrane is a metabolically active, dynamic, and fluid microenvironment where pericellular proteolysis plays a critical role. Membrane-anchored proteases may be expressed by endothelial cells as well as mural cells and leukocytes with distribution both inside and outside of the vascular system. Here, we will review the recent advances in our understanding of the direct and indirect roles of membrane-anchored proteases in vascular biology and the possible conservation of their extravascular functions in endothelial cell biology. Membrane-anchored proteases belonging to the serine or metalloprotease families contain amino-terminal or carboxy-terminal domains, which serve to tether their extracellular protease domains directly at the plasma membrane. This architecture enables protease function and substrate repertoire to be regulated through dynamic localization in distinct areas of the cell membrane. These proteases are proving to be key components of the cell machinery for regulating vascular permeability, generation of vasoactive peptides, receptor tyrosine kinase transactivation, extracellular matrix proteolysis, and angiogenesis. A complex picture of the interdependence between membrane-anchored protease localization and function is emerging that may provide a mechanism for precise coordination of extracellular signals and intracellular responses through communication with the cytoskeleton and with cellular signaling molecules.

Cell motility and ECM proteolysis regulate tumor growth and tumor relapse by altering the fraction of cancer stem cells and their spatial scattering

Tumors consist of multiple cell sub-populations including cancer stem cells (CSCs), transiently amplifying cells and terminally differentiated cells (TDCs), with the CSC fraction dictating the aggressiveness of the tumor and drug sensitivity. In epithelial cancers, tumor growth is influenced greatly by properties of the extracellular matrix (ECM), with cancer progression associated with an increase in ECM density. However, the extent to which increased ECM confinement induced by an increase in ECM density influences tumor growth and post treatment relapse dynamics remains incompletely understood. In this study, we use a cellular automata-based discrete modeling approach to study the collective influence of ECM density, cell motility and ECM proteolysis on tumor growth, tumor heterogeneity, and tumor relapse after drug treatment. We show that while increased confinement suppresses tumor growth and the spatial scattering of CSCs, this effect can be reversed when cells become more motile and proteolytically active. Our results further suggest that, in addition to the absolute number of CSCs, their spatial positioning also plays an important role in driving tumor growth. In a nutshell, our study suggests that, in confined environments, cell motility and ECM proteolysis are two key factors that regulate tumor growth and tumor relapse dynamics by altering the number and spatial distribution of CSCs.

The Arf-like GTPase Arl8b is essential for three-dimensional invasive growth of prostate cancer in vitro and xenograft formation and growth in vivo.

Cancer is a multistep process that requires cells to respond appropriately to the tumor microenvironment, both in early proliferative stages and in later invasive disease. Arl8b is a lysosome localized Arf-like GTPase that controls the spatial distribution of lysosomes via recruitment of kinesin motors. Common features of the tumor microenvironment such as acidic extracellular pH and various growthfactors stimulate lysosome trafficking to the cell periphery (anterograde), which is critical for tumor invasion by facilitating the release of lysosomal proteases to promote matrix remodeling. Herein we report for the first time that Arl8b regulates anterograde lysosome trafficking in response to hepatocyte growth factor, epidermal growth factor, and acidic extracellular pH. Depletion of Arl8b results in juxtanuclear lysosome aggregation, and this effect corresponds with both diminished invasive growth and proteolytic extracellular matrix degradation in a three-dimensional model of prostate cancer. Strikingly, we found that depletion of Arl8b abolishes the ability of prostate cancer cells to establish subcutaneous xenografts in mice. We present evidence that Arl8b facilitates lipid hydrolysis to maintain efficient metabolism for a proliferative capacity in low nutrient environments, suggesting a likely explanation for the complete inability of Arl8b-depleted tumor cells to grow in vivo. In conclusion, we have identified two mechanisms by which Arl8b regulates cancer progression: 1) through lysosome positioning and protease release leading to an invasive phenotype and 2) through control of lipid metabolism to support cellular proliferation. These novel roles highlight that Arl8b is a potential target for the development of novel anti-cancer therapeutics.

Role of gene expression profiling in defining indeterminate thyroid nodules in addition to BRAF analysis.

Fine-needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules. However, 15% to 30% of aspirations yield indeterminate cytologic findings. Because the assessment of BRAF mutations seems to improve the diagnostic accuracy, this study evaluated BRAF mutations with Sanger sequencing and real-time methods in 650 consecutive thyroid aspirates. In addition, the expression of a large number of genes involved in basement membrane remodeling, extracellular matrix proteolysis, and cell adhesion was studied in both benign and malignant nodules to identify new diagnostic tools. In this prospective series, despite the use of a very sensitive BRAF mutational testing method, the frequency of a BRAF alteration being identified in indeterminate FNA samples was 3 of 68. Expression analysis revealed several genes that were differentially expressed between benign and malignant nodules (transforming growth factor, cadherin 1, collagen α1, catenin α1, integrin α3, and fibronectin 1 [FN1]), between follicular adenomas and follicular variant of papillary thyroid carcinoma (FN1, laminin γ1, integrin β2, connective tissue growth factor, catenin δ1, and integrin αV), and between BRAF-wild-type and BRAF-mutated papillary thyroid carcinomas (TIMP metallopeptidase inhibitor 1; catenin α1; secreted phosphoprotein 1; FN1; ADAM metallopeptidase with thrombospondin type 1 motif, 1; and selectin L). These data were partially confirmed with real-time polymerase chain reaction analysis and immunohistochemistry. When the cost/benefit ratio of the procedures was taken into account, BRAF mutational testing failed to increase diagnostic accuracy in cytologically indeterminate nodules. However, the additional analysis of the expression of specific molecular markers could have possible utility as a diagnostic tool, although further evidence based on a large series of samples is needed before definitive conclusions can be drawn. Cancer Cytopathol 2016;124:340-9. © 2015 American Cancer Society.

Modeling Proteolytically Driven Tumor Lymphangiogenesis.

With the exception of a limited number of sites in the body, primary tumors infrequently lead to the demise of cancer patients. Instead, mortality and a significant degree of morbidity result from the growth of secondary tumors in distant organs. Tumor survival, growth and dissemination are associated with the formation of both new blood vessels (angiogenesis) and new lymph vessels (lymphagenesis or lymphangiogenesis). Although intensive research in tumor angiogenesis has been going on for the past four decades, experimental results in tumor lymphangiogenesis began to appear only in the last 10years. In this chapter we expand the models proposed by Friedman, Lolas and Pepper on tumor lymphangiogenesis mediated by proteolytically and un-proteolytically processed growth factors (Friedman and Lolas G, Math Models Methods Appl Sci 15(01):95-107, 2005; Pepper and Lolas G, Selected topics in cancer modeling: genesis, evolution, immune competition, and therapy. In: The lymphatic vascular system in lymphangiogenesis invasion and metastasis a mathematical approach. Birkhäuser Boston, Boston, pp1-22, 2008). The variables represent different cell densities and growth factors concentrations, and where possible the parameters are estimated from experimental and clinical data. The results obtained from computational simulations carried out on the model equations produce dynamic heterogeneous ("anarchic") spatio-temporal solutions. More specifically, we observed coherent masses of tumor clusters migrating around and within the lymphatic network. Our findings are in line with recent experimental evidence that associate cluster formation with the minimization of cell loss favoring high local extracellular matrix proteolysis and thus protecting cancer invading cells from an immunological assault driven by the lymphatic network.

Improvement of Psychotic Symptoms and the Role of Tissue Plasminogen Activator.

Tissue plasminogen activator (tPA) mediates a number of processes that are pivotal for synaptogenesis and remodeling of synapses, including proteolysis of the brain extracellular matrix, degradation of adhesion molecules, activation of neurotrophins, and activation of the N-methyl-d-aspartate receptor. Abnormalities in these processes have been consistently described in psychotic disorders. In this paper, we review the physiological roles of tPA, focusing on conditions characterized by low tPA activity, which are prevalent in schizophrenia. We then describe how tPA activity is influenced by lifestyle interventions and nutritional supplements that may ameliorate psychotic symptoms. Next, we analyze the role of tPA in the mechanism of action of hormones and medications effective in mitigating psychotic symptoms, such as pregnenolone, estrogen, oxytocin, dopamine D3 receptor antagonists, retinoic acid, valproic acid, cannabidiol, sodium nitroprusside, N-acetyl cysteine, and warfarin. We also review evidence that tPA participates in the mechanism by which electroconvulsive therapy and cigarette smoking may reduce psychotic symptoms.

MMP‐9 null mice display elevated IOP due to reduced aqueous humor drainage from the trabecular meshwork

PurposeAqueous humor outflow resistance depends on a complex equilibrium of extracellular matrix biosynthesis versus proteolysis in the trabecular meshwork. Several members of the MMP family, including MMP‐9, are essential to the regulation of aqueous humor outflow resistance, i.e. to intraocular pressure (IOP) homeostasis. Here, we characterized the baseline ocular phenotype of MMP‐9 null mice, with emphasis on the dysregulation of IOP homeostasis.MethodsMMP‐9 null and wild type mice, from 3 to 13 months of age, were studied. IOP and central corneal thickness were measured via rebound tonometry and pachymetry, resp. Anterior chamber morphology and trabecular meshwork organization were studied (1) in vivo with OCT, (2) via light microscopy and (3) by means of transmission electron microscopy (TEM), and its collagen composition was studied using Sirius Red and immunostainings. Integrity of the retina and optic nerve were evaluated with OCT and histological stainings on tissue sections and retinal flatmounts.ResultsMMP‐9 null mice present with early‐onset ocular hypertension, and fluorophotometric measurements of aqueous humor turnover revealed a reduced aqueous humor drainage. While OCT, light microscopy and TEM analysis did not disclose any abnormalities in the cellular organization of the trabecular meshwork, collagen staining indicated that there is an aberrant extracellular matrix composition in MMP‐9 null mice. Remarkably, the observed IOP elevation in MMP‐9 null mice did not result in a glaucomatous phenotype at the level of the retina and optic nerve at the ages studied.ConclusionsOur observations corroborate the role of MMP‐9 as an important remodeler of the trabecular meshwork, and evidence for a causal link between MMP‐9 deficiency, trabecular meshwork composition and IOP elevation is revealed.

Proteolytic regulation of synaptic plasticity in the mouse primary visual cortex: analysis of matrix metalloproteinase 9 deficient mice.

The extracellular matrix (ECM) is known to play important roles in regulating neuronal recovery from injury. The ECM can also impact physiological synaptic plasticity, although this process is less well understood. To understand the impact of the ECM on synaptic function and remodeling in vivo, we examined ECM composition and proteolysis in a well-established model of experience-dependent plasticity in the visual cortex. We describe a rapid change in ECM protein composition during Ocular Dominance Plasticity (ODP) in adolescent mice, and a loss of ECM remodeling in mice that lack the extracellular protease, matrix metalloproteinase-9 (MMP9). Loss of MMP9 also attenuated functional ODP following monocular deprivation (MD) and reduced excitatory synapse density and spine density in sensory cortex. While we observed no change in the morphology of existing dendritic spines, spine dynamics were altered, and MMP9 knock-out (KO) mice showed increased turnover of dendritic spines over a period of 2 days. We also analyzed the effects of MMP9 loss on microglia, as these cells are involved in extracellular remodeling and have been recently shown to be important for synaptic plasticity. MMP9 KO mice exhibited very limited changes in microglial morphology. Ultrastructural analysis, however, showed that the extracellular space surrounding microglia was increased, with concomitant increases in microglial inclusions, suggesting possible changes in microglial function in the absence of MMP9. Taken together, our results show that MMP9 contributes to ECM degradation, synaptic dynamics and sensory-evoked plasticity in the mouse visual cortex.

Matrix metalloproteinases and the vascular smooth muscle cell migration in hypertension

Vascular smooth muscle cells (VSMC) migration and proliferation may be the main precursors of the chronic and maladaptive vascular remodeling in hypertension. The proteolysis of extracellular matrix and the phenotype switch of VSMC need to occur in order to allow VSMC migration and the vascular remodeling and hypertrophy. The matrix metalloproteinases (MMPs) are well known to degrade many extracellular matrix and non-extracellular matrix components to favor VSMC migration and their phenotype switch. Targeting MMPs may reduce VSMC migration and the arterial maladaptation caused by hypertension and its cardiovascular events.

ADAMTS9-Mediated Extracellular Matrix Dynamics Regulates Umbilical Cord Vascular Smooth Muscle Differentiation and Rotation.

Despite the significance for fetal nourishment in mammals, mechanisms of umbilical cord vascular growth remain poorly understood. Here, the secreted metalloprotease ADAMTS9 is shown to be necessary for murine umbilical cord vascular development. Restricting it to the cell surface using a gene trap allele, Adamts9(Gt), impaired umbilical vessel elongation and radial growth via reduced versican proteolysis and accumulation of extracellular matrix (ECM). Both Adamts9(Gt) and conditional Adamts9 deletion revealed that ADAMTS9 produced by mesenchymal cells acted non-autonomously to regulate smooth muscle cell (SMC) proliferation, differentiation, and orthogonal reorientation during growth of the umbilical vasculature. In Adamts9(Gt/Gt), we observed interference with PDGFRβ signaling via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which regulates cytoskeletal dynamics during SMC rotation. In addition, we observed disrupted Shh signaling and perturbed orientation of the mesenchymal primary cilium. Thus, ECM dynamics is a major influence on umbilical vascular SMC fate, with ADAMTS9 acting as its principal mediator.

Abstract P1-07-32: Activated thrombin activatable fibrinolysis inhibitor is a novel anti-metastatic factor in breast cancer

Abstract Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen initially known for its role in attenuating fibrinolysis. Activated TAFI (TAFIa) is formed through proteolytic cleavage by thrombin, plasmin or, its most effective activator, thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase, which acts by cleaving carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen binding sites on cell surface receptors. Carboxyl terminal lysine residues play a vital role in accelerating plasminogen activation to plasmin on the cell surface. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. In addition, plasmin can activate matrix metalloproteinases (MMPs), which also play a role in degrading the ECM. Furthermore, the expression of TM in tumours is inversely correlated to metastasis. Studies have shown that the anti-metastatic effects of TM result from its ability to bind thrombin. Given that the thrombin/TM complex is responsible for the activation of TAFI, the anti-metastatic effects of TM may be modulated by TAFIa. We therefore hypothesize that the activation of TAFI on the cell surface inhibits plasminogen activation and decreases breast cancer cell invasion and migration. Expression of TAFI and TM were assessed in breast cancer cells with varying degrees of metastatic potential. Although TAFI mRNA levels did not correlate with malignancy, TM mRNA levels were found to be inversely correlated with breast cancer cell malignancy. Moreover, cell invasion and migration of MDA-MB-231 and SUM149 cells were assessed upon treatment with potato tuber carboxypeptidase inhibitor (PTCI), which is a specific inhibitor of TAFIa. Inhibition of TAFIa resulted in a significant increase in cell invasion and migration of both cell lines. Cell invasion and migration of MDA-MB-231 and SUM149 cells were also assessed upon treatment with TM. Treatment with TM significantly decreased cell invasion and migration of both MDA-MB-231 and SUM149 cells. Additionally, experiments using a fluorogenic collagen substrate showed an increase in extracellular collagen cleavage after PTCI treatment, using both MDA-MB-231 and SUM149 cells. Furthermore, the ability of TAFIa to inhibit pericellular plasminogen activation was evaluated. Plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa. Taken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis. Enhancement of TAFI activation in the breast cancer tumour microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells. Citation Format: Zainab A Bazzi, Deborah Rudy, Lisa A Porter, Dora Cavallo-Medved, Michael B Boffa. Activated thrombin activatable fibrinolysis inhibitor is a novel anti-metastatic factor in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-32.

Role of metalloproteinases and their inhibitors in intervertebral disc degeneration: research progress

Intervertebral disc degeneration(IVDD)is characterized by the excessive degradation of extracellular matrix(ECM),which underlies many spine-related disorders.Matrix metalloproteinases(MMPs)and a disintegrin metalloproteinases with thrombospondin motifs(ADAMTSs)are believed to be the major proteolytic enzymes responsible for ECM degradation.This review summarizes the current literatures on gene expression and regulation of MMPs,ADAMTSs,and tissue inhibitors of metalloproteinases(TIMPs)in IVDD.Reports have showen that specific MMPs(MMP-1,-2,-3,-7,-8,-10,and-13)and ADAMTS(ADAMTS-1,-4,and-15)are upregulated in human degenerated intervertebral discs.Tissue inhibitor of metalloproteinase-3is downregulated and TIMP-1 is upregulated in human degenerated intervertebral discs relative to nondegenerated intervertebral discs.Regulation of the MMP and ADAMTS expression is affected by many factors including mechanical,inflammatory,oxidative stress,and so on.Genetic predisposition also plays an important role in expression of MMP-1,-2,-3,and-9.Upregulation of MMP and ADAMTS expression is implicated in ECM destruction,which can lead to the development of IVDD.Future IVDD therapeutics may target specific MMPs and ADAMTSs which is essential in the pathological proteolysis of ECM.