Matrix Proteolysis Research Articles

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.

IL-1beta mediates MMP secretion and IL-1beta neosynthesis via upregulation of p22phox and NOX4 activity in human articular chondrocytes

Osteoarthritis (OA) is characterized by a progressive alteration of the biochemical properties of the articular cartilage. Inflammation plays a major role in OA, particularly through the cytokine Interleukine-1β, promoting reactive oxygen species (ROS) generation and matrix metalloproteinases (MMP) synthesis by the chondrocytes, orchestrating matrix proteolysis. NADPH oxidases (NOX) are membrane enzymes dedicated to the production of ROS. Role of oxidative stress is well established in OA; however, contribution of NOX in this process is still poorly documented. In this study, we addressed the role of NOX in primary human articular chondrocytes (HAC) upon inflammatory conditions--namely IL-1β and OA. HAC were collected from patients undergoing hip surgery. Chondrocytes were treated with IL-1β and NOX inhibitors Diphenylene Iodonium, GKT136901, Tiron and Heme oxygenase-1 before MMP expression and NOX activity assessment. Finally, NOX4 expression was compared between OA and non OA parts of hip cartilage (n = 14). This study establishes for the first time in human that NOX4 is the main NOX isoform expressed in chondrocytes. We found a significant upregulation of NOX4 mRNA in OA chondrocytes. Expression of NOX4/p22(phox) as well as ROS production is enhanced by IL-1β. On the other hand, the use of NOX4 inhibitors decreased IL-1β-induced collagenase synthesis by chondrocytes. Moreover, our study support the existence of a redox dependant loop sustaining pro-catabolic pathways induced by IL-1β. This study points out NOX4 as a new putative target in OA and suggests that NOX-targeted therapies could be of interest for the causal treatment of the pathology.

Biofilm Recruitment of Vibrio cholerae by Matrix Proteolysis

The appearance of bacterial biofilms involves secretion of polysaccharides and proteins that form an extracellular matrix embedding the bacteria. Proteases have also been observed, but their role has remained unclear. Smith and co-workers have now found that proteolysis can contribute to further recruitment of bacteria to Vibrio cholerae biofilms.

The Foreign Body Giant Cell Cannot Resorb Bone, But Dissolves Hydroxyapatite Like Osteoclasts.

Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones—cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K.

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.

HIV-1 reprograms the migration of macrophages

AbstractMacrophages are motile leukocytes, targeted by HIV-1, thought to play a critical role in host dissemination of the virus. However, whether infection impacts their migration capacity remains unknown. We show that 2-dimensional migration and the 3-dimensional (3D) amoeboid migration mode of HIV-1–infected human monocyte-derived macrophages were inhibited, whereas the 3D mesenchymal migration was enhanced. The viral protein Nef was necessary and sufficient for all HIV-1–mediated effects on migration. In Nef transgenic mice, tissue infiltration of macrophages was increased in a tumor model and in several tissues at steady state, suggesting a dominant role for mesenchymal migration in vivo. The mesenchymal motility involves matrix proteolysis and podosomes, cell structures constitutive of monocyte-derived cells. Focusing on the mechanisms used by HIV-1 Nef to control the mesenchymal migration, we show that the stability, size, and proteolytic function of podosomes are increased via the phagocyte-specific kinase Hck and Wiskott-Aldrich syndrome protein (WASP), 2 major regulators of podosomes. In conclusion, HIV-1 reprograms macrophage migration, which likely explains macrophage accumulation in several patient tissues, which is a key step for virus spreading and pathogenesis. Moreover, Nef points out podosomes and the Hck/WASP signaling pathway as good candidates to control tissue infiltration of macrophages, a detrimental phenomenon in several diseases.

Fibronectin remodelling: cell-mediated regulation of the microenvironment.

The biophysical, mechanical and chemical characteristics of extracellular matrixes influence many cellular functions to control tissue homoeostasis and drive progression of cancer and inflammatory diseases. To maintain normal tissue function, fibronectin-rich matrixes are subject to dynamic cell-mediated structural and chemical modification. In this article, we discuss how localized application of mechanical force, heterodimer-specific integrin engagement and matrix proteolysis regulate fibronectin assembly and turnover. We also speculate that recently identified integrin trafficking, syndecan signalling and adhesion receptor-growth factor receptor cross-talk mechanisms might dynamically control the function, assembly and mechanical properties of a viable, and mechanoresponsive, fibronectin network.

Differential expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in avian tibial dyschondroplasia

Tibial dyschondroplasia (TD) is an avian bone disorder of different aetiologies that may be associated with lameness. The disorder is characterized by focal disruption of endochondral bone formation, with a lack of matrix proteolysis and an accumulation of non-mineralized avascular cartilage. The aim of this study was to determine the expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in normal, thiram-induced TD lesions and in the process of recovery from TD in broiler chickens. An extracellular matrix (ECM) degrading enzyme, matrix metalloproteinase-9 (MMP-9), was selected to investigate the effects of CD147 in the degradation of ECM. Gene expression was analysed by quantitative real-time polymerase chain reaction and protein levels by immunohistochemistry and western blotting. The birds were divided into three groups: thiram fed; recovery; and controls. Genes encoding CD147 and MMP-9 were down-regulated during the development of the disease, and were up-regulated during recovery. Western blotting also showed lower protein levels of CD147 in TD, which increased during the recovery phase associated with ECM degradation and growth plate repair. The findings of this study suggest that ECM has a crucial role in the occurrence of TD and that CD147 appears to play a pivotal role in matrix proteolysis in the chicken, similar to that in other species.

Plasminogen activator inhibitor-1 inhibits angiogenic signaling by uncoupling vascular endothelial growth factor receptor-2-αVβ3 integrin cross talk.

Plasminogen activator inhibitor-1 (PAI-1) regulates angiogenesis via effects on extracellular matrix proteolysis and cell adhesion. However, no previous study has implicated PAI-1 in controlling vascular endothelial growth factor (VEGF) signaling. We tested the hypothesis that PAI-1 downregulates VEGF receptor-2 (VEGFR-2) activation by inhibiting a vitronectin-dependent cooperative binding interaction between VEGFR-2 and αVβ3. We studied effects of PAI-1 on VEGF signaling in human umbilical vein endothelial cells. PAI-1 inhibited VEGF-induced phosphorylation of VEGFR-2 in human umbilical vein endothelial cells grown on vitronectin, but not on fibronectin or collagen. PAI-1 inhibited the binding of VEGFR-2 to β3 integrin, VEGFR-2 endocytosis, and intracellular signaling pathways downstream of VEGFR-2. The anti-VEGF effect of PAI-1 was mediated by 2 distinct pathways, one requiring binding to vitronectin and another requiring binding to very low-density lipoprotein receptor. PAI-1 inhibited VEGF-induced angiogenesis in vitro and in vivo, and pharmacological inhibition of PAI-1 promoted collateral arteriole development and recovery of hindlimb perfusion after femoral artery interruption. PAI-1 inhibits activation of VEGFR-2 by VEGF by disrupting a vitronectin-dependent proangiogenic binding interaction involving αVβ3 and VEGFR-2. These results broaden our understanding of the roles of PAI-1, vitronectin, and endocytic receptors in regulating VEGFR-2 activation and suggest novel therapeutic strategies for regulating VEGF signaling.

HGF-Met Pathway in Regeneration and Drug Discovery.

Hepatocyte growth factor (HGF) is composed of an α-chain and a β-chain, and these chains contain four kringle domains and a serine protease-like structure, respectively. Activation of the HGF–Met pathway evokes dynamic biological responses that support morphogenesis (e.g., epithelial tubulogenesis), regeneration, and the survival of cells and tissues. Characterizations of conditional Met knockout mice have indicated that the HGF–Met pathway plays important roles in regeneration, protection, and homeostasis in various cells and tissues, which includes hepatocytes, renal tubular cells, and neurons. Preclinical studies designed to address the therapeutic significance of HGF have been performed on injury/disease models, including acute tissue injury, chronic fibrosis, and cardiovascular and neurodegenerative diseases. The promotion of cell growth, survival, migration, and morphogenesis that is associated with extracellular matrix proteolysis are the biological activities that underlie the therapeutic actions of HGF. Recombinant HGF protein and the expression vectors for HGF are biological drug candidates for the treatment of patients with diseases and injuries that are associated with impaired tissue function. The intravenous/systemic administration of recombinant HGF protein has been well tolerated in phase I/II clinical trials. The phase-I and phase-I/II clinical trials of the intrathecal administration of HGF protein for the treatment of patients with amyotrophic lateral sclerosis and spinal cord injury, respectively, are ongoing.

Stimulators of mineralization limit the invasive phenotype of human osteosarcoma cells by a mechanism involving impaired invadopodia formation.

BackgroundOsteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis).ResultsIn this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization.ConclusionsOur study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma.

Abstract 3149: Targeting ERBB2-induced, lysosome-mediated invasion

Abstract ErbB2 overexpression is strongly associated with invasive cancer with high recurrence and poor prognosis. Similarly, increased expression of lysosomal proteases, cysteine cathepsins, is observed in several cancers and correlates with enhanced angiogenesis, invasion and metastasis. We recently showed that ErbB2 level correlates positively with cysteine cathepsin B and L levels in primary breast cancer and that ErbB2 regulates the expression of cathepsins B and L leading to their increased activity in several ErbB2-positive breast cancer cell lines. The increased cysteine cathepsin activity is essential for the invasion of ErbB2 overexpressing breast cancer cells in 3-dimensional (3D) Matrigel cultures and is mediated by novel signaling network that involves TGFβR, ERK2-MAPK, PAK4, PKCα and CDC42bpβ kinases and two transcription factors, MZF1 and ETS1. With a pharmacological protein kinase inhibitor screen, using the ErbB2 inhibitor Lapatinib as positive control, we have now identified three inhibitors putatively targeting this novel ErbB2-induced signaling network. Our data shows that the treatment of ErbB2-positive, highly invasive breast or ovarian cancer cells with any of these inhibitors specifically reduces cysteine cathepsin expression, activity and invasion of these cancer cells in 3D Matrigel cultures. It also reverses lysosomal distribution from the invasion supporting pericellular position to less-invasive perinuclear position, where the lysosomal exocytosis and secretion of lysosome contents to extracellular space to assist extracellular matrix proteolysis becomes less likely. In this study we have identified novel inhibitors for ErbB2-induced invasion and discovered a novel, function for clinically used ErbB2 inhibitor Lapatinib in the regulation of lysosomal function and trafficking. Citation Format: Tuula Kallunki, Ditte M. Brix, Bo Rafn, Knut Kristoffer Bundgaard Clemmensen, Sofie Hagel Andersen, Noona Ambartsumian, Marja Jäättelä. Targeting ERBB2-induced, lysosome-mediated invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3149. doi:10.1158/1538-7445.AM2014-3149

Regulation of Fibulin-2 Gene Expression by Integrin α3β1 Contributes to the Invasive Phenotype of Transformed Keratinocytes

The laminin-binding integrin α3β1 is highly expressed in epidermal keratinocytes where it regulates both cell-autonomous and paracrine functions that promote wound healing and skin tumorigenesis. However, roles for α3β1 in regulating gene expression programs that control the behaviors of immortalized or transformed keratinocytes remain underexplored. In the current study, we used a microarray approach to identify genes that are regulated by α3β1 in immortalized keratinocytes. α3β1-responsive genes included several that are involved in extracellular matrix proteolysis or remodeling, including fibulin-2 and SPARC. However, α3β1-dependent induction of specific target genes was influenced by the genetic lesion that triggered immortalization, as α3β1-dependent fibulin-2 expression occurred in cells immortalized by either SV40 large T antigen or p53-null mutation, while α3β1-dependent SPARC expression occurred only in the former cells. Interestingly, qPCR arrays did not reveal strong patterns of α3β1-dependent gene expression in freshly isolated primary keratinocytes, suggesting that this regulation is acquired during immortalization. p53-null keratinocytes transformed with oncogenic RasV12 retained α3β1-dependent fibulin-2 expression, and RNAi-mediated knockdown of fibulin-2 in these cells reduced invasion, although not their tumorigenic potential. These findings demonstrate a prominent role for α3β1 in immortalized/transformed keratinocytes in regulating fibulin-2 and other genes that promote matrix remodeling and invasion.

E-cadherin loss alters cytoskeletal organization and adhesion in non-malignant breast cells but is insufficient to induce an epithelial-mesenchymal transition.

BackgroundE-cadherin is an adherens junction protein that forms homophilic intercellular contacts in epithelial cells while also interacting with the intracellular cytoskeletal networks. It has roles including establishment and maintenance of cell polarity, differentiation, migration and signalling in cell proliferation pathways. Its downregulation is commonly observed in epithelial tumours and is a hallmark of the epithelial to mesenchymal transition (EMT).MethodsTo improve our understanding of how E-cadherin loss contributes to tumorigenicity, we investigated the impact of its elimination from the non-tumorigenic breast cell line MCF10A. We performed cell-based assays and whole genome RNAseq to characterize an isogenic MCF10A cell line that is devoid of CDH1 expression due to an engineered homozygous 4 bp deletion in CDH1 exon 11.ResultsThe E-cadherin-deficient line, MCF10A CDH1-/- showed subtle morphological changes, weaker cell-substrate adhesion, delayed migration, but retained cell-cell contact, contact growth inhibition and anchorage-dependent growth. Within the cytoskeleton, the apical microtubule network in the CDH1-deficient cells lacked the radial pattern of organization present in the MCF10A cells and F-actin formed thicker, more numerous stress fibres in the basal part of the cell. Whole genome RNAseq identified compensatory changes in the genes involved in cell-cell adhesion while genes involved in cell-substrate adhesion, notably ITGA1, COL8A1, COL4A2 and COL12A1, were significantly downregulated. Key EMT markers including CDH2, FN1, VIM and VTN were not upregulated although increased expression of proteolytic matrix metalloprotease and kallikrein genes was observed.ConclusionsOverall, our results demonstrated that E-cadherin loss alone was insufficient to induce an EMT or enhance transforming potential in the non-tumorigenic MCF10A cells but was associated with broad transcriptional changes associated with tissue remodelling.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-552) contains supplementary material, which is available to authorized users.

Actin-associated protein palladin promotes tumor cell invasion by linking extracellular matrix degradation to cell cytoskeleton.

Basal-like breast carcinomas, characterized by unfavorable prognosis and frequent metastases, are associated with epithelial-to-mesenchymal transition. During this process, cancer cells undergo cytoskeletal reorganization and up-regulate membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP14), which functions in actin-based pseudopods to drive invasion by extracellular matrix degradation. However, the mechanisms that couple matrix proteolysis to the actin cytoskeleton in cell invasion have remained unclear. On the basis of a yeast two-hybrid screen for the MT1-MMP cytoplasmic tail-binding proteins, we identify here a novel Src-regulated protein interaction between the dynamic cytoskeletal scaffold protein palladin and MT1-MMP. These proteins were coexpressed in invasive human basal-like breast carcinomas and corresponding cell lines, where they were associated in the same matrix contacting and degrading membrane complexes. The silencing and overexpression of the 90-kDa palladin isoform revealed the functional importance of the interaction with MT1-MMP in pericellular matrix degradation and mesenchymal tumor cell invasion, whereas in MT1-MMP-negative cells, palladin overexpression was insufficient for invasion. Moreover, this invasion was inhibited in a dominant-negative manner by an immunoglobulin domain-containing palladin fragment lacking the dynamic scaffold and Src-binding domains. These results identify a novel protein interaction that links matrix degradation to cytoskeletal dynamics and migration signaling in mesenchymal cell invasion.

Tuberculosis, Pulmonary Cavitation, and Matrix Metalloproteinases

Tuberculosis (TB), a chronic infectious disease of global importance, is facing the emergence of drug-resistant strains with few new drugs to treat the infection. Pulmonary cavitation, the hallmark of established disease, is associated with very high bacillary burden. Cavitation may lead to delayed sputum culture conversion, emergence of drug resistance, and transmission of the infection. The host immunological reaction to Mycobacterium tuberculosis is implicated in driving the development of TB cavities. TB is characterized by a matrix-degrading phenotype in which the activity of proteolytic matrix metalloproteinases (MMPs) is relatively unopposed by the specific tissue inhibitors of metalloproteinases. Proteases, in particular MMPs, secreted from monocyte-derived cells, neutrophils, and stromal cells, are involved in both cell recruitment and tissue damage and may cause cavitation. MMP activity is augmented by proinflammatory chemokines and cytokines, is tightly regulated by complex signaling paths, and causes matrix destruction. MMP concentrations are elevated in human TB and are closely associated with clinical and radiological markers of lung tissue destruction. Immunomodulatory therapies targeting MMPs in preclinical and clinical trials are potential adjuncts to TB treatment. Strategies targeting patients with cavitary TB have the potential to improve cure rates and reduce disease transmission.

Connective Tissue Growth Factor Inhibition Attenuates Left Ventricular Remodeling and Dysfunction in Pressure Overload–Induced Heart Failure

Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.

0.1 kilopascal difference for mechanophenotyping: soft matrix precisely regulates cellular architecture for invasion.

Current knowledge understands the mesenchymal cell invasion in a 3D matrix as a combined process of cell-to-matrix adhesion based cell migration and matrix remodeling. Excluding cell invasion stimulated by cytokines and chemokines, the basal cell invasion itself is a complicated process that can be regulated by matrix ligand type, density, geometry, and stiffness, etc. Understanding such a complicated biological process requires delicate dissections into simplified model studies by altering only one or two elements at a time. Past cell motility studies focusing on matrix stiffness have revealed that a stiffer matrix promotes 2D X-Y axis lateral cell motility. Here, we comment on two recent studies that report, instead of stiffer matrix, a softer matrix promotes matrix proteolysis and the formation of invadosome-like protrusions (ILPs) along the 3D Z axis. These studies also reveal that soft matrix precisely regulates such ILPs formation in the stiffness scale range of 0.1 kilopascal in normal cells. In contrast, malignant cells such as cancer cells can form ILPs in response to a much wider range of matrix stiffness. Further, different cancer cells respond to their own favorable range of matrix stiffness to spontaneously form ILPs. Thus, we hereby propose the idea of utilizing the matrix stiffness to precisely regulate ILP formation as a mechanophenotyping tool for cancer metastasis prediction and pathological diagnosis.

Role of Fas and Treg Cells in Fracture Healing as Characterized in the Fas-Deficient (lpr) Mouse Model of Lupus

Previous studies showed that loss of tumor necrosis factor α (TNFα) signaling delayed fracture healing by delaying chondrocyte apoptosis and cartilage resorption. Mechanistic studies showed that TNFα induced Fas expression within chondrocytes; however, the degree to which chondrocyte apoptosis is mediated by TNFα alone or dependent on the induction of Fas is unclear. This question was addressed by assessing fracture healing in Fas-deficient B6.MRL/Faslpr/J mice. Loss of Fas delayed cartilage resorption but also lowered bone fraction in the calluses. The reduced bone fraction was related to elevated rates of coupled bone turnover in the B6.MRL/Faslpr/J calluses, as evidenced by higher osteoclast numbers and increased osteogenesis. Analysis of the apoptotic marker caspase 3 showed fewer positive chondrocytes and osteoclasts in calluses of B6.MRL/Faslpr/J mice. To determine if an active autoimmune state contributed to increased bone turnover, the levels of activated T cells and Treg cells were assessed. B6.MRL/Faslpr/J mice had elevated Treg cells in both spleens and bones of B6.MRL/Faslpr/J but decreased percentage of activated T cells in bone tissues. Fracture led to ∼30% to 60% systemic increase in Treg cells in both wild-type and B6.MRL/Faslpr/J bone tissues during the period of cartilage formation and resorption but either decreased (wild type) or left unchanged (B6.MRL/Faslpr/J) the numbers of activated T cells in bone. These results show that an active autoimmune state is inhibited during the period of cartilage resorption and suggest that iTreg cells play a functional role in this process. These data show that loss of Fas activity specifically in chondrocytes prolonged the life span of chondrocytes and that Fas synergized with TNFα signaling to mediate chondrocyte apoptosis. Conversely, loss of Fas systemically led to increased osteoclast numbers during later periods of fracture healing and increased osteogenesis. These findings suggest that retention of viable chondrocytes locally inhibits osteoclast activity or matrix proteolysis during cartilage resorption. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

UPA‐uPAR molecular complex is involved in cell signaling during neuronal migration and neuritogenesis

In the development of the central nervous system (CNS), neuronal migration and neuritogenesis are crucial processes for establishing functional neural circuits. This relies on the regulation exerted by several signaling molecules, which play important roles in axonal growth and guidance. The urokinase-type plasminogen activator (uPA)-in association with its receptor-triggers extracellular matrix proteolysis and other cellular processes through the activation of intracellular signaling pathways. Even though the uPA-uPAR complex is well characterized in nonneuronal systems, little is known about its signaling role during CNS development. In response to uPA, neuronal migration and neuritogenesis are promoted in a dose-dependent manner. After stimulation, uPAR interacts with α5- and β1-integrin subunits, which may constitute an αβ-heterodimer that acts as a uPA-uPAR coreceptor favoring the activation of multiple kinases. This interaction may be responsible for the uPA-promoted phosphorylation of focal adhesion kinase (FAK) and its relocation toward growth cones, triggering cytoskeletal reorganization which, in turn, induces morphological changes related to neuronal migration and neuritogenesis. uPA has a key role during CNS development. In association with its receptor, it orchestrates both proteolytic and nonproteolytic events that govern the proper formation of neural networks.