Lung Inflammatory Phenotype in Mice Deficient in Fibulin-2 and ADAMTS-12.

Interaction between extracellular matrix (ECM) components plays an important role in the regulation of cellular behavior and hence in tissue function. Consequently, characterization of new interactions within ECM opens the possibility of studying not only the functional but also the pathological consequences derived from those interactions. We have previously described the interaction between fibulin2 and ADAMTS-12 in vitro and the effects of that interaction using cellular models of cancer. Now, we generate a mouse deficient in both ECM components and evaluate functional consequences of their absence using different cancer and inflammation murine models. The main findings indicate that mice deficient in both fibulin2 and ADAMTS12 markedly increase the development of lung tumors following intraperitoneal urethane injections. Moreover, inflammatory phenotype is exacerbated in the lung after LPS treatment as can be inferred from the accumulation of active immune cells in lung parenchyma. Overall, our results suggest that protective effects in cancer or inflammation shown by fibulin2 and ADAMTS12 as interactive partners in vitro are also shown in a more realistic in vivo context.

ADAMTS-12: Functions and Challenges for a Complex Metalloprotease.

Nineteen members of the ADAMTS family of secreted zinc metalloproteinases are present in the human degradome. A wide range of different functions are being attributed to these enzymes and the number of their known substrates is considerably increasing in recent years. ADAMTSs can participate in processes such as fertility, inflammation, arthritis, neuronal and behavioral disorders, as well as cancer. Since its first annotation in 2001, ADAMTS-12 has been described to participate in different processes displayed by members of this family of proteinases. In this sense, ADAMTS-12 performs essential roles in modulation and recovery from inflammatory processes such as colitis, endotoxic sepsis and pancreatitis. ADAMTS-12 has also been involved in cancer development acting either as a tumor suppressor or as a pro-tumoral agent. Furthermore, participation of ADAMTS-12 in arthritis or in neuronal disorders has also been suggested through degradation of components of the extracellular matrix. In addition, ADAMTS-12 proteinase activity can also be modified by interaction with other proteins and thus, can be an alternative way of modulating ADAMTS-12 functions. In this review we revised the most relevant findings about ADAMTS-12 function on the 20th anniversary of its identification.

Hyalectanase Activities by the ADAMTS Metalloproteases.

The hyalectan family is composed of the proteoglycans aggrecan, versican, brevican and neurocan. Hyalectans, also known as lecticans, are components of the extracellular matrix of different tissues and play essential roles in key biological processes including skeletal development, and they are related to the correct maintenance of the vascular and central nervous system. For instance, hyalectans participate in the organization of structures such as perineural nets and in the regulation of neurite outgrowth or brain recovery following a traumatic injury. The ADAMTS (A Disintegrin and Metalloprotease domains, with thrombospondin motifs) family consists of 19 secreted metalloproteases. These enzymes also perform important roles in the structural organization and function of the extracellular matrix through interactions with other matrix components or as a consequence of their catalytic activity. In this regard, some of their preferred substrates are the hyalectans. In fact, ADAMTSs cleave hyalectans not only as a mechanism for clearance or turnover of proteoglycans but also to generate bioactive fragments which display specific functions. In this article we review some of the physiological and pathological effects derived from cleavages of hyalectans mediated by ADAMTSs.

Identification of relevant genetic alterations in cancer using topological data analysis.

Large-scale cancer genomic studies enable the systematic identification of mutations that lead to the genesis and progression of tumors, uncovering the underlying molecular mechanisms and potential therapies. While some such mutations are recurrently found in many tumors, many others exist solely within a few samples, precluding detection by conventional recurrence-based statistical approaches. Integrated analysis of somatic mutations and RNA expression data across 12 tumor types reveals that mutations of cancer genes are usually accompanied by substantial changes in expression. We use topological data analysis to leverage this observation and uncover 38 elusive candidate cancer-associated genes, including inactivating mutations of the metalloproteinase ADAMTS12 in lung adenocarcinoma. We show that ADAMTS12-/- mice have a five-fold increase in the susceptibility to develop lung tumors, confirming the role of ADAMTS12 as a tumor suppressor gene. Our results demonstrate that data integration through topological techniques can increase our ability to identify previously unreported cancer-related alterations.

New Insights into ADAMTS Metalloproteases in the Central Nervous System.

Components of the extracellular matrix (ECM) are key players in regulating cellular functions throughout the whole organism. In fact, ECM components not only participate in tissue organization but also contribute to processes such as cellular maintenance, proliferation, and migration, as well as to support for various signaling pathways. In the central nervous system (CNS), proteoglycans of the lectican family, such as versican, aggrecan, brevican, and neurocan, are important constituents of the ECM. In recent years, members of this family have been found to be involved in the maintenance of CNS homeostasis and to participate directly in processes such as the organization of perineural nets, the regulation of brain plasticity, CNS development, brain injury repair, axonal guidance, and even the altering of synaptic responses. ADAMTSs are a family of "A disintegrin and metalloproteinase with thrombospondin motifs" proteins that have been found to be involved in a multitude of processes through the degradation of lecticans and other proteoglycans. Recently, alterations in ADAMTS expression and activity have been found to be involved in neuronal disorders such as stroke, neurodegeneration, schizophrenia, and even Alzheimer's disease, which in turn may suggest their potential use as therapeutic targets. Herein, we summarize the different roles of ADAMTSs in regulating CNS events through interactions and the degradation of ECM components (more specifically, the lectican family of proteoglycans).

Novel Associations Within the Tumor Microenvironment: Fibulins Meet ADAMTSs.

The maintenance of tissue homeostasis in any organism is a very complex and delicate process in which numerous factors intervene. Cellular homeostasis not only depends on intrinsic factors but also relies on external factors that compose the microenvironment or cellular niche. Thus, extracellular matrix (ECM) components play a very important role in maintaining cell survival and behavior, and alterations in the ECM composition can lead to different pathologies. Fibulins and ADAMTS metalloproteases play crucial roles in the upkeep and function of the ECM in different tissues. In fact, members of both of these families of secreted multidomain proteins can interact with numerous other ECM components and thus shape or regulate the molecular environment. Individual members of both families have been implicated in tumor-related processes by exhibiting either pro- or antitumor properties. Recent studies have shown both an important relation among members of both families and their participation in several pathologies, including cardiogenesis or cancer. In this review, we summarize the associations among fibulins and ADAMTSs and the effects elicited by those interactions on cellular behavior.

Antitumor Potential of Fibulin-5 in Breast Cancer Cells Depends on Its RGD Cell Adhesion Motif.

BACKGROUND/AIMS: Different components of the tumor microenvironment can be either tumor-promoting or tumor-suppressive agents depending on factors which are not fully understood. Fibulins are components of the extracellular matrix from different tissues and constitute a clear example of this dual function. In fact, fibulins may either support tumor growth or abolish progression of malignant cells depending on the crosstalk between tumor cells and their surrounding stroma through mechanisms that remain to be elucidated. Among all fibulins, fibulin-5 contains a particular structural hallmark which consists in the presence of a RGD motif within its architecture. Previous reports have highlighted the importance of the interaction of this motif with integrins, and not only in normal functions but also in a tumor context. METHODS: Site-Directed Mutagenesis technique was employed to introduce the change RGD to RGE (RGD-to-RGE) within Fbln5 cDNA sequence. Cell proliferation was measured using the MTT assay or by counting Ki-67 positive cell nuclei. Cell adhesion was analysed using culture plates coated with different extracellular matrix components. Cell invasion was evaluated using 24-well Matrigel-coated invasion chambers, and mammosphere formation was monitored using ultralow attachment culture plates. BALB/c mice were employed to induce subcutaneous tumors. RESULTS: The RGD-to-RGE change alters the capacity of breast cancer cells to adhere to different extracellular matrix proteins as well as to αvß3 and α5ß1 integrins, and promotes protumor effects using different cell-based assays. Moreover, 4T1 cells, a mouse breast cancer cell line model, shows an increased capacity to generate tumors when exogenously expresses fibulin-5 with a RGD-to-RGE change, and such capacity is similar to that shown for 4T1 cells with an interfered Fbln5 gene. CONCLUSION: These data highlight the importance of the RGD motif of fibulin-5 to induce antitumor effects and provide new insights into the involvement of fibulins in tumor processes.

Neurocan is a New Substrate for the ADAMTS12 Metalloprotease: Potential Implications in Neuropathies.

BACKGROUND/AIMS: The composition of the extracellular matrix (ECM) in the central nervous system (CNS) has several features that make it unique. For instance, it is remarkable for the presence of proteoglycans such as versican, brevican, and neurocan, some of which have been identified as substrates of different members of the ADAMTS family of secreted metalloproteases. Previous studies have associated ADAMTSs with the repair of the CNS, including recovery following degradation of glial scar tissue and the stimulation of axonal growth after brain injury. However, the involvement of ADAMTSs in diseases of the CNS is complex and not understood fully, and a current challenge is unraveling the precise roles of these metalloproteases in the brain. METHODS: ADAMTS12 and neurocan gene expression was examined by quantitative PCR. Western blot analysis was employed to detect ADAMTS12 and neurocan protein expression in cell lines, and immunostaining techniques were used to detect neurocan in mouse brain tissues. Neurocan cleavage using recombinant ADAMTS1, ADAMTS4, ADAMTS5, and ADAMTS12 metalloproteases was evaluated by western blotting. Cell adhesion and migration were assessed using uncoated culture dishes or dishes coated with Matrigel or ECM components. RESULTS: We identified neurocan as a novel component of brain ECM that can be cleaved by ADAMTS12. In addition, we showed that neurocan cleavage by ADAMTS12 altered the adhesive properties of the human neuroglioma H4 cell line. Moreover, immunohistochemical analysis of Adamts12-deficient mice revealed the significant accumulation of neurocan in the brain of neonatal mice. CONCLUSION: Overall, our results suggest that ADAMTS12 could be involved in the repair of the CNS through its ability to degrade neurocan. Moreover, it can be inferred that alterations in neurocan degradation processes could be associated with the pathogenesis of neurological disorders.

An altered gene expression profile in tyramine-exposed intestinal cell cultures supports the genotoxicity of this biogenic amine at dietary concentrations.

Tyramine, histamine and putrescine are the most commonly detected and most abundant biogenic amines (BA) in food. The consumption of food with high concentrations of these BA is discouraged by the main food safety agencies, but legal limits have only been set for histamine. The present work reports a transcriptomic investigation of the oncogenic potential of the above-mentioned BA, as assessed in the HT29 human intestinal epithelial cell line. Tyramine had a greater effect on the expression of genes involved in tumorigenesis than did histamine or putrescine. Since some of the genes that showed altered expression in tyramine-exposed cells are involved in DNA damage and repair, the effect of this BA on the expression of other genes involved in the DNA damage response was investigated. The results suggest that tyramine might be genotoxic for intestinal cells at concentrations easily found in BA-rich food. Moreover, a role in promoting intestinal cancer cannot be excluded.

Tissue-engineering approaches in pancreatic islet transplantation.

Pancreatic islet transplantation is a promising alternative to whole-pancreas transplantation as a treatment of type 1 diabetes mellitus. This technique has been extensively developed during the past few years, with the main purpose of minimizing the complications arising from the standard protocols used in organ transplantation. By using a variety of strategies used in tissue engineering and regenerative medicine, pancreatic islets have been successfully introduced in host patients with different outcomes in terms of islet survival and functionality, as well as the desired normoglycemic control. Here, we describe and discuss those strategies to transplant islets together with different scaffolds, in combination with various cell types and diffusible factors, and always with the aim of reducing host immune response and achieving islet survival, regardless of the site of transplantation.

Assessing the Influence of a Protease in Cell Migration Using the Barrier-Migration Assay.

Proteases play crucial roles in all steps of tumor progression including cancer cell migration. In fact, uncontrolled proteolytic activity could lead to the degradation of different components of the extracellular matrix which facilitates dissemination of tumor cells. However, numerous studies have revealed that proteases may also exert tumor-protective actions which could impede progression of malignant cells. Consequently, it is crucial to distinguish those situations in which proteases promote tumor growth from those in which exhibit tumor-suppressive effects. In this regard, analysis of the influence of a particular protease on the capacity of a cell line to migrate can be employed as an approach to better understand its involvement in tumorigenesis. Different experimental designs have been developed to investigate cell migration. Herein, we describe a barrier assay to monitor cell migration, which overcomes some disadvantages of traditional methods such as the Boyden chamber or the wound healing assays. The version of the barrier assay explained in this chapter allows to examine cell migration through the analysis of the closure of a premade 500 µm wound. This method also facilitates comparison between two different situations in a given cell line (i.e., gene up- or downregulation) in the same assay and under the same conditions. Additionally, migration can be monitored and measured using a time lapse microscope which facilitates further analysis through different softwares.

Cleavage of Fibulin-2 by the aggrecanases ADAMTS-4 and ADAMTS-5 contributes to the tumorigenic potential of breast cancer cells.

Fibulin-2 participates in the assembly of extracellular matrix components through interactions with multiple ligands and promotes contacts between cells and their surrounding environment. Consequently, identification of processes that could lead to an altered Fibulin-2 could have a major impact not only in the maintenance of tissue architecture and morphogenesis but also in pathological situations including cancer. Herein, we have investigated the ability of the secreted metalloproteases ADAMTS-4 and ADAMTS-5 to digest Fibulin-2. Using in vitro approaches and cultured breast cancer cell lines we demonstrate that Fibulin-2 is a better substrate for ADAMTS-5 than it is for ADAMTS-4. Moreover, Fibulin-2 degradation is associated to an enhancement of the invasive potential of T47D, MCF-7 and SK-BR-3 cells. We have also found that conditioned medium from MCF-7 cells that simultaneously overexpress Fibulin-2 and ADAMTS-5 significantly induced the migratory and invasive ability of normal breast fibroblasts using 3D collagen matrices. Immunohistochemical analysis highlights the close proximity or partial overlap of both Fibulin-2 and ADAMTS-5 in breast tumor samples. Additionally, proteolytic products derived from a potential degradation of Fibulin-2 by ADAMTS-5 were also identified in these samples. Finally, we also show that the cleavage of Fibulin-2 by ADAMTS-5 is counteracted by ADAMTS-12, a metalloprotease that interacts with Fibulin-2. Overall, our results provide direct evidence indicating that Fibulin-2 is a novel substrate of ADAMTS-5 and that this proteolysis could alter the cellular microenvironment affecting the balance between protumor and antitumor effects associated to both Fibulin-2 and the ADAMTSs metalloproteases.

Fibulin-5 downregulates Ki-67 and inhibits proliferation and invasion of breast cancer cells.

Fibulins not only function as molecular bridges within the cellular microenvironment but also influence cell behavior. Thus, fibulins may contribute to create a permissive microenvironment for tumor growth but can also stimulate different mechanisms that may impede tumor progression. This is the case with Fibulin-5, which has been shown to display both tumor-promoting and tumor-protective functions by mechanisms that are not totally defined. We show new evidence on the tumor-protective functions displayed by Fibulin-5 in MCF-7, T47D and MDA-MB-231 breast cancer cells including the inhibition of invasion and proliferation capacity and hampering the ability to form mammospheres. Reduction in the level of phosphorylation of Ser residues involved in the nuclear translocation of ß-catenin may underlie these antitumor effects. We also found that Fibulin-5 reduces the level of expression of Ki-67, a nuclear protein associated with cell proliferation. Moreover, reduction in Fibulin-5 expression corresponds to an increase of Ki-67 detection in breast tissue samples. Overall, our data provide new insights into the influence of Fibulin-5 to modify breast cancer cell behavior and contribute to better understand the connections between fibulins and cancer.

Role of Periostin in Adhesion and Migration of Bone Remodeling Cells.

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology.

Polyserase-1/TMPRSS9 induces pro-tumor effects in pancreatic cancer cells by activation of pro-uPA.

Polyserase-1/TMPRSS9 is a type II transmembrane serine protease showing a complex molecular architecture characterized by the presence of three tandem serine protease domains in its amino acid sequence. This protease is widely expressed in mouse and human tissues, however, its functional significance is unknown in both normal and pathological conditions. In the present study, we evaluated the possible role of polyserase-1 in cancer progression. First, we showed that polyserase-1 increased the invasive capacities of PANC-1 and SK-PC-3 pancreatic cancer cells. Moreover, the presence of polyserase-1 enhanced anchorage-independent growth and diminished the adhesion capability of PANC-1 cells to different extracellular matrix components. These effects were mediated by the efficient conversion of pro-uPA to active uPA and high phosphorylation levels of ERK detected in the PANC-1 cells expressing exogenous polyserase-1. Collectively, our data suggest that polyserase-1 may be involved in cancer progression and, similarly to what has been proposed for the closely related serine proteases matriptase and TMPRSS4, inhibition of TMPRSS9 activity may contribute to the inhibition of tumor growth.

Interaction between the ADAMTS-12 metalloprotease and fibulin-2 induces tumor-suppressive effects in breast cancer cells.

Balance between pro-tumor and anti-tumor effects may be affected by molecular interactions within tumor microenvironment. On this basis we searched for molecular partners of ADAMTS-12, a secreted metalloprotease that shows both oncogenic and tumor-suppressive effects. Using its spacer region as a bait in a yeast two-hybrid screen, we identified fibulin-2 as a potential ADAMTS-12-interacting protein. Fibulins are components of basement membranes and elastic matrix fibers in connective tissue. Besides this structural function, fibulins also play crucial roles in different biological events, including tumorigenesis. To examine the functional consequences of the ADAMTS-12/fibulin-2 interaction, we performed different in vitro assays using two breast cancer cell lines: the poorly invasive MCF-7 and the highly invasive MDA-MB-231. Overall our data indicate that this interaction promotes anti-tumor effects in breast cancer cells. To assess the in vivo relevance of this interaction, we induced tumors in nude mice using MCF-7 cells expressing both ADAMTS-12 and fibulin-2 that showed a remarkable growth deficiency. Additionally, we also found that ADAMTS-12 may elicit pro-tumor effects in the absence of fibulin-2. Immunohistochemical staining of breast cancer samples allowed the detection of both ADAMTS-12 and fibulin-2 in the connective tissue surrounding tumor area in less aggressive carcinomas. However, both proteins are hardly detected in more aggressive tumors. These data and survival analysis plots of breast cancer patients suggest that concomitant detection of ADAMTS-12 and fibulin-2 could be a good prognosis marker in breast cancer diagnosis.

Cooperation by fibroblasts and bone marrow-mesenchymal stem cells to improve pancreatic rat-to-mouse islet xenotransplantation.

Experimental and clinical experiences highlight the need to review some aspects of islet transplantation, especially with regard to site of grafting and control of the immune response. The subcutaneous space could be a good alternative to liver but its sparse vasculature is its main limitation. Induction of graft tolerance by using cells with immunoregulatory properties is a promising approach to avoid graft rejection. Both Fibroblasts and Mesenchymal Stem Cells (MSCs) have shown pro-angiogenic and immunomodulatory properties. Transplantation of islets into the subcutaneous space using plasma as scaffold and supplemented with fibroblasts and/or Bone Marrow-MSCs could be a promising strategy to achieve a functional extra-hepatic islet graft, without using immunosuppressive drugs. Xenogenic rat islets, autologous fibroblasts and/or allogenic BM-MSCs, were mixed with plasma, and coagulation was induced to constitute a Plasma-based Scaffold containing Islets (PSI), which was transplanted subcutaneously both in immunodeficient and immunocompetent diabetic mice. In immunodeficient diabetic mice, PSI itself allowed hyperglycemia reversion temporarily, but the presence of pro-angiogenic cells (fibroblasts or BM-MSCs) within PSI was necessary to improve graft re-vascularization and, thus, consistently maintain normoglycemia. In immunocompetent diabetic mice, only PSI containing BM-MSCs, but not those containing fibroblasts, normalized glycemia lasting up to one week after transplantation. Interestingly, when PSI contained both fibroblasts and BM-MSCs, the normoglycemia period showed an increase of 4-times with a physiological-like response in functional tests. Histology of immunocompetent mice showed an attenuation of the immune response in those grafts with BM-MSCs, which was improved by co-transplantation with fibroblasts, since they increased BM-MSC survival. In summary, fibroblasts and BM-MSCs showed similar pro-angiogenic properties in this model of islet xenotransplantation, whereas only BM-MSCs exerted an immunomodulatory effect, which was improved by the presence of fibroblasts. These results suggest that cooperation of different cell types with islets will be required to achieve a long-term functional graft.

Immune-dependent and independent antitumor activity of GM-CSF aberrantly expressed by mouse and human colorectal tumors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2) is a cytokine produced in the hematologic compartment that may enhance antitumor immune responses, mainly by activation of dendritic cells. Here, we show that more than one-third of human colorectal tumors exhibit aberrant DNA demethylation of the GM-CSF promoter and overexpress the cytokine. Mouse engraftment experiments with autologous and homologous colon tumors engineered to repress the ectopic secretion of GM-CSF revealed the tumor-secreted GM-CSF to have an immune-associated antitumor effect. Unexpectedly, an immune-independent antitumor effect was observed that depended on the ectopic expression of GM-CSF receptor subunits by tumors. Cancer cells expressing GM-CSF and its receptor did not develop into tumors when autografted into immunocompetent mice. Similarly, 100% of the patients with human colon tumors that overexpressed GM-CSF and its receptor subunits survived at least 5 years after diagnosis. These data suggest that expression of GM-CSF and its receptor subunits by colon tumors may be a useful marker for prognosis as well as for patient stratification in cancer immunotherapy.

ADAMTS-12 metalloprotease is necessary for normal inflammatory response.

ADAMTSs (a disintegrin and metalloprotease with thrombospondin domains) are a family of enzymes with both proteolytic and protein interaction functions, which have been implicated in distinct pathologies. In this work, we have investigated the putative role of ADAMTS-12 in inflammation by using a mouse model deficient in this metalloprotease. Control and mutant mice were subjected to different experimental conditions to induce colitis, endotoxic sepsis, and pancreatitis. We have observed that Adamts12-deficient mice exhibit more severe inflammation and a delayed recovery from these challenges compared with their wild-type littermates. These changes are accompanied by an increase in inflammatory markers including several cytokines, as assessed by microarray expression analysis and proteomic-based approaches. Interestingly, the clinical symptoms observed in Adamts12-deficient mice are also concomitant with an elevation in the number of neutrophils in affected tissues. Finally, isolation and in vitro culture of human neutrophils demonstrate that the presence of ADAMTS-12 induces neutrophil apoptosis. On the basis of these results, we propose that ADAMTS-12 is implicated in the inflammatory response by modulating normal neutrophil apoptosis.