Ladislas Robert by SINGER-POLIGNAC

Connective Tissues, phylogenetic considerations, origin of its concept, its progression from anatomy to cell and molecular biology

Ladislas Robert

The « tissue » concept was among the first important generalization – a metaphor – in medical-scientific literature. Metaphors always plaid an important role in the progress of Biology as stated by Evelyn Fox-Keller, epistemologist of Sciences at MIT [1]. It appears in the French medical literature, at least during the XVIII^th Century, as for intance in the tretease on chronic diseases by the Bordeu-s, a family of outsanding physicians in the Bearn Country, the south-west of France [2]. The Tissue concept found its full blown expansion in Medicine with the work of Xavier Bichat (1771-1802), used however before him, by the last attending physician of Queen Marie Antoinette, Felix Vicq d’Azyr (1748-1794)[3]. Bichat distinguished 22 sorts of Tissues, a restricted selection of these composing the organs, the only visible components of the body, as seen by the early anatomists [4, 5]. The macromolecular constituents of the « tissue cellulaire » as called in the French texts started to be recognized first by histological procedures from the last decades of the XIX^th Century, followed by their isolation and characterization all through the XX^th Century. Even in the 1950ies, when we started our experiments at the Paris Medical Faculty, the number of these constituents remained restricted to 3, collagen, elastin and acid mucopolysaccharides. « Tissue cellulaire » was progressively replaced by « subtance fondamentale » (ground substance), followed by Connective Tissues and Extracellular Matrix.

The phylogenetic origin of these tissues was also clarified dring the last Century. The « fibroblasts » appeared recognizably with Trichoplax adherens, a plaquozoan, close to but simpler than the sponges which exhibit several macromolecular components of connective tissues: collagens, fibronectin,with cell-matrix interactions [6, 7]. During the second half of the XX^th Century a large number of macromolecular constituents of the extracellular matrix were identified, belonging to four major clases, collagens, elastins, glycosaminoglycans – proteoglycans and structural or matrix glycoproteins. With the intense study of fibronectin started the ever increasing recognition of the dynamic nature of cell matrix interactions mediated by receptors, integrins, the elastin receptor and others. Progress in molecular genetics enables now teams specialized in this discipline to complete the description of connective tissues by the identification of sequential gene activation « programs » resulting in the fine tuning of macromolecular tissue composition and fonction. Started also during the last decades, the study at the molecular – genetic level of pathological modifications of connective tissues and more recently their age-related alterations [8], as seen for instance in malignant growth [9]. No doubt that in the coming decades, the molecular and genetic regulations of connective tissue structure and fonctions will continue to make progresses.. From an essentially medical science it became a « basic discipline » with however constant cross-talk between « basic » and medical scientists. In these respect, our Society, as also those of other countries will continue to play a crucial role by bringing together scientists of subspecialities as for instance the Meetings of the Gordon Conferences, or those of the Federation of European Connective Tissue Societies (FECTS), founded also in Paris in 1967, to exchange, discuss and refine emerging concepts. To finish I wish to thank my friends, colleagues and my close family, my wife Jacqueline and my brother Alexandre who helped so much to transform into a living reality my dreams which emerged during my stay at the Biochemical Deprtment and Eye Institute of Columbia University, New York City, in 1961. My gratitude goes also to my friend, Prof. Y. Pouliquen, President of the Singer Polignac Foundation who generously helped us to orrganize this celebration.

• 1.Fox-Keller E. Refiguring Life – Metaphors of Twentieth-Century Biology . 1995. Columbia University Press
• 2.de Bordeu A, de Bordeu T, de Bordeu F. Recherches sur les maladies chroniques. Tome I, Ruault, Paris 1775
• 3.Labat-Robert J, Robert L, Pouliquen Y. 2011. Les tissus conjonctifs, de l’origine du concept à sa « mutation » en matrice extracellulaire. Application aux tissus oculaires. Contribution à l’histoire des sciences médicales. Pathol Biol. 59 : 183-186
• 4.Bichat X. Traité des membranes. 1800. Imprimerie de Marchant, Paris
• 5.Bichat X. Anatomie Générale appliquée à la physiologie et à la Médecine 1801. Imprimerie de Marchant, Paris
• 6.Garrone R. Phylogenesis of Connective Tissue. 1978. Frontiers of Matrix Biology, vol 5, Karger, Basel.
• 7.Labat-Robert J et al. A fibronectin-like protein in porifera: its role in cell aggregation. Proc. Natl. Acad. Sc. USA. 78 : 6261-6265
• 8.Robert L, Labat-Robert J. 1991. Euro. J. Gerontol. 2 : 82-91
• 9.Labat-Robert J, Robert L. 2007. The effect of cell-Matrix Interactions and Aging on the Malignant Process. 2007. Advances Cancer Res. 98 : 221-259

François-Xavier Maquart by SINGER-POLIGNAC

Fifty years of connective tissue research: from the French Connective Tissue Club to the French Society for Extracellular Matrix Biology

François-Xavier Maquart

The history of connective tissue research began in the late 18th century. However, it is only 50 years later that the concept of connective tissue has formed. It took another fifty years before that, in the first half of the 20th century, the first biochemical knowledge of extracellular matrix macromolecules began to appear. In 1962, thanks to Ladislas and Barbara Robert, back from the United States, the first society called « French Connective Tissue Club » was created in Paris. The first board was constitued of Albert Delaunay, Suzanne Bazin and Ladislas Robert. Very quickly, under the influence of these pioneers, national and international meetings were organized and, in 1967, a “Federation of the European Connective Tissue Clubs” was created at the initiative of Ladislas Robert (Paris) and John Scott (Manchester). It spread rapidly to the major European nations. In 1982 the transformation of « Clubs » in « Societies » occurred, a name more in line with the requirements of the time. In 2008, the « French Connective Tissue Society » became the « French Society of Extracellular Matrix Biology » (“Société Française de Biologie de la Matrice Extracellulaire”, SFBMEc), to better highlight the importance of the extracellular matrix in the biology of living organisms. The SFBMEc today’s mission is to promote and develop scientific exchanges between academic, industrial, and hospital laboratories involved in research on the extracellular matrix. The SFBMEc organizes or subsidizes scientific meetings and awards scholarships to PhD students or post-docs to participate in international conferences. It includes 200 to 250 members from different disciplines, developing strong interactions between scientists, clinicians and pathologists. It is present all around the French territory in many research laboratories. During these last 50 years, the extraordinary advances made possible by the development of new investigation techniques, in particular molecular biology, cell and tissue imaging, molecular modeling, etc…, have permitted a considerable increase of the knowledge in the field of connective tissue.

Laminins in the basement membrane of the skin

Monique Aumailley

Laminins are building blocks for polymers and networks of basement membranes. At least three laminins (LM), LM-311, LM-332 and LM-511, are present in the basement membrane of the skin. LM-511 form “classical” polymers, while LM-332 provides the core of a unique network anchoring the epidermis to the dermis. The N-terminus of LM-332 associates with other components of the network, such as collagens VII and XVII, while the C-terminus interacts with cell surface receptors, in particular the a3b1 and a6b4 integrins. Genetic studies in human and mice indicated that tissue-bound LM-332 and a6b4 integrins are involved in the anchorage of the epidermis to the underlying dermis. In contrast, the physiological relevance of b1 integrins, soluble LM-332 and other extracellular matrix components is not clear. To address these issues, we used either RNA interference to down-regulate LM-332, b1 integrins and b1 integrin-associated proteins in cultivated keratinocytes or a mouse model with an inducible, keratinocyte-restricted silencing of the Lama3 gene. Interestingly, silencing either LM-332, a3 integrin or preventing full activation of b1 integrins compromises intercellular adhesion and collective migration, while enhancing single cell migration. Mechanistically this is linked to a role of b1 integrins in maintaining integrity of the cortical actin network and targeting b4 integrins to cell surface sites important for cell-matrix adhesions. Moreover, the keratinocytes respond by activating repair/regeneration mechanisms, including up-regulation of fibronectin, collagen receptor and pro-inflammatory cytokines.

Françoise Rédini by SINGER-POLIGNAC

The bone microenvironment as new target for primary bone tumor therapy

Françoise Rédini

Primitive bone tumours, mainly osteosarcoma and Ewing’s sarcoma are the two most frequent bony tumors in children and adolescents. Current therapeutic strategies are based on tumor resection associated with highly toxic chemotherapy. Unfortunately, a recurrence associated with osteolytic processes as well as an absence of response to anti-tumour drugs are observed in many cases, leading to the development of metastases and ultimately to the death of the patients. Survival is closely related to the response of the tumor cells to anti-mitotic drugs, reaching 70% at 5 years for the localized forms and only 30% when pulmonary (osteosarcoma) or bone (Ewing’s sarcoma) metastases are detected at diagnosis. These data demonstrate that the response to the conventional treatment (chemotherapy) is disappointing, showing questionable benefit in terms of improvement in disease-free survival. Therefore new therapeutic approaches are needed, less toxic and better tolerated than chemotherapy in primary bone tumours.

As the activators of bone resorption and tumor growth are involved in a common vicious cycle, the modulation of bone resorption by cytokines or any other anti-bone resorption drug could interfere with the growth of the primary tumour in bone site. Indeed, Tumor cells produce osteoclast activating factors and cytokines (such as TNF-, IL-6…) that directly or indirectly via osteoblasts induce the up-regulation of the main cytokine regulating bone resorption, the Receptor Activator of NF-B Ligand (RANKL, TNFSF11). When activated, the osteoclasts degrade the mineralized bone matrix allowing the release of growth factors stored in the matrix (such as TGF-, IGF-1, FGFs..) which in turn activate tumor cell proliferation. In order to target both aspects of this so called “vicious cycle”, our laboratory develops new therapeutic approaches of osteosarcoma and Ewing’s sarcoma, and more particularly bi-therapies associating an anti-bone resorption agent to an anti-tumor one.

The therapeutic proof-of-concept has been validated with the combination of zoledronic acid (one of the most powerful bisphosphonate) with conventional chemotherapy in original animal models of primary bone tumors (osteosarcoma, Ewing’s sarcoma), in terms of prevention of tumor recurrence, bone formation and quality of tissue repair. These original results constituted the rationale for the current OS2006 phase III clinical trial developed in France for pediatric and adult patients. We also demonstrated the relevance of using osteoprotegerin (OPG), the decoy receptor of RANKL as a therapeutic tool delivered by gene transfer. However, the OPG bioavailability and biological activity is modulated by the presence of glycosaminoglycans in the bone tumor, demonstrating the importance of bone microenvironment in target therapies. These data emphasize the relevance of new therapeutic approaches of osteosarcoma and Ewing’s sarcoma that could be adjuvant, even alternative to chemotherapy.

Stéphane Dedieu by SINGER-POLIGNAC

The endocytic receptor LRP-1 as a versatile integrator of extracellular signals

Stéphane Dedieu, Benoît Langlois, Hervé Emonard, Laurent Martigny.

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a large multifunctional endocytic receptor mediating the clearance of various biological molecules from the extracellular matrix (ECM). In the field of cancer, LRP1-mediated endocytosis was first reported to decrease the accumulation of extracellular proteinases (MMPs, serine proteases) and to reduce the excessive remodeling of the ECM. LRP-1 was then widely associated to anti-tumor properties during the past decade and suggested as an attractive receptor for targeting the invasive behavior of malignant cells. However, the real functionalities connected to LRP-1 appear now much more complex and multifaceted. Using a long-term vector-based short hairpin RNA strategy against LRP-1, we indeed demonstrated that LRP-1 silencing prevents cell invasion, despite the high accumulation of proteolysis events associated to the tumor microenvironment. We highlighted that LRP1 contributes to cell attachment at the leading edge and cell detachment at the trailing edge to support cancer cell migration and invasion. In addition to its role in ligand binding and endocytosis, LRP-1 also regulates the focal adhesion disassembly, especially by targeting FAK and paxillin to focal complexes. The actin-cytoskeleton organization and dynamics of fast-invading tumor cells appeared also highly regulated by this cargo receptor. The following investigation revealed that LRP-1 β-chain constitutes a main docking site for focal adhesion components and mitogen-activated protein kinase-containing complexes. By regulating recruitment, activation and targeting of ERK and JNK to adhesion complexes in response to extracellular signals, LRP-1 maintains an adhesive state favorable for invasion. From our more recent data, we propose that LRP1 may also coordinate cell-matrix interactions in tumor cells by mediating the internalization of the hyaluronan receptor CD44 via the clathrin-coated vesicule pathway. Finally, a more complex model than that originally proposed emerges in which LRP-1 regulates the membrane proteome composition and dynamics and constitutes a key sensor of pericellular environment.

Role of adam12 and adamts1 in liver fibrosis: News actors in TGF-β activity

Nathalie Theret

Members of the ADAM (A Disintegrin And Metalloprotease) family share a multi-domain organization that includes metalloprotease, disintegrin, cystein, transmembrane and cytoplasmic domains. They have been implicated in highly diverse biological processes, such as spermatogenesis/fertilization, neurogenesis, inflammatory responses and cancer. Unlike the mammalian ADAMs which are transmembrane proteins, the ADAMTSs (A Disintegrin And Metalloprotease with ThromboSpondin motifs) and ADAMTSL (ADAMTS-like molecules that lack proteolytic activity) are secreted molecules, characterized by the presence of one or more thrombospondin type 1 repeats (TSR) and are associated with extracellular matrix components. Their activity is modulated by the matrix.

Extracellular matrix remodeling plays a pivotal role in liver fibrosis and is associated with increases in the synthesis of matrix components as well as proteases, mainly synthesized by the activated hepatic stellate cells (HSCs). A search for new matrix metalloproteases involved in matrix remodeling has led our group to identify ADAM12 and ADAMTS1 as new proteases associated with liver fibrosis and cancer. In order to understand the role of ADAM12 and ADAMTS1 in chronic liver disease, we investigated their protein interaction network using both high-throughput and integrative approaches. We show that ADAM12, expressed by activated HSCs, interacts 1) with the type II receptor of TGF-β to promote TGF-β transcriptional activity; 2) with RACK1 to migrate to the cell surface where it affects the Notch signaling pathway; 3) with ILK to regulate cell adhesion/survival through a PI3K/Akt signaling pathway. These new interactions fully integrate ADAM12 within the complex TGF-β signaling network that includes Smad- and non Smad- dépendent pathways, the latter being exacerbated in liver fibrosis and cancer.

Outside the cells, we recently identified ADAMTS1 as a new interactor of the latent form of TGF- β, LAPTGF-β. Our results show that the thrombospondin motif-containing domain from ADAMTS1 induces TGF-β activation through the interaction of the ADAMTS1 KTFR peptide with the LAP-TGF-β LKSL peptide. Down-regulation of ADAMTS1 in HSCs decreases the release of TGF-β competent for transcriptional activation and KTFR competitor peptides block HSC-mediated release of active TGF-β. Using a mouse liver fibrosis model, we show that CCl4 treatment induces ADAMTS1 expression in parallel to that of type I collagen. Importantly, concurrent injection of the KTFR peptide prevents liver damage.

Taken together our data suggest that the up-regulation of ADAM12 and ADAMTS1 in liver fibrosis results in the increase of the activity of the major profibrogenic cytokine, TGF-β, thereby contributing to the progress of disease. Interestingly, the protease activity of ADAM12 and ADAMTS1 is not implicated in these new functions, in which both proteins act as scaffolding proteins. Targeting ADAM12 and ADAMTS1 might constitute a new, alternate and highly specific therapeutic strategy for the treatment of chronic liver diseases.

References

• Atfi A, Dumont E, Colland F, Bonnier D, L’helgoualc’h A, Cl.ment B, Wewer U And Th.ret N. The disintegrin and metalloproteinase ADAM12 contributes to TGF-beta signaling through interaction with the Type II Receptor. J. Cell. Biol. 2007. 178(2): 201-8.
• Bourd-Boittin K, Le Pabic H, Bonnier D, L’helgoualc’h A And Th.ret N. Rack1, a new ADAM12 interacting protein; contribution to liver fibrogenesis. J. Biol.Chem. 2008. 283(38):26000-9.
• Bourd-Boittin K, Bonnier D, Leyme A, Mari B, Tuffery P, Samson M, Ezan F, Baffet G, Theret N.Protease profiling of liver fibrosis reveals the ADAM metallopeptidase with thrombospondin type 1 motif, 1 as a central activator of transforming growth factor beta.Hepatology. 2011 Dec;54(6):2173-84.
• Leyme A, Bourd-Boittin K, Bonnier D, Falconer A, Arlot-Bonnemains Y, Th.ret N. Identification of ILK as a new partner of the ADAM12 disintegrin and metalloprotease in cell adhesion and survival (submitted). Grant support : Institut National de la Santé et de la Recherche Médicale and Ligue Nationale Contre le Cancer.

Richard Hynes – 1 by SINGER-POLIGNAC

Richard Hynes – 2 by SINGER-POLIGNAC

Extracellular Matrix and Adhesion Receptors in Cancer

Richard O. Hynes

The extracellular matrix (ECM) is an important constituent of the microenvironment of tumours – it provides both mechanical and biochemical support for both the tumour cells themselves and for non-tumour cells in the stroma of the tumour. ECM provides signals that support survival, proliferation and migration, acting both through ECM receptors such as integrins and by presenting growth factors bound to the ECM. Evidence from classical pathology and from modern methods such as expression profiling have shown that significant changes occur in ECM during tumour progression. The presence of elaborate ECM such as desmoplastic stroma has long been used as an indicator of poor prognosis and further investigation of ECM changes should allow increased precision in such prognoses. However, the complexity and insolubility of ECM have rendered biochemical analyses of ECM composition in vivo a challenging task. We have developed methods for enriching ECM from samples of normal and tumour tissues and for analysis of proteolytic digests of these enriched ECM preparations by mass spectrometry. We have combined these experimental analyses with bioinformatic annotation of the “matrisome,” which we define as the universe of ECM proteins and associated proteins such as growth factors and ECM modifying enzymes (crosslinkers, proteases, etc.). This has allowed us to define the ECM composition of various normal tissues – in each case comprising 100-150 ECM proteins. Furthermore, we have been able to define in detail the changes that occur in ECM composition as tumours progress and metastasize and to assign ECM proteins as tumour-derived or stroma-derived. We are also able to apply these methods to human patient samples and we are attempting to define signatures that may predict whether tumours will metastasize and whether or not they will respond to chemotherapy.

The ECM proteins defined by these protocols as well as ECM receptors can be further analyzed for their causal involvement in cancer progression using mouse models of cancer.

Naba, A., Clauser, K.R., Hoersch, S., Liu, H., Carr, S.A. and Hynes, R.O. (2011). The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices. Mol. Cell Proteomics. Published online Dec. 9, 2011.

Hynes, R.O. and Naba, A. (2011). Overview of the Matrisome – an inventory of extracellular matrix constituents and functions. In Extracellular Matrix Biology (eds. RO Hynes and KM Yamada). Cold Spring Harbor Perspectives in Biology. doi: 10.1101/cshperspect.a004903. Published online September 21, 2011.

The Elastic Fiber and Elastin-related Diseases

Robert P. Mecham, Ph.D

The extracellular matrix protein elastin is found in tissues that undergo stretch and require elastic recoil. It appeared in evolution concurrent with the closed circulatory system and allowed the heart to evolve into an efficient multi-chambered pump. In a similar way, elastin enabled the vertebrate lung to become a more efficient gas exchange organ by allowing the respiratory apparatus to store potential energy created by contraction of the diaphragm during inhalation and use that energy to drive lung recoil during exhalation (1). Elastic fiber assembly occurs on the cell surface and in the extracellular matrix where tropoelastin, the soluble, secreted form of elastin, interacts with microfibrils, the fibulins, and lysyl oxidase to form the functional crosslinked elastic polymer.

Mutations in elastic fiber genes adversely affect multiple organ systems, which reflect the widespread distribution of these proteins. Mutations in fibrillin-1 and fibrillin-2, for example, have been linked to Marfan syndrome and congenital contractural arachnodactyly, respectively. While fibrillin-3 is not as well characterized as the other fibrillins, recent evidence suggests that fib-3 mutations are associated with polycystic ovary syndrome. Fibrillins covalently bind the large latent form of TGF and proBMPs. Characterization of pathological mechanisms underlying fibrillin-related diseases suggest that a major function of the microfibril is to regulate TGF growth factor activity. The concept now generally accepted from this work is that phenotypes associated with fibrillin mutations result from misregulated TGF signaling.

Fibrillin forms the structural core of microfibrils, but other proteins associate with fibrillin and modify microfibril function. The microfibril-associated glycoprotein 1 (MAGP-1) is a small molecular weight protein that binds to fibrillin and is considered to be a constitutive component of microfibrils in vertebrates. Like fibrillin, MAGP1 interacts with TGF but binds the active, not the latent, form of the growth factor. Mice lacking MAGP1 have an age-dependent osteopenia due to an increased number of osteoclasts, obesity, impaired wound healing, a bleeding defect, and monocytopenia, among others. In all of these phenotypes, changes in TGFβ and macrophage number or monocyte/macrophage differentiation have been implicated in the tissue changes that occur. No human diseases have yet been directly linked to MAGP1, but its gene resides in a region of chromosome 1 with QTLs for bone mineral density, COPD, and obesity—traits that are evident when MAGP1 is deleted in mice. Mutations in the elastin gene (ELN) generally fall into two disease classes. Loss of function mutations, such as premature stop mutations, large intragenic deletions, and complete gene deletion, lead to supravalvular aortic stenosis (SVAS), an autosomal dominant disease that predominantly affects the large elastic vessels in the vascular system. A second class of elastin mutation is linked to an autosomal dominant form of cutis laxa (ADCL) and arises from nucleotide deletion, insertion, or exon-splicing errors. These mutations produce missense sequence, usually in the 3’ end of the transcript that encodes the sequences important for fiber assembly and elastin function. Using a mouse expressing the human elastin gene, we showed that alternative splicing and tissue-specific elastin misassembly act as biological modifiers of ADCL disease pathogenesis. We also found that ADCL-mutant elastin is incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein are incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in ADCL.

Inflammation: directions from the matrix

Liliana Schaefer

There is growing evidence that small leucine-rich proteoglycans (SLRPs, e.g. biglycan and decorin) of the ECM, commonly thought to function exclusively as structural elements, act as signaling molecules. The identification of SLRP as endogenous ligands of Toll-like receptors (TLRs) provides a new concept how an inflammatory response is triggered by tissue stress and injury.

In Lupus Nephritis (LN), a prototypical autoimmune disease of the kidney, soluble biglycan triggers the expression of the B cell chemoattractant CXCL13 by signaling through TLR2 and TLR4 in interstitial macrophages and dendritic cells. Elevated tissue levels of CXCL13 subsequently lead to the recruitment of CXCR5-positive B cells (preferentially the B1 subset) to the kidney. Furthermore, biglycan induces the synthesis of RANTES, MCP-1, and MIP-1 in macrophages, thereby attracting T cells and additional macrophages. Thus, by bridging the innate and adaptive immune systems endogenous soluble biglycan enhances the inflammatory response reaction and thereby aggravates the course of LN and other B cell-mediated inflammatory disorders as well (e. g. acute renal allograft rejection).

The mechanisms linking immunity and inflammation with tumor development are not well understood. We could show that the soluble form of decorin controls inflammation and tumor growth via PDCD4 (programmed cell death protein 4) and miR-21 by a dual signaling mechanism. First, decorin acts as an endogenous ligand of TLR2/4 and stimulates the synthesis of proinflammatory PDCD4, TNFα, and IL-12 in macrophages. Second, decorin prevents the translational repression of PDCD4 by attenuating the activity of TGFβ1 and the expression of oncogenic miR-21, a translational inhibitor of PDCD4. Moreover, enhanced PDCD4 downregulates anti-inflammatory IL-10, thereby further driving the cytokine profile towards a proinflammatory phenotype. Importantly, these mechanisms appear to operate in a broad biological context linking pathogen-mediated with sterile inflammation, as shown here for sepsis and growth retardation of established tumor xenografts. In sepsis, decorin is an early response gene evoked by septic inflammation and is markedly elevated in plasma of septic patients and mice. In cancer, decorin-mediated inhibition of TGFβ1, oncogene miR-21 and IL-10 along with stimulation of PDCD4, TNFα, and IL-12 shifts the immune response to a more proinflammatory state, which inhibits tumor growth and therefore might be attractive as a novel antitumorigenic strategy.

Collagens : a fascinating family

Sylvie Ricard-Blum

The collagen family comprises 28 members in mammals and its diversity is increased by the use of alternative promoters for some collagen genes, alternative splicing (e.g. collagen XIII) and the existence of several molecular isoforms for some collagen types (e.g. collagens IV and V). Collagens are trimeric molecules. The collagens are divided into subgroups based on their supramolecular assemblies: fibril-forming collagens, FACITs (Fibril-Associated Collagens with Interrupted Triple helices), network-forming collagens, beaded filaments and anchoring fibrils. Collagen fibrils are macromolecular alloys made of several collagens and of proteoglycans at their surface. They are stabilized by covalent cross-linking mediated by enzymes of the lysyl oxidase family. These supramolecular assemblies play a structural role in the organization of the extracellular matrix and confer mechanical properties (e.g. resistance to traction) to the tissues. Although the vast majority of collagens are found in the extracellular matrix, four collagens (XIII, XVII, XXIII and XXV) are transmembrane proteins.

All the collagens contain at least one triple-helical domain (10 to 96% of the molecule length). The FACITs and the multiplexins (multiple triple-helix domains with interruptions) subfamilies contain several triple-helical domains interspersed with non-collagenous domains. Von Willebrand Factor A, fibronectin III and thrombospondin domains are some non-collagenous domains found in collagens. In addition to their structural roles, collagens modulate cell behavior (proliferation, migration, adhesion) via several receptor families (integrins, discoidin domain receptors, glycoprotein VI and Leukocyte-Associated Immunoglobulin-like Receptor-1). New functions such as wiring of the central nervous system have been recently reported for non-fibril forming collagens, some of them being expressed by neurons. Collagen XIII plays a role in the regulation of neuromuscular junction, several alpha chains of collagen IV are required for synapse maintenance and collagen XVIII regulates axon outgrowth. Furthermore, several collagens are a source of bioactive fragments called matricryptins, which exhibit anti-angiogenic and anti-tumoral properties after release from the parent collagen molecule by proteases.Since biological functions are mediated by molecular interactions, the identification of new interactions should predict additional functions for collagens. We developed protein and glycosaminoglycan arrays probed by surface plasmon resonance imaging (SPRi) to identify new partners of collagens. We built a database to store extracellular interaction data (MatrixDB, http://matrixdb.ibcp.fr/) and to build interaction networks of a tissue, a biological or a pathological process. Kinetics and affinity of the interactions were calculated by SPR, whereas binding sites were determined by site-directed mutagenesis or predicted by molecular modeling. The integration of all these parameters provided dynamics to the networks where interactions were prioritized according to affinity, and where simultaneous interactions were discriminated from the sequential ones according to the localization of binding sites. Protein domains and intrinsically disordered regions (able to interact with numerous partners due to their structural plasticity) were also mapped to the network. These approaches were applied to the study of the C-terminal fragment of collagen XVIII, endostatin, which is an anti-angiogenic matricryptin. They allowed us to predict new biological roles for endostatin in host-pathogen interactions, Alzheimer’s disease and innate immunity. Furthermore, this integrative approach, taking into account all the partners of endostatin and their interactions, provided new clues on the molecular mechanisms underlying its anti-angiogenic functions.

• Faye C, Chautard E, Olsen BR, Ricard-Blum S. The first draft of the endostatin interaction network. J Biol Chem 2009 284:22041-7.
• Chautard E, Fatoux-Ardore M, Ballut L, Thierry-Mieg N, Ricard-Blum S. MatrixDB, the extracellular matrix interaction database. Nucleic Acids Res 2011 39:D235-40.
• Ricard-Blum S. The collagen family. Cold Spring Harb Perspect Biol. 2011 3:a004978.
• Ricard-Blum S, Ballut L. Matricryptins derived from collagens and proteoglycans. Front Biosci 2011 16:674-97.
• Peysselon F, Xue B, Uversky VN, Ricard-Blum S. Intrinsic disorder of the extracellular matrix. Mol Biosyst 2011 7:3353-65.

Clair Baldock by SINGER-POLIGNAC

Structural Studies on BMP Regulators

Clair Baldock

The bone morphogenetic proteins (BMPs) are powerful growth factor signalling molecules in the extracellular matrix. BMPs play important roles at multiple stages of skeletal development, from early patterning to regulation of chondrocyte proliferation and maturation in the growth plate. Extracellular regulation of BMPs is essential for correct development and is of therapeutic interest in relation to a broad range of pathologies including cancer, vascular disease and arthritis. Large extracellular proteins form inhibitory complexes with BMPs, thereby preventing receptor activation but the molecular details of these complexes are unknown. One such regulator, chordin, acts by binding to BMPs thereby preventing their association with BMP receptors on the cell surface. The chordin-BMP inhibitory complex is cleaved by the tolloid family of proteinases which have important roles in tissue assembly and developmental patterning, two fundamental processes in mammalian biology. They also process a diverse range of extracellular protein precursors instigating assembly or activation, in addition to releasing growth factors from inhibitory complexes regulating signalling important for developmental processes such as dorsal-ventral patterning. Using a combination of single particle electron microscopy, small angle X-ray scattering and other biophysical measurements in solution, we have analysed the nanoscale structures of tolloid proteinase family members and their substrate chordin. We show that TLL-1 and mTLD, but not BMP-1, form calcium-ion dependent dimers under physiological conditions and based on a combination of structural and functional data, we propose that TLD activity is regulated by a substrate exclusion mechanism. We also present a model for chordin showing a horseshoe-like structure which may serve to bring the BMP-binding domains into close proximity for optimal growth factor binding. Additionally, chordin exists in a state of dynamic equilibrium between monomer and end-to-end dimer under physiological conditions. Together these results provide a mechanistic insight into BMP regulation by chordin and tolloids, and have broad implications for regulation of growth factor signaling and tissue assembly.

Daniela Quaglino by SINGER-POLIGNAC

Pseudoxanthoma elasticum: more than an elastic fiber disorder

Daniela Quaglino, Federica Boraldi and Giulia Annovi

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder mainly affecting skin, eyes and the cardiovascular system due to progressive mineralization of elastic fibres in the presence of normal levels of calcium and phosphorus in blood and urine. Because PXE is characterized by calcification of elastic fibres, genes involved in the synthesis and assembly of the elastic fibre network (namely elastin, elastin-associated microfibrillar proteins, lysyl oxidase) were initially considered as primary candidates for mutations. Failure in demonstrating their pathogenetic role led to the discovery that the ABCC6 gene (16p.13.1) is actually the main site of mutations in PXE. So far, more than 300 different mutations have been reported in ABCC6, a gene that encodes for a transmembrane protein (MRP6), whose physiological substrate is still elusive, although it has been suggested that it may be involved in the transport of complex conjugated molecules. In spite of the high level of ABCC6 expression, liver and kidney do not suffer from mutations in this gene. By contrast, tissues as skin, retina and vessel walls, which are deeply altered in PXE, express very low levels of MRP6.

These findings raised a still puzzling dilemma concerning the pathogenesis of PXE: how do mutations in a gene expressed primarily in the liver result in the mineralization of a certain number of elastic fibres in peculiar areas of the body? Furthermore, elastic fibers, given their almost absent turnover, can be regarded as a mirror of body homeostasis, thus reflecting changes in complex metabolic pathways and being the result of more general matrix alterations? From in vivo and in vitro studies, it has been shown that, although the dramatic involvement of the elastic component, other abnormalities characterize the PXE phenotype. Patients, for instance, exhibit altered levels of circulating factors such as proteoglycans, lipoproteins and mineralization inhibitors (i.e. fetuin-A and Matrix Gla Protein). Moreover, a number of plasma molecules have been shown to be modified by effects of an altered redox balance that, directly through and epigenetic regulation or indirectly due to the high amount of oxidised proteins and lipids, might have relevant consequences on soft connective tissue cells. Consistently, numerous in vitro studies undoubtedly highlighted the permanently modified phenotype of mesenchymal cells and the active role of these cells in the pathogenesis of ectopic calcifications.

Nevertheless, a clear understanding of PXE pathogenesis is further complicated by the fact that the age of disease onset and the expression of clinical symptoms are highly variable and marked phenotypic variations have been observed in affected siblings bearing the same ABCC6 mutation. Although there is no evidence of involvement of other genes in the pathogenesis of PXE, however, a number of modifying factors, both genetic and environmental, have been suggested to play a role in the phenotypic expression of the disease. Moreover, even though no other phenotypes are known to be associated with mutations in the ABCC6 gene, PXE-like clinical features, including aberrant mineralization of elastic fibres, have been reported in a number of apparently unrelated acquired and genetic clinical conditions.

Among the acquired conditions, PXE-like cutaneous changes may be associated with multiple pregnancy, in longstanding end-stage renal disease, L-tryptophan induced eosinophilia myalgia syndrome and amyloid elastosis as well as after D-penicillamine treatment, cutaneous exposure to calcium salts and salpeter. In these cases, mineralization of skin is the result of metabolic abnormalities affecting calcium and/or phosphate homeostasis or of direct deposition of mineral salts on collagen or elastic fibres.

Unexpectedly, PXE-like cutaneous changes have also been found in approximately 20% of patients with beta-thalassemia and sickle cell anaemia, that are well known severe congenital forms of anemia resulting from the deficient or altered synthesis of haemoglobin chains. In addition, the recent observation that a PXE-like phenotype can be observed in patients with pronounced deficiency of the vitamin K-dependent clotting factors raised the intriguing and exciting possibility that there might additional pathway/s, independent of ABCC6, leading to the PXE phenotype.

Therefore, Pseudoxanthoma elasticum represents a very interesting example for investigating the complexity that regulates molecular pathways and the influence of metabolism on the extracellular matrix of several organs/systems.

Data from the lab of the presenting Author result from research studies supported by PXE International, PXE Italia Onlus and Fondazione Cassa di Risparmio di Modena (EctoCal).

Frédéric Mallein-Gerin by SINGER-POLIGNAC

Novel strategies for cell therapy and tissue engineering of cartilage

Frédéric Mallein-Gerin

Articular cartilage is not vascularized and presents poor intrinsic healing potential. Consequently, traumatic and degenerative lesions of articular cartilage often progress to osteoarhtritis (OA), a leading source of disability. Joint replacement is a short term therapy since knee prostheses have limited lives. Besides, common surgical treatments are not satisfactory since often leading to the production of fibrocartilage which does not present the mechanical properties of articular cartilage. In this context, regeneration of cartilage requires tissue engineering techniques.

Autologous Chondrocyte Implantation (ACI) was the first application of cell therapy to orthopaedic surgery and is used worldwide. However, this method faces important issues: damage of healthy cartilage at the collection site and dedifferentiation of chondrocytes during cell amplification required for mass cell production. In addition, an arthrotomy (open knee surgery) is necessary to implant the cells in the lesion. Today, the health agencies that survey ACI in several countries report that the method should be improved by using soluble factors to control the chondrocyte phenotype and biomaterials to extend the method to larger defects such as developing OA lesions. In parallel, investigations are needed to evaluate the possibility of using stem cells as an alternative to autologous chondrocytes. The choice of soluble factors relies on the basic knowledge of their role in cartilage development and homeostasis. Bone morphogenetic protein (BMP)-2 is a member of the TGF-b superfamily which offers strong chondrogenic properties to human adult chondrocytes cultured in vitro, thus revealing its potential as thereapeutic agent for cartilage repair. Regarding the concept of Matrix-associated ACI (MACI), collagen based biomaterials are good candidates as scaffolds. The use of collagen sponges is advantageous since they have a pore structure that allows cell colonization, present low immunogenicity, have good mechanical stability and sufficient biodegradability and are easy to shape and calibrate. What is more, they can be grafted by arthroscopy and sealed with fibrin glue into the defect. Importantly, BMP-2 and collagen sponges have been approved for clinical use therefore they can be envisaged as tissue engineering package for human cartilage therapy. Regarding cells, works are in progress to determine if adult stem cells of diverse origins can be used as a substitute for autologous chondrocytes. Optimism seems justified based on the degree of chondrogenic conversion that we obtained recently with progenitor cells. Still, ethical and legal issues need to be considered before their clinical application.

Contribution of matrix metalloproteinases to hallmarks of cancer

Erik Maquoi, Benoit Detry, Charlotte Erpicum, Jenny Paupert, Silvia Blacher, Catherine Maillard and Agnès Noel

The interactions between cancer cells and their microenvironment represent a major event of tumor progression. Many carcinomas and predominately breast cancers are characterized by the formation of a reactive stroma associated with extensive type I collagen and fibrin deposition. By enmeshing cells in a dense fibrillar network, type I collagen acts as a physical barrier for cell migration. To develop metastatic capabilities, invading tumor cells must acquire the capacity to negotiate this remodeled microenvironment. In addition, infiltrating endothelial cells are also confronted to collagen barrier. How cancer cells and endothelial cells deal with interstitial collagen is not only decisive for cell migration, but also for cell survival and morphogenesis. We will discuss the mechanisms used by tumor cells to evade collagen-induced apoptosis and by lymphatic endothelial cells to deal with collagen barrier to form new lymphatics. Our recent findings are shedding light on the key contribution of interstitial collagenases during cancer progression and lymphangiogenesis.

Jean-Claude Monboisse by SINGER-POLIGNAC

Matrikines: a new anticancer therapeutic strategy

Jean Claude Monboisse

Tumor microenvironment is a complex system of a largely altered extracellular matrix with different cell types that influence the angiogenic response to a tumor and the local invasion of the tumor. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. The proteases degrade the stromal ECM and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to control tumor invasion and metastasis dissemination. We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV. The putative targets of the matrikine action are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. For example, canstatin, tumstatin and tetrastatin, respectively derived from the NC1 domains of α2, α3 and α4 chains of collagen IV, inhibit in vivo tumor growth in various experimental cancer models. Their anti-cancer activity comprises an anti-proliferative effect on tumor cells and on endothelial cells by induction of cell apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype.

Matrikines constitute a new family of potent anticancer agents that could be used under various therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogs designed from the structure of the active sequences. These matrikines could be used in combination with conventional chemotherapy or radiotherapy.

The pathogenic consequences of chondrocyte endoplasmic reticulum stress

Ray Boot-Handford

Several chondrodysplasias are caused by mutations in cartilage extracellular matrix (ECM) proteins. The mechanisms by which these mutations cause the associated pathology was until recently thought to involve either a deficiency in the extracellular matrix if the mutant protein was retained intracellularly, or alternatively, due to the assembly of a defect cartilage if secreted. Work from several laboratories has now demonstrated that the synthesis of mutant cartilage proteins is, in many instances, associated with increased endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). We have recently demonstrated in vivo using mouse models that the targeted induction of ER stress in hypertrophic chondrocytes is sufficient to phenocopy metaphyseal chondrodysplasia type Schmid (MCDS) demonstrating the central role played by increased ER stress in the disease mechanism for this chondrodysplasia (Rajpar et al. 2009). COMP and matrilin-3 mutations causing pseudoachondroplasia (PSACH) / multiple epiphyseal dysplasia (MED) are often associated with increased ER stress and a UPR in proliferative zone chondrocytes (Nundall et al. 2010 and papers therein). In order to determine the effects of increased ER stress in proliferative zone chondrocytes, we targeted the expression of an ER stress-inducing protein (Tgcog) to these cells using the collagen II promoter. Proliferative zone chondrocytes of mice carrying the Col2Tgcog transgenic construct expressed and retained the Tgcog protein and exhibited increased ER stress and a UPR based on elevated levels of the ER chaperone BiP (Grp78). Mice expressing the ER stress-inducing transgene exhibited significant decreases in bone growth rates compared to their non-stressed controls. The decreases in bone growth rate related to decreased rates of chondrocyte proliferation as assessed by BrdU incorporation. The Col2Tgcog mouse did not exhibit any abnormalities in apoptosis within the growth plate or in growth plate organisation. Microarray analyses are being performed to determine the effects of increased ER stress upon chondrocyte gene expression. These investigations illustrate that increased levels of ER stress in proliferative zone chondrocytes are sufficient to reduce growth bone growth rates but do not induce all of the phenotypic changes seen in the growth plate of MED mice expressing a matrilin-3 mutation such as dysregulated apoptosis and changes in cell shape accompanying defects in column formation. Initial attempts to pharmacologically reduce ER stress caused by the collagen X and matrillin-3 mutations in cellulo will also be described.

Rajpar et al. 2009, PLoS Genet 5(10): e1000691

Nundall et al. 2010, Cell Stress Chaperones15(6):835-49

Lysyl oxidase mediates colon cancer metastasis in the skeleton

Caroline Reynaud, Floriane Pez, Delphine Goehrig, Géraldine Aimond, Pascal Sommer and Philippe Clezardin.

Metastasis is a multistep process involving dynamic interactions between tumor cells and the surrounding extracellular matrix (ECM), both at the primary tumor site and distant sites. ECM proteins are also known to participate in the formation of premetastatic niches in distant organs. In this respect, lysyl oxidase (LOX) is an ECM protein that could play a critical role during tumor progression. It catalyzes the cross-linking of collagens and elastin in the extracellular compartment, thereby regulating the tensile strength of tissues.

We have recently shown that LOX overexpression fosters primary tumor growth in a model of colorectal carcinoma (Pez et al., Cancer Research 2011). However, the impact of LOX on colon cancer metastasis incidence was still poorly understood. Here, using lentiviral transduction, LOX was either stably overexpressed or silenced in three different luciferase-expressing human colon carcinoma cell lines. Transduced cancer cell lines were then inoculated intravenously to immunodeficient mice and the occurrence of metastasis was monitored by noninvasive bioluminescence imaging of animals. In addition, animals were analyzed by radiography for the presence of osteolytic lesions in the skeleton. LOX overexpression in colon cancer cells substantially enhanced bone metastasis formation in animals. Conversely, LOX silencing drastically reduced the formation of osteolytic regions. Furthermore, we demonstrate that circulating and active LOX is involved both in the nidation of tumor cells in the bone marrow and in bone metastasis progression. We also provide evidence that LOX, by enhancing adhesion to COL1, regulates the attachment and colonization of colon cancer cells to the skeleton. In conclusion, our results suggest that LOX plays a critical role in mediating bone colonization by colon cancer cells.

Hypoxia-driven angiogenesis and vascular extracellular matrix

Laurent Muller, Stéphane Germain, Catherine Monnot

Angiogenesis is a highly coordinated tissue remodeling process leading to the formation of a perfused network of blood vessels. Hypoxia triggers angiogenesis by promoting the expression of numerous genes, thereby modifying the balance between pro and anti-angiogenic factors. The upregulation of growth factors such as vascular endothelial growth factor (VEGF) leads to the angiogenic switch initiating endothelial sprouting. Activated endothelial cells acquire specialized functions: – tip cells located at the front of the growing vascular network extend numerous filopodia and lead migration toward the hypoxic microenvironment; – stalk cells proliferate and organize into luminized structures. During these cellular events, hypoxia regulates extracellular matrix composition, posttranslational modifications and assembly. The vascular extracellular microenvironnement constitutes a reservoir for growth factors and matricellular proteins and initiates biochemical and biomechanical cues essential for endothelial cell migration and capillary formation. Hypoxia-driven matrix scaffolding is thus dynamically regulated through modulation of extracellular matrix-modifying enzyme activities.

Using transcriptomic and proteomic analysis of human endothelial cells maintained in hypoxia, we have characterized the up-regulation of two extracellular matrix cross-linking enzymes, lysyl oxidase-like protein-2 (LOXL2) and transglutaminase 2 (TG2). Through different catalytic mechanisms, both enzyme activities are known to stabilize the vascular microenvironment and thus participate to blood vessel maturation. We could however show that they display opposite regulation of the angiogenic process: whereas LOXL2 stimulates capillary formation, TG2 limits endothelial sprouting. Investigating the mechanisms responsible for these effects demonstrated that LOXL2 is required for type IV collagen assembly by endothelial cells and that default of local stiffening of the microenvironment in the absence of LOXL2 could mediate capillary growth arrest. On the other hand, TG2 does not regulate extracellular matrix assembly by these cells but is involved in the modulation of VEGF signaling. These results thus highlight the fine-tuning of the angiogenic process by cross-linking enzymes. They also point out the major role of extracellular matrix scaffolding and protein interaction rather than cross-linking.

Bignon M, Pichol-Thievend C, Hardouin J, Malbouyres M, Bréchot N, Nasciutti L, Barret A, Teillon J, Guillon E, Etienne E, Caron M, Joubert-Caron R, Monnot C, Ruggiero F, Muller L, Germain S. Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane. Blood 2011;118(14):3979-89.

Germain S, Monnot C, Muller L, Eichmann A. Hypoxia-driven angiogenesis: role of tip cells and extracellular matrix scaffolding. Curr Opin Hematol. 2010;17(3):245-51.

Role of Lumican, a small leucine-rich proteoglycan, in the control of tumour progression

Stéphane Brézillon, François-Xavier Maquart, Yanusz Wegrowski

Lumican, is a member of a small leucine-rich proteoglycan family. It is the major leucine-rich keratan sulfate proteoglycan of numerous extracellular matrices, such as muscle, cartilage and cornea. In skin, lumican is present as a glycoprotein. As evidenced by knocking out of its gene in mouse, it plays a critical role in collagen fibrillogenesis. The lumican null mice (Lum-/-) were found to have serious functional defects including corneal opacity as well as skin and tendon fragility associated with disorganized and loosely packed collagen fibers (Chakravarti et al, 2003).

Our data have demonstrated a direct link between lumican expression and cancer progression (Vuillermoz et al, 2004, Brézillon et al, 2007, D’Onofrio et al, 2008) and metastasis (Brézillon et al, 2009). Lumican was shown to impede tumor cell migration by directly interacting with the alpha2beta1 integrin inducing remodeling of actin network and focal adhesion complexes (Brézillon et al, 2009, Zeltz et al, 2010).

In addition, we identified an active sequence of the lumican core protein responsible for the inhibition of melanoma cell migration (Zeltz et al, 2009). Using different recombinant and synthetic peptides derived from lumican, we localized an active site in the leucine-rich repeat 9 domain of the lumican core protein. We propose the name lumcorin (fragment of lumican core protein) for the active peptide derived from this site. Lumcorin was able to inhibit melanoma cell migration in vitro. The mechanism of action of lumcorin is an ongoing study in our laboratory.

Lumican was also shown to present angiostatic properties (Albig et al, 2007, Brézillon et al, 2009). Cell migration, including nascent vessel sprouting, is a complex process in which matrix metalloproteinases (MMPs) play an essential role. Our recent results provided strong evidence that lumican affects angiogenesis both by interfering with integrin alpha2beta1 receptor activity and down-regulating proteolytic activity associated with surface membranes of endothelial cells. In addition to blocking integrins, lumican affected expression of metalloproteinases such as MMP-14 and MMP-9. Furthermore, we showed that lumican blocked pro-angiogenic activity of endothelial cells to the same extent as siRNA to beta1 mRNA and produced synergistic effect when used together with anti-beta1 siRNA (Niewiarowska et al, 2011).

A review of the above results will be presented and the perspectives of our work will be discussed.

Ladislas Robert and Sylvie Ricard-Blum by SINGER-POLIGNAC