Patricia Rousselle

Substrate-specific Modulation of a Multisubstrate Proteinase C-TERMINAL PROCESSING OF FIBRILLAR PROCOLLAGENS IS THE ONLY BMP-1-DEPENDENT ACTIVITY TO BE ENHANCED BY PCPE-1

Members of the bone morphogenetic protein-1/tolloid (BMP-1/Tld) family of metalloproteinases, also known as procollagen C-proteinases (PCPs), control multiple biological events (including matrix assembly, cross-linking, cell adhesion/migration and pattern formation) through enzymatic processing of several extracellular substrates. PCP activities on fibrillar procollagens can be stimulated by another family of extracellular proteins, PCP enhancers (PCPE-1, PCPE-2), which lack intrinsic enzymatic activity. While PCPs have multiple substrates, the extent to which PCPEs is involved in the processing of proteins other than fibrillar procollagens is unknown. In the experiments reported here, PCPE-1 was found to have no effect on the in vitro BMP-1 processing of procollagen VII, the procollagen V N-propeptide, the laminin 5 γ2 chain, osteoglycin, prolysyl oxidase, or chordin. In contrast, PCPE-1 enhanced C-terminal processing of human fibrillar procollagen III but only when this substrate was in its native, disulfide-bonded conformation. Surprisingly, processing of procollagen III continued to be enhanced when essentially all the triple-helical region was removed. These and previous results (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869; Bernocco, S., Steiglitz, B. M., Svergun, D. I., Petoukhov, M. V., Ruggiero, F., Ricard-Blum, S., Ebel, C., Geourjon, C., Deleage, G., Font, B., Eichenberger, D., Greenspan, D. S., and Hulmes, D. J. S. (2003) J. Biol. Chem. 278, 7199-7205) indicate that the mechanism of PCPE-1 action involves recognition sites in both the C-propeptide domain and in the C-telopeptide region of the procollagen molecule. PCPEs therefore define a new class of extracellular adaptor proteins that stimulate proteinase activity in a substrate-specific manner, thereby providing a new target for the selective regulation of PCP activity on fibrillar procollagen substrates.

Laminin 332 processing impacts cellular behavior.

Laminin 332, composed of the α3, β3 and γ2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial-mesenchymal cohesion in tissues exposed to external forces, including skin and stratified squamous mucosa. After being secreted and deposited in the extracellular matrix, laminin 332 undergoes physiological maturation processes consisting in the proteolytic processing of domains located within the α3 and the γ2 chains. These maturation events are essential for laminin 332 integration into the basement membrane where it plays an important function in the nucleation and maintenance of anchoring structures. Studies in normal and pathological situations have revealed that laminin 332 can trigger distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and structural characteristics of laminin 332 domains are presented and we discuss whether they trigger specific functions.

β4 Integrin and Laminin 5 Are Aberrantly Expressed in Polycystic Kidney Disease: Role in Increased Cell Adhesion and Migration

Extracellular matrix alterations have been suggested to be part of the early events occurring in Autosomal Dominant Polycystic Kidney Disease (ADPKD), a disease characterized by formation of renal cysts and progressive renal failure. Here we report that cDNA array analysis identified β 4 integrin aberrant expression in ADPKD cells. Furthermore, laminin 5 (Ln-5), the main α 6 β 4 integrin ligand, was also found to be abnormally expressed in ADPKD. Studies performed with ADPKD cyst-lining epithelial cells (CC) by comparison with normal tubular cells indicate that integrin α 6 β 4 -Ln-5 interactions are involved in cellular events of potential importance for cystogenesis: 1) laminin 5 is a preferential adhesion substrate for CC, mainly through α 6 β 4 interaction, 2) CC increased haptotactic and chemotactic motility depends on the presence of Ln-5 and requires integrin α 3 β 1 cooperation, and 3) CC haptotactic or chemotactic migration is specifically increased by mAb-mediated β 4 integrin ligation, through an α 3 β 1 integrin-dependent and independent pathway, respectively. These results highlight the role of Ln-5 and α 6 β 4 integrin in adhesive and motility properties of cyst-lining epithelial cells, and further suggest that integrins and extracellular matrix modifications may be of general relevance to kidney epithelial cell cyst formation.

Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support.

To reveal the functional intrinsic niche of human embryonic stem cells (hESC) we examined the production of basement membrane (BM) proteins and the presence of their receptors in feeder‐free cell culture conditions. In addition, we investigated binding of hESCs to purified human BM proteins and identified the receptors mediating these contacts. Also, we tested whether purified human laminin (Lm) isoforms have a role in hESC self‐renewal and growth in short‐term cultures. The results show that hESCs synthesize Lm α1 and Lm α5 chains together with Lm β1 and γ1 chains suggesting the production of Lms‐111 and ‐511 into the culture medium and deposits on cells. hESCs contain functionally important integrin (Int) subunits, Int β1, α3, α6, α5, β5 and αV, as well as the Lm α5 receptor, Lutheran (Lu) glycoprotein and its truncated form, basal cell adhesion molecule (B‐CAM). In cell adhesion experiments, Int β1 was crucial for adhesion to most of the purified human BM proteins. Lu/B‐CAM mediated adhesion to Lm‐511 together with Int α3β1, and was essential for the adhesion of hESCs to embryonic feeder cells. Adhesion to Lm‐411 was mediated by Int α6β1. Lm‐511 supported hESC growth in defined medium equally well as Matrigel. These results provide consequential information of the biological role of BM in hESCs, warranting further investigation of BM biology of human pluripotent stem cells.

In vitro evaluation of an RGD-functionalized chitosan derivative for enhanced cell adhesion

Tissue repair is a spontaneous process that is initiated on wounding. However, if this complex mechanism is impaired or not sufficient the use of biomaterials might increase the chance of successful healing. In this view, an RGD-functionalized polymer was developed to promote dermal healing. A water-soluble chitosan derivative, carboxymethyl-trimethylchitosan (CM-TM-chitosan) was synthesized and GRGDS-moieties were grafted to the backbone at a concentration of 59 nmol/mg polymer to increase cell-biomaterial interaction. Tested in vitro with cultured human dermal fibroblasts, the developed polymer showed good biocompatibility and the initial adhesion was increased by 3-5 times due to the GRGDS-moieties. Moreover, cell spreading was specific to the interaction with GRGDS, giving a 12-fold increase of cells showing a fully spread morphology within 30 min. Overall, CM-TM-chitosan conjugated with GRGDS-peptides may prove useful as a biomaterial in wound healing.

Melanoma cells produce multiple laminin isoforms and strongly migrate on α5 laminin(s) via several integrin receptors.

Melanoma cells express and interact with laminins (LMs) and other basement membrane components during invasion and metastasis. In the present study we have investigated the production and migration-promoting activity of laminin isoforms in melanoma. Immunohistochemistry of melanoma specimens and immunoprecipitation/western blotting of melanoma cell lines indicated expression of laminin-111/121, laminin-211, laminin-411/421, and laminin-511/521. Laminin-332 was not detected. In functional assays, laminin-111, laminin-332, and laminin-511, but not laminin-211 and laminin-411, strongly promoted haptotactic cell migration either constitutively or following stimulation with insulin-like growth factors. Both placenta and recombinant laminin-511 preparations were highly active, and the isolated recombinant IVa domain of LMα5 also promoted cell migration. Function-blocking antibodies in cell migration assays revealed α6β1 integrin as the major receptor for laminin-111, and both α3β1 and α6β1 integrins for laminin-332 and laminin-511. In contrast, isolated LMα5 IVa domain-promoted melanoma cell migration was largely mediated via αVβ3 integrin and inhibited by RGD peptides. Given the ubiquitous expression of α5 laminins in melanoma cells and in melanoma-target tissues/anatomical structures, as well as the strong migration-promoting activity of these laminin isoforms, the α5 laminins emerge as putative primary extracellular matrix mediators of melanoma invasion and metastasis via α3β1 and other integrin receptors.

α1β1-integrin engagement to distinct laminin-1 domains orchestrates spreading, migration and survival of neural crest cells through independent signaling pathways

Integrin engagement regulates cell adhesion, shape, migration, growth, and differentiation, but molecular mechanisms coordinating these functions in cells remain unclear. Because of their migratory and differentiation potential, neural crest cells constitute a powerful paradigm to address this question. Here, we describe that laminin-1, a major component of their migration routes, promotes crest cell spreading, migration and survival through two distinct integrin-binding domains that are situated on both sides of its α1 subunit and can be separated in the LN-1 elastase proteolytic fragments E1 and E8. Interaction with either domain was mediated by the same integrin α1β1 but produced distinct, complementary responses through specific signaling cascades. FAK activation upon E8 binding induced spreading, formation of actin bundles and focal adhesions, stimulated oriented migration, but failed to support survival. Conversely, Erk activation upon E1 binding promoted long-term survival and random migration without actin reorganization. Consistent with this, interaction with laminin-5 or laminin-10/11, which do not harbor integrin-binding domains in the N-terminal side of their α chains, failed to support survival. Thus, the signaling activity and function of integrins might depend on binding domains in their ligands, thereby revealing ligand control of integrin function as a possible mechanism for the modulation and coordination of cell response to adhesive signals.

Cell surface proteoglycans syndecan-1 and -4 bind overlapping but distinct sites in laminin α3 LG45 protein domain.

Background: The cell surface proteoglycans syndecan-1 and -4 interact with laminin 332 to participate in keratinocyte migration. Results: Syndecan-1 and -4 bind specific residues in the laminin 332 LG45 domain. Conclusion: The LG45 domain encompasses a major heparan sulfate binding domain containing distinctive syndecan-1 and -4 binding sequences. Significance: Identifying syndecan-1- and -4-binding sites in laminin 332 is crucial for elucidation of their function in keratinocyte migration. Keratinocyte migration during epidermal repair depends on interactions between cellular heparan sulfate proteoglycan receptors, syndecan-1 and -4, and the C-terminal globular domains (LG45) of the extracellular matrix protein laminin 332. This study investigates the molecular basis of the binding specificity of the syndecan-1 and -4 receptors expressed by human keratinocytes. We used site-directed mutagenesis to alter a recombinant LG45 protein by substituting the most critical basic residues with glutamine. All proteins were expressed in mammalian cells, purified, and characterized biochemically. We used in vitro binding assays, including surface plasmon resonance, to examine interactions between mutated LG45 and heparan sulfates, syndecan-1 and -4. We identify a major heparin binding domain on the outer edge of a β-strand of LG45 surrounded by a track of converging low affinity residues. This domain harbors distinctive syndecan-1 and -4 binding-specific sequences. This is the first study to demonstrate a binding specificity of two proteoglycans produced by a single cell type. In addition, we found that although syndecan-1 interacts exclusively through its glycosaminoglycan chains, syndecan-4 binding relies on both its core protein and its heparan sulfate chains. These results suggest that LG45 may trigger different signals toward keratinocytes depending on its interaction with syndecan-1 or -4.

Processing of Laminin α Chains Generates Peptides Involved in Wound Healing and Host Defense

Laminins play a fundamental role in basement membrane architecture and function in human skin. The C-terminal laminin G domain-like (LG) modules of laminin α chains are modified by proteolysis to generate LG1-3 and secreted LG4-5 tandem modules. In this study, we provide evidence that skin-derived cells process and secrete biologically active peptides from the LG4-5 module of the laminin α3, α4 and α5 chain in vitro and in vivo. We show enhanced expression and processing of the LG4-5 module of laminin α3 in keratinocytes after infection and in chronic wounds in which the level of expression and further processing of the LG4-5 module correlated with the speed of wound healing. Furthermore, bacterial or host-derived proteases promote processing of laminin α3 LG4-5. On a functional level, we show that LG4-5-derived peptides play a role in wound healing. Moreover, we demonstrate that LG4-derived peptides from the α3, α4 and α5 chains have broad antimicrobial activity and possess strong chemotactic activity to mononuclear cells. Thus, the data strongly suggest a novel multifunctional role for laminin LG4-5-derived peptides in human skin and its involvement in physiological processes and pathological conditions such as inflammation, chronic wounds and skin infection.

Laminin isoforms of lymph nodes and predominant role of α5-laminin(s) in adhesion and migration of blood lymphocytes

During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin α3, α4, α5, β1, β2, and γ1 chains and that the same chains, in addition to α2, are found in RFs. In functional studies with laminin isoforms covering all Lm α chains, α5‐laminin (Lm‐511) was the most adhesion‐ and migration‐promoting isoform for human blood lymphocytes, followed by α3‐ (Lm‐332) and α4‐ (Lm‐411) laminins, and the lymphocytes used the α6β1 integrin as the primary receptor for the α5‐laminin. Moreover, Lm‐511 strongly costimulated T cell proliferation, and blood lymphocytes were able to secrete α4‐ and α5‐laminins following stimulation. The LN cell number in laminin α4‐deficient mice compared with wild‐type did not differ significantly. This study demonstrates a predominant role for α5‐laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.