Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (y1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized. (C) 2002 Elsevier Science (USA)

Extracellular Matrix Protein Fibulin-2 Is Expressed in the Embryonic Endocardial Cushion Tissue and Is a Prominent Component of Valves in Adult Heart

AbstractFibulins-1 and -2 are two recently characterized extracellular matrix proteins of a novel protein family. They are found in many tissues in the adult stage, but several differences in their expression patterns were noted in cultured cells and adult tissues. Fibulin-1 is particularly well expressed in the endocardial cushion tissue (ECT) during embryogenesis and we therefore compared the expression patterns of fibulin-1 and -2 in the developing mouse heart. Antibodies against mouse fibulin-1 and -2 were used in immunofluorescence, and the expression of mRNAs were studied with synthetic oligonucleotides by in situ hybridization. A striking codistribution of fibulin-1 and -2 was seen at all stages of the development of the endocardial cushion tissue. Fibulin-2 was found to be a more specific marker for the embryonic ECT than fibulin-1, which was also transiently expressed in cardiac muscle. Immunofluorescence showed that the fibulins remained expressed in valves of the heart in the adult stage. The results suggest a role of the two fibulins in the development of the ECT and in the maintenance of the tensile strength of the cardiac valves

Opposing Roles of Integrin α6Aβ1 and Dystroglycan in Laminin-mediated Extracellular Signal-regulated Kinase Activation

Laminin–integrin interactions can in some settings activate the extracellular signal-regulated kinases (ERKs) but the control mechanisms are poorly understood. Herein, we studied ERK activation in response to two laminins isoforms (-1 and -10/11) in two epithelial cell lines. Both cell lines expressed β1-containing integrins and dystroglycan but lacked integrin α6β4. Antibody perturbation assays showed that both cell lines bound to laminin-10/11 via the α3β1and α6β1 integrins. Although laminin-10/11 was a stronger adhesion complex than laminin-1 for both cell lines, both laminins activated ERK in only one of the two cell lines. The ERK activation was mediated by integrin α6β1 and not by α3β1 or dystroglycan. Instead, we found that dystroglycan-binding domains of both laminin-1 and -10/11 suppressed integrin α6β1-mediated ERK activation. Moreover, the responding cell line expressed the two integrin α6 splice variants, α6A and α6B, whereas the nonresponding cell line expressed only α6B. Furthermore, ERK activation was seen in cells transfected with the integrin α6A subunit, but not in α6B-transfected cells. We conclude that laminin-1 and -10/11 share the ability to induce ERK activation, that this is regulated by integrin α6Aβ1, and suggest a novel role for dystroglycan-binding laminin domains as suppressors of this activation

Laminin-121-Recombinant expression and interactions with integrins

Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm Swarm tumor (EHS-laminin) but its T(m) value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6β1 and α7β1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the β2 laminins have higher affinity for integrins than the β1 laminins