Elsevier

Matrix Biology

Volume 21, Issue 7, November 2002, Pages 611-621
Matrix Biology

Evidence of nidogen-2 compensation for nidogen-1 deficiency in transgenic mice

https://doi.org/10.1016/S0945-053X(02)00070-7Get rights and content

Abstract

Previous studies have shown that inhibition of nidogen–laminin binding interferes with basement membrane stabilization in various mouse organ cultures while no overt phenotype has been observed following inactivation of the nidogen-1 gene in mice. We have now used recombinant mouse nidogen-1 and nidogen-2 in order to evaluate a possible compensation between the two isoforms in the knock-out mice. Essentially, a comparable in vitro binding of nidogens-1 and -2 to the same laminin γ1 chain structure and to several other basement membrane proteins has been revealed. Quantitative radioimmuno-assays have demonstrated high concentrations of nidogen-1 exceeding those of laminin γ1 and nidogen-2 by factors of 5 and 20–50, respectively, in tissue extracts of wild-type mice. A three- to sevenfold increase in nidogen-2 was observed in heart and muscle of mice with nidogen-1 deficiency and confirmed by a similar increase in the intensity of immunogold staining of these tissues. However, a few of the tissues from mice with the gene knock-out still contained some nidogen-1-like immunoreactivity (1% of wild-type). Furthermore, both nidogen isoforms showed a similar distribution in various organs during embryonic development which, however, as shown previously, changed in some adult tissues. The data support the nidogen-2 compensation hypothesis to explain the limited phenotype observed following elimination of the nidogen-1 gene.

Introduction

Nidogens represent an ancient small family of basement membrane proteins and are considered to have major functions in the supramolecular organization of these extracellular matrices and in nerve guidance (Erickson and Couchman, 2000, Hutter et al., 2000, Kim and Wadsworth, 2000). Two isoforms, nidogen-1 and nidogen-2, have been identified in mammalian species and consist of three globular domains, G1–G3, connected by elongated segments (Fox et al., 1991, Kohfeldt et al., 1998). Domain G3 of nidogen-1 binds tightly to laminins while G2 has high affinity binding sites for collagen IV and perlecan which allow for the efficient formation of ternary complexes between major basement membrane proteins (Fox et al., 1991, Mayer et al., 1995, Hopf et al., 1999). Several of these interactions have been examined in more detail and show for instance a narrow ridge of three amino acid residues on the surface of a single LE-module of laminin γ1 chain which are mainly responsible for the nidogen-1 association (Pöschl et al., 1996, Stetefeld et al., 1996). The crystal structure of domain G2 of nidogen-1 has been recently elucidated and a set of hydrophobic and polar residues on the surface of a β barrel involved in the binding of perlecan has been found (Hopf et al., 2001a, Hopf et al., 2001b). These residues are highly conserved among the nidogen G2 sequences known so far. A comparable binding to perlecan and collagen IV has been demonstrated for recombinant human nidogen-2 while the affinity for the laminin γ1 chain structure is two to three orders of magnitude lower compared to human and mouse nidogen-1 (Kohfeldt et al., 1998).

Nidogen-1 is expressed at pre-implantation stages of mouse development and at subsequent stages it is very often produced by mesenchymal cells (Dziadek and Timpl, 1985, Dziadek, 1995). This has led to the concept that nidogen binding to its major ligands occurs in the extracellular space and depends on cellular cooperation as also shown in co-cultures of skin and mammary gland cells (Fleischmajer et al., 1995, Pujuguet et al., 2000). Support for this interpretation came from experiments with antibodies blocking nidogen-1 attachment to the laminin γ1 chain which perturbs basement membrane formation in organ cultures of embryonic kidney, lung and salivary gland (Ekblom et al., 1994, Kadoya et al., 1997). The elimination of the γ1 chain gene in mice caused early embryonic lethality but since this interfered with the assembly and secretion of most laminin isoforms it abolished more functions than just nidogen binding (Smyth et al., 1999). However, the selective removal of the nidogen-binding LE module from the γ1 chain gene in murine embryonic stem cells caused no large defects in laminin secretion but impaired tissue deposition of nidogen-1 (Mayer et al., 1998). The same gene deficiency in homozygous mice resulted in perinatal mortality due to severe defects in the organogenesis of many tissues (Willem et al., 2002). All these data support a distinct role of laminin–nidogen interactions in the stabilization of basement membranes. A recent study of mouse lines deficient in nidogen-1 showed, however, no overt abnormalities in the formation of organs and the basement membranes appeared normal (Murshed et al., 2000). Nidogen-2 was apparently not up-regulated, as shown by immunoblotting, but showed some changes in its tissue localization. This left the question of whether nidogen-2 could compensate for nidogen-1, which could explain for the lack of an obvious phenotype, still unanswered.

In the present study we have used recombinant mouse nidogen-2 in ligand binding assays and the quantitative analyses of tissue contents. These revealed a high affinity for the laminin γ1 chain not expected from previous studies of human nidogen-2 (Kohfeldt et al., 1998) and a significant up-regulation of nidogen-2 in some tissues of the transgenic animals. These results provide strong support for the compensation hypothesis.

Section snippets

Animals and tissues

Transgenic C57BL/6 mice heterozygous (+/−) or homozygous (−/−) for the nidogen-1 gene as well as wild-type littermates have been described previously (Murshed et al., 2000). Adult animals (3–6 months) were used as donors for the various organs kindly provided by Roswitha Nischt which were stored at −80 °C prior to tissue extraction. Six-month-old (+/−) and (−/−) mice and, in addition, NMRI mice were used for immunohistological studies and immunogold staining. In pregnant mice, day 0 of

Binding properties of recombinant mouse nidogen-2 to laminin γ1 chain and other ligands

The two nidogen isoforms known from mammalian species have been previously shown to bind with high affinity to collagen IV and perlecan but to differ considerably in their binding to laminin-1 (Fox et al., 1991, Kohfeldt et al., 1998, Hopf et al., 1999). Since these comparisons were carried out with mouse nidogen-1 and human nidogen-2 it was not possible to distinguish whether the different laminin affinities were due to a species or isoform-specific variability. We have now prepared

Discussion

Over the past decade, the targeted disruption of mouse genes encoding proteins involved in cell–cell adhesion, cell–matrix interactions and the supramolecular organization of extracellular matrices has become a widely used approach to examine biological functions (Hynes, 1996, Aszódi et al., 1998). Several of the mouse lines generated are viable and of these some do not show any overt or only a minimal phenotype. This was the case despite expected disturbances of important functions known from

Acknowledgements

We thank Vera van Delden and Mischa Reiter for excellent technical assistance and Roswitha Nischt, Peter Ekblom, Ulrike Mayer, Utz Fischer, Reinhard Fässler and Bernhard Bader for providing samples or helpful discussions. The study was supported by grants of the Deutsche Forschungsgemeinschaft Mi 573/1–3 (to NM).

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