Collagen types XII and XIV are present in basement membrane zones during human embryonic development

The collagens constitute a large group of proteins in the extracellular matrix that can be divided into several distinct families. Collagen types XII and XIV belong to a subgroup of non-fibrillar-collagens termed (fibril-associated collagens with interrupted triple-helices) (FACIT) and may be involved in basement membrane regulation providing specific molecular bridges between fibrils and other matrix components. However, the tissue distribution of the two proteins during human embryogenesis is still unclear. As a first step toward the elucidation of their possible cell biological functions, we compared the distribution of the two collagens during human organogenesis at the light microscopical level. We detected specific differences between the expression patterns of the two molecules, which may be related to their respective function within the basement membrane zones during human embryonic development. For example, in the developing intestine, collagen type-XII was present in the basement membrane zones of epithelia and endothelia. However, collagen type-XIV was restricted to the mesothelial basement membrane zones. We conclude that both collagens might well be able to serve different functions during human embryonic development although their structures are highly similar.     

Nidogen-1. Expression and ultrastructural localization during the onset of mesoderm formation in the early mouse embryo.

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen Type IV networks and is expressed by mesenchymal cells during embryonic and fetal development. It is not clear which cells produce nidogen-1 in early developmental stages when no mesenchyme is present. We therefore localized nidogen-1 and its corresponding mRNA at the light and electron microscopic level in Day 7 mouse embryos during the onset of mesoderm formation by in situ hybridization, light microscopic immunostaining, and immunogold histochemistry. Nidogen-1 mRNA was found not only in the cells of the ectoderm-derived mesoderm but also in the cytoplasm of the endoderm and ectoderm, indicating that all three germ layers express it. Nidogen-1 was localized only in fully developed basement membranes of the ectoderm and was not seen in the developing endodermal basement membrane or in membranes disrupted during mesoderm formation. In contrast, laminin-1 and collagen Type IV were present in all basement membrane types at this developmental stage. The results indicate that, in the early embryo, nidogen-1 may be expressed by epithelial and mesenchymal cells, that both cell types contribute to embryonic basement membrane formation, and that nidogen-1 might serve to stabilize basement membranes in vivo. (J Histochem Cytochem 48:229-237, 2000)     

Ultrastructural colocalization of nidogen-1 and nidogen-2 with laminin-1 in murine kidney basement membranes

Nidogen-1, a key component of basement membranes, is considered to function as a link between laminin and collagen type IV networks. Recently a new member of the nidogen family, nidogen-2, has been characterized. Preliminary immunohistochemical data indicated that nidogen-1 and nidogen-2 show a similar tissue distribution at the light microscopic level. We have now localized nidogen-1 and nidogen-2, as well as their corresponding mRNAs, at the light and electron microscopic levels in adult mouse kidney, by in situ hybridization and immunogold histochemistry, as well as carrying out double labeling with laminin-1. Both nidogen-1 and nidogen-2 mRNAs are found not only in mesenchymal cells of embryonic tissues, but also in all epithelial and endothelial cells in adult mouse kidney. Both nidogens are ubiquitous basement membrane components in the mouse kidney, being found in glomerular, tubular, and capillary compartments and Bowman’s capsule. Furthermore, a substantial fraction of nidogen-1 and nidogen-2 colocalizes with laminin-1. The results indicate that nidogen-1 and nidogen-2 could well substitute for one another in some of their biological activities in kidney, for example, stabilizing basement membrane networks in vivo. Accepted: 8 December 1999     

Gene structure and functional analysis of the mouse nidogen-2 gene: nidogen-2 is not essential for basement membrane formation in mice

Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.     

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 (gamma1III4) 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.     

The absence of one or both nidogens does not alter basement membrane composition in adult murine kidney

Nidogen-1 and nidogen-2 are major components of all basement membranes and are considered to function as link molecules between laminin and collagen type IV networks. Surprisingly, the knockout of one or both nidogens does not cause defects in all tissues or in all basement membranes. In this study, we have elucidated the appearance of the major basement membrane components in adult murine kidney lacking nidogen-1, nidogen-2, or both nidogens. To this end, we localized laminin-111, perlecan, and collagen type IV in knockout mice, heterozygous (+/-) or homozygous (-/-) for the nidogen-1 gene, the nidogen-2 gene, or both nidogen genes with the help of light microscopic immunostaining. We also performed immunogold histochemistry to determine the occurrence of these molecules in the murine kidney at the ultrastructural level. The renal basement membranes of single knockout mice contained a similar distribution of laminin-111, perlecan, and collagen type IV compared to heterozygous mice. In nidogen double-knockout animals, the basement membrane underlying the tubular epithelium was sometimes altered, giving a diffuse and thickened pattern, or was totally absent. The normal or thickened basement membrane of double-knockout mice also showed a similar distribution of laminin-111, perlecan, and collagen type IV. The results indicate that the lack of nidogen-1, nidogen-2, or both nidogens, plays no crucial role in the occurrence and localization of laminin-111, collagen type IV, and perlecan in murine tubular renal basement membranes.     

Laminin α5 guides tissue patterning and organogenesis

Laminins (LM) are extracellular matrix molecules that contribute to and are required for the formation of basement membranes. They participate in the modulation of epithelial/mesenchymal interactions and are implicated in organogenesis and maintenance of organ homeostasis. Among the LM molecules, the LM α5 chain (LMα5) is one of the most widely distributed LM in the developing and mature organism. Its presence in some basement membranes during embryogenesis is absolutely required for maintenance of basement membrane integrity and thus for proper organogenesis. LMα5 also regulates the expression of genes important for major biological processes, in part by repressing or activating signaling pathways, depending upon the physiological context.     

Structural elucidation of full-length nidogen and the laminin–nidogen complex in solution

Nidogen-1 is a key basement membrane protein that is required for many biological activities. It is one of the central elements in organizing basal laminae including those in the skin, muscle, and the nervous system. The self-assembling extracellular matrix that also incorporates fibulins, fibronectin and integrins is clamped together by networks formed between nidogen, perlecan, laminin and collagen IV. To date, the full-length version of nidogen-1 has not been studied in detail in terms of its solution conformation and shape because of its susceptibility to proteolysis. In the current study, we have expressed and purified full-length nidogen-1 and have investigated its solution behavior using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). The ab initio shape reconstruction of the complex between nidogen-1 and the laminin γ-1 short arm confirms that the interaction is mediated solely by the C-terminal domains: the rest of the domains of both proteins do not participate in complex formation.     

Structural basis for the high-affinity interaction of nidogen-1 with immunoglobulin-like domain 3 of perlecan

Nidogen and perlecan are large multifunctional basement membrane (BM) proteins conserved in all metazoa. Their high-affinity interaction, which is likely to contribute to BM assembly and function, is mediated by the central G2 domain in nidogen and the third immunoglobulin (IG)-like domain in perlecan, IG3. We have solved the crystal structure at 2.0 A resolution of the mouse nidogen-1 G2-perlecan IG3 complex. Perlecan IG3 belongs to the I-set of the IG superfamily and binds to the wall of the nidogen-1 G2 beta-barrel using beta-strands C, D and F. Nidogen-1 residues participating in the extensive interface are highly conserved, whereas the corresponding binding site on perlecan is more variable. We hypothesize that a second, as yet unidentified, activity of nidogen overlaps with perlecan binding and accounts for the unusually high degree of surface conservation in the G2 domain.     

Tissue distribution of the laminin β1 and β2 chain during embryonic and fetal human development

Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin β1 and β2 chains in various developing fetal organs is already available, a systematic localization of the laminin β1 and β2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin β1 and β2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin β1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin β2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development.     

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

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 gamma1 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 gamma1 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.     

Recombinant domains of mouse nidogen-1 and their binding to basement membrane proteins and monoclonal antibodies.

The basement membrane protein, nidogen-1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin-1.     

Angiogenesis inhibitor endostatin is a distinct component of elastic fibers in vessel walls.

Theendothelial cell inhibitor endostatin (22 kDa) is part of the carboxyl-terminal globular domain of collagen XVIII and shows a widespread tissue distribution. Immunohistology of adult mouse tissues demonstrated a preferred localization in many vessel walls and some other basement membrane zones. A strong immunogold staining was observed across elastic fibers in the multiple elastic membranes of aorta and other large arteries. Staining was less strong along sparse elastic fibers of veins and almost none was observed in the walls of arterioles and capillaries. Strong evidence was also obtained for some intracellular and basement membrane associations. Immunogold double staining of elastic fibers showed a close colocalization of endostatin with fibulin-2, fibulin-1, and nidogen-2, but not with perlecan. Reasonable amounts of endostatin could be extracted from aorta and skin by EDTA, followed by detergents, with aorta being the richest source of the inhibitor identified so far. Solubilizations with collagenase and elastase were approximately fivefold less efficient. Immunoblots of aortic extracts detected major endostatin components of 22-25 kDa whereas skin extracts also contained some larger components. Solid-phase assays demonstrated distinct binding of recombinant mouse endostatin to the fibulins and nidogen-2, consistent with their tissue colocalization. Together, the data indicate several different ways for endostatin to be associated with the extracellular matrix, and its release may determine biological activation. This also defines a novel function for some elastic tissues.     

Expression of nidogens in rat uterus and embryo during decidualization and implantation

The purpose of this study was to demonstrate the expression of nidogen-1 and nidogen-2 and their possible role in decidualization and implantation events during early pregnancy in rats. The tissue samples were examined from pregnant animals between gestational days 1-8 using immunocytochemistry. The uterine luminal epithelium, the glandular epithelium, and the myometrial smooth muscle cells stained strongly from gestational days 1-8 with both nidogen antibodies. At day 4 the decidual reaction areas began to appear in the stromal matrix and immunostaining of both nidogens revealed that the basement membrane of the surface epithelium was discontinuous. The differentiation of stromal cells into decidual cells was seen at gestational day 5 and both nidogens were weakly expressed in the decidualizing cells. At day 6, nidogen-2 immunoreactivity was higher in the primary decidual cells close to the embryo than nidogen-1, and during development of the decidual tissue both nidogens appeared in the endometrial stromal cells. At day 7, while expression of both nidogens declined in the primary decidual cells, their expression was markedly observed in the secondary decidual cells close to the myometrium. At day 8, expression of both nidogens was also observed to increase in the primary decidual cells. While nidogen-2 expression was seen in the parietal endoderm and primary ectoderm of the rat embryos at this developmental stage, nidogen-1 expression was only detected in the parietal endoderm. These results indicate that nidogen-1 and nidogen-2 could play important roles during embryogenesis, decidualization, and implantation in the endometrium of rat uterus.     

The ultrastructural composition of basement membranes in vivo

The ultrastructure of basement membranes has a homogeneous appearance. The enormous cell biological importance of basement membranes and their components for cell proliferation, migration and differentiation implies that their composition is more complex than their structure suggests. To elucidate the molecular composition of basement membranes in vivo, we optimised immunogold histochemistry to allow the determination of the molecular arrangement of matrix molecules. Basically, we apply a mild fixation and embed the tissues in the hydrophilic LR-Gold. This preserves the basement membrane with a quality similar to freeze substitution. The application of two antibodies directed toward the C- and N-terminal ends of a molecule and coupled to gold particles of different sizes allows determination of the orientation of a molecule within the basement membrane. We were able to demonstrate that the molecular orientation of the laminin-1 molecule changes in the basement membrane according to cell biological needs. We also showed that ultrastructurally identical basement membranes like the ones of the proximal and distal tubules of the kidney have a differing molecular arrangement. Integrin alpha7 influences the molecular composition of the basement membranes at the myotendinous junction. With the help of double labelling at the ultrastructural level we could show that nidogen-1 is co-localised with laminin-1 and only found in fully developed, mature basement membranes. In general, laminin-1, nidogen-1 and collagen type IV are localised over the entire width of basement membranes, with laminin-1 and nidogen-1 co-localised, in accordance with the current basement membrane models. Incidentally, our investigations warn us, that not every matrix protein found at the light microscopic level as a linear staining pattern underneath an epithelium (basement membrane zone) is a real basement membrane component when investigated at the ultrastructural level. Instead, one and the same molecule, e.g. endostatin, can be a basement membrane component in one organ and a matrix molecule in another.     

The growth factor-like adipokine tartrate-resistant acid phosphatase 5a interacts with the rod G3 domain of adipocyte-produced nidogen-2

The adipokine tartrate resistant acid phosphatase (TRAP) 5a isoform exerts a growth factor-effect on pre-adipocytes. This study aimed to identify potential TRAP 5a interacting proteins in pre-adipocytes using pull down assays in combination with mass spectrometry. Nidogen-2, a protein shown to be expressed intracellularly and for secretion by pre-adipocytes, was shown to interact, through its globular G3 domain, with TRAP 5a in vitro. In vivo, TRAP 5a interacted with nidogen-2 in cultured 3T3-L1 mouse pre-adipocytes, as well as with transforming growth factor-β (TGF-β) interacting protein (TRIP-1), which is a protein that has previously been suggested to interact with TRAP in bone. In addition, TRAP 5a and nidogen-2 co-localized in adipose tissue cells in situ. These results indicate that TRAP 5a interacts with nidogen-2 and TRIP-1 in pre-adipocytic cells.     

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin α5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin α5 chain and Lutheran glycoprotein, a receptor for the laminin α5 chain. In normal lymphatic capillaries, the presence of primarily α4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin α5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.     

Laminin 5 processing and its integration into the ECM.

Laminins are a family of multi-functional basement membrane proteins. Their C-terminal domain binds to cell surface receptors and is thereby responsible for cell anchorage and the initiation of specific outside-in and inside-out signals. With their N-terminal parts, laminins interact with proteins of the extracellular matrix scaffold to secure the basement membrane to the underlying mesenchymal tissue. Laminins 5A (alpha3Abeta3gamma2), 5B (alpha3Bbeta3gamma2) and 6 (alpha3Abeta1gamma1) are isoforms specific of the basement membrane underneath the epidermis and they undergo a sequential series of extracellular proteolytic changes, which might successively turn on and off one or several of their biological and mechanical functions. Under physiological conditions, such as in adult human skin, epithelial laminins have lost part of the C- and N-terminal domains of the alpha3 and gamma2 chains, respectively. In contrast, in cylindromatosis, a rare inherited disease characterised by major ultrastructural alterations of the basement membrane and altered expression/distribution of integrin receptors, laminin processing has not been completed. Together, these results suggest that laminin processing may regulate signalling pathways and the architecture of the basement membrane by restricting the repertoire of interactions with cell surface receptors and extracellular matrix components.