Comparison of non-collagenous type IV collagen components in the human glomerulus and EHS tumor

A method for the isolation of the NC1 domain of type IV collagen has been developed using the EHS sarcoma, a basement membrane-producing mouse tumor. This NC1 domain has been compared to the NC1 of human glomerular basement membrane (hGBM) to assess its usefulness in the biochemical characterization of the Goodpasture antigen which is associated with NC1. Both NC1 isolates appeared to migrate by gel filtration as hexamers (Mr 160,000) and in SDS-polyacrylamide gel electrophoresis as dimers and monomers (Mr 54,000 and 26,000), and were shown to share biochemical identity by amino acid analysis. The hGBM NC1 showed greater complexity in the monomer region, and when compared by two-dimensional gel electrophoresis was found to contain more components in both regions than EHS NC1. Anti-GBM autoantibodies from patients with Goodpasture's syndrome reacted with the EHS NC1 by immunoblotting of two-dimensional gels. The EHS NC1 isolated by reverse phase HPLC partially inhibited the reactivity of the anti-GBM autoantibodies against hGBM NC1 by inhibition ELISA assay. Reverse phase HPLC elution of EHS and hGBM NC1 showed differences in subunit composition and interaction; complete dissociation of the EHS monomers and dimers in 0.1% trifluoroacetic acid was observed, whereas hGBM monomers and dimers eluted together. Rotary shadowing of hGBM NC1 domains revealed size heterogeneity of globular domains, compared with greater uniformity of EHS NC1 hexamers. We conclude that EHS NC1 contains an epitope(s) that is reactive with human autoantibodies to hGBM NC1. However, the immune response in Goodpasture's syndrome may involve antibodies directed against epitopes which are present in greater density and on a more complex array of peptides in the hGBM NC1 than in EHS NC1.     

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.     

Wogonin suppresses tumor growth in vivo and VEGF-induced angiogenesis through inhibiting tyrosine phosphorylation of VEGFR2

Previous studies revealed that wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, possessed anticancer activity both in vitro and in vivo. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we found for the first time that wogonin inhibited the growth and tumor angiogenesis of human gastric carcinoma in nude mice. We explored the inhibitory effect of wogonin on angiogenesis stimulated by vascular endothelial growth factor (VEGF) in vitro. Wogonin suppressed the VEGF-stimulated migration and tube formation of human umbilical vein endothelial cells (HUVECs). It also restrained VEGF-induced tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This inhibition of receptor phosphorylation was correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that wogonin might be a promising antitumor drug.     

Differential modulation of human melanoma cell metalloproteinase expression by alpha2beta1 integrin and CD44 triple-helical ligands derived from type IV collagen

Tumor cell binding to components of the basement membrane is well known to trigger intracellular signaling pathways. Signaling ultimately results in the modulation of gene expression, facilitating metastasis. Type IV collagen is the major structural component of the basement membrane and is known to be a polyvalent ligand, possessing sequences bound by the alpha1beta1, alpha2beta1, and alpha3beta1 integrins, as well as cell surface proteoglycan receptors, such as CD44/chondroitin sulfate proteoglycan (CSPG). The role of alpha2beta1 integrin and CD44/CSPG receptor binding on human melanoma cell activation has been evaluated herein using triple-helical peptide ligands incorporating the alpha1(IV)382-393 and alpha1(IV)1263-1277 sequences, respectively. Gene expression and protein production of matrix metalloproteinases-1 (MMP-1), -2, -3, -13, and -14 were modulated with the alpha2beta1-specific sequence, whereas the CD44-specific sequence yielded significant stimulation of MMP-8 and lower levels of modulation of MMP-1, -2, -13, and -14. Analysis of enzyme activity confirmed different melanoma cell proteolytic potentials based on engagement of either the alpha2beta1 integrin or CD44/CSPG. These results are indicative of specific activation events that tumor cells undergo upon binding to select regions of basement membrane collagen. Based on the present study, triple-helical peptide ligands provide a general approach for monitoring the regulation of proteolysis in cellular systems.     

Affinity of integrin alpha 1 beta 1 from liver sinusoidal membranes for type IV collagen.

Hepatic sinusoidal membranes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N-acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS-PAGE revealed two glycoproteins with apparent molecular weights of 180,000 and 117,000. These were identified immunologically by Western blotting as the alpha and beta subunits of integrin alpha 1 beta 1.     

Crystal structure of NC1 domains. Structural basis for type IV collagen assembly in basement membranes

Type IV collagen, which is present in all metazoan, exists as a family of six homologous alpha(IV) chains, alpha1-alpha6, in mammals. The six chains assemble into three different triple helical protomers and self-associate as three distinct networks. The network underlies all epithelia as a component of basement membranes, which play important roles in cell adhesion, growth, differentiation, tissue repair and molecular ultrafiltration. The specificity of both protomer and network assembly is governed by amino acid sequences of the C-terminal noncollagenous (NC1) domain of each chain. In this study, the structural basis for protomer and network assembly was investigated by determining the crystal structure of the ubiquitous [(alpha1)(2).alpha2](2) NC1 hexamer of bovine lens capsule basement membrane at 2.0 A resolution. The NC1 monomer folds into a novel tertiary structure. The (alpha1)(2).alpha2 trimer is organized through the unique three-dimensional domain swapping interactions. The differences in the primary sequences of the hypervariable region manifest in different secondary structures, which determine the chain specificity at the monomer-monomer interfaces. The trimer-trimer interface is stabilized by the extensive hydrophobic and hydrophilic interactions without a need for disulfide cross-linking.     

Differential effects of laminin, intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration

Laminin and type IV collagen were compared for the ability to promote aortic endothelial cell adhesion and directed migration in vitro. Substratum-adsorbed IV promoted aortic endothelial cell adhesion in a concentration dependent fashion attaining a maximum level 141-fold greater than controls within 30 min. Aortic endothelial cell adhesion to type IV collagen was not inhibited by high levels (10(-3) M) of arginyl-glycyl-aspartyl-serine. In contrast, adhesion of aortic endothelial cells on laminin was slower, attaining only 53% of the adhesion observed on type IV collagen by 90 min. Type IV collagen when added to the lower well of a Boyden chamber stimulated the directional migration of aortic endothelial cells in a concentration dependent manner with a maximal response 6.9-fold over control levels, whereas aortic endothelial cells did not migrate in response to laminin at any concentration (.01-2.0 X 10(-7) M). Triple helix-rich fragments of type IV collagen were nearly as active as intact type IV collagen in stimulating both adhesion and migration whereas the carboxy terminal globular domain was less active at promoting adhesion (36% of the adhesion promoted by intact type IV collagen) or migration. Importantly, aortic endothelial cells also migrate to substratum adsorbed gradients of type IV collagen suggesting that the mechanism of migration is haptotactic in nature. These results demonstrate that the aortic endothelial cell adhesion and migration is preferentially promoted by type IV collagen compared with laminin, and has a complex molecular basis which may be important in angiogenesis and large vessel repair.     

Synthesis of heterotrimeric collagen peptides containing the alpha1beta1 integrin recognition site of collagen type IV.

Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the alpha1beta1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the alpha2 and alpha1 chains of the collagen molecule, respectively. Since the stagger of the three alpha chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457-469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to alpha1beta1 integrin is apparently less affected as indicated by preliminary experiments.     

Evidence for the involvement of type II domains in collagen binding by 72 kDa type IV procollagenase

The fibronectin-related region of the 72 kDa type IV procollagenase has been expressed in E. coli as a beta-galactosidase fusion product. The fragment containing the three type II units of the protein was found to have affinity for denatured collagen, suggesting that these domains may be responsible for the collagen-affinity of type IV collagenase. We have also shown that segment Ala-Ala-His-Glu of type IV collagenase (residues 372-375), which is similar to a fibronectin-segment previously implicated in collagen-binding, is not essential for binding activity.     

Posttranslational modifications in the biosynthesis of type IV collagen by a human tumor cell line

Factors responsible for the high extent of intracellular posttranslational modifications in type IV collagens were studied in a cultured human tumor cell line, HT-1080. These cells do not synthesize any detectable amounts of interstitial collagens but produce type IV collagen at a high rate, corresponding to about one-third of the production of interstitial collagens by cultured human skin fibroblasts. Prolyl 4-hydroxylase activity was lower in the HT-1080 cells than in human skin fibroblasts, there being a rough correlation between this enzyme activity and the rate of 4-hydroxyproline formation in these two cell types. The differing extents of the respective modifications could largely be explained by differences in the activities of lysyl hydroxylase and the hydroxylysyl glycosyltransferases between the two cell types. No difference ws found in prolyl 3-hydroxylase activity, however, even though the extent of 3-hydroxylation of proline residues was about 6-fold in the type IV collagens. In experiments where the HT-1080 cells were studied in suspension, a lag of about 100 min was found before the secretion of type IV collagen from the cells became linear. Pulse-chase experiments in suspension indicated that all the intracellular enzyme reactions proceeded for about 40 min, presumably due to the slow triple-helix formation in type IV collagens. This slow helix formation apparently contributed to the high extent of all the intracellular modifications but was not a major factor.     

Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human alpha1 type IV collagen expressed in Sf9 cells

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed. In the present study, expression of alpha1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.     

The type X collagen gene. Intron sequences split the 5'-untranslated region and separate the coding regions for the non-collagenous amino-terminal and triple-helical domains

Type X collagen, expressed by hypertrophic chondrocytes, consists of homotrimeric molecules with subunits that are only about one-half the size of the polypeptides of fibrillar collagens. In this report we describe for the first time the complete primary structure of type X collagen, based on cloning and sequencing of cDNA and genomic DNA. A comparison between the nucleotide sequences of the cDNA and genomic DNA clones has also allowed determination of the complete exon structure of the type X collagen gene. Our results demonstrate that the primary translation product of the chicken type X collagen mRNA is 682 amino acid residues long with a calculated molecular mass of 67,317 Da for the nonhydroxylated form. This calculated molecular mass is in excellent agreement with the observed electrophoretic mobility of cell-free translation products with both poly(A)+ RNA isolated from chondrocytes as well as RNA transcribed in vitro from a full length cDNA construct. It is also in agreement with the observed size of type X collagen polypeptides isolated from the media of cultured hypertrophic chondrocytes. Thus, our data exclude the possibility of a high molecular weight precursor form of type X collagen. Our results also confirm that the chicken type X gene has a most unusual exon structure when compared to other vertebrate collagen genes. The gene has only three exons. One exon (97 base pairs (bp)), codes for most of the 5'-untranslated region of the mRNA, a second exon (159 bp) codes for the signal peptide and a short non-triple-helical domain, while the third exon (2136 bp) contains the coding region for the entire triple-helix and a large non-triple-helical carboxyl domain.     

Evaluation of radiolabeled type IV collagen fragments as potential tumor imaging agents.

The objective of this study was to examine radiopharmaceuticals that target the alpha3beta1 integrin to determine if these agents target tumors for diagnostic imaging and/or targeted radiotherapy of cancer. Prior studies had shown that residues 531-542 from the alpha1 chain of type IV collagen bind a variety of tumor cell alpha3beta1 integrins. A peptide mimic of this sequence containing all D-amino acids (designated D-Hep-III) was synthesized by solid-phase methods. The tetraazamacrocyclic chelator, TETA, was conjugated to the peptide while it was resin-bound. TETA-D-Hep-III and D-Hep-III were radiolabeled with 64Cu and 125I, respectively, in high specific activity and radiochemical purity. Heterologous competitive binding assays between D-Hep-III and either 125I-D-Hep-III or 64Cu-TETA-D-Hep-III indicated low micromolar affinity of D-Hep-III. The biodistribution of each radiolabeled analogue of D-Hep-III was carried out in rats and tumor-bearing mice. Both analogues were rapidly cleared from the blood in normal rats, with the kidneys receiving the highest accumulation of each. SKOV3 human ovarian tumor cells, known to strongly express alpha3beta1, were xenografted in SCID mice. Localization of 125I-D-Hep III and 64Cu-TETA-D-Hep III in the xenografts were low (<2% ID/g), and in the case of 125I-D-Hep III, not inhibited by a competitive dose of D-Hep III. The low tumor accumulation is likely not due to receptor down-regulation, but rather due to the weak affinity of the radioligands for the alpha3beta1 integrin.     

Regulation of type IV collagen degrading enzyme by cortisol during human skin fibroblast growth

The effects of cortisol on type IV collagenolytic enzyme activity were studied in human skin fibroblast cultures. Cortisol reduced the production of the enzyme, both in normally dividing fibroblasts, and also when the secretion was induced by the administration of the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the subconfluent culture. TPA decreased the glucocorticoid receptor activity and augmented the cGMP content of subconfluent fibroblasts. Type IV collagenolytic activity appeared to be correlated positively with the cGMP/cAMP ratio, and negatively with the glucocorticoid receptor activity, and the activity was increased by the addition of dibutyryl cGMP. This suggests that this enzyme may at least partly be under glucocorticoid control during fibroblast growth, and that TPA may to some degree act via the inactivation of glucocorticoid receptors.     

Tumstatin, the NC1 domain of alpha3 chain of type IV collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth

Angiogenesis, the formation of new blood vessels, is required for physiological development of vertebrates and repair of damaged tissue, but in the pathological setting contributes to progression of cancer. During tumor growth, angiogenesis is supported by up-regulation of angiogenic stimulators (pro-angiogenic) and down-regulation of angiogenic inhibitors (anti-angiogenic). The switch to the angiogenic phenotype (angiogenic switch) allows the tumors to grow and facilitate metastasis. The bioactive NC1 domain of type IV collagen alpha3 chain, called tumstatin, imparts anti-tumor activity by inducing apoptosis of proliferating endothelial cells. Tumstatin binds to alphaVbeta3 integrin via a mechanism independent of the RGD-sequence recognition and inhibits cap-dependent protein synthesis in the proliferating endothelial cells. The physiological level of tumstatin is controlled by matrix metalloproteinase-9, which most effectively cleaves it from the basement membrane and its physiological concentration in the circulation keeps pathological angiogenesis and tumor growth in check. These findings suggest that tumstatin functions as an endogenous inhibitor of pathological angiogenesis and functions as a novel suppressor of proliferating endothelial cells and growth of tumors.     

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.     

Isolation of a type IV collagen binding protein from human platelets.

In order to study the molecular basis of platelet interaction with collagen IV of the basement membrane separating the arterial endothelium from the underlying subendothelial connective tissue, the possibility of presence of platelet membrane protein with affinity to type IV collagen was examined by subjecting the platelet membrane extract to affinity chromatography on collagen IV-sepharose. Urea (4 M) eluate was found to contain a protein with an apparent mol. wt of 68 kDa. The radioiodinated protein was isolated and used to test its specificity. By dot blot assay on nitrocellulose disks and solid-phase assays, the 68 kDa protein was found to bind with high affinity to collagen IV. Lack of significant binding to fibronectin and laminin when compared to albumin control indicated its high specificity for collagen. The radioiodinated protein was inserted into egg yolk lecithin liposomes. While these liposomes attached to microtitre plates coated with collagen IV, there was no significant binding to fibronectin or laminin coated wells, suggesting the membrane associated character of the protein as well as its specificity for collagen. These results indicate that presence of a 68 kDa protein in platelet membrane which interacts with very high specificity to collagen IV.     

An abundant chick gizzard integrin is the avian alpha 1 beta 1 integrin heterodimer and functions as a divalent cation-dependent collagen IV receptor.

The alpha-subunit of an abundant chick gizzard integrin was isolated (T. Kelly, L. Molony, and K. Burridge, 1987, J. Biol. Chem. 262, 17,189-17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin alpha 1-subunit. Monoclonal antibodies to the chick integrin beta 1-chain react on immunoblots with the gizzard integrin beta-subunit (U. Hofer, J. Syfrig, and R. Chiquet-Ehrismann, 1990, J. Biol. Chem. 265, 14,561-14,565). The chain composition of the abundant chick gizzard integrin is therefore alpha 1 beta 1. Polyclonal antibodies to the avian integrin alpha 1-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated alpha 1 beta 1 integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian alpha 1 beta 1 integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian alpha 1 beta 1 integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.     

The complete primary structure for the alpha 1-chain of mouse collagen IV. Differential evolution of collagen IV domains

We report here the complete nucleotide and amino acid sequences for the alpha 1-chain of mouse collagen IV which is 1669 amino acids in length, including a putative 27-residue signal peptide. In comparison with the amino acid sequence for the alpha 2-chain (Saus, J., Quinones, S., MacKrell, A. J., Blumberg, B., Muthkumaran, G., Pihlajaniemi, J., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6318-6324), the two chains of collagen IV are 43% identical. Most of the interruptions of the Gly-X-Y repeat are homologously placed but strikingly show no sequence similarity between the two chains. Availability of the amino acid sequences for human collagen IV allows a detailed comparison of the primary structure of collagen IV and reveals evolutionarily conserved domains of the protein. Between the two species, the alpha 1 (IV) chains are 90.6% and the alpha 2 (IV) chains are 83.5% identical in sequence. We discuss these data with respect to differential evolution between and within the collagen IV chain types.     

The localization and secretion of type IV collagen in synovial capillaries by immunohistochemistry using a monoclonal antibody against human type IV collagen

The localization and the secretion of type IV collagen in synovial capillaries have been investigated by detecting the antigenic determinant of the major triple helix of human type IV collagen. Type IV collagen was indicated to be localized mainly in the lamina densa of basement membranes (BM) and to be secreted by both endothelial cells and pericytes. The pericytes secreted this collagen to both surfaces facing endothelial cells and the interstitial connective tissue. On the contrary, the direction of type IV collagen secretion by the endothelial cells was strictly confined to one side, namely towards the surface facing the BM. The absence of the antigenic determinant in rough endoplasmic reticulum and Golgi apparatus of the endothelial cells and pericytes indicated that the major triple helix of type IV collagen is mainly formed in the secretory vesicles after budding from the Golgi apparatus.