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PubMed1982

Collagen metabolism in rat incisor predentine in vivo: synthesis and maturation of type I, alpha 1 (I) trimer, and type V collagens.

Collagen synthesis and deposition in the predentine of the continuously erupting rat incisor was analyzed in vivo following a single intraperitoneal injection of [14C]glycine. Newly synthesized collagen was extracted from the dissected predentine with a solution of 1.0 m sodium chloride containing proteolytic enzyme inhibitors. Mature, cross-linked collagen was solubilized by limited pepsin digestion following extractions with 0.5 M acetic acid or 0.1 M penicillamine. Analysis of the radiolabeled collagens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that over 97% of collagen synthesized migrated in the alpha 1 (I) and alpha 2 positions following pepsin digestion; the remaining 3% migrated in the positions of alpha 1 (V) and alpha 3 (V) collagens. However, from the ratio of the alpha 1 (I): alpha 2 it was estimated that approximately 30% of the salt-extractable collagen was alpha 1 (I) trimer. The presence of this collagen was confirmed by salt-fractionation and cyanogen bromide digestion patterns. Type I, alpha 1 (I) trimer, and type V collagens were also found in the salt-insoluble tissue residue. In this fraction the alpha 1 (I) trimer comprised 10-15% of the collagen measured as radioactivity but was difficult to discern colorimetrically. Type III collagen could not be detected in any of the fractions analyzed. From the profiles of isotope incorporation into collagens and collagen precursors, it was evident that collagen synthesis and processing was rapid. Processing of type I collagen and probably also alpha 1 (I) trimer proceeded almost entirely through procollagen intermediates. Rapid maturation of the types I and V collagens in the salt-insoluble fraction and the appearance of beta and gamma chains as early as 30 min after isotope administration. Radiolabeled procollagens were also extracted with acetic acid and penicillamine, indicating that cross-linking of collagen precursors may be involved in fiber formation.

Animals↗

PubMed2001

The collagenous domain of class A scavenger receptors is involved in macrophage adhesion to collagens.

Class A macrophage scavenger receptors (MSRs) have a remarkably broad ligand specificity and are well-known for their roles in atherogenesis and host defense. Recently, we demonstrated that these receptors also recognize and mediate adhesion to denatured forms of type I collagen. In this study, the involvement of the collagenous domain of MSRs in binding to denatured type I collagen was investigated. Transient expression of full-length, native type II MSR in COS-1 cells conferred adhesion to denatured type I collagens, whereas expression of a truncated receptor lacking the distal portion of the collagenous domain did not. Further, a synthetic peptide derived from the collagenous domain was effective in abrogating Mphi adhesion to denatured forms of type I collagen. We also addressed collagen-type specificity by examining MSR affinity for type III and type IV collagens. As with type I collagen, Mphis adhered only to denatured forms of type III collagen. Moreover, the adhesion was mediated by MSRs. In contrast, adhesion to denatured type IV collagen was not shown to be MSR-dependent, but adhesion to the native form was. MSR-mediated adhesion to types III and IV collagens was also shown to be dependent on the collagenous domain. Taken together, these data strongly suggest that the collagenous domain is involved in MSR-mediated adhesion to denatured forms of types I and III collagens and native, but not denatured, type IV collagen.

Amino Acid Sequence↗

PubMed2004

Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen.

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.

Animals↗

PubMed1994

Deposition and selective degradation of structurally-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro.

Collagen matrix deposition and turnover were studied in skin fibroblasts from a control and from a patient with lethal perinatal osteogenesis imperfecta (OI) identified as a Gly667 to Arg substitution in the alpha 1(I) chain. A culture system where ascorbic acid was included to stimulate collagen matrix formation over extended culture periods was used. Serial extraction of the control cell collagen matrix confirmed that a substantial mature crosslinked collagen matrix was formed in the control fibroblast cell layer. In contrast, total collagen deposition by the OI fibroblasts was poor, with the quantity of collagen deposited only about a quarter of that of the control cells. Detailed analysis of the OI fibroblast matrix revealed that the mutant collagen chains were incorporated into the collagenous matrix. These data indicate that, when grown with ascorbate in long-term culture, OI fibroblasts reproduced the abnormal matrix deposition pattern of OI tissues in vivo. The overall dramatic reduction in collagen matrix formation was not accounted for by reduced collagen production, since during the period of matrix deposition (days 8-12) the rate of production by the OI cells was only slightly less than that of the control cells. The incorporation of the newly-synthesized OI collagen into the matrix was less efficient than in control cells, reflecting the cooperative nature of matrix deposition. The fate of this mutant collagen containing the Gly to Arg charge-change was followed in the matrix by a pulse-chase experiment and two-dimensional electrophoresis. These data demonstrated that the mutant incorporated into the matrix was unstable, with the proportion of mutant declining during the chase. The deposition of the mutant monomers into a pool more accessible to proteolytic degradation indicated that the mutant and normal collagens did not copolymerize to form collagen fibers of even collagen distribution, but rather the mutant collagen was either enriched on the exposed surfaces of mixed-composition fibers, or was unable to form copolymers efficiently and polymerized into mutant-only fibrillar assemblies more prone to proteolytic attack.

Collagen↗

PubMedDate not supplied

Dermal collagen fibrils are hybrids of type I and type III collagen molecules.

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.

Adult↗

PubMed1997

Inhibition of RPE cell-mediated matrix adhesion and collagen gel contraction by crovidisin, a collagen-binding snake venom protein.

PURPOSE: Cell-mediated collagen gel contraction plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). Anti-adhesion therapy has been suggested as a promising strategy in the treatment of PVR. Crovidisin, a snake venom protein isolated from Crotalus viridis, has been shown to bind selectively to collagen and to inhibit collagen-induced platelet aggregation. In the present study, the effectiveness of crovidisin in inhibiting the attachment of retinal pigment epithelial (RPE) cells to collagen, and RPE cell-mediated collagen gel contraction, was evaluated. METHODS: Fluorescein isothiocyanate (FITC)-conjugated crovidisin was prepared and used to evaluate its binding affinity for collagen type I, fibronectin, vitronectin, and laminin. The inhibitory effect of crovidisin on RPE cell-mediated extracellular matrix attachment and collagen gel contraction was evaluated by cell adhesion and type I collagen gel contraction assays. The cytotoxic effect of crovidisin was examined with a cell proliferation assay, using the Alamar blue method. Flavoridin, an Arg-Gly-Asp-containing peptide from viper venom, was used for comparison. RESULTS: FITC-conjugated crovidisin bound selectively to collagen type I with high affinity. It did not bind to other matrix proteins, including fibronectin, vitronectin and laminin, nor to RPE cells. Crovidisin inhibited RPE cell attachment to type I collagen in a dose-dependent manner. This inhibitory effect was enhanced by the presence of flavoridin. Crovidisin also dose-dependently inhibited RPE cell-mediated type I collagen gel contraction. Crovidisin was non-toxic to RPE cells. CONCLUSIONS: Crovidisin, a snake venom-derived collagen-binding protein, possessing an inhibitory activity on RPE cell-collagen interaction and RPE cell-mediated collagen gel contraction, may be a useful tool for studying cell-collagen interaction, and a potential anti-adhesion therapeutic agent for ocular disorders in which cell-collagen interaction in involved, such as PVR.

Animals↗

PubMed2005

Regulation of platelet guanylyl cyclase by collagen: evidence that Glycoprotein VI mediates platelet nitric oxide synthesis in response to collagen.

The molecular regulation of nitric oxide synthase (NOS) in blood platelets is an uncharacterised area of platelet biology. We investigated the mechanism of collagen-stimulated NO synthesis in platelets. Our aim was to identify the key collagen receptor and downstream signalling mechanisms linking collagen to NOS activation. Collagen and the GpVI-specific platelet activator collagen-related peptide (CRP-XL) stimulated NO synthesis, as evidenced by increased [(3)H]L-citrulline production, and cyclic GMP (cGMP) formation. After platelet activation by collagen and CRP-XL was normalised, we found no differences in NOS activation or cGMP formation in response to these agonists. Blocking the interaction of collagen with integrin alpha(2)beta(1), a second collagen receptor, failed to affect NOS activation by collagen. These data indicate that collagen-induced NO synthesis is linked to GpVI activation. cGMP formation in response to collagen and CRP-XL required increased intracellular Ca(2+), Src family kinases, phosphatidylinositol 3-kinase (PI3-K) and protein kinase C. By comparison, Gp VI-independent cGMP formation induced by thrombin was Src kinase-dependent, but was independent of PI3-K and PKC. Thus the mechanisms of collagen- and CRP-XL-induced NOS activation were identical, but distinct from that of thrombin. Platelet activation in response to collagen leads to secretion of adenosine diphosphate (ADP) and thromboxane A(2) (TxA(2)). Our results demonstrate that collagen-stimulated cGMP synthesis was enhanced significantly by platelet-derived ADP and TxA(2). These results reveal that collagen stimulates platelet NOS activation through a specific Ca(2+)-dependent GpVI receptor signalling cascade, and demonstrate that collagen-induced cGMP accrual requires the release of secondary platelet agonists.

Adenosine Diphosphate↗

PubMed1982

Nature and specificity of the immune response to collagen in type II collagen-induced arthritis in mice.

To determine the role of collagen-immunity in the development of collagen-induced arthritis, DBA/1 mice were immunized with type II collagen and observed for the development of polyarthritis. 96% of the mice immunized with native type II collagen developed inflammatory arthritis between 4 and 5 wk after primary immunization. Immunization with denatured type II collagen in exactly the same manner was not effective in inducing arthritis. Cell-mediated immunity in arthritic mice was assessed by measuring [3H]thymidine incorporation by mononuclear cells cultured in the presence of collagen. The maximal proliferative response to collagen occurred at 2 wk after immunization. Equally good incorporation of label occurred when cells were cultured with native or denatured type II collagen or type I collagen. The cellular response of nonarthritic mice immunized with denatured collagen was indistinguishable from that seen in arthritic mice. Humoral immunity was assessed by an ELISA assay for antibodies to collagen. The immunoglobulin M (IgM) response peaked at 2 wk and the IgG response at 5 wk after immunization. Antisera from arthritic mice immunized with native type II collagen were relatively specific for conformational determinants on the native type II molecule although some reactivity with denatured collagen was noted. Antisera from nonarthritic mice immunized with denatured collagen primarily recognized covalent structural determinants. It was concluded that native type II collagen was essential for the induction of arthritis and that an antibody response specific for native type II collagen may be important for the development of arthritis.

Animals↗

PubMed1986

Immunohistochemical distribution of collagens types IV, V, and VI and of pro-collagens types I and III in human alveolar bone and dentine.

The aim of the present study was to characterize the composition of the organic matrix in alveolar jaw bone and dentine using antibodies against pro-collagens Types I and III and collagens Types IV, V, and VI. After demineralization of oral hard tissues in 0.2 N HCl, antigenicity was well preserved and the distribution of the pro-collagens and collagens could be demonstrated. Staining for pro-collagen Type I was prominent around osteoblasts and in pre-dentine, indicating active de novo synthesis of Type I pro-collagen. Pro-collagen Type I was ubiquitous but was less abundant in bone and dentine, whereas pro-collagen Type III was seen only in areas of bone remodeling, in peritubular spaces, and in pre-dentine. Type IV collagen was limited to the basement membranes of vessels in osteons and bone marrow. Type V collagen was detected neither in pre-dentine nor in bone. In contrast, Type VI collagen was found in dentine and bone, showing a faint but homogeneous staining which, similarly to pro-collagen Type III, was pronounced around osteoblasts and in pre-dentine, areas of active bone and dentine formation. This study showed that the organic matrix of dentine and bone contains Type VI as well as Type I collagen. Pro-collagen Type III (and to a lesser extent collagen Type VI) is transiently produced during new formation and remodeling of oral hard tissues, and disappears once the matrix calcifies. Type I pro-collagen qualifies as a general marker protein for increased osteoblastic activity. We conclude that immunostaining for the different collagen/pro-collagen types can be used to assess normal or abnormal stages of bone/dentine formation.

Adolescent↗

PubMed1997

Association of type VI collagen with D-periodic collagen fibrils in developing tail tendons of mice.

The process of the arrangement of D-periodic collagen fibrils and their growth in maturing tail tendon of mice were studied with the association of type VI collagen, from fetal day 10 to 10 weeks after birth. In tail tendons, the amount of collagen fibers gradually increased along with the diameters of D-periodic collagen fibrils during maturation. Type VI collagens first appeared on fetal day 10, when D-periodic collagen fibrils were not recognizable. Type VI collagens were observed around the fibroblastic cells in early stages of development, but were among thick collagen fibrils in the adult tendon. While the periodic distances of type VI collagen fibrils were over 100 nm at fetal days, they were packed to 80-90 nm after birth. The periodic bands were stained well with ruthenium red in adult but not in young tendons, indicating the close association of proteoglycans or glycosaminoglycans (PGs/GAGs) with maturing type VI collagens. Since type VI collagen in native form is known to associate with D-periodic collagen fibrils via PGs/ GAGs, ruthenium red-stainability on the surface of D-periodic collagen fibrils was also examined; results showed that ruthenium red-stainable elements were D-periodically associated. When the surface morphology of D-periodic collagen fibrils in adult animals was examined by atomic force microscopy, a large depth of the groove between elevated and depressed surfaces became prominent when the fibril surface was digested with hyaluronidase. Thus, it is possible to observe topologically the association of PGs/GAGs and probably that of type VI collagens with D-periodic collagen fibrils.

Animals↗

PubMed1994

Self-assembly into fibrils of collagen II by enzymic cleavage of recombinant procollagen II. Lag period, critical concentration, and morphology of fibrils differ from collagen I.

A recently developed recombinant system for synthesis of human procollagen II by stably transfected host cells was used to prepare adequate amounts of protein to study the self-assembly of collagen II into fibrils. The procollagen II was cleaved to pCcollagen II by procollagen N-proteinase (EC 3.4.24.14), the pCcollagen II was chromatographically purified, and the pCcollagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase. The kinetics for assembly of collagen II fibrils were similar to those observed previously for the self-assembly of collagen I in that a distinct lag phase was observed followed by a sigmoidal propagation phase. However, under the same experimental conditions, the lag time for assembly of collagen II fibrils was 5-6-fold longer, and the propagation rate for collagen II fibrils was about 30-fold lower than for collagen I fibrils. The relatively long lag time for the assembly of collagen II into fibrils made it possible to demonstrate that most of the conversion of pCcollagen II to collagen II occurred in the solution phase. The critical concentration at 37 degrees C for collagen II was about 50-fold greater than the critical concentration for collagen I. The Gibbs free energy change for the assembly of collagen II into fibrils was -40 kJ/mol, a value that was about 14 kJ/mol less than the free energy change for collagen I and about the same as the free energy change for the homotrimer of collagen I. Dark-field light microscopy and negative-staining electron microscopy demonstrated that the collagen II fibrils were thin and formed network-like structures. The results demonstrated, therefore, that the structural information of the monomer is sufficient to explain the characteristically small diameters and arcade-like geometry of collagen II fibrils found in cartilage and other tissues.

Cell Line↗

PubMed2006

Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates.

Collagens are thought to represent one of the most important molecular innovations in the metazoan line. Basement membrane type IV collagen is present in all Eumetazoa and was found in Homoscleromorpha, a sponge group with a well-organized epithelium, which may represent the first stage of tissue differentiation during animal evolution. In contrast, spongin seems to be a demosponge-specific collagenous protein, which can totally substitute an inorganic skeleton, such as in the well-known bath sponge. In the freshwater sponge Ephydatia mülleri, we previously characterized a family of short-chain collagens that are likely to be main components of spongins. Using a combination of sequence- and structure-based methods, we present evidence of remote homology between the carboxyl-terminal noncollagenous NC1 domain of spongin short-chain collagens and type IV collagen. Unexpectedly, spongin short-chain collagen-related proteins were retrieved in nonsponge animals, suggesting that a family related to spongin constitutes an evolutionary sister to the type IV collagen family. Formation of the ancestral NC1 domain and divergence of the spongin short-chain collagen-related and type IV collagen families may have occurred before the parazoan-eumetazoan split, the earliest divergence among extant animal phyla. Molecular phylogenetics based on NC1 domain sequences suggest distinct evolutionary histories for spongin short-chain collagen-related and type IV collagen families that include spongin short-chain collagen-related gene loss in the ancestors of Ecdyzosoa and of vertebrates. The fact that a majority of invertebrates encodes spongin short-chain collagen-related proteins raises the important question to the possible function of its members. Considering the importance of collagens for animal structure and substratum attachment, both families may have played crucial roles in animal diversification.

Amino Acid Sequence↗

PubMed1989

Effects of interferon-gamma on expression of cell surface receptors for collagen and deposition of newly synthesized collagen by cultured human lung fibroblasts.

We used cultured human diploid lung fibroblasts as a model system to examine the effects of recombinant IFN-gamma on synthesis of collagen, matrix deposition of newly synthesized collagen, and the expression of cell surface receptors for collagen. Using [3H]proline-labeled cells we found that IFN-gamma resulted in dose-dependent inhibition of fibroblast collagen synthesis. Pulse-chase experiments to analyze compartmentalization of newly synthesized collagen showed that the decrease in collagen synthesis was confined to the soluble pool of procollagen in the medium, while extracellular matrix associated collagen was not changed, indicating that a larger proportion of newly synthesized collagen was deposited into the matrix in IFN-gamma exposed fibroblasts (34.2 vs. 25.3%). This increase in the efficiency of collagen matrix deposition was associated with enhanced expression of a cell surface receptor for collagen as detected by indirect immunofluorescence labeling and analysis by flow cytometry. Fibroblasts (IMR-90) cultured in the presence of IFN-gamma (1,000 U/ml) exhibited a twofold increase in mean linear fluorescence intensity compared with cells cultured under control conditions. The distribution of log fluorescence intensity in both control and IFN-gamma exposed cells was normally distributed about the mean, indicating that discrete subpopulations with respect to receptor expression were not present. Increased fluorescence intensity and log normal distribution of fluorescence intensity also were identified in IFN-gamma-treated lung fibroblasts from a normal adult individual and two strains obtained from patients with pulmonary fibrosis. These results indicate that IFN-gamma modulates fibroblast collagen matrix deposition as well as collagen synthesis. The associated increase in collagen receptors suggests that cytokine-mediated modulation of the cell surface maybe a contributing factor in regulation of fibroblast collagen accumulation in the extracellular matrix or in cellular interaction with collagen-containing matrix. Such an effect could modulate the interaction of fibroblasts with extracellular matrix at sites of inflammation and play an important role in the remodeling of matrix during repair from tissue injury.

Adult↗

PubMed1994

Degradation of insoluble bovine collagen and human dentine collagen pretreated in vitro with lactic acid, pH 4.0 and 5.5.

The purpose of the study was to test the hypothesis that both insoluble pure type I collagen from bovine Achilles tendon and dentine collagen in root dentine powder from human teeth required acid pretreatment for subsequent degradation by trypsin, a non-specific protease. Pure type I collagen or dentine powder was treated with lactic acid, at pH 4 or 5.5, or distilled, deionized water (pH 7) as a negative control. After incubation at 37 degrees C for 24 h, extracts of pure type I collagen solutions were analysed for soluble collagen with the hydroxyproline assay. Extracts of dentine powder solution were analysed for Ca2+, total protein, final pH, and hydroxyproline. Residual, undegraded pellets were washed and then treated with trypsin or collagenase. After 24 h of incubation, the soluble fractions from the enzyme-treated pure type I collagen and dentine powder solutions were analysed for hydroxyproline. Results showed that almost no pure type I collagen was degraded during acid pretreatment. Trypsin degraded significantly more pure type I collagen in the pH 4-treated group than in the other groups. Collagenase degraded about 70% of the pure type I collagen irrespective of acid pretreatment. While acid pretreatment at pH 4 did not degrade dentine collagen, data from Ca2+ analyses and collagen breakdown by trypsin suggested that pretreatment at pH 4 demineralized and denatured dentine collagen so that the collagen could be subsequently degraded by enzymes. After pretreatment at pH 4, about 27 and 57% of the dentine collagen was degraded by trypsin and collagenase, respectively, in contrast to minimal degradation of non-acid-treated dentine collagen by the same enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Achilles Tendon↗

PubMed1983

Comparison of the growth of normal and neoplastic mouse mammary cells on plastic, on collagen gels and in collagen gels.

The growth of normal and neoplastic mouse mammary cells was compared in primary cultures on plastic, on rat tail collagen gels and in rat tail collagen gels. Cells on plastic grew for the first few days, then stopped with only a 1- to 3-fold increase in cell number after 2 weeks in culture. Cells grown on or in collagen gels grew continuously over the 2-week culture period with up to 10-fold increase in cell number for cultures on collagen gels and a 20-fold increase for cells embedded in collagen gels. The difference in growth rates between cells grown in collagen gel and those grown on collagen gels was due, in part, to the three-dimensional growth of the colonies in collagen gel their two-dimensional growth on collagen gel. Cells grown on and in collagen gel can produce an electron-dense basal lamina-like structure which is associated with collagen IV and laminin as judged by immunofluorescence. Cells grown on plastic do not form this structure. Cis-OH-proline blocks the production of collagen and inhibits the growth of the cultured cells indicating collagen production to be involved in growth. Rat tail collagen gels are a superior substratum for the growth of mouse mammary cells and this may be related to the cells' ability to form a collagen IV-containing basal lamina-like structure.

Animals↗

PubMed1987

Collagen metabolism and basement membrane formation in cultures of mouse mammary epithelial cells. Induction of 'assembly' on fibrillar type I collagen substrata.

Collagen metabolism was compared in cultures of mouse mammary epithelial cells maintained on plastic or fibrillar type I collagen gel substrata. The accumulation of dialysable and non-dialysable [3H]hydroxyproline and the identification of the collagens produced suggest no difference between substrata in the all over rates of collagen synthesis and degradation. The proportion of the [3H]collagen which accumulates in the monolayers of cultures on collagen, however, markedly exceeds that of cultures on plastic. Cultures on collagen deposit a sheet-like layer of extracellular matrix materials on the surface of the collagen fibres. Immunoprecipitation of the labelled extracts, electrophoresis, indirect immunofluorescence and immunoperoxidase techniques reveal the presence of type IV collagen, along with laminin and heparan sulfate proteoglycan in this layer, in excess over the amounts detectable on cells cultured on plastic. Transformed cells on collagen produce and accumulate more [3H]collagen, yet are less effective in basement membrane formation than normal cells, indicating that the accumulation of collagen alone and the effect of interstitial collagen thereupon do not suffice. Thus, exogenous fibrillar collagen appears to enhance, but is not sufficient for proper assembly of collagenous basement membrane components near the basal epithelial cell surface.

Animals↗

PubMed2004

Measurement of antibodies to collagen II by inhibition of collagen fibril formation in vitro.

Antibodies to type II collagen (collagen II) are pathogenic in experimental collagen-induced arthritis (CIA) and possibly also in rheumatoid arthritis (RA). Hitherto, results of assays for anti-collagen II have proven to be inconsistent. We tested whether mouse monoclonal antibodies (mAbs) to collagen II inhibit the natural self-assembly of soluble triple-stranded collagen II monomers to form insoluble polymeric fibrils. A spectrophotometric assay of self-assembly was based on change in absorbance at 313 nm, observed over 0-60 min after neutralisation and warming of a solution of monomeric collagen II. Two mAbs to collagen II (CII-CI and M2.139) strongly inhibited self-assembly of collagen II but not collagen I, whereas another antibody, CII-F4, and an irrelevant control mAb did not. Notably, CII-CI and M2.139, but not CII-F4, induce arthritis on passive transfer to naïve mice. The arthritogenic effects of mAbs CII-CI and M2.139 in vivo, and inhibition of collagen II self-assembly in vitro, may be attributable to interference with critical epitopes at sites essential for the stabilisation of the mature polymeric collagen II fibril, and, hence, the integrity of the entire cartilage matrix. This assay for inhibition of self-assembly of collagen II could be developed for routine measurement of anti-collagen II in body fluids as a marker of early RA, and perhaps also to distinguish populations of antibodies to collagen II that either have or lack the capacity to perpetuate arthritis.

Animals↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗