The biosynthesis of cartilage type collagen during limb regeneration in the larval salamander

In order to determine the relationships between the biosynthesis of cell-specific products and the morphological and cytological appearance of cells, the synthesis of cartilage type collagen was examined during different stages of regeneration of larval amphibian limbs. We have found that during blastemal formation, chondrocytes cease their synthesis of detectible levels of cartilage type collagen. This was accomplished by analyzing the radioactively labeled collagens synthesized in short-term culture by pieces of limbs containing a cross section of all limb tissues present, and comparing these collagens to the collagens synthesized by blastemas from corresponding limbs. The labeled collagens were extracted, purified, and analyzed by chromatography on carboxymethyl cellulose columns. Whereas all of the pieces of limbs analyzed, either before regeneration was initiated or after redifferentiation of cartilage had begun, synthesized clearly detectible levels of cartilage type collagen, none of the blastemas produced detectible levels of this cartilage-specific molecule. Thus, it seems that the normal production of (α1)₃, cartilage type collagen, is inhibited during the blastemal stage of amphibian limb regeneration.     

Procollagen and collagen produced by a teratocarcinoma-derived cell line,TSD4: Evidence for a new molecular form of collagen

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of θ chains (150,000 daltons), pro α chains (130,000 daltons), and α chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (α1)₃, as did collagen isolated from the cell layer. Each α1 chain appears to be slightly larger than α1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type α1(X)₃, although similar to that of the α1 chain of type I collagen, [α1(I)]₂α2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [α1(I)]₃.     

In vitro cartilage formation: effects of 6-diazo-5-oxo-L-norleucine (DON) on glycosaminoglycan and collagen synthesis.

To determine the relationships between glycosaminoglycan (GAG) synthesis and type-specific collagen synthesis, we have investigated mouse limbs cultured in the presence of antiglutamine DON (6-diazo-5-oxo-l-norleucine). When compared to control limbs, ultrastructural examination of the DON-treated limbs shows that newly formed cartilage lacks matrix granules and the collagen fibrils have an altered morphology. Using [³⁵S]sulfate as a precursor, we have found that DON (5 μg/ml) suppresses chondroitin sulfate synthesis to less than 15% of the control level. We have also examined the collagen synthesized in equivalent limbs labeled with [³H]proline. The α-chain patterns from CM-cellulose chromatography were very similar for control and experimental limbs (α1:α2 ～ 7), suggesting that both (α1)₃- and (α1)₂α2-type molecules were being produced. The (α1)₃ molecules in both cases were identified as type II collagen by fractional salt separation and cyanogen bromide peptide mapping on CM-cellulose columns. We conclude that (1) the synthesis of type II collagen can be dissociated from the production of GAG, and (2) environmental influences can be involved in controlling the fibrillogenesis of collagen.     

Termination of RNA by nucleotides of 9-beta-D-xylofuranosyladenine

The protease susceptibilities of recently identified cartilage collagens HMW, 1α, 2α, and 3α were investigated. Mammlian skin collagenase cleaved the 3α chain under conditions where HMW, 1α and 2α were not degraded. A tumor cell derived type V collagenolytic metalloproteinase degraded HMW, 1α and 2α, but not 3α. Plasmin or leucocyte elastase failed to significantly degrade any of the cartilage collagens when digestion was performed at 25°C (15 hours, enzyme to substrate ratio 1:100). At 36°C but not 33°C α thrombin degraded HMW, 1α and 2α, with little or no degradation of 3α. This pattern of protease susceptibility for HMW, 1α and 2α is therefore similar to type V collagen. The cleavage of 3α by skin collagenase but not by elastase is similar to type II collagen. These results suggest that HMW, 1α and 2α are part of the type V collagen family.     

Improved chromatographic purification of human and bovine type V collagen sub-molecular species and their subunit chains from conventional crude preparations Application to cell-substratum adhesion assay for human umbilical vien endothelial cell

Two human type V collagen sub-molecular species, designated [α1(V)]₂α2(V) and α1(V)α2(V)α3(V), were purified chromatographically from a commercially available preparation, in which cystine-rich collagenous contaminants were contained, with a column packed with Fractogel EMD SO₃⁻. From bovine crude preparations, the [α1(V)]₂α2(V) form free from the collagenous contaminants was purified. Type V collagen subunit chains were isolated from each type V collagen molecule by anion-exchange HPLC with a Bakerbond PEI Scout column. The highly purified human type V collagen molecules and their subunit chains were used to examine the inhibitory effect on human umbilical vein endothelial cell proliferation. It was confirmed that the α1(V) chain has inhibitory activity and it was found that the inhibitory effect of the [α1(V)]₂α2(V) form is stronger than that of the α1(V)α2(V)α3(V) form and that the α3(V) chain has no inhibitory activity.     

Collagen synthesis by regenerating rabbit ear tissue

The principal collagen types synthesized during two distinct phases of regeneration in rabbit ears have been investigated, in order to relate altered phenotypic expression in connective tissue cells to regeneration of cartilage. To do this, radioactively labeled collagens synthesized in short-term culture by selected regenerating ear tissues were analyzed by ion-exchange chromatography and SDS-gel electrophoresis of the intact collagens and of the cyanogen bromide peptides derived from them. Prior to the appearance of cartilage, rabbit ear holes are filled by an outgrowth of mesenchyme-like cells derived locally from adjacent tissues. These cells produce a mixture of collagens including type I, [α1(I)]₂α2, and the type I trimer, [α1(I)]₃, but not type II collagen. Trimer production represents about one-fourth of the collagen synthesized in either a 4-, 10-, or a 24-hr incubation. Trimer is not made by tissues from healing skin wounds nor is it present in normal, uninjured ear tissues. Type II collagen synthesis was detected in tissues taken from late regenerates containing histologically recognizable cartilage, and direct analysis of regenerated cartilage confirmed the presence of type II collagen in the matrix. Thus, regenerated cartilage in the rabbit ear system contains the normal cartilage collagen, type II, while the proliferating cell mass from which the cartilage develops synthesizes the unusual collagen, [α1(I)]₃.     

Isolation and native characterization of cysteine-rich collagens from bovine placental tissues and uterus and their relationship to types IV and V collagens

A simplified procedure for the fractionation and purification of different collagen types from various tissues is described which is particularly efficient in separating type-V from type-IV collagen, and highmol.-wt. (HMW) aggregates from 7 S collagen. Uterus and maternal villi contain 2 forms of type-V collagen -{α1(V)}₂α₂(V) and {α1(V)₂α₂(V)α₃(V)}—which have been separated on DEAE-cellulose. Uterus however appears to be the richest source of both HMW aggregates and the {α1(V)₂α₂(V)α₃(V)} collagen, and a probable relationship between these collagens is discussed.     

Biosynthesis of type IX collagen during chick limb development

We analyzed the collagens synthesized by developing chick limbs (stages 22 to 34). Type IX collagen synthesis started at stage 26, concurrently with the chondrogenic differentiation of limb mesenchyme, and gradually increased during subsequent stages. By stage 34, the central cartilaginous region of the limbs substantially synthesized type IX collagen, in addition to cartilage-specific type II collagen, while the outer non-cartilaginous region of the limbs synthesized predominantly type I collagen. The present study indicates that type IX collagen is cartilage-specific and can be used as a marker for the chondrogenic phenotype.     

Identification of collagen alpha1(I) trimer and normal type I collagen in a polyoma virus-induced mouse tumor.

A bone- and cartilage-forming mouse tumor, induced by transforming salivary epithelial cells with polyoma virus, contained large quantities of collagen. Two types of collagen molecules were isolated which had different solubilities in salt. One was type I collagen with a chain composition [α1(I)]₂ α2 and the other was an unusual form of type I collagen with a chain composition [α1(I)]₃. This would appear to be the first in vivo demonstration of α1 type I trimer.     

Effects of lysosomal enzymes on the type of collagen synthesized by bovine articular cartilage

Bovine articular cartilage normally synthesizes a collagen containing three identical α-chains. After pre-incubation with rat liver lysosomal enzymes, it begins to synthesize significant amounts of the more ubiquitous collagen of the (α₁)₂α₂ type. Since lysosomes are increased in osteoarthritis, it is possible that the abnormal biosynthetic patterns exhibited by cells in areas of degeneration are caused by such enzymes.Articular cartilage is an avascular tissue with very low cell density, composed primarily of extracellular substances such as collagen, proteoglycans, and glycoproteins. The structural integrity of this tissue depends on the relative proportion, nature, and structural organization of these components. Until recently, the destruction of cartilage seen in osteoarthritis was considered to result from a “wear and tear” process. This concept is not substantiated by recent ultrastructural and biochemical findings. Cellular activity in the involved areas leads to enlarged clones of chondrocytes containing increased numbers of intracellular organelles reflecting synthetic and secretory activity (1). There is an inverse correlation between the severity of the degenerative changes and the glycosaminoglycan content of the tissue (2–7). On the other hand, radioactive sulfate incorporation increases in osteoarthritis, an indication of the attempts made by the cells involved to repair the lesion (2). The nature of the proteoglycans synthesized under these conditions (less keratan sulfate and more chondroitin-4-sulfate) reflect the behaviour of immature chondroblasts (3–8). Lysosomal proteases have been associated with the degradation of the matrix (9–12). Cathepsin-D and a neutral protease which degrade proteoglycans at pH 5.0 and 7.0 respectively are considerably increased in early osteoarthritic lesions (13–15). Although the collagen content of cartilage does not change in osteoarthritis, qualitative differences may exist. Recently, we have shown that whereas normal human cartilage synthesizes only cartilage type collagen or (α₁-Type II)₃, osteoarthritic cartilage synthesizes in addition significant amounts of (α₁)₂α₂ collagen (skin type) (16). Articular cartilage collagen is quite different from other ubiquitous forms of mammalian collagens. In addition to containing three identical α-chains, it has four to five times more hydroxylysine and glycosidically associated carbohydrate than collagen from other tissues (17). It is quite possible that the abnormal collagen deposited by the cells at the site of degeneration may give rise to a mechanically weaker structure and lead to a loss of cartilage. While attempting to elucidate the mechanism underlying this abnormal metabolic pattern, it became apparent that lysosomal enzymes can alter the function of normal cartilage cells causing them to synthesize non-specific collagen molecules.     

The effect of embryo extract on the types of collagen synthesized by cultured chick chondrocytes.

Growth of embryonic chick chondrocytes in dialyzed embryo extract results in both a change in morphology of the cells toward that of a fibroblast and a change in the type of collagen synthesized from the cartilage-specific Type II collagen (chain composition [α1(II)]₃) to a mixture of Type I collagen (chain composition [α1(I)]₂α2) and the Type I trimer (chain composition [α1(I)]₃). Analyses after 6 days of growth in embryo extract show that the synthesis of only Type I collagen and the Type I trimer can be detected. However, on subculturing the cells to a low density and allowing a period of growth without embryo extract, colonies of chondrocytes reappear and the synthesis of Type II collagen apparently resumes. It is suggested that the observed changes represent a “modulation” in cell behavior, this being expressed not only by the morphological changes but also by changes in cell-specific protein synthesis as demonstrated by the changes in the type of collagen synthesized.     

The NC2 Domain of Type IX Collagen Determines the Chain Register of the Triple Helix

Precise mapping and unraveling the mechanism of interaction or degradation of a certain type of collagen triple helix requires the generation of short and stable collagenous fragments. This is a great challenge especially for hetero-trimeric collagens, where chain composition and register (stagger) are important factors. No system has been reported that can be efficiently used to generate a natural collagenous fragment with exact chain composition and desired chain register. The NC2 domain (only 35–50 residues) of FACIT collagens is a potent trimerization domain. In the case of type IX collagen it provides the efficient selection and hetero-trimerization of three distinct chains. The ability of the NC2 domain to determine the chain register of the triple helix is studied. We generated three possible sequence combinations (α1α1α2, α1α2α1, α2α1α1) of a type I collagen fragment (the binding region for the von Willebrand factor A3 domain) attached to the NC2 domain. In addition, two control combinations were produced that constitute homo-trimers of (α1)₃ or (α2)₃. For the hetero-trimeric constructs, α1α1α2 demonstrated a higher melting temperature than the other two. Binding experiments with the von Willebrand factor A3 domain revealed the homo-trimer of (α1)₃ as the strongest binding construct, whereas the homo-trimer of (α2)₃ showed no binding. For hetero-trimers, α1α1α2 was found to be the strongest binding construct. Differences in thermal stability and binding to the A3 domain unambiguously demonstrate that the NC2 domain of type IX collagen determines not only the chain composition but also the chain register of the adjacent triple helix.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition by collagen chains of lysyl hydroxylase of chick embryo and of WI-38 human lung fibroblasts.

Lysyl hydroxylase from chick embryos was strongly inhibited by heat-denatured collagens from various vertebrate sources, and by separated a chains and β components of rat tail tendon collagen. The kinetics exhibited with this enzyme when heat-denatured calf or rabbit skin collagens was used showed a mixed type of inhibition. On the other hand, a preparation of homologous heat-denatured 4,5-³H-L-lysine-labeled collagen, in itself an extremely poor substrate for the hydroxylase, showed non-competitive inhibition with a K_i of about 8–9 μM. Finally, the lysyl hydroxylase preparations from WI-38 fetal human lung fibroblasts and from transformed WI-38 cells (WI-38 VA 13) were also inhibited by heat-denatured collagens or, where tested, by separated collagen chains.     

Quantitation of collagen in polyacrylamide gels by fluorescent scanning of MDPF1-labeled proteins: An improvement over densitometric scanning of gels stained by coomassie blue

Purified α chains of collagen were made fluorescent by coupling with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) and then electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. The migration of MDPF-labeled collagen was similar to unlabeled collagen α chains except that MDPF-α1(I) migrated closer to MDPF-α2. The area under the peaks recorded from fluorometric scanning of MDPF-labeled α1(I), α2, and α1(III) was linear from 10^?5 to 10^?8 g. The standard curves for the three α chains were similar. The results from nonreplicate determinations had ±6% SE. This method is an improvement over staining with Coomassie blue. It has a greater sensitivity, peak area is independent of migration distance, has a wider range of linearity, and permits observation of bands during electrophoresis with quantitation immediately after electrophoresis.     

Quantitation of collagen types I,III and V in tissue slices by capillary electrophoresis after cyanogen bromide solubilization

A method for the determination of the proportions of major fiber-forming collagens (types I, III and V) in soft connective tissue was elaborated. The method is based on the release of insoluble collagen by CNBr with subsequent separation of the arising peptides. For routine application the peptides are separated by capillary electrophoresis (50 mM phosphate pH 2.5, 15 kV, 50°C, 70/60 cm×70 μm I.D. capillary with UV detection at 200 nm). Quantitation of collagen type I can be done either on the basis of spiking the sample with a peptide mixture obtained from a known amount of collagen type I, or by spiking the sample with an equimolar mixture of the two peptides [α₁(I)CB₂ and α₁(I)CB₄] (constituting a fused peak) along with α₁(III)CB₂ and α₁(V)CB₁. Compared to the previously published methods the procedure is faster and does not require isolation of marker peptides by tedious chromatographic procedures in a preceding preparatory step. Good results are obtained within a wide range of run buffer concentrations and applied voltages; conversely, intensive cleaning of the capillary after every three runs is recommended with a new capillary after 20–30 runs.