Non-cartilaginous Tissues Research Articles

Structural and functional analysis of the promoter of the human alpha 1(XI) collagen gene.

In order to eventually elucidate the mechanisms regulating alpha 1(XI) collagen expression in cartilaginous and non-cartilaginous tissues, we performed an initial analysis of the structural-functional features of the promoter of the human gene (COL11A1). After cloning and sequencing the 5' portion of COL11A1, primer extension and nuclease protection assays identified several minor transcriptional start sites clustered around a major one located 318 base pairs from the ATG codon. Consistent with this finding, analysis of the upstream sequence revealed the absence of a TATA motif and the presence of several GC boxes. Transient transfection experiments delineated the smallest promoter sequence directing relatively high expression of a reporter gene in a cell type-specific manner. Nine nuclear protein-bound areas were located within this promoter sequence of the COL11A1 gene. Sequence homologies suggested that the majority of the footprints correspond to potential binding sites for ubiquitous nuclear proteins, such as AP2 and Sp1. Additional experimental evidence indicated that one of the protected areas may bind a transcriptional complex that is identical or closely related to the one that regulates tissue specificity in the coordinately expressed alpha 2(V) collagen gene.

Link protein is ubiquitously expressed in non-cartilaginous tissues where it enhances and stabilizes the interaction of proteoglycans with hyaluronic acid.

Link protein (LP) is an abundant protein of cartilage which stabilizes the interaction of aggrecan with hyaluronic acid (HA). In this study we report that LP is also present in a large number of embryonic non-cartilaginous tissues. We demonstrate, using RNase protection experiments, that the coding region of the LP mRNAs isolated from these tissues is identical to that present in cartilage. Furthermore, we show that the LP mRNAs are translated in non-cartilaginous tissues by the identification of LP polypeptides with monoclonal antibody 4B6/A5 in Western blots. LP is localized in the extracellular matrix of the mesoderm along the entire digestive tract and in the dermis of the embryonic skin as revealed by immunofluorescence analysis. Investigations on the interactions between LP and proteoglycans from skin and proventriculus demonstrate that LP can enhance the binding of proteoglycans from these tissues to HA. In addition, we find that the same proteoglycans bound to HA in the presence of LP are always more resistant to competition by soluble HA than in the absence of LP. Our results suggest that LP is involved in the stabilization of extracellular matrices of a wide variety of non-cartilaginous tissues.

Differentiation Antigens of Human Articular Chondrocytes and their Tissue Distribution as Assessed by Monoclonal Antibodies

Five monoclonal antibodies (HuMC1, HuMC2, HuMC3, HuMC4 and HuMC5) raised against intact human articular chondrocytes were tested with chondrocyte samples from arthritic and non-arthritic patients by surface immunofluorescence and by immunoperoxidase labeling of fixed cells. All acetone-fixed samples reacted strongly with the monoclonal antibodies but some variation in the percentages of intact chondrocytes positive by immunofluorescence was noted. Under culturing conditions which induced de-differentiation, epitopes recognized by HuMC1, HuMC3 and HuMC4 disappeared with time in culture. In contrast, reactivities to HuMC2 and HuMC5 either persisted or increased as the culture became more fibroblastic and these antibodies bound to antigens on human fibroblast cell lines. HuMC1, HuMC3 and HuMC4 reacted with purified adult and fetal proteoglycan. HuMC2 and HuMC5 exhibited only slight or no reactivity to either proteoglycan. All five monoclonal antibodies reacted with chondrocytes in frozen articular cartilage but HuMC2 and HuMC5 failed to react to chondrocytes in paraffin-embedded cartilage sections. Only HuMC1, HuMC3 and HuMC4 recognized matrix components in both frozen and paraffin-embedded cartilage. When tested against 29 different non-cartilaginous tissues, each of the monoclonal antibodies had distinctive reactivity patterns, suggesting that each reacted to different epitopes. HuMC3 reacted with neurons in the cerebral cortex and cerebellum, indicating that it may recognize epitopes shared on the S-100 protein. HuMC1 showed the greatest specificity for chondrosarcomas. These antibodies are useful for identifying differentiated chondrocytes, providing information on the distribution of chondrocyte antigens shared by other human tissue, assessing the extent of chondrocyte heterogeneity in a population and aiding in the classification of chondrosarcomas.

Co-expression of collagens II and XI and alternative splicing of exon 2 of collagen II in several developing human tissues.

Northern analyses, RNAase protection assays and in situ hybridizations were used to study the expression of the mRNA for the alpha 2 chain of collagen XI and the two different mRNAs generated from the collagen II gene through alternative splicing of exon 2 in several different tissues of 15-19-week-old fetuses. The highest expression levels of procollagen alpha 2(XI) and alpha 1(II) mRNAs were detected in cartilage, but, using long exposure times, Northern hybridization revealed the presence of the approximately 5.3 kb procollagen alpha 1(II) mRNA in most tissues analysed: calvarial and diaphyseal bone, striated and cardiac muscle, skin, brain, lung, kidney, liver, small intestine and colon. Both alternatively spliced forms of the mRNA were present in these tissues. In cartilage, the short form of the procollagen alpha 1(II) mRNA (without exon 2 sequences) was clearly more abundant, whereas in most of the non-cartilaginous tissues the long form was the predominant one. Low levels of procollagen alpha 2(XI) mRNA were also seen in non-cartilaginous tissues: calvarial and diaphyseal bone, kidney, skin, muscle, intestine, liver, brain, and lung. In all the other positive tissues except brain cortex, both collagen II and XI transcripts were observed. The localization of collagen II and XI signals was identical in cartilage, kidney and skin. However, in cartilage the signal with collagen II probe was much higher than that with the collagen alpha 2(XI) probe. In epidermis the situation was reversed. Our results show considerable co-expression and co-localization of procollagen alpha 1(II) and alpha 2(XI) mRNAs in many tissues of developing human fetuses. Since the collagen alpha 1(II) gene also codes for the alpha 3(XI) chain of collagen XI we propose that some, but not all, of the expression of the collagen II gene in non-cartilaginous tissues relates to collagen XI production.

Comparison of Tissue Reaction With Chondroitinase ABC and Chymopapain in Rabbits as the Basis of Clinical Application in Chemonucleolysis

Although chemonucleolysis with chymopapain is an approved treatment for lumbar intervertebral disk herniation, recent serious complications have raised doubt concerning its safety. It is therefore necessary to search for a safer and more selective agent than chymopapain for chemonucleolysis. Experimental chemonucleolysis with chondroitinase ABC was thus tested and compared with chymopapain. Acute tissue reactions to chondroitinase ABC were investigated and compared with chymopapain. The epidural space, yellow ligament, sciatic nerve, knee joint, and Achilles tendon were examined. Chymopapain damaged nervous and ligamentous tissues as well as cartilaginous tissue. Chondroitinase ABC did not damage nervous and ligamentous tissue. Chondroitinase ABC affected only cartilaginous tissue, and its action was chiefly limited to digestion of the matrix. Chondroitinase ABC has high enzymatic specificity for matrix in vivo. In addition, chondroitinase ABC is less toxic to noncartilaginous tissues than chymopapain. Chondroitinase ABC might be a more suitable and safer enzyme than chymopapain for chemonucleolysis.

The specific hydraulic conductivity of bovine serum albumin

Previous studies of extracellular matrix hydraulic conductivity have characterized the flow resistance of glycosaminoglycans, proteoglycans and collagen. This work focuses on serum albumin, present in significant quantities in many connective tissues, but not previously considered for its role in determining connective tissue flow resistance. The specific hydraulic conductivity of bovine serum albumin solutions, as a function of concentration, was calculated from sedimentation and ultrafiltration data available in the literature. A rigid particle hydrodynamic model compared favorably with these results. Experimental measurements on an albumin ultrafiltration cell were in agreement with this model (within experimental error); furthermore, the experimental data confirmed the theoretical prediction that there is no (or negligible) pressure drop through the concentration polarization layer. Use of the hydrodynamic model for albumin specific hydraulic conductivity with literature values for the hindrance of albumin when passing through a glycosaminoglycan (GAG) matrix allows an estimate of the relative importance of the albumin on tissue hydraulic conductivity: in non-cartilaginous tissues with moderate GAG concentrations, tissue levels of albumin can generate flow resistance effects comparable to those of the GAGs, although well less than the flow resistance of these tissues.

Epithelio--mesenchymal interactions are critical for Quox 7 expression and membrane bone differentiation in the neural crest derived mandibular mesenchyme.

In higher vertebrates, branchial arch mesenchyme (ectomesenchyme) is derived from the cephalic neural crest. The ectomesenchyme of the mandibular arch yields the Meckel's cartilage and several membrane bones. We previously reported the isolation of a quail homeobox gene, Quox 7. In common with its mouse counterpart Hox 7, Quox 7 is highly expressed in the medioventral part of the mandibular arch and later in the precursor cells of the membrane bones. Since bone differentiation from ectomesenchyme is strictly dependent upon a signal provided by the mandibular epithelium, we decided to see whether the regulation of Quox 7 gene activity might be correlated with epithelio--mesenchymal interactions. Quox 7 expression was studied in E3 mandibular ectomesenchyme cultured in vitro or grafted on the chick chorioallantoic membrane either alone or recombined with the homotopic and heterotopic epithelia. We found that Quox 7 mRNA was undetectable after 48 h in cultures of mesenchyme alone while it remained abundant in non-cartilaginous tissue of the mandibular arch ectomesenchyme recombined with its own epithelium. The signal provided by the mandibular epithelium for Quox 7 expression can also arise from various heterotopic epithelia, e.g. of dorsal or ventral body wall and of limb bud. Thus the effect of the epithelium on Quox 7 expression in mesenchymal cells strictly parallels that on bone formation. These results strongly suggest that the epithelio-mesenchymal interactions have an essential role on the regulation of Quox 7 gene, the product of which seems to be, in turn, necessary for the execution of the skeletal developmental program in the facial area.

Bovine cartilage types VI and IX collagens. Characterization of their forms in vivo

1. Collagens were extracted from bovine cartilage by 4 M-guanidinium chloride in the presence of proteinase inhibitors and identified by immunoblotting with specific anti-collagen sera. 2. The collagens retained their native conformations (shown by the resistance of their triple-helical domains to pepsin digestion), and the molecular masses of their component alpha-chains indicated that the chains were intact. 3. Type VI collagen was extracted as a large-molecular-mass disulphide-bonded aggregate composed of components of molecular mass 140 kDa and 200-240 kDa, and was therefore similar to type VI collagen identified in noncartilaginous tissues. Immunoblotting established the 200-240 kDa components as intact forms of the alpha 3(VI) chain. 4. Type IX collagen consisted of three clearly separable components of molecular mass 84 kDa, 72 kDa and 66 kDa, which were assigned to the alpha 1(IX)-, alpha 3(IX)- and alpha 2(IX)-chains respectively, and a large proportion of this collagen had no covalently bound glycosaminoglycan attached to the alpha 2(IX)-chain. 5. Differences between the type IX collagen extracted from bovine cartilage and that identified in biosynthetic studies on chick cartilage are discussed.

Cloning and sequencing of pro-alpha 1 (XI) collagen cDNA demonstrates that type XI belongs to the fibrillar class of collagens and reveals that the expression of the gene is not restricted to cartilagenous tissue.

We have isolated several overlapping cDNA clones encoding alpha 1(XI) collagen chains from human and rat cDNA libraries. Together the human cDNAs code for 335 uninterrupted Gly-X-Y triplets, and a 264-amino acid C-propeptide, while the rat cDNAs cover the entire C-propeptide and about a third of the triple-helical domain. Comparison of the human and rodent nucleotide sequences showed a 95% sequence similarity. The identification of the clones as alpha 1(XI) cDNAs was based on the complete identity between the amino acid sequences of three human alpha 1(XI) cyanogen bromide peptides and the cDNA-derived sequence. Examination of and the cDNA-derived amino acid sequence showed a variety of structural features characteristic of fibrillar-forming collagens. In addition, nucleotide sequence analysis of a selected portion of the corresponding human gene revealed the characteristic 54-base pair exon motif. We conclude therefore that pro-alpha 1 (XI) collagen belongs to the group of fibrillar collagen genes. We also suggest that the expression of this gene is not restricted to cartilage, as previously thought, since the cDNA libraries from which the clones were isolated, originated from both cartilagenous and noncartilaginous tissues.

Complex carbohydrates in cartilaginous and other tissues of cartilage matrix deficiency (cmd/cmd) mice as studied by light microscopic histochemical methods

Mice homozygous for the autosomal recessive gene, cartilage matrix deficiency (cmd/cmd) are characterized by disproportionate dwarfism and cleft palate and are suffered from genetic failure in biosynthesis of cartilage-type proteoglycans. To investigate histochemical aspects of the defects in cartilaginous and other tissues of the mutant mice, complex carbohydrates in the tissues have been studied by methods of light microscopy. As a result of the present investigations, the intercellular matrix of cartilaginous tissues from the mutant was shown to exhibit weaker positive reactions for sulfated and acidic complex carbohydrates, as compared with the corresponding tissues from the control. In certain muscular and nervous tissues from the mutant, the reactions for sulfated complex carbohydrates tended to be weaker in intensity than those in the control. Furthermore, the dermal tissues of the mutant showed apparent mastocytosis. All these results indicate that there exist accessory effects in non-cartilaginous tissues in addition to the defects of cartilaginous tissues in the mutant mice.

Regulation of Type I Collagen Fibril Assembly by Link Protein and Proteoglycans

Link protein, a glycoprotein, that is present both in cartilaginous and non-cartilaginous tissues, has previously been shown to bind to collagen and to proteoglycan. Here, we have examined the effects of link protein and proteoglycans, both alone and in combination, on the assembly of type I collagen fibrils in vitro. Link protein alone had no effect on the kinetics of fibril formation or on the size of the fibrils. Link protein, however, modulated the effects of various proteoglycans including those from bone, cartilage, cornea and sclera. Link protein had the most significant effect on fibril assembly in the presence of the low molecular weight bone proteoglycan. Although the bone proteoglycan alone had no effect on fibril formation, the fibrils were wider in the presence of link protein and proteoglycan. Cartilage proteoglycan alone increased the extent of fibril formation and the resultant fibrils were wider in diameter with a complement of incompletely assembled fibrils. In the presence of both link protein and cartilage proteoglycan, the fibrils were fully formed with the characteristic banding pattern. Further, corneal and scleral proteoglycans alone decreased the extent of fibril formation and the width of the fibrils was either unaltered or slightly decreased in the presence of the link protein. Our results indicate that both link protein and tissuespecific proteoglycans may regulate the organization of collagen fibrils in tissues.

Characteristics of chondroitin sulfate proteoglycans of differentiating nonchondrogenic tissues.

The chondroitin 4- and 6-sulfate proteoglycan monomers from two differentiating tissues (brain and muscle of 14-day chick embryos) were compared by indirect means, including use of chondroitinase AC, with the major class of cartilage chondroitin sulfate proteoglycan monomers. In addition to slower sedimentation in sucrose density gradients, the monomers from both noncartilaginous tissues have longer chondroitin sulfate chains and exhibit little difference in the susceptibilities of their core proteins to papain and trypsin, in contrast to the cartilage monomer. However, slight differences in average sizes and polydispersity of fragments formed by enzyme treatments and beta-elimination were found between the proteochondroitin sulfate monomers of embryonic brain and muscle. Although the chondroitin sulfate chains are attached to separate tryptic peptides from the majority of other sulfated glycosaminoglycans in the noncartilaginous tissues, it is still uncertain whether these units form part of separate proteoglycan monomers.

Radioimmunoassay of the 148-kilodalton cartilage protein. Distribution of the protein among bovine tissues.

A sensitive and specific radioimmunoassay for the 148 kDa cartilage protein (previously referred to as 'cartilage matrix protein') was developed. The protein is insoluble in conventional buffers and was therefore dissolved in sodium dodecyl sulphate. Excess sodium dodecyl sulphate was bound in mixed micelles with Triton X-100 before assay. Antibodies raised against the 148 kDa cartilage protein did not react with other cartilage macromolecules. Neither did they react with guanidinium chloride extracts of a number of non-cartilaginous tissues. The protein, then, is unique for cartilage. It was demonstrated in tracheal, nasal-septum, xiphisternal, auricular and epiphysial cartilage. Surprisingly, the protein was detected neither in extracts of articular cartilage nor in extracts of the anulus fibrosus or the nucleus pulposus of the intervertebral disc.

Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues.

To test the value of Streptomyces hyaluronidase in carbohydrate histochemistry, the effects of digestion with the enzyme on the staining of cartilage and non-cartilaginous tissues by Alcian Blue (AB) pH 1.0, AB pH 2.5, high iron diamine, low iron diamine, aldehyde fuchsin, dialysed iron-ferrocyanide and AB pH 2.5-periods acid-Schiff were studied by light microscopy. The results obtained lead to the conclusion that the Streptomyces enzyme releases not only hyaluronic acid but also chondroitin sulphates and keratan sulphates in cartilage. Since hyaluronic acid is known to be linked to chondroitin sulphate proteoglycans, the enzyme is of limited value in localizing hyaluronic acid in cartilage. However, it is useful in localizing hyaluronic acid in most non-cartilaginous tissues.

Proteoglycans of bovine periodontal ligament and skin. Occurrence of different hybrid-sulphated galactosaminoglycans in distinct proteoglycans.

A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.

Tissue sampling as a potential source of error in experimental studies of cartilage.

Although it might seem trite to point out that tissue sampling is a potential source of experimental error, this survey disclosed that even experienced investigators in fact often work with cartilage that is contaminated by non-cartilaginous tissue of which they were unaware. Twenty-two specimens ranging from chick embryo sternum to bovine nasal septum were studied by serial sectioning. Eighteen of the 22 contained extraneous tissue comprising from 3 to 50% of the cross-sectional area. The impact of the contamination depends on the use being made of the material and probably is greatest in cell culture studies because chondrocytes and fibroblasts have large differences in population doubling time. Several approaches for minimizing the error are suggested by the findings. Histological examination of specimen material is thus a desirable quality control procedure in the design and interpretation of experiments on cartilage as well as other tissues.