Specific hybridization probes for mouse alpha 2(IX) and alpha 1(X) collagen mRNAs.

We have used polymerase chain reaction (PCR) technology and available cross-species sequence information to construct cDNA probes for mouse alpha 2(IX) and alpha 1(X) collagen transcripts. Sequencing confirmed the identification of the clones. Northern analysis proved sufficient divergence of the cloned sequences from other collagen transcripts: specific detection of the mouse 2.9 kb alpha 2(IX) and 3.3 kb alpha 1(X) collagen mRNAs was seen under normal hybridization and washing conditions.

Expression of mRNAs for collagens and other matrix components in dedifferentiating and redifferentiating human chondrocytes in culture.

Cell cultures were initiated from epiphyseal cartilages, diaphyseal periosteum, and muscle of 16-week human fetuses. Total RNAs isolated from these cultures were analyzed for the levels of mRNAs for major fibrillar collagens, two proteoglycan core proteins and osteonectin. In standard monolayer cultures the differentiated chondrocyte phenotype was replaced by a dedifferentiated one: the mRNA levels of cartilage-specific type II collagen decreased upon subculturing, while those of types I and III collagen, and the core proteins increased. When the cells were transferred to grow in agarose, redifferentiation (reappearance of type II collagen mRNA) occurred. Fibroblasts grown from periosteum and muscle were found to contain mRNAs for types I and III collagen and proteoglycan cores. When these cells were transferred to agarose they acquired a shape indistinguishable from chondrocytes, but no type II collagen mRNA was observed.

Identification of messenger RNA for human type II collagen.

Total RNA was purified from human fetal calvaria and articular cartilage. Messenger RNAs for type I and II collagens were identified by hybridization using cDNA clones for chicken pro alpha 1(I)-, pro alpha 2(I)- and pro alpha 1(II)collagen mRNAs and by analysis of cell-free translation products of these RNAs by polyacrylamide gel electrophoresis. The size of human pro alpha 1(II)collagen mRNA is approx. 5100 bases. Translatability of cartilage specific type II collagen mRNA was found to be concentration dependent: with increasing total RNA concentrations the relative translation of type II collagen mRNA was reduced with respect to type I mRNAs.

Molecular cloning of the human alpha 2(IX) collagen cDNA and assignment of the human COL9A2 gene to chromosome 1.

Type IX collagen, a heterotrimer of alpha 1(IX), alpha 2(IX) and alpha 3(IX) chains, is a cartilage-specific fibril-associated collagen. In the process of characterizing genomic clones for the mouse alpha 2(IX) collagen gene four pairs of oligonucleotide primers designed for amplification of murine exon sequences were also utilized to construct cDNA clones for human alpha 2(IX) collagen spanning > 90% of the coding region. The amino acid and nucleotide sequence identities between human and chick are 78% and 71%, respectively. Localization of the COL9A2 gene to human chromosome 1 was subsequently performed using a panel of DNAs from human/rodent somatic cell hybrids.

Osteoinductive proteins.

Cartilage and bone tissues are rich in different polypeptide factors (growth factors) which participate in the regulation of skeletal development and growth. Parallels between the embryonal endochondral ossification, callus formation during fracture repair, and ectopic bone induction in postnatal life have encouraged scientists to search for common mechanisms underlying these processes. A set of polypeptide factors belonging to the TGF-beta superfamily called the bone morphogenetic proteins (BMPs), have been found to be of fundamental importance both in bone formation and mesenchymal-epithelial interactions in early embryogenesis. Thus, this group of proteins is a common denominator in all the above-mentioned processes involving osteoinduction and there is great potential for their clinical application as bone-inducing factors.

Determination of the single polyadenylation site of the human pro alpha 1(II) collagen gene.

Several cDNA clones for the human type II procollagen mRNA were isolated from a cartilage cDNA library. Six of the clones containing the longest inserts were subjected to restriction site mapping for alignment. All these six clones extended to the poly A tail. The longest clone, containing a 1470 bp insert, was named pHCAR3. Sequencing of pHCAR3 made it clear that neither of the two canonical AATAAA sequences of the human type II collagen gene is used as the polyadenylation signal. Two 60 bp stretches of high interspecies homology terminating in a hexanucleotide ATTAAA, located 23 nucleotides upstream of the poly A tail, apparently have an important role in determining the single polyadenylation signal for this gene. S1 protection experiments confirmed these observations.

Specific collagen mRNAs elucidate the histogenetic relationship between the growth plate, the tissue in the ossification groove of Ranvier, and the cambium layer of the adjacent periosteum. A preliminary report.

In situ hybridization was employed to study cellular phenotypes in the ossification groove of Ranvier in young rabbits. The expression of Type II collagen mRNA was seen in chondrocytes in all layers of the growth plate. Messenger RNA for the cartilage-specific Type II collagen was also detected in cells of the inner layer in the ossification groove and of the cambium layer of the adjacent periosteum, providing strong evidence for the views of Ranvier that these cells originate in the growth plate. These findings modify the interpretation of the pathogenesis of diseases of the growing skeleton.

Construction and identification of a cDNA clone for human type II procollagen mRNA.

Double-stranded cDNA was constructed for poly(A)-containing RNA isolated from foetal human articular cartilage known to contain small amounts of pro alpha 1 (II) collagen mRNA. A 585 base pair PstI-EcoRI cDNA fragment was isolated and cloned into plasmid pBR322. A resulting recombinant plasmid pHCAR1 was shown to hybridize specifically to a 5.4 kilobase mRNA in cartilage but not in calvarial RNA. Definite identification of clone pHCAR1 was based on sequence analysis; marked homology with the corresponding chick gene and complete agreement with the human gene sequences available were observed.

Exclusion of the COL2A1 gene as the mutation site in diastrophic dysplasia.

The involvement of the cartilage specific type II collagen gene (COL2A1) was studied in nine patients with diastrophic dysplasia in the Finnish population, where the prevalence of this chondrodystrophy clearly exceeds that reported for other populations. COL2A1 was chosen as the candidate gene based on previous morphological and chemical studies which suggested abnormal structure of type II collagen in diastrophic dysplasia. Southern analysis of the patients' DNA showed no disease related differences in any of the restriction fragments covering the 30 kb COL2A1 gene. As a second approach, the nine patients and their 74 relatives were studied for the inheritance of the type II collagen gene. Three of the patients with diastrophic dysplasia were not homozygous for the intragenic RFLP markers, which suggests that the disease is not linked to the type II collagen gene. Multipoint linkage analysis gave a lod score of -2.95, which conclusively excluded the COL2A1 gene as the mutation site in diastrophic dysplasia in these families.

Polymorphic restriction sites of type II collagen gene: their location and frequencies in the Finnish population.

Restriction fragment length polymorphism (RFLP) of the cartilage-specific type II collagen gene has been studied in the Finnish population. Two high-frequency alleles, also reported in other populations, were detected. The HindIII allele had a frequency of 0.33, and that detected with PvuII a frequency of 0.46. Both of these frequencies resembled the ones reported for other populations. Also one BamHI allele, not earlier reported, was found at a low frequency. Two other previously reported polymorphisms for BamHI and EcoRI were not detected in the Finnish population. The RFLPs showed a fair agreement with the Hardy-Weinberg equilibrium. A linkage disequilibrium was found between PvuII and HindIII markers. The alpha 1(II) collagen gene seems to be more conserved in populations of various origins than the alpha 2(I) collagen gene. These polymorphic collagen markers would be useful in linkage studies of various inherited cartilage disorders.

The exon structure of the mouse alpha 2(IX) collagen gene shows unexpected divergence from the chick gene.

One cosmid and two overlapping phage clones covering the entire mouse alpha 2(IX) collagen gene including 12 kilobase pairs (kb) of 5'- and 8 kb of 3'-flanking sequences were isolated from two genomic libraries. The overall gene structure was determined by restriction mapping and nucleotide sequencing. The gene spans 16 kb from the start of transcription to the polyadenylation site and contains 32 exons. It codes for a mRNA of 3 kb that translates into a polypeptide of 688 amino acids. The intron-exon junctions and mRNA structure were confirmed by amplification of cDNA made for mouse cartilage RNA. The coding sequence of the mouse alpha 2(IX) collagen gene shows marked similarities to those for other type IX collagen chains. Although the overall exon-intron organization of the mouse gene is very similar to the chick alpha 2(IX) gene, some unexpected differences were observed at the splice junctions. Split codons characteristic for the central triple helical domain of the chick were not found in the mouse gene that thus exhibited a long stretch of exons with sizes that are multiples of 9 base pairs in this domain. The promoter of the mouse alpha 2(IX) collagen gene contains some G + C-rich elements including three Sp1 consensus recognition sites and a far upstream CCAAT box but no TATAA box. Both primer extension and RNase protection assays revealed several transcription start sites within 418 base pairs of the promoter. The present study reports the first complete nucleotide sequence of any type IX collagen gene and forms the basis for comparative structural studies on this collagen type and for experiments involving transgenic mice.

Nuclear factor binding to an AP-1 site is associated with the activation of pro-alpha 1(I)-collagen gene in dedifferentiating chondrocytes.

Isolated chondrocytes grown on plastic gradually lose their differentiated phenotype upon subculturing. This dedifferentiation is manifested by an altered production of extracellular-matrix molecules (ECM): e.g., the cartilage specific type II collagen is replaced by types I and III. We have studied the regulation of ECM gene expression in dedifferentiating human and murine fetal chondrocytes. Nuclear extracts from dedifferentiated cells, human fetal fibroblasts and 3T3 cells contained a protein that bound in an electrophoretic mobility shift assay to an AP-1 site in the first intron of the human alpha 1(I) collagen gene. This binding activity was not present in freshly isolated human or murine chondrocytes, which produced type II, but not type I, collagen mRNA in culture. Thus the binding activity was induced simultaneously with alpha 1(I)-collagen-gene expression during dedifferentiation. The specific interaction was sensitive to dephosphorylation of the nuclear extract and to chemical modification of reduced cysteine residues. The AP-1 site we studied had previously been shown to be a positive transcriptional contributor in the first intron to the expression of the alpha 1(I) collagen gene. In transient transfections into dedifferentiating chondrocytes, an alpha 1(I) collagen expression plasmid carrying a mutated AP-1 site in the first intron resulted in three-times-lower reporter gene RNA levels than a plasmid carrying the respective functional AP-1 site. These data suggest that the AP-1 sequence and its respective trans-acting factors may play a role in the transcriptional regulation of the alpha 1(I) collagen gene during dedifferentiation of chondrocytes.

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.

Incorporation of cortical bone allografts and autografts in rats: expression patterns of mRNAs for the TGF-betas.

Healing of bone grafts is dependent on the rate of new bone formation. To understand better the regulation of new bone formation in the graft we have studied local production of TGF-beta1, 2 and 3, and of the small proteoglycans by determining their mRNA levels in a rat bone graft model. These mRNA levels were compared to the healing rates of autografts and allografts, as determined by histology, UV-microscopic evaluation of tetracycline-labeled new bone formation, microradiography and mechanical testing at 1, 2, 4 and 8 weeks of healing. Analyses showed that, analogous to slower bone formation in allografts, the induction of TGF-beta1 gene expression was slower than in allografts, when compared with autografts. A similar delay was seen in decorin gene expression. The results agree with the suggested role of TGF-beta1 in induction of type I collagen and osteonectin production. Our findings thus support the view that locally produced TGF-beta1 plays a role in normal graft incorporation, while local production of TGF-beta3, and particularly TGF-beta2, may be less important in this respect.

Predisposition to familial osteoarthrosis linked to type II collagen gene.

The genetic background of two families, in whom a predisposition to primary osteoarthrosis is inherited as a dominant trait, was investigated. Use of restriction fragment length polymorphisms within and around the type II collagen gene on chromosome 12 revealed a linkage between this cartilage-specific gene and primary osteoarthrosis.

The mouse collagen X gene: complete nucleotide sequence, exon structure and expression pattern.

Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3'-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3'-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice.