Biomedical subjects

E Vuorio

Publications and source records attributed to E Vuorio.

At least 91 records · Page 5Linked to original sources

PubMed1994

Normal long bone growth and development in type X collagen-null mice.

To investigate the role of type X collagen in skeletal development, we have generated type X collagen-null mice. Surprisingly, mice without type X collagen were viable and fertile and had no gross abnormalities in long bone growth or development. No differences were detected between the type X collagen-null mice and controls when growth plates of both newborn and 3-week old mice were examined by histology and by immunostaining for extracellular matrix components of bone including osteopontin, osteocalcin and type II collagen. Our results suggest that type X collagen is not required for long bone development. However, mice and humans with dominant acting type X collagen mutations have bone abnormalities, suggesting that only the presence of abnormal type X collagen can modify bone growth and development.

Animals↗

PubMed1993

Assembly of cartilage collagen fibrils is disrupted by overexpression of normal type II collagen in transgenic mice.

Cartilage collagen fibrils, which are characterized by their thin, uniform diameters, are formed of a multicomponent system of three collagen types (II, IX, and XI) and interacting proteoglycans. We have used a genetic approach to test whether the proper assembly of this multiprotein structure was altered by overexpression of one of its normal components. Here we show that in transgenic mice in which the normal mouse alpha 1(II) collagen is overexpressed, thick abnormal collagen fibrils are generated. Mice that showed the highest expression of the transgene also displayed a larger proportion of abnormal fibrils and died at birth. We propose that an imbalance among the constituents of the cartilage collagen fibrils disrupts the mechanism that controls their assembly. The results show the applicability of the transgenic mice system to studies of complex multicomponent protein assemblies in intact animals.

Animals↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1993

A standardized experimental fracture in the mouse tibia.

The increased use of transgenic mice as experimental animals provides new opportunities to study the biology of fracture repair. We have developed a technique for the production of a standard closed experimental fracture in the mouse tibia. A 0.2 mm stainless-steel rod was introduced into the medullary cavity and the pre-nailed tibial shaft was fractured by an impact device, which resulted in a reproducible transverse or slightly oblique fracture pattern. The intramedullary rod maintained axial alignment, and the fractures united without displacement. On the basis of measurements of callus geometry, four-point bending tests, biochemical analyses, and quantitative histology, the progress of callus formation and remodeling occurred in a predictable sequence of healing phases. The ultimate bending loads of the fractures increased with time, reaching 74% of the strength of intact control tibias in 4 weeks. The stiffness values of the fractures returned to normal levels and, as determined radiographically, the fractures united by external callus in 4 weeks. Radiographically, callus size, cross-sectional callus area, and callus mass peaked at 2 weeks and decreased thereafter, indicating the start of external remodeling. Histologically, the amount of mesenchymal tissue was maximal at days 5 and 7. The callus cartilage area peaked at day 9; at its maximum, it accounted for 46% of the total callus area. Early periosteal formation of membranous new bone, followed by endochondral ossification, resulted in a linear increase of callus bone during the healing process. The healing sequence of the mouse tibial fracture was similar to that seen in the rat tibia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Expression of types I, III and IV collagen genes in fibrotic skin and nerve lesions of toxic oil syndrome patients.

We have studied the skin and nerve fibrosis in toxic oil syndrome by in situ hybridization using specific cDNA probes for types I, III, and IV collagens. Fibroblasts with high levels of type I and III collagen mRNA were observed in biopsies from fibrotic skin areas. Similarly, type IV collagen mRNA was abundant in cells within the fibrotic process of the nerves. These results suggest that the excessive accumulation of collagen in toxic oil syndrome results from transcriptional activation of collagen genes in a subpopulation of fibroblasts.

Autoradiography↗

PubMed1993

Autoimmune recognition of cartilage collagens.

Recent advances in basic research on the immune system and molecular biology of cartilage components have greatly increased our understanding of the role of autoimmunity in inflammatory diseases affecting joints, particularly rheumatoid arthritis. Many of these diseases are common and their complex pathogenesis probably involves a large number of genes polymorphic in the population as well as environmental factors. Characteristic features of inflammatory arthritis include expansion of the synovial tissue into a pannus containing lymphocytes and macrophages, autoimmune reactions against cartilage antigens, and erosion of cartilage. Since hyaline cartilage of the articular surfaces is the only structure within the joint known to contain joint-specific antigens this tissue is the prime suspect as the target of the autoimmune This review will first present the capacity of the immune system to discriminate between self and non-self structures, and then summarize our current understanding of the structures of cartilage collagens. Subsequently we will discuss how the immune system normally interacts with cartilage and how such interactions can lead to arthritis. We propose that collagen-induced arthritis (CIA) is valuable for understanding the autoimmune recognition of cartilage collagen which precedes the outbreak of arthritis and may perpetuate its chronicity, and serves as an animal model of rheumatoid arthritis.

Animals↗

PubMed1993

Gene expression at graft-host interfaces of cortical bone allografts and autografts.

The healing processes of autogenous and frozen allogeneic cortical bone grafts were compared in rats. Two unicortical defects created in the proximal metaphysis of the tibia were autografted or allografted with blocks of cortical bone. The biomechanical properties of the graft-host junction and the time-related changes of the expression of genes coding for Type I, II, III, and X collagens and osteonectin were determined at one, two, four, and eight weeks. The strength of the allograft-host bone union was lower than that of autografts at the early stage of incorporation but the difference diminished by eight weeks. Northern analysis of graft mRNAs demonstrated a strong expression of Type I collagen and osteonectin genes at the beginning of autograft incorporation. The allografts showed a lower expression of the corresponding genes at one and two weeks, but the difference diminished thereafter. The gene coding for Type III collagen showed a temporary expression at two weeks both in allografts and autografts, probably because of the formation of inflammatory fibrovascular stroma provided by the host bone at the graft-host interface. No expression of cartilage-specific Type II and Type X collagens were observed, demonstrating that the healing in both grafts proceeded through primary bone healing. Compared with cortical-cortical junctions of autografts, the graft-host interfaces of allografts exhibited a reduced osteoinductive process and a slower increase of union strength.

Animals↗

PubMed1993

Regulation of extracellular matrix genes during fracture healing in mice.

The expression of several collagens, cartilage proteoglycan core protein (aggrecan), and osteonectin was investigated during healing of standardized fractures of the mouse tibia. Total RNA was extracted from fracture calluses and normal bone, and analyzed by Northern hybridization using specific cDNA probes. Based on the predominant fibrillar collagen type expressed, three phases of fracture healing were observed. Type III collagen expression characteristic for the inflammatory stage increased first, followed by Type II collagen expression indicative of the cartilaginous reparative phase, and by Type I collagen production, characteristic for the ossification and remodelling stages. Type IX collagen and aggrecan expression coincided with Type II collagen expression during the chondrogenesis. The mRNA for Type X collagen, a marker for hypertrophic chondrocytes during endochondral ossification, occurred somewhat later than that of other cartilage-specific genes. Osteonectin mRNA was present throughout the healing process and peaked during rapid new bone growth. This study confirms that the pattern of gene expression in the murine system is similar to, but somewhat faster than, other species analyzed (rat, rabbit, human).

Aggrecans↗

PubMed1993

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.

Animals↗

PubMed1992

Structural analysis of the regulatory elements of the type-II procollagen gene. Conservation of promoter and first intron sequences between human and mouse.

Transcription of the type-II procollagen gene (COL2A1) is very specifically restricted to a limited number of tissues, particularly cartilages. In order to identify transcription-control motifs we have sequenced the promoter region and the first intron of the human and mouse COL2A1 genes. With the assumption that these motifs should be well conserved during evolution, we have searched for potential elements important for the tissue-specific transcription of the COL2A1 gene by aligning the two sequences with each other and with the available rat type-II procollagen sequence for the promoter. With this approach we could identify specific evolutionarily well-conserved motifs in the promoter area. On the other hand, several suggested regulatory elements in the promoter region did not show evolutionary conservation. In the middle of the first intron we found a cluster of well-conserved transcription-control elements and we conclude that these conserved motifs most probably possess a significant function in the control of the tissue-specific transcription of the COL2A1 gene. We also describe locations of additional, highly conserved nucleotide stretches, which are good candidate regions in the search for binding sites of yet-uncharacterized cartilage-specific transcription regulators of the COL2A1 gene.

Amino Acid Sequence↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1992

Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain.

We have generated transgenic mice by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7. This mutation was expected to disturb the assembly and processing of the homotrimeric type II collagen molecule in cartilage. Expression of transgene mRNA at levels equivalent or higher than the endogenous mRNA in the offspring of two founder animals resulted in a severe chondrodysplastic phenotype with short limbs, hypoplastic thorax, abnormal craniofacial development, and other skeletal deformities. The affected pups died at birth due to respiratory distress. Light microscopy of epiphyseal growth plates of transgenic pups demonstrated a marked reduction in cartilaginous extracellular matrix and disruption of the normal organization of the growth plate. The zone of proliferating chondrocytes was greatly reduced whereas the zone of hypertrophic chondrocytes was markedly increased extending deep into the diaphysis suggestive of a defect in endochondral ossification. Electron microscopic examination revealed chondrocytes with extended RER, a very severe reduction in the amount of cartilage collagen fibrils, and abnormalities in their structure. We postulate that the deletion in the alpha 1(II) collagen acts as a dominant negative mutation disrupting the assembly and secretion of type II collagen molecules. The consequences of the mutation include interference with normal endochondral ossification. These mice constitute a valuable model to study the mechanisms underlying human chondrodysplasias and normal bone formation.

Animals↗

PubMed1992

Transgenic mice as models for heritable diseases.

The development of methods for introduction of foreign DNA stably into genome of experimental animals has opened new possibilities to study the effects of mutations in complex gene-protein systems at the level of the entire organism. Information from such experiments is directly applicable to understanding of pathogenetic mechanisms in hereditary diseases and to designing of new therapeutic approaches. Techniques are currently available for studying both dominant mutations, introduced usually by microinjection, and recessive mutations introduced by homologous recombination employing the pluripotent embryonic stem cells. It remains to be emphasized, however, that the information which can be obtained with these techniques is optimal when sufficient background information is available on the protein system in question. The purpose of this review is to describe how the transgenic mouse methodology has increased our understanding of molecular basis of diseases both at the level of protein function and gene regulation.

Animals↗

PubMed1991

Reduced amounts of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycine-to-cysteine mutation in the mouse type II procollagen alpha 1-chain gene.

We have generated transgenic mice harboring a glycine-to-cysteine mutation in residue 85 of the triple helical domain of mouse type II collagen. The offspring of different founders displayed a phenotype of severe chondrodysplasia characterized by short limbs and trunk, cranio-facial deformities, and cleft palate. The affected pups died of acute respiratory distress caused by an inability to inflate lungs at birth. Staining of the skeleton showed a severe retardation of growth for practically all bones. Light microscopic examination indicated a decrease in cartilage matrix density, a severe disorganization of growth plate architecture, and the presence of streaks of fibrillar material in the cartilage matrix. Electron microscopic analysis showed a pronounced decrease in the number of typical thin cartilage collagen fibrils, distension of the rough endoplasmic reticulum of chondrocytes, and the presence of abnormally large banded collagen fibril bundles. The level of expression of the mutant type II procollagen alpha 1 chain transgene in cartilage tissues was approximately equal to that of the endogenous gene in two of the strains. We propose that the principal consequence of the mutation is a considerable reduction in density of the typical thin cartilage collagen fibrils and that this phenomenon causes the severe disorganization of the growth plate. We also postulate that the abnormal thick collagen fibrils are probably related to a defect in crosslinking between the collagen molecules. The cartilage anomalies displayed by these transgenic mice are remarkably similar to those of certain human chondrodysplasias.

Animals↗

PubMed1991

Mouse type II collagen gene. Complete nucleotide sequence, exon structure, and alternative splicing.

Several overlapping clones covering the entire mouse type II collagen gene including 10 kilobases (kb) of 5'- and 15 kb of 3'-flanking sequences were isolated from a cosmid library. The overall gene structure was determined by restriction mapping and sequencing. The gene spans 28.9 kb from the start of transcription to the polyadenylation site and contains 54 exons. It codes for a major mRNA species of 4910 bases which translates into a polypeptide of 1419 amino acids. A less abundant RNA species of 5110 bases contains additional sequences corresponding to an alternatively spliced exon 2. Except for the amino-terminal propeptide (N-propeptide) domain the exon-intron organization of the mouse pro alpha 1(II) collagen gene is remarkably similar to genes for other fibrillar collagen types. The overall identity of the coding sequences of the mouse and human type II collagen genes is 89% at the nucleotide level, but only 37 amino acid changes occur within the mature alpha 1(II) collagen chains between mouse and man. Intron sizes are also conserved between the mouse and human genes but not with the chick alpha 1(II) gene. The promoter of the mouse type II collagen gene is similar to those of the rat and human genes containing a TATA box and several G + C-rich elements but no CCAAT box. The 3'-untranslated sequence contains two regions of high homology between chick, mouse, bovine, and human genes preceding the major polyadenylation site. Additional size variation in the mRNA arises from the use of a minor polyadenylation signal. Information on conserved noncoding sequences will help in studies on the regulation of the pro alpha 1(II) collagen gene. Detailed knowledge of the gene is also necessary for site-directed mutagenesis and work with transgenic mice.

Amino Acid Sequence↗

PubMed1991

Specific hybridization probes for mouse type I, II, III and IX collagen mRNAs.

We have constructed DNA probes for the specific detection of mouse pro alpha 1(I), pro alpha 1(II), pro alpha 1(III) and alpha 1(IX) collagen transcripts. To avoid cross-hybridization the probes for fibrillar collagens cover mainly sequences in the 3' untranslated region of the gene. Sequencing and Northern analysis confirmed that the clones share minimal sequence similarity and detect only the specific mRNAs under normal hybridization and washing conditions. The clone for mouse alpha 1(IX) collagen covers coding sequences but is sufficiently divergent from other collagen transcripts to allow specific detection of the corresponding mRNA.

Amino Acid Sequence↗

PubMed1991

Comparison on collagen gene expression in the developing chick embryo tendon and heart. Tissue and development time-dependent action of dexamethasone.

Glucocorticoids modulate various cellular functions such as proliferation, energy metabolism and the synthesis of proteins. In the present study, the response of collagen genes to dexamethasone in different stages of chick embryo development was studied in tendon and heart using Northern blot analysis and specific cDNA probes. The changes in collagen gene expression were compared to alterations in two reference mRNAs: actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The levels of specific mRNAs measured per ribosomal RNA in tendon and heart varied markedly during normal development. In tendon the relative levels of alpha 1(I), alpha 2(I) and alpha 1(III) collagen mRNAs were highest between days 14-16 when also the synthesis of matrix proteins is most active. In heart the levels of these mRNAs peaked at day 12. In addition, qualitative differences were observed in the expression of actin genes between tendon and heart. Dexamethasone in high dose decreased collagen mRNA levels in tendons, while in heart a stimulatory effect was noted. Dexamethasone also decreased GAPDH mRNA levels in tendons. The alterations in gene expression after dexamethasone treatment in tendon and heart did not correlate with the level of specific glucocorticoid receptors, which varied markedly during the development of chick embryos. The cDNA for pro alpha 1(I) collagen hybridized to two transcripts corresponding to 6.2 and 5.1 kb in tendon and heart. During normal development of chick embryos the ratio of 6.2/5.1 kb mRNAs decreased markedly in heart, but no such change was observed in tendons. Dexamethasone, however, decreased the ratio of 6.2/5.1 kb transcripts in tendons. There was a significant correlation between the ratio 6.2/5.1 kb transcripts and total alpha 1(I) mRNA both in tendon and heart, suggesting that the 6.2 kb transcript may be associated with the rate of synthesis of type I collagen.

Actins↗