Up regulation of cathepsin K expression in articular chondrocytes in a transgenic mouse model for osteoarthritis.

OBJECTIVES: To study the expression of cysteine proteinases, particularly cathepsin K, and their extracellular inhibitor cystatin C in articular cartilage of transgenic Del1 mice which harbour a short deletion mutation in a type II collagen transgene and are predisposed to early onset osteoarthritis. METHODS: Northern analysis was used to measure mRNA levels of cathepsins B, H, K, L, and S, and cystatin C in total RNA extracted from knee joints of Del1 mice, using their non-transgenic litter mates as controls. Immunohistochemistry and morphometry was used to study the distribution of cathepsin K and cystatin C in the knee joints. RESULTS: Up regulation of cathepsin K mRNA expression was seen in the knee joints of transgenic Del1 mice at the onset of cartilage degeneration. Cathepsin K was found near sites of matrix destruction in articular chondrocytes, particularly in clusters of proliferating cells, and in calcified cartilaginous matrix. In intact articular cartilage of control animals, cathepsin K was only seen in a small number of chondrocytes. Upon aging, control animals also developed osteoarthritis, which was accompanied by increased cathepsin K expression. Cystatin C was mostly localised in and around chondrocytes located in calcified cartilage, with no obvious association with the onset of cartilage degeneration. CONCLUSION: The temporospatial distribution of cathepsin K in osteoarthritic cartilage suggests a role for this enzyme in the pathogenesis of osteoarthritis. Because cathepsin K can digest cartilage matrix components it may contribute to the development of osteoarthritic lesions. These data may provide new clues for the development of treatments aimed at preventing cartilage degeneration.

Differential expression patterns of matrix metalloproteinases and their inhibitors during development of osteoarthritis in a transgenic mouse model.

OBJECTIVE: To characterise the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during degeneration of articular cartilage in a transgenic Del1 mouse model for osteoarthritis. METHODS: Northern analysis was used to measure mRNA levels of MMP-2, -3, -8, -9, -13, and -14, and TIMP-1, -2, and -3 in total RNA extracted from knee joints of transgenic Del1 mice, harbouring a 15 amino acid deletion in the triple helical domain of the alpha1(II) collagen chain, using their non-transgenic littermates as controls. Immunohistochemistry was used to study the presence of cleavage products (neoepitopes) of type II collagen, and the distribution of MMP-13 and TIMP-1 in degenerating cartilage. RESULTS: Each of the MMP and TIMP mRNAs analysed exhibited distinct expression patterns during development and osteoarthritic degeneration of the knee joint. The most striking change was up regulation of MMP-13 mRNA expression in the knee joints of Del1 mice at the onset of cartilage degeneration. However, the strongest immunostaining for MMP-13 and its inhibitor TIMP-1 was not seen in the degenerating articular cartilage but in synovial tissue, deep calcified cartilage, and subchondral bone. The localisation of type II collagen neoepitopes in chondrocytes and their pericellular matrix followed a similar pattern; they were not seen in cartilage fibrillations, but in adjacent unaffected cartilage. CONCLUSION: The primary localisation of MMP-13 and TIMP-1 in hyperplastic synovial tissue, subchondral bone, and calcified cartilage suggests that up regulation of MMP-13 expression during early degeneration of articular cartilage is a secondary response to cartilage erosion. This interpretation is supported by the distribution of type II collagen neoepitopes. Synovial production of MMP-13 may be related to removal of tissue debris released from articular cartilage. In the deep calcified cartilage and adjacent subchondral bone, MMP-13 probably participates in tissue remodelling.

Collagens I and III in a porcine bronchial model of obliterative bronchiolitis.

The main extracellular matrix components of the lung, type I and III collagens, were studied in chronic allograft rejection developing in a porcine heterotopic bronchial transplantation model. Specific porcine complementary DNA probes were constructed for detection of the expression of type I and III procollagen messenger RNAs in the bronchial wall structures and in the obliterative plug by in situ hybridization. In autografts, and in allografts immunosuppressed with 40-O-(2-hydroxyethyl)-rapamycin, cyclosporine A, and methylprednisolone, no histological changes of obliterative bronchiolitis (OB) developed, and the number of fibroblast-like cells expressing type I and III procollagen mRNA remained low. In nontreated allografts obliterating within 21 d, a preponderance of fibroblast-like cells showing positivity for type III procollagen mRNA existed in the obliterative plug and bronchial wall. This study shows for the first time the temporal and spatial activation of type I and III procollagen genes during the course of obliterative bronchiolitis. The number of cells expressing procollagen III mRNA increased parallel to developing obliteration and fibrosis in nontreated allografts, whereas autografts and immunosuppressed allografts exhibited no such trend. This finding suggests a positive association between type III collagen mRNA expression in fibroblast-like cells and development of obliterative bronchiolitis.

Murine fibromodulin: cDNA and genomic structure, and age-related expression and distribution in the knee joint.

The genomic structure of murine fibromodulin was determined, and its age-related expression and distribution were characterized in knee epiphyses, with decorin studied for reference. Fibromodulin, as well as decorin, have roles in collagen fibrillogenesis both in vitro and in vivo. The murine fibromodulin gene, Fmod, was similar with that in other species, with three exons and 86% of the translated sequence in exon 2. The 2.7 kb long cDNA contains an open reading frame of 1131 nt. Fibromodulin mRNA levels were highest in tissues rich in fibrillar collagens type I or type II. During growth, the distribution of fibromodulin mRNA was similar with that of type II collagen, with the highest levels between 5 days and 1 month of age. Thereafter, the expression of type II collagen declined to a level near the detection limit, whereas the fibromodulin expression decreased less markedly to a level of approx. 35% of maximum, and remained constant throughout the rest of the observation period. In contrast, decorin mRNA levels were the highest in old animals. Pericellular deposition of fibromodulin was strong around the late-hypertrophic chondrocytes of the secondary ossification centre and in the growth plate. In young epiphyses, both fibromodulin and decorin were found interterritorially, mainly in the uncalcified and deep-calcified cartilage. In the old mice, calcified cartilage became enriched with regard to fibromodulin, while, in contrast, decorin deposition diminished, particularly near the tidemark. In the subchondral bone trabeculae, decorin was found in the endosteum of growing, but not in the mature, epiphyses. Differences in the expression and distribution profiles suggest different roles for fibromodulin and decorin in the regulation of collagen fibrillogenesis, maintenance of the fibril organization and matrix mineralization. As fibromodulin is deposited closer to cells than decorin, it may have a primary role in collagen fibrillogenesis, whereas decorin might be involved in the maintenance of fibril structures in the interterritorial matrix.

Osteoarthritis-like lesions in transgenic mice harboring a small deletion mutation in type II collagen gene.

OBJECTIVE: The present study was conducted on transgenic Del1 (+/-) mice harboring six copies of a transgene with small deletion mutation engineered into mouse type II collagen gene. Incorporation of transgene into mouse genome was predicted to cause reduced mechanical strength of articular cartilage with deposition of structurally inferior collagen network and consequently to predispose the animal to early-onset joint degeneration. DESIGN: Progression of degenerative chances in the knee joints of Del1 (+/-) and control mice was followed by macroscopic and histologic analyses at 3-5 month intervals between 3 and 22 months of age. Expression and distribution of type II collagen was studied with Northern hybridization, RNase protection assay and immunohistochemistry. RESULTS: Articular cartilage degeneration began with superficial fibrillation at the age of 3 months in Del1 (+/-) mice. These changes coincided with a significant reduction in the expression of both endogenous and transgene-derived type II collagen mRNA. The defects gradually progressed into erosions penetrating the articular cartilage, bony sclerosis, degeneration of menisci, mineralization of various joint structures, cyst formation and exposure of subchondral bone. Nontransgenic controls also developed osteoarthritic lesions, but these appeared significantly later and were less severe. Increased transcription of type IIA procollagen mRNA, typical for chondroprogenitor cells and cartilage repair was also observed at six months in Del1 (+/-) mice. CONCLUSION: These findings suggest that the impact of truncated type II collagen transgene, together with maturation-related reduction in type II collagen production significantly contribute to the early-onset degeneration of knee joints in Del1 (+/-) mice. These mice with osteoarthritis-like phenotype should provide a useful model for studies on the early pathogenic mechanisms involved in articular cartilage degeneration.

Tumor necrosis factor-alpha inhibits collagen synthesis in human and rat granulation tissue fibroblasts.

The purpose of the study was to examine the effects of tumor necrosis factor-alpha (TNF-alpha) on collagen gene expression in rat and human granulation tissue fibroblast cultures. The cells were exposed to 0.1, 1, 10, or 100 ng/ml of TNF-alpha, and the rate of collagen synthesis was measured as synthesis of protein-bound 3H-hydroxy-proline. Total cellular RNA was isolated from fibroblasts, and measurements of specific cellular RNAs from fibroblasts were performed by Northern blot hybridizations using 32P-labeled cDNA probes. In cultures of rat granulation tissue fibroblasts TNF-alpha decreased 3H-hydroxyproline production to about 75% of that in controls and it also decreased pro alpha 1(I) and pro alpha 1(III) collagen mRNA levels, maximally to 33% and 23% of the control levels, respectively. In cultures of human granulation tissue fibroblasts a similar inhibiting effect in the production of collagen was seen. TNF-alpha decreased the production of 3H-hydroxyproline to 56% of the control value with a dose of 100 ng/ml also having an inhibiting effect on pro alpha 1(I) collagen mRNA levels of up to 43% of the control level. However, no effect was seen on pro alpha 1(III) collagen mRNA levels.

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.

Gene expression during bone repair.

Detailed understanding of the basic events in fracture healing constitutes a foundation for the development of new approaches to stimulate bone healing. Since the fracture healing process repeats, in an adult organism, several stages of skeletal growth in the same temporal order, it offers an interesting model for developmental regulation of cellular phenotypes and tissue-specific genes. Molecular biology has introduced new methods to study the regulatory phenomena during the process of fracture repair. Gene technology has also produced purified growth factors for research, which will help to understand their roles in fracture healing. This review summarizes data on the regulation of genes coding for extracellular matrix components and growth regulatory molecules during fracture healing. The information available focuses on the sequential expression of genes coding for collagens, proteoglycans, and some other matrix proteins during secondary (callus) healing. The temporal and spatial appearance of the different connective tissue components, mesenchyme, cartilage, and bone, are closely linked to the expression of genes coding for their characteristic constituents. Members of the transforming growth factor-beta superfamily, such as the bone morphogenetic proteins (BMP), are currently the most interesting ones among the factors that regulate chondrogenesis and osteogenesis. In the coming years, the availability of new cloned probes combined with sensitive analytical methods, as reviewed here, will add greatly to our understanding of the various aspects of gene expression during bone repair. This information should provide answers to some of the unresolved questions in fracture callus development.

Characterization of excessive collagen production during development of pulmonary fibrosis induced by chronic silica inhalation in rats.

The activation of collagen synthesis during development of silicotic fibrosis was studied in rats exposed, in dusting chambers, to respirable SiO2 for periods of 2, 4, 6 or 12 months. Control animals were exposed similarly to clean air or TiO2. Development of fibrosis was followed by histological examination, measurement of lung weight and determination of lung collagen content (as hydroxyproline). A steady increase in lung weight and collagen content together with changes in cellularity and metabolic activity of the lungs, as ascertained by chemical determination of DNA and RNA, were measured in the lungs of the SiO2-exposed animals. Hybridization of total lung RNA, extracted at each time point, with cDNA probes specific for type I and type III procollagen mRNA levels showed that the development of fibrosis was associated with increased levels, as compared to age matched controls, of pulmonary procollagen mRNAs. Interestingly, the highest levels of procollagen mRNAs were observed in young (pretreatment control) animals, suggesting that during pulmonary development collagen metabolism in lungs is even greater than during development of fibrosis. In rats exposed to SiO2 the increase in type III procollagen mRNA occurred earlier than the increase in type I procollagen mRNAs. These observations demonstrate both age-dependent and silicosis-related changes in pulmonary procollagen mRNA levels. The results suggest that development of silicosis is associated with an altered capacity of the lungs to regulate collagen accumulation.

Modulation of collagen metabolism by glucocorticoids. Receptor-mediated effects of dexamethasone on collagen biosynthesis in chick embryo fibroblasts and chondrocytes.

The steroid modulation of collagen metabolism was studied by injecting chick embryos with dexamethasone in vivo, and collagen synthesis was subsequently assayed by pulse-labeling the tissue with [14C]proline in vitro. The synthesis of [14C]hydroxyproline in tendons and sterna from chick embryos treated with dexamethasone was markedly reduced as compared with untreated controls. The inhibition of [3H]hydroxyproline synthesis was accompanied by a similar reduction in type I and II procollagen mRNA levels, as detected by Northern blot and dot blot hybridizations with chick pro alpha 1(I), pro alpha 2(I) and pro alpha 1(II) sequence specific cDNAs. The reduction in type II procollagen mRNA level was shown to be dose dependent. Control experiments indicated that the post-translational hydroxylation of prolyl residues was only slightly decreased in dexamethasone treated animals, and that the specific activity of the intracellular free proline pool and the intracellular degradation of collagen were unchanged. To address the mechanisms of the inhibition of collagen biosynthesis, specific binding of dexamethasone to glucocorticoid receptors in chick embryo tendon and cartilage cells was studied in a whole cell assay using [3H]dexamethasone as the ligand. Matrix-free tendon and cartilage cells had approximately 19,000 and 15,000 receptor sites per cell, respectively, and the binding affinities (Kd) for dexamethasone in tendon and cartilage cells were 2.9 x 10(-9) and 2.3 x 10(-9) M. Comparable values were obtained using a cytosol binding assay. The nuclear binding of dexamethasone in tendon and cartilage cells were similar. The results suggest that the dexamethasone-induced inhibition of collagen production is primarily due to decreased levels of functional procollagen mRNA, possibly resulting from receptor-mediated inhibition of the gene expression on the transcriptional level.

Activated monocytes induce arthritis-associated changes in mitochondria of cultured synovial fibroblasts.

We have recently shown that synovial fibroblasts cultured from patients with reactive or rheumatoid arthritis exhibit increased autofluorescence when compared with controls. Morphological studies suggested that this increase was related to the anomalous structure of mitochondria in cells cultured from rheumatoid or non-rheumatoid inflammatory synovial tissue. The present study describes attempts to find an explanation for these observations. The effects of conditioned media of cultured mononuclear cells were tested on normal synovial fibroblasts. Conditioned media of monocytes stimulated with lipopolysaccharide or poly-IC induced an increase in the cellular autofluorescence and changes in the morphology of mitochondria in normal fibroblasts. These changes were indistinguishable from those seen in synovial fibroblasts cultured from various arthritides. Indomethacin or gold salts did not abolish the effects of monocyte-conditioned media. Abnormal mitochondria could not be induced in the presence of cycloheximide. This study describes a new aspect of monocyte-fibroblast interactions during rheumatoid and non-rheumatoid inflammation of synovial tissue.

Construction and partial characterization of recombinant cDNA clones for chicken type I collagen messenger RNAs.

Messenger RNAs for the alpha 1 and alpha 2 chains of type I procollagen were partially purified from total embryonic chicken calvaria using gel chromatography on Sepharose 4B and used to construct recombinant cDNA clones corresponding to both mRNAs. Restriction site mapping, nucleotide sequencing and hybridization to RNA blots were used to show that clones pCAL1 and pCAL2 contain inserted sequences corresponding to the mRNAs for chicken alpha 1 and alpha 2 procollagen chains, respectively.

Fibroblast activation in scleroderma.

Skin fibroblast cultures started from 6 scleroderma patients and 5 controls were compared with respect to synthesis of collagen and glycosaminoglycans. Fibroblast strains started from skin biopsy material from patients with active localized disease exhibited appreciably increased synthesis of collagen, whereas cultures from old sclerodermatous areas showed normal or decreased rates for collagen synthesis. During subculturing, all fibroblast strains with a high initial rate of collagen synthesis exhibited a gradual decline to near-normal values by the 12th in vitro passage. No similar alterations were detected in glycosaminoglycan metabolism. The results suggest that fibroblast activation in scleroderma is due to exogenous factors; in cell culture their effects are lost and activation disappears.