COL2A1
Collagen alpha-1(II) chain precursor (Alpha-1 type II collagen) [Contains: Collagen alpha-1(II) chain; Chondrocalcin]
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Osteoarthritis (OA) is a degenerative joint disease involving both cartilage and synovium. The canonical Wnt/β-catenin pathway, which is activated in OA, is emerging as an important regulator of tissue repair and fibrosis. This study seeks to examine Wnt pathway effects on synovial fibroblasts and articular chondrocytes as well as the therapeutic effects of Wnt inhibition on OA disease severity. Mice underwent destabilization of the medial meniscus surgery and were treated by intra-articular injection with XAV-939, a small-molecule inhibitor of Wnt/β-catenin signaling. Wnt/β-catenin signaling was highly activated in murine synovial fibroblasts as well as in OA-derived human synovial fibroblasts. XAV-939 ameliorated OA severity associated with reduced cartilage degeneration and synovitis in vivo. Wnt inhibition using mechanistically distinct small-molecule inhibitors, XAV-939 and C113, attenuated the proliferation and type I collagen synthesis in synovial fibroblasts in vitro but did not affect human OA-derived chondrocyte proliferation. However, Wnt modulation increased COL2A1 and PRG4 transcripts, which are downregulated in chondrocytes in OA. In conclusion, therapeutic Wnt inhibition reduced disease severity in a model of traumatic OA via promoting anticatabolic effects on chondrocytes and antifibrotic effects on synovial fibroblasts and may be a promising class of drugs for the treatment of OA.
MeSH Terms
- Animals
- Cartilage, Articular
- Cell Proliferation
- Cells, Cultured
- Chondrocytes
- Collagen Type II
- Disease Models, Animal
- Heterocyclic Compounds, 3-Ring
- Humans
- Injections, Intra-Articular
- Male
- Mice
- NIH 3T3 Cells
- Osteoarthritis
- Primary Cell Culture
- Proteoglycans
- Synovial Membrane
- Wnt Signaling Pathway
- beta Catenin
Keywords
- Aging
- Arthritis
- Cartilage
- Fibrosis
- Inflammation
Endoplasmic reticulum stress (ERS) has been shown to participate in many disease pathologies. Recent reports have reported that ERS exists in human osteoarthritis (OA) chondrocytes. During OA, chondrocytes exhibit increased level of some senescence marker, such as senescence-associated β-galactosidase (SA β-gal) activity. However, the persistence and accumulation of senescent cells in various tissues can also impair function and have been involved in the pathogenesis of many age-related diseases, including OA. In this present study, we used IL-1β (10 ng/ml) to mimic OA chondrocytes and we found that IL-1β stimulated chondrocytes caused the increasing expression of ADAMTS5 and MMP13, decreasing COL2A1 expression, which were in accord with OA chondrocytes changes. Our data also showed that ERS is involved in the OA chondrocytes, SA β-gal activity significantly increases and inhibition of ERS can decrease the SA β-gal activity, apoptosis of OA chondrocytes and increase cell viability. These results help us to open new perspectives for the development of molecular-targeted treatment approaches and thus present an effective novel therapeutic approach for OA.
MeSH Terms
- ADAMTS5 Protein
- Apoptosis
- Cartilage, Articular
- Cell Survival
- Cellular Senescence
- Chondrocytes
- Collagen Type II
- Endoplasmic Reticulum Stress
- Gene Expression Regulation
- Humans
- Interleukin-1beta
- Matrix Metalloproteinase 13
- Models, Biological
- Osteoarthritis
- Phenylbutyrates
- Primary Cell Culture
- Signal Transduction
- beta-Galactosidase
Keywords
- Apoptosis
- Endoplasmic reticulum stress
- Interleukin-1β
- Osteoarthritis chondrocytes
- Senescence
Articular cartilage undergoes substantial age-related changes in molecular composition, matrix structure, and mechanical properties. These age-related differences between juvenile and adult cartilage manifest themselves as markedly distinct potentials for tissue repair and regeneration. To compare the biological properties and tissue regeneration capabilities of juvenile and adult bovine articular cartilage. Controlled laboratory study. Articular cartilage harvested from juvenile (age, 4 months) and adult (age, 6-8 years) bovine femoral condyles was cultured for 4 weeks to monitor chondrocyte migration, glycosaminoglycan content conservation, and new tissue formation. The cartilage cell density and proliferative activity were also compared. Additionally, the effects of age-related changes on cartilage gene expression were analyzed using the Affymetrix GeneChip array. Compared with adult cartilage, juvenile bovine cartilage demonstrated a significantly greater cell density, higher cell proliferation rate, increased cell outgrowth, elevated glycosaminoglycan content, and enhanced matrix metallopeptidase 2 activity. During 4 weeks in culture, only juvenile cartilage was able to generate new cartilaginous tissues, which exhibited pronounced labeling for proteoglycan and type II collagen but not type I collagen. With over 19,000 genes analyzed, distinctive gene expression profiles were identified. The genes mostly involved in cartilage growth and expansion, such as COL2A1, COL9A1, MMP2, MMP14, and TGFB3, were upregulated in juvenile cartilage, whereas the genes primarily responsible for structural integrity, such as COMP, FN1, TIMP2, TIMP3, and BMP2, were upregulated in adult cartilage. As the first comprehensive comparison between juvenile and adult bovine articular cartilage at the tissue, cellular, and molecular levels, the results strongly suggest that juvenile cartilage possesses superior chondrogenic activity and enhanced regenerative potential over its adult counterpart. Additionally, the differential gene expression profiles of juvenile and adult cartilage suggest possible mechanisms underlying cartilage age-related changes in their regeneration capabilities, structural components, and biological properties. The results of this comparative study between juvenile and adult bovine articular cartilage suggest an enhanced regenerative potential of juvenile cartilage tissue in the restoration of damaged articular cartilage.
MeSH Terms
- Aging
- Animals
- Cartilage, Articular
- Cattle
- Cell Count
- Cell Proliferation
- Chondrocytes
- Gene Expression Profiling
- Glycosaminoglycans
- Matrix Metalloproteinase 2
- Oligonucleotide Array Sequence Analysis
- Regeneration
Keywords
- adult
- aging
- articular cartilage
- biology of cartilage
- bovine
- cartilage regeneration
- cartilage repair
- chondrocyte
- gene expression
- juvenile
- knee
- migration
Growth plate cartilage is responsible for longitudinal growth of the long bone in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. Whilst earlier studies with a rat growth plate injury repair model have identified inflammatory, mesenchymal infiltration, osteogenesis and remodeling responses, the molecular mechanisms involved in the bony repair remain unknown. Since our recent microarray study has strongly suggested involvement of Wnt-β-catenin signalling pathway in regulating the growth plate repair and the pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, the current study investigated the potential roles of Wnt-β-catenin signalling pathway in the bony repair of injured tibial growth plate in rats. Immunohistochemical analysis of the growth plate injury site revealed β-catenin immunopositive cells within the growth plate injury site. Treatment of the injured rats with the β-catenin inhibitor ICG-001 (oral gavage at 200mg/kg/day for 8days, commenced at day 2 post injury) enhanced COL2A1 gene expression (by qRT-PCR) and increased proportion of cartilage tissue (by histological analysis), but decreased level of osterix expression and amount of bone tissue, at the injury site by day 10 post-injury (n=8, P<0.01 compared to vehicle controls). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that β-catenin inhibitor ICG-001 dose dependently inhibited expression of Wnt target genes Cyclin D1 and survivin (P<0.01). At 25mM, ICG-001 suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt/β-catenin signalling pathway is involved in regulating growth plate injury repair by promoting osteoblastogenesis, and that intervention of this signalling could represent a potential approach in enhancing cartilage repair after growth plate injury.
MeSH Terms
- Aging
- Animals
- Bone Marrow Cells
- Bridged Bicyclo Compounds, Heterocyclic
- Cartilage
- Chondrogenesis
- Gene Expression Regulation
- Growth Plate
- Immunohistochemistry
- Male
- Mesenchymal Stem Cells
- Osteogenesis
- Pyrimidinones
- Rats
- Rats, Sprague-Dawley
- Reverse Transcriptase Polymerase Chain Reaction
- Salter-Harris Fractures
- Wnt Signaling Pathway
- Wound Healing
- beta Catenin
The present study was conducted to address whether the intervertebral disc of rabbit could be considered (i) as a valuable model to provide new insights into the tissue and cellular changes of Nucleus pulposus aging and (ii) as an appropriate tool to investigate the efficacy of Nucleus pulposus cell-based biotherapies. Lumbar intervertebral disc from rabbits with increasing ages (1, 6 and 30 month-old) were compared by MRI and histological observation using Pfirrmann's grading and Boos' scoring respectively. The expression of transcripts (COL2A1, AGC1, COL1A1, MMP13, BMP2, MGP and p21) in Nucleus pulposus cells were analysed by quantitative real-time PCR. MRI analysis indicated an early age-dependent increase in the Pfirrmann's grading. Histological Boos' scoring was also increased. The analysis of transcript expression levels showed that COL2A1 and AGC1 were down-regulated as a function of age. Conversely, COL1A1, MMP-13, BMP-2, MGP and p21 were significantly up-regulated in the Nucleus pulposus cells of aged rabbit intervertebral disc. Our study describes the consistency of the rabbit as a model of intervertebral disc changes as a function of age by correlating tissue alteration with cellular modification measured.
MeSH Terms
- Aging
- Animals
- Animals, Newborn
- Disease Models, Animal
- Down-Regulation
- Extracellular Matrix Proteins
- Gene Expression Regulation, Developmental
- Intervertebral Disc
- Magnetic Resonance Imaging
- Rabbits
- Up-Regulation
Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1beta (IL-1beta) signaling in chondrocytes. Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. The Hsp90 inhibitor geldanamycin (50 nM, 100 nM, or 500 nM) was used to assess age-related responses to Hsp90 with concurrent IGF-1 or IL-1beta stimulation of chondrocytes. Quantitative real-time PCR was used to measure COL2A1 and matrix metalloproteinase 13 (MMP13) gene expression; Western blot analysis was performed to determine the phosphorylation status of p42/44 and Akt/protein kinase B. The effects of Hsp90 inhibition with geldanamycin were concentration dependent. Inhibition of Hsp90 with 100 nM or 500 nM geldanamycin blocked IGF-1-induced cell proliferation, Akt and p42/44 activation, and COL2A1 expression. Basal and IL-1beta-induced up-regulation of MMP13 mRNA was blocked by all concentrations of geldanamycin tested. Gain-of-function assays with Hsp90 resulted in increased expression of MMP13 mRNA. These results suggest that Hsp90 is involved in opposing signaling pathways of cartilage homeostasis, and that catabolic responses are more sensitive to Hsp90 inhibition than are anabolic responses. Further studies are needed to determine the role of Hsp90 inhibition in osteoarthritis in order to assess its potential as a therapeutic target.
MeSH Terms
- Aging
- Animals
- Benzoquinones
- Cartilage, Articular
- Chondrocytes
- Collagen Type II
- Gene Expression Regulation, Developmental
- HSP90 Heat-Shock Proteins
- Horses
- Insulin-Like Growth Factor I
- Interleukin-1beta
- Lactams, Macrocyclic
- Matrix Metalloproteinases
- Polymerase Chain Reaction
- RNA, Messenger
- Signal Transduction
Molecular consequences of long-term deformation and altered mechanical loading of intervertebral disc (IVD) tissue in scoliosis have yet to be elucidated. We hypothesized that histological disc degeneration is faster in scoliosis than in normal ageing and that this is reflected by an altered gene expression profile. A semiquantitative histodegeneration score (HDS) revealed significantly enhanced degeneration in scoliosis (HDS 5.3) versus age-matched control IVDs (HDS 2.25; p = 0.001). Gene expression analysis by cDNA array and RT-PCR demonstrated higher mRNA levels for extracellular-matrix molecules like aggrecan, biglycan, decorin, lumican, chondromodulin, and COL2A1 in scoliotic discs versus normal discs of identical degeneration score. No differences were evident for catabolic molecules like MMP3, MMP13, MMP17, and TIMP1. In sum, morphologic disc degeneration was accelerated by about 2 decades in scoliosis versus physiological ageing and developed against a background of stronger anabolic matrix metabolism at younger age or in response to the altered mechanical environment of the tissue.
MeSH Terms
- Adolescent
- Adult
- Aged
- Aging
- Case-Control Studies
- Child
- Collagenases
- Female
- Gene Expression
- Humans
- Intervertebral Disc
- Intervertebral Disc Displacement
- Male
- Matrix Metalloproteinase 13
- Matrix Metalloproteinases
- Matrix Metalloproteinases, Membrane-Associated
- Middle Aged
- Oligonucleotide Array Sequence Analysis
- RNA, Messenger
- Scoliosis
- Tissue Inhibitor of Metalloproteinase-1
Human intervertebral disc anulus tissue was obtained in a prospective study of immunolocalization of SOX9, a protein that plays a role in chondrogenesis and Type II collagen expression. The Human Subjects Institutional Review Board approved experimental studies. Discs were obtained from surgical specimens and from control donors. To determine whether SOX9 could be detected in discs of Thompson Grades I-IV using immunohistochemistry and to quantify the percentage of cells with SOX9 expression. SOX9 is involved with cell-specific activation of COL2A1 in chondrocytes. Recent studies have used adenoviral delivery vectors expressing SOX9 to infect a chondroblastic cell line and human disc cells; SOX9 and Type II collagen production increased. The AdSOX9 virus has also been injected directly into rabbit discs in which disc architecture was preserved for 5 weeks. Despite current interest in SOX9 for gene therapy, there have been few studies of SOX9 in normal or degenerated discs. Discs from 12 normal donors and 25 surgical subjects 15-76 years old were examined for SOX9 immunolocalization. Eight Thompson Grade I discs, 7 Grade II discs, 10 Grade III discs, and 12 Grade IV discs were studied. In Thompson Grade I discs, SOX9 was uniformly localized throughout the anulus and in some cells of the nucleus. However, in discs from adult donors, anulus cells were present that showed no SOX9 localization, although neighboring cells might be positive. Mean percent localization was 74% for Grade II discs, 69% for Grade III, and 71.6% for Grade IV. Cervical sites showed significantly greater localization than lumbar sites. Findings showed a uniform expression of SOX9 in the newborn healthy anulus. With aging and disc degeneration, some anulus cells no longer express this transcription product. These observations suggest that the loss of expression of SOX9 in some disc cells may play a role indisc aging and disc degeneration by resulting in decreased expression and production of Type II collagen.
MeSH Terms
- Adolescent
- Adult
- Aged
- Aging
- Cell Count
- Child
- Child, Preschool
- Chondrogenesis
- Collagen Type II
- Female
- Fluorescent Antibody Technique, Direct
- High Mobility Group Proteins
- Humans
- Infant
- Infant, Newborn
- Intervertebral Disc
- Intervertebral Disc Displacement
- Male
- Middle Aged
- Prospective Studies
- SOX9 Transcription Factor
- Transcription Factors
Skeletal tissues of mice with an inactivated allele of the Col2a1 gene for Type II collagen ("heterozygous knockout") were studied. To determine whether a heterozygous inactivation of the Col2a1 gene has a role in the etiology of spine disorders such as disc degeneration. Mutations in the COL2A1, COL11A1, COL11A2, and COL9A2 genes have been linked to spine disorders. However, the mechanism by which genetic factors lead to disc degeneration still are largely unknown. Spine tissues were studied using radiograph analyses; conventional, quantitative, and polarized light microscopy; immunohistochemistry for the major extracellular components, and in situ hybridization for procollagens alpha1(I) and alpha1(II). Voluntary running activity also was monitored in half of the mice. As the findings showed, 1-month-old heterozygous knockout mice had shorter limb bones, skulls, and spines, as well as thicker and more irregular vertebral endplates, which calcified earlier than in the control mice. They also had a lower concentration of glycosaminoglycans in the anulus fibrosus, in the endplates, and in the vertebral bone than the controls. These features in the heterozygous knockout mice were compensated by the age of 15 months. However, the long bones and skulls of the mature heterozygous mice remained shorter than those of the controls. Gene-deficient mice used the running wheel less. However, physical exercise did not induce any marked structural changes in the skeleton. Mice with heterozygous knockout of Col2a1 show subtle early skeletal manifestations that bear some resemblance to those of human spine disorders.
MeSH Terms
- Aging
- Alleles
- Animals
- Bone and Bones
- Collagen Type II
- Gene Silencing
- Glycosaminoglycans
- Heterozygote
- Intervertebral Disc
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Motor Activity
- Ossification, Heterotopic
- Radiography
- Reference Values
- Skull
- Spinal Diseases
- Spine
- Tissue Distribution
Type II collagenopathies consist of chondrodysplasias ranging from lethal to mild in severity. A large number of mutations has been found in the COL2A1 gene. Glycine substitutions have been the most common types of mutation. Genotype-phenotype correlations in type II collagenopathies have not been established, partly because of insufficient clinical and radiographic description of the patients. We found a glycine-to-arginine substitution at position 154 in type II collagen in two unrelated isolated propositi with spondyloepimetaphyseal dysplasia and provide a comparative clinical and radiographic analysis from birth to young adulthood for this condition. The clinical phenotype was disproportionate short stature with varus/valgus deformities of the lower limbs requiring corrective osteotomies, and lumbar lordosis. The skeletal radiographs showed an evolution from short tubular bones, delayed epiphyseal development, and mild vertebral involvement to severe metaphyseal dysplasia with dappling irregularities, and hip "dysplasia." The metaphyseal abnormalities disappeared by adulthood.
MeSH Terms
- Adult
- Aging
- Amino Acid Sequence
- Arginine
- Base Sequence
- Bone Development
- Bone and Bones
- Collagen
- Female
- Genotype
- Glycine
- Humans
- Infant, Newborn
- Male
- Molecular Sequence Data
- Osteochondrodysplasias
- Pedigree
- Phenotype
- Point Mutation
- Polymerase Chain Reaction
- Radiography
- Spine
Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. Natl. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that approximately 15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d- and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis.
MeSH Terms
- Aging
- Animals
- Base Sequence
- Body Weight
- Bone Development
- Bone and Bones
- Cartilage
- Cleft Palate
- Collagen
- Cosmids
- Exons
- Extracellular Matrix
- Female
- Gene Deletion
- Genes, Lethal
- Growth Plate
- Humans
- Male
- Mice
- Mice, Inbred Strains
- Mice, Transgenic
- Microscopy, Electron
- Molecular Sequence Data
- Oligodeoxyribonucleotides
- Pedigree
- Polymerase Chain Reaction
- Procollagen
- Reference Values
- Restriction Mapping
- Sex Factors