Human hyaluronidase-2 is localized intracellularly in articular chondrocytes and other cultured cell lines.
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Cartilage, Articular↗
Biomedical subjects
C B Knudson
Publications and source records attributed to C B Knudson.
Expression of superficial zone protein in mandibular condyle cartilage.
OBJECTIVE: Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle. METHODS: The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta) on SZP expression were examined in condyle explants and fibroblasts derived from the fibrous zone of condyle cartilage. RESULTS: SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-beta in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1beta. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-beta. CONCLUSIONS: These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself, and exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies.
Animals↗
Expression and cellular localization of human hyaluronidase-2 in articular chondrocytes and cultured cell lines.
OBJECTIVE: There is debate whether hyaluronan (HA) can be enzymatically degraded within the extracellular matrix of cartilage and other tissues or whether its catabolism occurs strictly within the lysosomal compartment of chondrocytes and other cell types. Previous studies have suggested that one of the lysosomal hyaluronidases (hyaluronidase-2) can be expressed as a functionally-active glycosyl phosphatidylinositol-linked protein at the surface of mammalian cells. If this form of hyaluronidase expression occurs in chondrocytes, this could represent a possible mechanism for extracellular HA cleavage. Thus, which hyaluronidases are expressed and where was the objective of this study. METHODS: mRNA for hyaluronidases was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and enzymatic activity by HA zymograms. Recombinant forms of hyaluronidase-2 were generated and expressed in model cell lines. A peptide-specific polyclonal antiserum was prepared to localize endogenous human hyaluronidase-2 in human articular chondrocytes. RESULTS: Hyaluronidase-2 is the principal mRNA transcript expressed by primary human articular chondrocytes as well as various model cell lines. Recombinant hyaluronidase-2, containing N-terminal or C-terminal epitope tags, was strictly localized intracellularly and not released by treatment with a phosphatidylinositol-specific phospholipase. Endogenous hyaluronidase-2 expressed by human chondrocytes as well as HeLa cells could only be detected following detergent permeabilization of the plasma membranes. CONCLUSIONS: These data suggest that on chondrocytes and other cell types examined, hyaluronidase-2 is not present or functional at the external plasma membrane. Thus, local turnover of HA is dependent on receptor-mediated endocytosis and delivery to low pH intracellular organelles for its complete degradation.
Animals↗
Induction of MMP-3 by hyaluronan oligosaccharides in temporomandibular joint chondrocytes.
Low-molecular-weight hyaluronan (LMW-HA) is often increased in osteoarthritic joints; however, its biological function in cartilage has not been clarified. We hypothesize that LMW-HA causes the catabolic activation of chondrocytes through its interaction with CD44. Cartilage explants and chondrocytes, derived from bovine temporomandibular joints (TMJ), were examined for matrix loss and the expression of matrix metalloproteinase-3 (MMP-3) following treatment with hyaluronan oligosaccharides (HA(oligos)). Hyaluronan and CD44 were uniformly distributed throughout the fibrous and cartilaginous zones of the TMJ condyle. Treatment of cartilage explants with HA(oligos) resulted in cartilage matrix loss with increased secreted caseinolytic activity. HA(oligos) treatment of TMJ chondrocytes resulted in enhanced MMP-3 expression, whereas wash-out of the HA(oligos) in the middle of the experimental period reduced this induction. These results suggest that HA(oligos) activate chondrocytes, resulting in a substantial enhancement of proteinase expression, and the removal of HA(oligos) by wash-out reverses this catabolic activation.
Animals↗
Antisense inhibition of CD44 tailless splice variant in human articular chondrocytes promotes hyaluronan internalization.
OBJECTIVE: To determine whether alternatively spliced variants of CD44, in particular a short, intracellular tail CD44 isoform, are used by articular chondrocytes to modulate the functions of this matrix receptor. METHODS: Normal human articular chondrocytes were cultured with or without interleukin-1alpha (IL-1alpha), and the relative expression of CD44 exon 19 and CD44 exon 20, hyaluronan synthase 2, aggrecan, and GAPDH messenger RNA (mRNA) was determined using reverse transcriptase-polymerase chain reaction. Next, CD44 exon 19 mRNA was selectively inhibited by the use of antisense oligonucleotides. The effects of exon 19 loss were analyzed by matrix assembly and hyaluronan internalization assays. RESULTS: Human articular chondrocytes express varying levels of exon 19 (short tail)- and exon 20 (long tail)-containing CD44 mRNA. Both CD44 mRNA are up-regulated by IL-1alpha. Selective inhibition of CD44 exon 19 results in enhanced hyaluronan internalization and smaller cell-associated matrices. CONCLUSION: The expression of a natural CD44 decoy-like receptor by articular chondrocytes modulates the function of this matrix receptor.
Aggrecans↗
Cartilage proteoglycans.
The predominant proteoglycan present in cartilage is the large chondroitin sulfate proteoglycan 'aggrecan'. Following its secretion, aggrecan self-assembles into a supramolecular structure with as many as 50 monomers bound to a filament of hyaluronan. Aggrecan serves a direct, primary role providing the osmotic resistance necessary for cartilage to resist compressive loads. Other proteoglycans expressed during chondrogenesis and in cartilage include the cell surface syndecans and glypican, the small leucine-rich proteoglycans decorin, biglycan, fibromodulin, lumican and epiphycan and the basement membrane proteoglycan, perlecan. The emerging functions of these proteoglycans in cartilage will enhance our understanding of chondrogenesis and cartilage degeneration.
Aggrecans↗
Mechanisms of chondrocyte adhesion to cartilage: role of beta1-integrins, CD44, and annexin V.
The initial adhesion of transplanted chondrocytes to surrounding host cartilage may be important in the repair of articular defects. Adhesion may position cells to secrete molecules that fill the defect and integrate repair tissue with host tissue. While chondrocytes are known to become increasingly adherent to cartilage with time, the molecular basis for this is unknown. The objective of this study was to investigate the role of beta1-integrin, CD44, and annexin V receptors in chondrocyte adhesion to cartilage. Chondrocytes were cultured in high density monolayer, released with trypsin, and allowed to recover in suspension for 2 h at 37 degrees C. Under these conditions, flow cytometry analysis showed that chondrocytes expressed beta1-integrins, CD44, and annexin V. In a rapid screening assay to assess chondrocyte adhesion to cartilage, cell detachment decreased from 79% at 10 min following transplantation to 10% at 320 min. Treatment of cells with a monoclonal antibody to block beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls. Similarly, results from a parallel-plate shear flow adhesion assay showed that blocking beta1-integrins significantly increased chondrocyte detachment from cartilage compared to untreated controls at each level of applied shear (0-70 Pa). In both assays, treatment of cells with reagents that block CD44 (hyaluronan oligosaccharides or monoclonal Ab IM7) or annexin V (polyclonal Ab #8958) had no detectable effect on adhesion. With cartilage treated with chondroitinase ABC, blocking beta1-integrins also increased chondrocyte detachment, while blocking CD44 and annexin V also had no detectable effect. Under the conditions studied here, beta1-integrins appear to mediate chondrocyte adhesion to a cut cartilage surface. Delineation of the mechanisms of adhesion may have clinical implications by allowing cell manipulations or matrix treatments to enhance chondrocyte adhesion and retention at a defect site.
Animals↗
Osteogenic protein 1 stimulates cells-associated matrix assembly by normal human articular chondrocytes: up-regulation of hyaluronan synthase, CD44, and aggrecan.
OBJECTIVE: To determine the effects of osteogenic protein 1 (OP-1) on hyaluronan (HA), CD44, and aggrecan biosynthesis as well as the contribution of these molecules in promoting matrix assembly by human articular chondrocytes. METHODS: Normal human chondrocytes were cultured with or without OP-1 treatment. Changes in the relative expression of messenger RNA (mRNA) for HA synthases 2 and 3 (HAS-2 and HAS-3), CD44, and aggrecan were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Accumulation of HA was characterized by indirect staining, CD44 by flow cytometry, and aggrecan biosynthesis by 35SO4 incorporation. RESULTS: OP-1 stimulated the expression of HAS-2, CD44, and aggrecan mRNA in a time-dependent manner, resulting in increased expression of HA, CD44, and aggrecan. Prominent increases in HA-rich cell-associated matrices were also observed. CONCLUSION: OP-1 stimulates not only the synthesis of matrix macromolecules such as aggrecan, but also the synthesis of other molecules required for matrix retention, namely, HA and CD44.
Aggrecans↗
Hyaluronan oligosaccharides perturb cartilage matrix homeostasis and induce chondrocytic chondrolysis.
OBJECTIVE: To determine the role of CD44-mediated matrix assembly in maintaining cartilage homeostasis. METHODS: Articular cartilage tissue slices as well as isolated chondrocytes were treated with hyaluronan (HA) hexasaccharides. Tissues and cells were processed for histology, immunohistochemistry, colorimetric assay, reverse transcriptase-polymerase chain reaction, and zymography. RESULTS: HA oligosaccharides induced a dose-dependent state of chondrocytic chondrolysis, including near-total loss of stainable proteoglycan-rich matrix, activation of gelatinolytic activity, and exhibition of the NITEGE epitope. However, HA oligosaccharides also induced an increase of proteoglycan synthesis, including elevation of aggrecan and HA synthase 2 messenger RNA. CONCLUSION: Uncoupling chondrocytes from the matrix results in deleterious changes in matrix structure and modifications in chondrocyte metabolism. The close interaction of chondrocytes with their matrix and their ability to "sense" changes in receptor occupancy are needed for maintenance of cartilage homeostasis.
Aggrecans↗
Osteogenic protein-1 promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures.
OBJECTIVE: We have used recombinant osteogenic protein-1 to investigate our hypothesis that proper repair and maintenance of cartilage requires not only enhanced biosynthesis and replenishment of the extracellular matrix but also the enhancement of components necessary for matrix retention. DESIGN: The effects of osteogenic protein-1 were examined on bovine articular cartilage slices as well as isolated chondrocytes grown in alginate beads. Cartilage slices were examined for accumulation of proteoglycan by incorporation of 35S-sulfate and staining using Safranin O or, a biotinylated probe specific for hyaluronan. Bovine chondrocytes were characterized by use of a particle exclusion assay, in-situ hybridization, quantitative-competitive RT-PCR and a hyaluronan-binding assay. RESULTS: Osteogenic protein-1 treatment substantially enhanced the accumulation of hyaluronan and proteoglycan within cartilage tissue slices. As with the tissue, osteogenic protein-1 enhanced the size of cell-associated matrices assembled and retained by chondrocytes in vitro. This enhanced matrix assembly was paralleled by an increased expression of mRNA for aggrecan, hyaluronan synthase-2 and CD44. Of the two hyaluronan synthase genes expressed by chondrocytes, only hyaluronan synthase-2 was upregulated by osteogenic protein-1. Coupled with the increase in the CD44 mRNA was an increase in functional hyaluronan binding activity present at the chondrocyte cell surface. CONCLUSIONS: These results demonstrate that osteogenic protein-1 stimulates not only the synthesis of the major cartilage extracellular matrix component aggrecan, but also two associated molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.
Aggrecans↗
Internalization of the hyaluronan receptor CD44 by chondrocytes.
Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.
Animals↗
Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly.
In order to define the role of cell-associated hyaluronan in cartilage matrix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antisense to the mRNA of human HA synthase-2 (HAS-2). As a prerequisite for these studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, or HAS-3, is primarily responsible for HA synthesis in human articular chondrocytes. The copy number of each HAS mRNA expressed in cultured human articular chondrocytes was determined using quantitative (competitive) reverse transcription-polymerase chain reaction (RT-PCR). Only HAS-2 and HAS-3 mRNA expression was detected. The level of HAS-2 mRNA expression was 40-fold higher than that of HAS-3. Cultures of human articular chondrocytes and cartilage tissue slices were then transfected with HAS-2-specific antisense oligonucleotides. This treatment resulted in time-dependent inhibition of HAS-2 mRNA expression, as measured by quantitative RT-PCR, and a significant loss of cell-associated HA staining. Sense and reverse HAS-2 oligonucleotides showed no effect. The consequences of reduced HA levels (due to HAS-2 antisense inhibition) were a decrease in the diameter of the cell-associated matrix and a decreased capacity to retain newly synthesized proteoglycan. These results suggest that HA synthesized by HAS-2 plays a crucial role in matrix assembly and retention by human articular chondrocytes.
Base Sequence↗
The chondrocyte pericellular matrix: a model for hyaluronan-mediated cell-matrix interactions.
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Animals↗
Antisense inhibition of chondrocyte CD44 expression leading to cartilage chondrolysis.
OBJECTIVE: To better define critical functions of the hyaluronan receptor CD44 in cartilage. METHODS: Articular chondrocytes and cartilage tissue slices were treated with CD44 sequence-specific antisense phosphorothioate oligonucleotides. CD44 expression was probed by immunofluorescence microscopy, enzyme-linked immunosorbent assay, and Western blotting. RESULTS: Antisense oligonucleotides demonstrated a dose- and time-dependent inhibition of CD44 protein expression; negative controls showed no effect. Similar to osteoarthritic cartilage, antisense-treated cartilage slices displayed a near-total loss of stainable proteoglycan-rich matrix. CONCLUSION: CD44 expression is needed for maintenance of cartilage homeostasis.
Animals↗
CD44-anchored hyaluronan-rich pericellular matrices: an ultrastructural and biochemical analysis.
The chondrocyte pericellular matrix is an essential zone for cartilage matrix assembly and turnover. Electron micrographs of native endogenous and composition-defined exogenous pericellular matrices, both preserved via ruthenium hexaminetrichloride fixation procedures, depict strikingly similar networks of hyaluronan and proteoglycan extending out from the cell surface. Biochemical and morphological analyses of matrix regrowth show that monoclonal antibodies directed against the hyaluronan receptor CD44 blocked chondrocyte pericellular matrix assembly. Immunoperoxidase electron microscopy was used to display regular repeating spacing patterns of hyaluronan/proteoglycan assembly at the cell surface. These patterns compared well with the ultrastructural immunolocalization of CD44 at the cell surface. All of these data suggest that the hyaluronan receptor CD44 retains and participates in the assembly of the chondrocyte pericellular matrix.
Aggrecans↗
Hyaluronan-mediated aggregation of limb bud mesenchyme and mesenchymal condensation during chondrogenesis.
Cell condensations are the initial structures in the formation of proper cartilage and skeletal patterning in the developing vertebrate limb. Chondrogenic differentiation is dependent upon the cell-cell and/or cell-matrix interactions which take place during the condensation process. Coincident with the onset of condensation is the expression by limb mesenchyme of specific cell surface binding sites for the extracellular matrix macromolecule hyaluronan. The association of hyaluronan with the cell surface can influence the behavior of cells, especially cell aggregation. In this study the possible involvement of hyaluronan as an extracellular linker molecule in the cell-cell adhesion event during mesenchymal condensation was investigated in the avian limb model. Hyaluronan hexasaccharides were used to prevent the multivalent interactions that occur between native hyaluronan macromolecules and the cell surface. Our studies show that hyaluronan is required for early adhesive cell-cell interactions of limb bud mesenchyme and that perturbations of hyaluronan-cell interactions with hyaluronan hexasaccharides result in a delay in the formation of condensations as well as a delay in chondrogenic differentiation of mesenchymal cells in micromass cultures.
Animals↗
Matrix accumulation and retention in embryonic cartilage and in vitro chondrogenesis.
Since hyaluronan anchors the proteoglycan-rich pericellular matrix to chondrocytes, hyaluronan-cell interactions may direct cartilage matrix assembly. To test this hypothesis, the competitive binding of hyaluronan hexasaccharides for native hyaluronan during matrix assembly, accumulation and retention in embryonic cartilage was studied. Chondrocytes released from explants with collagenase P retained pericellular matrices, but chondrocytes appeared "matrix-free" when released from hexasaccharide-treated explants. Decreased safranin O staining was also observed in the hexasaccharide-treated explants. This loss of proteoglycan retention was demonstrated quantitatively in the cartilage extracts and recovered in the media. The continual presence of hexasaccharides in micromass cultures resulted in decreased proteoglycan deposition. Increased proteoglycan retention, indicative of matrix repair, occurred following hexasaccharide wash-out. Thus, native hyaluronan-chondrocyte interactions are important for the assembly and maintenance of cartilage matrix.
Aggrecans↗
Increased expression of CD44 in bovine articular chondrocytes by catabolic cellular mediators.
Bovine articular chondrocytes cultured in alginate beads were used to study the effect of catabolic cellular mediators on CD44 expression. Treatment with either the 29-kDa fragment of fibronectin or interleukin-1 alpha results in a time- and dose-dependent inhibition of proteoglycan synthesis as well as a stimulation in the expression of CD44 mRNA level as determined by semi-quantitative polymerase chain reaction following reverse transcription. No noticeable effect at 6 h was observed. By 24 h, the major CD44 product (CD44H) from fibronectin fragment-treated cultures showed an 8-fold increase; CD44H from interleukin-1 alpha-treated cultures showed a 6-fold increase as compared to control cultures. In addition, a minor band, determined to be an isoform of CD44, was also shown to be up-regulated by both mediators. Stimulation of CD44 mRNA via interleukin-1 was also evident by in situ hybridization studies of bovine as well as human articular cartilage in organ culture. The increased in CD44 mRNA is matched by an increase at the protein level as determined by Western blot analysis. The Western blot reveals a doublet protein band at 80-90 kDa that corresponds to the molecular mass of CD44H. Cultures incubated with fibronectin fragments for 24 h had an 8.0-fold increase in CD44, while a 6.6-fold was observed for interleukin-1 alpha. Fluorescein-conjugated hyaluronan binding and internalization studies indicate that the increase in CD44 protein, induced by interleukin-1 alpha, closely correlates with an increase in functional hyaluronan receptors present at the chondrocyte cell surface. Taken together these results indicate that conditions that up-regulate chondrocyte catabolism also up-regulate the expression of CD44, a cell surface hyaluronan receptor involved in hyaluronan endocytosis.
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