Shigeru Ohno

Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen

A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.

The effects of high magnitude cyclic tensile load on cartilage matrix metabolism in cultured chondrocytes

Excessive mechanical load is thought to be responsible for the onset of osteoarthrosis (OA), but the mechanisms of cartilage destruction caused by mechanical loads remain unknown. In this study we applied a high magnitude cyclic tensile load to cultured chondrocytes using a Flexercell strain unit, which produces a change in cell morphology from a polygonal to spindle-like shape, and examined the protein level of cartilage matrixes and the gene expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and proinflammatory cytokines such as IL-1beta and TNF-alpha. Toluidine blue staining, type II collagen immunostaining, and an assay of the incorporation of [35S]sulfate into proteoglycans revealed a decrease in the level of cartilage-specific matrixes in chondrocyte cultures subjected to high magnitude cyclic tensile load. PCR-Southern blot analysis showed that the high magnitude cyclic tensile load increased the mRNA level of MMP-1, MMP-3, MMP-9, IL-1beta, TNF-alpha and TIMP-1 in the cultured chondrocytes, while the mRNA level of MMP-2 and TIMP-2 was unchanged. Moreover, the induction of MMP-1, MMP-3 and MMP-9 mRNA expression was observed in the presence of cycloheximide, an inhibitor of protein synthesis. These findings suggest that excessive mechanical load directly changes the metabolism of cartilage by reducing the matrix components and causing a quantitative imbalance between MMPs and TIMPs.

Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes.

Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the underlying molecular events that become activated. In this study, hyaluronan oligosaccharides induced the loss of extracellular matrix proteoglycan and collagen from cultured slices of normal adult human articular cartilage. This loss was coincident with an increased expression of matrix metalloproteinase (MMP)-13. MMP-13 expression was also induced in articular chondrocytes by hyaluronan (HA) hexasaccharides but not by HA tetrasaccharides nor high molecular weight hyaluronan. MMP-13 promoter-reporter constructs in CD44-null COS-7 cells revealed that both CD44-dependent and CD44-independent events mediate the induction of MMP-13 by hyaluronan oligosaccharides. Electromobility gel shift assays demonstrated the activation of chondrocyte NFκB by hyaluronan oligosaccharides. NFκB activation was also documented in C-28/I2 immortalized human chondrocytes by luciferase promoter assays and phosphorylation of IKK-α/β. The link between activation of NFκB and MMP-13 induction by HA oligosaccharides was further confirmed through the use of the NFκB inhibitor helenalin. Inhibition of MAP kinases also demonstrated the involvement of p38 MAP kinase in the hyaluronan oligosaccharide induction of MMP-13. Our findings suggest that hyaluronan-CD44 interactions affect matrix metabolism via activation of NFκB and p38 MAP kinase.

RGD-CAP (βig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin α1β1

Abstract In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human βig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-β. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or β-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg 2+ or Mn 2+ ), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin α 1 or β 1 subunit, but not to the α 2 , α 3 , α 5 or β 2 subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the α 5 subunit, but not the mAb to the α 1 subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin α 1 β 1 that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.

Enhancement of Cell Adhesion and Spreading by a Cartilage-specific Noncollagenous Protein, Cartilage Matrix Protein (CMP/Matrilin-1), via Integrin α1β1

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10–20 μg/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin α1 or β1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin α2, α3, α5, β2, α5β1, or αVβ5had little effect on cell adhesion or spreading. The antibody to integrin α1, but not to other subunits, coprecipitated125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin α1 subunit. Unlabeled CMP competed for the binding to integrin α1with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin α1β1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.

Proinflammatory Cytokines Regulate the Gene Expression of Hyaluronic Acid Synthetase in Cultured Rabbit Synovial Membrane Cells

To elucidate the mechanism of accumulation and fragmentation of hyaluronic acid (HA) under inflammatory conditions, we investigated the effect of proinflammatory cytokines on hyaluronic acid synthetase (HAS) mRNA expression using cultured rabbit synovial membrane cells. HASs mRNA levels were determined by real-time PCR. HAS2 mRNA expression was maximally enhanced 3.3- and 2.8-fold after 3-hour stimulation with IL-1β (1 ng/ml) and after 1-hour stimulation with TNF-α (10 ng/ml). HAS3 mRNA expression was increased by a maximum of 4.3 times after 3-hour stimulation with IL-1 β (10 ng/ml), whereas 1-hour stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) induced around a 2.5-fold increase in HAS3 mRNA. Although IFN-γ (1–100 ng/ml) alone showed little effect on HAS2 mRNA expression, the effect was synergized by combined with both IL-lβ and TNF-α, substantially increasing HAS2 mRNA expression. These results suggest that proinflammatory cytokines regulate the HAS expression, and consequently may contribute to the accumulation and fragmentation of HA.

Cyclic Mechanical Strain Regulates the PTHrP Expression in Cultured Chondrocytes via Activation of the Ca2+ Channel

The association between mechanical stimulation and chondrocyte homeostasis has been reported. However, the participation of PTHrP (parathyroid-hormone-related protein) in the mechano-regulation of chondrocyte metabolism remains unclear. We determined whether mechanical stimulation of chondrocytes induces the expression of PTHrP and, further, whether the mechano-modulation of PTHrP is dependent on the maturational status of chondrocytes. Cyclic mechanical strain was applied to rat growth plate chondrocytes at the proliferating, matrix-forming, and hypertrophic stages at 30 cycles/min. Cyclic mechanical strain significantly increased PTHrP mRNA levels in chondrocytes at the proliferating and matrix-forming stages only. The induction of PTHrP was dependent on loading magnitude at the proliferating stage. Using specific ion channel blockers, we determined that mechano-induction of PTHrP was inhibited by nifedipine, a Ca2+ channel blocker. These results suggest that mechanical induction of PTHrP possibly provides the environment for greater chondrocyte replication and matrix formation that would subsequently affect cartilage formation.

RGD-CAP (βig-h3) Exerts a Negative Regulatory Function on Mineralization in the Human Periodontal Ligament

In our previous studies, RGD-CAP/βig-h3 was isolated from a fiber-rich fraction of cartilage and was found to have a negative function on mineralization of hypertrophic chondrocytes. However, the expression and biological function of RGD-CAP in the periodontal ligament (PDL) are not known. We hypothesized that RGD-CAP could be expressed in the PDL and regulate its mineralization. To test this, we investigated the expression of RGD-CAP in human PDL and the effects of RGD-CAP on mineralization of cultured PDL cells. RGD-CAP was detected in the human PDL as multimeric proteins greater than 200 kDa. The RGD-CAP mRNA level decreased in cultured PDL cells exposed to 10−8 M dexamethasone or 10−8 M 1α,25-dihydroxyvitamin D3 when these steroids increased alkaline phosphatase (ALP) activity. Furthermore, exogenous RGD-CAP suppressed the ALP activity and bone nodule formation of cultured PDL cells. These findings suggest that RGD-CAP in the PDL modulates the mineralization which affects adjacent alveolar bone metabolism.

RGD-CAP (βig-h3) is expressed in precartilage condensation and in prehypertrophic chondrocytes during cartilage development

RGD-CAP ((beta)ig-h3), isolated from cartilage as a collagen-associated protein, was demonstrated to have a binding ability to collagen and to enhance the adhesion of chondrocytes via integrin alpha(1)beta(1). However, the role of this protein in cartilage development remains unclear. In this study, we investigated the expression of RGD-CAP ((beta)ig-h3) in chick embryos and cultured mesenchymal stem cells (MSCs) during the differentiation to chondrocytes. The effects of recombinant RGD-CAP on adhesion and DNA synthesis of MSCs and mineralization were also examined. Tissue sections from chick embryos at Hamburger-Hamilton (HH) stages 19-37 were immunostained with anti-chick RGD-CAP antibodies. The expression of RGD-CAP was slightest in chick embryos at HH stage 19, whereas a considerable expression of RGD-CAP was observed in the developing vertebrae and precartilage aggregate in the limb bud of chick embryos at HH stage 26. The expression of RGD-CAP was significantly reduced in vertebrae of chick embryo at HH stage 32. Reverse transcriptional polymerase chain reaction (RT-PCR) analysis showed that RGD-CAP was highly expressed in cultured MSCs and decreased by 4-day treatment with 10(-8) M dexamethasone when MSCs proliferated to adipocyte-like cells, whereas it was recovered by co-treatment with 3 ng/ml TGF-beta for 8-12 days when MSCs proliferated to hypertrophic chondrocyte-like cells. The adhesion and DNA synthesis of MSCs cultured on RGD-CAP-coated dishes increased significantly compared with the controls. RGD-CAP was distributed in the prehypertrophic zone in matured cartilage of the vertebrae of chick embryos at HH stage 37. Recombinant RGD-CAP inhibited the mineralization of hypertrophic chondrocytes. These results suggest that RGD-CAP ((beta)ig-h3) exerts an essential role in the early cartilage development by enhancing the adhesion and growth of the pre-chondrogenic cells, and functions as a negative regulator for mineralization at the terminal stage of the chondrogenic differentiation.

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 (HAoligos). Hyaluronan and CD44 were uniformly distributed throughout the fibrous and cartilaginous zones of the TMJ condyle. Treatment of cartilage explants with HAoligos resulted in cartilage matrix loss with increased secreted caseinolytic activity. HAoligos treatment of TMJ chondrocytes resulted in enhanced MMP-3 expression, whereas wash-out of the HAoligos in the middle of the experimental period reduced this induction. These results suggest that HAoligos activate chondrocytes, resulting in a substantial enhancement of proteinase expression, and the removal of HAoligos by wash-out reverses this catabolic activation.