ABSTRACT
Hyaluronan (HA) is one of the major extracellular matrix components in cartilage. In addition to the biomechanical functions, HA has various important roles in the differentiation of chondrocytes. The purpose of this study was to clarify the nature of HA synthesis during chondrocyte differentiation. Growth plate chondrocytes were isolated from rabbit ribs and cultured in chondrocyte differentiation medium. The amount of HA and HA synthase (HAS) mRNA levels were analyzed for each stage of chondrocyte differentiation by means of high-performance liquid chromatography (HPLC) and real-time PCR, respectively. The distribution of HA in cultured chondrocytes was observed by histochemical staining. The amount of HA, ranging widely in size, was increased substantially during the hypertrophic stage. The expression levels of HAS2 and HAS3 mRNAs were low during the matrix-forming stage. HAS2 mRNA level was substantially enhanced at the pre-hypertrophic stage, whereas HAS3 mRNA level exhibited a slight increase. HAS1 mRNA was not detected. The intensity of HA staining was high around the hypertrophic chondrocytes. These results suggest that HA metabolism in chondrocyte differentiation is regulated by the selective expression of HASs, and HAS2 and the related large size-HA may have a certain association with the hypertrophic changes of chondrocytes.
Subject(s)
Chondrocytes/metabolism , Growth Plate/cytology , Hyaluronic Acid/metabolism , Animals , Cell Differentiation , Cell Proliferation , Chondrocytes/cytology , Chromatography, Gel , Chromatography, High Pressure Liquid , Collagen Type II/metabolism , Collagen Type X/metabolism , Extracellular Matrix/metabolism , Glucuronosyltransferase/metabolism , Hyaluronan Synthases , Immunohistochemistry , RNA, Messenger/metabolism , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
Hyaluronan (HA) synthesis in the synovial membrane is affected by various chemical mediators. It is hypothesized that transforming growth factor-beta 1 (TGF-beta 1) would be a mediator to modulate HA synthesis in cultured synovial membrane fibroblasts of the temporomandibular joint (TMJ). Fibroblasts were extracted from the TMJ synovial membrane of four-week-old Japanese white rabbits. The amount of HA and expression levels of HA synthase (HAS) mRNAs induced by TGF-beta 1 treatment were analyzed by means of high-performance liquid chromatography and real-time polymerase chain-reaction, respectively. Both medium and large amounts of HA were enhanced by the stimulation of TGF-beta 1. HAS2 mRNA expression was enhanced 13-fold after six-hour stimulation with TGF-beta 1 (10 ng/mL), whereas HAS3 mRNA expression was not changed significantly. These results suggest that TGF-beta 1 enhances the expression of HAS2 mRNA in the TMJ synovial membrane fibroblasts and may contribute to the production of high-molecular-weight HA in the joint fluid.
Subject(s)
Glycosyltransferases , Hyaluronic Acid/analysis , Membrane Proteins , Synovial Membrane/drug effects , Temporomandibular Joint/drug effects , Transferases , Transforming Growth Factor beta/pharmacology , Xenopus Proteins , Animals , Cells, Cultured , Chromatography, High Pressure Liquid , Fibroblasts/drug effects , Fibroblasts/pathology , Glucuronosyltransferase/drug effects , Hyaluronan Synthases , Isoenzymes/analysis , Isoenzymes/drug effects , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/drug effects , Rabbits , Synovial Membrane/pathology , Temporomandibular Joint/pathology , Time Factors , Transforming Growth Factor beta1ABSTRACT
RGD-CAP, a member of the fasciclin family, is expressed in the periodontal ligament (PDL). Since the PDL is continually subjected to mechanical forces from such orofacial functions as mastication, biting, speech and swallowing, the mechanical stimuli is thought to be associated with the expression of RGD-CAP. Furthermore, the adhesive functions of RGD-CAP may contribute to the maintenance or regeneration of PDL architecture. The objective of this study was to examine whether mechanical stimuli modulate the expression of RGD-CAP in the human PDL, and to examine the effects of recombinant RGD-CAP on the adhesion of PDL cells. During experimental tooth movement, the expression of RGD-CAP was significantly enhanced in the PDL. In vitro experiments with cultured PDL cells showed that the expression of RGD-CAP mRNA was significantly enhanced by mechanical tensile force of 15.4kPa for 48h. The induction of RGD-CAP mRNA, meanwhile, was completely inhibited by cycloheximide which is an inhibitor of protein synthesis. Furthermore, neutralising antibody against TGF-beta also suppressed the mechanical induction of RGD-CAP. The adhesion of cultured PDL cells onto plates coated with recombinant RGD-CAP increased significantly compared with the controls. These findings suggest that RGD-CAP, induced by TGF-beta expressed in response to mechanical stimuli, plays an important role in modulating the homeostasis of PDL.
Subject(s)
Extracellular Matrix Proteins/metabolism , Mechanotransduction, Cellular/physiology , Periodontal Ligament/metabolism , Transforming Growth Factor beta , Blotting, Western , Cell Adhesion/drug effects , Cells, Cultured , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/pharmacology , Gene Expression Regulation , Humans , Periodontal Ligament/cytology , Physical Stimulation/methods , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , Recombinant Proteins/pharmacology , Stress, Mechanical , Tooth Movement TechniquesABSTRACT
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.
