Nobuaki Tanaka

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.

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.

Effects of TGF-β on Hyaluronan Anabolism in Fibroblasts Derived from the Synovial Membrane of the Rabbit Temporomandibular Joint:

Hyaluronan (HA) synthesis in the synovial membrane is affected by various chemical mediators. It is hypothesized that transforming growth factor-beta1 (TGF-β1) 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-β1 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-β1. HAS2 mRNA expression was enhanced 13-fold after six-hour stimulation with TGF-β1 (10 ng/mL), whereas HAS3 mRNA expression was not changed significantly. These results suggest that TGF-β1 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.

Molecular cloning of rabbit hyaluronic acid synthases and their expression patterns in synovial membrane and articular cartilage.

cDNAs for hyaluronic acid synthases (HAS2 and HAS3) were cloned from a cDNA library of cultured rabbit synovial membrane cells. The cDNA encoding the open reading frame of rabbit HAS2 and HAS3 was 1659 nucleotides in length with a predicted molecular mass of about 63 kDa. The amino acid sequence showed that the rabbit HAS2 was 98.7 and 98.4%, and HAS3 was 98.2 and 97.5% identical with human and mouse forms of the proteins, respectively. The predicted sequences for hyaluronic acid (HA) binding motifs and the catalytic domains related to beta 1-4 and beta 1-3 linkages, essential for HA synthesis, were almost conserved in both rabbit HAS2 and HAS3, similarly to human and mouse HASs. RT-PCR analysis and in situ hybridization revealed that the mRNA of HAS2 was highly expressed in the synovial membrane and articular cartilage, whereas the expression of HAS3 mRNA was slightest in these tissues. Thus, it is demonstrated that rabbit HASs are highly conserved in sequence content as compared to the human and mouse homologues described previously, and that HAS2 is predominantly expressed in the synovial membrane and articular cartilage, but HAS3 is not.

Mechanical Regulation of Terminal Chondrocyte Differentiation via RGD-CAP/βig-h3 Induced by TGF-β

RGD-CAP (βig-h3), initially cloned as a transforming growth factor (TGF)-β inducible gene in human lung adenocarcinoma cells, was demonstrated to have a negative regulatory function in mineralization in hypertrophic chondrocytes, and the expression was shown to be associated with mechanical stimulation. We hypothesized that mechanical stimulation may regulate the terminal chondrocyte differentiation through the TGF-β pathway by enhancing the RGD-CAP expression. To test this hypothesis, we investigated the effects of mechanical strain on the terminal differentiation and mineralization of growth-plate chondrocytes and assessed the mechanical regulation of TGF-β and RGD-CAP expression. A cyclic mechanical strain of 12% elongation was applied to the cultured prehypertrophic chondrocytes isolated from the rib cartilage of 4-week-old male rats at 30 cycles/min (loading and relaxation on every alternate second). The terminal differentiation and mineralization of chondrocytes were assessed by alkaline phosphatase (ALP) activity assay and alizarin red staining. The gene expressions of TGF-β and RGD-CAP, as well as chondrocytic terminal differentiation markers such as type X collagen and ALP, were examined with real-time RT-PCR. Cyclic mechanical strain decreased the ALP activity and intensity of alizarin red staining in prehypertrophic chondrocytes, as well as the gene expressions of type X collagen and ALP. TGF-β and RGD-CAP were upregulated in the prehypertrophic chondrocytes subjected to mechanical strain, whereas the level of PTHrP receptor mRNA was not affected by the mechanical strain. The neutralizing antibody for TGF-β suppressed the reduction of the mineralization of chondrocyte cultures with the downregulation of RGD-CAP. These results suggest that mechanical strain negatively regulates the terminal differentiation of chondrocytes through the signal pathway of TGF-β with the induction of RGD-CAP.

Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system.

Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen, aggrecan, Sry-related HMG box (SOX-9), and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen and bone sialoprotein (BSP) genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3 and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. These results demonstrate that alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA.

Mechanical stimuli enhances the expression of RGD-CAP/βig-h3 in the periodontal ligament

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.

Modulation of hyaluronan catabolism in chondrocytes by mechanical stimuli

Hyaluronan (HA) is a component of the extracellular matrices of cartilage contributing to the structural and functional integrity. HA metabolism is regulated by both anabolic and catabolic processes; however, a great deal more of the detail has been unknown yet. The purpose of this study was to clarify the effect of excessive mechanical load on the expression and activity of hyaluronidase (HYAL) in chondrocytes with a special reference to the expressions of IL-1beta and tumor necrosis factor (TNF)-alpha. A cyclic tensile load of 22.8% cell elongation, regarded as an excessive mechanical stimulus, was applied to cultured rabbit knee articular chondrocytes. HYAL1, HYAL2, IL-1beta, and TNF-alpha mRNA levels were examined by quantitative real-time PCR analysis. The HYAL activity in culture medium was examined by HA zymography. Both HYAL1 and HYAL2 mRNA levels were upregulated significantly by the loading in cultured chondrocytes. HYAL activity was also enhanced as compared with unloaded controls. The IL-1beta mRNA level was upregulated significantly by the loading, and TNF-alpha mRNA level was slightly upregulated. HYAL1 and HYAL2 mRNA levels were upregulated significantly by IL-1beta treatment, resulting in a slight increase in HYAL activity. These results show that the expression of HYAL1 and HYAL2 in articular chondrocytes is enhanced by excessive mechanical stimuli and affected in part by induction of IL-1beta, leading to HA catabolism in articular cartilage.