Shigeru Miyaki

Expression of microRNA-146 in rheumatoid arthritis synovial tissue

OBJECTIVE Several microRNA, which are approximately 22-nucleotide noncoding RNAs, exhibit tissue-specific or developmental stage-specific expression patterns and are associated with human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in synovial tissue from patients with rheumatoid arthritis (RA). METHODS The expression of miR-146 in synovial tissue from 5 patients with RA, 5 patients with osteoarthritis (OA), and 1 normal subject was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization and immunohistochemistry of tissue sections. Induction of miR-146 following stimulation with tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR. RESULTS Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFalpha, but the 2 microRNA were less highly expressed in OA and normal synovial tissue. In situ hybridization showed primary miR-146a expression in cells of the superficial and sublining layers in synovial tissue from RA patients. Cells positive for miR-146a were primarily CD68+ macrophages, but included several CD3+ T cell subsets and CD79a+ B cells. Expression of miR-146a/b was markedly up-regulated in RASFs after stimulation with TNFalpha and IL-1beta. CONCLUSION This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFalpha and IL-1beta. Further studies are required to elucidate the function of miR-146 in these tissues.

MicroRNA-140 plays dual roles in both cartilage development and homeostasis

Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140 is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however, its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function in tissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140(-/-) mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilage appeared grossly normal in 1-mo-old miR-140(-/-) mice. Interestingly, miR-140(-/-) mice showed age-related OA-like changes characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) mice overexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We show that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

Expression of MicroRNA-146a in osteoarthritis cartilage.

OBJECTIVE A role of microRNA, which are approximately 22-nucleotide noncoding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146a (miR-146a) in cartilage from patients with osteoarthritis (OA). METHODS The expression of miR-146a in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146a by cultures of normal human articular chondrocytes following stimulation with interleukin-1beta (IL-1beta) was examined by quantitative RT-PCR. RESULTS All cartilage samples were divided into 3 groups according to a modification of the Mankin score (grade I = mild OA scored 0-5, grade II = moderate OA scored 6-10, and grade III = severe OA scored 11-14). In grade I OA cartilage samples, the expression of miR-146a and COL2A1 was significantly higher than that in the other groups (P < 0.05). In grades II and III OA cartilage, the expression of miR-146a and COL2A1 was decreased, whereas the expression of matrix metalloproteinase 13 (MMP-13) was elevated in grade II OA cartilage. These data showed that miR-146a is expressed intensely in cartilage with a low Mankin grade and that miR-146a expression decreases in parallel with the level of MMP-13 expression. Tissue section in situ hybridization of primary miR-146a (pri-miR-146a) revealed that pri-miR-146a was expressed in chondrocytes residing in all tissue layers, especially in the superficial layer, where it was intensely expressed. The expression of miR-146 was markedly elevated by IL-1beta stimulation in human chondrocytes in vitro. CONCLUSION This study shows that miR-146 is intensely expressed in low-grade OA cartilage and that its expression is induced by stimulation of IL-1beta. Thus, miR-146 might play a role in OA cartilage pathogenesis.

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

IntroductionRecent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage.MethodsExpression of the markers was analyzed by immunohistochemistry (IHC) and flow cytometry. Hoechst 33342 dye was used to identify and sort the cartilage side population (SP). Multilineage differentiation assays including chondrogenesis, osteogenesis and adipogenesis were performed on SP and non-SP (NSP) cells.ResultsA surprisingly high number (>45%) of cells were positive for Notch-1, Stro-1 and VCAM-1 throughout normal cartilage. Expression of these markers was higher in the superficial zone (SZ) of normal cartilage as compared to the middle zone (MZ) and deep zone (DZ). Non-fibrillated OA cartilage SZ showed reduced Notch-1 and Sox9 staining frequency, while Notch-1, Stro-1 and VCAM-1 positive cells were increased in the MZ. Most cells in OA clusters were positive for each molecule tested. The frequency of SP cells in cartilage was 0.14 ± 0.05% and no difference was found between normal and OA. SP cells displayed chondrogenic and osteogenic but not adipogenic differentiation potential.ConclusionsThese results show a surprisingly high number of cells that express putative progenitor cell markers in human cartilage. In contrast, the percentage of SP cells is much lower and within the range of expected stem cell frequency. Thus, markers such as Notch-1, Stro-1 or VCAM-1 may not be useful to identify progenitors in cartilage. Instead, their increased expression in OA cartilage implicates involvement in the abnormal cell activation and differentiation process characteristic of OA.

Autophagy activation by rapamycin reduces severity of experimental osteoarthritis

Objectives Osteoarthritis is associated with cell death and extracellular matrix degradation in articular cartilage. Autophagy is an essential cellular homeostasis mechanism that was found to be deficient in ageing and osteoarthritic cartilage. This study determined whether pharmacological inhibition of the mammalian target of rapamycin (mTOR), a key inhibitor of autophagy, has disease-modifying activity in experimental osteoarthritis. Methods Experimental osteoarthritis was induced by transection of the medial meniscotibial ligament and the medial collateral ligament in 2-month-old C57Bl/6 mice (n=36). Rapamycin (1 mg/kg weight/day) (n=18 mice) or dimethyl sulphoxide vehicle control (n=18 mice) was administered intraperitoneally for 10 weeks. Histopathological changes in articular cartilage and synovium were examined by using semiquantitative scoring systems. Rapamycin effects on mTOR signalling, autophagy, cartilage homeostasis and inflammation were analysed by immunohistochemistry and immunofluorescence staining. Results Rapamycin affected the mTOR signalling pathway in mouse knee joints as indicated by the inhibition of ribosomal protein S6 phosphorylation, a target of mTOR and activation of LC3, a main marker of autophagy. The severity of cartilage degradation was significantly (p<0.01) reduced in the rapamycin-treated group compared with the control group and this was associated with a significant (p<0.05) decrease in synovitis. Rapamycin treatment also maintained cartilage cellularity and decreased ADAMTS-5 and interleukin-1β expression in articular cartilage. Conclusions These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental osteoarthritis. Pharmacological activation of autophagy may be an effective therapeutic approach for osteoarthritis.

The Mohawk homeobox gene is a critical regulator of tendon differentiation

Mohawk (Mkx) is a member of the Three Amino acid Loop Extension superclass of atypical homeobox genes that is expressed in developing tendons. To investigate the in vivo functions of Mkx, we generated Mkx−/− mice. These mice had hypoplastic tendons throughout the body. Despite the reduction in tendon mass, the cell number in tail tendon fiber bundles was similar between wild-type and Mkx−/− mice. We also observed small collagen fibril diameters and a down-regulation of type I collagen in Mkx−/− tendons. These data indicate that Mkx plays a critical role in tendon differentiation by regulating type I collagen production in tendon cells.

Macro view of microRNA function in osteoarthritis

Osteoarthritis (OA), the most common musculoskeletal disorder, is complex, multifaceted, and characterized by degradation of articular cartilage and alterations in other joint tissues. Although some pathogenic pathways have been characterized, current knowledge is incomplete and effective approaches to the prevention or treatment of OA are lacking. Understanding novel molecular mechanisms that are involved in the maintenance and destruction of articular cartilage, including extracellular regulators and intracellular signalling mechanisms in joint cells that control cartilage homeostasis, has the potential to identify new therapeutic targets in OA. MicroRNAs control tissue development and homeostasis by fine-tuning gene expression, with expression patterns specific to tissues and developmental stages, and are increasingly implicated in the pathogenesis of complex diseases such as cancer and cardiovascular disorders. The emergent roles of microRNAs in cartilage homeostasis and OA pathogenesis are summarized in this Review, alongside potential clinical applications.

Mesenchymal‐stem‐cell‐derived exosomes accelerate skeletal muscle regeneration

Mesenchymal stem cell (MSC) transplantation is used for treatment of many diseases. The paracrine role of MSCs in tissue regeneration is attracting particular attention. We investigate the role of MSC exosomes in skeletal muscle regeneration. MSC exosomes promote myogenesis and angiogenesis in vitro, and muscle regeneration in an in vivo model of muscle injury. Although MSC exosomes had low concentrations of muscle‐repair‐related cytokines, a number of repair‐related miRNAs were identified. This study suggests that the MSC‐derived exosomes promote muscle regeneration by enhancing myogenesis and angiogenesis, which is at least in part mediated by miRNAs such as miR‐494.

Translational Suppression of Atrophic Regulators by MicroRNA-23a Integrates Resistance to Skeletal Muscle Atrophy

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3′-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.