Osteoarthritis (OA) 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 aging and OA 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 OA.
Experimental OA 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 DMSO 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 signaling, autophagy, cartilage homeostasis and inflammation were analyzed by immunohistochemistry and immunofluorescence staining.
Rapamycin affected the mTOR signaling pathway in mouse knee joints as indicated by 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 to 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 IL-1β expression in articular cartilage.
These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental OA. Pharmacological activation of autophagy may be an effective therapeutic approach for OA.
mTOR; rapamycin; autophagy; cartilage; osteoarthritis
The development and patterns of spontaneous aging-related changes in the anterior cruciate ligament (ACL) and their relationship to articular cartilage degeneration are not well characterized. The aim of this study was to investigate the types and temporal sequence of aging-related ACL changes and establish the correlation with cartilage lesion patterns at all stages of OA development in human knee joints without prior joint trauma.
Human knee joints (n=120; 65 donors; age 23-92) were obtained at autopsy and ACL and cartilage were graded macroscopically and histologically. Inflammation surrounding the ACL was assessed separately.
Histological ACL substance scores and ligament sheath inflammation scores increased with aging. Collagen fiber disorganization was the earliest and most prevalent change. The severity of mucoid degeneration and chondroid metaplasia in the ACL increased with development of cartilage lesions. A correlation between ACL and cartilage degeneration was observed, especially in the medial compartment of the knee joint.
ACL degeneration is highly prevalent in knees with cartilage defects, and may even precede cartilage changes. Hence, ACL deficiencies may not only be important in post-traumatic OA, but also a feature associated with knee OA pathogenesis in general.
anterior cruciate ligament; aging; osteoarthritis
Osteoarthritis (OA) is the most common musculoskeletal disorder. It is a complex and multifaceted disease, characterized by the degradation of articular cartilage and joint inflammation. Although few pathogenesis pathways have been characterized, current knowledge is incomplete and has not led to effective approaches for prevention or treatment. These limitations can be overcome by advances in the understanding of molecular mechanisms that are involved in the maintenance and destruction of articular cartilage. Understanding extracellular regulators and intracellular signaling mechanisms in joint cells that control cartilage homeostasis has the potential to lead to the identification of new therapeutic targets for OA. Recently, non-coding RNAs, miRNAs, was noted to act as novel regulatory molecules that regulated the expression of several target genes. The major role of miRNAs is to control development and tissue homeostasis through the ‘fine-tuning’ of the gene expression. Several miRNAs exhibit a tissue- or developmental stage-specific expression pattern and have been associated with diseases such as cancer and cardiovascular disorders. This review is based on our observations that miRNAs play an important role in cartilage homeostasis, and is to summarize the information on miRNA involved in OA pathogenesis, and its clinical approach.
Osteoarthritis; Cartilage; Chondrocytes; microRNA; miR-140
Glucocorticoids, such as dexamethasone (Dex), have been used as in vitro inducers of adipogenesis. However, the roles of the glucocorticoid receptor (GR) in adipogenesis have not been well characterized yet. Here we show that inhibition of GR activity using the GR antagonist RU486 prevents human mesenchymal stem cell (hMSC) and mouse embryonic fibroblast (MEF) differentiation into adipocytes. Moreover, in MEFs isolated from GR knockout (GRnull) and GRdim mice deficient in GR DNA-binding activity, adipogenesis was blocked. We identified GRE sites in the first intron of KLF15 by bioinformatical promoter analysis and confirmed their functional relevance by demonstrating GR interaction by chromatin immunoprecipitation. Moreover transfection of MEFs with siRNA for KLF15 significantly attenuated the expressions of adipogenic-marker genes and the lipid accumulation. Our results provide a new mechanism for understanding glucocorticoids dependent adipogenesis and that GR promotes adipogenesis via KLF15 gene expression as a transcriptional direct target.
adipogenesis; glucocorticoid receptor; human mesenchymal stem cell; mouse embryonic fibroblast; bioinformatics analysis; Krüppel-like factor 15
We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos—a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoD’s ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.
Recent studies demonstrate that histone deacetylase (HDAC) inhibitors therapeutically prevent cartilage degradation in osteoarthritis (OA). Matrix metalloproteinase-13 (MMP-13) plays an important role in the pathogenesis of this disease and in the present study we investigated the correlation between HDACs and MMP-13. We found that HDAC inhibitor trichostatin A (TSA) could suppress both IL-1 dependent and independent MMP-13 mRNA expression (real time PCR) in human knee chondrocytes. Comparing the expression of different HDACs in cartilage from OA patients and healthy donors, HDAC7 showed a significant elevation in cartilage from OA patients. These results were confirmed by immunohistochemistry. Knockdown of HDAC7 by siRNA in SW 1353 human chondrosarcoma cells strongly suppressed IL-1 dependent induction of MMP-13 gene expression.
In conclusion, elevated HDAC7 expression in human OA may contribute to cartilage degradation via promoting MMP-13 gene expression and inhibition of HDACs by TSA or the selective inhibition of HDAC7 could be used therapeutically to stop OA progression.
This study determined the potential for neo tissue formation and the role of STRO-1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with Safranin O, for type II collagen and STRO-1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months and surgically injured cartilage were analyzed for changes in STRO-1 expression patterns. Results show that immature explants contained more STRO-1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO-1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neo tissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO-1+ staining as compared to pellets from mature chondrocytes. The frequency of STRO-1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO-1+ cells in immature cartilage changes with joint maturation. STRO-1+ cells have the potential to form new cartilage spontaneously and after tissue injury.
Tissue neogenesis; cartilage; stem cells; STRO-1; aging
MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. The miRNAs exhibit tissue-specific expression patterns and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNAs expressed in articular chondrocytes, determine changes in osteoarthritic cartilage and address the function of miR-140.
To identify miRNAs specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative PCR with human articular chondrocytes compared to human mesenchymal stem cells (MSC). The expression pattern of miR-140 was monitored during chondrogenic differentiation of hMSC in pellet cultures and in human articular cartilage from normal and osteoarthritic knee joints. We tested effects of IL-1β on miR-140 expression. Double-strand (ds) miR-140 was transfected into chondrocytes to analyze changes in the expression of genes associated with osteoarthritis.
Microarray analysis showed that miR-140 has the largest difference in expression between chondrocytes and MSC. During chondrogenesis cultures of MSC miR-140 expression increased in parallel with Sox9 and Col2a1. Normal human articular cartilage expressed miR-140 and this was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1β suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 downregulated IL-1β-induced ADAMTS-5 expression and rescued the IL-1β –dependent repression of Aggrecan gene expression.
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-1β may contribute to the abnormal gene expression pattern characteristic of OA.
microRNA; chondrocytes; mesenchymal stem cells; cartilage; osteoarthritis
The transcription factor, Sry-related High Mobility Group (HMG) box containing gene 9 (Sox9), plays a critical role in cartilage development by initiating chondrogenesis and preventing the subsequent maturation process called chondrocyte hypertrophy. This suppression mechanism by Sox9 on late-stage chondrogenesis partially results from the inhibition of Runt-related transcription factor 2 (Runx2), the main activator of hypertrophic chondrocyte differentiation. However, the precise mechanism by which Sox9 regulates late chondrogenesis is poorly understood.
In the present study, the transcriptional repressor vertebrate homolog of Drosophila bagpipe (Bapx1) was found to be a direct target of Sox9 for repression of Runx2 expression in chondrocytes. We identified a critical Sox9 responsive region in the Bapx1 promoter via a luciferase reporter assay. Analysis by chromatin immunoprecipitation and electrophoretic mobility shift assays indicated that Sox9 physically bound to this region of the Bapx1 promoter. Consistent with the notion that Bapx1 and Sox9 act as negative regulators of chondrocyte hypertrophy by regulating Runx2 expression, transient knockdown of Sox9 or Bapx1 expression by shRNA in chondrocytes increased Runx2 expression, as well as expression of the late chondrogenesis marker, Col10a1. Furthermore, while over-expression of Sox9 decreased Runx2 and Col10a1 expressions, simultaneous transient knockdown of Bapx1 diminished that Sox9 over-expressing effect.
Our findings reveal that the molecular pathway modulated by Bapx1 links two major regulators in chondrogenesis, Sox9 and Runx2, to coordinate skeletal formation.
Sox9; Bapx1; Runx2; chondrocyte; chondrogenesis; shRNA
MicroRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression through sequence-specific interactions with the 3′ untranslated regions (UTRs) of target mRNA and play various biological roles. miR-133 was identified as a muscle-specific miRNA that enhanced the proliferation of myoblasts during myogenic differentiation, although its activity in myogenesis has not been fully characterized. Here, we developed a novel retroviral vector system for monitoring muscle-specific miRNA in living cells by using a green fluorescent protein (GFP) that is connected to the target sequence of miR-133 via the UTR and a red fluorescent protein for normalization. We demonstrated that the functional promotion of miR-133 during myogenesis is visualized by the reduction of GFP carrying the miR-133 target sequence, suggesting that miR-133 specifically down-regulates its targets during myogenesis in accordance with its expression. Our cell-based miRNA functional assay monitoring miR-133 activity should be a useful tool in elucidating the role of miRNAs in various biological events.
Myogenesis; miR-133; Retroviral vector; Fluorescent protein; Live-cell imaging
A role of microRNAs, which are ∼22- nucleotide non coding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in cartilage from patients with osteoarthritis (OA).
The expression of miR-146 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-146 by cultures of normal human articular chondrocytes following stimulation with interleukin-1β (IL-1β) was examined by quantitative RT-PCR.
All cartilage samples were divided into three groups according to a modified Mankin scale; grade I: 0 - 5, grade II: 6 - 10, grade III: 11 - 14. In OA cartilage samples of grade I, the expression of miR-146a and Col2a1 was significantly higher than that of other groups (p<0.05). In OA cartilage of grades II and III, the expression of miR-146a and Col2a1 decreased while the expression of MMP13 was elevated in grade II. These data show that miR-146a is expressed intensely in cartilage with a low Mankin grade, and that miR-146a expression decreases in accordance with level of MMP13 expression. Section in situ hybridization of pri-miR-146a revealed that pri-miR-146a is expressed in chondrocytes in all layers, especially in the superficial layer where it is intensely expressed. The expression of miR-146 was markedly elevated by IL-1β stimulation in human chondrocytes in vitro.
This study shows that miR-146 is intensely expressed in low grade OA cartilage, and that its expression is induced by stimulation of IL-1β. MiR-146 might play a role in OA cartilage pathogenesis.
Several microRNA, which are ~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).
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 α (TNFα) and interleukin-1β (IL-1β) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR.
Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFα, 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 TNFα and IL-1β.
This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFα and IL-1β. Further studies are required to elucidate the function of miR-146 in these tissues.
Recent 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.
Expression 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.
A 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.
These 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.