Weishen Chen

The Role of MicroRNA-381 in Chondrogenesis and Interleukin-1-β Induced Chondrocyte Responses.

Aim: The molecular pathways regulating cartilage degradation are unclear. miR-381 was identified as a putative regulator of chondrogenesis related genes. Here, we examined its role in chondrogenesis and osteoarthritic cartilage degeneration. Methods: miR-381 expression was assessed in vitro in response to IL-1β stimulation in primary human (PHC) and mouse (PMC) chondrocytes, and ATDC5 derived chondrocytes; and in vivo in mouse embryos and human osteoarthritic cartilage. The effects of miR-381 on chondrogenesis and NF-kB signaling were assessed using a synthetic RNA mimic or inhibitor and luciferase assay, respectively. Upstream regulators of miR381 were probed using siRNA or overexpression plasmids for Sox9 and Runx2. Results: miR-381 expression was elevated in chondrogenic and hypertrophic ATDC5 cells. miR-381 was induced in vitro by IL-1β in ATDC5 cells, PMCs, and PHCs, and was expressed in areas of cartilage degradation or absorption in vivo. Overexpression of Runx2 or Sox9 increased miR-381 expression in ATDC5 cells. miR-381 suppressed expression of collagen, type II, alpha 1, and enhanced expression of metalloproteinase-13 (MMP-13), but did not regulate NFKBIA and NKRF activity. Conclusion: miR-381 was highly expressed during chondrogenesis and in arthritic cartilage. It may contribute to absorption of the cartilage matrix by repressing type II collagen and inducing MMP-13.

MicroRNA-381 Regulates Chondrocyte Hypertrophy by Inhibiting Histone Deacetylase 4 Expression.

Chondrocyte hypertrophy, regulated by Runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase 13 (MMP13), is a crucial step in cartilage degeneration and osteoarthritis (OA) pathogenesis. We previously demonstrated that microRNA-381 (miR-381) promotes MMP13 expression during chondrogenesis and contributes to cartilage degeneration; however, the mechanism underlying this process remained unclear. In this study, we observed divergent expression of miR-381 and histone deacetylase 4 (HDAC4), an enzyme that directly inhibits RUNX2 and MMP13 expression, during late-stage chondrogenesis of ATDC5 cells, as well as in prehypertrophic and hypertrophic chondrocytes during long bone development in E16.5 mouse embryos. We therefore investigated whether this miRNA regulates HDAC4 expression during chondrogenesis. Notably, overexpression of miR-381 inhibited HDAC4 expression but promoted RUNX2 expression. Moreover, transfection of SW1353 cells with an miR-381 mimic suppressed the activity of a reporter construct containing the 3′-untranslated region (3′-UTR) of HDAC4. Conversely, treatment with a miR-381 inhibitor yielded increased HDAC4 expression and decreased RUNX2 expression. Lastly, knockdown of HDAC4 expression resulted in increased RUNX2 and MMP13 expression in SW1353 cells. Collectively, our results indicate that miR-381 epigenetically regulates MMP13 and RUNX2 expression via targeting of HDAC4, thereby suggesting the possibilities of inhibiting miR-381 to control chondrocyte hypertrophy and cartilage degeneration.

Identification and Characterization of Long Non-Coding RNAs in Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Background/Aims: Long noncoding RNAs (lncRNAs) play important roles in stem cell differentiation. However, their role in osteogenesis of human adipose-derived stem cells (ASCs), a promising cell source for bone regeneration, remains unknown. Here, we investigated the expression profile and potential roles of lncRNAs in osteogenic differentiation of human ASCs. Methods: Human ASCs were induced to differentiate into osteoblasts in vitro, and the expression profiles of lncRNAs and mRNAs in undifferentiated and osteogenic differentiated ASCs were obtained by microarray. Bioinformatics analyses including subgroup analysis, gene ontology analysis, pathway analysis and co-expression network analysis were performed. The function of lncRNA H19 was determined by in vitro knockdown and overexpression. Quantitative reverse transcription polymerase chain reaction was utilized to examine the expression of selected genes. Results: We identified 1,460 upregulated and 1,112 downregulated lncRNAs in osteogenic differentiated human ASCs as compared with those of undifferentiated cells (Fold change ≥ 2.0, P < 0.05). Among these, 94 antisense lncRNAs, 85 enhancer-like lncRNAs and 160 lincRNAs were further recognized. We used 12 lncRNAs and 157 mRNAs to comprise a coding-non-coding gene expression network. Additionally, silencing of H19 caused a significantly increase in expression of osteogenesis-related genes, including ALPL and RUNX2, while a decrease was observed after H19 overexpression. Conclusion: This study revealed for the first time the global expression profile of lncRNAs involved in osteogenic differentiation of human ASCs and provided a foundation for future investigations of lncRNA regulation of human ASC osteogenesis.

Resveratrol Protects against Titanium Particle-Induced Aseptic Loosening Through Reduction of Oxidative Stress and Inactivation of NF-κB

Aseptic implant loosening is closely associated with chronic inflammation induced by implant wear debris, and reactive oxygen species (ROS) play an important role in this process. Resveratrol, a plant compound, has been reported to act as an antioxidant in many inflammatory conditions; however, its protective effect and mechanism against wear particle-induced oxidative stress remain unknown. In this study, we evaluated resveratrol’s protective effects against wear particle-induced oxidative stress in RAW 264.7 macrophages. At non-toxic concentrations, resveratrol showed dose-dependent inhibition of nitric oxide (NO) production, ROS generation, and lipid peroxidation. It also downregulated the gene expression of oxidative enzymes, including inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-1 and NOX-2, and promoted the gene expression and activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). This protective effect against wear particle-induced oxidative stress was accompanied by a reduction of gene expression and release of tumor necrosis factor-α (TNF-α), and decreased gene expression and phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These findings demonstrate that resveratrol can inhibit wear particle-induced oxidative stress in macrophages, and may exert its antioxidant effect and protect against aseptic implant loosening.

Wear Particles Promote Reactive Oxygen Species-Mediated Inflammation via the Nicotinamide Adenine Dinucleotide Phosphate Oxidase Pathway in Macrophages Surrounding Loosened Implants

Background/Aims: Prosthesis loosening is closely associated with chronic inflammatory cytokine secretion by macrophages, which are activated by wear particles or inflammatory stimulants such as lipopolysaccharide (LPS). Reactive oxygen species (ROS) are critical regulators of inflammation, but their enzymatic sources in response to wear particles and their effects on peri-implant LPS-tolerance remain unclear. Methods: Three ROS-related enzymes—nicotinamide adenine dinucleotide phosphate oxidase (NOX)-1 and -2 and catalase—were investigated in interface membrane tissues and in titanium (Ti) particle-stimulated macrophages in vitro. The generation of ROS and downstream inflammatory effects were measured with or without pre-incubation with apocynin, an NOX inhibitor. Results: Pre-exposure to Ti particles attenuated NF-κB activation in LPS-stimulated macrophages, indicating that wear particles suppress immune response, which may lead to chronic inflammation. NOX-1 and -2 were highly expressed in aseptically loosened interface membranes and in macrophages stimulated with Ti particles; the particles induced a moderate amount of ROS generation, NF-κB activation, and TNF-a secretion in macrophages, and these effects were suppressed by apocynin. Conclusion: Wear particles induce ROS generation through the NOX signaling pathway, resulting in persistent inflammation and delayed loosening. Thus, the suppression of NOX activity may be a useful strategy for preventing prosthesis loosening.

Advanced glycation end products biphasically modulate bone resorption in osteoclast-like cells

Advanced glycation end products (AGEs) disturb bone remodeling during aging, and this process is accelerated in diabetes. However, their role in modulation of osteoclast-induced bone resorption is controversial, with some studies indicating that AGEs enhance bone resorption and others showing the opposite effect. We determined whether AGEs present at different stages of osteoclast differentiation affect bone resorption differently. Based on increased levels of tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CTSK), we identified day 4 of induction as the dividing time of cell fusion stage and mature stage in RAW264.7 cell-derived osteoclast-like cells (OCLs). AGE-modified BSA (50-400 μg/ml) or control BSA (100 μg/ml) was then added at the beginning of each stage. Results showed that the presence of AGEs at the cell fusion stage reduced pit numbers, resorption area, and CTSK expression. Moreover, expression of receptor activator of nuclear factor-κB (RANK) as well as the number of TRAP-positive cells, nuclei per OCL, actin rings, and podosomes also decreased. However, the presence of AGEs at the mature stage enlarged the resorption area markedly and increased pit numbers slightly. Intriguingly, only the number of nuclei per OCL and podosomes increased. These data indicate that AGEs biphasically modulate bone resorption activity of OCLs in a differentiation stage-dependent manner. AGEs at the cell fusion stage reduce bone resorption dramatically, mainly via suppression of RANK expression in osteoclast precursors, whereas AGEs at the mature stage enhance bone resorption slightly, most likely by increasing the number of podosomes in mature OCLs.

Wear Particles Impair Antimicrobial Activity Via Suppression of Reactive Oxygen Species Generation and ERK1/2 Phosphorylation in Activated Macrophages.

Implant-related infection (IRI) is closely related to the local immunity of peri-implant tissues. The generation of reactive oxygen species (ROS) in activated macrophages plays a prominent role in the innate immune response. In previous studies, we indicated that implant wear particles promote endotoxin tolerance by decreasing the release of proinflammatory cytokines. However, it is unclear whether ROS are involved in the damage of the local immunity of peri-implant tissues. In the present study, we assessed the mechanism of local immunosuppression using titanium (Ti) particles and/or lipopolysaccharide (LPS) to stimulate RAW 264.7 cells. The results indicate that the Ti particles induced the generation of a moderate amount of ROS through nicotinamide adenine dinucleotide phosphate oxidase-1, but not through catalase. Pre-exposure to Ti particles inhibited ROS generation and extracellular-regulated protein kinase activation in LPS-stimulated macrophages. These findings indicate that chronic stimulation by Ti particles may lead to a state of oxidative stress and persistent inflammation, which may result in the attenuation of the immune response of macrophages to bacterial components such as LPS. Eventually, immunosuppression develops in peri-implant tissues, which may be a risk factor for IRI.