Changhe Hou

MiR‐193b regulates early chondrogenesis by inhibiting the TGF‐beta2 signaling pathway

Cartilage generation and degradation are regulated by miRNAs. Our previous study has shown altered expression of miR‐193b in chondrogenic human adipose‐derived mesenchymal stem cells (hADSCs). In the current study, we investigated the role of miR‐193b in chondrogenesis and cartilage degradation. Luciferase reporter assays showed that miR‐193b targeted seed sequences of the TGFB2 and TGFBR3 3′‐UTRs. MiR‐193b suppressed the expression of early chondrogenic markers in chondrogenic ATDC5 cells, and TNF‐alpha expression in IL‐1b‐induced PMCs. In conclusion, MiR‐193b may inhibit early chondrogenesis by targeting TGFB2 and TGFBR3, and may regulate inflammation by repressing TNF‐alpha expression in inflamed chondrocytes.

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.

MiR-455-3p regulates early chondrogenic differentiation via inhibiting Runx2.

The expression of miR‐455‐3p has been shown to be up‐regulated in chondrogenesis of mesenchymal stem cell, but its role in different stages during chondrogenesis remains unknown. Here, we show that miR‐455‐3p is increased in ATDC5 cells from 0 d to 21 d, but rapidly decreases at 28 d, and a similar expression kinetic is detected in the development of mouse embryos. We show that miR‐455‐3p functions as an activator for early chondrogenic differentiation, most likely by inhibiting the expression of Runt‐related transcription factor 2 (Runx2) as indicated by luciferase reporter assays. In conclusion, miR‐455‐3p may activate early chondrogenesis by directly targeting Runx2.

IRAK-M in macrophages around septically and aseptically loosened hip implants†

The most common long-term complication of joint arthroplasty is loosening, which is mediated by chronic inflammatory cytokines produced by macrophages stimulated by implant-derived debris and eventually bacterial components adherent to such debris. In this study, antiinflammatory interleukin-1 receptor-associated kinase-M (IRAK-M) was studied in macrophages in interface membranes in vivo using immunohistochemical staining and in titanium particle-stimulated macrophages in vitro using reverse transcriptase-polymerase chain reaction. Results show that the interface membranes of septically and aseptically loosened prosthesis express more IRAK-M protein than control membranes from osteoarthritic patient and that IRAK-M mRNA-levels increase upon particle stimulation. These findings suggest that, the upregulation of IRAK-M in macrophages is involved in the local immunosuppression around implants, and may contribute to septic and aseptic implant loosening.

Wear particles promote endotoxin tolerance in macrophages by inducing interleukin‐1 receptor‐associated kinase‐M expression

Toll-like receptors (TLRs) recognizing pathogen-associated molecular patterns (PAMP) play a role in local immunity and participate in implant-associated loosening. TLRs-mediated signaling is regulated by interleukin-1 receptor-associated kinase-M (IRAK-M). Our previous studies have proved that IRAK-M is induced by wear particles in macrophages from periprosthetic tissues. In this study, the IRAK-M-related mechanisms were further explored by lipopolysaccharide (LPS) and/or titanium (Ti) particles stimulations and small interfering RNAs (siRNAs). The protein level of IRAK-M was studied using western blotting and tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) levels were measured using ELISA. Results showed that in RAW264.7 cells stimulated by LPS after Ti particle pre-exposure, IRAK-M was slightly changed, compared with LPS stimulation. And levels of TNF-α and IL-1β in cultures stimulated by LPS first after Ti particle pre-exposure were lower than in the other two groups which were stimulated by LPS with or without Ti particles (p < 0.001), whereas there were no statistic differences between the later two (p > 0.05). The cytokines were lowest in Ti particles alone stimulation. After siRNAs silenced, IRAK-M-deficient cells exhibited increased expression of the cytokines in LPS stimulation after Ti particle pre-exposure and when stimulated with Ti particles alone. Our findings suggest that debris-induced IRAK-M decreases foreign body reactions, but at the same time, the over-expression of IRAK-M may also be detrimental on local intrusion of PAMPs or bacteria, negatively regulates the LPS-induced and TLRs-mediated inflammation and results in immunosuppression in periprosthetic tissue, which may predispose to implant-associated infections.

Presence and function of microRNA-92a in chondrogenic ATDC5 and adipose-derived mesenchymal stem cells

The aim of the present study was to investigate the presence and biological function of microRNA-92a (miR-92a) in chondrogenesis and cartilage degeneration. Human adipose-derived mesenchymal stem cells (hADSCs) in micromass and chondrocyte-like ATDC5 cells were induced to chondrogenesis, and primary human/mouse chondrocytes (PHCs/PMCs) and chondrogenic ATDC5 cells were stimulated with interleukin-1β (IL-1β). An miR-92a mimic/inhibitor was transfected into the ATDC5 cells using lipofectamine 2000. Gene expression was analyzed using reverse transcription-quantitative polymerase chain reaction. Alcian blue was used to stain the cartilage nodules and chondrogenic micromass. The potential target genes, signaling pathways and functions of miR-92a were examined using miRanda, miRDB, CLIP-Seq, TargetScan and Kyoto Encyclopedia of Genes and Genomes. The expression of miR-92a was elevated in the chondrogenic ATDC5 cells and hADSCs, and also in the IL-1β-induced ATDC5 cells, PMCs and PHCs. Forced expression of miR-92a enhanced the expression levels of col9a2 and aggrecan. A total of 279 genes were predicted as potential target genes of miR-92a. The phosphoinositide 3-kinase/PI3K)-Akt, ErbB and focal adhesion kinase pathways, extracellular matrix (ECM)-receptor interaction and the mammalian target of rapamycin (mTOR) signaling pathway were suggested to mediate the effects of miR-92a on chondrogenesis and cartilage degeneration. These results demonstrated that miR-92a was involved in chondrogenesis and the chondrocyte response induced by IL-1β. miR-92a positively contributed to the expression of col9a2 and of aggrecan.

Resistin Stimulates Expression of Chemokine Genes in Chondrocytes via Combinatorial Regulation of C/EBPβ and NF-κB

To further investigate the regulation role of two chemokine genes CCL3 and CCL4 in chondrocytes in response to resistin, human primary chondrocytes and T/C-28a2 cells were cultured. The function of resistin on the chemokine genes, and the expression of C/EBPβ, NF-κB isoforms were tested using qPCR. The methods used to investigate timed co-regulation of C/EBPβ and NF-κBwere NF-κB inhibitor (IKK-NBD) and C/EBPβ inhibitor (SB303580) treatments, and subcellular localization, with or without resistin stimulation. Results showed that resistin could increase the up-regulation of chemokine genes independently. Resistin increased the expression of C/EBPβ and NF-κB isoforms. C/EBPβ regulated basal activity and steadily increased over time up to 24h with resistin. NF-κB was up-regulated upon induction with resistin, peaking at 4 h. C/EBPβ and NF-κB co-enhanced the chemokines expression; inhibition of their activity was additive. The timing of activation in chondrocytes was confirmed by subcellular localization of C/EBPβ and c-rel. Chondrocytes react to resistin in a non-restricted cell-specific manner, utilizing C/EBPβ and NF-κB in a combinatorial regulation of chemokine gene expression. The activity of C/EBPβ is augmented by a transient increase in activity of NF-κB, and both transcription factors act independently on the chemokine genes, CCL3 and CCL4. Thus, resistin stimulates CCL3 and CCL4 through combinatorial regulation of C/EBPβ and NF-κB in chondrocytes.

Presence of interleukin-17C in the tissue around aseptic loosened implants.

PurposeThe most common long-term complication of joint arthroplasty is aseptic loosening. The proinflammatory cytokines secreted by macrophages are involved in aseptic loosening. Recently, a novel proinflammatory cytokine IL-17C was reported to participate in inflammatory diseases by synergising with proinflammatory cytokines. However, the relationship between IL-17C and the aseptic loosening is unclear.MethodsThe tissues around aseptic loosened implants were collected during revision surgery and handled by formalin fixation and embedded in paraffin. The presence of IL-17C in the tissues around the aseptic loosened implants was investigated in 12 aseptic loosening patients using immunofluorescence.ResultsThe presence of IL-17C protein in the tissues around aseptic loosened implants was detected by immunofluorescence. There are no statistical differences between optical density of IL-17C in aseptic loosening samples and in rheumatoid arthritis samples (positive control).ConclusionsThese results suggest the presence of IL-17C in aseptic loosening. Interleukin-17C was related to the inflammation of aseptic loosening, possibly by contributing to the inflammation and osteolysis in the tissues surrounding aseptic loosened implants.