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PR11-364P22.2/ATF3 protein interaction mediates IL-1β-induced catabolic effects in cartilage tissue and chondrocytes.

ABSTRACT: Osteoarthritis (OA) is a degenerative joint disease which lacks effective medical treatment due to ill-defined molecular mechanisms underlying the pathology. Inflammation is a key factor that induces and aggravates OA. Therefore, the current study aims to explore roles of the dysregulated long non-coding RNAs in the pro-inflammatory cytokine IL-1β-mediated catabolic effects in cartilage tissue and chondrocytes. We identified RP11-364P22.2 as dysregulated in OA patient-derived cartilage tissues and highly responsive to IL-1β stimulus. RNA pull-down coupled with mass spectrometry demonstrated that RP11-364P22.2 physically binds to activating transcription factor 3 (ATF3) and thus increases the protein stability and facilitates its nuclear translocation. Loss- and gain-of-function assays indicated that the interaction between RP11-364P22.2 and ATF3 is indispensable for the detrimental effects of IL-1β including growth inhibition, apoptosis induction as well as degradation of the key chondrocyte structural proteins of type II collage and Aggrecan and synthesis of the extracellular matrix-degrading enzyme MMP13 in chondrocytes. In vivo, depletion of the RP11-364P22.2 effector ATF3 drastically prevented OA development in the rats with surgical destabilization of the medial meniscus (DMM). These results highlight the important roles of lncRNAs in the pathogenesis of OA and indicate the RP11-364P22.2/ATF3 regulatory axis as a potential therapeutic target of inflammation-induced OA.

SUBMITTER: Li X

PROVIDER: S-EPMC8256372 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) provide a promising therapeutic approach in the cell-based therapy of osteoarthritis (OA). However, several disadvantages evolved recently, including immune responses of the host and regulatory hurdles, making it necessary to search for alternative treatment options. Extracellular vesicles (EVs) are released by multiple cell types and tissues into the extracellular microenvironment, acting as message carriers during intercellular communication. Here, we investigate putative protective effects of hBMSC-derived EVs as a cell-free approach, on IL-1β-stimulated chondrocytes obtained from OA-patients. Methods: EVs were harvested from the cell culture supernatant of hBMSCs by a sequential ultracentrifugation process. Western blot, scanning electron microscopy (SEM), and nanoparticle tracking analysis (NTA) were performed to characterize the purified particles as EVs. Intracellular incorporation of EVs, derived from PHK26-labeled hBMSCs, was tested by adding the labeled EVs to human OA chondrocytes (OA-CH), followed by fluorescence microscopy. Chondrocytes were pre-stimulated with IL-1β for 24 h, followed by EVs treatment for 24 h. Subsequently, proliferation, apoptosis, and migration (wound healing) were analyzed via BrdU assay, caspase 3/7 assay, and scratch assay, respectively. With qRT-PCR, the relative expression level of anabolic and catabolic genes was determined. Furthermore, immunofluorescence microscopy and western blot were performed to evaluate the protein expression and phosphorylation levels of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB as components of pro-inflammatory signaling pathways in OA-CH. Results: EVs from hBMSCs (hBMSC-EVs) promote proliferation and reduce apoptosis of OA-CH and IL-1β-stimulated OA-CH. Moreover, hBMSC-EVs attenuate IL-1β-induced reduction of chondrocyte migration. Furthermore, hBMSC-EVs increase gene expression of PRG4, BCL2, and ACAN (aggrecan) and decrease gene expression of MMP13, ALPL, and IL1ß in OA-CH. Notably, COL2A1, SOX9, BCL2, ACAN, and COMP gene expression levels were significantly increased in IL-1β+ EV groups compared with those IL-1β groups without EVs, whereas the gene expression levels of COLX, IL1B, MMP13, and ALPL were significantly decreased in IL-1β+ EV groups compared to IL-1β groups without EVs. In addition, the phosphorylation status of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling molecules, induced by IL-1β, is prevented by hBMSC- EVs. Conclusion: EVs derived from hBMSCs alleviated IL-1β-induced catabolic effects on OA-CH via promoting proliferation and migration and reducing apoptosis, probably via downregulation of IL-1ß-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways. EVs released from BMSCs may be considered as promising cell-free intervention strategy in cartilage regenerative medicine, avoiding several adverse effects of cell-based regenerative approaches.

Project description:Shenmai injection (SMI) has been widely used as a therapy to treat a number of diseases. However, its anti-osteoarthritic properties have not yet been fully investigated. In the present study, the protective effect of SMI on knee articular cartilage of anterior cruciate ligament transected rabbits and interleukin-1β (IL-1β)-stimulated human chondrocytes was investigated. For the in vivo study, knee osteoarthritis (KOA) was induced in female New Zealand white rabbits by anterior cruciate ligament transection (ACLT) in the knee of right hind limb. Rabbits either underwent sham surgery or ACLT surgery. Out of the rabbits receiving ACLT surgery, half of the rabbits received one 0.3 ml Shenmai intra-articular injection in the knee per week for four weeks, following ACLT surgery. The other rabbits received the same volume of normal saline solution. The cartilage was subsequently collected for histological evaluation. For the in vitro study, cultured human chondrocytes were treated with 10 ng/ml IL-1β in the presence or absence of 5 and 2% (v/v) SMI for 24 h. Nitric oxide (NO) and prostaglandin E2 (PGE2) levels in cell culture supernatant were assessed using a Griess reaction and ELISA respectively. The mRNA expression of cyclooxgenase-2 (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-1, MMP-13 and tissue inhibitors of metalloproteinase-1 (TIMP-1) in chondrocytes were detected by reverse transcription-quantitative polymerase chain reaction. The results of the current study revealed that treatment with SMI ameliorated cartilage degradation in the ACLT rabbit model, and decreased levels of NO and PGE2. Furthermore, treatment with SMI decreased levels of COX-2, iNOS, MMP-1 and MMP-13 mRNA expression and increased TIMP-1 mRNA expression in IL-1β-stimulated human chondrocytes. These results indicate that SMI suppresses inflammation and ameliorated cartilage degradation, making it a potential and promising therapeutic option to treat KOA.

Project description:PURPOSE:The significant shortcomings associated with current autologous reconstructive options for auricular deformities have inspired great interest in a tissue engineering solution. A major obstacle in the engineering of human auricular cartilage is the availability of sufficient autologous human chondrocytes. A clinically obtainable amount of auricular cartilage tissue (ie, 1 g) only yields approximately 10 million cells, where 25 times this amount is needed for the fabrication of a full-scale pediatric ear. It is thought that repeated passaging of chondrocytes leads to dedifferentiation and loss of the chondrogenic potential. However, little to no data exist regarding the ideal number of times that human auricular chondrocytes (HAuCs) can be passaged in a manner that maximizes the cellular expansion while minimizing dedifferentiation. METHODS:Human auricular chondrocytes were isolated from discarded otoplasty specimens. The HAuCs were then expanded, and cells from passages 3, 4, and 5 were encapsulated into discs 8 mm in diameter made from type I collagen hydrogels with a cell density of 25 million cells/mL. The constructs were implanted subcutaneously in the dorsa of nude mice and harvested after 1 and 3 months for analysis. RESULTS:Constructs containing passages 3, 4, and 5 chondrocytes all maintained their original cylindrical geometry. After 3 months in vivo, the diameters of the P3, P4, and P5 discs were 69 ± 9%, 67 ± 10%, and 73 ± 15% of their initial diameter, respectively. Regardless of the passage number, all constructs developed a glossy white cartilaginous appearance, similar to native auricular cartilage. Histologic analysis demonstrated development of an organized perichondrium composed of collagen, a rich proteoglycan matrix, cellular lacunae, and a dense elastin fibrin network by Safranin-O and Verhoeff stain. Biochemical analysis confirmed similar amounts of proteoglycan and hydroxyproline content in late passage constructs when compared with native auricular cartilage. CONCLUSIONS:These data indicate that late passage HAuCs (up to passage 5) form elastic cartilage that is histologically, biochemically, and biomechanically similar to native human elastic cartilage and have the potential to be used for auricular cartilage engineering.

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PR11-364P22.2/ATF3 protein interaction mediates IL-1β-induced catabolic effects in cartilage tissue and chondrocytes.

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