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GABARAP ameliorates IL-1β-induced inflammatory responses and osteogenic differentiation in bone marrow-derived stromal cells by activating autophagy.

ABSTRACT: Bone mesenchymal stem cells (BMSCs) are the most commonly investigated progenitor cells in bone defect repair and osteoarthritis subchondral bone regeneration; however, these studies are limited by complex inflammatory conditions. In this study, we investigated whether pro-autophagic γ-aminobutyric acid receptor-associated protein (GABARAP) promotes BMSCs proliferation and osteogenic differentiation by modulating autophagy in the presence or absence of interleukin-1 beta (IL-1β) in vitro. The expression levels of all relevant factors were evaluated by qRT-PCR or western blotting where appropriate. BMSCs differentiation were assessed by Alizarin Red, alkaline phosphatase, safranin O, and Oil Red O staining. Furthermore, the interactions between autophagy and osteogenic differentiation were investigated by co-treatment with the autophagy inhibitor 3-methyladenine (3-MA). As the results, we found that treatment with recombinant human His6-GABARAP protein promoted cell proliferation, inhibited apoptosis, and reduced ROS generation by increasing autophagic activity, particularly when co-cultured with IL-1β. Moreover, His6-GABARAP could effectively increase the osteogenic differentiation of BMSCs. The expression levels of inflammatory factors were significantly decreased by His6-GABARAP treatment, whereas its protective effects were attenuated by 3-MA. This study demonstrates that GABARAP maintains BMSCs survival and strengthens their osteogenic differentiation in an inflammatory environment by upregulating mediators of the autophagy pathway.

SUBMITTER: Guo X

PROVIDER: S-EPMC8172545 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes erosion of articular cartilage and bone and has adverse effects on both patients and livestock animals. The aim of the present study was to investigate the role of interleukin-1β (IL-1β) in the pathogenesis of RA, and to further determine whether injection of IL-1β small interfering RNA (siRNA) or transplantation of IL-1β siRNA + bone marrow mesenchymal stem cells (BMSCs) can ameliorate RA in rats. A collagen-induced arthritis (CIA) rat model was established by injecting type II collagen for 4 weeks. Next, CIA rats were randomly divided into three groups and injected or transplanted with PBS, IL-1β siRNA and IL-1β siRNA + BMSCs for another 4 weeks. The CIA rat model was successfully established, as demonstrated by the higher toe swelling value, thymus and spleen/body weight, immobility time and serum IL-1β concentration, as well as lower body weight, climbing time and mRNA expression of programmed death-1 (PD-1), transforming growth factor-β1 (TGF-β1) and forkhead box protein 3 (Foxp3) in the spleen, compared with control rats. Furthermore, histopathology results demonstrated that joint swelling and redness were observed in the knee joints of CIA rats. H&E results revealed that CIA rats presented erosive destruction of the bone and ulceration of the articular cartilage. In addition, in vitro results demonstrated that IL-1β expression was successfully silenced after IL-1β siRNA transfection in lipopolysaccharide-stimulated BMSCs. When compared with the results of PBS rats, both IL-1β siRNA injection and IL-1β siRNA + BMSC transplantation significantly increased the body weight, climbing time and mRNA expression of PD-1, TGF-β1 and Foxp3 in the spleen, while significantly reduced the immobility time and serum IL-1β concentration. In addition, when compared with that of IL-1β siRNA injection, IL-1β siRNA + BMSC transplantation exhibited markedly higher therapeutic efficacy against CIA. These results demonstrated that higher IL-1β contributed to the pathogenesis of CIA, and that IL-1β siRNA injection ameliorated CIA, while its combination with BMSCs exerted synergistic effects, which may be beneficial against RA.

Project description:FK506 (also known as tacrolimus) is a potent immunosuppressive agent that is widely used in the treatment of graft-rejection and autoimmune diseases. FK506 has attracted additional attention owing to its potential role in osteogenic differentiation and bone formation. MicroRNAs (miRNAs) have been demonstrated to serve important roles in the regulation of osteogenic differentiation; however, identification of specific miRNAs and their roles in regulating FK506‑induced osteogenic differentiation have been poorly examined. In the present study, osteodifferentiation of rat bone marrow stromal cells (BMSCs) was induced with varying concentrations of FK506 (5‑5,000 nM) for 3, 7 and 14 days. Differentially expressed miRNAs were profiled using miRNA array, verified by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and subjected to gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results from the present study identified a subset of miRNAs that were differentially expressed, of which five upregulated miRNAs (miR‑106b‑5p, miR‑101b‑3p, miR‑193a‑3p, miR‑485‑3p and miR‑142‑3p) and four downregulated miRNAs (miR‑27a‑3p, miR‑207, miR‑218a‑2‑3p and let‑7a‑5p) were confirmed by RT‑qPCR. GO and KEGG analysis revealed that the predicted target genes of these miRNAs are involved in multiple biological processes and signaling pathways, including cell differentiation and the mitogen‑activated protein kinase (MAPK) signaling pathway. Verification of the miRNA‑target genes revealed that Smad5, Jagged 1 and MAPK9 were significantly upregulated, whereas Smad7, BMP and activin membrane‑bound inhibitor, and dual‑specificity phosphatase 2 were significantly downregulated during FK506‑induced osteodifferentiation. The present study may provide an experimental basis for further research on miRNA functions during FK506‑induced osteogenic differentiation in rat BMSCs.

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GABARAP ameliorates IL-1β-induced inflammatory responses and osteogenic differentiation in bone marrow-derived stromal cells by activating autophagy.

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