Glycogen Synthase Kinase 3 β inhibits BMSCs Chondrogenesis in Inflammation via the Cross-Reaction between NF- κ B and β-Catenin in the Nucleus

Abstract

Inflammation can influence the pluripotency and self-renewal of mesenchymal stem cells (MSCs), thereby altering their cartilage regeneration ability. Sprague-Dawley (SD) rat bone marrow mesenchymal stem cells (BMSCs) were isolated and found to be defective in differentiation potential in the interleukin-1β- (IL-1β-) induced inflammatory microenvironment. Glycogen synthase kinase-3β (GSK-3β) is an evolutionarily conserved serine/threonine kinase that plays a role in numerous cellular processes. The role of GSK-3β in inflammation may be related to the nuclear factor-κB (NF-κB) signaling pathway and the Wnt/β-catenin signaling pathway, whose mechanism remains unclear. In this study, we found that GSK-3β can inhibit chondrogenesis of IL-1β-impaired BMSCs by disrupting metabolic balance and promoting cell apoptosis. By using the inhibitors LiCl and SN50, we demonstrated that GSK-3β regulates the chondrogenesis via the NF-κB and Wnt/β-catenin signaling pathways and possibly mediates the cross-reaction between NF-κB and β-catenin in the nucleus. Given the molecular mechanisms of GSK-3β in chondrogenic differentiation in inflammation, GSK-3β is a crucial target for the treatment of inflammation-induced cartilage disease.

Figure 1

Chondrogenic differentiation of BMSCs in an IL-1β-induced inflammatory microenvironment. (a) and (b) Representative images and intensity quantification of Alcian blue staining. (c) The GAG content in the culture medium. The expression level of Sox9, Collagen 2a and Aggrecan were detected by qRT-PCR (d) and western blotting (e) and (f). All results are expressed as the mean ± SD (n = 3). NS: not significant, ^∗P < 0.05 and ^∗∗P < 0.01. Bar = 800 μm.

Figure 2

GSK-3β disrupts metabolic balance of IL-1β-impaired BMSCs. (a) Representative immunofluorescence images of collagen 2a. (b) The GAG content in the culture medium. (c) The expression level of Sox9, C and Aggrecan were detected by qRT-PCR (c) and western blotting (d) and (e). All results are expressed as the mean ± SD (n = 3). NS: not significant and ^∗P < 0.05. Bar = 1000 μm.

Figure 3

GSK-3β promotes apoptosis of IL-1β-impaired BMSCs. (a)-(d) The expression level of caspase 9, cleaved caspase 9, caspase 3, cleaved caspase 3, Bax, Bcl-2, and Survivin were detected by western blotting. Flow cytometric analysis (e) and (f) and TUNEL staining (g) were performed to assess the number of apoptotic cells. All results are expressed as the mean ± SD (n = 3). NS: not significant, ^∗P < 0.05. Bar = 800 μm.

Figure 4

GSK-3β regulates the NF-κB signaling pathway in IL-1β-induced inflammation. Phosphorylation and/or total expression of IKKβ, IKKα/β, IκBα, and NF-κB p65 (a) and (b) were detected by western blotting. Visualization and quantification of the expression of cytoplasmic (c) and (d) and nuclear (e) and (f) NF-κB p65. All results are expressed as the mean ± SD (n = 3). NS: not significant, ^∗P < 0.05, and ^∗∗P < 0.01.

Figure 5

GSK-3β regulates the Wnt/β-catenin signaling pathway in IL-1β-induced inflammation. Phosphorylation and total expression of GSK-3β (a) and (b) and β-catenin (c) and (d) were detected by western blotting. (e)-(h) visualization and quantification of the expression of nuclear β-catenin. All results are expressed as the mean ± SD (n = 3). NS: not significant, ^∗P < 0.05, and ^∗∗P < 0.01.

Figure 6

GSK-3β mediates the cross-reaction between NF-κB and β-catenin in the nucleus. Immunofluorescence was used to observe the distribution of NF-κB p65 (red) and β-catenin (green). The nuclei were DAPI-stained (blue). Three independently repeated experiments were performed, and a representative image is shown. Bar = 100 μm.

Figure 7

Schematic diagram of the mechanisms. GSK-3β regulates the chondrogenesis via NF-κB signaling and Wnt/β-catenin signaling and possibly mediates the cross-reaction between NF-κB and β-catenin in the nucleus.