Activation of kappa opioid receptor suppresses post-traumatic osteoarthritis via sequestering STAT3 on the plasma membrane

Abstract

Objective: Kappa opioid receptor (KOR) signaling is involved in joint development and inflammation in Osteoarthritis (OA), while the biochemical mechanism remains unclarified. This study aims to investigate downstream molecular events of KOR activation, to provide novel perspectives in OA pathology.

Methods: U50,488H, a selective KOR agonist, was intra-articularly injected in mice upon destabilization of the medial meniscus (DMM) as OA models, with PBS injection as control. The behavioral and histological evaluation was assessed by hot plate test and red solid green staining, respectively. Alterations in mRNA and protein expression were assessed by RNA-seq, RT-qPCR, immunohistochemistry and western blotting (WB) in chondrocytes treated with TNF-α or TNF-α + U50,488H. Proteins interacted with KOR were explored using proximity labeling followed by mass spectrometry and then testified by co-immunoprecipitation (Co-IP) assay and immunofluorescence (IF).

Results: OA-induced pain was reduced and cartilage degeneration was alleviated upon KOR activation in DMM mice. In chondrocytes, activation of KOR reversed the upregulation of MMPs, IL-6, IL-1β and phosphorylated(p-) STAT3, stimulated by TNF-α, while the expression of NF-κB, MAPKs and AKT signaling weren't reversed. RNA-seq and IF results presented that KOR activation evidently reduced STAT3 nuclear translocation in chondrocytes upon TNF-α stimuli. The reduction may be resulted from the binding of KOR and STAT3 in the plasma membrane, revealed by proximity labeling and Co-IP results.

Conclusions: KOR activation protects cartilage from OA, and this protective effect is mainly exerted via sequestering STAT3 on the plasma membrane, resulting in inactivation of STAT3-dependent immune responses which otherwise contributes to OA.

Keywords: Kappa opioid receptor; Osteoarthritis; STAT3; TNF-α.

Fig. 1

KOR activation decelerates the progression of osteoarthritis. A The schematic diagram of the in vivo experiment. B Results of the hot plate assessment (n = 9–12 mice each group). C Results of the Red Solid green dyeing and quantification in items of medial femoral condyle (MFC) and medial tibial plateau (MTP) according to OARSI (n = 9–12 mice each group). Scale bar: 200 μm. D Immunochemical staining and quantification results of ACAN, COL2 and COMP, respectively (n = 9–12 mice each group). Scale bar: 50 μm. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. U50 is short for U50,488H. Data are mean ± SD or median and defined ranges

Fig. 2

KOR activation reduces the expression of cartilage-catabolic markers. A Genes expression of cartilage-degrading enzymes in the RT-qPCR results. B Immunohistochemical results and quantification of MMP3 and MMP13 in mice (n = 6 mice each group). Scale bar: 50 μm. C Expression and quantification of MMP3 and MMP13 in the WB results. One-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. U50 is short for U50,488H. Error bars indicate ± SD

Fig. 3

Activation of KOR down-regulated the inflammatory reactions in chondrocytes upon TNF-α stimuli. A PCA analysis result of three groups. B Differentially expressed genes in the three groups presented in the volcano plot. We mainly focused on the proteins up-regulated on TNF-α stimuli and reversed by KOR activation as shown in the purple and yellow box, respectively. C Venn diagram showed the proteins up-regulated on TNF-α stimuli and reversed by KOR activation through paired comparison. D Heatmap shows the quantification of differentially expressed proteins. Results from three biological replicates. (E) KEGG analysis results showed the pathway enrichment of differentially expressed proteins in the three groups. U50 is short for U50,488H. TNF-α VS CON represented TNF-α group compared to the control group and TNF-α + U50 VS TNF-α represented TNF-α + U50,488H group compared to the TNF-α group

Fig. 4

KOR activation limits STAT3-mediated immune responses in chondrocytes. A RT-qPCR results showed the expression of IL-6 and IL-1β. One-way ANOVA analysis, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate ± SD. B WB results of STAT3 signaling pathway. C WB results of AKT signaling pathway. D WB results of NF-κB signaling pathway. E WB results of MAPK signaling pathway. F WB tested the impacts of stattic, a selective STAT3 inhibitor, on expression of MMP3 and MMP13. G Enrichment of STAT3 pathway by GSEA. H WB results of STAT3 signaling pathway. I RT-qPCR results showed the expression of IL-6 and IL-1β. One-way ANOVA analysis, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate ± SD. J IF tested the location of STAT3 in mice. K IF tested the location of STAT3 in primary chondrocytes. Scale bar: 5 μm. STAT3 (red), DAPI (blue), U50 is short for U50,488H

Fig. 5

KOR interacts with and sequesters STAT3 on the cell plasma. A. The sketch map of plasmid construction. Chondrocytes 293 T cells and ATDC5 cells were transfected with pLV-KOR-TurboID-expression plasmid for 48 h; then expression of TurboID protein were detected using (B) western blotting and (C) immuno-fluorescence assay, respectively. Scale bar: 5 μm. D Workflow for sample preparation and mass spectrometry analysis. E The results of mass spectrometry detection of STAT3 expression. Scale bar: 5 μm. F After plasmid transfection in 293 T cells for 48 h, immunofluorescence was used to detect changes in the of STAT3 localization. ATDC5 cells (H) and transfected 293 T cells (G) and were cultured with TNF-α or TNF-α + U50,488H for 24 h and Co-IP was used to confirm the binding of KOR and STAT3. U50 is short for U50,488H

Fig. 6

Schematic diagram demonstrating that activation of KOR restrains the STAT3 pathway via binding to STAT3 and thereby protects chondrocytes against OA. ↑: up-regulation and ↓: down-regulation (Created with BioRender. com.). U50 is short for U50,488H