Inhibition of a signaling modality within the gp130 receptor enhances tissue regeneration and mitigates osteoarthritis

Abstract

Adult mammals are incapable of multitissue regeneration, and augmentation of this potential may shift current therapeutic paradigms. We found that a common co-receptor of interleukin 6 (IL-6) cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) were resistant to surgically induced osteoarthritis as reflected by reduced loss of proteoglycans, reduced synovitis, and synovial fibrosis. The F814 mice also exhibited enhanced regenerative, not reparative, responses after wounding in the skin. In addition, pharmacological modulation of gp130 Y814 upstream of the SRC and MAPK circuit by a small molecule, R805, elicited a protective effect on tissues after injury. Topical administration of R805 on mouse skin wounds resulted in enhanced hair follicle neogenesis and dermal regeneration. Intra-articular administration of R805 to rats after medial meniscal tear and to canines after arthroscopic meniscal release markedly mitigated the appearance of osteoarthritis. Single-cell sequencing data demonstrated that genetic and pharmacological modulation of Y814 resulted in attenuation of inflammatory gene signature as visualized by the anti-inflammatory macrophage and nonpathological fibroblast subpopulations in the skin and joint tissue after injury. Together, our study characterized a molecular mechanism that, if manipulated, enhances the intrinsic regenerative capacity of tissues through suppression of a proinflammatory milieu and prevents pathological outcomes in injury and disease.

Fig. 1.. Inhibition of gp130 Y814 signaling leads to down-regulation of prodegenerative and profibrotic pathways.

(A) Western blot data for pSRC (active) in pig articular chondrocytes stimulated with the IL-6 family of cytokines (10 ng/ml), including LIF, ciliary neurotrophic factor (CNTF), OSM, IL-11, hyper IL-6, and IL-6. Total SRC was used for normalization. Data represent means ± SD. n = 4 biological replicates per group. (B) Schematic of modified amino acids within the gp130 812 to 827 domain. Western blot of pSRC in Ba/F3 cells after transfection with the modified or wild-type (WT) gp130 plasmids stimulated with IL-6 (10 ng/ml) for 24 hours. Histone H3 was used for normalization. Horizontal lines with bars show the means ± SD. n = 4 biological replicates (C) Western blots for gp130 pY814 in WT and F814 mouse splenocytes treated with and without OSM, LIF, IL-11, or IL-6 (10 ng/ml) for 24 hours. Total gp130 was used for normalization. Graphs show means ± SD. n = 4 biological replicates. (D) Western blots for immunoprecipitated protein complex formation between gp130 and pSRC in WT and F814 mouse splenocytes stimulated with or without OSM (10 ng/ml) for 24 hours. Total gp130 was used for normalization. Graphs show means ± SD. n = 4 biological replicates. (E) Western blot pSRC in WT and F814 mouse splenocytes stimulated with or without OSM (10 ng/ml) for 24 hours. Total SRC was used for normalization. Graphs show the means ± SD. n = 4 biological replicates. Statistical analysis was performed using one-way ANOVA followed by the Tukey test to compare more than two groups. P values less than 0.05 were considered significant. (F) Distribution of differentially expressed genes in WT and F814 mouse synovial fibroblasts stimulated with or without OSM (10 ng/ml). Log ratio/mean average plots (log₂fold change versus log₂mean expression) for WT-OSM versus WT (left) and Y814-OSM versus Y814 (right) mouse synovial fibroblasts are shown. Each dot represents a gene. Red dots represent up-regulated genes (log₂fold change >1 and P < 0.05). Blue dots represent down-regulated genes (log₂fold change < −1 and P < 0.05). Genes that do not qualify any of these thresholds are indicated by gray dots.

Fig. 2.. Gp130 Y814-deficient mice show improved responses to degenerative joint disease and wound healing.

(A) Histological staining and quantitative assessment of cartilage degradation in WT and F814 mouse knee joints 6 weeks after destabilization of the medial meniscus surgery after 3 months. Safranin O delineates proteoglycans (pink). The OARSI scoring system was used to quantify the extent of cartilage damage. Scale bars, 100 μm. Horizontal lines with bars show the means ± SD. n = 6 biological replicates. (B) WT and F814 mouse post-wound day (PWD) 21 wound sections after wound excision at 6 weeks. Representative images are shown for H&E, Picrosirius red, and COL1 staining of PWD 21 wound sections from WT and F814 mice. n = 5 biological replicates. Scale bars,100 μm. (C) Reanalysis of macrophage clusters from WT and F814 mouse skin wounds PWD 14 after wound excision at 6 weeks. Clusters are color coded in each analysis. Dot plots depict gene expression in each macrophage cluster. Dot sizes are proportional to the percentage of cells in each cluster expressing the indicated gene. The contribution of each sample to each cluster is shown as a stacked bar graph (WT, red; F814, blue). Cluster 6 (mainly WT cells) is highlighted by the red dotted line. (D) Reanalysis of fibroblast clusters from WT and F814 skin wounds PWD 14 after wound excision at 6 weeks. Clusters are color coded in each analysis. Dot plots depict gene expression in each fibroblast cluster. Dot sizes are proportional to the percentage of cells in each cluster expressing the indicated gene. The contribution of each sample to each cluster is shown as a stacked bar graph (WT, red; F814, blue). Statistical analysis was performed using two-tailed Student’s t test to compare two groups. P values less than 0.05 were considered significant.

Fig. 3.. QQpYF peptide prevents gp130-SRC signaling to ameliorate degenerative outcomes in human and porcine samples.

(A) Western blots for immunoprecipitated complex formation between gp130 and pSRC in pig articular chondrocytes stimulated with or without OSM (10 ng/ml) in presence or absence of control scrambled peptide (300 μg/ml) or peptide QQpYF (300 μg/ml) for 24 hours. Total gp130 was used for normalization. Graphs show the means ± SD. n = 4 biological replicates. (B) Computational analysis using GOLD software predicted a high-affinity binding of peptide QQpYF to the regulatory site of SRC (c-SRC). c-SRC as visualized by crystallography. The structure of the indicated c-SRC domains is shown in ribbon diagram representation (left) and with electrostatic potential (blue, positive charge; red, negative charge; white, neutral) mapped onto the molecular surface (right). Peptide QQpYF is shown in stick representation. (C) Western blots in pig articular chondrocytes stimulated with or without OSM (10 ng/ml) in presence or absence of control scrambled peptide (300 μg/ml) or peptide QQpYF (300 μg/ml) for 24 hours. Total respective proteins were used for normalization. Graphs show the means ± SD. n = 4 biological replicates. (D) qPCR analysis of human adult osteoarthritic articular chondrocytes treated with or without peptide QQpYF (300 μg/ml) for 48 hours. Graphs show the means ± SD. n = 3 biological replicates. (E) Pig knee cartilage explants were stimulated with or without OSM (10 ng/ml) and treated with or without peptide QQpYF at indicated doses for 72 hours, followed by a neoepitope assay and Western blot. Amounts of cleaved aggrecan (ACAN) and collagen II (COL2) neoepitopes in the supernatant were quantified with respect to the wet weight of the explant. Graphs show the means ± SD. n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA followed by the Tukey test to compare more than two groups or two-tailed Student’s t test to compare two groups. P values less than 0.05 were considered significant.

Fig. 4.. The small-molecule R805 targets gp130 Y814 signaling to promote wound healing in mice.

(A) Chemical structure of R805. (B) Western blots for gp130 pY814, pY905, and pY759 in pig articular chondrocytes treated with or without OSM (10 ng/ml) and R805 (1 or 10 μM/ml) for 24 hours and normalized to total gp130. Graphs show the means ± SD. n = 4 biological replicates. (C) Mouse vehicle-treated (saline) or R805-treated (20 μM/ml) PWD 21 skin wound sections after wound excision at 6 weeks. Representative images are shown for alkaline phosphatase, H&E, and Picrosirius red staining. Magnification, ×10. Scale bars, 100 μm. n = 8 biological replicates. (D) Hair follicle (n = 3 biological replicates) and fiber length (n = 10 biological replicates) in R805-treated (10 μM or 20 μM/ml) mouse PWD 14 and untreated control wounds after wound excision at 6 weeks. Graphs show the means ± SD. (E) Reanalysis of macrophage clusters from skin wounds of vehicle-treated (saline) or R805-treated (20 μM/ml) mice PWD 14 after wound excision at 6 weeks. Clusters are color coded in each analysis. Dot plots depict gene expression in each macrophage cluster. Dot sizes are proportional to the percentage of cells in each cluster expressing the indicated gene. The contribution of each sample to each cluster is shown as a stacked bar graph (WT, red; R805, blue). Cluster 6 (mainly WT cells) is highlighted by the red dotted line. (F) Reanalysis of fibroblast from skin wounds of vehicle-treated (saline) or R805-treated (20 μM/ml) mice PWD 14 after wound excision at 6 weeks. Clusters are color coded in each analysis. Dot plots depict gene expression in each fibroblast cluster. Dot sizes are proportional to the percentage of cells in each cluster expressing the indicated gene. The contribution of each sample to each cluster is shown as a stacked bar graph (WT, red; R805, blue). Statistical analysis was performed using one-way ANOVA followed by the Tukey test to compare more than two groups. P values less than 0.05 were considered significant.

Fig. 5.. R805 demonstrates disease-modifying effects in large animal models of OA.

(A) Representative images of Safranin O staining and OARSI scoring of canine joint sections after 18 weeks of R805 treatment in surgical OA model. Scale bars, 100 μm. Horizontal lines with bars show the means ± SEM. n = 5. (B) Synovial inflammation, determined by fibrillations and immune infiltration observed in H&E staining after R805 treatment in canine joint. Representative images are shown. Scale bars, 100 μm. Horizontal lines with bars show the means ± SEM. n = 5 or 6 biological replicates. (C) Assessment of canine bone mineral density (BMD) in the medial compartment of the operated stifle and contralateral, non-operated medial compartment by microcomputed tomography (CT). Horizontal lines with bars show the means ± SEM. n = 5 or 6 biological replicates. (D) Representative images of canine knee joint shape change from baseline as determined by serial CT imaging to visualize ectopic bone formation (red, left); quantification on right. Horizontal lines with bars show the means ± SEM. n = 5 or 6 biological replicates. (E) Canine Colorado pain scores were measured daily and totaled in R805 and vehicle-treated animals starting after the first intra-articular injection. Linear regression analysis of daily scores is presented with P values. n = 5 or 6 biological replicates. For (A) to (E), x axes indicate dose used for intra-articular injections. (F) Linear regression analysis of lameness scores of R805-treated (10 μg/ml) and vehicle-treated (saline) canines. n = 5 or 6 biological replicates. Statistical analysis was performed using one-way ANOVA followed by the Tukey test to compare more than two groups. P values less than 0.05 were considered significant.

Fig. 6.. R805 promotes an anti-inflammatory, antifibrotic milieu in canine joints after surgical induction of OA.

(A) Reanalysis of macrophage clusters from synovial joints of R805-treated (10 μg/ml) and vehicle-treated (saline) canines. Clusters are color coded in each analysis. The contribution of each sample to each cluster is shown as a stacked bar graph (WT, red; R805, blue). Cluster 3 (mainly WT cells) is highlighted by the red dotted line. (B) Dot plots depicting gene expression in each macrophage cluster from synovial joints of R805-treated and vehicle-treated (saline) canines. Color-coded clusters correspond to colors in (A). Dot sizes are proportional to the percentage of cells in each cluster expressing the indicated gene. (C) Reanalysis of fibroblast clusters from synovial joints of R805-treated and vehicle-treated (saline) canines. Cells are color coded by their sample of origin. The dashed oval indicates clusters of fibroblasts dominated by R805-treated cells, whereas the solid oval denotes a cluster mainly derived from control cells. Table shows gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are enriched (false discovery rate <0.05 and fold change >1.5) when comparing cells in the solid versus dashed ovals.