Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis

Abstract

Objectives: Dysregulated chondrocyte metabolism is closely associated with the pathogenesis of osteoarthritis (OA). Suppressing chondrocyte catabolism to restore cartilage homeostasis has been extensively explored, whereas far less effort has been invested toward enhancing chondrocyte anabolism. This study aimed to repurpose clinically approved drugs as potential stimulators of chondrocyte anabolism in treating OA.

Methods: Screening of a Food and Drug Administration-approved drug library; Assays for examining the chondroprotective effects of digoxin in vitro; Assays for defining the therapeutic effects of digoxin using a surgically-induced OA model; A propensity-score matched cohort study using The Health Improvement Network to examine the relationship between digoxin use and the risk of joint OA-associated replacement among patients with atrial fibrillation; identification and characterisation of the binding of digoxin to low-density lipoprotein receptor-related protein 4 (LRP4); various assays, including use of CRISPR-Cas9 genome editing to delete LRP4 in human chondrocytes, for examining the dependence on LRP4 of digoxin regulation of chondrocytes.

Results: Serial screenings led to the identification of ouabain and digoxin as stimulators of chondrocyte differentiation and anabolism. Ouabain and digoxin protected against OA and relieved OA-associated pain. The cohort study of 56 794 patients revealed that digoxin use was associated with reduced risk of OA-associated joint replacement. LRP4 was isolated as a novel target of digoxin, and deletion of LRP4 abolished digoxin's regulations of chondrocytes.

Conclusions: These findings not only provide new insights into the understanding of digoxin's chondroprotective action and underlying mechanisms, but also present new evidence for repurposing digoxin for OA.

Keywords: chondrocytes; osteoarthritis; therapeutics.

Fig. 1.. Ouabain and Digoxin enhance chondrogenesis and stimulate chondrocyte anabolism.

(A) C3H10T1/2 mesenchymal stem cells were incubated in the absence or presence of 50 nM Ouabain or 100 nM Digoxin for 1, 7, 14 days, followed by Alcian blue staining. (B) Quantification of (A). (C) C3H10T1/2 cells were incubated in the absence or presence of 50 nM Ouabain or 100 nM Digoxin for 2 or 7 days, qRT-PCR was performed to examine the expression of Col2a1, Comp, Acan, Sox5, Sox6 and Sox9. (D, E) mRNA levels of COL2A1, ACAN and COMP in human C28I2 chondrocytes treated with a series of Ouabain or Digoxin for 24 h, assayed by qRT-PCR analysis. (F) mRNA levels of Col2a1 and Acan in murine primary chondrocytes treated with various concentrations of Ouabain or Digoxin for 24 h, assayed by qRT-PCR analysis. (G) mRNA levels of COL2A1, ACAN and COMP in human primary normal and OA chondrocytes treated with or without Ouabain or Digoxin for 24 h, assayed by qRT-PCR analysis. The values are mean±SEM of at least 3 independent experiments; *p<0.05, **p<0.01, ***p<0.001 versus control group.

Fig. 2.. Ouabain and Digoxin protect against OA in a surgically induced model in vivo.

(A) Experimental flow chart. DMM surgery was performed in 12-week-old male C57BL/6J mice. Ouabain or Digoxin was administered 3 days after DMM surgery (n=8; a mouse in the PBS control group died of unknown causes 10 weeks after surgery). (B) The severity of OA-like phenotype 12 weeks after surgery was analyzed by grading histological sections using the Osteoarthritis Research Society International (OARSI) score system. (C) Representative images of Safranin O/Fast Green stained sections of knee joints from mice treated with or without Ouabain or Digoxin for 12 weeks. Scale bar=800 μm (top panel) and 200 μm (bottom panel). (D, E) Representative images of immunohistochemical staining for type II collagen, aggrecan neoepitope, MMP13 and ADAMTS5 in knee joint sections of mice treated with or without Ouabain or Digoxin for 12 weeks. Scale bar=100 μm. Positive staining for type II collagen, aggrecan neoepitope, MMP13 and ADAMTS5 were quantified. (F) Three-dimensional mirco-CT images of pathological structural changes in the mouse knee 12 weeks after surgery. (G) Osteophyte number (Op.N) and (I) size (Op.TV) in the knee of mice after DMM surgery. (H) Three-dimensional mirco-CT images of osteophyte formation between the groups. The region marked in red shows osteophyte. (J) Three-dimensional mirco-CT images of calcified meniscus and synovial tissue between the groups. (K) The volume of calcified meniscus and synovial tissue (Cal Tis.V) was quantified. *p<0.05, **p<0.01, ***p<0.001 versus PBS control group.

Fig. 3.. Effect of Ouabain and Digoxin on pain-related behaviors in the DMM-induced OA mice model.

(A) Mechanical sensitivity was measured using von Frey filaments twice a week after DMM surgery. Statistical analysis was conducted using two-way analysis of variance (ANOVA) and multiple t tests. P values were compared between Ouabain (*) or Digoxin (#) group and PBS control group. (B) Representative track plots show decreased spontaneous activity of mice in open field tests after DMM surgery. Changes in spontaneous activity, including (C) travel distance, (D) max speed, (E) active time and (F) absolute turn angle were evaluated 4, 8 and 12 weeks after DMM surgery. * or #p<0.05, ** or ##p<0.01, *** or ###p<0.001 versus PBS control group.

Fig. 4.

Cumulative incidence of knee or hip OA-associated joint replacement in 28397 Digoxin users and 28397 non-users, matched by propensity-score.

Fig. 5.. LRP4 is the target of Ouabain and Digoxin.

(A) Coomassie blue staining of DARTS assay. The band with molecular weight around 200 kDa was protected by Ouabain. (B) LRP4 adapted image from mass spectrometry. (C) C28I2 cells were digested with several dosages of protease with or without various concentrations of drugs, as indicated, then the level of LRP4 was assayed using Western blot. (D) C28I2 cell lysate was denatured under various temperatures and the protein level of LRP4 in control, Ouabain-treated and Digoxin-treated groups were assayed using Western blot and densitometry analysis curve. (E) Isothermal dose response with serial concentrations of drug. Protein level of LRP4 was measured via Western blot with associated curve. (F) Overview of IFD-predicted binding positions of Ouabain and Digoxin in LRP4 aa 146-737 monomer. LRP4 is shown by ribbons along with yellow surface of 70% transparency. Ouabain and Digoxin are shown by CPK representation with the following color scheme: carbon-faded orange (Ouabain) or teal (Digoxin), oxygen-red, polar hydrogen-white. Non-polar hydrogen atoms are not shown. (G, H) IFD-predicted docked LRP4-Ouabain complex (G) and LRP4-Digoxin complex (H) with the ligand depicted in ball and stick and the important interacting residues depicted as sticks. Hydrogen bonds are represented by dotted yellow lines and the distance of hydrogen bonds are measured in Å. (I, J) The 2D interaction diagrams of Ouabain (I) and Digoxin (J) docked with LRP4 aa 146-737. The amino acids within 4 Å to the ligand are shown as colored bubbles, where polar residues are cyan, hydrophobic residues are green, positively charged residues are purple, and negatively charged residues are red. Hydrogen bonds are shown by magenta arrows. (K) Schematic view of LRP4 receptor domain organizations and localization of mutations tested. (L, M) DARTS assay for serial point mutants of LRP4. C28I2 cells were transfected with the plasmid expressing various Flag-tagged LRP4 mutants, as indicated. Mutants of LRP4 were detected by Flag antibody. The values are mean±SEM of at least 3 independent experiments.

Fig. 6.. LRP4 is down-regulated in OA cartilage and required for Ouabain and Digoxin regulation of chondrocyte anabolism.

(A) Human non-arthritic and OA cartilage stained with Safranin O/Fast Green (left) and detected LRP4 by immunohistochemical staining (right). The red arrow and the inserted image indicate the expression of LRP4 in a single chondrocyte. Scale bar=400 μm (top panel) and 200 μm (bottom panel). (B) Expression of LRP4 was measured in healthy, early OA and late OA patients via Western blot. (C) Densitometry analysis of immunoblotting results shown in (B). (D, E) Immunohistochemical staining of LRP4 and quantification of LRP4 positive cells in knee joint sections of C57BL/6J mice treated with or without cardenolides for 12 weeks after DMM surgery. Scale bar=100 μm; n≥7. (F) Workflow of generating LRP4 knockout C28I2 cells using CRISPR/Cas9 technology. (G) Western blot confirmation of LRP4 knockout in C28I2 cells. (H) Immunoblotting of ERK1/2 and AKT signaling activation in control and LRP4 knockout C28I2 cells with or without re-expression of LRP4 treated with 50 nM Ouabain or 100 nM Digoxin for 30 min. (I, J) mRNA levels of COL2A1, ACAN and COMP in control or LRP4 knockout C28I2 cells treated with a series of Ouabain or Digoxin concentrations for 24 h, assayed by qRT-PCR analysis. (K, L) Control and LRP4 knockout C28I2 cells with or without re-expression of wildtype or Glu343 site mutated LRP4 were treated with 50 nM Ouabain or 100 nM Digoxin for 24 h, mRNA levels of COL2A1, ACAN and COMP were detected by qRT-PCR. LRP4 mu: Glu343 site mutation of LRP4. The values are mean±SEM of at least 3 independent experiments; *p<0.05, **p<0.01, ***p<0.001 versus control group.