Fig 2: EP4 regulates chondrocyte differentiation and chondrocyte anabolism.a, b H&E staining of femoral articular cartilage from EP4f/f and Ep4Col2 mice at 3 months of age (a). Scale bar, 50 µm. Chondrocyte numbers (CH) were calculated in superficial zone (SZ), transition, and middle zones (TZ + MZ), calcified zone (CZ), and entire femoral articular cartilage quantified (Total CH) (b). n = 5 mice per group. c, d Primary articular chondrocytes were isolated from Ep4Col2 knee joints of new born pups. Micromass of the chondrocytes cultured in the chondrogenic media for 3, 7, and 14 days were stained with Alcian blue (c). Alcian blue-stained cultures were quantified (d). Error bars are means ± s.d., n = 6. **P < 0.01, ***P < 0.001, ****P < 0.0001 by by two-way ANOVA with multiple comparison using Sidak method. e–g qRT-PCR analyses of Acan, Col2a1, and Sox9 expression in primary articular chondrocytes of EP4f/f and Ep4Col2 and 7 days after chondrogenic differentiation. Error bars are means ± s.d., *P < 0.05, **P < 0.01 by student t-test. h, i qRT-PCR analyses of Acan and Sox9 expression in primary articular chondrocytes from EP4f/f and Ep4Col2mice treated with IL-1ß for 24 h. Error bars are means ± s.d., n = 6, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with multiple comparison using Tukey method.

Fig 3: EP4 deletion in osteoclasts reduces pain in OA by suppressing Netrin-1 secretion and CGRP-positive sensory neuron recruitment to subchondral bone. Von Frey assay and thermal hyperalgesia test conducted at the right hind paw of the EP4fl/fl or littermate EP4LysM mice 2 weeks (a) or 8 weeks (b) after ACLT surgery. Error bars are the mean ± s.d. Two-way ANOVA followed by Tukey’s t tests. n = 6 for each group. c qRT-PCR analysis of the mRNA levels of Ngf and Netrin-1 in osteoclasts generated from BMMs stimulated with 10 ng·mL-1 M-CSF and 50 ng·mL-1 RANKL and incubated with 100 nmol·L-1 PGE2 for 5 days. The experiments were performed with three biological replicates. Error bars are the mean ± s.d. **P < 0.01 and ns, not significant by one-way ANOVA followed by Tukey’s t tests. d Representative images of Netrin-1 protein by western blotting of osteoclasts generated using BMMs from the EP4fl/fl or littermate EP4LysM mice stimulated with 10 ng·mL-1 M-CSF and 50 ng·mL-1 RANKL and incubated with 100 nmol·L-1 PGE2 for 5 days. The experiments were performed with three biological replicates. Representative images of IHF staining of Netrin-1 (e, red) and CGRP-positive sensory nerve fibers (f, red) in the subchondral bone of the EP4fl/fl or littermate EP4LysM mice 2 weeks after ACLT surgery (left) and corresponding quantitative analysis (right). The white arrows indicate Netrin-1 or CGRP IHF signals. Error bars are the mean ± s.d. Two-way ANOVA followed by Tukey’s t tests. n = 3 for each group. Scale bars, 20 µm

Fig 4: EP4 expression level increased in human and mouse injured articular cartilage.a Representative images of safranin O/Fast Green (S.O.) and immunofluorescence staining for EP4 in the uninjured or injured human adult articular cartilage from human OA patients (left). The EP4 expression was quantified (right). Nucleus were shown by DAPI staining. Scale bar, S.O. staining, 200 µm, immunofluorescence staining; 50 µm. Error bars are means ± s.d., n = 3, *P < 0.05 by student t-test. b Scatter plot showing the linear correlation between MMP3 expression and EP4 in articular cartilage of human OA patients. Two to three articular cartilage explants from each patient were collected. Samples from ten patients were subject to qRT-PCR. Pearson’s correlation analysis was performed. c Representative images of S.O. staining on sham operated or MF at 4 weeks after surgery. Scale bar, 200 µm. EP4 expression level increased in the mouse articular cartilage defect region 4 weeks after MF surgery, co-stained with Acan. Sham-operated mice were used as controls. Scale bar, 50 µm. The EP4 expression was quantified (right). Error bars are means ± s.d., n = 3. *P < 0.05 by student t-test. White dotted lines indicated the surface of articular cartilage and dotted boxes indicated the defect region. d Sham or DMM surgery was carried out at 10-week-old mice. Representative images of S.O. staining of sham-operation or DMM group at 6 weeks after surgery. EP4 expression levels increased in the mouse articular cartilage defect region 6 weeks after the DMM surgery (left). Scale bar, 50 µm. The EP4 expression was quantified, co-stained with Acan (right). Error bars are means ± s.d., n = 3. *P < 0.05 by student t-test. White dotted lines indicated the surface of articular cartilage. e, f qRT-PCR analysis for human EP4 in primary chondrocyte cells from human OA patients’ uninjured articular cartilage treated with or without IL-1ß (1 ng/ml) (e) or TNF-a (10 ng/ml) (f) stimulation for 24 h. Error bars are means ± s.d., n = 3. *P < 0.05, **P < 0.01 by student t-test. g, h qRT-PCR analysis for mouse EP4 in primary mouse articular chondrocyte cells with IL-1ß (1 ng/ml) (g) or TNF-a (10 ng/ml) (h) stimulation for 24 h. Error bars are means ± s.d., n = 3. *P < 0.05, ***P < 0.001 by student t-test.

Fig 5: EP4 antagonists prevented cartilage ECM breakdown by promoting ECM generation and inhibiting cartilage catabolism in human articular cartilage.a HL-43 prevented cartilage ECM degradation. The relative intact non-weight-bearing explants of human cartilage were isolated from knee OA patients undergoing total knee replacement. S.O. staining of OA patient cartilage explants after 7 days of treatment with Celecoixb, Grapiprant, and HL-43 at indicated dosage. Scale bars, 200 µm. Veh, vehicle. b Proteoglycan staining area (%) was measured by S.O. staining. Error bars are means ± s.d., n = 4 for each group. c Release of total glycosaminoglycan (GAG) into the media of human articular cartilage explant was measured by 1,9-dimethylmethylene blue (DMMB) assays. Human articular cartilage explants were treated with the indicated chemical compounds for 7 days. The secreted GAG was normalized to the wet weight of the cartilage explant to calculate the release of GAG, then normalized to vehicle. Error bars are means ± s.d., n = 3 for each group. d qRT-PCR analysis of anabolic factors COL2A1, ACAN, SOX9, and MMP13 expressed in the human articular cartilage explants treated with 1 ng/ml IL-1ß and indicated chemical compound for 24 h (Celecoxib 10 µM, Grapiprant and HL-43 30 µM). e Representative images of immunohistochemistry staining of indicated antibodies in vehicle, Celecoxib, Grapiprant, HL-43 treated human cartilage explants, scale bars, 100 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with multiple comparison using Tukey method. ns, not significant; Veh, vehicle.