Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice Q. Wang, Q.Y. Tan, W. Xu, H.B. Qi, D. Chen, S.

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Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice Q. Wang, Q.Y. Tan, W. Xu, H.B. Qi, D. Chen, S. Zhou, Z.H. Ni, L. Kuang, J.Y. Guo, J.L. Huang, X.X. Wang, Z.Q. Wang, N. Su, L. Chen, B. Chen, W.L. Jiang, Y. Gao, H.G. Chen, X.L. Du, Y.L. Xie, L. Chen Osteoarthritis and Cartilage Volume 25, Issue 11, Pages 1868-1879 (November 2017) DOI: 10.1016/j.joca.2017.07.010 Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Inducible cartilage-specific Alk5 cKO mice exhibit a progressive OA–like phenotype. A, Immunohistochemistry (IHC) analysis of ALK5 and pSmad3 protein expressions in articular cartilage of 2 month-old Alk5 cKO mice and Cre-negative control mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice was defined as 1, student's unpaired t-test, n = 4 mice per group). B, Knee joint samples were dissected from 2-month-old mice, and Safranin O/Fast green staining was performed. Results showed articular cartilage lesions (black arrow), increased number of hypertrophic chondrocytes (yellow arrows) and increased thickness (blue double-ended arrows) in articular cartilage of Alk5 cKO mice. Quantitative data were shown in the right panel (Student's unpaired t-test, n = 4 mice per group). C, Knee joint samples were dissected from 3-month-old mice, and Safranin O/Fast green staining was performed. Results showed an early features of OA-like phenotype in articular cartilage of 3-month-old Alk5 cKO mice, including tears and clefts in the articular surface (black arrows) and increased number of hypertrophic chondrocytes in articular cartilage (yellow arrows). D, Knee joint samples were dissected from 6-month-old mice, and Safranin O/Fast green staining was performed. Results showed more severe loss of articular cartilage tissue (black arrows), osteophyte formation (red arrow), and subchondral sclerosis (yellow arrows) in 6-month-old Alk5 cKO mice compared with Cre-negative control mice. E and F, OARSI scoring system showed more severe articular cartilage destruction in 3- and 6-month-old Alk5 cKO mice compared with Cre-negative mice (Mann–Whitney U test, n = 8 mice per group). MFC: medial femoral condyle; MTP: medial tibial plateau. Scale bar: 100 μm. Data in were expressed as the mean ± 95% confidence intervals. In A, B, E and F, symbols represent individual mice. Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10.1016/j.joca.2017.07.010) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Loss of ALK5 signaling alters the expressions of ECM related genes in chondrocytes. A, Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of Alk5 and Serpine1 mRNA expressions in femoral head cartilage isolated from Alk5Col2ERT2 and Cre-negative control mice after 4-OH TM treatment (Student's unpaired t-test, n = 4 femoral head cartilage per group). B, Western blotting analysis of ALK5 and pSmad3 protein expressions in femoral head cartilage isolated from Alk5Col2ERT2 and Cre-negative control mice after 4-OH TM treatment. Quantitative data were shown in the right panel (Student's unpaired t-test, n = 3 femoral head cartilage per group). C, qRT-PCR analysis of Mmp13, Adamts5, Col10, Aggrecan and Col2 mRNA expressions in femoral head cartilage isolated from Alk5Col2ERT2 and Cre-negative mice after 4-OH TM treatment (Student's unpaired t-test, n = 4 femoral head cartilage per group). D, Western blotting analysis of MMP13, ADAMTS5 and ACAN protein expressions in femoral head cartilage isolated from Alk5Col2ERT2 and Cre-negative control mice after 4-OH TM treatment. Quantitative data were shown in the lower panel (Student's unpaired t-test, n = 3 femoral head cartilage per group). E, IHC analysis of MMP13, ADAMTS5 and ACAN protein expressions in articular cartilage of 2-month-old Alk5 cKO mice and Cre-negative control mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice was defined as 1, Mann–Whitney U test, n = 4 mice per group). Scale bar: 100 μm. Data were expressed as the mean ± 95% confidence intervals. In A, B, C and D, symbols represent three technical replicates. In E, symbols represent individual mice. Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10.1016/j.joca.2017.07.010) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Alk5 deficiency increases articular chondrocyte apoptosis. Knee joint samples were dissected from 2-month-old mice, TUNEL assay and cleaved caspase 3 IHC were performed. A, TUNEL staining showed a significant increase in the number of TUNEL positive cells (arrowheads) in articular cartilage of Alk5 cKO mice compared with that in Cre-negative control mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice were defined as 1, Mann–Whitney U test, n = 4 mice per group). B, IHC analysis showed a significant increase in the number of cleaved caspase 3 positive cells in articular cartilage of Alk5 cKO mice compared with that in Cre-negative control mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice was defined as 1, student's unpaired t-test, n = 4 mice per group). Scale bar: 100 μm. Data were expressed as the mean ± 95% confidence intervals. In A and B, symbols represent individual mice. Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10.1016/j.joca.2017.07.010) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Alk5 cKO mice exhibit enhanced synovial hyperplasia and reduced PRG4 expression. A, Knee joint samples were dissected from 3-month-old mice, and hematoxylin and eosin staining (H&E) was performed. H&E staining showed increased synovial thickness in Alk5 cKO mice compared with that in Cre-negative mice (student's unpaired t-test, n = 4 mice per group). B, IHC analysis of PRG4 protein expression in articular cartilage of 2 month-old Alk5 cKO mice and Cre-negative mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice were defined as 1, student's unpaired t-test, n = 4 mice per group). C, qRT-PCR analysis of Prg4 mRNA expression in femoral head cartilage isolated from Alk5Col2ERT2 and Cre-negative mice after 4-OH TM treatment (Student's unpaired t-test, n = 4 femoral head per group). D and E, Femoral head cartilage isolated from 1-month-old Cre-negative control mice was treated with increasing concentrations of SB-505124 (0.1, 0.5 and 1 μM) for 24 h (D) or treated with 1 μM SB-505124 for different periods of time (6, 12 and 24 h) (E). qRT-PCR was performed to detect the Prg4 mRNA expression (One-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group). Scale bar: 100 μm. Data were expressed as the mean ± 95% confidence intervals. In A and B, symbols represent individual mice. In C, D and E, symbols represent three technical replicates. Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10.1016/j.joca.2017.07.010) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 TGF-β1 induces the expression of Prg4 via ALK5 signaling pathways in chondrocytes. A and B, Femoral head cartilage isolated from Prg4GFPCreERT2/+ mice was treated with increasing concentrations of TGF-β1 (5, 10 and 20 ng/ml) for 24 h (A) or treated with 10 ng/ml TGF-β1 for different periods of time (6, 12 and 24 h) (B). qRT-PCR was performed to detect the Prg4 mRNA expression (One-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group). D and E, Femoral head cartilage isolated from Prg4GFPCreERT2/+ mice were treated with increasing concentrations of TGF-β1 (5, 10 and 20 ng/ml) for 24 h (D) or treated with 10 ng/ml TGF-β1 for different periods of time (6, 12 and 24 h) (E). Western blotting was performed to detect the GFP protein expression. Quantitative data were shown in the lower panel (One-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group). C and F, Femoral head cartilage isolated from Prg4GFPCreERT2/+ mice was pretreated with 1 μM SB-505124 for 30 min, followed by an additional 24 h incubation of 10 ng/ml TGF-β1, 1 μM SB-505124, or a combination of both. qRT-PCR and western blotting were performed to detect Prg4 mRNA expression (C, two-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group) and the GFP protein expression (F, quantitative data were shown in the lower panel, two-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group), respectively. G, Femoral head cartilage isolated from Alk5Col2ERT2 and Cre-negative control mice was treated with 4-OH TM for 72 h, followed by an additional 24 h incubation of 10 ng/ml TGF-β1. qRT-PCR was performed to detect the Prg4 mRNA expression (two-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group). H, Primary chondrocytes isolated from Prg4GFPCreERT2/+ mice were transfected with constitutively activated ALK5 (CA-ALK5) expression plasmid or pcDNA3.1/Myc empty vector. 12 h after transfection, cells were treated with 1 μM SB-505124 for another 12 h qRT-PCR was performed to detect the Prg4 mRNA expression (Two-way ANOVA followed by Tukey's test, n = 4 mice per group). Data were expressed as the mean ± 95% confidence intervals. In A-H, symbols represent three technical replicates. Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10.1016/j.joca.2017.07.010) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 TGF-β1/ALK5 signaling induces Prg4 expression via the PKA-CREB signaling pathway in chondrocytes. A, B, G and H. Femoral head cartilage isolated from Prg4GFPCreERT2/+ mice was pretreated with selective inhibitor for 30 min, followed by an additional 24 h incubation with TGF-β1, inhibitor, or a combination of both. qRT-PCR and western blotting were performed to detect the expression of Prg4 mRNA (A and G, two-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group) and the GFP protein expression (B and H, quantitative data were shown in the lower panel, two-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group), respectively. C and D, Primary chondrocytes isolated from Cre-negative mice were pretreated with 10 μM H89 (C) or 1 μM SB-505124 (D) for 30 min, followed by an additional 30 min incubation of 10 ng/ml TGF-β1, 10 μM H89/1 μM SB-505124, or a combination of both. Western blotting was performed to detect the pCREB and pSmad3 protein expressions. Quantitative data were shown in the right panel (two-way ANOVA followed by Tukey's test, n = 4 mice per group). E, Western blotting analysis of pCREB in Cre-negative or Alk5-deficient primary chondrocytes treated with TGF-β1 for 30 min. Quantitative data were shown in the right panel (two-way ANOVA followed by Tukey's test, n = 4 mice per group). F, IHC analysis of pCREB protein expression in articular cartilage of 2-month-old Alk5 cKO mice and Cre-negative control mice. Quantitative data were shown in the right panel (The percentage of positive cells in Cre-negative mice was defined as 1, student's unpaired t-test, n = 4 mice per group). I, qRT-PCR analysis of Prg4 mRNA expression in Cre-negative or Alk5-deficient femoral head cartilage treated with increasing concentrations of forskolin (two-way ANOVA followed by Tukey's test, n = 3 femoral head cartilage per group). J. Schematic illustration of the mechanisms of ALK5 signaling in the maintenance of articular cartilage. Data were expressed as the mean ± 95% confidence intervals. In F, symbols represent individual mice. In A, B, C, D, E, G and H, symbols represent three technical replicates. Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10.1016/j.joca.2017.07.010) Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10. 1016/j. joca Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10. 1016/j. joca Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10. 1016/j. joca Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

Osteoarthritis and Cartilage 2017 25, 1868-1879DOI: (10. 1016/j. joca Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions