Fig 1.
Upregulation of CRTAC1 in the articular cartilage of human osteoarthritis (OA).
A, Representative safranin-O/fast green stain of lesional and macroscopically normal (non-lesional) articular cartilage from knee OA patients undergoing total joint replacement. Scale bars, 200 μm. B, Real-time PCR analysis of CRTAC1 expression in lesional and non-lesional cartilage biopsies from advanced OA patients (n = 19). Paired two-tailed t-test was performed. C, Immunohistochemistry of CRTAC1 on lesional and macroscopically normal (non-lesional) articular cartilage from OA patients (results are representative of n = 6). Normal sheep IgG was used as a negative control. Scale bars, 200 μm (left panels), 100 μm (right panels).
Fig 2.
IL-1β and TNF-α stimulate CRTAC1 expression in human articular chondrocytes and synovial fibroblasts.
A, Real-time PCR analysis of CRTAC1 expression in human articular chondrocytes isolated from macroscopically normal parts of OA cartilage treated by IL-1β for 24 hrs. Two-tailed t-test was performed. B, Real-time PCR analysis of CRTAC1 expression in human OA synovial fibroblasts after treatment with IL-1β for indicated times. C, Real-time PCR analysis of CRTAC1 in human OA synovial fibroblasts treated with TNF-α for indicated times. Two-Way ANOVA followed by Sidak’s test for multiple comparisons was performed for B and C. All data are presented as means ± SDs.
Fig 3.
Crtac1 knockout mice demonstrate normal bone and cartilage development at 12 weeks of age.
A, Micro-CT (μCT) analyses of trabecular (BV/TV, Tb.N, Tb.Th, Tb.Sp) and cortical (Cortical Th., Cortical BV/TV) bone parameters in female wild type and Crtac1-/- mice of 12 weeks age. All data are means ± SDs. Two-tailed t-tests were performed. B, Three-D μCT images (upper panels) and safranin-O/fast green stain (middle and lower panels) of mouse knee joints showing bone or articular cartilage of wild type and Crtac1-/- female mice at 12 weeks of age. The local lack of safranin-O staining in articular cartilage of the femoral condyle of Crtac1-/- mouse is an artifact of cutting/staining, not a real lesion. The artifact is also observed in the femoral condyle of WT mouse in S3B Fig. Scale bars, 1 mm (upper panels), 800 μm (middle panels), 200 μm (lower panels).
Fig 4.
Similar histologic changes of OA in wild type and Crtac1-/- mice 8 weeks after DMM surgery.
A and B, Safranin-O/fast green stain of right knee joints of wild type and Crtac1-/- mice showing changes of articular cartilage (arrowheads) and osteophyte formation (B, arrows) 8 weeks after DMM surgery. C and D, The maximum and summed OA histologic scores for articular cartilage (C) and osteophytes (D) in wild type and Crtac1-/- mice 8 weeks after DMM surgery. Data were pooled from male (5 wild type, 3 Crtac1-/-) and female (3 wild type, 3 Crtac1-/-) animals. The maximum cartilage scores for male mice: 5 WT: 1, 1, 2, 2, 3; 3 Crtac1-/-: 0.5, 1, 3; and for females: 3 WT: 1, 1, 3; 3 Crtac1-/-: 0.5, 1, 1. The summed cartilage scores for male mice: 5 WT: 3.5, 4.5, 5, 5.5, 10; 3 Crtac1-/-: 2.5, 5.5, 7; and for females: 3 WT: 3.5, 4, 9.5; 3 Crtac1-/-: 2.5, 3.5, 5. The maximum osteophyte scores for male mice: 5 WT: 1, 1, 1, 1, 2; 3 Crtac1-/-: 1, 1, 2; and for females: 3 WT: 1, 1, 1; 3 Crtac1-/-: 1, 1, 2. The summed osteophyte scores for male mice: 5 WT: 3, 3, 5, 6, 6; 3 Crtac1-/-: 3, 4, 8; and for females: 3 WT: 4, 5, 5; 3 Crtac1-/-: 4, 5, 6. All data are means ± SDs. Nonparametric Mann-Whitney tests were performed. Scale bars: 200 μm (A), 400 μm (B).
Fig 5.
Ablation of CRTAC1 inhibits post-traumatic OA in female mice.
A, Safranin-O/fast green stain of knee articular cartilage of wild type and Crtac1-/- female mice 16 weeks after DMM surgery showing decreased structural destruction of articular cartilage after deletion of Crtac1. B, The maximum and summed OA histologic scores for articular cartilage 16 weeks after DMM surgery in wild type and Crtac1-/- female mice. C, Safranin-O/fast green stain of right knee joints showing that ablation of Crtac1 reduce osteophyte formation at the margin of articular cartilage (arrows) 16 weeks after DMM surgery. D, Maximum and summed histologic scores of osteophytes in wild type and Crtac1-/- female mice 16 weeks after DMM surgery. Scale bars: 200 μm (A), 400 μm (C). All data are means ± SDs. Nonparametric Mann-Whitney tests were performed.
Fig 6.
Deletion of CRTAC1 inhibits pain of post-traumatic OA in female mice.
A, Gait analyses of right hind limb (surgical) of wild type and Crtac1-/- female mice 16 weeks after DMM surgery displaying decreased brake time and paw angle, and increased paw area and midline distance after ablation of Crtac1. B, Gait analyses showing no significant difference about these parameters in contralateral left hind limb of wild type and Crtac1-/- female mice 16 weeks after DMM surgery. All data are means ± SDs. Two-tailed t-tests were performed.