Expression of the cadherin-11 gene is a discriminative factor between articular and growth plate chondrocytes Dr T. Matsusaki, M.D., T. Aoyama, M.D.,

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Presentation transcript:

Expression of the cadherin-11 gene is a discriminative factor between articular and growth plate chondrocytes Dr T. Matsusaki, M.D., T. Aoyama, M.D., Ph.D., K. Nishijo, M.D., Ph.D., T. Okamoto, M.D., Ph.D., T. Nakayama, M.D., Ph.D., T. Nakamura, M.D., Ph.D., Dr J. Toguchida, M.D., Ph.D. Osteoarthritis and Cartilage Volume 14, Issue 4, Pages 353-366 (April 2006) DOI: 10.1016/j.joca.2005.10.008 Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Expression of Cdh-11 in MMR14 and MMR17. The expression of Cdh-11 in MMR14 and MMR17 cells during culture was examined by RT-PCR (A) and Western blotting (B). Localization of Cdh-11 was analyzed by immunocytochemistry (C). Counterstained with DAPI (original magnification, ×400; bar=10μM). Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 Expression of Cdh-11 in primary cultured articular chondrocytes, growth plate chondrocytes and COs. The mRNA expression of Cdh-11 and bone/cartilage-related genes in primary cultured articular chondrocytes, growth plate chondrocytes and COs was examined by RT-PCR (A). The expression of Cdh-11 protein in these primary cultured cells was analyzed by Western blotting (B) and immunocytochemistry (C). Counterstained with DAPI (original magnification, ×400; bar=10μM). Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 Expression of Cdh-11 in growth plate. Localization of Cdh-11 in growth plate was analyzed by immunohistochemistry. Decalcified specimens were stained with antibody for Cdh-11 (A) or nonimmune IgG (B). Same specimens were also stained with Safranin-O (C). Each specimen was counterstained with hematoxylin (original magnification, ×400; bar=50μM). Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Expression of cadherin family genes. The expression of cadherin family genes in cell lines and primary cultured articular chondrocytes, growth plate chondrocytes and COs was examined by RT-PCR. Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Cell–cell adhesion of MMR14 requires Ca2+. MMR14 cells were cultured for 2 weeks to form nodules, and then treated with 0.01% trypsin with (TC treatment) (A, a and B, a) or without (TE treatment) (A, b and B, b) 1mM CaCl2. (A, a and b) Phase contrast view of nodules. (B, a and b) Alizarin red staining of nodules. (C) The number of alizarin red-positive nodules after TC or TE treatment. Experiments were performed individually two times. *P<0.01. MMR17 cells (D, a and b), MMR14 cells (D, c and d), and primary cultured chondrocytes from growth plate (D, e and f), were dispersed with 0.01% trypsin without CaCl2. After a wash with HCMF, cells were allowed to aggregate in HCMF with (D, a, c, and e), or without (D, b, d, and f) 1mM CaCl2 (original magnification, ×100). Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 Knockdown of the expression of Cdh-11 by siRNA reduced the nodular formation of MMR14. The mRNA expression of the Cdh-11, Cdh-2, Cdh-3 and Alp genes was analyzed by RT-PCR in MMR14, MMR14 transduced with lentiviral expression vectors containing siRNA for the firefly luciferase gene (MMR14/LV-FFi), or the murine cadherin-11 gene (MMR14/LV-Cdh11i) (A). Calcified nodules produced by each cell were stained with alizarin red (B) and the number (C) and percent area (D) of alizarin red-positive nodules were calculated. *P<0.01, *P<0.01. Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 7 Association of expression level of the Cdh-11 gene with the number of calcified nodules in siRNA-transduced clones. MMR14 cells transduced with LV-Cdh11i were cultured under drug selection, and single cell cloning by the limited dilution method was performed. Four clones were randomly selected and further analyzed. The mRNA expression of the Cdh-11 gene of each clone was analyzed by RT-PCR (A, upper), and the relative expression level was digitalized as described in the Materials and methods section (A, lower). Each clone was cultured for 2 weeks to produce nodules, which were then stained with alizarin red (B, upper). The number and percent area of alizarin red-positive nodules were calculated (B, lower). Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions

Fig. 8 Introduction of Cdh-11 expression in MMR17. The mRNA expression of cadherin and calcification-related genes was analyzed by RT-PCR in MMR17 transduced with a retroviral expression vector containing the murine Cdh-11 gene (MMR17/QCXIH-Cdh11) or an empty vector (MMR17/QCXIH) (A). The expression of Cdh-11 protein in these cells was analyzed by Western blotting (B) and immunocytochemistry (C) (original magnification, ×400; bar=10μM). Calcified nodule formation of these cells was analyzed by alizarin red staining (D). Osteoarthritis and Cartilage 2006 14, 353-366DOI: (10.1016/j.joca.2005.10.008) Copyright © 2005 OsteoArthritis Research Society International Terms and Conditions