Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes

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Calcitonin directly attenuates collagen type II degradation by inhibition of matrix metalloproteinase expression and activity in articular chondrocytes B.C. Sondergaard, M.Sc., H. Wulf, K. Henriksen, M.Sc., S. Schaller, Ph.D., S. Oestergaard, M.Sc., P. Qvist, Ph.D., L.B. Tankó, M.D., Ph.D., Y.Z. Bagger, M.D., Ph.D., C. Christiansen, M.D., Ph.D., M.A. Karsdal, Ph.D. Osteoarthritis and Cartilage Volume 14, Issue 8, Pages 759-768 (August 2006) DOI: 10.1016/j.joca.2006.01.014 Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 1 Calcitonin receptors are found in articular chondrocytes. Monoclonal antibody against the calcitonin receptor was used to localize calcitonin receptor. (A) Bovine articular cartilage explants positive for calcitonin receptor localization, (B) human OA articular cartilage positive for calcitonin receptor localization, and (C) immunohistochemistry performed with a negative control mouse antibody of the same isotype, IgG2a, on bovine articular cartilage explants. Tissue sections were counterstained with Ehrlich's hematoxylin, and the scale bar represents 20μM and the pictures 60× magnification of the sections. Osteoarthritis and Cartilage 2006 14, 759-768DOI: (10.1016/j.joca.2006.01.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 2 The calcitonin receptor is expressed in articular chondrocytes. PCR fragments were amplified from ssDNA reverse transcribed from total RNA of isolated bovine articular chondrocytes. Human osteoclasts were used as positive control for the calcitonin receptor. As internal positive control, oligonucleotides for actin were used (Actin). As negative control (Neg. Co), calcitonin receptor oligonucleotides were used and the reverse transcriptase left out. CT-R, oligonucleotides for the calcitonin receptor. Osteoarthritis and Cartilage 2006 14, 759-768DOI: (10.1016/j.joca.2006.01.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 3 The calcitonin receptor on articular chondrocytes signals through the cAMP pathway. Articular chondrocytes were isolated and sub-cultured for 5 days, lifted and cultured under serum-free conditions for 24h. The chondrocytes were hydrolyzed after 20min stimulation with (1) IBMX alone, (2) calcitonin in combination with IBMX, (3) forskolin and (4) forskolin in combination with IBMX. The levels of cAMP were quantified as described in Materials and methods. Untreated cells were used as negative control. The asterisks indicate significant differences (**P<0.01, ***P<0.001). Osteoarthritis and Cartilage 2006 14, 759-768DOI: (10.1016/j.joca.2006.01.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 4 Direct concentration-dependent chondroprotective effect of calcitonin. Bovine articular cartilage explants were cultured under serum-free conditions with a combination of the cytokines TNF-α and OSM to induce degradation of collagen type II. Every 2nd or 3rd day conditioned medium was replaced with cytokines and calcitonin were indicated. Control explants were cultured in medium only. The bars show the concentration-dependent inhibition of CTX-II release by calcitonin in the conditioned medium on day 19, adjusted for the amount of cultured cartilage. Each bar represents the mean value+s.e.m. from six individual wells. The asterisk indicates significant difference (*P≤0.05). Osteoarthritis and Cartilage 2006 14, 759-768DOI: (10.1016/j.joca.2006.01.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 5 Calcitonin attenuates MMP activity. Bovine articular cartilage explants were cultured under serum-free conditions in the presence of the cytokines TNF-α and OSM. Every 2nd and 3rd day the conditioned medium was replaced with cytokines, calcitonin, or vehicle. The conditioned medium collected at day 18 was used for gelatinase zymography. Lane A: vehicle, lane B: cytokines, lane C: cytokines+1μM calcitonin, and lane D: cytokines+100nM calcitonin. A corresponding gel was incubated in buffer containing the general MMP inhibitor, GM6001, as control. The arrows indicate the molecular weight of pro- and active-MMP-9. Osteoarthritis and Cartilage 2006 14, 759-768DOI: (10.1016/j.joca.2006.01.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions

Fig. 6 Calcitonin inhibits cartilage degradation in vivo. Four groups of rats: Sham, ovariectomy (OVX)+salmon calcitonin (CT)+vehicle, OVX+vehicle, and OVX+17-β-estradiol, entered the study at baseline, week 0. OVX+vehicle induced a significant increase in CTX-II levels after 2 weeks, compared to sham levels, +++P<0.001, except at week 9. Oral administration of 2.0mg/kg salmon calcitonin in combination with the vehicle 5-CNAC 150mg/kg, resulted in a significant decline in the CTX-II release compared to vehicle-treated, ***P<0.001, at all time points. Values are mean+s.e.m., and the CTX-II levels are compared by unpaired t test with Welch's correction. Osteoarthritis and Cartilage 2006 14, 759-768DOI: (10.1016/j.joca.2006.01.014) Copyright © 2006 OsteoArthritis Research Society International Terms and Conditions