BackgroundPreclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA). However, the presence of the calcitonin receptor (CTR) in articular cartilage chondrocytes is yet to be identified. In this study, we sought to further investigate the expression of the CTR in naïve human OA articular chondrocytes to gain further confirmation of the existents of the CTR in articular cartilage.MethodsTotal RNA was purified from primary chondrocytes from articular cartilage biopsies from four OA patients undergoing total knee replacement. High quality cDNA was produced using a dedicated reverse transcription polymerase chain reaction (RT-PCR) protocol. From this a nested PCR assay amplifying the full coding region of the CTR mRNA was completed. Western blotting and immunohistochemistry were used to characterize CTR protein on protein level in chondrocytes.ResultsThe full coding transcript of the CTR isoform 2 was identified in all four individuals. DNA sequencing revealed a number of allelic variants of the gene including two potentially novel polymorphisms: a frame shift mutation, +473del, producing a shorter form of the receptor protein, and a single nucleotide polymorphism in the 3' non coding region of the transcript, +1443 C>T. A 53 kDa protein band, consistent with non-glycosylated CTR isoform 2, was detected in chondrocytes with a similar size to that expressed in osteoclasts. Moreover the CTR was identified in the plasma membrane and the chondrocyte lacuna of both primary chondrocytes and OA cartilage section.ConclusionsHuman OA articular cartilage chondrocytes do indeed express the CTR, which makes the articular a pharmacological target of salmon calcitonin. In addition, the results support previous findings suggesting that calcitonin has a direct anabolic effect on articular cartilage.

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BackgroundIt is important to establish translational methods that aid in pre-clinical go/no-go decision points to increase the success rate of approved DMARDs. The Spleen tyrosine kinase (Syk) inhibitor Fostamatinib (Fosta) was terminated for development for RA, due to insufficient effect on joint structure in phase III.ObjectivesThe objective was to use a translational system of ex vivo cultures to back-translate the insufficient effect on joint structure described in clinical studies.MethodsHuman mature osteoclasts (HOC) seeded on bone, bovine cartilage explants (BEX) and human synovial explants (SME) were treated with R406 (API of Fosta) at 5μM-0.05μM. Osteoclasts were co-stimulated with 25 ng/ml M-CSF and RANKL, while BEX and SME were co-stimulated with TNFα 2 ng/mL and OSM 10 ng/mL (O+T) or TNFα 10 ng/mL, respectively. CTX-1 and Ca2+ were measured in conditioned medium (CM) from HOC. Metabolic activity of HOC was assessed with Alamar Blue. C2M and AGNx1 were measured in CM from BEX, while acMMP3, C1M, and C3M were measured in CM from SME. The biomarkers in BEX and SME CM were measured at 4 time points and the total release were quantified by area under the curve (AUC). CTX-1, C2M, AGNx1, acMMP3, C1M, C2M and C3M were measured with ELISA. Ca2+was measured with ADVIA Chemistry system. Statistical differences of metabolic activity was calculated with One-way ANOVA with Dunnett's multiple comparison test. Statistical differences between biomarkers levels or AUC were calculated with Kruskall Wallis test with Dunn's multiple comparision test.ResultsR406 decreased the release of CTX-1 (Fig 1A) and Ca2+ in a dose-dependent manner, with a significant decrease at 1μM (P<0.01). This might be due to a toxic effect of R406 on HOC (Fig 1B) (P<0.05). R406 decreased the total release of C2M and AGNx1 in a dose-dependent manner in BEX. C2M was inhibited a concentrations down to 1.25μM (P=0.034), and AGNx1 down to 5μM (P=0.012). In SME R406 only decreased the release of C1M and C3M (Fig 1e) significantly at 5 μM (C1M: P=0.03, C3M: P=0.046), and tended to decrease release of acMMP3 (Fig 1f) at 5μM. The later was however not significant.ConclusionsSerum-based biomarkers of the joint ECM turnover were measured in CM from HOC, cartilage and synovial explant cultures. R406 decreased bone resorption and HOC metabolic activity in a dose-dependent manner, together with the MMP-mediated degradation of type II (C2M) collagen and aggrecanse degradation of aggrecan (AGNx1) in cartilage. However, R406 had limited effect on the inflammation driven MMP-mediated degradation of type I (C1M) and III (C3M) collagen and activation of MMP-3. CTX-1, C2M, and C3M have previously been measured in OSKIRA-1, with a profile identical to the ex vivo measurements here [1].References Platt A, Bay-Jensen AC, Braddock M, et al. The Effect of Fostamatinib with Methotrexate on Circulating Biomarkers of Synovium, Cartilage and Bone Metabolism: Potential Utility for Clinical Development Decision Making in Rheumatoid Arthriti [abstract]. Arthritis Rheumatol. 2016...