Abstract
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.
Highlights
Preclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA)
The physiological effects of salmon calcitonin (sCT) in humans are mediated through high-affinity CT receptors (CTR) - a class B of the G-protein coupled receptor (GPCR) family
Human chondrocytes express isoform 2 of the calcitonin receptor (CTR) Chondrocytes isolated by enzymatic digestion were used to obtain high quality cartilage RNA for reverse transcription polymerase chain reaction (RT-polymerase chain reaction (PCR)) analysis, which was converted to Complementary DNA (cDNA)
Summary
Preclinical and clinical studies have shown that salmon calcitonin has cartilage protective effects in joint degenerative diseases, such as osteoarthritis (OA). A different splice variant lacking both the 16-amino acid insert in the first intracellular domain as well as the first 47 amino acids of the amino-terminus extracellular domain was reported to bind to [125I] sCT with high affinity and responding to human CT with increases in cAMP [26]. A further human CTR variant results from a T-to-C base mutation producing a leucine 447 to proline (L447P) amino acid change. This substitution has no apparent effect on ligand binding or receptor function in vitro [28], but has been associated with decreased fracture risk in postmenopausal women in different studies [29,30]. The occurrence, expression regulation and physiological significance of those variants are largely unknown
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