Investigation of the direct effects of salmon calcitonin on human osteoarthritic chondrocytes

Abstract

Background: Calcitonin has been demonstrated to have chondroprotective effects under pre-clinical settings. It is debated whether this effect is mediated through subchondral-bone, directly on cartilage or both in combination. We investigated possible direct effects of salmon calcitonin on proteoglycans and collagen-type-II synthesis in osteoarthritic (OA) cartilage.

Methods: Human OA cartilage explants were cultured with salmon calcitonin [100 pM-100 nM]. Direct effects of calcitonin on articular cartilage were evaluated by 1) measurement of proteoglycan synthesis by incorporation of radioactive labeled 35SO4 [5 microCi] 2) quantification of collagen-type-II formation by pro-peptides of collagen type II (PIINP) ELISA, 3) QPCR expression of the calcitonin receptor in OA chondrocytes using four individual primer pairs, 4) activation of the cAMP signaling pathway by EIA and, 5) investigations of metabolic activity by AlamarBlue.

Results: QPCR analysis and subsequent sequencing confirmed expression of the calcitonin receptor in human chondrocytes. All doses of salmon calcitonin significantly elevated cAMP levels (P < 0.01 and P < 0.001). Calcitonin significantly and concentration-dependently [100 pM-100 nM] induced proteoglycan synthesis measured by radioactive 35SO4 incorporation, with a 96% maximal induction at 10 nM (P < 0.001) corresponding to an 80% induction of 100 ng/ml IGF, (P < 0.05). In alignment with calcitonin treatments [100 pM-100 nM] resulted in 35% (P < 0.01) increased PIINP levels.

Conclusion: Calcitonin treatment increased proteoglycan and collagen synthesis in human OA cartilage. In addition to its well-established effect on subchondral bone, calcitonin may prove beneficial to the management of joint diseases through direct effects on chondrocytes.

Figure 1

Calcitonin stimulates proteoglycan formation in human osteoarthritic cartilage. Human OA articular cartilage explants were cultured with or without doses of salmon calcitonin, and on day 16 pulsed with 5 μCi ³⁵SO₄ for 24 hours. The proteoglycans were extracted and the incorporation of ³⁵SO₄ were followed by liquid scitillation counting. Insulin-like growth factor (IGF) was used as a positive control to induce formation in articular cartilage. The bars represent the mean value of count pr. minute, cpm, from 6-replicates and the indivdual values were adjusted for the amount of cultured cartilage. The errorbars are the standard error of mean, SEM. The asterisks symbolize the levels of statistical difference to the vehicle control, * P < 0.05, ***P < 0.001.

Figure 2

Stimulation of human OA cartiage explants with salmon caltionin increase the release of pro-peptides of type II collagen. Human OA cartilage explants were cultured for 16 days with different doses of salmon calcitonin and IGF as positiv control. The released neoepitopes in the supernatant from day 16, were quatifiened by an ELISA detecting the N-terminal collagen type II pro-peptides, PIINP. The bars represent the mean value from 6-replicates and the indivdual values were adjusted for the amount of cultured cartilage. The errorbars are the standard error of mean, SEM. The asterisk symbolize the levels of statistical difference to the vehicle control, * P < 0.05. **P < 0.01.

Figure 3

Human chondrocytes express the calcitonin receptor. A. Calcitonin receptor expression in human osteoclasts and cartilage was analyzed using QPCR. QPCR products were visualized on a 2% agarose gel. Lanes: 1+19 NEB 100 bp DNA ladder, 2-4 CalcR1, 5-7 CalcR2, 8-10 CalcR 3, 11-13 CalcR 4, 14-16 Surf 1, 17-18 Coll2a. Abbreviations: CalcR - calcitonin receptor primer pair 1-4; Coll2a - Collagen type II; HOC - human osteoclasts; HCC - human chondrocytes; NTC - no template control. B. Intracellular cAMP levels after stimulation with salmon calcitonin: Human OA chondrocytes were stimulated with IBMX for one hour, followed by 15 minutes stimulation in presence or absence of salmon calcitonin as specified in the figure legends. C. Binding competition by the calcitonin receptor antagonist 8-32sCT. Forskolin was used as positive control. The asterisks indicate significant differences (** P < 0.01, *** P < 0.001).

Figure 4

Chondrocyte viability. The chondrocyte viability was investigated by the metabolic dye AlamarBlue and was by quantified by fluorescence. The bars represent the mean value from 6-replicates at the last day of culture and the indivdual values were adjusted for the amount of cultured cartilage. The errorbars are the standard error of mean, SEM, and the asteriks indicate ***, P < 0.001. Salmon calcitonin: sCT.