Calcitonin and prednisolone display antagonistic actions on bone and have synergistic effects in experimental arthritis

Abstract

We tested here the hypothesis that calcitonin and glucocorticoids, known to modulate bone metabolism, could have opposite actions on bone cells regulating expression of cytokine receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG). In the U2OS osteosarcoma cell line, calcitonin (10(-11) to 10(-9) mol/L) reduced RANKL and augmented OPG both at the mRNA and protein levels. Cell incubation with prednisolone (10(-8) to 10(-6) mol/L), the glucocorticoid chosen for this study, produced opposite results. These molecular studies prompted more functional analyses whereby osteoclast bone resorptive activity was determined. Calcitonin (10(-10) mol/L) abrogated the stimulating effect of 10 ng/ml RANKL or 10(-9) mol/L prednisolone; similar results were obtained with OPG. Assessment of calcitonin and prednisolone effects in an in vivo model of rheumatoid arthritis revealed partially surprising results. In fact, calcitonin not only preserved bone morphology (as assessed on day 18) in rats subjected to arthritis and treated with prednisolone (0.8 to 4 mg/kg daily from day 13) but also synergized with the steroid to elicit its antiarthritic effects. These results suggest that calcitonin could be used as a novel cotreatment to augment efficacy and reduce side effects associated with the prolonged use of steroids.

Figure 1

GC receptor binding assay in U2OS cells. A: Scatchard plot analysis with different concentrations of the tracer [³H]dexamethasone indicates the existence of a single specific binding site in U2OS cells. The parameters calculated from this analysis are the following: affinity constant (K_D) of 10.4 ± 1.3 nmol/L, with a maximal number of binding sites (B_max) of 53.6 ± 3.8 pmol/L, corresponding to 32,532 ± 2993 sites per cell. The results are expressed as the means ± SEM of three experiments performed in triplicate. B: Dexamethasone binding was displaced with unlabeled prednisolone in a concentration-related manner with a calculated IC₅₀ of 87.8 ± 3.4 nmol/L, demonstrating that U2OS cells are also capable of binding prednisolone. The results are expressed as the means ± SEM of three experiments. All experiments were performed in triplicate. **P < 0.01.

Figure 2

Expression of a functional calcitonin receptor in U2OS cells. A: Expression of the specific mRNA for the calcitonin receptor as detected by RT-PCR. U2OS cells show a positive band at the expected size (386 bp) (lane 1), whereas another human osteosarcoma cell line, Saos-2, did not express CTR (lane 2). The breast cancer cell line T47D was used as positive control for the receptor expression (lane 3). B: FACS histogram showing immunofluorescence due to the anti-CT receptor mAb compared with control antibody. C: Increase in intracellular cAMP accumulation in U20S cell following 30 minutes of stimulation with eCT. Values for C are mean ± SEM of three experiments performed in triplicate.

Figure 3

CT modulates RANKL and OPG expression in U2OS cells. A: Cell incubation with CT (10⁻¹⁰ mol/L, 2 hours) down-regulates RANKL mRNA expression and up-regulates OPG mRNA expression, as detected by RT-PCR. The representative blot shows the RANKL product (441 bp), the OPG product (412 bp), and the 18S (324 bp), used as internal control. Graph on the right presents these data in a semiquantitative manner, with normalization for each specific product versus 18S RNA band. The values obtained in U2OS cells in the absence of treatment are taken as 100%, with a calculated decrease in RANKL mRNA of 79% and increase in OPG mRNA of 59%. Data are means ± SEM of three experiments. B: RANKL protein levels as measured in U2OS cells by EIA. Cells were incubated with eCT for 4 or 24 hours. C: OPG protein released by U2OS cells as measured by EIA. Cell incubation as in B. For B and C, results are expressed as the means ± SEM of three experiments performed in triplicate. *P < 0.05 versus control (concentration 0).

Figure 4

Prednisolone modulates RANKL and OPG expression in U2OS cells. A: Cells were incubated with prednisolone (10⁻⁸ mol/L, 2 hours) and analysis of mRNA expression conducted as detailed in Figure 3. Blot is representative of three experiments whose densitometric analysis after normalization with 18S RNA is shown on the right. B: RANKL protein levels as measured in U2OS cells by EIA. Cells were incubated with prednisolone for 4 or 24 hours. C: OPG protein released by U2OS cells as measured by EIA. Cell incubation as in B. For B and C, results are expressed as the means ± SEM of three experiments performed in triplicate. *P < 0.05 versus control (concentration 0).

Figure 5

CT and prednisolone modulate the bone resorptive activity of rat primary osteoclasts. A: Bone resorption after overnight cell incubation with single compounds (each concentration given in the legend). Prednisolone (10⁻⁹ mol/L) and RANKL (10 ng/ml) potentiated bone resorption, whereas OPG (100 ng/ml) and eCT (10⁻¹⁰ mol/L) diminished it. Data are means ± SEM of three experiments. *P < 0.05 and **P < 0.01 versus control (unstimulated cells). B: Cell incubation with prednisolone (10⁻⁹ mol/L) and the reported concentrations of OPG reduced bone resorption. Data are means ± SEM of three experiments. **P < 0.01 versus control (unstimulated cells); ^#P < 0.05 and ^##P < 0.01 versus prednisolone alone. C: eCT (10⁻¹⁰ mol/L) addition to cells abolishes the effect of both RANKL (10 ng/ml) and prednisolone (10⁻⁹ mol/L). Data are means ± SEM of three experiments. **P < 0.01 versus control (unstimulated cells) and ^##P < 0.01 versus RANKL or prednisolone alone.

Figure 6

CT and prednisolone synergize in the CIA model. Collagen II injection to rats provoked arthritis from day 11 onwards when treatment with CT (2 μg/kg s.c.) alone or with prednisolone (0.8 or 4 mg/kg s.c.) started and continued on a daily basis. A: Profiles of disease incidence. B: Changes in right hind paw volume. C: Arthritic scores. Data are means ± SEM of 10 rats per group. *P < 0.05 versus vehicle; ^#P < 0.05 versus prednisolone (0.8 mg/kg/day) alone.

Figure 7

Histological analyses of arthritic joints. Selected digit joints were subjected to histological preparation as described in Materials and Methods and analyzed for monitoring synovitis and bone. a: Representative picture taken from a naïve rat, showing normal bone structure and adjacent tissue with no apparent signs of inflammation. b: Arthritic rat joint with clear focal bone resorption (arrowheads) and severe active inflammation (asterisks) in surrounding tissue. After treatment with after 0.8 mg/kg s.c. prednisolone as detailed in Figure 6, intense inflammation in periosseous tissues is evident (c, asterisks) accompanied by marked bone resorption (d, arrowheads). e and f: Animals treated with prednisolone together with CT (2 μg/kg s.c.) displayed minimal inflammation and no significant bony abnormalities. All pictures are representative of more than five sections prepared from two to three different rats. Original magnifications, ×200.