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From The William Harvey Research Institute, London, United Kingdom
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Abstract |
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B 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.
ß ligand (RANKL) and its receptor RANK are key regulators of bone remodeling and are essential for osteoclast development and activation.1,2
In the presence of macrophage colony-stimulating factor, RANKL promotes osteoclastogenesis and activates mature osteoclasts in vitro.1
Consistently, RANKL-null mice display severe osteopetrosis, stunted growth, and a defect in tooth eruption.3
Some bone-resorbing factors including glucocorticoids (GCs), vitamin D, interleukin-1, interleukin-6, and parathormone up-regulate RANKL expression.4
Osteoprotegerin (OPG), a secreted protein with high homology to the tumor necrosis factor receptor family, is a soluble decoy receptor to RANKL. It therefore acts as an effective inhibitor of osteoclast maturation and activation both in vitro and in vivo.5
OPG-null mice display severe osteoporosis associated with a high incidence of fractures.6,7
All these data indicate that the balance between RANKL-RANK signaling and the levels of biologically active OPG are crucial in regulating the development and activation of osteoclasts and bone metabolism. Further, many factors that inhibit or enhance bone resorption can act, directly or indirectly, through regulation of RANKL-RANK and/or OPG. Besides specific bone pathologies, enhanced bone resorption is also observed in rheumatoid arthritis (RA), a condition characterized by progressive synovial inflammation and joint destruction. Patients with RA have lower bone mineral density and are at risk of pathological fractures.8,9 Bone erosion in RA is caused by osteoclast activation triggered by the production of RANKL by synovial fibroblasts and T lymphocytes and is therefore susceptible to OPG inhibition.3,10 However, OPG has no major anti-inflammatory effects on synovitis or pannus.11
GCs are potent immunosuppressive and anti-inflammatory agents widely used in all forms of chronic inflammation although their long-term use is associated with secondary osteoporosis in vivo. Throughout the years, GC action on bone cells has been associated with an increase in parathormone release leading to an increase in bone resorption as well as a decreased number of bone-forming cells.12,13 Furthermore, GC’s effect on the osteoblast has been linked to RANKL up-regulation and OPG down-regulation.14,15 More recently, a direct action of GCs on osteoclast cytoskeletal rearrangements resulting in suppression of the whole bone-remodeling process has also been reported.16
Calcitonin (CT), a peptide hormone secreted in response to hypercalcemia, has the dual effect of inhibiting osteoclast recruitment as well as their resorptive activity.17 Despite CT efficacy for the treatment of bone metabolic disorders, including osteoporosis, a limitation to its therapeutic application is attributable to CT receptor down-regulation after continuous treatment.18 It is of interest that a dual relationship between GC and CT is emerging, with the former being able to restore or retain CT receptor expression in osteoclasts18 and the latter being effective in preventing GC-induced spine fractures.19,20 No studies have yet addressed if and how GC and CT actions could converge on the RANKL system or the potential functional consequences on this interaction on bone cells, as well as in vivo, and this was the main aim of the present investigation.
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Materials and Methods |
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Cell Culture
The osteosarcoma cell lines U2OS and Saos-2 and the breast cancer cell line T47D (all from the American Type Culture Collection catalog; distributed by LGC Promochem, Teddington, Middlesex, UK) were cultured in McCoy’s medium with 10% fetal calf serum (Gibco, Paisley, UK), 100 U/ml penicillin, and 100 µg/ml streptomycin in an atmosphere of 5% CO2/95% air at 37°C and passaged two times a week.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
Subconfluent cells were incubated with RPMI without phenol red and with 1% stripped bovine serum for 24 hours before stimuli addition. Plates were washed with phosphate-buffered saline (PBS) and RNA extracted with a phenol-free total RNA isolation kit (RNAqueous; Ambion Inc., Austin, TX); total RNA was then reverse-transcribed with random primers (RETROscript; Ambion Inc.) and cDNA used for PCRs (Hybaid; Thermofisher Scientific Inc., Waltham, MA). The sequences of the primers and the PCR conditions used are summarized in Tables 1 and 2
. Primers for 18S RNA (QuantumRNA; Ambion Inc.) (expected size, 324 bp) were used as internal control to normalize the results obtained with densitometry analysis, as performed using Scion Image from NIH Software, Bethesda, MD.
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To quantify OPG and RANKL protein contents, cells (105/well) were incubated for 4 or 24 hours with 10–8 to 10–6 mol/L prednisolone (prednisolone 21-hemisuccinate, sodium salt) or 10–11 to 10–9 mol/L eCT. OPG was measured with enzyme immunoassay (EIA) development reagents from R&D Systems (Abingdon, UK) following the supplier instructions, and RANKL was measured by EIA using paired antibodies from Peprotech (London, UK) as previously described.21 Detection limits for these assays were as follows: 25 pg/ml for OPG and 50 pg/ml for RANKL. Whereas OPG was readily detected in the cell incubation media, RANKL was not detected in the media (data not shown). Thus, cell pellets were incubated with 2 mmol/L Triton in PBS for 10 minutes, centrifuged, and the supernatant analyzed. Relevantly activated T cells produce soluble RANKL, whereas cell-associated RANKL is a feature of osteoblasts and fibroblasts and is functionally active in inducing efficient osteoclastogenesis.22
CT Receptor
The expression of CT receptor on U2OS cell surface was monitored by flow cytometry. In brief, 5 x 105 treated cells were washed twice with PBC (PBS containing 0.2% bovine serum albumin and 1.3 mmol/L CaCl2) and labeled with 20 µg/ml mouse anti-human CT receptor antibody (1 hour at 4°C; Serotec, Abingdon, UK). Then, cells were washed with ice-cold PBC and incubated for 30 minutes at 4°C with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (Serotec). Finally, cells were washed and resuspended in PBC before flow cytometry analysis with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Receptor functionality was confirmed by measuring intracellular accumulation of cAMP.23 For this purpose, cells were plated in 24-well plates (5 x 105/well), serum-starved for 24 hours, and then stimulated for 30 minutes with 10–12 to 10–9 mol/L eCT in the presence of 10–3 mol/L isobuthylmethylxanthine. Samples were then lysed and processed for cAMP EIA determination following the kit manufacturer procedure (Amersham Biosciences, Buckinghamshire, UK).
GC Receptor Binding Assay
Subconfluent U2OS cultures were kept for 24 hours in RPMI medium without phenol red and with 2% bovine serum (charcoal stripped to remove endogenous GC). The binding assay was performed as previously described.24 Briefly, triplicate samples of 106 cells were incubated with 1.57 to 50 pmol [3H]dexamethasone (Amersham Biotechnology, Abingdon, Oxon, UK) at 37°C for 1 hour, in the presence or absence of 50 x 10–6 mol/L unlabeled dexamethasone, to determine the values for nonspecific binding. Samples were then washed three times with ice-cold PBS and analyzed with a ß-counter (LS6000IC; Beckman). Scatchard plot analysis was performed to measure the affinity constant KD and the maximal binding Bmax. To construct displacement curves, cells were prepared as above and incubated with 50 pmol of [3H]dexamethasone in the presence or absence of 10–9 to 10–5 mol/L unlabeled prednisolone for 1 hour at 37°C. Samples were then processed as above, and count per minute (cpm) values were used to calculate the IC50.
Rat Osteoclast Isolation and Bone Resorption Assay
Newborn Sprague-Dawley rats were killed by cervical dislocation and legs removed for isolating osteoclast-rich cell population as previously described.25 The ability of osteoclasts to resorb bone was measured using 24-well plates coated with a film of calcium phosphate apatite (OAAS; OCT Inc., Chunan, Korea). The osteoclast suspension was applied to the 24-well plates and allowed to sediment, and the unattached cells were washed away with minimal essential medium. After 18 hours of incubation with the compounds to be tested, the 24-well plates were treated with a sodium hypochlorite solution [10% (v/v)], washed with distilled water, and dried. The 24-well plates were then analyzed with an inverted microscope interfaced with an Argus-10 image-processing system (Hamamatsu Photonics, Enfield, UK). The number of excavation pits was counted for each well, and the pit area was recorded. The resorbed area on the bone surface was calculated as the sum of the areas of individual excavations and expressed as a percentage of control values.
Collagen II-Induced Arthritis (CIA)
Female Lewis rats (150 ± 20 g body weight; Harlan UK Ltd., Bicester, UK) were fed on a standard chow pellet diet and had free access to water and maintained on a 12-hour light/dark cycle. Animal work was performed under license from the Home Office in accordance with the Animals (Scientific Procedures) Act, 1986. Bovine nasal collagen II (4 mg/ml) was dissolved in acetic acid (0.01 mol/L) and then emulsified with the same volume of ice-cold Freund’s incomplete adjuvant. On day 0, rats were anesthetized with halothane and injected intradermally at the base of the tail with collagen II/adjuvant suspension (400 µg of collagen II per rat). The first signs of arthritis were evident between days 11 and 13, with maximal inflammation observed at days 16 to 18.26
Salmon CT was dissolved in PBS with 0.1% bovine serum albumin and given daily intraperitoneally at the dose of 2 µg/kg per rat. Prednisolone was dissolved in PBS with 0.1% bovine serum albumin and given daily intraperitoneally at doses of 0.6 and 3 mg/kg (corresponding to 1.2 and 6.2 µmol/kg), alone or with sCT. Control treatments consisted of PBS with 0.1% bovine serum albumin. CIA-induced inflammation was confined to ankle joints and footpads of the hind legs (with digit involvement in severe cases). Hind ankles were scored clinically on an arbitrary scale, ranging from 0 (no inflammation) to 3 (severe inflammation, involving ankles, footpads, and digits). In addition, between days 0 and 18, hind paw volumes were measured using a plethysmometer (Ugo Basile, Milan, Italy), and values were averaged to give a measurement of inflammation for each animal. Body weight was also monitored during the study.
Tissue slices for histology were prepared along classical protocols. In brief, on day 18 animals were killed by cervical dislocation and the hind limbs removed, fixed in buffered formol-saline solution, and then placed in formic acid-formal saline solution for 
1 week for decalcification. X-ray analysis by Faxitron imaging confirmed decalcification. Tissue was then sampled, using cross sections of the digits, and tissue blocks were processed to paraffin wax using a Shandon hypercenter with standard protocol (Thermo Electron Corporation, Waltham, MA). Slides were prepared from paraffin blocks at 5-µm thickness; these were dewaxed, rehydrated through graded alcohols, and stained using hematoxylin and eosin. Sections were mounted in Canada balsam and viewed by standard transmission light microscopy. Slides were prepared and examined blind to the treatment group by a histopathologist.
Statistical Analysis
Experiments were repeated at least three times, each in triplicate. In vivo experiments were performed with 10 rats per group. In all cases, data are reported as means ± SEM and were analyzed by analysis of variance followed by Dunnet post hoc analysis. A P value less than 0.05 was taken as significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Initially, we validated the presence of specific receptors for CT and GC in the osteosarcoma cell line U2OS. To detect the GC receptor, we used [3H]dexamethasone as tracer for experiments of binding. Scatchard plot analysis revealed that U2OS cells possess 32,532 ± 2993 binding sites per cell, with an affinity for the receptor of 10.4 ± 1.3 nmol/L (KD) and a Bmax of 53.6 ± 3.8 pmol/L (n = 3 experiments) (Figure 1A)
. Because prednisolone was the GC selected for the majority of the functional experiments, we verified its ability to compete for [3H]dexamethasone binding to GC receptor. Increasing concentrations of unlabeled prednisolone produced a significant displacement with a calculated IC50 of 87.8 ± 3.4 nmol/L (n = 3 experiments) (Figure 1B)
. Complete displacement of [3H]dexamethasone binding was observed at 10–5 mol/L prednisolone.
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. The assay was validated by using the human breast cancer cell line T47D as positive control. Thus, U2OS cells were selected for further experiments. Flow cytometry analysis shows specific and marked presence of the CT receptor in U2OS cells (Figure 2B)
. The functionality of this receptor was assessed by measuring cAMP accumulation, which was augmented after cell incubation with 10–9 mol/L eCT (Figure 2C)
.
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RT-PCR analysis showed that after 2 hours of incubation of U2OS cells with 10–10 mol/L eCT, there was a decrease in RANKL mRNA coupled with an increase in OPG mRNA (Figure 3A)
. This was paralleled by changes at the protein level: eCT produced a decrease in the cellular content of RANKL as seen after 24 hours of incubation with U2OS cells, although significantly different only at the concentration of 10–11 mol/L (Figure 3B)
. The release of OPG in the medium was also modulated by eCT such that significant increases were detected at the 4-hour, but not 24-hour, time point (Figure 3C)
. U2OS cell incubation with prednisolone produced opposite effects on the RANKL/OPG system. In fact, early (2 hours) incubation with this GC yielded an increase in RANKL mRNA level, and a decrease in OPG mRNA (Figure 4A)
. In terms of protein expression, cell-associated RANKL content was not modified by any of the concentrations used (Figure 4B)
, whereas a significant reduction in OPG cellular output was produced both at 4 and 24 hours of incubation with prednisolone (Figure 4C)
.
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The functional relevance of the cellular changes described above was tested by means of bone resorption assay using an osteoclast-enriched cell population. Data in Figure 5
illustrate this set of experiments. Both 10–9 mol/L prednisolone and 10 ng/ml RANKL (equivalent to 4 x 10–10 mol/L) increased bone resorption to a similar extent. Equally, OPG and 10–10 mol/LM eCT inhibited almost completely osteoclast resorptive activity. OPG was active at 100 ng/ml (equivalent to 1.4 x 10–9 mol/L) but not at 30 ng/ml (Figure 5A)
. However, either OPG concentration was effective in significantly attenuating the stimulating effect of 10–9 mol/L prednisolone on resorption (Figure 5B)
. Likewise, 10–10 mol/L eCT was able to challenge both RANKL-induced and prednisolone-induced bone resorption producing a significant reduction of osteoclast activity (Figure 5C)
. None of these actions was consequent on widespread toxicity because no changes in trypan blue uptake could be measured when the cells were incubated with or without 100 ng/ml OPG or 10–10 mol/L eCT (data not shown).
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Rat immunization with collagen provoked a delayed arthritic response localized at the foot and digit level, with kinetics similar to our previous studies (eg, Paul-Clark et al26
). Likewise, administration of a full dose of prednisolone (4 mg/kg/day) markedly inhibited disease incidence and its aggressive malformations, monitored by measuring paw volume and arthritis score: these data are illustrated in Figure 6
. Interestingly, daily administration of CT or a subtherapeutic dose of prednisolone (0.8 mg/kg) failed to impact on disease incidence and arthritic indices, whereas a marked synergistic response was observed in the group of rats co-treated with CT plus 0.8 mg/kg prednisolone. Figure 6A
shows the marked attenuation of disease incidence in the CT plus 0.8 mg/kg prednisolone group, and this was mirrored by reductions in paw volume (Figure 6B)
and arthritic score (Figure 6C)
. The synergistic effect of CT could not be observed when the higher dose of prednisolone was used because it is fully therapeutic on its own (Figure 6)
. All these effects were not secondary to alteration in the health status of the animals: vehicle-treated rats gained 4 g throughout the 26-day period, and this was reflected in all groups with changes ranging from 4 to 8 g of body weight gain (not significantly different for any group).
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. In the rats treated with the subtherapeutic dose of prednisolone, no major protective effects were observed with respect to both the inflammatory reaction and bone alterations (Figure 7, c and d)
. However, analysis of the joints of the rats treated with CT plus this subtherapeutic dose of prednisolone showed a marked attenuated degree of synovitis (Figure 7e)
, in line with the macroscopic analysis (Figure 6)
, and a nearly intact bone profile (Figure 7f)
. Thus, histological morphology reinforced the synergistic protective role of CT when accompanied by the low-dose prednisolone.
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Discussion |
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CT is a 32-amino acid peptide hormone with potent inhibitory effects on osteoclast activity: on one hand, it down-regulates osteoclast recruitment and, on the other hand, exerts potent inhibition on the resorptive activity of mature osteoclasts.17 Whereas these actions of CT are well described, not many studies have investigated the possibility that these effects could be, at least partially, indirect. For instance, CT can produce an anabolic effect on osteoblasts in vitro,27,28 and moreover, CT receptors have been found in cells of osteoblast lineage as well as in an osteosarcoma cells line.29 Here, we demonstrated that the osteoblast-like cell line U2OS expressed both message and protein for the CT receptor. In addition, the receptor was functional because marked increases in cAMP23 could be measured after addition of low eCT concentrations. U2OS cells also expressed the GC receptor. Analysis of the experiments of binding indicated an affinity constant similar to one previously found in human osteosarcoma cell lines30 as well as in our previous studies with other cellular systems.24 The coexpression of these two receptors in this osteoblastic cell line prompted the second part of the study, also in view of the preclinical and clinical opposing effects exerted by GC and CT on bone resorption.12,13,17 In the in vitro experimentation, we focused on the RANKL/RANK system shown to have crucial primary effects on bone metabolism.3
GCs increase RANKL and suppress OPG production in primary osteoblasts and osteosarcoma cells.14,15 In our hands, U2OS cell mRNA levels of RANKL were significantly augmented after 2 hours of incubation with prednisolone. Interestingly, and congruently with the proresorptive action of this GC, OPG mRNA levels were significantly decreased in the same culture conditions. This set of results is in line with what reported by other studies in which micromolar concentrations of hydrocortisone or dexamethasone affected OPG and RANKL mRNA.14 These actions of prednisolone on transcription were only partially reflected on translation. U2OS cell incubation with prednisolone significantly decreased OPG release as measured at both 4 and 24 hours after GC addition, but RANKL protein expression was only slightly, and not significantly, increased. The reason for this discrepancy is unclear and may warrant more detailed analysis. In any case, it was important to compare the effects of prednisolone with those of CT on these cells because we found that CT was also able to modulate these two cytokines. Short (2 hours) U2OS cell incubation with eCT markedly decreases RANKL mRNA expression counteracted by an increase in OPG mRNA expression to a similar degree (–79 versus +50%). Interestingly, these two effects related to changes in protein expression with a distinct time profile: whereas OPG protein expression was augmented at 4 hours after eCT addition, a reduction in RANKL content was significant only at the 24-hour time point. These data demonstrate that the U2OS osteoblast cell line is a sensitive target for the actions of CT. Whereas this conclusion apparently contrasts with the classic views of mechanism of action of CT,17 it is noteworthy that more recent observations provide support to our findings. For instance, CT displays antiapoptotic effects on osteocytic cells as well as mature osteoblasts.27 This hormone stimulates proliferation, calcium uptake, and appearance of alkaline phosphatase activity (a distinct marker of osteoblast differentiation) in osteoblast-like cells.28 In addition, the procalcitonin amino-terminal cleavage peptide stimulates U2OS cell proliferation.31 However, the relationship of CT on osteoblast function is complex and may be bidirectional because CT receptor heterozygous knockout mice show an increase in bone formation.32
Because the relative local expression of RANKL and OPG is instrumental in determining the degree of osteoclast-mediated resorption,22 a balance of these cytokines in favor of OPG will yield a protective effect on the skeleton. Therefore, we next tested the effects that eCT and prednisolone would exert on OPG and RANKL-regulated bone resorption. In a widely used in vitro model of bone resorption, eCT prevented RANKL-induced bone resorption, producing an almost complete abolition of osteoclast activity. These effects were not attributable to toxicity and were evident at the low concentration, congruent with the effects on de novo OPG synthesis and release. Then, as we had reported its ability to potently stimulate bone resorption in vitro,26 we added prednisolone to the system. At nanomolar concentrations, the GC displayed a stimulating effect on osteoclast activity, abolished by CT.
Finally, we wished to translate these findings to a more complex and pathologically relevant condition, and the choice fell to a model of RA. This pathology is characterized by increased bone erosion in addition to progressive synovial inflammation and joint destruction. Such bone degradation is caused by osteoclast activation as triggered by the production of RANKL by synovial fibroblasts and T lymphocytes.3,33,34 In addition, a highly effective therapy for RA is GC treatment;35 however, its long-term use is associated with secondary osteoporosis.19,36 Besides their catabolic action on bone-forming cells, GCs also increase bone resorption via augmented parathormone release and more recently have been shown to up-regulate RANKL and decrease OPG levels,12-14 as well as directly affect the osteoclast activation process by preventing optimal microfilament formation.16 Therefore, the beneficial anti-inflammatory action of GCs can be impaired by an exacerbating effect on bone erosion.
In the present study, we evaluated the effect of administration of CT in combination with prednisolone in a model of RA. Whereas CT alone did not modify the incidence or severity of disease in terms of inflammation signs, there was a marked improvement in bone erosion as evident from the histological analysis. Interestingly, co-administration of CT and a subtherapeutic dose of prednisolone had a dramatic synergistic effect and reduced inflammation symptoms to the same levels of those attained by the high-dose prednisolone; importantly, CT beneficial effects on bone erosion were maintained. It is unfortunate that, in our hands, the immunoassays for OPG and RANKL did not determine the rat homologs (L.M., data not shown), and therefore we were unable to measure any changes in their plasma levels in response to prednisolone and/or CT in the rat.
A potential reason for the synergic action associated with the combined treatment of CT and prednisolone could be proposed. It is known that after repeated stimulation with CT there is an escape phenomenon due to receptor down-regulation, consequent to reduced mRNA stability and receptor binding activity. For instance, treatment of human osteoblast-like cells with GC increases transcription of CT receptor gene expression, thereby preventing loss of CT receptor function even in the presence of the agonist.18 However, other molecular mechanisms behind these synergistic anti-inflammatory properties could also be postulated and deserve systematic investigation, although some of the interrelationship discussed here, converging on the OPG/RANKL system, might represent a logical starting point. In addition, early indications that CT might augment glycosaminoglycan, possibly aggrecan, synthesis from chondrocytes37 could also have implications for our findings, adding this cell type to the list of potential CT targets. It is therefore possible that multiple actions of CT are responsible for its remarkable in vivo effects in experimental arthritis.
In conclusion, starting from analyses of CT and prednisolone effects on bone-regulating cytokines, we have revealed the novel notion that CT could be an effective anti-arthritic treatment complementary to GC, such that the latter drug would be effective in controlling inflammatory diseases without producing the side effects on the bone compartment. Moreover, our data in the CIA model suggest that CT could potentiate the anti-inflammatory activity of prednisolone itself.
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Footnotes |
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Supported by the Joint Research Board of the St. Bartholomew’s Hospital (grant XMNH) and the Pinewood Foundation (to I.M.).
M.P. is a senior fellow of the Arthritis Research Campaign UK.
Accepted for publication November 21, 2006.
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  B. R. Becklund, D. W. Hansen Jr., and H. F. DeLuca Enhancement of 1,25-dihydroxyvitamin D3-mediated suppression of experimental autoimmune encephalomyelitis by calcitonin PNAS, March 31, 2009; 106(13): 5276 - 5281. [Abstract] [Full Text] [PDF]
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