Divergent effects of strontium and calcium-sensing receptor positive allosteric modulators (calcimimetics) on human osteoclast activity

Abstract

Background and purpose: Strontium ranelate, a drug approved and until recently used for the treatment of osteoporosis, mediates its effects on bone at least in part via the calcium-sensing (CaS) receptor. However, it is not known whether bone-targeted CaS receptor positive allosteric modulators (PAMs; calcimimetics) represent an alternative (or adjunctive) therapy to strontium (Sr²⁺ _o ).

Experimental approach: We assessed three structurally distinct calcimimetics [cinacalcet, AC-265347 and a benzothiazole tri-substituted urea (BTU-compound 13)], alone and in combination with extracellular calcium (Ca²⁺ _o ) or Sr²⁺ _o , in G protein-dependent signalling assays and trafficking experiments in HEK293 cells and their effects on cell differentiation, tartrate-resistant acid phosphatase (TRAP) activity and hydroxyapatite resorption assays in human blood-derived osteoclasts.

Key results: Sr²⁺ _o activated CaS receptor-dependent signalling in HEK293 cells in a similar manner to Ca²⁺ _o , and inhibited the maturation, TRAP expression and hydroxyapatite resorption capacity of human osteoclasts. Calcimimetics potentiated Ca²⁺ _o - and Sr²⁺ _o -mediated CaS receptor signalling in HEK293 cells with distinct biased profiles, and only cinacalcet chaperoned an endoplasmic reticulum-retained CaS mutant receptor to the cell surface in HEK293 cells, indicative of a conformational state different from that engendered by AC-265347 and BTU-compound 13. Intriguingly, only cinacalcet modulated human osteoclast function, reducing TRAP activity and profoundly inhibiting resorption.

Conclusion and implications: Although AC-265347 and BTU-compound 13 potentiated Ca²⁺ _o - and Sr²⁺ _o -induced CaS receptor activation, they neither replicated nor potentiated the ability of Sr²⁺ _o to inhibit human osteoclast function. In contrast, the FDA-approved calcimimetic, cinacalcet, inhibited osteoclast TRAP activity and hydroxyapatite resorption, which may contribute to its clinical effects on bone mineral density LINKED ARTICLES: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.

Figure 1

Structures of the calcimimetics used in this study.

Figure 2

Cooperativity (log αβ) and affinity (pK_B) values for the modulation by cinacalcet, AC‐265347 and BTU‐compound 13 of Ca²⁺ _o‐ and Sr²⁺ _o‐mediated activation of multiple signalling endpoints in Flp‐In HEK293‐TREx‐c‐myc‐CASR cells (graphed by calcimimetic). Affinity and cooperativity estimates were derived from analysis of grouped datasets using an operational model of allosterism and agonism (see Methods). Data are mean ± SEM for 5–29 individual experiments performed in duplicate (individual ‘n’ numbers are shown in Table 1). *P < 0.01, one‐way ANOVA with Tukey's multiple comparisons test for differences between pathways.

Figure 3

Effect of calcimimetics (3 μM) on the cell surface expression of either c‐myc‐tagged wild‐type or mutant (G670E) CaS receptors expressed in HEK293 cells. Cinacalcet, but not AC‐256347 or BTU‐compound 13, was able to chaperone the intracellular‐retained G670E mutant to the cell surface as detected by flow cytometry (*P < 0.01 vs. respective vehicle control; two‐way ANOVA with Dunnett's multiple comparisons test, for five individual experiments).

Figure 4

Characterization of human osteoclast cultures. (A) mRNA expression of markers of mature osteoclasts including the CaS receptor (CASR), TRAP (ACAP5), the receptor for RANK‐L (RANK), TNF receptor associated factor‐6 (TRAF6), calcitonin receptor (CALCR), cathepsin K (CTSK) and integrin subunit α V (ITGAV). (B) Expression of CaS receptor protein as detected by Western blotting in osteoclasts with varying days of culture with RANK‐L and (C) comparison with human CaS receptors expressed in HEK293 cells. Human osteoclasts are characteristically multinucleated (Hoechst, blue) and express calcitonin receptors (mouse‐anti human calcitonin receptor 1^o with anti‐mouse IgG‐Alexa 594 2^o, yellow; D) and TRAP (ELF‐97®, green; E) and have distinct actin ring morphology typical of active, resorbing osteoclasts (Phaloidin‐647, red; F). Von Kossa staining of osteoclast‐mediated hydroxyapatite surface resorption after culture in the absence (G) and presence (H) of RANK‐L.

Figure 5

Sr²⁺ _o (20 mM) inhibits osteoclast differentiation and activity. (A) Sr²⁺ _o had no significant effect on the proportion of multi‐nucleated cells within the population and (B) significantly decreased TRAP activity (fewer TRAP foci per cell) in higher‐order osteoclasts (bins of cells containing greater than 10 nuclei; *P < 0.01 vs. vehicle; repeated measures two‐way ANOVA with Dunnett's multiple comparisons test). (C) Unsurprisingly, osteoclast‐mediated hydroxyapatite surface resorption was impaired in the presence of increasing concentrations of Sr²⁺ _o (*P < 0.01 vs. vehicle, one‐way ANOVA with Dunnett's multiple comparisons test). Data are mean ± SEM from 11 individual experiments with analysis of four fields of view per experiment per treatment.

Figure 6

Calcimimetics do not modulate osteoclast differentiation, either alone (3 μM) or in combination with a sub‐effective (5 mM) concentration of Sr²⁺ _o. Data are mean ± SEM from 10 individual experiments with four fields of view analysed per experiment per treatment.

Figure 7

Quantitative analysis of TRAP activity (average number of TRAP foci per cell) in osteoclasts treated with calcimimetics alone (3 μM) or in the presence of a sub‐effective concentration of Sr²⁺ (5 mM), binned by nuclei number. Cinacalcet (A; in the presence of Sr²⁺ _o) significantly reduced TRAP activity in the highest‐order osteoclasts (18+ nuclei), whereas AC‐265347 (B) was without effect. BTU‐compound 13 (C) paradoxically appeared to increase TRAP activity alone in osteoclasts with 18+ nuclei, although there was no effect when combined with Sr²⁺. Data are mean ± SEM from 10 individual experiments with four fields of view analysed (*P < 0.01 vs. vehicle; repeated measures two‐way ANOVA with Dunnett's multiple comparisons test).

Figure 8

Osteoclasts were differentiated with calcimimetics alone (3 μM) or in the presence of a sub‐effective concentration of Sr²⁺ _o (5 mM) for 8 days. Cinacalcet alone (and in combination with Sr²⁺ _o) significantly inhibited osteoclast‐mediated hydroxyapatite surface resorption; BTU‐compound 13 had a small inhibitory effect alone (which was not seen in combination with Sr²⁺ _o) and AC‐256347 was without significant effect. Data are mean ± SEM from 10 individual experiments performed in duplicate (*P < 0.01 vs. vehicle; repeated measures two‐way ANOVA with Dunnett's multiple comparisons test).