High bone mass in mice can be linked to lower osteoclast formation, resorptive capacity, and restricted in vitro sensitivity to inhibition by stable sulforaphane

Abstract

Mouse strains can have divergent basal bone mass, yet this phenotype is seldom reflected in the design of studies seeking to identify new modulators of bone resorption by osteoclasts. Sulforaphane exerts inhibitory effects on in vitro osteoclastogenesis in cells from C57BL/6 mice. Here, we explore whether a divergent basal bone mass in different mouse strains is linked both to in vitro osteoclastogenic potential and to SFX-01 sensitivity. Accordingly, osteoclasts isolated from the bone marrow (BM) of C57BL/6, STR/Ort and CBA mice with low, high, and intermediate bone mass, respectively, were cultured under conditions to promote osteoclast differentiation and resorption; they were also treated with chemically stabilised sulforaphane (SFX-01) and respective sensitivity to inhibition evaluated by counting osteoclast number/resorption activity on dentine discs. We observed that osteoclastogenesis exhibited different macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-Β ligand sensitivity in these mouse strains, with cells from C57BL/6 and CBA generating higher osteoclast numbers than STR/Ort; the latter formed only half as many mature osteoclasts. We found that 100 nM SFX-01 exerted a potent and significant reduction in osteoclast number and resorptive activity in cells derived from C57BL/6 mice. In contrast, 10-fold higher SFX-01 concentrations were required for similar inhibition in CBA-derived cells and, strikingly, a further 2.5-fold greater concentration was required for significant restriction of osteoclast formation/function in STR/Ort. These data are consistent with the notion that the BM osteoclast precursor population contributes to the relative differences in mouse bone mass and that mice with higher bone mass exhibit lower in vitro osteoclastogenic potential as well as reduced sensitivity to inhibition by SFX-01.

Keywords: SFX-01; STR/Ort mice; bone mass; osteoclastogenesis; osteoclasts; sulforaphane.

Figure 1

Schematic illustration of murine osteoclast differentiation and activation. (A) Bone marrow‐derived cells undergo differentiation into osteoclast precursors in the presence of M‐CSF which primarily promotes preosteoclast proliferation and survival. M‐CSF and RANKL, in combination, are required for differentiation of mononuclear precursors into multinucleated and mature osteoclasts. Osteoclast‐mediated bone resorption is initiated 5 days after the initial marrow isolation by acidifying the S2MEM medium in which the cells were maintained to pH 6.9. When included, SFX‐01 was added to the medium from Days 2 to 7. (B) Range of RANKL and M‐CSF concentrations used in supplementation of the S2MEM medium for the determination of optimal primary osteoclast formation from precursor cells derived from three different mice strains in culture. M‐CSF, macrophage colony‐stimulating factor; RANKL, receptor activator of nuclear factor kappa‐Β ligand.

Figure 2

(A) Cell yield from C57BL/6, CBA and STR/Ort mouse BM differs. BM cell yield extracted from STR/Ort mice is significantly lower than from C57BL/6 (p < .001) and CBA mice (p < .01). **p ≤ .01 ***p ≤ .001. Combined data from n = 5 experiments, 6–8 replicates/condition. (B) The heatmap, represents the number of mice required for each mouse strain and the yield of BM cells that can be extracted in each case. As indicated, STR/Ort yield extraction requires double the number of mice, compared with C57BL/6 and CBA. BM, bone marrow.

Figure 3

There is lower sensitivity to M‐CSF‐/RANKL‐induced in vitro capacity in BM sources from the STR/Ort mice with the highest bone mass. (A–C) Representative reflective light images of osteoclasts extracted from STR/Ort (highest bone mass), CBA (high bone mass), and C57BL/6 (low bone mass) indicate that STR/Ort mice failed to produce mature osteoclasts and resorb using the well‐established culture conditions of S2MEM (1) (3 ng/ml RANKL and 200 ng/ml M‐CSF). (D–F) Representative toluidine blue images indicating BM cells extracted from the high bone mass STR/Ort need raised cytokines (S2MEM (2) (5 ng/ml RANKL and 250 ng/ml M‐CSF) to differentiate and form mature osteoclasts. (G–I) Quantitative analysis indicates reduced osteoclast numbers (TRAP+) in BM‐derived from high bone mass STR/ort mice compared with C57BL/6 and CBA using the optimal conditions of S2MEM (2) (5 ng/ml RANKL and 250 ng/ml M‐CSF) and no effect in resorption or area resorbed/OC. Each experiment was repeated 4–5 times, with 6–8 replicates within each experiment. Numbers are also expressed as mean osteoclast per dentine disc ± SEM, *Statistical p ≤ .05. BM, bone marrow; M‐CSF, macrophage colony‐stimulating factor; RANKL, receptor activator of nuclear factor kappa‐Β ligand; TRAP, tartrate‐resistant acid phosphatase.

Figure 4

Effect of different SFX‐01 concentrations on TRAP+ osteoclast numbers, area resorbed, area resorbed/OC from C57BL/6, CBA, and STR/Ort mice. 100 nM was sufficient to inhibit osteoclastogenesis (A) and total area resorbed (B), but not area rersobed per/OC (C) in cells derived from C57BL/6; while for CBA 1 μΜ was required to achieve similar inhibition levels (D, E, F); STR/Ort derived osteoclasts were completely insensitive up to this concentration(G, H, I). *p ≤ .05, **p ≤ .01. Combined data from n = 5 experiments, 6–8 replicates/condition. TRAP, tartrate‐resistant acid phosphatase.

Figure 5

(A–I) Representative reflective light images of osteoclasts extracted from STR/Ort, CBA, and C57BL/6 mice treated with 100 nM to 1 μM SFX‐01. BM cells extracted from C57BL/6, CBA, and STR/Ort were treated with PBS (controls A, D, and G), 100nM SFX‐01 (B, E, and G, respectively), and 1 μΜ SFX‐01 (C, F, and I). Highlighted images (purple and grey boxes) show the concentration of SFX‐01 required to inhibit osteoclastogenesis. 100 nM of SFX‐01 was required for BM cells extracted from C57BL/6 and 1 μΜ for BM cells extracted from CBA. Scale bar = 200 μm. BM, bone marrow.

Figure 6

BM cells extracted from STR/Ort mice require higher concentration of SFX‐01 to inhibit osteoclastogenesis. (A) Effect of 2.5 μΜ SFX‐01 on TRAP+ osteoclast number, area resorbed and area resorbed/OC from STR/Ort mice. (B) Representative reflective light images of osteoclasts extracted from BM of STR/Ort mice; 2.5 μΜ of SFX‐01 was required for inhibition of osteoclastogenesis/resorption. Resoprtion pits are the brown areas highlighted by the red arrows Combined data from n = 4 experiments, six–eight replicates for each group. *p ≤ .05. BM, bone marrow; TRAP, tartrate‐resistant acid phosphatase.