Fig 1: GC Treatment suppresses osteoclast formation but does not directly regulate ANG gene expression in osteoclasts.a Schematic diagram showing the procedure of the in vitro experiments. Bone marrow mononuclear cells/macrophages were isolated from 3-week-old C57BL/6J mice and cultured with M-CSF and RANKL for 5 days to induce the formation of mature osteoclasts. The cells were treated with vehicle or MPS with or without RU486 for the entire 5 days (i) or only the last day (ii). The cells were subjected to TRAP staining or lysed to measure the mRNA expression level of ANG. TRAP staining of the cells is shown in (b). Quantified number of TRAP+ spreading multinucleated cells/well is shown in (c). The levels of ANG expression were analyzed by qRT-PCR (d). Experiments were performed in triplicate and were repeated three times. Data are represented as mean ± s.e.m. **p < 0.01, ns, not significant as determined by one-way ANOVA with post hoc Tukey test.
Fig 2: Schematic representation of proposed and theorized mechanism leading to morphine-induced hypersensitivity and enhanced osteolysis through TLR4 receptor activation. Chronic morphine administration is able to activate TLR4 primarily on immune cells within the bone-tumor microenvironment leading to downstream activation of NF-κB transcription factor. In the bone tumor microenvironment where there is an increased inflammatory response, this process can exacerbate production of inflammatory cytokines such as IL-6, TNFα, and IL-1β. These have differential effects on osteoclasts and osteoblasts. Chronic inflammation has a negative effect on osteoblastic differentiation and activity. Acting through multiple receptors including IL-6R and TNFR, the upregulation of genes such as sclerostin and RANKL lead to enhanced osteoclast activity, whereas the downregulation of Runx2 leads to decreased osteoblast differentiation.13,14,21,22,33 Alternatively, on osteoclasts there these same inflammatory factors lead to the upregulation of osteoclastic genes such as Ca2, Trap, Sost, and Csk, which results in enhanced bone resorption.38,45,47,71 In addition, this upregulation in inflammatory genes leads to the promotion of inflammatory pain states and the enhanced bone loss leads to greater pain and risk of pathologic fracture. These effects are observed to be blocked by the TLR4 antagonist, TAK242. Ca2, carbonic anhydrase 2; Csk, cathepsin K; Ctr, calcitonin receptor; IL-1β, interleukin 1β; IL-6, interleukin 6; Opg, osteoprotegerin; Pthr, parathyroid hormone receptor; Rankl, receptor activator of NF-κB ligand; Runx2, runt-related transcription factor 2; Sost, sclerostin; TLR4, toll-like receptor-4; Trap, tartrate-resistant acid phosphatase.
Fig 3: TLR4 receptor inhibition prevents morphine-enhanced osteoclastogenesis of RAW264.7. (A) RAW264.7 cells were treated with RANKL (20 ng/mL) in the presence or absence of morphine (30 µM) or the TLR4 antagonist, TAK242 (500 nM) for 7 days to allow for differentiation and then stained for tartrate resistant acid phosphatase (TRAP). Representative images are displayed (×10). (B) Quantification of TRAP activity in conditioned media. Data are expressed as means ± SEM. One-way ANOVA, Tukey post hoc correction. (*P < 0.05, ****P < 0.0001. n = 4 cultures/condition). TLR4, toll-like receptor-4.
Fig 4: iSN40 inhibits the RANKL-induced osteoclastogenesis of RAW264.7 cells. (A) Representative images of the TRAP staining of RAW264.7 cells treated with 30 ng/mL RANKL and 0.1–3.0 μM iSN40 for 7 days. Scale bar, 100 μm. (B) The number of nuclei in the TRAP+ osteoclasts was quantified. No TRAP+ cells were observed in any of the RANKL(−) groups. ** p < 0.01 vs. RANKL(+)/control. n = 4 fields. (C) Representative images of the pits generated in the bone matrix resorption assay of RAW264.7 cells treated with 100 ng/mL RANKL and 0.3 μM iSN40 for 6 days. Scale bar, 100 μm. (D) The pit area was quantified. ** p < 0.01 vs. RANKL(−)/control; †† p < 0.01 vs. RANKL(+)/control. n = 3 fields.
Fig 5: Time course of anti-osteoclastogenic effect of iSN40. (A) Representative images of TRAP staining of cocultured RAW264.7 and MC3T3-E1 cells treated with 100 ng/mL RANKL for 4 days and 0.3 μM iSN40 for defined periods. d, differentiation days with iSN40 treatment. Scale bar, 100 μm. (B) The number of nuclei in TRAP+ osteoclasts was quantified. ** p < 0.01 vs. control/RANKL(−); †† p < 0.01 vs. control/RANKL(+); ‡‡ p < 0.01 vs. d0–3, d0–4, d1–3, and d1–4; §§ p < 0.01 vs. d2–3 and d2–4. n = 3 fields.
Supplier Page from Abcam for Recombinant mouse RANKL protein (Active)