Irisin prevents trabecular bone damage and tumor invasion in a mouse model of multiple myeloma

Abstract

Bone disease associated with multiple myeloma (MM) is characterized by osteolytic lesions and pathological fractures, which remain a therapeutic priority despite new drugs improving MM patient survival. Antiresorptive molecules represent the main option for the treatment of MM-associated bone disease (MMBD), whereas osteoanabolic molecules are under investigation. Among these latter, we here focused on the myokine irisin, which is able to enhance bone mass in healthy mice, prevent bone loss in osteoporotic mouse models, and accelerate fracture healing in mice. Therefore, we investigated irisin effect on MMBD in a mouse model of MM induced by intratibial injection of myeloma cells followed by weekly administration of 100 μg/kg of recombinant irisin for 5 wk. By micro-Ct analysis, we demonstrated that irisin improves MM-induced trabecular bone damage by partially preventing the reduction of femur Trabecular Bone Volume/Total Volume (P = .0028), Trabecular Number (P = .0076), Trabecular Fractal Dimension (P = .0044), and increasing Trabecular Separation (P = .0003) in MM mice. In cortical bone, irisin downregulates the expression of Sclerostin, a bone formation inhibitor, and RankL, a pro-osteoclastogenic molecule, while in BM it upregulates Opg, an anti-osteoclastogenic cytokine. We found that in the BM tibia of irisin-treated MM mice, the percentage of MM cells displays a reduction trend, while in the femur it decreases significantly. This is in line with the in vitro reduction of myeloma cell viability after 48 h of irisin stimulation at both 200 and 500 ng/mL and, after 72 h already at 100 ng/mL rec-irisin. These results could be due to irisin ability to downregulate the expression of Notch 3, which is important for cell-to-cell communication in the tumor niche, and Cyclin D1, supporting an inhibitory effect of irisin on MM cell proliferation. Overall, our findings suggest that irisin could be a new promising strategy to counteract MMBD and tumor burden in one shot.

Keywords: Opg; RankL; bone damage; irisin; multiple myeloma; sclerostin.

Graphical Abstract

Figure 1

Irisin reduces femoral BM malignant cell invasion and serum levels of IgG2b after intratibial injection in mice. Experimental design (A). Percentage of 5TGM1-eGFP cells detected within the BM from the tibia and femur of 5TGM1-eGFP-bearing female mice treated for 5 wk with vehicle (MM-vehicle) or 100 μg/kg rec-irisin (MM-irisin) (B). IgG2b serum levels in Sham-vehicle (n = 5), MM-vehicle (n = 6), and MM-irisin mice (n = 6) (C). Kruskal–Wallis test or one-way ANOVA with Tukey’s multiple comparisons tests was performed. Data are presented as a box-and-whisker plot with median and interquartile ranges, from max to min, with all data points shown.

Figure 2

Irisin prevents trabecular bone loss in the femur of MM mice. Representative micro-CT-generated section images (A) and calculated trabecular parameters (B) of femur harvested from Sham-vehicle mice, MM-vehicle mice, and MM-irisin mice after 5 wk of treatment. Trabecular bone parameters included Trabecular Bone Volume/Total Volume (BV/TV), Trabecular Number (Tb. Number); Trabecular Separation (Tb. Separation), and Trabecular Fractal Dimension (Tb. FD) evaluated on Sham-vehicle (n = 4), MM-vehicle (n = 4), and MM-irisin (n = 5) mice (B, C, D, E). A one-way ANOVA with Tukey’s multiple comparisons tests was performed. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.

Figure 3

Irisin modulates RankL, Opg, and Sclerostin expression in MM mice. Quantitative PCR (qPCR) showing modulation of mRNA expression levels of RankL (A), Opg (B), and RankL/Opg ratio (C) assayed on the femoral BM of Sham-vehicle mice (n = 4 or n = 5), MM-vehicle mice (n = 5 or n = 6) and MM-irisin mice (n = 5 or n = 6) after 5 wk of treatment. Gene expression was normalized to Gapdh and plotted as a fold increase from the Sham-vehicle group of mice. Kruskal–Wallis test (RankL and RankL/Opg ratio) or one-way ANOVA with Tukey’s multiple comparisons tests (Opg) were performed. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown. Western immunoblotting (D, E, I, J) and densitometric analysis (F, G, K, L) showing RankL, Opg, Sclerostin, and Dkk1 expression normalized to β-actin in the femur of Sham-vehicle mice (n = 4), MM-vehicle mice (n = 4), and MM-irisin mice (n = 4) after 5 wk of treatment. RankL/Opg ratio of densitometric analysis (H). A one-way ANOVA with Tukey’s multiple comparisons tests was performed. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.

Figure 4

Irisin increases PINP serum levels in MM mice. N-terminal propeptide of type I procollagen (PINP) (A), and C-terminal telopeptides of type I collagen (CTX), (B) in Sham (n = 5), MM-vehicle (n = 5), and MM-irisin (n = 5 or n = 6) mice. A one-way ANOVA with Tukey’s multiple comparisons tests was performed. Data are presented as box-and-whisker with median and interquartile ranges, from max to min, with all data points shown.

Figure 5

Irisin improves MM mice explorative abilities in the OFT. Evaluation of digging frequency in Sham-vehicle, MM-vehicle, and MM-irisin mice in the OFT. A one-way ANOVA with Tukey’s multiple comparisons tests was performed. Data are presented as box-and-whisker with median and interquartile ranges, from max to min, with all data points shown (n = 5 mice per group).

Figure 6

In vitro Irisin reduces 5TGM1 cell viability and Notch 3 and Cyclin D1 gene expression. MTT assay performed on 5TGM1 cells treated or not with different rec-irisin concentrations (100, 200, or 500 ng/mL) for different time periods (24, 48, or 72 h) (n = 6 for each group of treatment) (A). An ordinary one-way ANOVA with Tukey’s multiple comparisons tests was performed. Data are shown as a box-and-whisker plot with median and interquartile ranges, from max to min, with all data points shown. MTT assay performed on 5TGM1 cells pre-treated for 10 min with 20 nM of CycloRGDyK or DMSO, and then stimulated with 0 or 200 ng/mL rec-irisin for 72 h (n = 6 for each group of treatment) (B). Kruskal–Wallis test was performed. Data are shown as a box-and-whisker plot with median and interquartile ranges, from max to min, with all data points shown. Quantitative PCR (qPCR) showing modulation of mRNA expression levels of Notch 3 (C) and Cyclin D1 (D) and assayed on 5TGM1 cells treated or not (n = 4) with 100 ng/mL (n = 4) or 200 ng/mL (n = 4) of rec-irisin for 8 h. A one-way ANOVA with Tukey’s multiple comparisons tests was performed. Data are presented as box-and-whisker plots with median and interquartile ranges, from max to min, with all data points shown.