Ovariectomy-induced bone loss in TNFα and IL6 gene knockout mice is regulated by different mechanisms

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   Figure 1

   Changes in metabolic parameters after OVX. OVX induced greater weight gain only in WT mice, and gene knockout mice could correct the effect of OVX on body weight (A). The high bone turnover was confirmed in all mice, although a repair effect was observed in gene-knockout mice (B and C). OVX significantly lowered wet uterine weight (D) in all genotypes and resulted in uterus atrophy, reflected by representative uterus sample (E). Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, compared to the same genotype. ^#P < 0.05, ^##P < 0.01, compared to WT mice subjected to similar treatment; n = 9. A full colour version of this figure is available at https://doi.org/10.1530/JME-17-0218.
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   Figure 2

   Retained bone mass in TNFα^−/− and IL6^−/− mice. The volume of selected region of interest (ROI) maintained equal among groups (A). OVX led to a noticeable decline in trabecular thickness (Tb. Th) (B), bone volume to tissue volume ratio (BV/TV) (D), trabecular number (Tb. N) (E), and total trabecular bone mineral density (BMD) (F), while an appreciable increase in thickness separation (Tb. Sp) (C) in WT mice was observed. TNFα^−/− and IL6^−/− mice prevented OVX-induced bone loss and a slight increase in Tb. N (E) could be observed in OVX TNFα^−/− mice. Representative three-dimensional micro-CT images of femoral trabecular bone (G). Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, compared to the same genotype. ^#P < 0.05, ^##P < 0.01, compared to WT mice with the same treatment. ^$P < 0.05, ^$$P < 0.01, compared to IL6^−/− mice with the same treatment, n = 9.
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   Figure 3

   TNFα and IL6 knockout antagonized trabecular architectural deterioration. Representative hematoxylin eosin (H&E) images for bone microarchitecture of the distal femora in WT, TNFα^−/− and IL6^−/− mice after SHAM (A, B and C) and OVX (D, E and F) respectively. The first left and right panel show the original magnification ×10, scale bar = 100 μm. Images besides show equivalent staining at 5× and 20× magnification respectively, scale bar = 200 and 50 μm. A full colour version of this figure is available at https://doi.org/10.1530/JME-17-0218.
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   Figure 4

   Evaluation of osteoclastogenesis. OVX significantly increased the number and size of TRAP-positive multinucleated cells (indicated by red arrows), whereas TNFα and IL6 knockout acted reduced osteoclastogenesis (A, B and C). Original magnification ×40, scale bar = 20 μm. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, compared to the same genotype. ^#P < 0.05, ^##P < 0.01, compared to WT mice with the same treatment, n = 6. A full colour version of this figure is available at https://doi.org/10.1530/JME-17-0218.
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   Figure 5

   Relative mRNA expressions in the distal metaphyses of right femurs, including Col1a1 (A), Runx2 (B), DKK1 (C), Sost (D), TRAP (E), MMP9 (F), CTSK (G) and TRAF6 (H). While TNFα and IL6 knockout could inhibit osteoclastogenesis and stimulate osteogenesis, the extent of their reversal was different (A, D and H). Data are expressed as mean ± s.e.m. and the relative expression level of each gene is normalized to GAPDH. *P < 0.05, **P < 0.01, compared to the same genotype. ^#P < 0.05, ^##P < 0.01, compared to WT mice with the same treatment. ^$P < 0.05, ^$$P < 0.01, compared to IL6^−/− mice with the same treatment, n = 9.
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   Figure 6

   Effects of estrogen, TNFα and IL6 on osteoblast and osteoclast differentiation in vitro. E2 treatment and knockout of TNFα and IL6 enhanced osteoblast differentiation by increasing osteoblasts number, size and ALP activity (A, B, C and D), and inhibited osteoclast differentiation by decreasing the number and size of TRAP-positive multinucleated cells (E, F and G). Original magnification ×20. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, compared to the same genotype. ^#P < 0.05, ^##P < 0.01, compared to WT mice with the same treatment, n = 6. A full colour version of this figure is available at https://doi.org/10.1530/JME-17-0218.
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   Figure 7

   Relative mRNA expressions in vitro. E2 treatment and knockout of TNFα and IL6 enhanced osteoblastogenesis via upregulation of Col1a1 and Runx2 (A and B) and downregulation of DKK1 and Sost (C and D), suppressed osteoclastogenesis by decreasing the mRNA levels of TRAP, MMP9, CTSK, TRAF6, c-Fos and NFATc1 (E, F, G, H, I and J). The differences between TNFα^−/− and IL6^−/− BMSCs in regulating osteoblast- and osteoclast-related gene expressions was also found (A, B, C, D and H). Data are expressed as mean ± s.e.m. and the relative expression level of each gene is normalized to GAPDH mRNA. *P < 0.05, **P < 0.01, compared to the same genotype. ^#P < 0.05, ^##P < 0.01, compared to WT mice with the same treatment. ^$P < 0.05, ^$$P < 0.01, compared to IL6^−/− mice with the same treatment, n = 6.
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   Figure 8

   Schematic working model for regulatory roles of TNFα and IL6 in mediating bone loss induced by estrogen deficiency. The knockout of TNFα and IL6 restore the bone-wasting effect of estrogen deficiency and preserve osteoporotic bone mass, possibly via the inhibition of NF-κB, AP1 component and WNT signaling inhibitors. However, TNFα potentially appears to exert an important role in the inhibition of bone formation and enhancing TRAF6-mediated osteoclastogenesis when compared to IL6. It is implied that although the regulatory mechanisms of TNFα and IL6 in bone metabolism may be different, both could be potential therapeutic targets for osteoporosis induced by estrogen deficiency. A full colour version of this figure is available at https://doi.org/10.1530/JME-17-0218.

• © 2018 Society for Endocrinology