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. 2018 Aug 23;3(1):14-22.
doi: 10.1002/jbm4.10066. eCollection 2019 Jan.

Manipulation of the Alternative NF-κB Pathway in Mice Has Sexually Dimorphic Effects on Bone

Allahdad Zarei  1 , Chang Yang  1 , Jesse Gibbs  1 , Jennifer L Davis  1 , Anna Ballard  1 , Rong Zeng  1 , Linda Cox  1 , Deborah J Veis  1   2
Affiliations

Manipulation of the Alternative NF-κB Pathway in Mice Has Sexually Dimorphic Effects on Bone

Allahdad Zarei et al. JBMR Plus. .

Abstract

Alternative NF-κB signaling promotes osteoclastogenesis and pathological bone loss, but the effect of sex on phenotype has not been explored. We disrupted alternative NF-κB signaling by deletion of upstream kinase NF-κB-inducing kinase (NIK) or NF-κB subunit RelB and found that both NIK-deficient and RelB-deficient female mice possessed more than twofold higher trabecular bone mass compared to controls, whereas no differences were observed in males. In vitro, RelB-deficient precursors from female mice showed a more severe osteoclast (OC) differentiation defect than male, while WT had no sex bias. Next, we asked whether pharmacologic activation of alternative NF-κB by inhibitor of apoptosis (IAP) antagonist BV6 has sex-dependent effects on bone. Unlike male mice that lost bone, female mice on BV6 for 4 weeks showed no changes in either trabecular bone mass or OC number. Because estrogen generally suppresses NF-κB, we hypothesized that estrogen protects bone from BV6 effects in vivo. Thus, we performed ovariectomy or sham surgery in female mice, then treated with BV6 or vehicle for 4 weeks. Although ovariectomy caused bone loss, BV6 did not have any additional impact, suggesting that direct estrogen effects do not cause resistance to BV6 in vivo. The osteopenic effects of IAP antagonists in males may have implications for their use in cancer therapy. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Keywords: ALTERNATIVE NF‐κB PATHWAY; IAP ANTAGONIST; NIK; RelB; SEXUAL DIMORPHISM.

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Figures

Figure 1
Figure 1
Increased trabecular bone volume and bone mineral density in RelB‐KO and NIK‐KO female mice. μCT was performed on tibiae of RelB WT, HT, and KO mice (A–C) and femurs of NIK‐WT, HT, and KO mice (D–F), all at 10 weeks of age. Representative three‐dimensional μCT reconstructions of median RelB WT and KO (A) and NIK WT and KO mice (D). Trabecular BV/TV (B,E) and BMD (C,F). Data was analyzed by two‐way ANOVA, with comparisons within each sex. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. BV/TV = bone volume/total volume; BMD = bone mineral density.
Figure 2
Figure 2
Increased severity of osteoclast defect in female RelB‐KO mice in vitro. (A) RANKL‐induced (top row) osteoclast formation from male and female RelB‐KO mice compared to WT littermates, demonstrate a more dramatic decrease in OCs in female RelB‐KO. Addition of TGF‐β with RANKL (bottom row) does not normalize the difference between male and female RelB‐KO. (B) TRAP positive cells with ≥3 nuclei in A were counted in each well and plotted, white indicates RANKL only, black RANKL + TGFβ. OC differentiation markers (C) Acp5 (Trap), (D) CatK, (E) DC‐Stamp, and (F) MMP9 were assessed at transcript levels from BMMs of 3 male WT (M‐WT), 3 male knockout (M‐KO), 3 female WT (F‐WT), and 3 female knockout (F‐KO) RelB mice after 3 days of culture and plotted against BMM as baseline. (G) Representative images of bone resorption from samples in C–F after 5 days of cell culture at magnification ×10. Data presented as mean ± SD, two‐way ANOVA, *p < 0.05, **p < 0.01 and ***p < 0.001. Significant differences for comparison between RANKL and RANKL + TGFβ data within each group is not shown, * for comparison between male and female.
Figure 3
Figure 3
Only male mice treated with IAP antagonist BV6 lose bone. (A) WT BALB/c mice, age 6 weeks, male and female (n = 7–8/group) were given 4 weekly IP injections of BV6 (orange) (10 mg/kg) or vehicle (black). vivaCT was used to calculate fold changes from 6 to 10 weeks for each animal in (B) BV/TV and (C) BMD. Mean ± SD, two‐way ANOVA, **p < 0.01 and ***p < 0.001. n.s. = not significant.
Figure 4
Figure 4
Female osteoclast number and surface do not change with BV6 treatment. (A) Tibia of the mice in Fig. 3 were prepared for histomorphometry of TRAP‐stained sections. (B) Representative double labeling images of male and female mouse on vehicle or BV6, left to right. (C) Histomorphometric analysis of TRAP sections in A and bone formation parameters in B: Oc.S/BS, N.Oc/BS, MAR, and BFR. Data presented as mean ± SD, **p < 0.01 and ***p < 0.001, two‐way ANOVA. n.s. = not significant; Oc.S/BS = osteoclast surface per bone surface; N.Oc/BS = number of osteoclasts per bone surface; MAR = mineral apposition rate; BFR = bone formation rate.
Figure 5
Figure 5
BV6 does not exacerbate bone loss in response to OVX. (A) Three‐month‐old WT BALB/c female mice (10/group) were ovariectomized or sham‐operated, then treated with 4 weekly doses of BV6 (orange) (10 mg/kg) or vehicle (black). Mice were euthanized 8 days after the last dose of BV6 and bone parameters were determined. (B) μCT three‐dimensional tibia reconstructions of the median mouse in each group. (C) BV/TV and (D) BMD, (E) Tb.N, (F) Tb.Th measurements show that OVX mice have lower bone mass than SHAM controls, but BV6 has no additional effect. (G) An increase in Tb. Sp was observed in OVX mice on vehicle/BV6 compared to SHAM controls. Data presented as mean ± SD, *p < 0.05, **p < 0.01, and ***p < 0.001, two‐way ANOVA. n.s. = not significant.
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