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. 2019 Nov;34(11):2087-2100.
doi: 10.1002/jbmr.3819. Epub 2019 Jul 31.

Conditional Activation of NF-κB Inducing Kinase (NIK) in the Osteolineage Enhances Both Basal and Loading-Induced Bone Formation

Jennifer L Davis  1 , Linda Cox  1 , Christine Shao  1 , Cheng Lyu  2 , Shaopeng Liu  2 , Rajeev Aurora  3 , Deborah J Veis  1   4   5
Affiliations

Conditional Activation of NF-κB Inducing Kinase (NIK) in the Osteolineage Enhances Both Basal and Loading-Induced Bone Formation

Jennifer L Davis et al. J Bone Miner Res. 2019 Nov.

Abstract

Studies from global loss-of-function mutants suggest that alternative NF-κB downstream of NF-κB inducing kinase (NIK) is a cell-intrinsic negative regulator of osteogenesis. However, the interpretation of the osteoblast and/or osteocyte contribution to the bone phenotype is complicated by simultaneous osteoclast defects in these models. Therefore, we turned to a transgenic mouse model to investigate the direct role of NIK in the osteolineage. Osx-Cre;NT3 animals (NT3-Cre +), which bear a constitutively active NIK allele (NT3) driven by Osx-Cre, were compared with their Cre-negative, Control (Ctrl) littermates. NT3-Cre + mice had elevated serum P1NP and CTX levels. Despite this high turnover state, µCT showed that constitutive activation of NIK resulted in a net increase in basal bone mass in both cortical and cancellous compartments. Furthermore, NT3-Cre + mice exhibited a greater anabolic response following mechanical loading compared with controls. We next performed RNA-Seq on nonloaded and loaded tibias to elucidate possible mechanisms underlying the increased bone anabolism seen in NT3-Cre + mice. Hierarchical clustering revealed two main transcriptional programs: one loading-responsive and the other NT3 transgene-driven. Gene ontology (GO) analysis indicated a distinct upregulation of receptor, kinase, and growth factor activities including Wnts, as well as a calcium-response signature in NT3-Cre + limbs. The promoters of these GO-term associated genes, including many known to be bone-anabolic, were highly enriched for multiple κB recognition elements (κB-RE) relative to the background frequency in the genome. The loading response in NT3-Cre + mice substantially overlapped (>90%) with Ctrl. Surprisingly, control animals had 10-fold more DEGs in response to loading. However, most top DEGs shared between genotypes had a high incidence of multiple κB-RE in their promoters. Therefore, both transcriptional programs (loading-responsive and NT3 transgene-driven) are modulated by NF-κB. Our studies uncover a previously unrecognized role for NF-κB in the promotion of both basal and mechanically stimulated bone formation. © 2019 American Society for Bone and Mineral Research.

Keywords: ANABOLIC; CONSENSUS MOTIF; MOUSE MODEL; NF-KAPPAB; OSTEOBLAST; OSTEOCYTE.

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Conflict of interest statement

Disclosures

All authors state that they have no conflicts of interest.

Figures

Figure 1:
Figure 1:. Basal bone mass is enhanced in both male and female NT3-Cre+ mice.
(A) MicroCT analysis (ex-vivo) of the femur in male mice at 16 wks showing increased cortical thickness and cortical total area in NT3 transgenic mice. n=12–14 per group; black circles = male Ctrl and green circles = male NT3-Cre+. (B) Cancellous BV/TV and vBMD are higher in male NT3-Cre+ animals. (C) Representative images for cancellous (left) and cortical (right) femoral bone indicating heightened bone mass in male NT3-Cre+ mice. (D) MicroCT analysis (ex-vivo) of the femur in female mice at 16 wks also shows increased cortical thickness and cortical total area in NT3 transgenic mice. n=13–22 per group; black circles = female Ctrl and pink circles = female NT3-Cre+. (E) Cancellous BV/TV and vBMD are also elevated in female NT3-Cre+ animals. (F) Representative images for cancellous (left) and cortical (right) femoral bone illustrating greater bone mass in female NT3-Cre+ mice. Data are represented as mean ± SD. Unpaired, student’s two-tail t-test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Ct.Th = cortical thickness; BV/TV = bone volume/total volume; vBMD = volumetric bone mineral density
Figure 2:
Figure 2:. Both osteoblast and osteoclast activity are upregulated in NT3-Cre+ mice.
(A) Representative images of calcein and alizarin 5-day double-labeling for each genotype at 6 wks. (B) Dynamic histomorphometry at the tibial endocortical surface shows increased BFR/BS, (C) MS/BS, and (D) MAR in 6-wk-old NT3 transgenic animals (n=9–10 per group). Serum P1NP (E) and CTX (F) levels are markedly elevated in 12-wk-old NT3-Cre+ mice (n=12–15 per group). (G) qPCR showing increases in Rankl expression and the Rankl/Opg ratio in flushed, crushed bone of 12-wk-old NT3-Cre+ mice (n=4–7 per group). Results are presented as mean ± SD. black = male Ctrl and green = male NT3-Cre+. Unpaired, student’s two-tail t-test: *p<0.05, **p<0.01, ***p<0.001. BFR/BS = bone formation rate per bone surface; MS/BS = mineralizing surface per bone surface; MAR = mineral apposition rate; OPG (Tnfrsf11b); RANK (Tnfrsf11a); RANKL (Tnfsf11).
Figure 3:
Figure 3:. NT3-Cre+ mice have an enhanced anabolic response to mechanical loading.
(A) Study design and adaptive mechanical loading protocol for 16-week-old Ctrl and NT3-Cre+ mice. One bout of cyclic loading per day was performed a total of 9 times. Calcein green and alizarin red fluorochromes were given on study days 4 and 11, respectively. Strain gauging was used to determine the force required to achieve an equivalent microstrain in each genotype, as shown. (B) (top) Representative cross-section of the loaded tibia for each genotype and (bottom) inset view of the area of peak compressive strain highlighting the enhanced bone formation seen in NT3-Cre+ mice. Quantification of fluorochrome labeling shown as (C) Ps.MS/BS, (D) Ps.MAR, and (E) Ps.BFR/BS (n=10–12 per group). Results are presented as mean ± SD. black = male Ctrl and green = male NT3-Cre+. Right tibiae were loaded (LOAD, triangles) and left tibiae served as non-loaded (NL, circles) controls. Student’s, paired, two-tailed t-test (LOAD vs NL within each genotype) or 2-way ANOVA followed by Tukey’s multiple comparison test (cross-genotype comparisons): n.s. - not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Ps.BFR/BS = periosteal bone formation rate per bone surface; Ps.MS/BS = periosteal mineralizing surface per bone surface; Ps.MAR = periosteal mineral apposition rate
Figure 4:
Figure 4:. RNAseq analysis reveals two main transcriptional programs: one loading-responsive and the other driven by the NT3 transgene.
(A) Schematic representing the tibial region of interest harvested for RNA-Seq analysis (top line: 2 mm distal to the articular surface and bottom line: distal tibia-fibula junction). (B) Heatmap displaying z-scores across all samples highlighting two main transcriptional programs: one induced by loading (blocks 1–2) and the other by the NT3 transgene (blocks 3–4). (C) Table listing RNAseq analysis comparisons and description of resulting differentially expressed genes (DEGs). (D-G) Volcano plots for each of the comparisons in (C) showing number of upregulated DEGs in each pairwise comparison. DEGs were defined as having a fold-change > 1.5 together with an adjusted p-value ≤ 0.05. n=4 per genotype/loading condition. light purple = Ctrl NL; dark purple = Ctrl LOAD; light green = NT3-Cre+ NL; dark green = NT3-Cre+ LOAD
Figure 5:
Figure 5:. Gene ontology analysis reveals a bone anabolic signature in NT3-Cre+ mice.
(A) Venn diagram for the gene ontology (GO) analysis of molecular function across all 4 experimental groups using a multi-way partitioning algorithm. The number of unique GO terms is listed for each bin. The top GO terms (p<10−3) in NT3-Cre+ mice unique to the (B) NL side or (C) shared between loading conditions is enriched for receptor, kinase, and growth factor activities. (D) Top GO terms unique to NT3-Cre+ loaded tibiae indicate involvement of calcium signaling. NL = non-loaded tibia; LOAD = loaded tibia
Figure 6:
Figure 6:. The transcriptional response to mechanical loading is augmented in NT3-Cre+ mice.
(A) Venn diagram depicting the extensive overlap in DEG lists between Ctrl vs NT3-Cre+ in response to loading (adjusted p-value <0.01). Fold change (expressed as Log2FC) for each genotype and/or loading condition compared to Ctrl NL samples for (B) Wnt genes and (C) other loading-related genes of interest. Significance (adjusted p-value): *p<0.05, #p<0.01, †p<0.001, ‡p<0.0001. Tnfrsf11b (OPG); Tnfrsf11a (RANK); Tnfsf11 (RANKL).
Figure 7:
Figure 7:. κB-RE are enriched in response to the NT3 transgene and with loading.
(A) Log FC for each genotype and/or loading condition compared to Ctrl NL samples for NF-κB signaling pathway components. Significance (adjusted p-value): *p<0.05, ‡p<0.0001. (B) (left) Incidence of genes with ≥2 κB recognition elements (κB-RE) in the top 20 upregulated DEG lists and (right) NT3 transgenic GO bins is much higher than the background frequency in the genome in both loading and NT3 transgene settings. Genome background frequency is defined as the probability of at least 1 out of 121 randomly selected genes (size of largest NT3 transgenic GO bin) containing ≥2 κB-RE in 10 million iterations. (C) Number of genes with the indicated number of κB-RE in the top 50 Ctrl NL vs LOAD upregulated DEG list, indicating an enrichment of κB-RE in the normal loading response. (D) Both Ptgs2 and Wnt7b contain multiple κB-RE. Genomic coordinates are listed relative to the transcription start site (position zero). Bold genomic location indicates sequence is found across all 3 motifs tested. R = A/G; N = A/G/C/T; Y= C/T; W = A/T
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