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

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.

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma

“…Sex and location of tumors: mixed male and female, 2 facial and 1 perineal for Osx-Cre;NT3 or 2 perineal and 1 trunk for FSP1-Cre;NT3. gDNA was extracted from tissue samples as previously described [26]. PCR cycling conditions using GoTaq polymerase (M7123, Promega, USA) are listed in S3 Table . GelRed (41003, Biotium) was added at a concentration of 1:10,000 to agarose gels for DNA visualization as well as a 1Kb Plus DNA Ladder (10787018, Invitrogen, USA) for band size determination.…”

“…Tumors were excised to remove any gross overlaying tissue, and either stored at -20C˚in RNAlater (R0901; Sigma, USA) or flash frozen in liquid nitrogen, and stored at -80C˚. Frozen tumors were pulverized in liquid nitrogen followed by RNA extraction as previously described [26]. Samples were screened by quantitative real-time PCR (qPCR) for blood-(Hba1, Hba2, and Hbb) and monocyte-specific (CD68) markers to assess low peripheral blood content.…”

“…Osx-Cre;NT3 and FSP1-Cre;NT3 tumors were grouped together as NT3 tumors due to their high degree of similarity on principal component analysis. Normalized reads per gene of NT3 tumors (group 1) were compared to a panel of benign tissue datasets (group 2) including non-loaded cortical bone from Ctrl and Osx-Cre;NT3 mice (GSE133212) [26], skeletal muscle (GSE83541) [39], white adipose tissue (GSE112999), mature cartilage (GSE110051), vein (GSE128870), dermal fibroblast (GSE60569) [40], blood (GSE116630), and liver (GSE92364 [41], GSE93380 [42]). Individual sample files used for each tissue type are listed in S5 Table.…”

“…In the course of characterizing the bone phenotype of the Osx-Cre;NT3 mice [26], we observed spontaneous soft tissue tumors. Based on their subcutaneous location and histological appearance, we considered that a mesenchymal origin was likely, and therefore crossed the NT3 transgenic mice with a FSP1-Cre (S100a4) line [31], which predominantly shows expression in the fibroblast population of multiple organs but has also been documented in the myeloid lineage [31,32,[43][44][45].…”

“…Our laboratory has recently shown that constitutive activation of NIK in the osteolineage in Osterix-Cre;NT3 (Osx-Cre;NT3) mice enhances both basal and loading-induced bone formation [26]. Unexpectedly, these Osx-Cre;NT3 mice also developed malignant subcutaneous, soft tissue tumors in early adulthood.…”