Increased NF-κB activity in osteoprogenitor-lineage cells impairs the balance of bone/bone marrow fat in skeletal
mature mice
1
Tzu-hua Lin1, Jukka Pajarinen1, Akira Nabeshima1, Laura Lu1, Karthik Nathan1, Zhenyu Yao1, Joy Wu3, and Stuart Goodman1, 2
Department of Orthopaedic Surgery, Stanford University, Stanford, CA, USA. 2Department of Bioengineering, Stanford University, Stanford, CA, USA.
3
Department of Medicine, Stanford University, Stanford, CA, USA
Disclosure: The authors have no conflicts of interest to disclose
INTRODUCTION: Aging is associated with significant bone loss and delayed fracture healing. NF-κB is a master regulator of inflammation and the bone
remodeling process. We previously reported that NF-κB activity is increased in mesenchymal stem cells (MSCs) and osteoprogenitor (OP)-lineage cells
during the natural aging process, and is associated with reduced osteogenesis in vitro. In the current study, we have generated a doxycycline-inducible
transgenic mouse model that activates NF-κB signaling in OP-lineage cells upon doxycycline withdrawal in skeletally mature mice. The effects of NF-κB
activation on bone formation and the balance between bone/bone marrow fat were examined.
METHODS: Male transgenic mice (iNF-κB/OP, Cre+/- & floxed/-) with inducible NF-κB activation in OPs were generated by breeding the Tet-Osx-Cre
male mice (Cre +/-, Jackson Laboratory, 006361, C57BL/6) with aIKK-EGFP female mice (floxed/floxed, Jackson laboratory, 008242, C57BL/6). Primary
MSCs were isolated from the transgenic or control mice (Cre-/- & floxed/-) at 8 weeks of age, and were differentiated in osteogenic medium (α-MEM
supplemented with 10% FBS, 100 nM dexamethasone, 10 mM β-glycerol phosphate and 50 µM ascorbate-2-phosphate) for 3 weeks. Bone mineralization
was quantified by Alizarin red stain. To delay the activation of NF-κB in OPs until bone development is mature, the mice were supplied with doxycycline in
their drinking water at a dose of 10µg/ml from breeding period until 12 weeks of age. Each group included 6 male mice. The mice were euthanized 24 weeks
after doxycycline withdrawal. Femurs and tibias were scanned by µCT using a GE RS150 µCT scanner with 49 µm resolution. Regions of interests (ROI)
were created at the epiphysis (4x4x1mm3), metaphysis (4x4x3mm3), and diaphysis (4x4x3mm3). The bone mineral density (BMD), bone volume fraction
(BVF), and cortical thickness were quantified using GEMS MicroView (threshold: 700 HU). Femurs and tibias were dissected and decalcified in EDTA for
2 weeks, and the bone marrow fat was stained by osmium tetroxide and scanned by µCT (threshold: 2000HU). The percentage of osmium in mm3/total
volume, and the stained osmium density at the epiphysis, metaphysis, and diaphysis were quantitated and compared. Statistical analysis was performed
using Graph-Pad Prism. Data is presented as mean ± SEM.
RESULTS SECTION: The bone mineralization was reduced 44.4% in the cells isolated from iNF-κB/OP mice compared to the controls (Fig.1). The
reduction was reversed by continuous doxycycline treatment, which inhibited Cre recombinase expression and NF-κB activation in OPs. The metaphyseal
trabecular BMD in the femurs and tibias was reduced in iNF-κB/OP mice (Fig.2a). No significant differences in BMD or BVF were observed in the
epiphysis and diaphysis in femurs and tibias. The iNF-κB/OP mice showed a trend for decreased diaphyseal cortical thickness (p=0.08) in femurs, but no
differences were observed in the tibias (Fig.2b). The osmium (bone marrow fat) density in iNF-κB/OP mice was increased at the epiphysis in femurs and
tibias and showed a tendency to increase at metaphysis in tibias (p=0.059, Fig.3a). The fat volume fraction (fat/total volume) was increased at the epiphysis
and metaphysis in tibias but not in the femurs from iNF-κB/OP mice (Fig.3b).
DISCUSSION: The preliminary study suggests that NF-κB activation in OP-lineage cells reduces the bone-forming ability in vitro and in vivo, and impairs
the balance between bone and bone marrow fat. Histology and immunohistochemistry staining is currently being performed to dissect the potential
mechanism of increased bone marrow fat in iNF-κB/OP mice.
SIGNIFICANCE: Increased NF-κB activity in OP-lineage cells in skeletally mature mice leads to aging-associated osteoporosis-like phenotypes in bone.
Targeting the NF-κB activity as a therapeutic strategy may improve bone healing and prevent aging-associated bone loss in aged patients.
ACKNOWLEDGEMENTS: This work was Supported by NIH grants 2R01 AR055650 and 1R01AR063717 and the Ellenburg Chair in Surgery.
Fig.2
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Fig.1 Decreased osteogenesis in MSCs/OPs with constitutive NF-κB activation. The MSCs isolated from the iNF-κB/OP or control mice was
differentiated in osteogenic media with/without doxycycline (100ng/ml). Bone mineralization was quantified at day 21. ***p<0.005
Fig.2 Bone analysis in the iNF-κB/OP transgenic and control mice. Metaphyseal trabecular BMD (a) and diaphyseal cortical thickness (b) were
evaluated by µCT scan and analysis. **p<0.01
Fig.3 Quantification of bone marrow fat stained with osmium. Quantification of osmium density (a) and fat volume fraction (b) in femurs and tibias
at epiphyseal, metaphyseal, and diaphyseal regions. *p<0.05, **p<0.01
ORS 2017 Annual Meeting Poster No.0667