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P&F Grant Awards

Year 10

Grant # 22

The role of NF-κB signaling in Osteogenesis

PI: Gaurav Swarnkar, PhD


Specific Aims

Normal skeletal development is a coordinated process of bone formation by osteoblasts (OB) and bone resorption by osteoclasts (OC), which is tightly regulated by metabolic and bio-energetic circuits. However, pathological conditions, such as chronic inflammation and aging, compromise the rate of bone formation and delay healing of bone fractures1-11. Various inflammatory signals such as TNF and IL-1β elicit bone catabolic responses through increased bone resorption via osteoclasts concurrent with reduced bone formation resulting in net bone loss12-14. However, the mechanisms underlying the effect of inflammation on bone formation and on bone cells such as OB and osteocytes (OCy) are not fully understood owing to pro-inflammatory cytokine redundancy and to paucity of relevant genetic models. This gap of knowledge offers a unique opportunity to identify new intervention targets to improve bone health.

Inflammatory responses are bio-energetically costly, hence they impede metabolic homeostasis. Recent reports reveal an intimate connection between NF-κB and metabolism 15-17. The transcription factor NF-κB has been implicated as crucial mediator of immune/inflammatory responses and is required for skeletal development 18-23. We have shown that constitutive activation of IKK2 (IKK2ca), mimicking unresolved inflammation, in the mesenchymal compartment using Col2-cre, disrupts OB and chondrocyte differentiation and impairs skeletal development. In this proposal, our preliminary results using OB specific cre (SP7 and Col1 cre) to drive expression of IKK2ca in mice resulted in marked decrease in serum glucose levels and significant bone abnormalities illustrated by defective OB differentiation and elevated osteocyte markers. Mechanistically, we further found that primary calvarial OBs (cOB) expressing IKK2ca or treated with IL1β or TNFα cultured under osteogenic conditions, displayed significant decrease in OB differentiation-associated genes including ALP, Col1, OCN, OPG and Runx2. Most surprisingly, these cells assumed an osteocytic/dendritic morphology, expressed bona fide osteocyte markers (E11, FGF23, SOST, DKK1, RANKL), and exhibited high metabolic rate evident by accelerated glucose consumption and increased ECAR (extracellular acidification rate) values compared with control cells. Furthermore, Western blot analysis showed an increase in mTOR expression, which is well established as central sensor of cellular metabolism. Intriguingly, mTOR inhibitors significantly reversed the IKK2ca-induced osteocytic and glycolytic gene expression profiles. These observations, suggested that IKK2ca abnormally upregulated mTOR signaling and cellular energy metabolism in OBs leading to reprogramming and accelerated cell differentiation of the highly anabolic OB into terminal OCY. Taken together, these novel observations lead us to hypothesize that inflammatory responses alter osteoblast differentiation program and fate decision leading to their rapid conversion into osteocytes resulting in abnormal bone formation and bone loss. Mechanistically, the process is mediated by IKK2 activation of mTOR pathway leading to abnormal cellular metabolic activity by OBs resulting with accelerated differentiation of OBs to OCy.

Aim 1: Investigate the role of activation of IKK2/NF-κB on accelerated differentiation of osteoblast to osteocyte, and subsequent bone loss, in animal model of chronic inflammation. We will use genetic and pharmacologically-induced chronic inflammatory models to analyze the accelerated differentiation of OB to OCy.

Aim 2: Determine if OB-specific attenuation of NF-κB activity, inhibition of mTOR, or inhibition of glycolysis under inflammatory conditions attenuate bone loss and improves fracture healing in mice. Genetic and pharmacologic inhibition of glycolysis, NF-κB and mTOR pathways will be employed to accelerate and improve bone defect healing.

We expect that these approaches will identify novel therapeutic intervention targets to combat inflammation-induced bone loss.