Mechanical stimulation is critical for the maintenance of skeletal integrity and bone mass.[1, 2] Especially in a super-aged society, disuse osteoporosis is a critical issue in patients with reduced locomotor function in bedridden patients due to cardiovascular, brain, and skeletal diseases. In such an unloading-induced osteoporosis, the loss of mechanical stress is responsible for impairments in the maintenance of bone mass. Even in a general physical point of view, reduced physical activity per se in aged patients also significantly reduces bone mass. This disuse-induced bone loss and osteoporosis is not limited to the aged population but also observed in young adults who are suffering from joint immobilization due to fractures and dislocations. Disuse-induced bone loss clearly occurs even in healthy astronauts. This phenomenon is acutely observed during spaceflights and continues to persist long after the astronauts return to the Earth. Thus, the link between disuse and bone loss is well-established, and unloading suppresses bone formation and activates bone resorption. These phenomena are exerted by many types of bone cells, including osteoblasts, osteocytes, and osteoclasts.[1-4] However, the molecular mechanism underlying the alterations of cellular activity and disuse-induced bone loss is not fully understood.
The transcription factor nuclear factor κB (NF-κB) plays a key role in immune and inflammatory responses, proliferation, tumorigenesis, and survival.[5, 6] Currently, five proteins with a conserved homology in the Rel domain (p65 [RelA], RelB, cRel, NF-κB1 [p50/p105], and NF-κB2 [p52/p100]) have been identified. These members can homodimerize and heterodimerize in numerous combinations, with the predominant cellular species being p50:p65, p50:cRel, and NF-κB2:RelB. All five members share an N-terminal domain of 300 amino acids, designated the Rel homology domain (RHD), which is responsible for DNA binding, dimerization, and interactions with the inhibitory IκB proteins. NF-κB1 and NF-κB2 are synthesized as the large precursors of p105 and p100, respectively, and contain long COOH-terminal domains with multiple ankyrin repeats, rendering these precursors functionally similar to the inhibitor IκB. The inhibitory effect of p105 and p100 is relieved when these proteins are processed into p50 and p52, respectively. Three members, p65, c-Rel, and RelB, contain C-terminal transcriptional activation domains (TADs) that are crucial for their ability to induce target gene expression, whereas homodimers of p50 and p52 lack TADs and therefore have no intrinsic ability to drive transcription. In unstimulated cells, NF-κB is predominantly localized to the cytoplasm as part of a complex with inhibitory IκB proteins, including IκBα, IκBβ, IκBϵ, and IκBγ. In response to a variety of stimuli, such as tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β), IκBs are phosphorylated (Ser-32 and Ser-36 for IκBα and Ser-19 and Ser-21 for IκBβ) by the activated IκB kinase (IKK) complex. Phosphorylated IκBs are ubiquitinated and subsequently degraded by the 26S proteasome. The IKK complex consists of two catalytic kinase subunits, specifically, IKKα (IKK1) and IKKβ (IKK2), as well as a regulatory subunit, NF-κB essential modulator (NEMO), also known as IKKγ. IKKβ is critical for the classical (canonical) NF-κB pathway that depends on IκB degradation. In this pathway, the p50/p65 heterodimer enters the nucleus and binds to NF-κB-responsive elements to regulate the expression of genes.[5, 6]
The importance of NF-κB in both bone formation and bone resorption is well-known. In osteoclast development, two groups have reported that mice lacking both the NF-κB1 (p50) and NF-κB2 (p52) subunits develop typical osteopetrosis, which is accompanied by a dramatic reduction in the number of osteoclasts due to the defective tracking of the osteoclast lineage.[7, 8] Certain inhibitors of the NF-κB pathway suppress the receptor activator of the NF-κB ligand (RANKL), inducing osteoclastogenesis and animal models of inflammatory bone destruction, such as collagen-induced arthritis or ovariectomized mice.[9, 10] A recent study indicates that the inhibition of NF-κB in mature mice osteoblasts expressing a dominant-negative form of IKKβ increases bone mineral density and bone volume, due to the increased activity of the osteoblasts. Furthermore, the selective inhibition of NF-κB restores the inhibitory effect of TNF on bone morphogenic protein 2 (BMP2)-induced osteoblast differentiation and prevents bone loss in ovariectomized mice.[12, 13] We have previously reported that the activation of the classical NF-κB pathway induced by TNFα inhibits BMP2-induced osteoblast differentiation by inhibiting the DNA-binding activity of the Smad protein to its target genes. These results strongly suggest that the activation of NF-κB induces osteoclastogenesis and suppresses bone formation.
Although NF-κB transcription factors are activated by mechanical loading and inflammatory cytokines,[14, 15] the role that NF-κB exerts on the maintenance of bone mass with disuse-induced osteoporosis still remains unclear. Shear stress stimulates osteoblastic differentiation via the prostaglandin synthesis induced by NF-κB.[14, 16] Conversely, the activity of the NF-κB-dependent reporter gene is markedly increased in unloaded muscles.[17, 18] Thus, there are many unknown mechanisms with respect to whether the NF-κB activation induced by mechanical unloading produces a positive or negative regulation of bone formation. In this study, our objectives were to elucidate (1) the NF-κB activation mechanism regulated by mechanical unloading, and (2) the role of NF-κB activation on bone formation using gene targeting for molecules that are important for NF-κB signaling.