TNF‐α–induced NF‐κB signaling reverses age‐related declines in VEGF induction and angiogenic activity in intervertebral disc tissues

We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF‐α induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF‐α–induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF‐α–induced VEGF using organ disc cultures with wild type, TNF receptor 1‐null (TNF‐RI^null), or TNF receptor 2‐null (TNF‐RII^null) mice. VEGF induction was inhibited when we used TNF‐RI^null‐derived disc tissues. NF‐κB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF‐α induced VEGF expression in disc cells primarily through the NF‐κB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF‐α stimulation. TNF‐α treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF‐κB inhibitors or anti‐VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF‐κB pathway, both of which are induced by TNF‐α. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patient's age. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:229–235, 2009     

Endogenous TGF‐β activity limits TSLP expression in the intervertebral disc tissue by suppressing NF‐κB activation

Thymic stromal lymphopoietin (TSLP), an IL‐7‐like cytokine, is highly expressed in herniated disc (HD) tissue and may act as a key molecule for the initiation of macrophage recruitment into the tissue and natural resorption of HD. However, it remains unclear how TSLP expression is regulated in the intervertebral discs. This study showed that expression of TSLP and phosphorylated NF‐κB in HD tissue samples was inversely correlated with expression of phosphorylated Smad2/3 (an indicator of active TGF‐β signaling) and vice versa in posterior lumbar spinal fusion samples. The pharmacological blockades of endogenous TGF‐β activity induced TSLP expression in mouse intervertebral disc tissue culture, which was inhibited by NF‐κB inhibitors. Additionally, phosphorylation of Smad2/3 was constitutively detected in mouse intervertebral disc tissue in the steady states. Collectively, these results suggest that endogenous TGF‐β activity limits TSLP expression in intervertebral disc tissue in the steady states by suppressing NF‐κB activation. The findings reveal a regulatory mechanism how TSLP expression is induced in the intervertebral disc tissue and suggest a novel role of TGF‐β in maintaining the homeostasis of intervertebral disc tissue. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1144–1149, 2013     

Silent information regulator (Sir)T1 inhibits NF‐κB signaling to maintain normal skeletal remodeling

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF‐κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF‐κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast‐ or osteoblast‐specific SirT1 deletion using the Sirt^flox/flox mice crossed to lysozyme M‐cre and the 2.3 kb col1a1‐cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF‐κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF‐κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF‐κB. In further support of the critical role of NF‐κB signaling in bone remodeling, elevated NF‐κB activity in IκBα^+/? mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell‐autonomous manner in both osteoblasts and osteoclasts, through control of NF‐κB and bone cell differentiation. © 2013 American Society for Bone and Mineral Research.     

Combined effects of TNF‐α, IL‐1β, and HIF‐1α on MMP‐2 production in ACL fibroblasts under mechanical stretch: An in vitro study

The dynamics between inflammatory factors, mechanical stress, and healing factors, in an intra‐articular joint, are very complex after injury. Injury to intra‐articular tissue [anterior cruciate ligament (ACL), synovium] results in hypoxia, accumulation of various pro‐inflammatory factors, cytokines, and metalloproteases. Although the presence of increased amounts of matrix‐metalloproteinases (MMP) in the joint fluid after knee injury is considered the key factor for ACL poor healing ability; however, the exact role of collective participants of the joint fluid on MMP‐2 activity and production has not been fully studied yet. To investigate the combined effects of mechanical injury, inflammation and hypoxia induced factor‐1α (HIF‐1α) on induction of MMP‐2; we mimicked the microenvironment of joint cavity after ACL injury. The results show that TNF‐α and IL‐1β elevate the activity of MMP‐2 in a dose‐ and time‐dependent manner. In addition, mechanical stretch further enhances the MMP‐2 protein levels with TNF‐α, IL‐1β, and their mixture. CoCl₂‐induced HIF‐1α (100 and 500 µM) also increases the levels and activity of MMP‐2. Mechanical stretch has a strong additional effect on MMP‐2 production with HIF‐1α. Our results conclude that mechanical injury, HIF‐1α and inflammatory factors collectively induce increased MMP‐2 production in ACL fibroblasts, which was inhibited by NF‐κB pathway inhibitor (Bay‐11‐7082). © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1008–1014, 2011     

Maslinic acid suppresses osteoclastogenesis and prevents ovariectomy‐induced bone loss by regulating RANKL‐mediated NF‐κB and MAPK signaling pathways

Activation of NF‐κB and MAPK/activator protein 1 (AP‐1) signaling pathways by receptor activator NF‐κB ligand (RANKL) is essential for osteoclast activity. Targeting NF‐κB and MAPK/AP‐1 signaling to modulate osteoclast activity has been a promising strategy for osteoclast‐related diseases. In this study we examined the effects of maslinic acid (MA), a pentacyclic triterpene acid that is widely present in dietary plants, on RANKL‐induced osteoclastogenesis, osteoclast function, and signaling pathways by in vitro and in vivo assay systems. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, MA inhibited RANKL‐induced osteoclastogenesis in a dose‐dependent manner within nongrowth inhibitory concentration, and MA decreased osteoclastogenesis‐related marker gene expression, including TRACP, MMP9, c‐Src, CTR, and cathepsin K. Specifically, MA suppressed osteoclastogenesis and actin ring formation at early stage. In ovariectomized mice, administration of MA prevented ovariectomy‐induced bone loss by inhibiting osteoclast activity. At molecular levels, MA abrogated the phosphorylation of MAPKs and AP‐1 activity, inhibited the IκBα phosphorylation and degradation, blocked NF‐κB/p65 phosphorylation, nuclear translocation, and DNA‐binding activity by downregulating RANK expression and blocking RANK interaction with TRAF6. Together our data demonstrate that MA suppresses RANKL‐induced osteoclastogenesis through NF‐κB and MAPK/AP‐1 signaling pathways and that MA is a promising agent in the treatment of osteoclast‐related diseases such as osteoporosis. © 2011 American Society for Bone and Mineral Research.