Activation of NF-κB by fluid shear stress, but not TNF-α, requires focal adhesion kinase in osteoblasts

Abstract

When bone is mechanically loaded fluid shear stress (FSS) is generated as a result of the movement of interstitial fluid across the membranes of osteoblasts and osteocytes. This external mechanical loading stimulates changes in the activity of cytoplasmic signaling molecules and alters gene expression in bone cells. This process, referred to as mechanotransduction, is vital for maintaining bone health in vivo by regulating the balance between bone formation and bone resorption. This current study focuses on the role of focal adhesions, sites of integrin-mediated cellular attachment to the extracellular matrix, and their proposed function as mechanosensors in bone cells. We examined the role of a key component of focal adhesions and of mechanotransduction, focal adhesion kinase (FAK) in regulation of FSS- and tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-kappa B (NF-κB) signaling in osteoblasts. Immortalized FAK+/+ and FAK−/− osteoblasts were exposed to periods of oscillatory fluid shear stress (OFF) and NF-κB activation was analyzed. We determined that FAK is required for OFF-induced nuclear translocation and activation of NF-κB in osteoblasts. In addition we found that OFF induced phosphorylation of the IκB kinases (IKKα/β) in both FAK+/+ and FAK−/− osteoblasts, but only FAK+/+ osteoblasts demonstrated the resulting degradation of NF-κB inhibitors IκBα and IκBβ. OFF did not induce the degradation of IκBε or the processing of p105 in either FAK+/+ and FAK−/− osteoblasts. To compare the role of FAK in mediating OFF-induced mechanotransduction to the well characterized activation of NF-κB by inflammatory cytokines, we exposed FAK+/+ and FAK−/− osteoblasts to TNF-α. Interestingly, FAK was not required for TNF-α induced NF-κB activation in osteoblasts. In addition we determined that TNF-α treatment did not induce the degradation of IκBβ as did OFF. These data indicate a novel relationship between FAK and NF-κB activation in osteoblast mechanotransduction and demonstrates that the mechanism of FSS-induced NF-κB activation in osteoblasts differs from the well characterized TNF-α-induced activation.

Focal adhesion kinase signaling regulates anti-inflammatory function of bone marrow mesenchymal stromal cells induced by biomechanical force

“…NF-κB and AP-1 transcriptional complexes have well-documented roles downstream of shear stress and have been found to be regulated by FAK and Src [57–59]. Consistent with the notion that NF-κB may contribute to flow-induced COX2 regulation by a FAK signaling cascade, FAK has been shown to be required for fluid shear stress-induced degradation of NF-κB inhibitors IκBα and IκBβ and subsequent NF-κB nuclear localization in osteoblasts and endothelium [59,60]. Future studies will be required to fully elucidate the key transcriptional regulators in the FAK-COX2 pathway.…”

From tissue mechanics to transcription factors

“…In fibroblasts from cardiac valves, it was shown that TGF-β induced β-catenin nuclear translocation, leading to myofibroblast differentiation, and that this was dependent on ECM stiffness (Chen et al, 2011). Finally, NF-κB, a factor that has an important role linking inflammation and fibrosis (Elsharkawy and Mann, 2007), has been shown to be regulated by mechanical signals in some tissues (Nam et al, 2009; Young et al, 2010). Adipocytes also activate NF-κB and upregulate MCP-1 with increased ECM density (Li et al, 2010)…”

Role of NF-κB in the skeleton

“…The downstream NF-κB targets include COX2 and eNOS as well as TNFα and IL-1β. Fluid shear stress-induced NF-κB in OBs depends on focal adhesion kinase (FAK), a component of the focal adhesion complex that mediates signaling downstream of integrin engagement with extracellular matrix [49]. Since mechanical forces play an important role in the maintenance of bone mass, this NF-κB-mediated response to sheer stress might be an important component.…”