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-alpha (TNF-alpha)-induced activation of nuclear factor-kappa B (NF-kappaB) signaling in osteoblasts. Immortalized FAK(+/+) and FAK(-)(/)(-) osteoblasts were exposed to periods of oscillatory fluid shear stress (OFF) and NF-kappaB activation was analyzed. We determined that FAK is required for OFF-induced nuclear translocation and activation of NF-kappaB in osteoblasts. In addition we found that OFF-induced phosphorylation of the IkappaB kinases (IKKalpha/beta) in both FAK(+/+) and FAK(-/-) osteoblasts, but only FAK(+/+) osteoblasts demonstrated the resulting degradation of NF-kappaB inhibitors IkappaBalpha and IkappaBbeta. OFF did not induce the degradation of IkappaBepsilon 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-kappaB by inflammatory cytokines, we exposed FAK(+/+) and FAK(-/-) osteoblasts to TNF-alpha. Interestingly, FAK was not required for TNF-alpha induced NF-kappaB activation in osteoblasts. In addition we determined that TNF-alpha treatment did not induce the degradation of IkappaBbeta as did OFF. These data indicate a novel relationship between FAK and NF-kappaB activation in osteoblast mechanotransduction and demonstrates that the mechanism of FSS-induced NF-kappaB activation in osteoblasts differs from the well characterized TNF-alpha-induced activation.