Abstract
BackgroundNFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis.Methodology/Principal FindingsHere, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit), an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α) to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling.Conclusions/SignificanceThese results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFκB signaling will not be limited to FGFR4 alone.
Highlights
NFkB is a transcription factor of pivotal importance as a regulator of genes that control cell differentiation, survival, and inflammatory responses in mammalian cells
FGF19 treatment reduced the amount of cleaved Poly(ADP-ribose) Polymerase (PARP) induced by TNFa by approximately 35% (Figs. 5E and F). These results indicate that activation of FGFR4 signaling pathways in DU145 cells by FGF19 is able to negatively regulate apoptosis induced by TNFa stimulation
We characterize a novel interaction between a receptor tyrosine kinase, FGFR4, and a key regulatory protein in the NFkB pathway, IKKb
Summary
NFkB is a transcription factor of pivotal importance as a regulator of genes that control cell differentiation, survival, and inflammatory responses in mammalian cells. Regulatory interactions between FGFR4 and NFkB signaling pathways have not previously been reported, both pathways represent major axes of cell signaling. We describe the discovery of a twohybrid interaction between the receptor tyrosine kinase FGFR4 and IKKb, an important regulatory protein in the NFkB signaling pathway, and confirm this interaction in mammalian cells. NFkB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. A direct relationship between growth factor signaling pathways and NFkB activation has not been previously described, FGFs have been known to antagonize TNFa-induced apoptosis
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