2012 Theses Doctoral

Exploring a Novel Mechanism of Reguation of the TNFR Family Member FN14

Gurunathan, Sujatha

Fibroblast growth factor-inducible 14 (FN14) is a highly inducible cytokine receptor linked to a number of intracellular signaling pathways, including the nuclear factor-kappaB (NFkB) and mitogen-activated protein kinase (MAPK) pathways, as well as to physiological processes, such as angiogenesis, tissue repair, and regeneration. Although FN14 is expressed at low levels in normal tissue, it is highly expressed after tissue injury and appears to mediate activities crucial to wound repair and regeneration. In vitro, FN14 expression is induced by numerous growth factors, as well as by its own ligand TNF-like weak inducer of apoptosis (TWEAK). Given these results, it is thought that FN14 plays an important physiological role in the response to acute injury, and that FN14 activity is primarily regulated at the level of ligand and receptor expression. Various pathological states, however, also exhibit increased expression and activity of FN14. Tumor progression, chronic autoimmune disease, and neuroinflammation are just a few of the conditions in which increased expression or function of FN14 have been implicated. In addition, it has been shown, in vitro, that overexpression of FN14 can result in ligand-independent signaling by the receptor. This signaling in the absence of TWEAK has been associated with increased proliferation and invasiveness of several types of cancer cells. Thus, although FN14 plays an important physiological role after acute injury, it appears that dysregulation of FN14 expression may contribute to various chronic pathological states. Since FN14 protein expression is so highly inducible, and TWEAK-FN14 signaling can further amplify receptor levels, this begs the question whether unregulated receptor levels can influence the pathological switch through altered signaling and resultant cell proliferation, migration, or chemokine expression. It seems likely that there are cellular mechanisms in place to prevent excess accumulation of FN14. To date, however, the stability of FN14 or mechanisms of FN14 downregulation, factors which would presumably have dramatic effects on the sustained activity of the receptor, have not been explored. The main focus of this project was to explore the cellular mechanisms involved in downregulation of FN14 levels or signaling capacity which are likely important for preventing inappropriate and uncontrolled signaling. One mechanism through which receptor signaling can be attenuated is by ligand-induced downregulation. In some cases, as with the receptor Notch, ligand-independent turnover can occur as well. During the course of this project, both of these turnover mechanisms were evaluated for FN14. It was determined that the receptor undergoes both rapid ligand-dependent and ligand-independent turnover, and that these two processes are distinct and synergistic. A described endocytic motif in the FN14 cytoplasmic tail does not seem to be required for either method of turnover, which means that the endocytic mechanism remains to be elucidated. We focused on dissecting the ligand-independent turnover of FN14 due to the novel nature of this regulatory mechanism. It appears that FN14 is constitutively expressed, trafficked to the cell surface, endocytosed, and degraded in lysosomes. This constitutive trafficking to sites of degradation seems to require only the extracellular domain of the receptor, suggesting that FN14 may interact with a trafficking partner using this domain. Although FN14 seems to be constitutively degraded in the steady-state, growth factor treatment stabilizes protein levels, in a manner independent of transcriptional upregulation. This finding supports the notion that FN14 levels are regulated not only at the level of expression, but also at the level of protein stability. Ultimately, this work describes previously unrecognized aspects of FN14 biology, which may enhance our understanding of how FN14 activity is dysregulated in chronic pathological states. Using this information, it might be possible to design peptides to alter FN14 trafficking patterns in a therapeutically applicable manner.