Soluble TNF-a interacts with the TNF-a receptor, TNF-R1 (CD120a (p55)), and plays an essential role in the development of acute and chronic inflammatory diseases of the lung via its ability to stimulate NF-kB-dependent pro-inflammatory cytokine production and induce apoptosis. How these mutually-antagonistic functions are regulated is not well understood. In work supported by this grant, we showed that TNF-R1 is inducibly phosphorylated on Ser and Thr residues by the mitogen-activated protein kinase, p42mapk/erk2. In addition, we have shown that phosphoryation of TNF-R1 renders the receptor incapable of stimulating apoptosis, but does not affect its ability to activate NF-kB. We propose that TNF-R1 phosphorylation represents a survival mechanism that promotes the preservation of cells during pulmonary inflammation leading to prolonged NF-kB-dependent cytokine expression. In this competitive renewal, we propose to address 3 fundamental questions about the mechanism of TNF-R1 phosphorylation and the consequences of this event on pulmonary inflammation and host defense. First, what is the mechanism of p42mapk/erk2 activation by soluble TNF-a ? Second, how does phosphorylation of TNF-R1 inhibit apoptosis? Third, what are the physiologic consequences of TNF-R1 phosphorylation on the development of pulmonary inflammation? Based on our preliminary findings we will test the hypotheses that: (i) p42mapk/erk2 activation is dependent on the endocytosis and translocation of soluble TNF-( to an intracellular pool of TNF-R1 wherein TNF-R1 is phosphorylated and (ii) physiologic phosphorylation of TNF-R1 will enhance pulmonary inflammation while inhibiting lung cell apoptosis. These hypotheses will be addressed with three specific aims.
In Aim 1 we will investigate the role of macropinocytosis in translocating soluble TNF-( to the intracellular pool of TNF-R1 in the trans-Golgi network and in promoting p42maPk/erk2 activation and TNF-R1 phosphorylation.
Aim 2 will address the mechanisms through which phosphorylation of TNF-R1 inhibits the apoptotic activity of the receptor. Lastly, in Aim 3, we will address the physiologic consequences of TNF-R1 phosphorylation on pulmonary inflammation and host defense in mice bearing germline mutations in the tnf-r1 locus that will prevent or mimic TNF-R1 phosphorylation. The outcome of this work will have broad implications for understanding how signaling by TNF-R1 is regulated in pulmonary inflammation and injury.
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