Description of Project:

Tumor Necrosis Factor (TNF) is an effector of several different physiological processes such as tumor regression, inflammation, apoptosis, and proliferation. How TNF is able to signal to such a wide array of responses is the focus of numerous studies. In addition to signaling through the trimeric TNF receptors 1 and 2, we have found that TNF is able to utilize the EGFR (erbB1) in a hepatocyte cell line to induce proliferation. AML12 cells are a non-transformed, differentiated hepatocyte cell line which was developed from hepatocytes isolated from a TGF? transgenic mouse. The cells express the human TGF? transgene on the cell surface and in the culture medium. In the absence of exogenously added co-factors, we found that TNF induces proliferation as indicated by DNA synthesis. Our hypothesis is that TNF trans-activates the EGFR to induce signaling required for proliferation. Consistent with other reports of TNF trans-activation of the EGFR, TNF-induced proliferation in AML12 cells is dependent on matrix-metalloprotease (MMP) activtiy, suggesting TNF induces cleavage of a membrane bound ligand which activates EGFR. Using neutralizing antibodies to hTGFa, we determined that the ligand cleaved from the surface is the TGFa transgene. To study the downstream effects of TNF transactivation of EGFR, we utilized an inhibitor to the EGFR kinase activity, AG1478. When the TGFa-EGFR signal pathway is blocked in this manner, TNF's ability to induce proliferation is significantly reduced. Furthermore, some of the signaling induced by TNF that is required for proliferation is also blocked. TNF-induced activation of ERK1/2, the classical MAPK, is completely inhibited by AG1478 whereas TNF-induced activation of PKB/AKT in the PI-3 kinase pathway is only partially affected. These results indicate that TNF requires the EGFR for some of the signaling required for proliferation, but not all. Current studies are examining differences in phosporylation of EGFR in response to TNF and growth factor stimulation in order to determine the mechanism of activation of the receptor.

Pyridinyl imidazole inhibitors of p38 MAPK have been used extensively in vitro and in vivo to draw conclusions about the physiological role of p38 MAPK. While these inhibitors have proven to be fairly specific for p38? and ?, several groups have reported non-specific targets. We have found that RIP2 is another target for the p38 inhibitors. Receptor Interacting Protein 2 (RIP2) is a signaling protein that has an N-terminal S/T kinase domain and a C-terminal CARD domain. The kinase domain is believed to be important for NF-?B and ERK activation by specific stimuli. We have found that the kinase activity of RIP2 is inhibited in vitro by several of the pyridinyl imidazole inhibitors of p38 MAPK. The IC50 of these inhibitors for RIP2 is similar to the reported IC50 values for p38. Furthermore, we have determined that the mechanism of RIP2 inhibition is similar to the inhibition of p38. As was shown for p38, a conserved threonine in the kinase domain of RIP2 is required for sensitivity to the inhibitors. Mutation of this threonine residue to methionine blocks access of the inhibitor to the ATP binding site, and confers resistance of RIP2 to the p38 inhibitors. Current studies utilize the inhibitor-resistant RIP2 mutant to assess the contribution of RIP2 to pyridinyl imidadole-inhibitable events in cultured cells.