The study of p38gamma invasion pathways

Mitogen activated protein kinases regulate gene expression through transcription factors and precise mechanisms in this signal event however are largely unknown. Our recent studies showed that a member of the p38 family, p38gamma, is induced by the Ras oncogene and is antagonizing p38a activity in regulating Ras transformation and stress-induced cell death. These studies suggest that p38gamma may be an oncogene by stimulating cell growth, inhibiting cell death, increasing cell invasion and/or transforming cells by itself. The goal of this thesis was to test the hypothesis that p38gamma may itself act as an oncogene to transform cells through increasing cell proliferation, inhibiting cell death, increasing cell invasion and/or conferring anchorage independent growth.;The Tet-on inducible p38gamma expression system in IEC-6 cells were used to assess cell proliferation by cell counting, [3H]-thymidine incorporation and MTT assays. In IEC-6 cells, Tet induced p38gamma expression was found to increase cell proliferation in all three assays. Moreover, wild-type p38gamma had higher activity than the phosphorylation mutant p38gamma/AGF in stimulating cell proliferation, suggesting that p38gamma increases cell proliferation by phosphorylation dependent mechanisms. However, the transformation assay with soft-agar revealed that p38gamma, either alone or together with the co-oncogenes E1A or Wip1, failed to induce anchorage-independent growth. Consistent with its proliferative activity, p38gamma was found to inhibit stress-induced cell death in trypan blue and Apo-One caspase 3/7 assays. The results together show that p38gamma is both proliferative and anti-apoptotic, albeit it fails to transform cells.;In analyzing p38gamma invasive activity, experiments were performed to examine whether p38gamma was capable of inducing cellular invasion using the Matrigel invasion chambers. WT p38gamma, but not its AGF or C-terminal deleted mutants, was found to increase cell matrigel invasion. Mechanism analysis with luciferase promoter and RT-PCR assays showed that p38gamma stimulates the promoter activity of MMP-9, a well-established invasive protein. Moreover, p38gamma was found to stimulate the MMP-9 promoter by AP-1 dependent mechanisms to increase MMP-9 RNA expression dependent of p38gamma's phosphorylation and C-terminus. Furthermore, p38gamma was shown to increase MMP-9 protein expression by Western analysis and stimulate MMP-9 activity by Zymographic assays. Importantly, inhibition of MMP-9 activity by two pharmacological inhibitors blocks p38gamma-induced invasion. These results together establish a novel p38gamma/MMP-9 invasion pathway.;Previous studies have shown that Hog1 (a p38 homologue in yeast) and p38alpha are capable of binding to the chromatin complex. Experiments were next performed to examine if p38gamma stimulates MMP-9 transcription by binding to the DNA. Results from the Chromatin Immunoprecipitation (ChIP) assays showed that p38gamma is indeed capable of binding to a specific AP-1 site of the MMP-9 promoter as compared with the negative IgG control and positive c-Jun IP control. Co-IP analysis further showed that p38gamma and c-Jun form a protein complex and Western blot showed that p38gamma may be inducing c-Jun expression, indicating that p38gamma may be recruited to the MMP-9 promoter via c-Jun binding. To demonstrate if the p38gamma/MMP-9 invasive pathway exists in human colon cancer a group of human colon cell lines was analyzed for p38gamma, c-Jun and MMP-9 expression by Western and/or RT-PCR. Results showed that endogenous p38gamma protein-expression positively correlates with levels of c-Jun protein and MMP-9 RNA expression. Importantly, p38gamma depletion by lentiviral shRNA infection decreases both c-Jun/MMP-9 expression and cell invasion. This result suggests that this p38gamma/c-Jun/MMP-9 pathway functionally exists in human colon cancer cells.;In conclusion, this study reveals a novel p38gamma/c-Jun/MMP-9 invasion pathway. p38gamma was shown further to stimulate MMP-9 transcription through c-Jun mediated promoter binding, leading to increased MMP-9 protein expression and an increased cell invasion. Disruption of this pathway may be a new approach to control colon cancer invasion.