v-Rel is the acutely oncogenic member of the NF-[kappa]B family of transcription factors and transforms cells through the altered regulation of pathways normally controlled by cellular NF-[kappa]B. Initial studies revealed that expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAP kinases. This induction is critical for the v-Rel transformed phenotype, as suppression of MAPK activity with chemical inhibitors or siRNA severely limited colony formation of v-Rel transformed cell lines of hematopoietic origin. However, signaling must be maintained within a certain range in these cells, as strong additional activation of either pathway through expression of constitutively active MKK mutants also attenuated the transformed phenotype. Studies in primary spleen cells revealed that MAPK signaling is also required for the early stages of v-Rel-mediated transformation. However, constitutive MAPK activity further enhanced the transformation efficiency of v-Rel in primary cells. These studies, as well as analogous experiments in DT40 cells, indicate distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation. The proto-oncoprotein, c-Rel, only weakly activates ERK and JNK signaling compared to v-Rel. Importantly, elevated MAPK activity enhanced transformation by c-Rel, indicating that the ability of v-Rel to induce MAPK signaling is a major contributor to its oncogenic potential. Taken together, this work demonstrates an important role for ERK and JNK activity in transformation by v-Rel. Additional studies examined mechanisms through which MAPK activity is regulated in v-Rel transformed cells. Feedback regulation of the ERK activator, MKK1, at T292 was shown to limit ERK activation in v-Rel transformed cells, preventing the detrimental effects of constitutive activity. This result is the first indication that this regulation may have a role in the maintenance of transformation. Further, several v-Rel induced cytokines were identified that activate ERK and JNK signaling in v-Rel transformed cells, revealing one means by which v-Rel-dependent transcriptional changes lead to MAPK activation. These studies demonstrate the integration of multiple mechanisms in achieving the optimal levels of MAPK activity that are essential for v-Rel-mediated transformation. / text

The v-rel oncogene is the most efficiently transforming member of the Rel/NF-κB family of transcription factors. Identification of genes or signal transduction pathways that contribute to v-Rel transformation provide insight into the mechanisms of tumorigenesis by Rel/NF-κB proteins. In these studies, the contribution of TGF-β/Smad signaling to v-Rel transformation was assessed. TGF-β/Smad signaling regulates several cellular processes, including growth, differentiation, and apoptosis and has been implicated in a number of different cancers. Using microarray technology and Northern blot analysis, key components of the TGF-β/Smad pathway (tgf-β2 and tgf-β3 ligands, TGF-β type II receptor, and receptor-activated smad3) were identified with upregulated mRNA expression in v-Rel-transformed fibroblasts and lymphoid cells relative to control cells. A corresponding change in their protein levels was also observed. Further analysis revealed elevated levels of the phosphorylated, active form of Smad3, which correlated with its increased DNA-binding activity in v-Rel transformed cells. In contrast, the overexpression of c-Rel resulted in little to no alteration in the RNA and protein expression of members of the TGF-β/Smad pathway. Further studies demonstrated that elevated TGF-β/Smad signaling is required for the transforming ability of v-Rel. Blocking TGF-β signaling with a kinase inhibitor of TGF-β type I receptor inhibited the activation of Smad3 and dramatically reduced the ability of v-Rel transformed cells to form colonies in soft agar. Overexpression of a constitutively active form of Smad3 in the inhibitor-treated cells restored their ability to form colonies in soft agar close to the levels seen in untreated cells. Additional experiments with dominant negative Smad3 also revealed its ability to hinder the oncogenic potential of v-Rel. In complementary experiments, a stimulatory effect on v-Rel transformation was observed with cells treated with recombinant TGF-β2 ligand or overexpressed with wild-type Smad3. Taken together, these studies demonstrate that TGF-β signaling is crucial for the transformation potential of v-Rel and is primarily mediated by Smad3 activity. / text