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ERK and JNK activation is essential for oncogenic transformation by v-Rel.

Kralova J, Sheely JI, Liss AS, Bose HR - Oncogene (2010)

Bottom Line: The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways.MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified.We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

ABSTRACT
v-Rel is the acutely oncogenic member of the NF-κB family of transcription factors. Infection with retroviruses expressing v-Rel rapidly induces fatal lymphomas in birds and transforms primary lymphocytes and fibroblasts in vitro. We have previously shown that AP-1 transcriptional activity contributes to v-Rel-mediated transformation. Although v-Rel increases the expression of these factors, their activity may also be induced through phosphorylation by the mitogen-activated protein kinases (MAPKs). The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways. This induction is critical for the v-Rel-transformed phenotype, as suppression of MAPK activity with chemical inhibitors or small interfering RNA severely impairs colony formation of v-Rel-transformed lymphoid cell lines. However, signaling must be maintained within an optimal range in these cells, as strong additional activation of either pathway beyond the levels induced by v-Rel through the expression of constitutively active MAPK proteins attenuates the transformed phenotype. MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified. We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

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CA MKK mutants enhance colony formation of DT40 cells infected with helper virus or retroviruses encoding c-Rel. DT40 cells were co-infected with CSV alone or with retroviruses expressing c-Rel (REV-C) as well as with retroviruses expressing the CA MKK mutants. Infections were expanded in liquid culture for 8–14 days and cells were harvested for protein and plated into soft agar. (a) The expression of c-Rel and the CA MKK mutants were examined by Western blot. The levels of phosphorylated and total ERK in cells expressing CA MKK1 and CA MKK2 and phosphorylated and total JNK in cells expressing CA MKK7 relative to cells infected with empty DS viruses were also determined. (b) Soft agar colonies were scored microscopically 7–9 days later. Colony numbers for cells infected with CSV and with empty DS retroviruses were standardized to 100. Statistically significant differences from cells infected with CSV and empty DS vector are indicated (*P<0.05). The average of at least three independent experiments is shown with standard error.
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Figure 7: CA MKK mutants enhance colony formation of DT40 cells infected with helper virus or retroviruses encoding c-Rel. DT40 cells were co-infected with CSV alone or with retroviruses expressing c-Rel (REV-C) as well as with retroviruses expressing the CA MKK mutants. Infections were expanded in liquid culture for 8–14 days and cells were harvested for protein and plated into soft agar. (a) The expression of c-Rel and the CA MKK mutants were examined by Western blot. The levels of phosphorylated and total ERK in cells expressing CA MKK1 and CA MKK2 and phosphorylated and total JNK in cells expressing CA MKK7 relative to cells infected with empty DS viruses were also determined. (b) Soft agar colonies were scored microscopically 7–9 days later. Colony numbers for cells infected with CSV and with empty DS retroviruses were standardized to 100. Statistically significant differences from cells infected with CSV and empty DS vector are indicated (*P<0.05). The average of at least three independent experiments is shown with standard error.

Mentions: v-Rel is much more oncogenic than c-Rel. Spleen cells infected with retroviruses expressing v-Rel readily form colonies in soft agar, whereas cells overexpressing c-Rel can only grow in liquid culture. Our initial observations showed that v-Rel expression activates MAPK signaling to a much greater extent than c-Rel (Figure 1a, Figure 1b). To determine whether the difference in c-Rel and v-Rel oncogenicity results from their differential activation of MAPK signaling, we examined whether additional induction of MAPK activity in cells expressing c-Rel would enhace their ability to grow in soft agar. These experiments were performed in DT40 cells, in which expression of v-Rel results in a 2.3-fold increase in colony formation relative to CSV-infected cells (Figure 4c). DT40 cells were co-infected with helper virus (CSV) or with retroviruses expressing c-Rel (REV-C) and with DS retroviruses expressing the CA MKK mutants. Western analysis demonstrated c-Rel overexpression in REV-C infected cells and confirmed similar expression of the CA MKK constructs in all infections (Figure 7a). c-Rel overexpression alone caused a slight increase in MAPK activation. In both CSV- and REV-C-infected cells, expression of the CA MKK mutants resulted in elevated levels of ERK and JNK activity. Notably, when CA MKKs were expressed in REV-C-infected cells, the levels of ERK and JNK signaling were higher than in CSV-infected cells expressing the same MKK constructs. Moreover, CA MKK2 expression, either alone or in the context of c-Rel overexpression, resulted in stronger ERK activation than CA MKK1.


ERK and JNK activation is essential for oncogenic transformation by v-Rel.

Kralova J, Sheely JI, Liss AS, Bose HR - Oncogene (2010)

CA MKK mutants enhance colony formation of DT40 cells infected with helper virus or retroviruses encoding c-Rel. DT40 cells were co-infected with CSV alone or with retroviruses expressing c-Rel (REV-C) as well as with retroviruses expressing the CA MKK mutants. Infections were expanded in liquid culture for 8–14 days and cells were harvested for protein and plated into soft agar. (a) The expression of c-Rel and the CA MKK mutants were examined by Western blot. The levels of phosphorylated and total ERK in cells expressing CA MKK1 and CA MKK2 and phosphorylated and total JNK in cells expressing CA MKK7 relative to cells infected with empty DS viruses were also determined. (b) Soft agar colonies were scored microscopically 7–9 days later. Colony numbers for cells infected with CSV and with empty DS retroviruses were standardized to 100. Statistically significant differences from cells infected with CSV and empty DS vector are indicated (*P<0.05). The average of at least three independent experiments is shown with standard error.
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Related In: Results  -  Collection

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Figure 7: CA MKK mutants enhance colony formation of DT40 cells infected with helper virus or retroviruses encoding c-Rel. DT40 cells were co-infected with CSV alone or with retroviruses expressing c-Rel (REV-C) as well as with retroviruses expressing the CA MKK mutants. Infections were expanded in liquid culture for 8–14 days and cells were harvested for protein and plated into soft agar. (a) The expression of c-Rel and the CA MKK mutants were examined by Western blot. The levels of phosphorylated and total ERK in cells expressing CA MKK1 and CA MKK2 and phosphorylated and total JNK in cells expressing CA MKK7 relative to cells infected with empty DS viruses were also determined. (b) Soft agar colonies were scored microscopically 7–9 days later. Colony numbers for cells infected with CSV and with empty DS retroviruses were standardized to 100. Statistically significant differences from cells infected with CSV and empty DS vector are indicated (*P<0.05). The average of at least three independent experiments is shown with standard error.
Mentions: v-Rel is much more oncogenic than c-Rel. Spleen cells infected with retroviruses expressing v-Rel readily form colonies in soft agar, whereas cells overexpressing c-Rel can only grow in liquid culture. Our initial observations showed that v-Rel expression activates MAPK signaling to a much greater extent than c-Rel (Figure 1a, Figure 1b). To determine whether the difference in c-Rel and v-Rel oncogenicity results from their differential activation of MAPK signaling, we examined whether additional induction of MAPK activity in cells expressing c-Rel would enhace their ability to grow in soft agar. These experiments were performed in DT40 cells, in which expression of v-Rel results in a 2.3-fold increase in colony formation relative to CSV-infected cells (Figure 4c). DT40 cells were co-infected with helper virus (CSV) or with retroviruses expressing c-Rel (REV-C) and with DS retroviruses expressing the CA MKK mutants. Western analysis demonstrated c-Rel overexpression in REV-C infected cells and confirmed similar expression of the CA MKK constructs in all infections (Figure 7a). c-Rel overexpression alone caused a slight increase in MAPK activation. In both CSV- and REV-C-infected cells, expression of the CA MKK mutants resulted in elevated levels of ERK and JNK activity. Notably, when CA MKKs were expressed in REV-C-infected cells, the levels of ERK and JNK signaling were higher than in CSV-infected cells expressing the same MKK constructs. Moreover, CA MKK2 expression, either alone or in the context of c-Rel overexpression, resulted in stronger ERK activation than CA MKK1.

Bottom Line: The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways.MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified.We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

ABSTRACT
v-Rel is the acutely oncogenic member of the NF-κB family of transcription factors. Infection with retroviruses expressing v-Rel rapidly induces fatal lymphomas in birds and transforms primary lymphocytes and fibroblasts in vitro. We have previously shown that AP-1 transcriptional activity contributes to v-Rel-mediated transformation. Although v-Rel increases the expression of these factors, their activity may also be induced through phosphorylation by the mitogen-activated protein kinases (MAPKs). The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways. This induction is critical for the v-Rel-transformed phenotype, as suppression of MAPK activity with chemical inhibitors or small interfering RNA severely impairs colony formation of v-Rel-transformed lymphoid cell lines. However, signaling must be maintained within an optimal range in these cells, as strong additional activation of either pathway beyond the levels induced by v-Rel through the expression of constitutively active MAPK proteins attenuates the transformed phenotype. MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified. We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

Show MeSH
Related in: MedlinePlus