Polyamine Depletion Associated With Increase in JunDAP1 Activity

JunD is a member of the jun family proto-oncogenes, which are primary c o mp o-nents of the activator protein-1 (AP-1) transcription factor (37,38). The Jun proteins (c-Jun, JunB, and JunD) are basic leucine transcription factors that can form either AP-1 homodimers (Jun/Jun) or AP-1 heterodimers with members of related the Fos family (c-Fos, FosB, Fra-1, and Fra-2) or the ATF family (ATF2, ATF3, and ATF4) (3 8). Jun/Jun and Jun/Fos dimers bind to the TPA responsive element TGACTCA present in many gene promoters, whereas Jun/ATF dimers bind preferentially to the cyclic AMP responsive element TGACGTCA (39). The three Jun proteins are similar in their DNA-binding affinity that is determined by the C-terminal leucine zipper dimerization domain (40). The N-terminal transactivation domain is less conserved and may account for the different transactivation characteristics of the Jun proteins. There is increasing evidence that individual AP-1 dimers play distinct functions in different cellular contexts, including cell proliferation, growth arrest, differentiation, and apoptosis (37-40). For example, c-jun and junB function as immediate early response genes and activation of these two genes enhances the transition from a quiescent state to a proliferating state, indicating that c-Jun and JunB are positive AP-1 factors for cell proliferation. In contrast, the activation of junD gene expression slows cell proliferation in some cell types and increases the percentage of population of cells arrested in the G0/Gj phase of

Fig. 4. Schematic diagram depicting the regulation in expression of the p53 gene by cellular polyamines and the proposed role of p53 in growth inhibition of the small intestinal mucosa after polyamine depletion. Cellular polyamine levels are decreased by either inhibition of their biosynthesis by blocking ornithine decarboxylase activity, stimulation of the catabolism through the activation of spermidine/spermine-N-acetyltransferase activity, or suppression of polyamine uptake. Decreased cellular polyamines stabilize p53 messenger RNA and result in the accumulation of p53 through the induction of newly synthesized p53 protein. Polyamine depletion also increases nucleophosmin that interacts with and stabilizes p53 protein. The resultant increases in p53 initiate or enhance transcription of cell-cycle arrest genes such as p21, inhibits cell proliferation, and suppresses mucosal growth of the small intestine. SPD, spermidine; SPM, spermine; Put, putrescine; DFMO, a-difluoromethylornithine; ODC, ornithine decarboxylase; SSTA, sper-midine/spermine-N-acetyltransferase; NPM, nucleophosmin.

Fig. 4. Schematic diagram depicting the regulation in expression of the p53 gene by cellular polyamines and the proposed role of p53 in growth inhibition of the small intestinal mucosa after polyamine depletion. Cellular polyamine levels are decreased by either inhibition of their biosynthesis by blocking ornithine decarboxylase activity, stimulation of the catabolism through the activation of spermidine/spermine-N-acetyltransferase activity, or suppression of polyamine uptake. Decreased cellular polyamines stabilize p53 messenger RNA and result in the accumulation of p53 through the induction of newly synthesized p53 protein. Polyamine depletion also increases nucleophosmin that interacts with and stabilizes p53 protein. The resultant increases in p53 initiate or enhance transcription of cell-cycle arrest genes such as p21, inhibits cell proliferation, and suppresses mucosal growth of the small intestine. SPD, spermidine; SPM, spermine; Put, putrescine; DFMO, a-difluoromethylornithine; ODC, ornithine decarboxylase; SSTA, sper-midine/spermine-N-acetyltransferase; NPM, nucleophosmin.

the cell cycle (37,41), suggesting that JunD is a negative AP-1 factor and downregu-lates the G1 to S phase transition.

Patel and Wang (9) have demonstrated that depletion of cellular polyamines is associated with an increase in JunD/AP-1 activity in IECs. It has been shown that exposure of IEC-6 cells or Caco-2 (a human colon carcinoma cell line) to DFMO for 4 and 6 d significantly increases AP-1-binding activity as measured by electrophoretic mobility shift assays. Spermidine, when given with DFMO, restores AP-1-binding activity toward

Fig. 5. Effect of addition of the antibody against JunD protein on AP-1 composition in cells exposed to DFMO alone or DFMO plus SPD. Gel supershift assays were performed by initially incubating nuclear protein with 32p end-labeled oligonucleotides containing a single AP-1-bind-ing site. The antibody against JunD protein was then added to the binding reaction mixture. The positions of supershifted AP-1 components, the total AP-1-binding activity, and freely migrating probes are indicated. No antibody: the lane with nuclear extract and no antibody. Control antibody: the reaction mixture with nuclear extracts and the control antibody (anti-Myc antibody). Three experiments were performed that showed consistent results.

Fig. 5. Effect of addition of the antibody against JunD protein on AP-1 composition in cells exposed to DFMO alone or DFMO plus SPD. Gel supershift assays were performed by initially incubating nuclear protein with 32p end-labeled oligonucleotides containing a single AP-1-bind-ing site. The antibody against JunD protein was then added to the binding reaction mixture. The positions of supershifted AP-1 components, the total AP-1-binding activity, and freely migrating probes are indicated. No antibody: the lane with nuclear extract and no antibody. Control antibody: the reaction mixture with nuclear extracts and the control antibody (anti-Myc antibody). Three experiments were performed that showed consistent results.

normal. As can be seen in Fig. 5, the anti-JunD antibody, when added to the binding reaction mixture, dramatically supershifted the AP-1 complexes present in the IEC-6 cell exposed to DFMO for 4 and 6 d. The AP-1 activity attributed to JunD in the DFMO-treated cells was approximately one-third of the total AP-1-binding activity on d 4, and about half on d 6, respectively. In control cells and cells exposed to DFMO and spermidine, the AP-1-binding activity was slightly supershifted by the anti-JunD anti-b o dy. On the other hand, addition of antibodies against c-Jun or JunB to the binding reaction mixture had an effect on the AP-1-binding activity in all three treatment groups. There was no stimulation of the AP-1-activity attributed to c-Jun or JunB by DFMO (5,9). Although the anti-Fos antibody also partially supershifted the AP-1 complexes, there were no significant differences in the AP-1 activity attributed to Fos between control cells and cells exposed to DFMO or DFMO plus spermidine. The increased A P - 1 -

binding activities in polyamine-deficient cells were not supershifted by the anti-Myc antibo dy. These results indicate that the increase in AP-1 activity in polyamine-deficient cells is primarily contributed by an increase in JunD/AP-1, whereas c-Jun/AP-1 and JunB/AP-1 activity remains essentially unchanged or decreased.

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