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The rapid migration of intestinal epithelial cells is important to the healing of mucosal ulcers and wounds. This cell migration requires the presence of polyamines and the activation of RhoA. RhoA activity, however, is not sufficient for migration because polyamine depletion inhibited the migration of IEC-6 cells expressing constitutively active RhoA. The current study examines the role of Rac1 and Cdc42 in cell migration and whether their activities are polyamine-dependent. Polyamine depletion with alpha-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of alpha-difluoromethylornithine. IEC-6 cells transfected with constitutively active Rac1 and Cdc42 migrated more rapidly than vector-transfected cells, whereas cells expressing dominant negative Rac1 and Cdc42 migrated more slowly. Polyamine depletion had no effect on the migration of cells expressing Rac1 and only partially inhibited the migration of those expressing Cdc42. Although polyamine depletion caused the disappearance of actin stress fibers in cells transfected with empty vector, it had no effect on cells expressing Rac1. Constitutively active Rac1 increased RhoA and Cdc42 activity in both normal and polyamine-depleted cells. These results demonstrate that Rac1, RhoA, and Cdc42 are required for optimal epithelial cell migration and that Rac1 activity is sufficient for cell migration in the absence of polyamines due to its ability to activate RhoA and Cdc42 as well as its own effects on the process of cell migration. These data imply that the involvement of polyamines in cell migration occurs either at Rac1 itself or upstream from Rac1.

The proliferation of the rat intestinal mucosal IEC-6 cell line requires polyamines, whose synthesis is catalyzed by the enzyme ornithine decarboxylase (ODC). ODC inhibition leads to polyamine depletion, as well as inhibition of both cell proliferation and apoptosis by regulating gene expression. The NF-kappa B transcription factor regulates genes involved in apoptotic, immune, and inflammatory responses. In the present study we tested the hypothesis that NF-kappa B is activated following ODC inhibition. We found that the inhibition of ODC by alpha-difluoromethylornithine (DFMO) resulted in a approximately 50% decrease in intracellular putrescine levels within 1 h. NF-kappa B is activated by DFMO through the degradation of the inhibitory protein I kappa B alpha that sequesters NF-kappa B in the cytoplasm. The DFMO-induced NF-kappa B complexes contain the p65 and p50 members of the Rel protein family. DFMO-induced NF-kappa B activation was accompanied by the translocation of p65 from the cytoplasm into the nucleus. DFMO selectively inhibited a gene reporter construct dependent on the kappa B site present in the HLA-B7 gene. In contrast, DFMO had no effect on a gene reporter construct dependent on the kappa B site present in the interleukin-8 gene. Thus, we report that ODC inhibition activates the NF-kappa B transcription factor, which may mediate the altered physiological state of intestinal cells that occurs following polyamine depletion.