Abstract
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.
Highlights
Cell migration maintains the organization and integrity of the mucosa of the gastrointestinal tract [1]
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
We have shown that the early phase of mucosal healing, which is due to cell migration, requires polyamines [21, 22] and that polyamine depletion inhibits migration in the IEC-6 cell model and leads to numerous alterations in the cytoskeleton
Summary
Polyamine synthesis prevent concanavalin A-induced expression of ␣-tubulin and -actin mRNAs in mouse splenocytes [19]. Polyamines are necessary for RhoA synthesis and activity, they are required for an additional step either upstream or downstream from RhoA that results in cell migration [27] Both Rac and Cdc are excellent candidates for additional steps in the process of cell motility to be regulated by polyamines. We demonstrate that the rates of migration of IEC-6 cells transfected with constitutively active Rac or Cdc are significantly more rapid than those of the corresponding wild type cells or those transfected with empty vectors. We demonstrate that Rac activation leads to RhoA and Cdc activation independent of polyamines, which explains the restoration of the cytoskeleton and migration rate in constitutively active Rac cells depleted of polyamines
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