The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1

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 ␣-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of ␣-difluoromethylornithine. IEC-6 cells transfected with constitutively active Rac1 and Cdc42 migrated more rapidly than vectortransfected 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.

MEK/ERK regulates adherens junctions and migration through Rac1

“…The expression of a DN-Rac mutant decreased cadherin-mediated cell-cell contacts in both primary keratinocytes and MDCKII cells [Hirano et al, 1992;Jou and Nelson, 1998]. We previously found that polyamine depletion decreased Rac1 activity [Ray et al, 2003], and the expression of CA-MEK1 restored Rac1 activity, migration and the ACS in these cells [Vaidya et al, 2005]. EGF also increased the activities of MEK1 and Rac1 and migration (Figs.…”

“…6). Since Rac1 activates RhoA and Cdc42 in these cells [Ray et al, 2003], these results suggest that Rho GTPases regulate adherens junctions and the ACS.…”

“…The general protocol for the experiments and the methods used were similar to those described previously [McCormack et al, 1993;Ray et al, 2003]. In brief, IEC-6 cells were plated in control medium consisting of DMEM supplemented with 5% dialyzed FBS, 10 lg/ml of insulin and 50 lg/ml gentamicin sulfate or in control medium containing 5 mM DFMO or DFMO plus 10 lM putrescine.…”

“…Stable clones were isolated and characterized as described earlier [Ray et al, 2003;Bhattacharya et al, 2004].…”

“…Rac1 activity was determined as described earlier [Ray et al, 2002[Ray et al, , 2003. Briefly, GST-PAK fusion protein bound with glutathione-agarose was mixed with cell lysates (100 lg).…”