Activation of Dbl restores migration in polyamine-depleted intestinal epithelial cells via Rho-GTPases.

Integrin binding to the extracellular matrix (ECM) activated Rho GTPases, Src, and focal adhesion kinase in intestinal epithelial cells (IEC)-6. Polyamine depletion inhibited activities of Rac1, RhoA, and Cdc42 and thereby migration. However, constitutively active (CA) Rac1 expression abolished the inhibitory effect of polyamine depletion, indicating that polyamines are involved in a process upstream of Rac1. In the present study, we examined the role of polyamines in the regulation of the guanine nucleotide exchange factor, diffuse B-cell lymphoma (Dbl), for Rho GTPases. Polyamine depletion decreased the level as well as the activation of Dbl protein. Dbl knockdown by siRNA altered cytoskeletal structure and decreased Rac1 activity and migration. Cells expressing CA-Dbl increased migration, Rac1 activity, and proliferation. CA-Dbl restored migration in polyamine-depleted cells by activating RhoA, Rac1, and Cdc42. CA-Dbl caused extensive reorganization of the F-actin cortex into stress fibers. Inhibition of Rac1 by NSC23766 significantly decreased migration of vector-transfected cells and CA-Dbl-transfected cells. However, the inhibition of migration was significantly higher in the vector-transfected cells compared with that seen in the CA-Dbl-transfected cells. Dbl localized in the perinuclear region in polyamine-depleted cells, whereas it localized with the stress fibers in control cells. CA-Dbl localized with stress fibers in both the control and polyamine-depleted cells. These results suggest that polyamines regulate the activation of Dbl, a membrane-proximal process upstream of Rac1.

Regulation of JNK activity in the apoptotic response of intestinal epithelial cells.

We have studied apoptosis of gastrointestinal epithelial cells by examining the receptor-mediated and DNA damage-induced pathways using TNF-α and camptothecin (CPT), respectively. TNF-α requires inhibition of antiapoptotic protein synthesis by cycloheximide (CHX). CHX also results in high levels of active JNK, which are necessary for TNF-induced apoptosis. While CPT induces apoptosis, the increase in JNK activity was not proportional to the degree of apoptosis. Thus the mechanism of activation of JNK and its role in apoptosis are unclear. We examined the course of JNK activation in response to a combination of TNF-α and CPT (TNF + CPT), which resulted in a three- to fourfold increase in apoptosis compared with CPT alone, indicating an amplification of apoptotic signaling pathways. TNF + CPT caused apoptosis by activating JNK, p38, and caspases-8, -9, and -3. TNF-α stimulated a transient phosphorylation of JNK1/2 and ERK1/2 at 15 min, which returned to basal by 60 min and remained low for 4 h. CPT increased JNK1/2 activity between 3 and 4 h. TNF + CPT caused a sustained and robust JNK1/2 and ERK1/2 phosphorylation by 2 h, which remained high at 4 h, suggesting involvement of MEKK4/7 and MEK1, respectively. When administered with TNF + CPT, SP-600125, a specific inhibitor of MEKK4/7, completely inhibited JNK1/2 and decreased apoptosis. However, administration of SP-600125 at 1 h after TNF + CPT failed to prevent JNK1/2 phosphorylation, and the protective effect of SP-600125 on apoptosis was abolished. These results indicate that the persistent activation of JNK might be due to inhibition of JNK-specific MAPK phosphatase 1 (MKP1). Small interfering RNA-mediated knockdown of MKP1 enhanced TNF + CPT-induced activity of JNK1/2 and caspases-9 and -3. Taken together, these results suggest that MKP1 activity determines the duration of JNK1/2 and p38 activation and, thereby, apoptosis in response to TNF + CPT.

The phosphorylation state of MRLC is polyamine dependent in intestinal epithelial cells.

Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.