Abstract: Cdc42 plays a central role in regulating the actin cytoskeleton and maintaining cell polarity. Here, we show that Cdc42 is crucial for epidermal growth factor (EGF)-stimulated protrusion in MTLn3 carcinoma cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the protruding edge of the cells. siRNA-mediated knockdown of Cdc42 expression caused a decrease in EGF-stimulated protrusion and reduced cell motility in time-lapse studies. These changes were correlated with a decrease in barbed-end formation and Arp2/3 localization at the cell edge, and a marked defect in actin filament branching, as revealed by rotary-shadowing scanning electron microscopy. Upstream of Arp2/3, Cdc42 knockdown inhibited EGF-stimulated activation of PI 3-kinase at early (within 1 minute) but not late (within 3 minutes) time points. Membrane targeting of N-WASP, WAVE2 and IRSp53 were also inhibited. Effects on WAVE2 were not owing to Rac1 inhibition, because WAVE2 recruitment is unaffected by Rac1 knockdown. Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac.

Abstract: We have investigated the effects of inhibiting the expression of cofilin to understand its role in protrusion dynamics in metastatic tumor cells, in particular. We show that the suppression of cofilin expression in MTLn3 cells (an apolar randomly moving amoeboid metastatic tumor cell) caused them to extend protrusions from only one pole, elongate, and move rectilinearly. This remarkable transformation was correlated with slower extension of fewer, more stable lamellipodia leading to a reduced turning frequency. Hence, the loss of cofilin caused an amoeboid tumor cell to assume a mesenchymal-type mode of movement. These phenotypes were correlated with the loss of uniform chemotactic sensitivity of the cell surface to EGF stimulation, demonstrating that to chemotax efficiently, a cell must be able to respond to chemotactic stimulation at any region on its surface. The changes in cell shape, directional migration, and turning frequency were related to the re-localization of Arp2/3 complex to one pole of the cell upon suppression of cofilin expression.

Abstract: Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P(3) production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P(3) production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P(3) production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.

Abstract: The Rho GTPases Rac and Cdc42 play a central role in the regulation of secretory and cytoskeletal responses in antigen-stimulated mast cells. In this study, we examine the kinetics and mechanism of Rac and Cdc42 activation in the rat basophilic leukemia RBL-2H3 cells. The activation kinetics of both Rac and Cdc42 show a biphasic profile, consisting of an early transient peak at 1 min and a late sustained activation phase at 20-40 min. The inhibition of phospholipase C (PLC)gamma causes a twofold increase in Rac and Cdc42 activation that coincides with a dramatic production of atypical filopodia-like structures. Inhibition of protein kinase C using bisindolylmaleimide mimics the effect of PLCgamma inhibition on Rac activation, but not on Cdc42 activation. In contrast, depletion of intracellular calcium leads to a complete inhibition of the early activation peak of both Rac and Cdc42, without significant effects on the late sustained activation. These data suggest that PLCgamma is involved in a negative feedback loop that leads to the inhibition of Rac and Cdc42. They also suggest that the presence of intracellular calcium is a prerequisite for both Rac and Cdc42 activation.

Abstract: Phosphoinositide 3-kinase (PI 3-kinase) activity is required for growth factor-induced cytoskeletal regulation and cell migration. We previously found that in MTLn3 rat adenocarcinoma cells, EGF-stimulated induction of actin barbed ends and lamellipod extension specifically requires the p85/p110alpha isoform of PI 3-kinase. To further characterize signaling by distinct PI 3-kinase isoforms, we have developed MTLn3 cells that transiently or stably overexpress either p110alpha or p110beta. Transient overexpression of p110beta inhibited EGF-stimulated lamellipod extension, whereas p110alpha-transfected cells showed normal EGF-stimulated lamellipod extension. Similar results were obtained by overexpression of kinase-dead p110beta, suggesting that effects on cytoskeletal signaling were due to competition with p85/p110alpha complexes. Stable overexpression of p110alpha appeared to be toxic, based on the difficulty in obtaining stable overexpressing clones. In contrast, cells expressing a 2-fold increase in p110beta were readily obtainable. Interestingly, cells stably expressing p110beta showed a marked inhibition of EGF-stimulated lamellipod extension. Using computer-assisted analysis of time-lapse images, we found that overexpression of p110beta caused a nearly complete inhibition of motility. Cells overexpressing p110beta showed normal activation of Akt and Erk, suggesting that overall PI 3-kinase signaling was intact. A chimeric p110 molecule containing the p85-binding and Ras-binding domains of p110alpha and the C2, helical, and kinase domains of p110beta, was catalytically active yet also inhibited EGF-stimulated lamellipod extension. These data highlight the differential signaling by distinct p110 isoforms. Identification of effectors that are differently regulated by p110alpha versus p110beta will be important for understanding cell migration and its role in metastasis.

Abstract: BACKGROUND: In inflammatory bowel disease (IBD), cytokine levels (such as interleukin-1 (IL-1)) are elevated. We have shown previously that IL-1 activates phospholipid signaling pathways in intestinal epithelial cells (EEC), leading to increased ceramide levels. AIM: To determine whether ceramide induces apoptosis in IEC. METHODS: Apoptosis was evaluated by annexin-V binding or Hoechst nuclear staining. Levels of bcl-2, bcl-x, bax, p53 and p21 were determined by Western blotting, and celi cycle analysis was determined by flow cytometry. RESULTS: IL-1 increased ceramide accumulation in a time-dependent and concentration-dependent manner with a peak response at 4 h, with [IL-1] = 30 ng/ml. Neither IL-1 nor ceramide induced apoptosis in EEC, but they increased bcl-2 levels and decreased bax and p21 levels without affecting bcl-x and p53 levels. They also caused a slight but significant increase in the G2/M phase. These data suggest a role for ceramide in IBD and suggest a possible mechanism for the enhanced tumorigenic activity in IBD patients.