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• W2077032767 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 α-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of α-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 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 α-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of α-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. α-difluoromethylornithine fetal bovine serum Dulbecco's modified Eagle's medium glutathione S-transferase green fluorescent protein guanosine 5′-3-O-(thio)triphosphate internal ribosome entry sequence p21-activated kinase protein kinase N formin-related diaphanous protein Cell migration maintains the organization and integrity of the mucosa of the gastrointestinal tract (1Edelman G.M. Dev. Dyn. 1992; 192: 2-10Crossref Scopus (106) Google Scholar). New cells migrate from the crypts of the small intestine onto the villi and differentiate as they move toward the tips. Mature cells are sloughed into the lumen and replaced from below. The damaged mucosa rapidly repairs itself through two basic processes (2Siley W. Ito S. Annu. Rev. Physiol. 1985; 47: 217-229Crossref PubMed Google Scholar). The early phase consists of the loss of damaged cells and the migration of remaining viable cells over the denuded lamina propria. This process of early mucosal restitution rapidly seals the wound and re-establishes the barrier to luminal contents (3Moore R. Carlson S. Madara J.L. Lab. Invest. 1989; 60: 227-284Google Scholar). The later phase of mucosal repair is the replacement of lost cells by mitosis and does not take effect until 24 h or so after injury. We have examined the early phase and the process of cell migration using IEC-6 cells, a non-transformed, putative crypt cell line derived from adult rat intestine and originally described by Quaroni et al. (4Quaroni A. Wands J. Trelstad R.L. Isselbacher K.J. J. Cell Biol. 1979; 80: 248-265Crossref PubMed Scopus (680) Google Scholar). This model resembles the early phase of mucosal healing in the gastrointestinal tract in that cell migration is independent from DNA synthesis, has a complete dependence on actin polymerization (5McCormack S.A. Viar M.J. Johnson L.R. Am. J. Physiol. Gastrointest. Liver Physiol. 1992; 263: 426-435Crossref PubMed Google Scholar), and depends on polyamines (6McCormack S.A. Viar M.J. Johnson L.R. Am. J. Physiol. Gastrointest. Liver Physiol. 1993; 264: 367-374Crossref PubMed Google Scholar). Cell migration requires an intact and functioning cytoskeleton, which for the most part consists of filamentous or F-actin, tubulin, and intermediate fibers. F-actin is formed by the polymerization of 420-kDa monomers termed G-actin. These filaments of actin with their associated binding proteins make up the actin cortex, a dense network just inside the inner surface of the plasma membrane (7Abraham V.C. Krishnamurthi V. Taylor D.L. Lanni F. Biophys. J. 1991; 77: 1721-1732Abstract Full Text Full Text PDF Scopus (214) Google Scholar). Long filaments of actin traverse the cell as stress fibers, and short filaments extend into the lamellipodia that are prominent during migration (8Pantoloni D. Le Clainche C. Carlier M.F. Science. 2001; 292: 1502-1506Crossref PubMed Scopus (565) Google Scholar). Focal adhesions provide the necessary attachments to the substrate and via the stress fibers allow the cytoskeleton to exert force on the extracellular matrix (9Beningo K.A. Dembo M. Kaverina I. Small J.V. Wang Y. J. Cell Biol. 2001; 153: 881-882Crossref PubMed Scopus (601) Google Scholar). Cells initiate migration in response to receptor signaling via integrins and the extracellular matrix (10Göke M. Zuk A. Podolsky D.K. Am. J. Physiol. Gastrointest. Liver Physiol. 1996; 271: 729-740Crossref PubMed Google Scholar) or in response to soluble factors (11Polk D.B. Gastroenterology. 1998; 114: 493-502Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Soluble factor and integrin signaling is relayed to the cytoskeleton by signal transduction pathways involving a subgroup of the Ras superfamily of small GTP-binding proteins (12Nobes C.D. Hall A. Cell. 1995; 8: 53-62Abstract Full Text PDF Scopus (3747) Google Scholar). The Rho (forRas homology) GTPases consist of three major types of small (21 kDa) proteins that bind and hydrolyze GTP. Their intrinsic GTPase activity is controlled by guanine nucleotide exchange factors and GTPase-activating proteins, known as GAPs. Rho guanine nucleotide dissociation inhibitor is an inhibitory guanine nucleotide exchange factor that prevents the dissociation of guanosine diphosphate from Rho as well as from Rac and Cdc42, the two other members of this family (13Fukumoto Y. Kaibuchi K. Hori Y. Fujioka H. Araki S. Ueda T. Kikuchi A. Takai Y. Oncogene. 1990; 5: 1321-1328PubMed Google Scholar, 14Mizuro T. Kaibuchi T. Yamamoto M. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Soc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (169) Google Scholar). Rho and Rac regulate the polymerization of actin to produce stress fibers and lamellipodia, respectively (15Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3843) Google Scholar, 16Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar). Cdc42 has been shown to be responsible for the formation of filopodia (12Nobes C.D. Hall A. Cell. 1995; 8: 53-62Abstract Full Text PDF Scopus (3747) Google Scholar). In NIH 3T3 fibroblasts, the Rho GTPases can be activated sequentially in that activation of Cdc42 activates Rac, which in turn activates Rho (12Nobes C.D. Hall A. Cell. 1995; 8: 53-62Abstract Full Text PDF Scopus (3747) Google Scholar). Thus, there appears to be a mechanism for the coordinated regulation of the cytoskeleton through activation of the Rho GTPases. Our laboratory has been interested in the cellular actions of polyamines, which are involved in many aspects of membrane function including stability, Ca2+ homeostasis, and ion transport (17Schuber F. Biochem. J. 1989; 260: 1-10Crossref PubMed Scopus (406) Google Scholar). Polyamines are also involved in the organization of the cytoskeleton and cell migration. Polyamine-deficient Chinese hamster ovary cells lack actin filaments and microtubules unless polyamines are supplied exogenously (18Pohjanpelto P. Virtanen I. Holtta E. Nature. 1981; 293: 475-477Crossref PubMed Scopus (124) Google Scholar). Inhibitors of polyamine synthesis prevent concanavalin A-induced expression of α-tubulin and β-actin mRNAs in mouse splenocytes (19Kaminska B. Kazmareck L. Grzelakowska B. FEBS Lett. 1992; 304: 198-200Crossref PubMed Scopus (39) Google Scholar). In vitro, polyamines stimulate the rapid polymerization of G-actin and formation of bundles from F-actin, indicating a possible direct effect of polyamines on cytoskeletal organization (20Grant N.J. Orial-Audit C. Eur. J. Cell Biol. 1983; 30: 67-73PubMed Google Scholar). We have shown that the early phase of mucosal healing, which is due to cell migration, requires polyamines (21Wang J.-Y. Johnson L.R. Am. J. Physiol. Gastrointest. Liver Physiol. 1990; 259: 584-592Crossref PubMed Google Scholar, 22Wang J.-Y. Johnson L.R. Gastroenterology. 1991; 100: 333-343Abstract Full Text PDF PubMed Scopus (0) Google Scholar) and that polyamine depletion inhibits migration in the IEC-6 cell model and leads to numerous alterations in the cytoskeleton. When cells were depleted of polyamines with α-difluoromethylornithine (DFMO),1 which inhibits ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, there was a significant decrease in actin stress fibers and a corresponding increase in the density of the actin cortex (23McCormack S.A. Wang J.-Y. Johnson L.R. Am. J. Physiol. Cell Physiol. 1994; 267: C715-C722Crossref PubMed Google Scholar). There was also a redistribution of tropomyosin from stress fibers to the actin cortex. Additional changes in response to polyamine depletion included a marked reduction in the formation of lamellipodia and a dissociation of actin from nonmuscle myosin II (24Wang J.-Y. McCormack S.A. Johnson L.R. Am. J. Physiol. Gastrointest. Liver Physiol. 1996; 270: G355-G362Crossref PubMed Google Scholar). Although no changes occurred in the absolute amounts of G- and F-actin, the association of actin with the sequestering protein thymosin β4 was inhibited (25McCormack S.A. Ray R.M. Blanner P.M. Johnson L.R. Am. J. Physiol. Cell Physiol. 1999; 276: C459-C468Crossref PubMed Google Scholar). All of the alterations in the cytoskeleton as well as the inhibition of migration caused by DFMO were prevented if exogenous polyamines were supplied in the presence of DFMO. Santos et al. (26Santos M.F. McCormack S.A. Guo Z. Okolicany J. Zheng Y. Johnson L.R. Tigyi G. J. Clin. Invest. 1997; 100: 216-225Crossref PubMed Scopus (142) Google Scholar) demonstrate that Rho was required for the actual migration of IEC-6 cells after wounding or in response to growth factor stimulation. Migration was inhibited after microinjection of Rho guanine nucleotide dissociation inhibitor Clostridium botulinum C3 ADP-ribosyltransferase toxin or Rho T19N, a dominant negative form of RhoA. Inactivation of Rho by these means not only inhibited migration but also altered the cytoskeleton in ways that were identical to those produced by polyamine depletion. With this in mind we recently examined the effects of polyamine depletion on RhoA (27Ray R.M. Patel A. Viar M.J. McCormack S.A. Zheng Y. Tigyi G. Johnson L.R. Gastroenterology. 2002; 123: 196-205Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We found that DFMO caused a significant decrease in RhoA levels in the cytoplasm and membranes of IEC-6 cells. This decrease was due to an approximate 50% inhibition in RhoA synthesis. Neither the half-life of RhoA nor the level of RhoA mRNA was affected. Constitutively active HA-V14 RhoA cells migrated much more rapidly than vector-transfected cells, and dominant negative HA-N19-RhoA cells exhibited almost no motility. Surprisingly, the depletion of polyamines almost totally inhibited the migration of the cells expressing constitutively active Rho. Polyamine depletion did not affect the activity of RhoA in the HA-V14-RhoA cells but inhibited it dramatically in the vector-transfected cells (27Ray R.M. Patel A. Viar M.J. McCormack S.A. Zheng Y. Tigyi G. Johnson L.R. Gastroenterology. 2002; 123: 196-205Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). After the loss of polyamines, constitutively active HA-V14-RhoA cells had fewer stress fibers and took on the appearance of the HA-N19-RhoA cells and the polyamine-depleted wild type cells. Thus, although RhoA activity is essential for the migration of intestinal epithelial cells, it is not sufficient. And although polyamines are necessary for RhoA synthesis and activity, they are also required for an additional step either upstream or downstream from RhoA that results in cell migration (27Ray R.M. Patel A. Viar M.J. McCormack S.A. Zheng Y. Tigyi G. Johnson L.R. Gastroenterology. 2002; 123: 196-205Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Both Rac1 and Cdc42 are excellent candidates for additional steps in the process of cell motility to be regulated by polyamines. There is considerable signaling cross-talk between Rho, Rac, and Cdc42, and each of these molecules regulates some aspect of cytoskeletal transformation (12Nobes C.D. Hall A. Cell. 1995; 8: 53-62Abstract Full Text PDF Scopus (3747) Google Scholar, 16Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar). In fact Rac1 has also been shown to be involved in stress fiber formation (16Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3084) Google Scholar). In the current paper, we show that the activities of Rac and Cdc42, like that of RhoA, are polyamine-dependent. We demonstrate that the rates of migration of IEC-6 cells transfected with constitutively active Rac or Cdc42 are significantly more rapid than those of the corresponding wild type cells or those transfected with empty vectors. We also demonstrate that Rac1 activation leads to RhoA and Cdc42 activation independent of polyamines, which explains the restoration of the cytoskeleton and migration rate in constitutively active Rac1 cells depleted of polyamines. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Media and other cell culture reagents were obtained from Invitrogen. Dialyzed fetal bovine serum (FBS) and other biochemicals were purchased from Sigma. Plasmids, pMX-IRES-GFP-V12-Rac1, pMX-IRES-GFP-N17-Rac1, pMX-IRES-GFP-F28L-Cdc42, pMX-IRES-GFP-N17-Cdc42, and pMX-IRES-GFP (vector) were used for the transfection experiments. FuGENE™ 6 transfection reagent was a gift from Roche Diagnostics. The primary antibodies, affinity-purified mouse monoclonal antibodies against RhoA and affinity-purified rabbit polyclonal antibodies against Rac1 and Cdc-42, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Matrigel was obtained from Collaborative Research Inc. (Bedford, MA). DFMO was the kind gift of Ilex Oncology Inc. (San Antonio, TX). The IEC-6 cell line was purchased from the American Type Culture Collection at passage 13. IEC-6 cells originated from intestinal crypt cells as judged by morphological and immunological criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells. Stock cell cultures were maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS, 10 μg insulin, and 0.05 mg of gentamicin sulfate/ml. The flasks were incubated at 37 °C in a humidified atmosphere of 90% air, 10% CO2. Stock cells were passaged once a week at 1:20; medium was changed 3 times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative. The general protocol for the experiments and the methods used were similar to those described previously (27Ray R.M. Patel A. Viar M.J. McCormack S.A. Zheng Y. Tigyi G. Johnson L.R. Gastroenterology. 2002; 123: 196-205Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In brief, IEC-6 cells were plated at 6.25 × 104 cells/cm2 in DMEM supplemented with 5% dialyzed FBS, 10 μg of insulin, and 50 μg of gentamicin sulfate/ml (DMEM/dialyzed FBS, control) or in DMEM/dialyzed FBS containing 5 mm DFMO or DFMO plus 10 μm putrescine. Cells were grown at 37 °C in a humidified atmosphere of 90% air, 10% CO2. They were fed every other day and serum-starved during the 24 h before harvesting or cell migration assay. Previously we have shown that maximal depletion of polyamines occurs after 4 day of DFMO treatment (6McCormack S.A. Viar M.J. Johnson L.R. Am. J. Physiol. Gastrointest. Liver Physiol. 1993; 264: 367-374Crossref PubMed Google Scholar, 28Pfeffer L.M. Yang C.H. Murti A. McCormack S.A. Viar M.J. Ray R.M. Johnson L.R. J. Biol. Chem. 2001; 276: 45909-45913Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and others also report an identical pattern of polyamine depletion (29Patel A.R. Li J. Bass B.L. Wang J.-Y. Am. J. Physiol. Cell Physiol. 1998; 275: C590-C598Crossref PubMed Google Scholar). In IEC-6 cells putrescine was completely absent after 6 h of exposure to DFMO and after 24 h to spermidine, and 40% of the spermine remained after 4 days. DFMO inhibits ornithine decarboxylase, preventing the synthesis of putrescine; therefore, exogenous addition of putrescine along with DFMO restores endogenous spermidine and spermine. Thus, cells grown in DFMO plus putrescine are an important control showing that effects are due to the depletion of polyamines and not to pharmacological effects of DFMO. Cells were grown in control medium for 4 days or in medium to which DFMO was added at the time of plating for 4 days or on days 1, 2, and 3 during feeding for 3-, 2-, and 1-day treatments, respectively. Another group of cells was grown in DFMO plus putrescine for 4 days. Cells were serum-starved for 24 h before the experiment (during day 4). For the cell migration assay, plates containing a confluent monolayer of cells were marked in the center by drawing a line along the diameter of the plate with a black marker. Wounding of the monolayer was carried out perpendicular to the marked line using a gel-loading micro-tip. Plates were washed, and the area of migration was photographed with a video camera system using NIH Image software (Version 1.58*) at the intersection of the marked line and wound edge at 0 h (WW0) and at desired time intervals (WWT). Cell migration was calculated as wound width covered at time t (WW0 − WWT) and expressed as % control. Each experiment was carried out three times in duplicate, and each plate was wounded twice. Therefore, n was considered to be 6 even though results are the means of 12 observations. Biological activities of RhoA, Rac1, and Cdc42 proteins were assayed by pull-down assays following the method of Kranenburg et al. (30Kranenburg O. Poland M. vanHorck F.P.G. Drechsel D. Hall A. Moolenaar W. Mol. Biol. Cell. 1999; 10: 1851-1857Crossref PubMed Scopus (275) Google Scholar). GST-Rho-kinase, GST-mDia, GST-PKN, and GST-PAK fusion proteins were prepared by lysing the bacteria (Escherichia coliBL21-DE-3pLysE strain transformed with GST fusion protein construct) in a buffer containing 1% Nonidet P-40, 50 mm Tris, pH 7.4, 100 mm NaCl, 5 mm MgCl2, and 10% glycerol supplemented with protease inhibitors. The lysates were then sonicated and cleared by centrifugation at 10,000 × gfor 15 min. The fusion proteins were recovered by the addition of glutathione beads to the supernatants. The beads were washed three times in cell lysis buffer and resuspended before the addition of the cell lysates (200 μg of protein for RhoA and Cdc42 and 100 μg of protein for Rac1). After 1 h of tumbling at 4 °C, beads were washed with lysis buffer, and the amounts of RhoA, Rac1, and Cdc42 proteins bound to GST fusion proteins were analyzed by SDS-PAGE and Western blot using RhoA-, Rac1-, and Cdc42-specific antibodies. Protein (20 μg) from each sample was resolved by SDS-PAGE to determine the levels of RhoA, Rac1, and Cdc42 proteins. Protein was separated on 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. The membranes were then probed with an antibody directed against one of the proteins (RhoA, Rac1, Cdc42, or actin). Immunocomplexes were visualized by the enhanced chemiluminescence detection system and quantitated by densitometric scanning. IEC-6 cells were transfected with constitutively active and dominant negative Rac1 and Cdc42 as well as vector. DNA was prepared by using a Qiagen (endotoxin-free) plasmid preparation kit. Cells were grown as mentioned earlier to 70–80% confluence in 60-mm culture dishes. FuGENE 6 reagent was mixed with constitutively active (pMX-IRES-GFP-V12-Rac1), dominant negative (pMX-IRES-GFP-N17-Rac1), constitutively active (pMX-IRES-GFP-F28L-Cdc42), dominant negative (pMX-IRES-GFP-N17-Cdc42), or vector (pMX-IRES-GFP) DNA (3 μl: 2 μg) in serum-free medium to a total volume of 100 μl and incubated 15 min at room temperature. The reaction mixture was added dropwise onto monolayers of cells containing 4.0 ml of serum-free medium and further incubated for 12 h at 37 °C. Cells were fed after 12 h with fresh medium. The limiting dilution technique was used to select stable clones. Selected clones were grown to confluence, and the characterization of clones was carried out by measuring cell migration and the direct visualization of green fluorescence protein (GFP). Transfected cells were grown for 4 days, trypsinized, replated in 35-mm dishes (each containing a matrigel-coated glass coverslip), and allowed to attach and spread. Cells were fixed with 4.0% formaldehyde, washed with DPBS, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 3% BSA for 20 min. Cell monolayers were stained with Rac1 antibody for 1 h followed by goat anti-rabbit IgG-Texas Red (Chemicon Intl., Temecula, CA) at a 1:60 dilution for 1 h. Images were captured by digital confocal microscopy and processed with NIH image. Control, DFMO, and DFMO plus putrescine-treated (4 day) V12- Rac1, F28L-Cdc42, and vector-transfected cells were plated in 35-mm dishes (each containing a matrigel-coated glass coverslip) and allowed to attach and spread for 24 h. Cells were fixed with 4.0% formaldehyde, washed with DPBS, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 3% BSA for 20 min. Cell monolayers were stained with Texas-red conjugated phalloidin for 1 h. Images were captured by digital confocal microscopy and processed with NIH image. All data are expressed as means ± S.E. from six dishes. Autoradiographic results were repeated three times. Densitometry of Western blots was done by NIH image analysis. The significance of the differences was determined using Dunnett's multiple-range test (R), and values of p < 0.05 were considered significant. To study the effect of polyamine depletion on activities of Rho family GTPases, it is important to determine the relative amounts of activated RhoA, Rac1, and Cdc42 in normal IEC-6 cells. Whole cell extracts equivalent to 25–200 μg of protein were subjected to pull-down assay using either GST-PKN and GST-mDia (for RhoA) or GST-PAK (for Rac1 and Cdc42) protein. Pull-down samples along with 25 μg of whole cell extract was resolved by SDS-PAGE and analyzed by Western blot analysis. Confluent IEC-6 cells grown in control medium showed high levels of active Rac1 protein in as low as 25 μg of total protein (Fig. 1A). In contrast, a significant amount of active RhoA protein was detected only in 200 μg of total protein (Fig. 1B). Although active Cdc42 protein was evident in as low as 50 μg of total protein, the total amount of activated Cdc42 was lower than amounts of active Rac1 and RhoA (Fig.1C). Total protein levels of RhoA and Rac1 were comparable and significantly higher than that of Cdc42. Thus, in subsequent experiments we used 100 μg of total protein for Rac1 and 200 μg of total protein for the determination of RhoA and Cdc42 activity. Polyamine depletion by treatment of cells with DFMO for 4 days significantly decreased the activities of all three Rho GTPases (Fig.2). Active RhoA decreased to 35 or 20% of control levels depending on whether binding to mDia or to PKN was used in the pull-down assay, respectively. There was also a 40% decrease in the level of RhoA protein when cells were grown for 4 days in the presence of DFMO. PAK1 was used to pull down both active Rac1 and active Cdc42. Rac1 activity was reduced to 40% and Cdc42 activity was reduced to 23% that of control levels. There was no change in the level of Rac1 protein, but Cdc42 levels decreased to 45% of normal. In each case supplying putrescine along with DFMO during growth prevented the decrease (Fig. 2).Figure 2Polyamine depletion inhibits activation of RhoA, Rac1, and Cdc42 in IEC-6 cells. Cells were grown in the presence of 5 mm DFMO or DFMO plus 10 μmputrescine for 4 days. Equal amounts of protein (200 μg for RhoA and Cdc42 and 100 μg for Rac1) were used for the pull-down assays as described under “Experimental Procedures.” 25 μg of protein was used to determine the levels of total RhoA, Rac1, and Cdc42 proteins. Western blot (WB) analysis using RhoA-, Rac1-, and Cdc42-specific antibodies was carried out. A, active RhoA protein bound to GST-mDia. B, active RhoA protein bound to GST-PKN. C, RhoA protein levels in whole cell extract.D, active Rac1 protein bound to GST-PAK. E, Rac1 protein levels in whole cell extract. F, active Cdc42 protein bound to GST-PAK. G, Cdc42 protein levels in whole cell extract. H, actin levels in whole cell extract. Representative Western blots and densitometry readings from three observations are shown.View Large Image Figure ViewerDownload (PPT) As shown in Fig. 3A the activities of RhoA, Rac1, and Cdc42 decreased little when cells were treated with the DFMO for 1, 2, and 3 days. There were dramatic decreases in the activities of all 3 to below 50% that of control on the 4th day of DFMO treatment. The levels of RhoA and Cdc42 proteins followed a similar pattern, and there was no significant decrease in Rac1 protein (Fig. 3A). Fig. 3B illustrates the means of the quantified data of the activities of all three Rho GTPases. As in the studies depicted in Fig. 2, the addition of putrescine to the incubation medium containing DFMO prevented all of the effects of ornithine decarboxylase inhibition. Clones of IEC-6 cells transfected with vector and with dominant negative and constitutively active genes of Rac1 and Cdc42 were characterized by the direct visualization of green fluorescent protein and by determining rates of cell migration. Digital confocal micrographs of one clone of each of the five types are shown in Fig.4. In each case, all cells expressed GFP, indicating successful transfection and clonal selection. Transfection with dominant negative constructs of both Rac1 and Cdc42 inhibited cell migration compared with vector-transfected cells, whereas transfection with constitutively active constructs significantly increased the rates of migration (Fig. 5A). As shown in Fig. 5B, by 8 h vector-transfected cells had migrated to cover ∼40% of the original wound. This number was reduced to 25% in cells carrying dominant negative Rac1 and to 10% in cells with dominant negative Cdc42. On the other hand, cells transfected with constitutively active Rac1 covered more than 85% of the wound width and those with constitutively active Cdc42 had migrated over 60% of the original wound.Figure 5Rac1 and Cdc42 protein expression influences IEC-6 cell migration. A, cells transfected with vector (pMX-IRES-GFP), constitutively active (CA) Rac1 and Cdc42, and dominant negative (DN) Rac1 and Cdc42 were grown in DMEM, 5% FBS for 4 days. Confluent monolayers were wounded with a gel-loading tip in the center of plates marked to localize the wound site. Plates were photographed immediately to record the wound width (0 h), washed, and incubated with fresh serum free medium. Plates were photographed at the marked wound location after 10 h of incubation. A plate containing vector cells at 0 and 10 h is shown as a representative control. Representatives of three experiments are shown. B, quantitative analysis of migration showing wound width covered as compared with initial scratch size (0 h) using NIH image analysis. Values are the mean ± S.E. of six observat" @default.
• W2077032767 created "2016-06-24" @default.
• W2077032767 creator A5000496429 @default.
• W2077032767 creator A5020584779 @default.
• W2077032767 creator A5038876202 @default.
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• W2077032767 creator A5075201965 @default.
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• W2077032767 date "2003-04-01" @default.
• W2077032767 modified "2023-10-10" @default.
• W2077032767 title "The Requirement for Polyamines for Intestinal Epithelial Cell Migration Is Mediated through Rac1" @default.
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