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Am J Physiol Gastrointest Liver Physiol 292: G983-G995, 2007. First published December 14, 2006; doi:10.1152/ajpgi.00356.2006
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MUCOSAL BIOLOGY

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

Ramesh M. Ray, Huazhang Guo, Minesh Patel, Shi Jin, Sujoy Bhattacharya, and Leonard R. Johnson

Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 2 August 2006 ; accepted in final form 9 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell-permeable, and broad-spectrum serine/threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr18/Ser19-MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr18- and pSer19-MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr18-MRLC without any effect on nuclear pSer19-MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyamine-depleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.

putrescine; {alpha}-difluoromethylornithine; F-actin; focal adhesion kinase; paxillin

POLYAMINES [spermidine, spermine, and their diamine precursor putrescine (PUT)] are found in all tissues of almost all living species. These polyamines are organic, aliphatic, cationic amines. At physiological pH, they bind to proteins, DNA, RNA, and other negatively charged molecules to regulate diverse biological effects. The first rate-limiting step in polyamine synthesis is the production of PUT from the amino acid ornithine. This reaction is catalyzed by ornithine decarboxylase (ODC), which has one of the shortest half-lives of any mammalian enzyme and is present at extremely low levels in quiescent cells. Its activity is highly regulated to accommodate cellular function (6, 7). In our previous studies (17, 37, 46–49), we showed that the early phase of mucosal healing due to cell migration requires polyamines and that polyamine depletion by means of treatment with {alpha}-difluoromethylornithine (DFMO), a specific inhibitor of ODC, inhibits migration in a rat stress ulcer model. Subsequently, we showed that the small intestinal epithelial crypt cell line IEC-6 could be used as an in vitro model to study the process of cell migration involved in the early restitution of mucosal erosions (16). Recently, we found that polyamine depletion inhibits the activities of RhoA and Rac1 by sequestration of Rac1 and RhoA to the nucleus as well as perinuclear regions (29, 30, 44).

Myosins are a superfamily of actin-activated Mg2+-ATPases that convert the energy of ATP hydrolysis into force between actin and myosin filaments. Myosin II consists of two heavy chains and two pairs of light chains, one essential and one regulatory. Myosin II plays important roles in many contractile-like cell functions including cell migration, adhesion, and retraction (36, 42). Myosin II is activated by myosin regulatory light chain (MRLC) phosphorylation on Ser19 (and Thr18), whereas MRLC dephosphorylation leads to myosin II inactivation (4, 5). It has been suggested that phosphorylated (p)MRLC might stabilize actomyosin, thus inhibiting the turnover of stress fibers (21, 43).

An interesting phenomenon in polyamine-depleted IEC-6 cells is the high level of serine/threonine phosphorylation of proteins due to the inhibition of serine/threonine protein phosphatase [protein phosphatase 2A (PP2A)] (28). We used staurosporine (STS), a broad-spectrum serine/threonine kinase inhibitor, to study its effect on migration in these cells. Another interesting characteristic of polyamine-depleted IEC-6 cells is the formation of a prominent thick F-actin cortex (18). The mechanism of formation of this cortex is unknown. In this study, we found that pMRLC localized with the F-actin cortex at the cell periphery in polyamine-depleted cells. We also found that STS caused dephosphorylation of MRLC, dissolved thick cortical F-actin, increased Rac1 activity, and increased cell migration. We envisage that polyamines may regulate MRLC dephosphorylation and the turnover of cytoskeletal components essential for the activation of Rac1 and migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The IEC-6 cell line (CRL-1592) was obtained from the American Type Culture Collection (Manassas, VA) at passage 13. This line was derived from normal rat intestinal crypt cells and was developed and characterized by Quaroni et al. (26). The cells are nontumorigenic and retain the undifferentiated character of epithelial stem cells. Cell culture medium, FBS, dialyzed FBS (1,000 mol. wt. cutoff), trypsin-EDTA, antibiotics, and insulin were obtained from GIBCO-BRL (Grand Island, NY). Mammalian protein extraction reagent (M-PER) and the bicinchoninic acid protein assay reagent kit were purchased from Pierce (Rockford, IL). Antibodies for pMRLC (Thr18/Ser19, Thr18, and Ser19) and total MRLC were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Rac1 and anti-focal adhesion kinase (FAK) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-paxillin antibody was purchased from Cell Signaling Technologies (Beverly, MA). Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies and rhodamine-phalloidin were purchased from Molecular Probes (Eugene, OR). 4',6-Diamidino-2-phenylindole (DAPI) and mouse anti-beta-actin were purchased form Sigma (St. Louis, MO). The enhanced chemiluminescence Western blot detection system was purchased from DuPont-NEN (Boston, MA). SDS sample loading buffer and the electrophoresis apparatus were obtained from Bio-Rad (Hercules, CA). DFMO was a gift from ILEX Oncology (San Antonio, TX). STS, cytochalasin D (CD), NSC-23766, and ML7 were purchased from Calbiochem and EMD Biosciences (La Jolla, CA). The Cell Death Detection ELISA Plus kit was purchased from Roche (Nutley, NJ). All other chemicals were of the highest purity commercially available.

Cell culture. The stock cell culture was maintained in DMEM supplemented with 5% FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate and incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were passaged once weekly at 1:20 dilution, and the medium was changed three times weekly. Cells were restarted from the original frozen stock after every seven passages.

General experimental protocols. The general protocol for the experiments and the methods used were similar to those described previously (30). In brief, IEC-6 cells were plated at ~6.25 x 104 cells/cm2 in control medium consisting of DMEM supplemented with 5% dialyzed FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate or in control medium containing 5 mM DFMO or DFMO + 10 µM PUT on day 0. Cells were grown at 37°C in a humidified atmosphere of 90% air-10% CO2. They were fed once on day 2 and serum starved on day 3. Polyamines were depleted by incubation in 5 mM DFMO. We (16) have previously reported that maximal polyamine depletion occurs after 4 days of treatment with 5 mM DFMO. Within 6 h of DFMO treatment, PUT was undetectable, spermidine was absent after 24 h, and 40% of spermine remained after 4 days. One group of cells was given exogenous PUT (10 µM) in addition to DFMO. This group acted as a control to indicate that all results were due to the depletion of polyamines and not to DFMO itself. Throughout this article, the term polyamine depletion and cells grown with DFMO are used interchangeably.

Quantitative DNA fragmentation ELISA. Cells were grown in 24-well culture plates for both DNA fragmentation ELISA and protein determination. On day 4, after being treated with different concentrations of STS for 3 h, cells were washed two times with Dulbecco's PBS (dPBS). Cells were lysed and centrifuged to remove nuclei. An aliquot of the nuclei-free supernatant was placed in streptavidin-coated wells and incubated with anti-histone-biotin antibody and anti-DNA peroxidase-conjugated antibody for 2 h at room temperature. After the incubation, the sample was removed, and the wells were washed three times with incubation buffer. Following the addition of 100 µl of the substrate 2,2'-azino-di(3-ethylbenzthiazolin-sulfonate), absorbance read at 405 nm using a plate reader. Results were expressed as absorbance at 405 nm·min–1·mg protein–1.

Attachment and spreading assay. The attachment and spreading assay was performed as previously described (19). Cells were harvested by trypsinization, counted by a Beckman Coulter counter, and resuspended in serum-free DMEM. Cells were conditioned in serum-free medium for 1 h and then plated on a 24-well plate (1 x 106 cells/well) in the presence and absence of 5 nM STS.

Attachment assay. After 3- or 6-h incubations at 37°C, nonadherent cells were removed by washing three times with dPBS, and attached cells were stained with 0.5% crystal violet dissolved in 20% methanol for 15 min. Plates were then washed three times with dPBS, and the stain was extracted with 0.1 M sodium citrate (pH 4.2). The amount of the stain was analyzed by measuring the optical density (OD) at 590 nm.

Spreading assay. Following incubations for 0.5, 1, and 3 h, plates were placed on ice, and cells were washed three times with ice-cold dPBS, fixed with 3.7% formaldehyde in PBS for 15 min, stained with rhodamine-phalloidin for 20 min, and washed three times with PBS. Cells were photographed using a Nikon Diaphot inverted microscope.

Migration assay. On day 4, six-well plates containing a confluent monolayer of cells grown under control, DFMO-containing, and DFMO + PUT-containing media were marked in the center by drawing a line along the diameter of the plate with a black marker. Wounding of the monolayer was performed perpendicular to the marked line using a gel-loading microtip. Plates were washed, and the respective medium with or without 5 nM STS was added. The area of migration was photographed with a charge-coupled device camera using NIH Image software (version 1.58) at the intersection of the marked line and the wounded edge at 0 h (WW0) and at 6 h (WW6). Cell migration was calculated as wound area covered at 6 h (WW0 – WW6). Each experiment was conducted three times in duplicate, and each plate was wounded twice. Therefore, the number of experiments conducted was considered to be 6 even though the results were the means of 12 observations.

Rac1 activation assay. A confluent monolayer of cells was wounded by a multitooth comb on day 4. Plates were washed, the corresponding medium with or without 5 nM STS was added, and plates were further incubated for 3 h. Rac1 activity was analyzed using a pulldown assay according to the method of Kranenburg et al. (11). Glutathione S-transferase (GST)-p21-activated kinase (PAK) fusion protein was prepared by lysing the bacteria (Escherichia coli BL21-DE-3pLysE strain transformed with GST-PAK plasmid 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 bacterial cell lysate was sonicated and clarified by centrifugation at 10,000 g for 15 min. The fusion protein was recovered by the addition of glutathione-agarose beads to the supernatant. Beads were washed three times in cell lysis buffer and resuspended before the addition of IEC-6 cell lysates (100 µg). After 1 h of being tumbled at 4°C, beads were washed with lysis buffer, and the amount of Rac1 protein bound to GST-PAK protein was analyzed by performing SDS-PAGE and Western blot analysis using a Rac1-specific antibody.

Western blot analysis. For the Western blot analysis of pMRLC and MRLC, cells were washed twice with ice-cold PBS and harvested in ice-cold cell lysis buffer (M-PER supplemented with protease and phosphatase inhibitor) by scraping with a rubber policeman. Cell lysates were centrifuged at 10,000 g for 10 min. The supernatant was used for protein measurement. The amount of protein was determined by following the bicinchoninic acid protein assay reagent manufacturer's instructions. Proteins were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were then probed with the appropriate primary and secondary antibodies. Immunocomplexes were visualized using enhanced chemiluminescence detection reagent.

Immunocytochemistry. Cells were seeded onto coverslips or 24-well plates coated with poly-L-lysine (BD Labware, Bedford, MA) and grown as described previously. Cells were fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and washed with PBS. Coverslips were blocked with 3% BSA in PBS for 20 min and then incubated with primary antibody for 2 h. Coverslips were then washed with 0.1% BSA in PBS for 20 min, followed by a 2-h incubation with an appropriate fluorescent dye-conjugated secondary antibody. Coverslips were mounted on glass slides and observed using a Nikon Diaphot inverted microscope.

Statistics. Data are means ± SE. All experiments were repeated three times (n = 3). Western blots are representative of three experiments. Student's t-test (for samples with unequal variances) or one-way ANOVA (for samples with equal variances) with appropriate post hoc testing was used to determine the significance of the differences between means. P < 0.05 was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
STS and apoptosis. STS inhibits several serine/threonine kinases that regulate migration including calmodulin kinase, myosin light chain kinase (MLCK), and PKC (10, 45). In IEC-6 cells, 1.0 µM STS induced apoptosis in control as well as polyamine-depleted cells (14). Polyamine depletion has been shown to inhibit PP2A and migration in IEC-6 cells (28, 30). Therefore, to study the effect of STS on events related to migration, it is essential to determine the concentration of STS that does not induce apoptosis. We found that 100 nM or higher concentrations of STS significantly increased DNA fragmentation (Fig. 1A). However, 10 nM STS did not induce apoptosis as evident by levels of DNA fragmentation similar to untreated cells. Therefore, we chose 5 nM STS for our migration experiments.


Figure 1
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  		Fig. 1. IEC-6 cells were grown in control medium (DMEM) supplemented with 5% dialyzed FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate for 3 days. After serum starvation for 24 h, they were treated with indicated concentrations of staurosporine (STS) for 7 h. Apoptosis was measured by DNA fragmentation assay (A). Cells were harvested, conditioned, and plated in serum-free DMEM with or without STS for the attachment assay as described in MATERIALS AND METHODS. The number of attached cells was determined by crystal violet staining after 3 and 6 h (B). IEC-6 cells were allowed to attach for the indicated times followed by the removal of floating cells. Cells were fixed, permeabilized, and stained for F-actin using rhodamine-conjugated phalloidin as a probe. Images were captured using a Nikon UV fluorescence microscope (C). a–f, Cells treated for 1, 2, or 3 h with or without 5 nM STS. g and h, Enlarged views of F-actin staining in untreated (UT) and 5 nM STS-treated cells, respectedly. Values are means ± SE. Images are representative of 3 experiments. OD, optical density. *P < 0.05, significantly different from the untreated group.

 
STS and attachment and spreading. Before assessing cell migration, we evaluated the effect of STS on cell attachment and spreading in IEC-6 cells. STS significantly increased the attachment of cells in a time-dependent manner compared with untreated cells (Fig. 1B). To measure spreading, cells were allowed to attach for 1 h followed by the removal of floating cells, followed by a further incubation for 3 h. The incubation of cells with STS increased attachment as judged by brightly stained cells (Fig. 1C, a and d), which further confirmed our data shown in Fig. 1B. In addition, attached cells spread significantly more compared with untreated cells (Fig. 1Cf). STS-treated cells began to spread as early as 1 h postplating, which was evident by the increases in the numbers of diffusely stained cells. At the time point when untreated cells began to spread, most of the STS-treated cells had spread, as indicated by the loss of brightly stained cells (Fig. 1C, c and f). The enlarged view of F-actin staining showed that STS (5 nM, 3 h) increased spreading with an organized F-actin structure compared with untreated cells (Fig. 1C, g and h). These results indicate that STS enhances the attachment and spreading of cells and suggest that it may enhance migration.

STS, migration, and Rac activation. Since polyamine depletion decreased the migration of IEC-6 cells by decreasing Rac1 activity (29), we studied the effect of STS on the migration of cells grown under control, DFMO, and DFMO + PUT conditions. The results in Fig. 2A show phase-contrast images of wound areas at 0 and 7 h following scratching. Cells treated with STS significantly covered the wound areas compared with those from untreated groups. STS increased the number of lamellipodia as evidenced by the elongated cell morphology compared with untreated cells. Consistent with our previous report (16), the results in Fig. 2B show that DFMO, an inhibitor of ODC, significantly inhibited migration, and supplementation of DFMO-containing medium with PUT, the product of the ODC-catalyzed reaction, restored migration to control levels. STS at 5 nM significantly increased migration in all three groups. STS-induced migration of polyamine-depleted cells was more or less similar to the untreated control and DFMO + PUT groups (Fig. 2B).


Figure 2
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  		Fig. 2. STS and migration. IEC-6 cells grown under control, {alpha}-difluoromethylornithine (DFMO), and DFMO + putrescine (PUT) conditions as described in MATERIALS AND METHODS were wounded, washed to remove damaged cells, and incubated with the respective serum-free media with or without 5 nM STS. Plates were photographed to capture the wound width at 0 and 7 h (A). Migration was calculated by measuring the area covered by the monolayer and expressed in µm2 (B). *P < 0.05, significantly different from the respective untreated group.

 
The results in Fig. 3A show that Rac1 protein levels were unaltered in control and polyamine-depleted cells as well as those treated with STS. Rac1 activity was significantly low in cells grown in DFMO-containing medium compared with control cells. However, STS significantly increased the Rac1 activity of cells grown in DFMO-containing medium and restored it to control levels within 1 h (Fig. 3A). In addition, Rac1 inhibition using NSC-23766 (120 µM), a Rac1-specific GEF Tiam1 inhibitor, totally abolished the stimulatory effect of STS and significantly inhibited the migration of polyamine-depleted cells (Fig. 3B). In IEC-6 cells, dominant negative Rac1 (DN-Rac1) expression significantly decreased migration compared with cells transfected with vector (Fig. 3C). Furthermore, polyamine depletion decreased the migration of vector-transfected cells to levels more or less similar to those of DN-Rac1-transfected cells grown under control conditions. STS significantly increased the migration of cells transfected with vector grown under control and DFMO conditions (Fig. 3C). However, STS failed to increase the migration of cells transfected with DN-Rac1 and grown under either control or DFMO conditions (Fig. 3C). We (8) have shown recently that 120 µM NSC-23766 and DN-Rac1 expression decreased apoptosis in IEC-6 cells by preventing JNK activation. NSC-23766 (120 µM) treatment alone had no effect on apoptosis (89.7 ± 3.5%) compared with untreated cells (100 ± 4.3%). STS (100 nM) significantly increased apoptosis (257 ± 12.4%) compared with untreated controls (100 ± 4.3%). NSC-23766 significantly decreased STS-induced apoptosis from 257 ± 12.4% to 128 ± 3.0%. Thus, NSC-23766 (120 µM) does not have any toxic effect. These results indicate that STS-induced migration requires Rac1 activation. ML7, a specific inhibitor of MLCK, inhibited the migration of cells grown under control and DFMO conditions by 39 ± 6.8% and 37 ± 7.5%, respectively, compared with both untreated groups. These results indicate that both phosphorylation and dephosphorylation of MRLC regulate migration.


Figure 3
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  		Fig. 3. STS, Rac1, and migration. A: cells were grown under control and DFMO conditions in 100-mm plates. Serum-starved cells were scratched with a multitooth comb, and damaged cells were removed by washing plates. Cells were further incubated with the respective medium for 3 h, during which 5 nM STS was added to the DFMO-containing medium for the specified times. Cells were harvested and processed for the Rac1 activity assay as described in MATERIALS AND METHODS. B: cells grown in DFMO-containing medium were wounded as described for the migration assay and incubated in DFMO-containing medium with the indicated concentrations of STS or STS + NSC-23766 (a Rac1 inhibitor) for 6 h. Migration was then calculated. Values are means ± SE. *P < 0.05, column 2 significantly different from column 1 and column 3 significantly different from column 2. C: IEC-6 cells transfected with vector and dominant negative Rac1 (DN-Rac) were grown under control and DFMO conditions as described in MATERIALS AND METHODS. Cell monolayers were wounded, washed to remove damaged cells, and incubated with the respective-serum free media with or without 5 nM STS. Plates were photographed to capture wound widths at 0 and 6 h. Migration was calculated by measuring the area covered by the monolayer and expressed in µm2. *P < 0.05, significantly different from the respective untreated group.

 
MRLC phosphorylation. Since STS is known to inhibit serine/threonine phosphorylation and increased migration of polyamine-depleted cells, we predicted that phosphorylation and dephosphorylation of myosin II by STS might be linked to reorganization of the actin cytoskeleton. We studied the localization of pMRLC (pThr18/Ser19-MRLC) and actin in cells grown under control, DFMO, and DFMO + PUT conditions. The results in Fig. 4 show that pThr18/Ser19-MRLC colocalized with a thick actin cortex at the cell periphery in polyamine-depleted cells (Fig. 4, C and D). It is noteworthy that pMRLC also localized to the nucleus in all three groups. However, the intensity of nuclear pMRLC staining in polyamine-depleted cells was greater compared with control and DFMO + PUT-supplemented cells. Control cells and those grown with DFMO + PUT showed less pMRLC at the cell periphery and contained stress fibers scattered throughout the cells (Fig. 4, A, B, E, and F). Since pThr18/Ser19-MRLC localized with stress fibers and in the nucleus, we investigated whether pThr18-MRLC and pSer19-MRLC had differential localizations using phosphorylation site-specific antibodies. pThr18-MRLC extensively localized with stress fibers; however, pSer19-MRLC localized in the nucleus of the cells (Fig. 5A). However, pThr18/Ser19-MRLC localized with stress fibers and in the nucleus. In addition to the localization of pSer19-MRLC in the nucleus, it appeared to associate with chromosomes during nuclear division as evident by the characteristic localization at equatorial plates in the nuclei of some cells undergoing mitosis (Fig. 5Ab, arrows). We also observed pSer19-MRLC localization with different stages of mitotic events, which was confirmed by its colocalization with DAPI (Fig. 5B). Thus mono- or diphosphorylated MRLC, depending on its localization, may have different functions in these cells.


Figure 4
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  		Fig. 4. Polyamine depletion and phosphorylated myosin regulatory light chain (pMRLC) (Thr18/Ser19) localization. IEC-6 cells were grown in control (A and B), DFMO (5 mM)-containing media (C and D), and DFMO + 10 µM PUT-containing media (E and F) for 3 days. After being serum starved for 24 h, cells were fixed and stained for pMRLC and F-actin. Arrowheads indicate thick cortical F-actin, and arrows indicate pMRLC around the cell periphery.

 

Figure 5
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  		Fig. 5. Localization of pThr18-, pSer19- and pThr18/Ser19-MRLC. A: IEC-6 cells grown under control conditions were washed with Dulbecco's PBS, fixed, permeablized, and incubated with pThr18- (a and b), pSer19- (c and d), or pThr18/Ser19-MRLC antibodies (e and f) for 2 h at room temperature. After being washed with Ca2+-free PBS, cells were stained with the respective secondary antibodies conjugated with Alexa Fluor 488 (AF-488) and rhodamine-conjugated phalloidin for 2 h. B: cells incubated with pSer19-MRLC antibody and rhodamine-conjugated phalloidin for 2 h were also stained with 4',6-diamidino-2-phenylindole (DAPI) for 30 min. Cells were washed and observed using a Nikon UV fluorescence microscope. Representative images from 3 independent experiments are shown.

 
MRLC phosphorylation and cytoskeletal structure. Since pThr18/Ser19-MRLC associates with the F-actin cortex in polyamine-depleted cells (Fig. 4) and STS increased migration (Fig. 2), we determined whether MRLC phosphorylation was associated with STS-induced reorganization of the actin cytoskeleton. The results in Fig. 6 clearly show that STS decreased the phosphorylation of MRLC, leading to a total loss of pMRLC localization at the cell periphery in all three groups of cells; however, nuclear pMRLC levels were not altered significantly (Fig. 6A). STS-induced extensive reorganization of the thick actin ring into stress fibers and the lamellipodia accompanied the loss of F-actin-associated pMRLC in polyamine-depleted cells (Fig. 6B). STS-induced spreading and lamellipodia formation accompanied loss of colocalization of F-actin and pMRLC, which was evident in the merged images (Fig. 6B). These results further confirm the localization of pMRLC to the cell periphery and nucleus in polyamine-depleted cells and that its dephosphorylation is associated with the loss of the F-actin cortex. The results in Fig. 7 show that pThr18/Ser19-MRLC levels were similar in control and DFMO + PUT-supplemented cells. However, STS significantly decreased the phosphorylation of MRLC in all three groups (Fig. 7). The levels of total MRLC were unchanged in all three groups. We also tested whether disruption of the actin cytoskeletal structure by cytochalasin D (CD) led to the dephosphorylation of MRLC. CD at 1.0 µM (3 h) disrupted F-actin stress fibers, leading to the formation of actin patches in control cells, and decreased the density of the actin cortex in polyamine-depleted cells (Fig. 8). However, CD had no dramatic effect on the localization of pMRLC at the cell periphery. These results suggest that dephosphorylation of MRLC plays an important role in the remodeling of the actin cytoskeleton and migration.


Figure 6
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  		Fig. 6. STS and pMRLC (Thr18/Ser19). A: IEC-6 cells were grown in control, DFMO (5 mM)-containing, and DFMO + 10 µM PUT-containing media for 3 days. After being serum starved for 24 h, monolayers were wounded. They were treated with STS for 3 h and fixed and stained with pThr18/Ser19-MRLC antibody. Cells were stained with secondary antibodies conjugated with AF-488 and observed using a Nikon UV fluorescence microscope. Representative images from 3 independent experiments are shown. B: IEC-6 cells were grown in medium containing 5 mM DFMO for 3 days. After being serum starved for 24 h, they were treated with STS for 3 h and fixed and incubated with pThr18/Ser19-MRLC antibody. Cells were stained with secondary antibodies conjugated with AF-488 and rhodamine-conjugated phalloidin and observed using a Nikon UV fluorescence microscope. Representative images from 3 independent experiments are shown.

 

Figure 7
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  		Fig. 7. STS and MRLC dephosphorylation. IEC-6 cells were grown in control (C), DFMO (5 mM)-containing (D), and DFMO + 10 µM PUT-containing (DP) media for 3 days. After being serum starved for 24 h, monolayers were wounded. They were treated with STS for 3 h, and the amounts of pMRLC (Thr18/Ser19) were determined by Western blot. Representative Western blots from 3 observations are shown along with the densitometric analysis. *P < 0.05, significantly different from the respective untreated group.

 

Figure 8
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  		Fig. 8. Cytochalasin D (CD) and pMRLC. IEC-6 cells were grown in control and DFMO (5 mM)-containing media for 3 days and serum starved for 24 h. Cells were treated with 1.0 µM CD for 3 h, fixed, and incubated with pThr18/Ser19-MRLC antibody. Cells were stained with secondary antibodies conjugated with AF-488 and rhodamine-conjugated phalloidin and observed using a Nikon UV fluorescence microscope. Representative images from 3 independent experiments are shown.

 
STS and focal adhesions. We determined whether STS mediates its effects through focal adhesions by monitoring the localization of FAK and phospho-paxillin. Control cells formed typical adhesion plaques as seen by the localization of FAK (Fig. 9A). FAK protein colocalized with phospho-paxillin in focal adhesions (data not shown). Polyamine depletion diminished focal adhesion plaques, and the morphology of these cells was distinctly different compared with that of control cells. FAK colocalized with F-actin in polyamine-depleted cells, which was evident by the intensity of FAK staining in conjunction with the actin cortex. STS significantly altered the size and distribution of FAK in both control and polyamine-depleted cells (Fig. 9A). STS-induced reorganization of the actin cortex was associated with the redistribution of FAK and phospho-paxillin at the tips of lamellipodial extensions in polyamine-depleted cells (Fig. 9B). However, phospho-paxillin remained firmly associated with the actin cortex in untreated cells. STS caused a similar effect on the paxillin distribution in control cells (data not shown). Furthermore, STS-induced reorganization of FAK and phospho-paxillin indicated the remodeling of focal contacts.


Figure 9
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  		Fig. 9. STS and focal adhesions. IEC-6 cells were grown in control and 5 mM DFMO-containing medium for 3 days. After being serum starved for 24 h, cells were treated with STS for 3 h and fixed and stained with focal adhesion kinase (FAK; A)-specific or phospho-paxillin (pY-paxillin; B)-specific antibodies. Cells were stained with secondary antibodies conjugated with rhodamine and/or AF-488 secondary antibodies and rhodamine and/or AF-488-conjugated phalloidin and observed using a Nikon UV fluorescence microscope. Representative images from 3 independent experiments are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The regulation of the migration of intestinal epithelial cells in response to wounding is important for the maintenance of gastrointestinal mucosal integrity. This regulation is unique because it not only depends on normal blood-borne factors but is also influenced by factors present within the digestive tract. Polyamines are known to play a critical role in the migration of intestinal epithelial cells (17, 29, 30, 35, 46, 47). Extracellular signals for cell migration converge to the Rho family of small GTPases in the cytoplasm (3, 32, 33, 38). In a previous study (29), we found that polyamine depletion inactivates Rac1 in IEC-6 cells and that the transfection of cells with constitutively active Rac1 completely restores cell migration in polyamine-depleted cells. We proposed that the involvement of polyamines in cell migration occurs either at Rac1 itself or upstream of Rac1. This hypothesis was supported by the findings that MEK increased Rac1 activity and reversed the inhibitory effect of polyamine depletion on cell migration (44). In this study, we further validated this hypothesis by showing that STS increased Rac1 activity and reversed the inhibitory effect of polyamine depletion on cell migration, whereas the stimulating effect of STS on migration was totally blocked by a Rac1 inhibitor.

Actin turnover and rearrangement play a paramount role in migration (2, 39). The dense actin filaments lies in the cortex, a narrow zone just beneath the plasma membrane. In this region, most actin filaments are arranged into a network with myosin II as a component that excludes most organelles from the cortical cytoplasm. Cortical actin networks help support and stiffen the fluid-like plasma membrane. When animal cells change shape or move, they generally disassemble older actin filaments, which are needed to generate actin monomers for polymerization. Moving cells use actin polymerization to push the plasma membrane outward, forming localized protrusions known as lamellipodia (24). These protrusions are stabilized by adhering to the extracellular matrix or adjacent cells via transmembrane receptors linked to actomyosin (3, 23). These adhesions serve as traction sites for migration as the cell moves forward over them, and they are disassembled at the rear of the cell, allowing it to detach. Previously, we showed that in control cells, actin filaments form stress fibers that traverse the cell, form a thin actin cortex, and extend into lamellipodia. In polyamine-depleted cells, actin stress fibers are less dense, whereas the actin cortex is greatly increased in density and lamellipodia are less extensive (18).

In this study, we showed that there was a colocalization of pMRLC and thick cortical F-actin in polyamine-depleted cells (Figs. 3–5). STS decreased pMRLC around the cell periphery, dissolved thick cortical F-actin, increased stress fibers and lamellipodia, and restored migration (Figs. 2 and 5). These results suggest that increased pMRLC around the cell periphery in polyamine-depleted cells stabilizes cortical F-actin, thereby inhibiting actin turnover and the formation of lamellipodia and stress fibers and inhibiting migration.

It is generally accepted that myosin II plays a role in the last step of cell migration, that is, the translocation of the cell body forward by the contraction of the posterior region (13, 20, 50). This notion is supported by the location of phosphorylated myosin II at the rear of migrating cells (25). Recently, Matsumura et al. (15) reported a localization-specific role for pMRLC in the regulation of membrane protrusions and cell migration. They found that phosphorylated myosin II is also localized in the anterior regions of motile fibroblasts (15). Using a newly developed inhibitor of MLCK and a inhibitor of ROCK (Y27632), they also found that the spatially differentiated reduction of MRLC phosphorylation produces strikingly opposing effects on the cell migration as well as on membrane protrusions and the dynamics of focal adhesions in free-moving fibroblasts (40). Our results also indicate that pMRLC stabilized cortical F-actin in polyamine-depleted cells and inhibited actin turnover, suggesting another role of pMRLC in migration. Dephosphorylation of MRLC in response to STS accompanied the reorganization of the actin cortex leading to the formation of lamellipodia (Figs. 6 and 9). These events correlated well with the reorganization of focal contacts as seen by the localization of FAK and phospho-paxillin (Fig. 9). The inhibition of MLCK by ML7 significantly inhibited the migration of cells grown under both control and DFMO conditions, emphasizing the importance of MRLC phosphorylation in migration. Unlike STS, disruption of actin stress fibers by CD had no effect on the localization or phosphorylation of MRLC (Fig. 8), which suggests that dephosphorylation of MRLC plays an important role in the reorganization of the actin cytoskeletal structure. Furthermore, STS decreased pThr18-MRLC without a significant effect on nuclear pSer19-MRLC in polyamine-depleted cells, indicating that pThr18-MRLC plays role in migration. The localization of pSer19-MRLC to the nucleus and its association with chromosomes during nuclear segregation (mitosis) indicates a localization-specific role for pMRLC (Fig. 5).

Inhibition of RhoA activity may lead to decreased pMRLC levels due to inhibition of ROCK and MRLC phosphatase (27). On the other hand, inhibition of Rac1 and Cdc42 activities can increase pMRLC by decreasing PAK and increasing MLCK activities (1, 34). Thus, Rho GTPases play a crucial role in the regulation of MRLC phosphorylation. In polyamine-depleted IEC-6 cells, RhoA, Rac1, and Cdc42 activities are inhibited. While we have shown that RhoA activation is essential, the expression of constitutively active RhoA is not sufficient to restore the migration of polyamine-depleted cells. However, constitutively active Rac1 completely restored the migration of these cells (29, 30). Since Rac1 regulates RhoA and Cdc42 in IEC-6 cells (29), inhibition of Rac1 by NSC-2376 or expression of dominant negative Rac1 should inhibit RhoA and Cdc42 activities. STS increased the migration of polyamine-depleted cells by activating Rac1 (Fig. 3, A and B), and we used NSC-2376 and DN-Rac1 to prevent Rac1 activation and studied their effect on STS-induced migration. The inhibition of Rac1 completely prevented STS-induced migration in both control and polyamine-depleted cells (Fig. 3, B and C). Although RhoA, Rac1, and Cdc42 activities are inhibited in polyamine-depleted cells, the levels of pMRLC were not altered significantly compared with control cells (Fig. 7), indicating the involvement of multiple signaling pathways. Other than MLCK (5) and MRLC phosphatase (50), phosphorylation of MRLC is also regulated by zipper-interacting protein kinase (21), citron kinase (51), death-associated protein kinase (9, 12), and PP2A (10, 22, 41). The increase in pMRLC at the cell periphery in polyamine-depleted cells might be due to a complex signal transduction network involving multiple pathways.

In summary, we found that polyamine depletion increased pMRLC (Thr18) around the cell periphery, which colocalized with a thick cortex of F-actin. STS decreased pMRLC (Thr18) at the cell periphery, dissolved the thick cortex, increased lamellipodia and stress fibers, reorganized focal contacts, and increased cell migration in polyamine-depleted IEC-6 cells. These data indicate that increased peripheral pThr18-MRLC in polyamine-depleted cells stabilizes the thick cortex of F-actin, thereby inhibiting actin turnover and migration.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52784 and the Thomas A. Gerwin Endowment.


   ACKNOWLEDGMENTS
 
We sincerely acknowledge Mary Jane Viar and Rebecca West for technical support and Gregg Short and Danny Morse for help in preparation of the figures.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Ray, Dept. of Physiology, The Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (e-mail: rray{at}physio1.utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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