Polyamines and Cytoskeletal Regulation During Intestinal Epithelial Restitution

Ramesh M. Ray and Leonard R. Johnson 1. Introduction

Damage to gastrointestinal epithelia can result from infection (ulcer), chemical agents (alcohol, drugs), or mechanical forces (stretching), and immediate repair is required to restore the epithelial barrier against luminal antigens. The mucosa of the gastrointestinal tract has the unique ability to repair itself rapidly after damage. Mucosal repair consists of two phases. Early mucosal restitution is the rapid re-establishment of epithelial integrity and continuity after superficial injury before cell proliferation or an extensive inflammatory response occurs (1). It comprises sloughing of the damaged cells and migration of remaining viable cells over the denuded lamina propria (2,3). The second phase involves replacement of the lost cells by mitosis and does not begin until 24 h or so after injury (1). The process of early mucosal restitution was originally described for the stomach, but later Fiel et al. (4) and Moore et al. (5) have shown a similar process for the small intestine.

The mucosal epithelium of the alimentary tract represents a crucial barrier to a broad spectrum of noxious and immunogenic substances within the intestinal lumen. Impairment of the integrity of the mucosal epithelial barrier is observed in the course of various intestinal disorders, including inflammatory bowel diseases, celiac disease, intestinal infections, and various other diseases. Furthermore, even under physiological conditions proteases, residential flora, dietary compounds, or other factors may cause temporary damage to the epithelial surface. Healing of the intestinal epithelium is regulated by a complex network of highly divergent factors, among them a broad spectrum of structurally distinct regulatory peptides that have been identified within the mucosa. These regulatory peptides, conventionally designated as growth factors and cytokines, play an essential role in regulating differential epithelial cell functions to preserve normal homeostasis and integrity of the intestinal mucosa. In addition, a number of other peptides, such as those in the extracellular matrix and blood clotting factors, as well as nonpeptides including phospholipids, short chain fatty acids, adenine

From: Polyamine Cell Signaling: Physiology Pharmacology and Cancer Research Edited by: J.-Y. Wang and R. A. Casero, Jr. © Humana Press Inc., Totowa, NJ

nucleotides, trace elements, and pharmacological agents, modulate intestinal epithelial repair. Some of these molecules may be released by platelets, adjacent stromal cells, inflammatory cells, or injured epithelial and nonepithelial cells. Enhancement of repair mechanisms by regulatory factors may provide future approaches for the treatment of diseases that are characterized by damage to the epithelial surface.

Polyamines have been shown to be essential for various processes, including cell proliferation, migration, and apoptosis both in animal models and in in vitro cell culture models (6-8). The role of polyamines in the regulation of cell proliferation and apoptosis has been discussed in detail elsewhere in this book. Because migration is involved in restitution, it is important to understand the mechanism by which polyamines affect this process. Using stress and hypertonic NaCl models for mucosal injury in rats, it has been shown that polyamines are essential for the healing of gastric and intestinal lesions (8-10). The polyamines spermidine, spermine, and their precursor putrescine, are found in virtually all cells of higher eukaryotes and are intimately involved in, and required for, cell growth and proliferation (11,12). Intracellular polyamine levels are highly regulated and depend on the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting step in polyamine biosynthesis (12,13). An increase in ODC activity is one of the earliest events associated with the induction of cellular proliferation and depletion of polyamines by DL-a-difluoromethyl-ornithine (DFMO), a specific inhibitor and irreversible inhibitor of ODC, attenuates trophic responses in tissues and cultured cells (14-17).

ODC activity in the mucosa of the small intestine is increased after partial resection (18), during lactation (19) during the third week of life at the time of weaning in rats (20), and after obstruction of the lumen (21)—all instances associated with mucosal growth. DFMO prevents the accumulation of polyamines and growth of the mucosa in each of these models (18-21). In both gastric (8,22) and duodenal (22,23) mucosa, ODC activity increases significantly in response to the production of stress ulcers. In the same models, the mucosal polyamine content also increases, and DFMO prevents the increases in ODC and polyamines (8,22,23). Although polyamine depletion did not increase the severity of the lesions, it prevented, almost entirely, the 80-90% healing that occurred within the 24-h period after damage (8,23-25). Oral administration of any of the polyamines immediately after the period of stress increased the rate of normal healing and prevented the inhibition of repair caused by DFMO in both gastric (24) and duodenal (25) mucosa. Because polyamines are required for cell division, some of their beneficial effects were a result of this process; however, substantial and significant healing occurred in polyamine-depleted rats given exogenous spermidine and spermine within 12 h after damage (24,25). Because regeneration by proliferation requires at least 24 h (1,26,27), it was obvious that the polyamines are essential to the process of early mucosal restitution and cell migration, as well as proliferation.

2. Mechanism of Cell Migration

Cell migration involves a coordinated cycle of plasma membrane protrusion at the leading edge, adhesion formation to stabilize the protrusion, destabilization of older adhesion sites at the rear of the cell, and stress fiber contraction against adhesions for cell body movement (28,29). The initial response of a cell to a migration-promoting stimulus is to polarize and extend protrusions in the direction of migration. These protrusions, which can be broad and large or spikelike structures, called lamellipodia and filopodia, respectively, are thought to result from actin filament (F-actin) polymerization at the plasma membrane. In lamellipodia, actin filaments form a branching network, whereas they form parallel bundles in the filopodia. These protrusive structures are stabilized by adhering to the extracellular matrix (ECM) or to adjacent cells via transmembrane proteins linked to the actin cytoskeleton. Adhesion to the ECM provides traction sites for the forward movement of the cell. Thus dynamic assembly and disassembly of these adhesions plays a crucial role in determining the direction and rate of cell motil-ity. Using fluorescence speckle microscopy, Ponti et al. (30) showed that assembly and disassembly of the lamellipodial network occurred within 1-3 |im of the leading edge. It was weakly coupled to the rest of the cytoskeletal structure and promoted random protrusions. However, productive cell movement resulted from a second network colocalized with actomyosin at focal adhesions in migrating epithelial cells (30).

Actin filaments consist of fast-growing "barbed ends" and slow-growing "pointed ends" imparting polarity, which is used to drive membrane protrusions. The Arp2/3 complex mediates actin polymerization in lamellipodia. Several actin-binding proteins also regulate the rate and organization of actin polymerization by modulating the pool of available monomers (G-actin) and free ends. Profilin prevents self-nucleation by binding to actin monomers and also is involved in targeting monomers to barbed ends. Filament elongation is prevented by capping proteins, resulting in decreased polymerization of new filaments in the vicinity of the plasma membrane. The ADF/cofilin group of proteins promotes disassembly of older filaments, which is required to generate actin monomers for polymerization at the leading edge. These proteins sever filaments and promote actin dissociation from the pointed end and are referred to as actin severing proteins (31,32).

3. Polyamines and Migration in Intestinal Epithelial Cells

The mechanism by which polyamines influence cell migration is not clear. Polyamines affect various types of cell migration, such as human sperm motility (33), metastasis of breast cancer cells (34), and cell attachment to fibronectin (35) in addition to epithelial cell motility. Our laboratory elucidated the role of polyamines in gastrointestinal healing using an in vivo rat model. McCormack et al. (36) established an in vivo cell culture model of restitution using the IEC-6 cell line, a well-characterized intestinal crypt cell line derived from the rat by Quaroni et al. (37). To determine the rate of migration, we used a modified model in which IEC-6 cells are plated on 35-mm plates and allowed to grow to confluence. Plates are marked at the bottom along the diameter and wounded twice with a fine, sterile, plastic microtip perpendicular to the marking. Plates are washed to remove damaged cells, replenished with fresh medium, and photographed at 0 h and at various times allowed for migration, usually 7-8 h (Fig. 1). The width of the wound covered by migrating cells is determined by National Institutes of Health imaging software and used for calculating the rate of migration (38). To study the effects of polyamines on migration, cells are grown for 4 d in control medium containing

Fig. 1. Wound healing model and effect of polyamine depletion. Cell migration wound assay of IEC-6 cells grown in control, a-difluoro-ornithine (DFMO), and DFMO plus putrescine containing media as described in ref. 41 showing size of wound at 0 and 7 h after wounding. DFMO-treated cells show decreased wound closer.

Fig. 1. Wound healing model and effect of polyamine depletion. Cell migration wound assay of IEC-6 cells grown in control, a-difluoro-ornithine (DFMO), and DFMO plus putrescine containing media as described in ref. 41 showing size of wound at 0 and 7 h after wounding. DFMO-treated cells show decreased wound closer.

5 mM DFMO and medium containing DFMO plus putrescine. In the presence of DFMO, intracellular putrescine disappears completely within 6 h, spermidine within 48 h, and spermine decreases to 30 to 40% by d 4 (39). Exogenous addition of putrescine to DFMO containing medium serves as a control to ascertain that the effects of DFMO are owing to depletion of polyamines and not to DFMO itself. In both animal and cell culture models, polyamine depletion decreased migration by 60-70%, and addition of putrescine to DFMO-containing medium restored migration to control levels (Fig. 1). Furthermore, inhibition of S-adenosylmethionine decarboxylase decreased intracellular spermidine and spermine, increased putrescine, and inhibited migration, suggesting that putrescine is not required for the migration of these cells (40). Examination of the actin cytoskeletal structure revealed a significant alteration in the organization of F-actin in response to polyamine depletion. In polyamine-depleted cells, F-actin localized to a heavy actin cortex with short stress fibers (Fig. 2A). Unlike cells grown under control conditions, cells grown in DFMO showed significantly less F-actin reorganization at the migrating edge (Fig. 2B). In control cells, long stress fibers of F-actin traversed the cell and lamellipodia, a characteristic feature of actively migrating cells, were evident. Total amounts of G-actin and F-actin were unchanged in control cells and those depleted of polyamines, suggesting that the remodeling of the actin cytoskeleton, rather than the ratios of the actins, was altered by polyamines (41).

Attachment to the ECM is the first step in migration. To migrate, cells must form and break attachments with the ECM. The ECM binds to cell surface receptors, the integrins, resulting in their aggregation and the formation of a signaling complex, which provides attachment sites for stress fibers (42). The reorganization of actin filaments into stress fibers causes a positive feedback, resulting in additional integrin clustering (43). The

Fig. 2. Effect of polyamine depletion on the F-actin cytoskeleton. IEC-6 cells grown in control, a-difluoro-ornithine (DFMO), and DFMO plus putrescine containing media were fixed after 7 h migration, stained with rhodamine-conjugated phalloidin and observed under ultraviolet fluorescence with an inverted microscope. Control and DFMO plus putrescine groups show stress fibers in the confluent area (A), and at the migrating edge (B). The DFMO group has a thick cortical localization of F-actin and fewer stress fibers.


Fig. 2. Effect of polyamine depletion on the F-actin cytoskeleton. IEC-6 cells grown in control, a-difluoro-ornithine (DFMO), and DFMO plus putrescine containing media were fixed after 7 h migration, stained with rhodamine-conjugated phalloidin and observed under ultraviolet fluorescence with an inverted microscope. Control and DFMO plus putrescine groups show stress fibers in the confluent area (A), and at the migrating edge (B). The DFMO group has a thick cortical localization of F-actin and fewer stress fibers.

resulting transmembrane complexes of extracellular matrix proteins, integrins, cytoskeletal proteins, focal adhesion kinase, and actin are called focal adhesion complexes. Both attachment and stress fiber formation depend on focal adhesion complexes and integrin signaling. Polyamine depletion of cells significantly decreased attachment to fibronectin (44), decreased clustering of integrin a1 with p2, and decreased tyrosine phosphorylation of focal adhesion kinase (FAK), specifically, the phosphorylation of tyrosine 925, the paxillin binding site (44). In control cells, FAK phosphorylation occurred rapidly after attachment to the ECM, whereas it was significantly delayed in attached cells depleted of polyamines. Autophosphorylation of FAK was also significantly inhibited, as was the phosphorylation of paxillin in polyamine-depleted cells. Polyamine-depleted cells failed to spread normally after attachment, and immunocytochemistry showed little colocalization of FAK and actin compared with controls. Focal adhesion complex formation was greatly reduced in the absence of polyamines, suggesting that decreased integrin signaling may, in part, account for the decreased rates of attachment, spreading, and migration that are observed in polyamine-depleted cells.

Soluble factor and integrin signaling is relayed to the cytoskeleton by signal transduction pathways involving a subgroup of the Ras superfamily of small guanosine 5'-triphos-phate-binding proteins (45). The Rho (Ras homology) guanosine 5'-triphosphatases (GTPases) consist of three major types of small (21 kDa) proteins that bind GTP. Their intrinsic GTPase activity is controlled by guanine nucleotide exchange factors and GTPase-activating proteins. Rho guanine nucleotide dissociation inhibitor is an inhibitory guanine nucleotide exchange factors that prevents the dissociation of guanosine diphos-phate from Rho as well as from Rac and Cdc42, the two other members of this family (46). Rho and Rac regulate the polymerization of actin and subsequent formation of stress fibers and lamellipodia, respectively (47-49). Cdc42 has been shown to be responsible for the formation of filopodia (50).

Polyamine-deficient Chinese hamster ovary cells lack actin filaments and microtubules unless polyamines are supplied exogenously (51). Inhibitors of polyamine synthesis prevent concanavalin A-induced expression of a-tubulin and ß-actin messenger RNAs in mouse splenocytes (52). In vitro, polyamines stimulate the rapid polymerization of G-actin and formation of F-actin, indicating a possible direct effect of polyamines on cytoskeletal organization (53). Because the early phase of mucosal healing, which is the result of cell migration, requires polyamines (23,24) and polyamine depletion inhibits migration in the IEC-6 cell model (Fig. 1) and leads to numerous alterations in the actin cytoskeleton (Fig. 2) (42), we examined the effect of polyamine depletion on the Rho proteins. Santos et al. (54) showed that Rho was required for migration in response to wounding or in response to growth factor stimulation. Migration was inhibited after microinjection of the Rho guanine nucleotide dissociation inhibitor, Clostridium botulinum C3 adenosine 5'-diphosphate-ribosyltransferase toxin, or a dominant-negative form of RhoA (Rho T19N). The actin cytoskeleton was also altered in these cells in ways that were identical to those produced by polyamine depletion. Polyamine depletion caused significant reduction in RhoA protein in the cytoplasm and membrane fractions of IEC-6 cells. An approx 50% reduction in the rate of synthesis of RhoA protein was observed in polyamine-depleted cells without a reduction in the halflife of RhoA or the levels of RhoA messenger RNA (55). Transfection of IEC-6 cells with constitutively active RhoA (HA-V14-RhoA) increased the rate of migration compared with cells transfected with empty vector, and dominant-negative RhoA cells exhibited almost no motility. Surprisingly, polyamine-depleted cells expressing constitutively active RhoA also had reduced rates of migration (55). Polyamine depletion inhibited RhoA activity in cells transfected with vector as it did in untransfected cells. In cells expressing dominant-negative RhoA, the cortical localization of F-actin was almost identical to that observed in polyamine-depleted cells. Constitutively active RhoA was unable to restore stress fiber formation in polyamine-depleted cells. Thus, although RhoA activity is essential for the migration of intestinal epithelial cells (IECs), it is not sufficient to reverse the effects of polyamine depletion.

There is considerable signaling cross talk between Rho, Rac, and Cdc42, and each of these molecules regulates a different aspect of the motility machinery of the cell. Transfection of IEC-6 cells with constitutively active Rac1 (V12-Rac1) completely reversed the effects of polyamine depletion on migration, restored the actin cytoskeletal structure, and returned the levels of RhoA protein to normal (38). This finding also implies that polyamines do not exert their effects by interacting directly with cytoskeletal proteins. IECs expressing constitutively active Racl had increased basal levels of active RhoA and Cdc42, indicating that Racl activates RhoA and Cdc42. Constitutively active RhoA cells had increased Cdc42 activity but Racl activities were unchanged. Activation of Cdc42 also increased the basal activity of Racl and RhoA. These observations clearly indicate that Racl is upstream of RhoA, and that RhoA may be downstream of Cdc42. Thus, Racl activation is not only essential but also sufficient for cell migration. This observation provides important insights into the involvement of polyamines in migration. Polyamines either activate Racl directly or, more likely, are required for an upstream event for the activation of Racl and not for the maintenance of levels of Racl protein. Racl protein levels were unaltered in response to polyamine depletion.

Epidermal growth factor (EGF) plays a critical role in the protection and repair of the gastrointestinal mucosa (56-58). EGF is produced by salivary glands (59) and by the gastric mucosa (60), and EGF receptors are present on the epithelial cells of the gastrointestinal tract (61,62). Injury-induced increases in the production of EGF and overexpression of EGF receptor (EGFR) have been observed during mucosal repair (63,64). We have shown that wounding transiently increased mitogen-activated protein kinase (MAPK) activity, and that EGF further increased migration in control and polyamine-depleted cells (65). However, in polyamine-depleted cells, EGF stimulated migration only to the levels observed in untreated control cells. U-0l26, a strong inhibitor of mitogen-activated protein kinase kinase (MEK) l, decreased basal as well as EGF-induced extracellular signal-regulated kinase (ERK) phosphorylation and inhibited migration, indicating that MAPK activity is required for migration. Xie et al. (66) reported that the EGF-mediated disassembly of focal adhesions depended on ERK activation. Klemke et al. (67) have shown that activated ERK can associate with, and phosphorylate, myosin light chain kinase, thereby increasing its activity and enhancing migration. Sustained ERK2 activation mediates scattering in SK-N-MC cells in response to Ret (rearranged during transfection) proto-oncogene and fibroblast growth factor (68), and stimulates colony dispersion in SCC-l2F cells (69). In fibroblasts, U-0l26 inhibits EGF-induced migration (70). These reports suggest that ERKl/2 inhibition alone is sufficient to prevent migration. We have also observed that on EGF treatment, EGFR downregulation occurs within l0-l5 min (unpublished observations). Therefore, we predicted that downregulation of EGFR and the transient nature of ERK activation might be rate-limiting factors in the migration of polyamine-depleted cells. We used stable IEC-6 cell lines expressing HA-tagged constitutively active (CA) and dominant-negative (DN) MEKl to examine the effects of sustained activation and inhibition of ERKl/2 on migration. Characteristic of actively migrating cells, those expressing CA-MEK exhibited significant spreading and stress fiber formation, which were less prominent in cells transfected with empty vector (65). In contrast, cells expressing DN-MEK were characterized by a significant loss of actin stress fibers essential for maintenance of cell shape, size, and migration. CA-MEK restored migration of polyamine-depleted cells to a level comparable to that of cells transfected with CA-MEK and grown in control medium. Migration of these cells was significantly higher compared with that observed in cells transfected with empty vector. Unlike cells trans-fected with empty vector, those transfected with CA-MEK displayed extensive actin cytoskeletal reorganization at the migrating edge in response to polyamine depletion.

The intracellular localization of Rho GTPases is crucial for their activation. RhoA and Rac1 proteins were localized throughout the cytoplasm in cells transfected with empty vector grown in control and DFMO plus putrescine media, and aggregates of RhoA were clearly distinguishable at the cell periphery. Interestingly, in polyamine-depleted cells, the subcellular localization of RhoA and Rac1 was significantly altered. Significant amounts of RhoA protein were found in the nucleus and in the perinuclear region with decreased concentrations at the cell periphery. In contrast, in cells trans-fected with CA-MEK, aggregates of RhoA protein were distributed throughout the cytoplasm with relatively higher amounts at cell periphery. Surprisingly, a significant fraction of Rac1 in polyamine-depleted cells transfected with empty vector was localized in the nucleus, unlike control and DFMO plus putrescine groups in which it localized throughout the cytoplasm. Expression of CA-MEK in polyamine-depleted cells prevented the nuclear accumulation of Rac1 and increased its distribution in cytoplasm. Unlike polyamine-depleted cells transfected with empty vector, Rac1 was localized in lamellipodia of DFMO-treated cells transfected with CA-MEK. We have shown that constitutively active RhoA increased cell proliferation by preventing the transcription of p21waf/kip in IECs (71) and that constitutive activation of Rac1 also increased proliferation (unpublished data), suggesting additional roles for these proteins, which might explain their localization in the nucleus.

Because constitutive activation of Rac1 and MEK proved to be necessary and sufficient for the migration of polyamine-depleted cells, we examined whether MEK activated Rac1 in polyamine-depleted cells. CA-MEK expression increased Rac1 activity in control and polyamine-depleted cells, unlike cells transfected with vector, which showed decreased Rac1 activity. These results clearly demonstrate that MAPK activation is necessary for actin cytoskeletal organization, which is essential for migration.

Nonmuscle myosin II has been shown to play an important role in the regulation of the dynamics of actin cytoskeletal structure and cell shape. These changes are associated with changes in intracellular Ca2+ levels and its distribution in organelles (72). Polyamine depletion significantly decreased levels of myosin II. Myosin II localized in the form of small patches throughout the cytoplasm instead of in its functional association with stress fibers (73). Decreased intracellular Ca2+ resulting from downregula-tion of voltage gated K+ (Kv1.1) channel expression has been shown to influence RhoA protein levels and migration in polyamine-depleted cells (74,75). Levels and distribution of myosin II and levels of intracellular Ca2+ and RhoA protein were restored to normal by exogenous addition of putrescine to cells grown in the presence of DFMO. Furthermore ionomycin, a Ca2+ ionophore, also increased RhoA protein and migration in polyamine-depleted cells. However, the enzymatic activity of RhoA, which is essential to migration, was not determined in this study. It is difficult to understand how intracellular K+ and Ca2+ homeostasis can be regulated by a single mechanism, which determines all or no effects on migration. Increased K+ channel activity has been shown to increase the migration of fibroblasts, however, inhibition of K+ channel activity also accelerates intestinal wound healing in the human colon carcinoma cell lines, T84 and Caco-2, via both Ca2+-dependent and constitutively active channels (76). Pharmacological activation of K+ channels had no effect on the repair process in those cell lines (76). Furthermore, spermine and spermidine have been shown to inhibit inward rectifying K (Kir) channel activity (77). Thus, in the absence of polyamines (polyamine-depleted cells), Kir channels should have higher activity because of the lack of gating by sper-midine and spermine.

Polyamine depletion decreases total RhoA protein, and significantly decreases the activities of RhoA, Rac1, and Cdc42 (54). Furthermore, expression of constitutively active RhoA does not restore migration in polyamine-depleted cells, indicating that normal RhoA protein levels or activity are not sufficient for migration. Therefore, increases in RhoA protein in polyamine-depleted cells in response to increased intra-cellular Ca2+ may not account for the restoration of migration. Ca2+ may actually regulate a step upstream from Rho that is sufficient for migration. Intracellular Ca2+ homeostasis and the desired levels of cytoplasmic-free Ca2+ depend on binding proteins known as calmodulins and storage in the endoplasmic reticulum. Sustained increases in free cytoplasmic Ca2+ have been shown to decrease migration and may induce apop-tosis (78-86). Thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor, increases Ca2+ in the cytoplasm by activating store operated Ca++ entry. Thapsigargin increased intracellular Ca2+ in both control and polyamine-depleted cells, but did not restore migration of polyamine-depleted cells (unpublished data). Because polyamine depletion decreases the cell number in confluent cultures of IEC-6 cells by about 40%, fewer cells occupy a unit area. Thus less Ca2+ might be measured in monolayers of polyamine-depleted cells without translating in to decreased Ca2+ levels per cell. Because intracellular Ca2+ has not been determined in normal and polyamine-depleted cells, its role in RhoA activation in DFMO treated cells is unclear.

4. Conclusions

The scheme in Fig. 3 depicts the signaling events reported to be associated with polyamine depletion in IECs. Inhibition of migration of these cells is associated with decreased integrin (a2/p1) heterodimerization (46), myosin distribution (76,78), myosin light chain phosphorylation (78), intracellular Ca2+ levels (78), expression and activity of Kv channels (77), cell attachment and spreading (46), phosphorylation of FAK and paxillin (46), RhoA protein levels (54), and activities of RhoA, Rac1, and Cdc42 (41,54). Constitutive activation of MEK1 or Rac1 (67) completely restores migration and RhoA protein. This suggests that MEK1 or an upstream activator is a sensor of intracellular polyamine levels. Intracellular Ca2+ may activate MEK1, which in turn may activate Rac1 leading to activation of RhoA and Cdc42 and restore migration in polyamine-depleted cells. Because wounding initiates migration, it is reasonable to believe that polyamines modulate signaling at a membrane receptor where most of the key molecules described are found to localize at some time and point during migration. However, it is not clear how polyamines, in response to wounding, integrate outside signals to the intracellular events leading to migration. Current evidence suggests that, after wounding, polyamines influence the intracellular events associated

Range Estimation Formula

Fig. 3. Wound-induced signaling during cell migration. Steps depicted in the signaling cascade are directly or indirectly altered in response to polyamine depletion. FAK, focal adhesion kinase; ERMs, ezrin, radixin, moesin actin-binding proteins of focal adhesion complexes; MLCK, myosin light chain kinase; ECM, extracellular matrix.

Fig. 3. Wound-induced signaling during cell migration. Steps depicted in the signaling cascade are directly or indirectly altered in response to polyamine depletion. FAK, focal adhesion kinase; ERMs, ezrin, radixin, moesin actin-binding proteins of focal adhesion complexes; MLCK, myosin light chain kinase; ECM, extracellular matrix.

with migration in two ways. First, they are involved in the activation of MEK and ERKs, which in turn activate Racl. Second, they play a role in the organization of the focal adhesion complex via integrin ligation.

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