Shirley A. McCormack

Rho proteins play a critical role in cell migration during the early phase of mucosal restitution.

In the intestine, several growth factors stimulate migration of epithelial cells, contributing to the maintenance of tissue integrity. The Ras-like GTPase Rho regulates a signal transduction pathway linking growth factor receptors to the formation of actin stress fibers and focal adhesions, presumed to be important for motility. Using an in vitro wound-induced migration assay, we have examined the role of Rho GTPases in the migration of IEC-6 and Caco-2 cells, and provide evidence that the Rho GTPases play an essential role in the initial phase of mucosal wound healing. Treatment of the cells with Clostridium difficile toxins A and B, inhibitors of the Rho family GTPases inhibited migration in a dose-dependent fashion. Microinjection of the inhibitory exchange factor Rho-guanine nucleotide dissociation inhibitor (GDI), or Clostridium botulinum C3 ADP-ribosyl transferase (C3) toxin, a Rho-ADP-ribosylating exoenzyme, potently inhibited migration. Microinjection of RhoT19N, a dominant negative form of RhoA, or in vitro ADP-ribosylated RhoA impaired the ability of cells to migrate. Rho-GDI and C3 exoenzyme also inhibited EGF-induced migration of IEC-6 cells. These results demonstrate that Rho is required for endogenous and EGF-induced migration of small intestinal crypt cells, and that Rho proteins are essential elements of a mechanism by which growth factors induce cell migration to restitute mucosal integrity.

Polyamine depletion arrests cell cycle and induces inhibitors p21Waf1/Cip1, p27Kip1, and p53 in IEC-6 cells

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence of DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21(Waf1/Cip1) and p27(Kip1). Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

Polyamines are required for microtubule formation during gastric mucosal healing

Polyamines are essential for the repair of gastric and duodenal erosions. Concentrated NaCl (3.4 M) given intragastrically damages the oxyntic gland mucosa and increases the activity of gastric mucosal ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis. The nature of the process of restitution of damaged mucosa is not well known, except that cell migration and the actin cytoskeleton play a prominent role. Microtubules are cytoskeletal components essential for cell migration. The present investigation determines the relationship between polyamines, the distribution of microtubules, and gastric healing in mucosa damaged with hypertonic NaCl solution. Rats were fasted for 22 h and then given 1.0 ml of 3.4 M NaCl intragastrically. Animals were killed 1, 2, 4, 8, and 10 h after 3.4 M NaCl. The oxyntic gland mucosa was removed, and tubulin was visualized by immunofluorescence. Microtubule density was increased around and below the damaged mucosa in the upper one-third of the glandular epithelium at 2 and 4 h and returned to near control levels by 10 h. In rats damaged with 3.4 M NaCl and pretreated intraperitoneally with α-difluoromethylornithine (DFMO), a specific inhibitor of ODC, microtubule content was reduced significantly at all time points after NaCl treatment. Addition of spermidine after pretreatment with DFMO and 3.4 M NaCl significantly prevented the effects of DFMO. Colchicine, a potent microtubule-disrupting drug, significantly delayed normal gastric mucosal healing with no effect on ODC activity. These data show that polyamines influence the distribution of microtubules during damage in vivo and indicate a partial mechanism for the dependency of mucosal healing on polyamines.

Polyamine depletion alters the relationship of F-actin, G-actin, and thymosin β4 in migrating IEC-6 cells

The cause of reduced migration ability in polyamine-deficient cells is not known, but their actin cytoskeleton is clearly abnormal. We depleted polyamines with α-difluoromethylornithine (DFMO) in migrating cells with or without stimulation by epidermal growth factor (EGF) and investigated filamentous (F-) actin, monomeric (G-) actin, and thymosin β4 (Tβ4), using immunofluorescent confocal microscopy, DNase assay, and immunoblot analysis. DFMO reduced F-actin in the cell interior, increased it in the cell cortex, redistributed G-actin, and increased nuclear staining of Tβ4. However, DFMO did not affect the amount of Tβ4 mRNA. EGF caused a rapid increase in the staining of F-actin in control cells, but DFMO prevented this response to EGF. Despite the visible changes shown by immunocytochemistry, statistically significant changes in the amount of either actin isoform or of total actin did not occur. We propose that DFMO reduces migration by interfering with the sequestration of G-actin by Tβ4 and the association of F-actin with activated EGF receptors.

Polyamine deficiency alters EGF receptor distribution and signaling effectiveness in IEC-6 cells

Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipatated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.

EGF induces nuclear translocation of STAT2 without tyrosine phosphorylation in intestinal epithelial cells

Signal transducers and activators of transcription (STATs) are cytoplasmic proteins that bind to activated membrane receptors, undergo ligand-dependent phosphorylation on tyrosine residues, and translocate to the nucleus, where they induce transcription of specific genes in response to a variety of ligands, including cytokines and some growth factors. Using immunocytochemical and biochemical techniques, we investigated the localization and responses of STAT1 and STAT2 to epidermal growth factor (EGF) stimulation in IEC-6 intestinal epithelial cells and HeLa cells. These studies provide the first description of STAT activation and localization in response to EGF in intestinal epithelial cells and some novel findings regarding the activation and localization of STATs in general. These include the following. First, EGF promoted the tyrosine phosphorylation of STAT1 in IEC-6 cells and caused its translocation to the nucleus. Second, in the absence of EGF stimulation both STAT1 and STAT2 were localized to the Golgi apparatus in IEC-6 cells. Third, EGF caused the translocation of STAT2 to the nucleus in both IEC-6 and HeLa cells without inducing the tyrosine phosphorylation of STAT2.

Altered distribution of the nuclear receptor rarβ accompanies proliferation and differentiation changes caused by retinoic acid in Caco-2 cells

SummaryAll epithelial cells require retinoic acid for growth, maintenance, and differentiation. Although the epithelial cells that line the gastrointestinal tract are exposed to extreme retinoid concentration fluctuations in luminal fluid, whether proliferation and differentiation in these cells are significantly affected is not known. We have investigated this question using Caco-2 cells as a model because, although they are derived from a colon adenocarcinoma, they differentiate spontaneously in a manner similar to enterocytes in the small intestine. We found that retinoic acid caused maximum inhibition of cell growth and ornithine decarboxylase activity during the proliferative period. Retinoic acid increased brush border enzyme activities only in differentiating cells but stimulated transglutaminase activity in cells at all stages. In untreated proliferating cells, we found an early peak of transglutaminase activity that has not been reported before. Retinoic acid in intestinal cells acts through its nuclear receptor, RARβ. The nuclear distribution of this receptor has not been demonstrated. In this study, we show that RARβ responds to increasing concentrations of retinoic acid with a shift to the nuclear membrane in undifferentiated cells and progressive aggregation, diffusion, and loss in differentiated cells. We conclude that retinoic acid can inhibit proliferation and stimulate differentiation in Caco-2 cells depending on concentration and cell stage, and that these effects are accompanied by changes in distribution, as well as by the loss of RARβ.

Putrescine uptake and release by a normal rat small intestine crypt cell line, IEC-6.

IEC-6 cells were cultured on permeable filter inserts with separate access to the apical and basolateral sides. [3H]Putrescine uptake favored the apical side and its release (in Earles balanced salt solution containing 0.1% bovine serum albumin) was six times greater in the apical-to-basolateral than in the basolateral-to-apical direction. Release in DMEM did not show this preference. The uptake of [3H]putrescine was stimulated approximately 1.3 times the basal level by 10 mM asparagine (ASN) or 5% dialyzed fetal bovine serum whether the [3H]putrescine was added at a concentration of 1 or 100 nM. The increased uptake was maintained for up to 6 h. When [3H]putrescine was removed after 4 h of uptake, the cells continued to release it into the medium on both sides for up to 4 h. Stimulated cells released only 50% as much as unstimulated cells. Unlabeled putrescine reduced the uptake of [3H]putrescine with an IC50 of 1.81 x 10(-6) M (r = 0.9476) and 1.02 x 10(-6) M (r = 0.9967) for unstimulated and ASN-stimulated cells, respectively. When the intracellular putrescine was reduced by difluoromethylornithine, the uptake of [3H]-putrescine was not changed, but its release was inhibited. Sodium was not required for [3H]putrescine uptake or release. Although the stimulated cells attained intracellular levels of [3H]putrescine which, if expressed as concentration based on cell volume, were up to 500 times the original extracellular concentration, a true concentration gradient could not be proven because 85% of the [3H]putrescine was probably bound to polyanions as shown by butanol extraction.