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.
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 ofdl-α-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 p21Waf1/Cip1 and p27Kip1. 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.
Cell migration is the principal force behind the early restitution of erosions of the mucosa of the gastrointestinal tract. Despite the importance of cell migration to healing, no attempts to study the process in culture have been reported. We have attempted to standardize conditions for migration and test the migration responses of the small intestinal epithelial crypt cell line IEC-6 in some experimental situations already well known in vivo. We found good correspondence between in culture and in vivo on the following points: 1) migration was independent of DNA synthesis; 2) DNA synthesis was not concentrated at the wound edge; and 3) inhibition of actin polymerization stopped migration altogether. In addition, the presence of an extracellular matrix maximized migration. Protein inhibitors with different modes of action inhibited cell migration to different degrees, not always commensurate with their inhibition of protein synthesis. Cell surface proteoglycans were important; hyaluronic acid had an effect, but the secretion of a migration-stimulating substance by wounded cells was equivocal. Significantly, alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase and polyamine synthesis, almost totally prevented cell migration. Because DFMO also prevents healing of mucosal erosions in vivo, we believe that this model can be used, keeping in mind its spatial limitations, to study the process of cell migration involved in the early restitution of mucosal erosions.
The polyamines have been under active investigation for nearly three decades. There is a great deal of evidence that they play an important role in gastrointestinal mucosal growth, but the mechanisms through which this role is carried out are still not fully explained. This review examines the role of the polyamines in the regulation of mucosal growth, the control of intracellular polyamine levels, the biosynthesis of the polyamines, and some known mechanisms of their action. Finally, we propose a model of polyamine action that reconciles the effects of various trophic agents and situations in which growth is stimulated along with concomitant changes in polyamine levels. It accounts for both humoral and gradient-oriented features of various adaptive responses of the gastrointestinal mucosa and is intended to provide a framework for the future investigation of the role of the polyamines in this tissue.
Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)
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 ␣-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of ␣-difluoromethylornithine. IEC-6 cells transfected with constitutively active Rac1 and Cdc42 migrated more rapidly than vectortransfected 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 separation of viable epithelial, stromal, and myometrial cells from immature rat uteri is described. The integrity of the separated cells was established by their ability to grow in culture and to exclude trypan blue and by transmission and scanning microscopy. Epithelial preparations contain insignificant contamination by other cells. Stromal fractions are estimated to contain 10% epithelial contamination. Myometrial fractions probably contain significant contamination by stroma, but the amount is difficult to quantify. The three cell types separated from untreated rats or from rats treated with 17 beta-estradiol (E2) differ from each other physiologically. Control epithelial cells incorporated more tritiated cytidine per microgram DNA than stromal or myometrial cells. 17 beta-Estradiol enhanced incorporation of tritiated cytidine into RNA of epithelial cells but not stromal or myometrial cells. RNA to DNA ratios of cells from untreated rats were 1.0 (epithelial), 0.5 (stromal), and 0.6 (myometrial). RNA to DNA ratios rose approximately 30% in all cell types after E2. Estrogen receptor (ER) concentrations, determined by Scatchard analysis under exchange conditions, also differed in the three cell types. Nuclear ER concentrations (as measured in crude nuclear fractions) were significantly higher (P less than 0.01) in epithelial and stromal cell fractions from untreated animals than in the myometrial fraction, i.e. 5.5, 3.8, and 1.2 fmol/microgram DNA, respectively. The chief effect of E2 administration was a significant increase (P less than 0.05) in cytosol ER of all cell types. Epithelial cells had the highest cytosol ER concentration, increasing from 0.5 to 3.1 fmol/microgram DNA 48 h after E2 administration. Although the myometrial cells had the lowest ER concentration, they contained 84-89% of the entire uterine ER by virtue of the proponderance of myometrium in the whole uterus. The differing responses of epithelial and stromal cells, therefore, are diluted or masked by the ER content of the myometrium in assays of whole uterus. Although the total number of ER sites in the cytosol and nucleus (16,900-32,100 sites/cell) in separated stromal cells at various times after E2 stimulation was similar to ER concentrations (20,000-30,000 sites/cell) previously reported for the whole uterus, the partition of ER between cytosol and nucleus (10:90) was different, favoring the nucleus even before E2 administration. ER began to appear in increased amounts in the cytosol only 24 h post E2 administration. When tritiated E2 was given in vivo and its specific binding was assessed in the uterine cells subsequently separated, ER partition between cytosol and nucleus was also heavily in favor of the nucleus. However, when whole uterus was assayed, the partition of ER between cytosol and nucleus (90:10) was exactly the opposite. Possible reasons for this unusual finding are discussed.
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