Mary Jane Viar

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.

Transglutaminase in response to hypertonic NaCl-induced gastric mucosal injury in rats

BACKGROUND Polyamines serve as substitutes for transglutaminase-catalyzed protein cross-linking and are essential to the healing of gastric mucosal lesions. This study determines whether transglutaminase and protein cross-linking have a role in the healing of hypertonic NaCl-induced gastric lesions. METHODS Rats were fasted 22 hours before given 1 mL 3.4 Mol/L NaCl intragastrically. Gastric mucosa was examined histologically and grossly, and transglutaminase activity was measured as the Ca(2+)-dependent covalent incorporation of [3H]putrescine into acid-precipitable protein. RESULTS Transglutaminase activity increased significantly from 2 to 8 hours, peaking between 4 and 6 hours after NaCl administration. Lesions were significantly produced after 2 hours, and damage paralleled transglutaminase activity. Dansylcadaverine (200 mg/kg orally), a specific inhibitor of protein cross-linking, prevented the increases in transglutaminase activity and significantly delayed healing but had no effect on lesion formation. CONCLUSIONS These results indicate that (1) hypertonic NaCl-induced gastric mucosal damage is associated with a significant increase in transglutaminase activity and (2) increased transglutaminase activity is involved in the mechanism of normal mucosal healing.

Amino Acids Regulate Expression of Antizyme-1 to Modulate Ornithine Decarboxylase Activity

Background: Amino acids regulate ornithine decarboxylase (ODC) activity through antizyme-1 (AZ1). Results: Asparagine or glutamine prevents AZ1 expression in response to amino acid starvation. AZ1 synthesis by amino acids is regulated by a differential activation of mTORC1/2. Conclusion: The inhibition of mTORC1 and activation of mTORC2 during amino acid starvation induces AZ1 synthesis. Significance: Putrescine and amino acids regulate ODC activity by stimulating AZ1 synthesis via mTORC2. In a glucose-salt solution (Earles balanced salt solution), asparagine (Asn) stimulates ornithine decarboxylase (ODC) activity in a dose-dependent manner, and the addition of epidermal growth factor (EGF) potentiates the effect of Asn. However, EGF alone fails to activate ODC. Thus, the mechanism by which Asn activates ODC is important for understanding the regulation of ODC activity. Asn reduced antizyme-1 (AZ1) mRNA and protein. Among the amino acids tested, Asn and glutamine (Gln) effectively inhibited AZ1 expression, suggesting a differential role for amino acids in the regulation of ODC activity. Asn decreased the putrescine-induced AZ1 translation. The absence of amino acids increased the binding of eukaryotic initiation factor 4E-binding protein (4EBP1) to 5′-mRNA cap and thereby inhibited global protein synthesis. Asn failed to prevent the binding of 4EBP1 to mRNA, and the bound 4EBP1 was unphosphorylated, suggesting the involvement of the mammalian target of rapamycin (mTOR) in the regulation of AZ1 synthesis. Rapamycin treatment (4 h) failed to alter the expression of AZ1. However, extending the treatment (24 h) allowed expression in the presence of amino acids, indicating that AZ1 is expressed when TORC1 signaling is decreased. This suggests the involvement of cap-independent translation. However, transient inhibition of mTORC2 by PP242 completely abolished the phosphorylation of 4EBP1 and decreased basal as well as putrescine-induced AZ1 expression. Asn decreased the phosphorylation of mTOR-Ser2448 and AKT-Ser473, suggesting the inhibition of mTORC2. In the absence of amino acids, mTORC1 is inhibited, whereas mTORC2 is activated, leading to the inhibition of global protein synthesis and increased AZ1 synthesis via a cap-independent mechanism.

Differences in transglutaminase mRNA after polyamine depletion in two cell lines

Polyamines serve as natural substrates for the transglutaminase that catalyzes covalent cross-linking of proteins and is involved in cellular adhesion and proliferation. This study tests the hypothesis that intracellular polyamines play a role in the regulation of transglutaminase expression in rat small intestinal crypt cells (IEC-6 cell line) and human colon carcinoma cells (Caco-2 cell line). Treatment with α-difluoromethylornithine (DFMO; a specific inhibitor of polyamine synthesis) significantly depleted the cellular polyamines putrescine, spermidine, and spermine in both cell lines. In IEC-6 cells, polyamine depletion was associated with a decrease in the levels of transglutaminase mRNA. In Caco-2 cells, however, polyamine depletion significantly increased the levels of transglutaminase mRNA and enzyme activity. In both cell lines, ornithine decarboxylase mRNA levels increased and protooncogene c- myc mRNA decreased in the presence of DFMO. Addition of polyamines to cells treated with DFMO reversed the effect of DFMO on the levels of mRNA for these genes in both lines. There was no significant change in the stability of transglutaminase mRNA between control and DFMO-treated IEC-6 cells. In contrast, the half-life of mRNA for transglutaminase in Caco-2 cells was dramatically increased after polyamine depletion. Spermidine, when given together with DFMO, completely prevented increased half-life of transglutaminase mRNA in Caco-2 cells. These results indicate that 1) expression of transglutaminase requires polyamines in IEC-6 cells but is inhibited by these agents in Caco-2 cells, 2) polyamines modulate transglutaminase expression at the level of mRNA through different pathways in these two cell lines, and 3) posttranscriptional regulation plays a major role in the induction of transglutaminase mRNA in polyamine-deficient Caco-2 cells.

DIFFERENTIAL EFFECT ON POLYAMINE METABOLISM IN MITOGEN- AND SUPERANTIGEN-ACTIVATED HUMAN T-CELLS

Polyamines are important for regulation of lymphocyte differentiation and proliferation. Mitogens induce synthesis of ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine biosynthesis. Since mitogens stimulate T-cells by non-physiological routes, the role of polyamine metabolism in T-cell receptor (TCR)-mediated T-cell activation has not been adequately evaluated. The effect of phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) on T-cell ODC and polyamine synthesis was compared. ODC activity was 6-11-fold higher in PHA compared to SEB stimulated T-cells. These differences were not attributed to differences in the magnitude of T-cell proliferation. Kinetics of ODC and polyamine synthesis were also different in PHA- and SEB-stimulated T-cells. In PHA-stimulated cells ODC levels and the induction of putrescine and spermidine synthesis peaked 6 h prior to peak IL-2 production, while in SEB-stimulated cells, ODC levels and polyamine synthesis peaked 6-12 h after IL-2 production. Differences in the temporal relationship between IL-2 production and polyamine induction in mitogen- versus superantigen-stimulated cells may account for the significant inhibition of the proliferative response by alpha-difluoromethylornithine following PHA but not SEB stimulation. Polyamine metabolism is regulated differently in T-cells stimulated via TCR engagement than with polyclonal mitogens.

Interaction of asparagine and EGF in the regulation of ornithine decarboxylase in IEC-6 cells.

Our laboratory has shown that asparagine (ASN) stimulates both ornithine decarboxylase (ODC) activity and gene expression in an intestinal epithelial cell line (IEC-6). The effect of ASN is specific, and other A- and N-system amino acids are almost as effective as ASN when added alone. In the present study, epidermal growth factor (EGF) was unable to increase ODC activity in cells maintained in a salt-glucose solution (Earles balanced salt solution). However, the addition of ASN (10 mM) in the presence of EGF (30 ng/ml) increased the activity of ODC 0.5- to 4-fold over that stimulated by ASN alone. EGF also showed induction of ODC with glutamine and α-aminoisobutyric acid, but ODC induction was maximum with ASN and EGF. Thus the mechanism of the interaction between ASN and EGF is important for understanding the regulation of ODC under physiological conditions. Therefore, we examined the expression of the ODC gene and those for several protooncogenes under the same conditions. Increased expression of the genes for c-Jun and c-Fos but not for ODC occurred with EGF alone. The addition of ASN did not further increase the expression of the protooncogenes, but the combination of EGF and ASN further increased the expression of ODC over that of ASN alone. Western analysis showed no significant difference in the level of ODC protein in Earles balanced salt solution, ASN, EGF, or EGF plus ASN. Addition of cycloheximide during ASN and ASN plus EGF treatment completely inhibited ODC activity without affecting the level of ODC protein. These results indicated that 1) the increased expression of protooncogenes in response to EGF is independent of increases in ODC activity and 2) potentiation between EGF and ASN on ODC activity may not be due to increased gene transcription but to posttranslational regulation and the requirement of ongoing protein synthesis involving a specific factor dependent on ASN.Our laboratory has shown that asparagine (ASN) stimulates both ornithine decarboxylase (ODC) activity and gene expression in an intestinal epithelial cell line (IEC-6). The effect of ASN is specific, and other A- and N-system amino acids are almost as effective as ASN when added alone. In the present study, epidermal growth factor (EGF) was unable to increase ODC activity in cells maintained in a salt-glucose solution (Earles balanced salt solution). However, the addition of ASN (10 mM) in the presence of EGF (30 ng/ml) increased the activity of ODC 0.5- to 4-fold over that stimulated by ASN alone. EGF also showed induction of ODC with glutamine and alpha-aminoisobutyric acid, but ODC induction was maximum with ASN and EGF. Thus the mechanism of the interaction between ASN and EGF is important for understanding the regulation of ODC under physiological conditions. Therefore, we examined the expression of the ODC gene and those for several protooncogenes under the same conditions. Increased expression of the genes for c-Jun and c-Fos but not for ODC occurred with EGF alone. The addition of ASN did not further increase the expression of the protooncogenes, but the combination of EGF and ASN further increased the expression of ODC over that of ASN alone. Western analysis showed no significant difference in the level of ODC protein in Earles balanced salt solution, ASN, EGF, or EGF plus ASN. Addition of cycloheximide during ASN and ASN plus EGF treatment completely inhibited ODC activity without affecting the level of ODC protein. These results indicated that 1) the increased expression of protooncogenes in response to EGF is independent of increases in ODC activity and 2) potentiation between EGF and ASN on ODC activity may not be due to increased gene transcription but to posttranslational regulation and the requirement of ongoing protein synthesis involving a specific factor dependent on ASN.

Regulation of transglutaminase activity by polyamines in the gastrointestinal mucosa of rats.

Abstract Transglutaminases catalyze the covalent cross-linking of protein and are involved in the mechanism of polyamine-dependent mucosal healing. The current study examined the effect of polyamines on transglutaminase activity in gastrointestinal mucosa. Rats were fasted 22 hr before experiments and enzyme activity was measured as the Ca++-dependent covalent incorporation of [3H]-putrescine into acid-precipitable protein. In some of the experiments, mucosal ornithine decarboxylase (ODC) activity and polyamine levels were also examined. Transglutaminase activity in both gastric and duodenal mucosa increased significantly after polyamine administration. Treatment with α-difluoromethylornithine (DFMO) decreased both basal ODC activity and putrescine levels in the duodenal mucosa. DFMO also significantly decreased mucosal transglutaminase activity. In stress or hypertonic NaCl-induced gastric mucosal injury models, increased polyamine biosynthesis was associated with increased transglutaminase activity, which was completely prevented by DFMO. Exogenous polyamines returned transglutaminase activity toward control levels in the presence of DFMO. In conclusion, these results indicate that: (i) luminal polyamines increase transglutaminase activity in gastric and duodenal mucosa; (ii) polyamine depletion caused by the inhibition of ODC is accompanied by a significant decrease in transglutaminase activity; and (iii) exogenous polyamines significantly reverse the decrease in transglutaminase activity caused by polyamine depletion.