Differences in transglutaminase mRNA after polyamine depletion in two cell lines

Jian-Ying Wang1, Mary Jane Viar2, Ji Li1, Hui-Jun Shi1, Anami R. Patel1, and Leonard R. Johnson2

1 Department of Surgery, University of Maryland Medical School and Veterans Affairs Medical Center, Baltimore, Maryland 21201; and 2 Department of Physiology and Biophysics, University of Tennessee College of Medicine, Memphis, Tennessee 38163

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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.

ornithine decarboxylase; IEC-6 cell line; Caco-2 cell line; posttranscription

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

TRANSGLUTAMINASES, A FAMILY of Ca2+-dependent enzymes, have attracted considerable interest in recent years because of their involvement in the posttranslational modification of proteins by promoting the formation of isopeptide bonds between glutamyl and lysyl residues (1, 5, 15). There are three biochemically and immunochemically different forms of transglutaminases in mammalian tissues: the extracellular form known as factor XIII, the membrane-bound intracellular enzyme, and the intracellular transglutaminase, which are encoded by three distinct genes (3, 6, 8, 12, 21, 34). The extracellular transglutaminase plays a role in the clotting reactions of plasma proteins (10, 34), and the expression of the membrane-bound intracellular enzyme is restricted to skin and squamous epithelia (6, 12). Although an exact role of the intracellular transglutaminase has not been established, increased activity of the cytosolic form of enzyme is implicated in a variety of phenomena, including changes in cellular morphology, growth arrest, terminal differentiation, apoptosis, and cellular adhesion (3, 10, 18, 23, 26).

The polyamines spermidine and spermine and their precursor, putrescine, are organic cations found in all eukaryotic cells and serve in both cellular and extracellular systems as natural substrates for transglutaminases (7, 13). In the normal gastrointestinal mucosa, increased tissue polyamine concentrations, either provided by ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, or supplied luminally, induce transglutaminase activity. Depletion of intracellular polyamines caused by the inhibition of ODC is accompanied by a significant decrease in enzyme activity (27, 32). These results strongly suggest that polyamines modulate the activity of the transglutaminase in gut mucosal epithelial cells. However, the precise molecular mechanisms responsible for the activation of transglutaminase by polyamines remain to be elucidated.

Using the standard biochemical method of measuring enzyme activity, we (17) recently demonstrated that depletion of intracellular polyamines by alpha -difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ODC, decreased the activity of cytosolic transglutaminase in rat small intestinal crypt cells (IEC-6) but increased the activity in human colon carcinoma cells (Caco-2). The present study tests the hypothesis that the effects of polyamines on the activities of transglutaminase in IEC-6 and Caco-2 cells are mediated by the modulation of levels of transglutaminase mRNA. We examined whether depletion of cellular polyamines by DFMO alters the levels of transglutaminase mRNA and whether the observed change in the mRNA levels is regulated posttranscriptionally in these cell lines. Some of these data have been published in abstract form (33).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals and supplies. Disposable culture ware was purchased from Corning (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were from GIBCO (Grand Island, NY). Biochemicals were purchased from Sigma (St. Louis, MO). The DNA probes used in these experiments included pGEM-32 containing mouse cytosolic transglutaminase cDNA (from Dr. Peter J. A. Davies, University of Texas, Houston, TX), pSVcmyc1 containing a rat genomic fragment coding for c-myc [American Type Culture Collection (ATCC) no. 41029], pBR322 containing mouse ODC cDNA (ATCC no. 63075), and pHcGAP containing human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (ATCC no. OM-11-904A). [alpha -32P]dCTP (3,000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Tritiated putrescine ([2,3-3H(N)]putrescine dihydrochloride; 1.2 TBq/mmol) was purchased from DuPont NEN (Boston, MA). DFMO was a gift from the Merrell Dow Research Institute of Marion Merrell Dow (Cincinnati, OH).

Cell culture and general experimental protocol. The IEC-6 cell line was purchased from the ATCC at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (24). IEC-6 cells are from intestinal crypt cells as judged by morphological and immunologic criteria, and are nontumorigenic, retaining the undifferentiated character of epithelial stem cells. Stock cells were maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 10 µg insulin, and 50 µg/ml gentamicin sulfate. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were subcultured once a week at 1:10; medium was changed three times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative. Passages 15-20 were used for all experiments. There were no significant changes of biological function and characterization from passage 15 to passage 20.

The Caco-2 cell line (a human colon carcinoma cell line) was obtained from ATCC at passage 16. It was maintained similarly to the IEC-6 cell line except that it was maintained in an atmosphere of 95% air-5% CO2. The medium used was Eagle's minimum essential medium (MEM) with 10% heat-inactivated FBS and 50 µg/ml gentamicin sulfate. The stock was passaged weekly at 1:5 and fed three times a week. Passages 18-23 were used for the experiments.

The general protocol of the experiments and the methods used were similar to those described previously (17, 29). The cells were detached from the flasks with 0.05% trypsin plus 0.53 mM Na4EDTA in Hanks' balanced salt solution without Ca2+ and Mg2+ and counted by hemocytometer. The IEC-6 cells were plated at 6.25 × 104 cells/cm2 in DMEM plus 5% dFBS plus 10 µg/ml insulin and 50 µg/ml gentamicin sulfate (supplemented DMEM). The Caco-2 cells were plated at 2.5 × 104 cells/cm2 in MEM containing 5% dFBS plus 10 µg/ml insulin and 50 µg/ml gentamicin sulfate (supplemented MEM).

In the first series studies, we examined the effect of polyamine depletion on levels of transglutaminase mRNA and enzyme activity in IEC-6 and Caco-2 cells. In most studies, one-half of the cultures contained 5 mM DFMO with or without spermidine (5 µM). Dosages were chosen from dose-response curves carried out previously (29). These cells were incubated in the same manner as the stock, and the media were changed every other day. At 6 and 14 days for IEC-6 cells or 8 and 15 days for Caco-2 cells, dishes were placed on ice, the monolayers were washed three times with ice-cold Dulbecco's phosphate-buffered saline (D-PBS), and then different solutions were added according to the assays to be conducted.

In the second series of studies, we selectively examined the mechanism possibly responsible for the observed change in transglutaminase mRNA following polyamine depletion in these two cell lines. The half-life of cytoplasmic mRNA for transglutaminase was measured in control and DFMO-treated cells. Actinomycin D (5 µg/ml) was added to cultures after cells had been exposed to 5 mM DFMO for 6 days in IEC-6 cells or for 8 days in Caco-2 cells. Control cells received actinomycin D without treatment with DFMO. Transglutaminase mRNA levels were assayed at different times after the addition of actinomycin D.

RNA isolation and Northern blot analysis. Total RNA was extracted with guanidinium isothiocyanate solution and purified by CsCl density gradient ultracentrifugation as described by Chirgwin et al. (4). Briefly, the monolayer of cells was washed in D-PBS and lysed in 4 M guanidinium isothiocyanate. The lysates were brought to 2.4 M CsCl concentration and centrifuged through a 5.7 M CsCl cushion at 150,000 g at 20°C for 24 h. After centrifugation, the supernatant was aspirated and the tube was cut ~0.5 cm from the bottom with a flamed scalpel. The resulting RNA pellet was dissolved in tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 7.5) containing 1 mM EDTA, 5% sodium laurylsarcosine, and 5% phenol (added just before use). The purified RNA was precipitated from the aqueous phase by the addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol in sequence. Final RNA was dissolved in water and estimated from its ultraviolet absorbance at 260 nm using a conversion factor of 40 units. In most cases, 30 µg total cellular RNA were denatured and fractionated electrophoretically, using a 1.2% agarose gel containing 3% formaldehyde, and transferred by blotting to nitrocellulose filters. Blots were prehybridized for 24 h at 42°C with 5× Denhardt's solution, 5× standard saline citrate (SSC), 50% formamide, 25 mM potassium phosphate, and 100 µg/ml denatured salmon sperm DNA. DNA probes for transglutaminase, ODC, c-myc, and GAPDH were labeled with [alpha -32P]dCTP using a standard nick translation procedure. All procedures were performed according to the supplier's instructions (DuPont NEN). Hybridization was carried out overnight at 42°C in the same solution containing 10% dextran sulfate and 32P-labeled DNA probes. Blots were washed in 1× SSC-0.1% sodium dodecyl sulfate (SDS) for 10 min at room temperature, followed by two washes of 0.25× SSC-0.1% SDS, one at 42°C and one at room temperature. After the final wash, the filters were subjected to autoradiography with intensifying screens at -70°C. The signals were quantitated by densitometry analysis of the autoradiographic results.

Measurement of transglutaminase activity. After three washes with ice-cold D-PBS, the cells were scraped into tubes in ice-cold collection buffer [10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 5 mM dithiothreitol (DTT) at pH 7.4], sonicated for 30 s on ice, and centrifuged at 51,000 g for 1 h at 40°C. The supernatant was used for the transglutaminase assay and protein determination. Protein was determined by the method of Bradford (2). The transglutaminase assay was a modification of the original method of Lorand et al. (14). Briefly, a reaction buffer consisting of 225 mM HEPES (pH 9.0) was prepared as the basis for a reaction mixture that contained (per tube) 50 mM HEPES, 1 mg/ml dimethylcasein, 1 mM CaCl2, 0.25 mM unlabeled putrescine, 3 µCi [3H]putrescine, 0.308 mg DTT, and water to a volume of 50 µl. The sample (50 µl) was then added so that the final volume was 100 µl. The tubes were incubated at 37°C for 15 min; 70 µl of the mixture were added to labeled 7-mm pieces of Whatman 3M filter paper, and the papers were kept in a beaker of cold 10% trichloroacetic acid (TCA) for 30 min with occasional mixing. After three washes in cold 5% TCA and two washes in 95% ethanol, the papers were dried overnight. Scintillation fluid was then added and the disintegrations per minute were counted 24 h later. The results were expressed as picomoles [3H]putrescine incorporated into dimethylcasein per hour per milligram protein.

Polyamine analysis. The cellular polyamine content was analyzed by high-performance liquid chromatography (HPLC) as described previously (29). In brief, after the monolayers had been washed three times with ice-cold D-PBS, 0.5 M perchloric acid was added, and the monolayers were frozen at -80°C until ready for dansylation, extraction, and HPLC. The standard curve encompassed 0.31-10 µM putrescine, spermidine, and spermine. Values that fell >25% below the curve were considered undetectable. Protein was determined by the Bradford method (2). The amount of polyamines was expressed as nanomoles per milligram of protein.

Statistics. All data are expressed as means ± SE from six dishes. Autoradiographic results were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined using the Dunnett's multiple range test (11).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of polyamine depletion on transglutaminase mRNA levels in IEC-6 and Caco-2 cells. Administration of DFMO completely depleted the intracellular polyamines putrescine and spermidine and dramatically decreased spermine in IEC-6 and Caco-2 cells. As shown in Table 1, the presence of 5 mM DFMO in IEC-6 cells decreased intracellular putrescine and spermidine content to undetectable levels on days 6 and 14. Spermine was less sensitive to the inhibition of ODC but was decreased by 56% on day 6 and by 81% on day 14. Approximately the same effects were produced in Caco-2 cells by administration of DFMO. The levels of putrescine and spermidine were decreased by 100% on days 8 and 15 after DFMO treatment. Spermine was decreased by ~55% on days 8 and 15 in Caco-2 cells treated with DFMO.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of DFMO on intracellular polyamine concentrations in IEC-6 and Caco-2 cells

In control IEC-6 cells, steady-state levels of transglutaminase mRNA significantly increased from day 2 and remained significantly elevated for >6 days after initial plating (Fig. 1A and 2). By day 14, transglutaminase mRNA returned to near basal levels. In the same cells, ODC mRNA levels increased on day 2 and then returned to normal. On the other hand, depletion of intracellular polyamines by DFMO (5 mM) not only prevented the increased mRNA for transglutaminase but also significantly reduced levels of transglutaminase mRNA below basal levels (Figs. 1B and 2). The lowest levels of transglutaminase mRNA occurred between 6 and 14 days in the DFMO-treated cells and were almost undetectable. The mRNA levels of ODC in DFMO-treated cells increased 4 days after polyamine depletion and peaked between 8 and 14 days. There were no changes in GAPDH mRNA levels in IEC-6 cells grown in the presence or absence of DFMO.


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of expression of transglutaminase (TGase) and ornithine decarboxylase (ODC) genes in IEC-6 cells treated without (control; A) and with (B) 5 mM alpha -difluoromethylornithine (DFMO). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 5% dialyzed fetal bovine serum (dFBS), and medium was changed every 2nd day. RNA was harvested at different time points and determined by Northern blotting analysis. Total cellular RNA (30 µg) was blotted and hybridized with 32P-labeled transglutaminase and ODC DNA probes. Hybridization to labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe served as a marker for equal loading of lanes.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Quantitative analysis of Northern blots by densitometry from IEC-6 cells described in Fig. 1. A: transglutaminase. B: ODC. Values are means of 3 separate experiments, and measured variability is <5%. Relative levels of mRNA species were corrected for RNA loading as measured by densitometry of GAPDH. * P < 0.05 compared with control groups.

In control Caco-2 cells (without DFMO), the levels of mRNAs for transglutaminase and ODC significantly increased on day 2 and then gradually decreased with the time after initial plating (Figs. 3 and 4). In contrast to the effects on IEC-6 cells, depletion of intracellular polyamines was associated with an increased level of transglutaminase mRNA in Caco-2 cells. Transglutaminase mRNA increased significantly on day 4, and the maximum increase observed on days 6 to 15 after exposure to DFMO was ~8-9 times the control level (Fig. 4). The levels of ODC mRNA in Caco-2 cells also increased after depletion of intracellular polyamines. The maximum increase in ODC mRNA levels occurred on day 15 after exposure to DFMO and was 4.5 times control. Polyamine depletion had no effect on the levels of GAPDH mRNA in Caco-2 cells.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 3.   Time course of expression of transglutaminase and ODC genes over 15 days in Caco-2 cells in control cultures (A) and cultures in which polyamines were depleted with 5 mM DFMO (B). Cells were grown in DMEM containing 5% dFBS, and medium was changed every 2nd day. Total cellular RNA (30 µg) was analyzed by Northern blotting, using transglutaminase and ODC DNA probes. Loading of RNA was monitored by hybridization to labeled GAPDH probe.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Quantitative analysis of Northern blots by densitometry from Caco-2 cells described in Fig. 3. A: transglutaminase. B: ODC. Values are means of 3 separate experiments, and measured variability is <5%. Relative levels of mRNA species were corrected for RNA loading as measured by densitometry of GAPDH. * P < 0.05 compared with control groups.

Effect of exogenous spermidine on transglutaminase mRNA levels. To exclude nonspecific effects of DFMO, we examined whether administration of exogenous spermidine substitutes for intracellular polyamines in the regulation of expression of transglutaminase in the DFMO-treated cells. Levels of protooncogene c-myc and ODC mRNA served as down- and upregulated controls, respectively, in polyamine-deficient cells. Figure 5 shows that exposure to 5 mM DFMO for 6 and 14 days significantly decreased the steady-state levels of transglutaminase and c-myc mRNAs but increased the level of ODC mRNA in IEC-6 cells. The addition of spermidine (5 µM) completely reversed the effects of DFMO on the mRNA levels of these genes. The reduced mRNA levels of transglutaminase and c-myc in cells treated with DFMO returned toward control levels in the presence of spermidine. The increased levels of ODC mRNA in DFMO-treated cells also returned to normal when spermidine was added.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of exogenous polyamines on expression of transglutaminase, ODC, and c-myc in IEC-6 cells treated with either DFMO (5 mM) or DFMO + spermidine (SPD; 5 µM) for 6 or 14 days. Total cellular RNA (30 µg) was analyzed by Northern blotting, using transglutaminase, ODC, and c-myc DNA probes. Loading of RNA was monitored by hybridization to labeled GAPDH probe.

In Caco-2 cells, polyamine depletion by DFMO increased transglutaminase and ODC mRNA levels and decreased the levels of c-myc mRNA (Fig. 6). Addition of exogenous spermidine to DFMO-treated cells significantly prevented the increases in transglutaminase and ODC mRNA and overcame the inhibitory effect on the levels of c-myc mRNA. When IEC-6 and Caco-2 cells were grown under standard culture conditions (without DFMO), the level of transglutaminase mRNA was not altered by addition of 5 µM exogenous spermidine (data not shown).


View larger version (K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of exogenous polyamines on expression of transglutaminase, ODC, and c-myc in Caco-2 cells treated with either DFMO (5 mM) or DFMO + spermidine (5 µM) for 8 or 15 days. Total cellular RNA (30 µg) was analyzed as described. Loading of RNA was monitored by hybridization to labeled GAPDH probe.

We also examined the effect of polyamine depletion on the levels of transglutaminase mRNA in HT-29 cell line, another line of human colon carcinoma cells. Administration of 5 mM DFMO completely inhibited ODC activity and depleted cellular polyamines in HT-29 cells (data not shown). In general, the response of expression of the transglutaminase to polyamine depletion in HT-29 cells was similar to that observed in Caco-2 cells exposed to DFMO. Treatment with DFMO for 6 and 12 days significantly increased the level of transglutaminase mRNA, which was completely prevented by exogenous spermidine (Fig. 7).


View larger version (67K):
[in this window]
[in a new window]
 
Fig. 7.   Expression of transglutaminase gene in HT-29 cells treated with either DFMO (5 mM) or DFMO + spermidine (5 µM) for 6 and 12 days. HT-29 cells were purchased from American Type Culture Collection and grown in McCoy's 5a medium modified to contain 10% dFBS, and medium was changed every 2nd day. Total cellular RNA (30 µg) was analyzed by Northern blotting, using transglutaminase cDNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. Three experiments were performed that showed similar results.

Effect of polyamines on the activity of transglutaminase. Because our previous studies (17) and others (6) have demonstrated that the amount of transglutaminase protein correlates well with the increase in transglutaminase activity, we examined the effect of polyamine depletion on the enzyme protein by measurement of transglutaminase activity. Consistent with the effect on mRNA levels, depletion of intracellular polyamines by DFMO lowered transglutaminase activity in IEC-6 cells, which was prevented if the cells were given spermidine with the DFMO (Fig. 8A). The activity of transglutaminase in Caco-2 cells, on the other hand, was significantly increased after exposure to DFMO for 8 and 15 days. Administration of spermidine prevented the increase in enzyme activity caused by polyamine depletion (Fig. 8B). Enzyme activity was not altered by the addition of spermidine to cultures in which cells were grown in the standard growth media (without DFMO).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8.   Transglutaminase enzyme activity in both IEC-6 cells (A) and Caco-2 cells (B) in control cultures (Con), cultures in which ODC was inhibited with 5 mM DFMO, and cultures that were inhibited but to which 5 µM spermidine had been added. Values are means ± SE from 6 flasks. * P < 0.05 compared with control groups.

Effect of polyamine depletion on the stability of transglutaminase mRNA. This study tested the possibility that intracellular polyamines play a role in posttranscriptional regulation of transglutaminase expression and that changes in steady-state levels of transglutaminase mRNA following polyamine depletion result from the alteration of the mRNA stability in IEC-6 and Caco-2 cells. We compared the half-life of transglutaminase mRNA in control cells with that of cells treated with DFMO. In IEC-6 cells, the levels of mRNA for transglutaminase declined rapidly after the addition of actinomycin D. There was no significant difference in the stability of mRNA for transglutaminase between control and DFMO-treated cells. The half-life of transglutaminase mRNA in control cells and cells treated with DFMO for 6 days were ~45 and 41 min, respectively (Fig. 9). These slight differences were not statistically significant. We also measured the half-lives of mRNA for transglutaminase in cells treated with DFMO for 14 days and demonstrated that the results were identical to those observed after 6 days of treatment (data not shown).


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 9.   Cytoplasmic half-life studies of transglutaminase mRNA from IEC-6 cells grown in absence or presence of DFMO. Cells were untreated (control; A) or treated with 5 mM DFMO for 6 days (B) and were incubated further with 5 µg/ml actinomycin D for indicated times. Total cellular RNA (30 µg) was analyzed by Northern blotting, using transglutaminase cDNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. Three experiments were performed that showed similar results.

In Caco-2 cells, however, the half-life of transglutaminase mRNA was dramatically extended after polyamine depletion. As can be seen in Figs. 10 and 11, basal levels of transglutaminase mRNA in control cells decreased after the addition of actinomycin D, with a half-life of 56 min. In cells exposed to DFMO for 8 days, transglutaminase mRNA decreased at a slower rate in the presence of actinomycin D, with a half-life of ~430 min. The prolonged half-life of transglutaminase mRNA in the DFMO-treated cells was prevented when spermidine (5 µM) was given together with DFMO. The half-life of transglutaminase mRNA in cells grown in the presence of DFMO plus spermidine for 8 days was indistinguishable from that of control cells. The half-life of transglutaminase mRNA in cells treated with DFMO for 15 days was identical to that in cells treated for 8 days (data not shown).


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 10.   Cytoplasmic half-life studies of transglutaminase mRNA in Caco-2 cells untreated (control; A) or treated with DFMO (B) or DFMO + spermidine (C). After cells had been grown in presence of 5 mM DFMO with or without spermidine (5 µM) for 8 days, actinomycin D (5 µg/ml) was given. Total cellular RNA was analyzed after addition of actinomycin D by Northern blotting, using transglutaminase cDNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 11.   Percent transglutaminase mRNA remaining in Caco-2 cells described in Fig. 10. Autoradiographs of RNA blots were quantitated by densitometric scanning and normalized with respect to density of GAPDH. Values are means ± SE from 3 separate experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The current results clearly show that intracellular polyamines are involved in the regulation of transglutaminase expression at the mRNA level in IEC-6 and Caco-2 cells and that these two cell lines respond differently to polyamines. Depletion of polyamines by DFMO in IEC-6 cells is associated with a decrease in levels of mRNA for transglutaminase (Figs. 1 and 2). Treatment with DFMO, however, increases transglutaminase mRNA levels and enzyme activity in Caco-2 cells (Figs. 3 and 4). The opposite effect of polyamine depletion is specific to transglutaminase expression, since the levels of ODC mRNA increase but protooncogene c-myc mRNA decreases in both cell lines. Although there is no significant difference in the stability of transglutaminase mRNA between control and DFMO-treated IEC-6 cells (Fig. 9), the half-life of the mRNA is dramatically increased following polyamine depletion in Caco-2 cells (Figs. 10 and 11). These results indicate that polyamines modulate the activation of various genes through different pathways and that their effects are cell type dependent. Increased level of transglutaminase mRNA in polyamine-deficient Caco-2 cells is predominantly regulated at the posttranscription level.

Although the physiological roles of transglutaminases have been extensively studied for many years (1, 3, 8, 10, 18, 21), little is known about the regulation of the expression of their genes. We are particularly interested in the effect of polyamines on the regulation of expression of the tissue transglutaminase in gastrointestinal epithelial cells, since polyamines are absolutely required for the normal repair of gastric and duodenal mucosal stress ulcers and since increased transglutaminase and protein cross-linking following increased polyamine biosynthesis is one of the mechanisms requiring polyamines for healing of the damaged mucosa (27, 31). We (32) have recently demonstrated that tissue transglutaminase activity in both gastric and duodenal mucosa increases significantly after oral administration of polyamines. Inhibition of polyamine synthesis by DFMO decreases the enzyme activity in the mucosa. In stress or hypertonic NaCl-induced mucosal injury models, increased polyamine synthesis is associated with increased transglutaminase activity, which is completely prevented by DFMO. Furthermore, depletion of intracellular polyamines by DFMO significantly decreases the activity of transglutaminase in cultured IEC-6 cells but increases the enzyme activity in Caco-2 cells (17). These results strongly suggest that polyamines play a role in the regulation of transglutaminase activity in the gastrointestinal mucosa, but the mechanism of the action of polyamines at the molecular level is still unknown.

The current study is the first report indicating that the effects of polyamines on the activity of transglutaminase are mediated at the level of mRNA in a normal small intestinal crypt cell line and a human colon carcinoma cell line. As can be seen in Figs. 1 and 2, the administration of DFMO prevents the increased levels of transglutaminase mRNA during the early phase after plating and significantly reduces the basal levels of the enzyme mRNA thereafter. In contrast to effects on the transglutaminase gene, the mRNA levels of ODC in DFMO-treated IEC-6 cells are increased. The reduced mRNA levels of transglutaminase in cells treated with DFMO are returned toward control levels when spermidine is given (Fig. 5). This finding indicates that the expression of transglutaminase in normal small intestinal crypt cells requires polyamines and demonstrates that exogenous spermidine effectively substitutes for intracellular polyamines in the modulation of the expression of transglutaminase. In Caco-2 cells, however, polyamine depletion has the opposite effect on the levels of mRNA for transglutaminase. In these cells, polyamines appear to downregulate the expression of transglutaminase.

The reasons for the different responses in expression of transglutaminase to polyamine depletion in IEC-6 and Caco-2 cells remain obscure and may be related to the fact that IEC-6 cells are derived from the small intestine and Caco-2 cells are from the colon. Although we did not examine the effect of polyamine depletion on the expression of transglutaminase in all types of cells available from small intestine and colon, the results presented in Fig. 7 clearly show that depletion of cellular polyamines also significantly increases levels of mRNA for transglutaminase in the HT-29 cell line, another line derived from the colon. On the other hand, there is no doubt that the difference in their responses does not result from growth inhibition, because IEC-6 cells have a pattern of growth identical to that observed in Caco-2 cells and because normal cell growth of both cell lines is dependent on polyamines (17, 29) in the same manner as in other eukaryotic cells (16, 28, 30).

As shown in Fig. 9, polyamine depletion has no effect on the stability of transglutaminase mRNA in IEC-6 cells, indicating that the downregulation of transglutaminase expression following polyamine depletion is not regulated posttranscriptionally in small intestinal crypt cells. Polyamines influence chromatin structure and sequence-specific DNA-protein binding activities that effectively alter the rate of transcription of some genes (9, 19). Panagiotidis et al. (21) have demonstrated that polyamines at physiological concentrations specifically enhance the binding of several proteins including upstream stimulating factor, transcription factor E3, immunoglobulin/enhacer binding protein, nuclear factor-interleukin-6, and Yin Yang binding site to DNA, but inhibit others such as octamerbinding protein 1 in cell-free systems. We (22) have recently reported that polyamines are involved in the regulation of the transcription of c-myc and c-jun in IEC-6 cells but have no effect on the posttranscriptional regulation of the mRNAs for these two genes in IEC-6 cells. It is postulated that changes in intranuclear polyamines may provide an ionic environment that affects the binding of transcriptional factors. Clearly additional studies are necessary at the transcriptional level to fully understand regulation of transglutaminase gene by polyamines in IEC-6 cells.

Figures 10 and 11 show that posttranscriptional stabilization appears to be the predominant factor increasing the levels of transglutaminase mRNA in Caco-2 cells exposed to DFMO. Polyamine depletion increases the half-life of the induced transglutaminase mRNA by more than seven times in the presence of actinomycin D. Although the exact mechanism involved in the process of mRNA turnover in general is unknown, several studies have indicated that many transiently expressed genes contain destabilizing sequences in the 3' untranslated regions of mRNAs, which are related to the maintenance of the mRNA stability (25). Whether there are destabilizing sequences in the 3' untranslated regions of transglutaminase mRNA and whether the action of polyamines involves this sequence in Caco-2 cells remain to be elucidated.

In summary, these results indicate that intracellular polyamines are involved in the regulation of transglutaminase expression at the level of mRNA in a rat small intestinal crypt cell line and a human colon carcinoma cell line. However, polyamines play completely different roles in the modulation of transglutaminase expression in these two cell lines. Inhibition of polyamine biosynthesis by DFMO is associated with a decrease in the mRNA levels of transglutaminase in IEC-6 cells but with a significant increase in the mRNA in Caco-2 cells. Although polyamine depletion has no effect on the stability of transglutaminase mRNA in IEC-6 cells, the half-life of the mRNA is increased sevenfold in Caco-2 cells exposed to DFMO. These results indicate that polyamines modulate the expression of transglutaminase through different pathways in these two cell lines and that increased levels of mRNA for transglutaminase in polyamine-deficient Caco-2 cells predominantly result from a delay in the rate of mRNA degradation.

    ACKNOWLEDGEMENTS

We thank Dr. Barbara L. Bass for helpful discussion, Dr. Peggy Swoveland for critically reading the manuscript, and Jordan Denner for illustrations.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R29-DK-45314 and P01-DK-37260.

Address for reprint requests: J.-Y. Wang, Dept. of Surgery, Baltimore VA Medical Center and University of Maryland, 10 N. Greene St., Baltimore, MD 21201.

Received 15 August 1996; accepted in final form 6 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Beninati, S., M. Piacentini, M. P. Argento-Ceru, S. Russo-Caia, and F. Autuori. Presence of di- and polyamines covalently bound to protein in rat liver. Biochim. Biophys. Acta 841: 120-126, 1985[Medline].

2.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

3.   Chiocca, E. A., P. J. A. Davies, and J. P. Stein. The molecular basis of retinoic acid action: transcriptional regulation of tissue transglutaminase gene expression in macrophages. J. Biol. Chem. 263: 11584-11589, 1988[Abstract/Free Full Text].

4.   Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299, 1979[Medline].

5.   Davies, P. J. A., E. A. Chiocca, J. P. Basilion, S. Poddan, and J. P. Stein. Transglutaminases and their regulation: implications for polyamine metabolism. Adv. Exp. Med. Biol. 250: 391-401, 1988[Medline].

6.   Floyd, E. E., and A. M. Jetten. Regulation of type I (epidermal) transglutaminase mRNA levels during squamous differentiation: down regulation by retinoids. Mol. Cell. Biol. 9: 4846-4851, 1989[Medline].

7.   Folk, J. E., M. H. Park, S. I. Chung, J. Schrode, E. P. Lester, and H. L. Copper. Polyamines as physiological substrates for transglutaminases. J. Biol. Chem. 255: 3695-3700, 1980[Abstract/Free Full Text].

8.   Gentile, V., V. Thomazy, M. Piacentini, L. Fesus, and P. J. A. Davies. Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion. J. Cell Biol. 119: 463-474, 1992[Abstract].

9.   Gross, D. S., and W. T. Garrard. Nuclease hypersensitive sites in chromatin. Annu. Rev. Biochem. 57: 159-197, 1988[Medline].

10.   Haddox, M. K., and D. H. Russel. Increased nuclear conjugated polyamines and transglutaminase during liver regeneration. Proc. Natl. Acad. Sci. USA 78: 1712-1716, 1981[Abstract].

11.   Harter, J. L. Critical values for Duncan's new multiple range test. Biometrics 16: 671-685, 1960.

12.   Ichinose, A., L. E. Hendrickson, K. Fujikawa, and E. W. Davie. Amino acid sequence of the subunit of human factor XIII. Biochemistry 25: 6900-6906, 1986[Medline].

13.   Janne, J., L. Alhonen, and P. Leinonen. Polyamines: from molecular biology to clinical applications. Ann. Med. 23: 241-259, 1991[Medline].

14.   Lorand, L., L. K. Campbell-Wilkes, and L. Cooperstein. A filter paper assay for transamidating enzymes using radioactive amine substrates. Anal. Biochem. 50: 623-631, 1972[Medline].

15.   Lorand, L., and S. M. Conrad. Transglutaminases. Mol. Cell. Biochem. 58: 9-35, 1984[Medline].

16.   McCormack, S. A., and L. R. Johnson. Role of polyamines in gastrointestinal mucosal growth. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G795-G806, 1991[Abstract/Free Full Text].

17.   McCormack, S. A., J.-Y. Wang, M. J. Viar, L. T. Tague, P. J. A. Davies, and L. R. Johnson. Polyamines influence transglutaminase activity and cell migration in two cell lines. Am. J. Physiol. 267 (Cell Physiol. 36): C706-C714, 1994[Abstract/Free Full Text].

18.   Melino, G., M. Annicchiarico-Petruzzelli, L. Piredda, E. Candi, V. Gentile, P. J. A. Davies, and M. Piacentini. Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells. Mol. Cell. Biol. 14: 6584-6596, 1994[Abstract].

19.   Moore, J. J., D. W. Lungren, R. M. Moore, and B. Andersen. Polyamines control human chorionic gonadotropin production in the JEG-3 choriocarcinoma cell. J. Biol. Chem. 263: 12765-12769, 1988[Abstract/Free Full Text].

20.   Nara, K., K. Nakanishi, H. Hagiwara, K.-I. Wakita, S. Kojima, and S. Hirose. Retinol-induced morphological changes of cultured bovine endothelial cells are accompanied by a marked increase in transglutaminase. J. Biol. Chem. 264: 19308-19312, 1989[Abstract/Free Full Text].

21.   Panagiotidis, C. A., S. Artandi, K. Calame, and S. J. Silverstein. Polyamines alter sequence-specific DNA-protein interactions. Nucleic Acids Res. 23: 1800-1809, 1995[Abstract].

22.   Patel, A. R., and J.-Y. Wang. Polyamines modulate the transcription but not posttranscription of c-myc and c-jun in IEC-6 cells. Am. J. Physiol 273 (Cell Physiol. 42): C1020-C1029, 1997[Abstract/Free Full Text].

23.   Piacentini, M., F. Autuori, L. Dini, M. G. Farrace, L. Ghibelli, L. Piredda, and L. Fesus. "Tissue" transglutaminase is specifically expressed in neonatal rat liver cells undergoing apoptosis upon epidermal growth factor-stimulation. Cell Tissue Res. 263: 227-235, 1991[Medline].

24.   Quaroni, A., J. Wands, R. L. Trelstad, and K. J. Isselbacher. Epithelial cell cultures from rat small intestine. J. Cell Biol. 80: 248-265, 1979[Abstract].

25.   Steeg, H. V., C. T. M. V. Oostrom, H. M. Hodemaekers, and C. F. V. Kreyl. Cloning and functional analysis of the rat ornithine decarboxylase-encoding gene. Gene 93: 249-256, 1990[Medline].

26.   Trejo-Skalli, A. V., P. T. Velasco, S. N. P Murthy, and L. Lorand. Association of a transglutaminase-related antigen with intermediate filaments. Proc. Natl. Acad. Sci. USA 92: 8940-8944, 1995[Abstract].

27.   Wang, J.-Y., and L. R. Johnson. Role of transglutaminase and protein cross-linking in the repair of mucosal stress erosions. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G818-G825, 1992[Abstract/Free Full Text].

28.   Wang, J.-Y., S. A. McCormack, and L. R. Johnson. Role of nonmuscle myosin II in polyamine-dependent intestinal epithelial cell migration. Am. J. Physiol. 270 (Gastrointest. Liver Physiol. 33): G355-G362, 1996[Abstract/Free Full Text].

29.   Wang, J.-Y., S. A. McCormack, M. J. Viar, H. Wang, C.-Y. Tzen, R. E. Scott, and L. R. Johnson. Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G331-G338, 1993[Abstract/Free Full Text].

30.   Wang, J.-Y., and K. Takeuchi. Polyamines in gastrointestinal mucosal cell proliferation. J. Gastrointest. Res. 4: 86-92, 1996.

31.   Wang, J.-Y., M. J. Viar, and L. R. Johnson. Transglutaminase in response to hypertonic NaCl-induced gastric mucosal injury in rats. Gastroenterology 104: 65-74, 1993[Medline].

32.   Wang, J.-Y., M. J. Viar, and L. R. Johnson. Regulation of transglutaminase activity by polyamines in the gastrointestinal mucosa of rats. Proc. Soc. Exp. Biol. Med. 205: 20-28, 1994[Abstract].

33.   Wang, J.-Y., M. J. Viar, and L. R. Johnson. Different responses of transglutaminase (TGA) gene expression to polyamine depletion in two cell lines (Abstract). FASEB J. 10: A761, 1996.

34.   Williams-Ashman, H. G. Transglutaminases and the clotting of mammalian seminal fluids. Mol. Cell. Biochem. 58: 51-61, 1984[Medline].


AJP Cell Physiol 274(2):C522-C530
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society