Effects of epidermal growth factor on diamine expression and cell growth in Caco-2 cells BRUNO

DANIELE

AND

ANDREA

oxidase

QUARONI

Section of Physiology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853

DANIELE, BRUNO, AND ANDREA QUARONI. Effects of epiderma1 growth factor on diamine oxidase expression and cell growth in Caco-2 cells. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G669-G676, 1991.-To investigate the role of diamine oxidase (DAO) in the intestinal mucosa, we compared its expression with cell proliferation and differentiation in the human colon carcinoma cell line Caco-2. DA0 synthesis was evaluated in subconfluent and confluent cultures and in the presence of epidermal growth factor (EGF), a polypeptide hormone known to have specific trophic effects on the small intestinal mucosa. EGF stimulated DNA synthesis, significantly increased cellular DA0 activity and the amount of enzyme secreted into the culture medium, but decreased expression of dipeptidyl peptidase IV, a marker of cell differentiation in confluent Caco-2 cells. Immunoprecipitation of DA0 from cells labeled metabolically with [““Slmethionine failed to demonstrate an increased enzyme synthesis in EGF-treated cells, suggesting that this hormone acted primarily at a posttranslational level by reducing DA0 degradation before intracellular storage or secretion. A possible relationship between changes in cellular DA0 activity and cell proliferation was also investigated by using aminoguanidine, a specific and potent DA0 inhibitor. Although DA0 activity was markedly suppressed, aminoguanidine had no significant effects on the rate of DNA synthesis. These results demonstrated that in Caco-2 cells EGF stimulated DNA synthesis and DA0 expression; however, cell proliferation and differentiation were not correlated with the levels of cellular DAO, suggesting that this enzyme does not play a major role in the regulation of intestinal epithelial cell turnover.

polyamines;

dipeptidyl

peptidase

IV

SPERMIDINE, spermine, and their precursor putrescine are ubiquitous low-molecular-weight polyamines whose concentrations within cells are tightly regulated. They have been implicated in the control of cell proliferation and differentiation in several tissues and cultured cells (29), where they have been shown to promote synthesis of nucleic acids and proteins (17, 22). The rate-limiting step in the biosynthesis of polyamines is the decarboxylation of ornithine by ornithine decarboxylase (ODC) to form putrescine. The catabolism of polyamines depends on interconversion and terminal oxidation: spermine can be converted into spermidine, and spermidine into putrescine (29); after terminal oxidation, the products of these reactions cannot be reconverted into polyamines and are irreversibly eliminated from their metabolic cycle (37). The enzyme diamine oxidase (DAO; diamine-oxygen 0193~1857/91

$1.50

Copyright

0 1991

oxidoreductase, EC 1.4.3.6) catalyzes the oxidation of putrescine (4) and is therefore involved in the regulation of its cellular concentration. The observation of high levels of DA0 activity in several tumors, cancer cell lines, and in normal tissues containing rapidly proliferating cell types (36) has led to the suggestion that this enzyme may play an important role in growing cells and tissues. DA0 is unevenly distributed in vertebrates and, since the small intestine is by far the organ with the highest activity (39), functional interrelations have been suspected between this enzyme and the rapid cell turnover characteristic of the epithelial cells in the adult intestinal mucosa. However, most of the immunologically detectable DA0 was localized in the upper region of the intestinal villi, mainly associated with the basolateral aspect of the absorptive cells (9), rather than with the crypt cells. These findings, together with the well-known ability of heparin to release the enzyme into the bloodstream (15), seem to contradict a possible role of DA0 in the regulation of polyamine metabolism in the rapidly proliferating crypt cells. To further examine the proposed role of DA0 in the regulation of intestinal cell proliferation and differentiation, we used the Caco-2 cell line as an experimental model. These cells have been characterized extensively and have been shown, upon reaching confluence, to be highly polarized with a well-formed brush border and to express several differentiated markers typical of adult enterocytes (16, 31, 33). They also behave like small intestinal villous cells with respect to DA0 expression (9): DA0 activity in Caco-2 cells was found to be very low during the proliferative phase, but to increase markedly as the cells reached confluence and cell proliferation was greatly reduced (7). Secretion of DA0 by confluent Caco-2 cells closely resembles the analogous process taking place in the intestinal mucosa in vivo, both in its predominantly basolateral direction and in its marked stimulation by heparin added to the basal culture medium (9). In this study, cell proliferation, expression of the differentiation-specific marker dipeptidyl peptidase IV (DPPIV), DA0 levels, and DA0 synthesis were compared in cells at different stages of proliferation and differentiation in the presence of epidermal growth factor (EGF) and of the specific DA0 inhibitor aminoguanidine. EGF has well-known growth stimulatory effects on many cell types in culture (5), and high-affinity EGF receptors have been demonstrated on Caco-2 cells (18). There is also increasing evidence for a role of this horthe American

Physiological

Society

G669

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G670

EFFECTS

OF

EGF

ON

INTESTINAL

mone in the development of the gut, where it has been shown to significantly alter expression of digestive enzymes, such as lactase, sucrase, trehalase, and glucoamylase (1, 2, 14, 21, 24-27, 32, 35). EGF given intraperitoneally was also found to stimulate ODC and S-adenosylmethionine activities (key enzymes in polyamine biosynthesis) along the entire crypt-villus column of adult rats (13). The results obtained in this study demonstrated significant effects of EGF on DA0 expression by Caco-2 cells, but no correlation could be made between enzyme levels and proliferation or differentiation of these intestinal cells. MATERIALS

AND

METHODS’

Materials. The Caco-2 cell line was obtained from the American Type Culture Collection (Rockville, MD). It was cloned by dilution plating, and a clone (clone 5) producing and secreting very small amounts of TGF-cu/ EGF-like activity (Cl0 ng/lO’ cells over a 24-h period, J. F. Beaulieu and A. Quaroni, unpublished observations) was used in these studies. Dulbecco’s modified Eagle’s Medium (DMEM), fetal bovine serum, and penicillinstreptomycin mixture were from Whittaker Bioproducts (Walkersville, MD). L-Glutamine was from Aldrich (Milwaukee, WI). EGF and putrescine dihydrochloride were obtained from Sigma (St. Louis, MO). Methylated 14Clabeled molecular weight markers [carbonic anhydrase, bovine serum albumin (BSA), phosphorylase b, globulins, and myosin], L-[35S]methionine (1,083-1,151 Ci/mmol), and [3H]thymidine (20 Ci/mmol) were obtained from New England Nuclear (Boston, MA). [ 1,4-14C]putrescine dihydrochloride (100-120 mCi/mmol) was obtained from Amersham (Arlington Heights, IL). Transwell clusters, 24.5 mm in diameter (surface area, 4.71 cm2) with a 3pm pore size, were from Costar (Cambridge, MA). AffiGel 10 beads, acrylamide, bis-acrylamide, and Dowex AG 5OW-X4 resin were from Bio-Rad (Richmond, CA). Sequenal grade sodium dodecyl sulfate (SDS) was from Pierce (Rockford, IL). Antibodies. The mouse monoclonal antibodies DAO-1 (9), produced against DA0 purified from the conditioned culture medium of Caco-2 cells, and DAO-7/219, specific for human DPPIV, have been described and characterized elsewhere. Both antibodies are of the IgGl subtype and were used in this study as immunoglobulins purified from ascites fluids by affinity chromatography on a Sepharose 4B-protein A column (33). Cell culture. The Caco-2 cells were routinely cultured in loo-mm diameter Petri dishes (Falcon; Becton, Dickinson Labware, Oxnard, CA) at 37°C in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 50 U/ml penicillin, and 50 ,ug/ml streptomycin in an atmosphere of 5% C02-95% air. They were refed every other day with 10 ml of fresh medium and were subcultured serially when -80% confluent. Cells to be used for measurement of DA0 and DPPIV [3H] thymidine incorporation into cellular activities, DNA, and metabolic labeling with [35S]methionine were seeded at a density of 8 x lo6 cells per filter in Transwell

DA0

AND

CELL

PROLIFERATION

clusters; 2.6 ml of medium per well was added to the cluster plates (lower chamber volume) and 1.5 ml medium was added to the Transwells (upper chamber volume). When required, the culture medium was supplemented with 50 rig/ml EGF or 10 PM aminoguanidine. DA0 and DPPIV assays. DA0 was assayed in the medium and in total cell homogenate by a previously described modification (9) of the method of Okuyama and Kobayashi (28); 1 mU of DA0 activity was defined as the amount of enzyme oxidizing 1 nmol of putrescine per hour at 37°C and at pH 7.2 (28). DPPIV was measured with glycyl-proline-p-nitroanilide-p-tosylate as substrate as described elsewhere (16). Total protein in the homogenates was determined by the method of Lowry et al. (20). Cell proliferation and DNA assays. Cell proliferation was assessed by measuring incorporation of [3H]thymidine into cellular DNA. Caco-2 cells were incubated for 1 h at 37°C with complete medium containing 4 &i/ml of [3H]thymidine; then the medium was aspirated, the cells were washed two times with PBS for 2 min, and subsequently fixed and extracted twice (10 min each time) with 5% trichloroacetic acid. After two washes with distilled water (5 min each) the cells were detached by scraping and solubilized in 1 ml of 1 N NaOH. After neutralization with 1 M acetic acid, the extracts were added to 10 ml Aquasol(New England Nuclear) and counted in a Beckman LS-3800 scintillation counter. DNA was assayed in total cell homogenates using a method based on the enhancement of fluorescence seen when bis-benzimidazole (Hoechst 33258) binds to DNA (19). Cells were homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) in phosphate-saline buffer (50 mM sodium phosphate, 2 M NaCl, pH 7.4). An aliquot of the homogenate (usually lo-50 ~1) was added to 2 ml of 10 mM tris(hydroxymethyl)aminomethane (Tris) l HCl buffer, 100 mM NaCl, pH 7.4, containing 0.1 pg/ml of Hoechst 33258 dye. Calf thymus DNA was used as a standard. Fluorescence was measured with a minifluorimeter (TKO 100, Hoefer Scientific Instruments, San Francisco, CA) according to the protocol suggested by the manufacturer. Labeling of Caco-2 cells with radioactive methionine.

Caco-2 cells cultured on Transwell filters were rinsed twice with methionine-free medium (DMEM methionine-free, GIBCO, Grand Island, NY) containing 5% dialyzed fetal calf serum, 10 mM HEPES, 2 mM Lglutamine, 50 U/ml penicillin, 50 pg/ml streptomycin, and incubated 1 h with the same medium to deplete intracellular methionine pools. Then fresh medium containing 50 &i/ml of [35S]methionine was added to both basal and apical chambers, and the Transwells were incubated at 37°C for 45-60 min (pulse-chase experiments) or 2-7 h (other metabolic labeling experiments). In pulse-chase experiments, after incubation with radioactive methionine the cells were rinsed twice with standard complete medium and further incubated with complete medium supplemented with 10 mM unlabeled methionine. After the indicated times the medium present in the lower chambers was collected, cleared of any suspended cells and cell debris by centrifugation at 50,000 revolutions/min for 30 min in a Beckman L8-

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EFFECTS

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70M centrifuge (FA-70 rotor), supplemented with 1% Triton X-100 and protease inhibitors [1 mM phenylmethylsulfonyl fluoride (PMSF), 50 pg/ml leupeptin, 50 pg/ml antipain, 0.1 mg/ml aprotinin], and then incubated with monoclonal antibodies bound to Affi-Gel 10 overnight. The cell layers were washed three times with PBS before they were suspended in homogenization buffer (10 mM sodium phosphate buffer, pH 7.4, 50 mM mannitol containing the same protease inhibitors listed above) by scraping with a rubber policeman, and then homogenized four times for 15 s with a Polytron (Brinkmann Instruments) at setting 10. Homogenates were centrifuged at 2,000 revolutions/min for 10 min to eliminate unbroken cells and nuclei, and the supernatants were spun at 50,000 revolutions/min for 30 min in a Beckman L870M centrifuge (FA-70 rotor). The supernatants (cytosols) were discarded, and the pellets (total cell membranes) were suspended in 20 mM sodium phosphate, pH 8.0, 1% Triton X-100, 0.2% sodium deoxycholate, 1 mM PMSF, and other protease inhibitors as above, sonicated (3 x 3 s), then left at 4°C for 2 h. Subsequently, NaCl (to 150 mM final concn) and galactose (to 100 mM final concn) were added, and the samples were spun in a Beckman centrifuge at 50,000 revolutions/min for 30 min (FA-70.1 rotor). The supernatants were incubated at 4°C with antibodies bound to Affi-Gel 10 beads overnight. Antigens bound to the antibody-Affi-Gel bead conjugates were eluted as previously described (33, 34) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (50 mM dithiothreitol). Gel electrophoresis. SDS-PAGE and detection of labeled proteins by fluorography were performed as previously described (33) using 7.5% acrylamide gels. The intensity of the bands was assessed by using a laser densitometer (Ultroscan XL, Pharmacia, Piscataway,

NJ) .

Statistical analysis. Student’s

t test for unpaired

data was used as a statistical test. Results, expressed as means -+ SD, were considered as significant when P < 0.05. RESULTS

Effects of EGF and aminoguanidine on DNA synthesis by Caco-2 cells. The addition of EGF (50 rig/ml) to the

culture medium stimulated significantly DNA synthesis in both subconfluent and confluent Caco-2 cell cultures; incorporation of [3H]thymidine into cellular DNA was increased 25-45%, varying within that range in different experiments (Table 1). Cells kept in a confluent state for longer periods of time (3 weeks or more) exhibited markedly reduced levels of DNA synthesis, and EGF stimulation, while still observed, did not reach statistical significance. The addition of aminoguanidine (10 PM) to the culture medium of Caco-2 cells at all stages of confluence had no significant effect on DNA synthesis and did not prevent the EGF-induced stimulation (Table 1). Effects of EGF on DA0 and DPPIVexpression

by Caco-

2 cells. In the small intestinal mucosa, both DA0 (9) and DPPIV (16) are predominantly expressed by the absorp-

DA0

AND

CELL

G671

PROLIFERATION

1. Effects of EGF and aminoguanidine on DNA synthesis in subconfluent and confluent Caco-2 cells TABLE

Subconfluent (4 days) Just confluent (7 days) Confluent (14 days) Confluent (24 days)

Control

+AG

+EGF

872k18 912t32 605t24 231rtll

882t,27 889t43 494k65 252t21

1,095+31* 1,135+_37* 818&15* 312t41

+AG,

EGF

1,162+74* 1,218+56* 831t22* 328k52

Values are expressed as cpm/pg DNA and are means t, SE; n/group: 5 cultures in all cases. Caco-2 cells were seeded on day I in 60-mm diameter dishes; on the indicated days after seeding, replicate cultures were incubated for 1 h at 37°C with complete medium containing 4 &i/ml of [3H]thymidine. Incorporation of [“Hlthymidine into cellular DNA and total cell DNA were determined as described in MATERIALS AND METHODS. Control cultures were fed standard complete medium. +AG, complete medium + 10 PM aminoguanidine; +EGF, complete medium + 50 rig/ml epidermal growth factor; +AG, EGF, complete medium + 10 PM aminoguanidine, 50 rig/ml epidermal growth factor. * Statistically significant difference between treated and control cul-

tures. 20

r

5

0 0

5

10

15

20

25

days of culture FIG. 1. Effects of epidermal growth factor (EGF; 50 rig/ml) and aminoguanidine (10 PM) on cellular diamine oxidase (DAO) acti vity. Caco-2 cells were analyzed at the indicated days after seeding; confluence was reached at 6 days. DA0 activity and protein were measured in total cell homogenates as described in MATERIALS AND METHODS. o, Control cultures in standard complete medium; A, complete medium + 10 PM aminoguanidine; l , complete medium + 50 rig/ml EGF; A, complete medium + 10 PM aminoguanidine, 50 rig/ml EGF. Each point represents mean ,t SE of 3 cultures. * Statistically significant difference between treated and control cultures.

tive villous cells, and can be therefore considered markers of intestinal cell differentiation. Similarly, their expression by Caco-2 cells grown under standard culture conditions is greatly enhanced as the cells reach confluence and undergo a series of morphological and functional changes paralleling those seen with cell differentiation in vivo (7, 16, 31). The addition of EGF (50 rig/ml) to the culture medium of Caco-2 cells had opposite effects on synthesis and expression of these two enzymes. As reported previously (7), very low levels of DA0 activity were detected intracellularly (Fig. 1) or in the culture medium (Fig. 2) of subconfluent and just-confluent Caco2 cells, and they were not affected by EGF (Figs. 1 and 2). After the cells remained confluent for 3-4 days or longer, DA0 levels increased markedly and were further

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G672

EFFECTS

OF EGF ON INTESTINAL

DA0 AND CELL PROLIFERATION 70

40

60 E

0: 0

5

10

15

20

25

days of culture

days of culture FIG. 2. Effect of EGF (50 rig/ml) on DA0 activity present in the culture medium of Caco-2 cells at the indicated days after seeding; confluence was reached at 6 days. Cultures were refed every 2-3 days, and culture medium left in contact with the cells for 2 days was used for analysis of DA0 activity (expressed as mu/ml conditioned medium). o, Control cultures in standard complete medium; l , complete medium + 50 rig/ml EGF. Each point represents mean + SE of 3 cultures. * Statistically significant difference between treated and control cultures.

significantly enhanced in the presence of EGF. Cells treated with EGF (Fig. 1) had cellular DA0 activities 50-75% and X50-200% higher than control cultures at, respectively, 8 and 18 days after confluence. At the same times, DA0 levels in the culture medium were four- to fivefold higher in EGF-treated cultures (Fig. 2). In contrast, DPPIV enzyme activities in cell homogenates and total cell membrane fractions were significantly lower in EGF-treated cells at both 8 and 18 days after confluence (Fig. 3). No DPPIV activity was detected in the culture medium at all times. Aminoguanidine (10 PM) was highly effective in inhibiting DA0 activity, confirming results from other studies conducted both in vivo and in vitro using various cell and tissue models (4, 22, 36). It completely inhibited DA0 activity in the culture medium (data not shown), and strongly reduced the enzyme levels in homogenates of cells cultured both in the presence and absence of EGF (Fig. 2). DPPIV activity was also slightly reduced in some cultures treated with aminoguanidine (Fig. 3). Effects of EGF on biosynthesis and secretion of DA0 by Caco-2 cells. To investigate the biosynthetic basis for the EGF-induced stimulation of DA0 expression, confluent Caco-2 cells were administered radioactive methionine; labeled DA0 and DPPIV were immunoprecipitated with specific antibodies from culture medium and total cell homogenates and analyzed by SDS-PAGE. The relative amounts of newly synthesized enzymes were evaluated by scanning the fluorographs with a laser densitometer. Pulse-chase experiments demonstrated that labeled DA0 was present in the cell layers at all chase times, but significant amounts of secreted enzyme could only be detected in the culture medium starting at 3 h after the pulse, reaching a peak by 6-9 h (Fig. 4). These results

FIG. 3. Effects of EGF (50 rig/ml) and aminoguanidine (10 PM) on dipeptidyl peptidase IV (DPPIV) expression. Caco-2 cells were analyzed at the indicated days after seeding; confluence was reached at 6 days. DPPIV activity and protein were measured in total cell homogenates as described in MATERIALS AND METHODS. o, Control cultures in standard complete medium; A, complete medium + 10 pM aminoguanidine; l , complete medium + 50 rig/ml EGF; A, complete medium + loPM aminoguanidine, 50 rig/ml EGF. Each point represents mean + SE of 3 cultures. * Statistically significant difference between treated and control cultures.

kDa 1

234567

8 9 10 11 12 13

200-

97.4-

69-

FIG. 4. Biosynthesis and secretion of DA0 by Caco-2 cells cultured in regular complete medium. Ten days after reaching confluence, Caco2 cells on Transwell filters were labeled with [?‘S]methionine for 45 min, rinsed with complete medium, and then incubated with the same medium containing 10 mM unlabeled methionine. At the indicated times (starting from the end of the pulse), DA0 was immunoprecipitated from culture medium and Triton X-lOO-solubilized cell homogenates, analyzed by SDS-PAGE under reducing conditions (50 mM dithiothreitol), and visualized by fluorography. Lane 1, molecular mass markers (myosin, 200 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin, 69 kDa). Lanes 2-7, DA0 immunoprecipitated from culture medium. Lanes 8-13, DA0 from total cell homogenates.

suggest that intracellular transport and secretion of DA0 through the basolateral membrane (9) are considerably slower than the transport from endoplasmic reticulum to the apical cell surface of brush-border enzymes such as DPPIV and aminopeptidase N, a process which under similar experimental conditions is completed in 90-120 min (16). This different rate of intracellular processing was also reflected in the much more rapid conversion of DPPIV from the “high mannose” to the complex-glycosylated form, present in the trans-Golgi and in the cell

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EFFECTS

OF EGF ON INTESTINAL

surface (16), which in the same cultures used to study DA0 biosynthesis was completed within 1 h of chase (data not shown). The above findings also suggested that to evaluate the effects of EGF on DA0 synthesis in Caco-2 cells 2 h of continuous labeling with [35S]methionine was the most appropriate incubation time, representing nearly a plateau in the level of intracellular labeled DA0 and preceding its secretion into the culture medium. The results of these studies are illustrated in Fig. 5 (top), and the corresponding densitometric analysis is presented in Table 2. As expected, DA0 synthesis was significantly higher in cultures kept in a confluent state for 10 or 20 days than in just-confluent cultures. However, in contrast with the results obtained in the DA0 assays (Figs. 1 and 2), EGF did not appear to affect significantly the amounts of enzyme synthesized at any time after confluence, suggesting that its stimulation of DA0 expression was not due to increased synthesis. It should also be noted that, in the same cultures, the amount of newly

DA0

G673

AND CELL PROLIFERATION

TABLE 2. Densitometric analysis of the fluorographs shown in Fig. 5

DA0

DPPIV

20 Days Confluent

10 Days

Confluence

Confluent DA0

DPPIV

DA0

DPPIV

Control 0.75 7.89 1.63 9.28 2.77 8.56 AG 0.83 8.27 2.16 9.45 3.27 9.40 EGF 0.78 5.91 1.66 6.11 3.05 6.76 EGF + AG 0.92 6.37 1.94 6.09 2.91 6.16 The output (peak areas) of the laser densitometer used is expressed in absorbance units (AU) times millimeters. Cultures were incubated 2 h with [35S]methionine (50 &i/ml) when just confluent, and at 10 or 20 days after reaching confluence. Diamine oxidase (DAO) and dipeptidy1 peptidase IV (DPPIV) were immunoprecipitated from total cell homogenates and analyzed by SDS-PAGE as described in the legend to Fig. 5. AG, aminoguanidine (10 PM); EGF, epidermal growth factor (50 rig/ml).

synthesized DPPIV varied much less than that of DA0 with the age of the cultures (Fig. 5, bottom), indicating that these two differentiation-specific enzymes are not coordinately expressed as Caco-2 cells reach confluence. In all of these experiments, aminoguanidine, added alone or together with EGF, did not appear to have any significant effect on the amounts of DA0 and DPPIV synthesized. To further elucidate the mechanism by which EGF stimulated DA0 expression in confluent Caco-2 cells, pulse-chase experiments were performed in the presence and absence of this hormone. The amount of labeled DA0 immunoprecipitated was essentially identical in control and treated cells during the first 2-3 h of chase (Fig. 6). Subsequently, total labeled DA0 in EGF-treated cells (culture medium and cell homogenates were combined before immunoprecipitation in these experiments) continued to increase until 5 h after the pulse, while in control cells a slow and steady decline was observed starting at 3 h. These results indicate that EGF acted predominantly by stabilizing the newly synthesized enzyme during intracellular processing and/or storage, possibly preventing its degradation before secretion into the extracellular medium. In contrast, and in accordance with the results obtained in the DPPIV assays (Fig. 3), a reduced amount of this enzyme was immunoprecipitated from EGF-treated cells at all chase times (Fig. 6, ‘bottom). DISCUSSION

69

1

L

5430

u

Y

1u

11

1z

1s

FIG. 5. Effects of EGF and aminoguanidine (AG) on DA0 (top) and DPPIV (bottom) synthesis by Caco-2 cells. Cells just confluent (lanes 2-5) or confluent for 10 (lanes 6-9) or 20 (lanes 10-13) days, cultured on Transwell filters in regular complete medium (C) or in medium containing 10 PM AG, 50 rig/ml EGF, or 50 rig/ml EGF + 10 pM AG were labeled with [%]methionine for 2 h. DA0 and DPPIV were immunoprecipitated from Triton X-100solubilized combined medium and cell homogenates, thus representing total enzyme synthesis over a 2-h period. This time period was chosen to avoid possible interference of extracellular degradation of DA0 after its secretion into the culture medium. Labeled antigens were analyzed by SDS-PAGE under reducing conditions (50 mM dithiothreitol) and visualized by fluorography.

The major findings of this work are 1) the ability of EGF to stimulate DNA synthesis and modulate expression of two differentiation-specific intestinal marker enzymes in Caco-2 cells and 2) the observation that DA0 activity in these cells was not correlated with their proliferative activity or degree of differentiation. Several studies have suggested an important physiological role for EGF in the development and function of the intestinal tract in suckling animals and fetal human intestine (1, 2, 13, 14, 21, 24, 27, 32, 35). This is also supported by the presence of EGF in high concentrations in biological fluids, such as saliva and milk, and in its ability to reach the small intestinal lumen, epithelial cells, and lamina propria in a biologically active form

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G674

EFFECTS

1I

21

3I

Time

o;chki I

OF

EGF

I

I

6

ON

1

7

INTESTINAL

a I

DA0

I

9

[hl

6. Pulse-chase labeling of DA0 and DPPIV synthesized by confluent Caco-2 cells cultured in the absence (0) and presence (0) of EGF (50 rig/ml). Caco-2 cells on Transwell filters, confluent since 10 days, were labeled with [““S]methionine for 45 min, rinsed, and then chased in complete medium containing 10 mM unlabeled methionine. At the indicated times (starting from the end of the pulse) culture medium and cells were combined and solubilized with 1% Triton X100. DA0 and DPPIV were immunoprecipitated, analyzed by SDSPAGE under reducing conditions (50 mM dithiothreitol), and visualized by fluorography. Fluorographs were scanned with a laser densitometer and output (peak areas) is expressed in absorbance units (AU) times millimeters. Each point represents mean k SE of 3 cultures. * Statistically significant difference between EGF-treated and control cultures. FIG.

(3235). However, conflicting results have been obtained with respect to the specific intestinal functions affected by endogenous or exogenous EGF, and in most studies the mechanism of action of this hormone in the intestinal cells has not been explored in any detail. In newborn rat jejunum, lactase activity and calcium uptake were found to be increased by EGF, with no effects on maltase and sucrase activities (27). In another study, none of the brush-border enzymes examined were significantly affected by EGF administered alone, while a synergistic effect on lactase expression and mRNA levels was observed in animals injected EGF together with hydrocortisone and thyroxine (14). In the colon of the same animals, EGF was found to inhibit the postnatal increase in lactase, maltase, and aminopeptidase activities observed in untreated controls (14). In suckling mice, proliferation and cell differentiation were found to be differentially sensitive to this hormone, which produced premature induction of sucrase and significant increases in

AND

CELL

PROLIFERATION

trehalase and lactase activities (21). In suckling New Zealand White rabbits, both systemic and oral administration of EGF resulted in increased sucrase and decreased lactase levels (26). In contrast, organ cultures of human fetal jejunum treated with EGF showed induction of lactase and a marked reduction in labeling indexes (24) Our data clearly demonstrated a significant stimulatory effect of EGF on DNA synthesis in both subconfluent and confluent Caco-2 cell cultures (Table 1). The EGF concentration used (50 rig/ml) was chosen because in preliminary experiments (data not shown) it produced maximal effects on stimulation of DA0 expression by confluent Caco-2 cells. It is comparable to that found in human colostrum and milk (35) and is within the range (lo-100 rig/ml) previously shown to influence expression of brush-border enzymes in intestinal organ cultures (2, 24, 25). Lower EGF concentrations (lo-20 rig/ml) also stimulated DNA synthesis in confluent Caco-2 cells (data not shown), as found for other intestinal cell lines (3), but were less effective in enhancing DA0 activity both in the cell layers and in the culture medium. Because fetal bovine serum was present at a 10% concentration in the culture medium, we cannot exclude a synergistic effect of other growth factors or hormones on the observed induction of DA0 expression and DNA synthesis by EGF. However, EGF previously has been found to influence DNA synthesis by human fetal colonic epithelial cells (25) and rat intestinal epithelial cells (3) cultured under serum-free conditions. In the intestinal mucosa in vivo DA0 and DPPIV are both markers of cell differentiation and are coexpressed in villous enterocytes (9, 16), but their synthesis and accumulation in Caco-2 cells were affected in opposite ways by the presence of EGF in the culture medium. A significant reduction in DPPIV-specific activity was found in EGF-treated cells (Fig. 3), paralleling the decrease in cellular biosynthetic ability demonstrated by [35S] methionine labeling and immunoprecipitation of newly synthesized enzyme (Figs. 5 and 6, Table 2). However, the intracellular processing of DPPIV and its conversion from high mannose to complex-glycosylated forms did not appear to differ in control and hormonetreated cultures. These results, and those obtained with 1 normal human fetal colonic mucosa in organ culture (25), suggest that EGF decreases expression of at least some brush-border enzyme activities in intestinal epithelial ceils. In contrast, EGF increased markedly DA0 activity both in Caco-2 cell layers (Fig. 1) and in their culture medium (Fig. 2) without apparently affecting its rate of synthesis (Figs. 5 and 6). This discrepancy may be explained by a greater intracellular stability of DA0 in EGF-treated cells. Pulse-chase experiments suggested that, in control cultures, a fraction of the newly synthesized enzyme was degraded intracellularly, a process that was absent or considerably reduced in the presence of EGF (Fig. 6), possibly accounting for the four- to fivefold greater DA0 activity secreted in the culture medium (Fig. 2). Overall, these findings provide evidence for different mechanisms of action of EGF in the intestinal mucosa, affecting the synthesis and expression of specific

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EFFECTS

OF

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ON

INTESTINAL

differentiated cell products at either the pre- or posttranslational levels. The hypothesis that DA0 may play a role in the regulation of cell growth is based on two observations: 1) that putrescine is an excellent substrate for this enzyme, and 2) that high levels of DA0 activity are present in rapidly growing tissues such as normal small intestine, regenerative liver (34), hypertrophic kidney (9) and heart (27). However, no firm evidence for a relationship between DA0 levels and cell proliferation has been obtained to date. On the contrary, in an experimental model of intestinal adaptation (6), DA0 activity failed to correlate with the indexes of mucosal proliferation. In Caco-2 cells cultured under standard conditions, DA0 activity was inversely related to cell proliferation and was much lower in exponentially growing subconfluent cultures than in confluent, differentiated cells (Figs. 1 and 2). This observation is reminiscent of the exclusive staining of the absorptive villous cells previously demonstrated with anti -DA0 monoclonal antibodies in the human intestinal mucosa (9). However, this association between reduced proliferation and increased DA0 levels was not observed in EGF-treated cells, in which both parameters were increased. Furthermore, suppression of DA0 activity by aminoguanidine did not affect the rate of cellular DNA synthesis (Table 1) and had only minor effects on cell differentiation measured as either DPPIV activity or amount of labeled DA0 synthesized in the presence of [35S]methionine. These latter findings must be taken with a note of caution, since in the presence of aminoguanidine the cells may have been able to regulate their intracellular levels of polyamines through other metabolic pathways (i.e., interconversion) or through an increased uptake from the culture medium (8, 23). It should also be noted that the present study was centered on DA0 expression and its correlation with proliferation and differentiation of Caco-2 cells, and no attempt was made to determ ine polyam ine levels, which could have been modulated bY other factors, such as uptake or release into the culture medium (23) and variations in cellular ODC activity. Previous studies have demonstrated that EGF increased cell growth in several colon tumor cell lines without affecting cellular ODC activity (II), and in Caco-2 cells ODC and DA0 activities were inversely affected by cell density and differentiation (7). However, our findings are consistent with the results of experiments conducted in vivo: prolonged admi nistration of several inhibitors of polyamine oxidation only led to an enhanced urinary excretion of both free and acetylated polyamines (36). In one study of intestinal adaptation (12) the inhibition of DA0 by aminoguanidine induced a greater mucosal proliferation with respect to controls; however, aminoguanidine also increased ODC activity so that the rel .ative importance of DA0 i nhibition remained unclear. In conclusion, the results obtained in this study indicate that EGF is a potent stimulant of intestinal cell and that it may affect expression of differ-proliferation entiation- !specific enzymes by intestinal cells through different mechan isms. The increase in DA0 activity induced by EGF in Caco-2 cells appeared to be unrelated to their greater cell growth, since nearly total inhibition

DA0

AND

CELL

G675

PROLIFERATION

of the enzyme by aminoguanidine in the both the absence and presence of EGF did not affect DNA synthesis. Our results therefore suggest that the main function(s) of DA0 in the small intestine is (are) unrelated to the regulation of epithelial cell growth and differentiation. This work was presented in part at the annual meeting of the American Gastroenterological Association, San Antonio, TX, May 1316, 1990, and was published in abstract form (Gustroenterology 98: A408, 1990). This study was supported by National Institute of Diabetes and D’igestive and Kidney Diseases Grant DK-32656. Present address of B. Daniele: Div. di Gastroenterologia, 2a Facolta’ di Medicina, via S. Pansini 5, 80131 Naples, Italy. Address for reprint requests: A. Quaroni, Section of Physiology, 724A Veterinary Research Tower, Cornell Univ., Ithaca, NY 14853. Received

12 March

1991; accepted

in final

form

5 June

1991.

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