Expression of the TGF-beta receptor gene and sensitivity to growth inhibition following polyamine depletion

Jaladanki N. Rao, Li Li, Barbara L. Bass, and Jian-Ying Wang

Department of Surgery, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our previous studies have shown that inhibition of polyamine biosynthesis increases the sensitivity of intestinal epithelial cells to growth inhibition induced by exogenous transforming growth factor-beta (TGF-beta ). This study went further to determine whether expression of the TGF-beta receptor genes is involved in this process. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Administration of alpha -difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (the rate-limiting enzyme for polyamine synthesis), for 4 and 6 days depleted cellular polyamines putrescine, spermidine, and spermine in IEC-6 cells. Polyamine depletion by DFMO increased levels of the TGF-beta type I receptor (TGF-beta RI) mRNA and protein but had no effect on the TGF-beta type II receptor expression. The induced TGF-beta RI expression after polyamine depletion was associated with an increased sensitivity to growth inhibition induced by exogenous TGF-beta but not by somatostatin. Extracellular matrix laminin inhibited IEC-6 cell growth without affecting the TGF-beta receptor expression. Laminin consistently failed to induce the sensitivity of TGF-beta -mediated growth inhibition. In addition, decreasing TGF-beta RI expression by treatment with retinoic acid not only decreased TGF-beta -mediated growth inhibition in normal cells but also prevented the increased sensitivity to exogenous TGF-beta in polyamine-deficient cells. These results indicate that 1) depletion of cellular polyamines by DFMO increases expression of the TGF-beta RI gene and 2) increased TGF-beta RI expression plays an important role in the process through which polyamine depletion sensitizes intestinal epithelial cells to growth inhibition induced by TGF-beta .

cell proliferation; transforming growth factor-beta receptor; laminin; retinoic acid; IEC-6


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE TRANSFORMING GROWTH factors-beta (TGF-beta ) are a family of multifunctional peptides involved in the regulation of epithelial cell growth and phenotype (1, 29). There are three distinct but highly related mammalian isoforms of TGF-beta : beta 1, beta 2, and beta 3. TGF-beta exert their multiple actions through heteromeric complexes of two types of transmembrane receptors (type I and type II) with a serine/threonine kinase domain in their cytoplasmic region (7, 14, 15, 27). To date, six different TGF-beta type I receptors (TGF-beta RI) have been identified in mammals, including Tbeta R-I, ActR-I, ActR-IB, BMPR-IA, BMPR-IB, and R3 (7, 14, 26). Sizes of the TGF-beta RI are similar to each other (502-532 amino acid residues) and 60-90% amino acid sequence identities in their kinase domains. The TGF-beta RI contain a conserved sequence known as the GS domain (also named type I box) in their cytoplasmic juxtamembrane region (14, 26). The TGF-beta RI are more similar to each other than they are to the known type II receptors (TGF-beta RII) and thus form a subgroup of mammalian type I receptors in the family of receptor serine/threonine kinases.

Exposure of epithelial cells to TGF-beta leads to inhibition of growth (1), induction of extracellular matrix protein formation (1, 29), modulation of proteolysis (5), and stimulation of cell migration (4, 31). To initiate the signaling of these responses, TGF-beta binds directly to the TGF-beta RII that is a constitutive active kinase, after which TGF-beta RI is recruited into the complex (31, 45). The TGF-beta RII in the complex phosphorylates the GS domain of TGF-beta RI, which leads to propagation of further downstream signals (45). Mutational analyses altering serine and threonine residues in the TGF-beta RI GS domain have indicated that the phosphorylation by TGF-beta RII is indispensable for TGF-beta signaling, although its signaling activity does not appear to depend on the phosphorylation of any particular serine or threonine residue in the TTSGSGSG sequence of the GS domain (42, 45).

The intestinal mucosa has the most rapid turnover rate of any tissue in the body and is continuously renewed from the proliferative zone of undifferentiated stem cells within the crypts (10). Polyamines spermidine and spermine and their precursor putrescine are absolutely required for cell proliferation in the crypts of the small intestinal mucosa, and decreasing the cellular polyamines inhibits epithelial cell renewal both in vivo (34, 35, 37) and in vitro (13, 38). We (21) have recently reported that depletion of cellular polyamines induces the activation of the TGF-beta gene through posttranscriptional regulation and that the increased gene product, TGF-beta , plays an important role in the process of growth inhibition after polyamine depletion. Furthermore, we observed that polyamine-deficient cells were more sensitive to growth inhibition when they were exposed to exogenous TGF-beta (21). However, the mechanism through which polyamine depletion increases the sensitivity to growth inhibition by TGF-beta has not been demonstrated.

Because intestinal epithelial cells can produce both TGF-beta receptors and ligand (1, 29), it is possible that the regulation of cellular responsiveness relies on the production of active TGF-beta and its presentation to signaling receptors. The present study was designed to address several questions regarding the involvement of expression of the TGF-beta R genes in the process by which polyamine-deficient intestinal epithelial cells are more sensitive to exogenous TGF-beta . First, we examined whether depletion of cellular polyamines by inhibition of ornithine decarboxylase (ODC; the rate-limiting enzyme in the biosynthesis of polyamines) with alpha -difluoromethylornithine (DFMO) increases expression of TGF-beta RI and TGF-beta RII genes in intestinal epithelial cells (IEC-6 line). Second, we examined whether inhibition of TGF-beta receptor expression by treatment with retinoic acid (RA) prevented the increased sensitivity of polyamine-deficient cells to growth inhibition by TGF-beta . Some of these data have been published in abstract form (25).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials

Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were obtained from GIBCO BRL (Gaithersburg, MD), and biochemicals were from Sigma Chemical (St. Louis, MO). The primary antibody, an affinity-purified rabbit polyclonal antibody against TGF-beta RI or TGF-beta RII, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit IgG-fluorescein isothiocyanate (FITC) isomer conjugate was purchased from Sigma. DFMO was a gift from Merrell Dow Research Institute of Marion Merrell Dow (Cincinnati, OH).

Methods

Cell culture and general experimental protocol. The IEC-6 cell line was purchased from the American Type Culture Collection 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 originated from intestinal crypt cells, as judged by morphological and immunologic criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells.

Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated 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 1:20, and medium was changed three times per week. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative, and passages 15-20 were used in the experiments. There were no significant changes of biological function and characterization from passages 15 to 20 (38).

The general protocol of the experiments and the methods used were similar to those described previously (21). Briefly, IEC-6 cells were plated at 4.25 × 104 cells/cm2 in DMEM plus 5% dFBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate (supplemented DMEM). Cells were incubated in a humidified atmosphere at 37°C in 90% air-10% CO2 (vol/vol) for 24 h; this was followed by a period of different experimental treatments.

In the first series of studies, we examined the effect of polyamine depletion on the TGF-beta RI and TGF-beta RII gene expression in IEC-6 cells. Cells were grown in control cultures and cultures containing either 5 mM DFMO alone or DFMO plus 5 µM spermidine for 4 and 6 days. The dishes were placed on ice, the monolayers were washed three times with ice-cold Dulbecco's PBS (D-PBS), and then different solutions were added according to the assays to be performed. In addition, the effects of extracellular matrix laminin on TGF-beta receptor expression and cell responsiveness to exogenous TGF-beta in the presence or absence of DFMO were also examined.

In the second series of studies, we determined whether decreasing TGF-beta receptor expression by treatment with RA altered the increased susceptibility of polyamine-deficient cells to growth inhibition induced by exogenous TGF-beta . Cells were initially grown in DMEM containing 5% dFBS in the presence or absence of 5 mM DFMO for 4 days, and then different concentrations of RA and TGF-beta were added. The levels of TGF-beta receptor protein and cell responsiveness to exogenous TGF-beta were measured 48 h after exposure to RA.

RT-PCR. Total RNA was extracted with guanidinium isothiocyanate solution and purified by CsCl density gradient ultracentrifugation as described by Chirgwin et al. (3). Briefly, the cells were washed with 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 · HCl (pH 7.5), containing 1 mM EDTA, 5% sodium lauryl sarcosine, and 5% phenol (added just before use). The addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol precipitated the purified RNA from aqueous phase in sequence. Final RNA was dissolved in water and estimated from its ultraviolet absorbance at 260 nm using a conversion factor of 40 units.

Ten micrograms of the total RNA were reversely transcribed using a first-strand cDNA synthesis kit (GIBCO BRL) and random hexamers [pd(N)6 primer]. The reaction mixture was incubated for 1 h at 42°C and then heated at 90°C for 5 min to inactivate the reverse transcriptase. The specific sense and antisense primer for TGF-beta RI included 5'-TACAGTGTTTCTGCCACCTCTGT-3' and 3'-ACACGTGGTAGAAGTTTTTGTCC-5'. The expected size of TGF-beta -RI fragments was 177 bp, within the 128- to 305-bp encoding region of the TGF-beta RI cDNA (7). The specific sense and antisense primer for TGF-beta RII included 5'-CACTGTCCACTTGTGACAACC-3' AND 3'-GGTAGTAGGACCTCCTGCTGGC-5'. The expected size of TGF-beta RII fragments was 503 bp, within the 421- to 922-bp encoding region of the cDNA (15). PCR was performed by a GeneAmp PCR system (Perkin-Elmer) using Taq polymerase. Two microliters of the first-strand cDNA reaction mixture were used in PCR reaction. The cDNA samples were amplified in the thermal cycler under the following conditions: the mixture was annealed at 59°C (1 min), extended at 72°C (2 min), and denatured at 94°C (1 min) for 35 cycles. This was followed by a final extension at 72°C (10 min) to ensure complete product extension. The PCR products were electrophoresed through a 1% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining.

To quantify the PCR products (the amounts of mRNA) of TGF-beta RI and TGF-beta RII, an invariant mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Immediately after each of the experiments, the optical density (OD) values for each band on the gel were measured by a gel documentation system (UVP, Upland, CA). The OD values in the TGF-beta RI and TGF-beta RII signals were normalized to the OD values in the GAPDH signals. The normalized values in the controls were expressed as 1 arbitrary unit for quantitative comparison (33).

Western immunoblotting analysis. Cell samples, dissolved in SDS sample buffer, were sonicated for 20 s and centrifuged at 2,000 rpm for 15 min. The supernatant was boiled for 5 min and then subjected to electrophoresis on 10% acrylamide gels according to Laemmli (12). Each lane was loaded with 20 µg of protein equivalents. After the transfer of protein to nitrocellulose filters, the filters were incubated overnight at 4°C in 5% nonfat dry milk in 10× PBS-Tween 20 [PBS-T: 15 mM NaH2PO4, 80 mM Na2HPO4, 1.5 M NaCl (pH 7.5), and 0.5% (vol/vol) Tween 20]. Immunologic evaluation was then performed for 90 min in 1% BSA-PBS-T buffer containing affinity-purified rabbit polyclonal antibody against TGF-beta RI or TGF-beta RII protein. The filters were subsequently washed with 1× PBS-T and incubated for 1 h with anti-rabbit IgG antibody conjugated to peroxidase by protein cross-linking with 0.2% glutaraldehyde. After an extensive washing with 1× PBS-T, the immunocomplexes on the filters were reacted for 1 min with chemiluminescence reagent (NEL-100. DuPont-NEN). Finally, the filters were placed in a plastic sheet protector and exposed to autoradiography film for 30 or 60 s.

Immunohistochemical staining. Cells were plated at 4.25 × 104/cm2 in chambered slides and incubated with a medium containing DMEM + 5% dFBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. DFMO at a dose of 5 mM with or without 5 µM spermidine was added as treatment. The immunofluorescence procedure was carried out according to the method of Hembrough et al. (9) with minor changes. Briefly, the cells were washed with D-PBS and incubated with rabbit anti-TGF-beta RI antibody at 1:50 dilution for 2 h at 4°C. This primary antibody recognizes the 55-kDa TGF-beta RI in immunoblots of IEC-6 cell extracts and does not cross-react with other proteins. The cells were then washed three times with D-PBS, incubated with anti-rabbit IgG-FITC conjugates (1:100 dilution) for 2 h at 4°C, rinsed three times again, and fixed in 4% paraformaldehyde. The slides were mounted with Vectashield mounting medium (Vector Laboratories) and viewed through a Zeiss confocal microscope (model LSM410).

Electron microscopy. After the cells were grown in the presence or absence of 5 mM DFMO for 4 days, they were washed with D-PBS and then fixed at room temperature in 2.5% glutaraldehyde-3.2% paraformaldehyde buffered with 0.1 M sodium cacodylate (pH 7.4). Cells were postfixed in 2% osmium tetroxide in the same buffer, dehydrated, and embedded in Epon. Ultrathin sections were examined in an electron microscope.

HPLC analysis of cellular polyamines. The cellular polyamine content was determined as previously described (38). Briefly, after the cells were washed three times with ice-cold D-PBS, 0.5 M perchloric acid was added, and the cells were frozen at -80°C until ready for extraction, dansylation, and HPLC. The standard curve encompassed 0.31-10 µM. Values that fell >25% below the curve were considered not detectable. Protein was determined by the Bradford method (2). The results are expressed as nanomoles of polyamines per milligram of protein.

Statistics. All data are expressed as means ± SE from six dishes. Autoradiographic and immunofluorescence labeling results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using Dunnett's multiple range test (8).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Inhibition of Polyamine Synthesis on the TGF-beta Receptor Expression

Exposure of IEC-6 cells to 5 mM DFMO for 4 and 6 days, which totally inhibits ODC activity (37, 38), almost completely depleted cellular polyamines. The levels of putrescine and spermidine were undetectable on days 4 and 6 after administration of DFMO. Spermine was less sensitive to the inhibition of ODC and was decreased by ~60% on days 4 and 6 in DFMO-treated cells (data not shown). Similar results have been published previously (21).

Depletion of cellular polyamines by DFMO resulted in a significant increase in expression of the TGF-beta RI gene in IEC-6 cells (Fig. 1). The increase in mRNA levels for TGF-beta RI was noted on day 4 and remained elevated on day 6 after exposure to DFMO. The levels of TGF-beta RI mRNA in cells exposed to DFMO for 4 and 6 days were ~2.5 times the normal values (without DFMO; Fig. 1, Aa and Ab). Spermidine at a dose of 5 µM given together with DFMO completely prevented the increased expression of the TGF-beta RI gene. The levels of TGF-beta RI mRNA in cells treated with DFMO plus spermidine were indistinguishable from those in cells grown in control cultures.


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Fig. 1.   Expression of the transforming growth factor-beta (TGF-beta ) type I receptor (TGF-beta RI) and type II receptor (TGF-beta RII) genes in control IEC-6 cells and cells treated with either alpha -difluoromethylornithine (DFMO) alone or DFMO plus spermidine (SPD). A: TGF-beta receptor mRNA expression. Cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO (5 mM) or DFMO + SPD (5 µM) for 4 and 6 days. mRNA levels for TGF-beta RI and TGF-beta RII were determined by semiquantitative RT-PCR analysis. a: Representative PCR-amplified products displayed in agarose gels for TGF-beta RI (177 bp) and TGF-beta RII (503 bp). b: Data normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [optical density (OD) of the TGF-beta RI mRNA/OD of the GAPDH] are expressed as means ± SE of data from 3 separate experiments. * P < 0.05 compared with controls (Con). B: TGF-beta receptor protein expression. a: Representative autoradiograms of Western blots in cell extracts from cells described in A. Whole cell lysates (20 µg) were subjected to electrophoresis on a 10% acrylamide gel. TGF-beta RI and TGF-beta RII proteins were identified by probing nitrocellulose with a specific anti-TGF-beta RI or TGF-beta RII antibody. Molecular mass markers are indicated on left in kDa. Molecular masses of TGF-beta RI and TGF-beta RII proteins are ~55 and 70 kDa, respectively. b: Quantitative analysis of TGF-beta RI immunoblots by densitometry from bands described in a. Values are means ± SE of data from 3 separate experiments. * P < 0.05 compared with controls. C: distribution of TGF-beta RI protein in IEC-6 cells from all 3 treatment groups on day 4. Slides were prepared in duplicate for each treatment and stained by immunofluorescence using a specific antibody to TGF-beta RI antibody as described in METHODS. Images were made as described in METHODS. a: Control. b: DFMO. c: DFMO + SPD. Original magnification, ×600.

Increased levels of TGF-beta RI mRNA in cells exposed to DFMO were paralleled by an increase in TGF-beta RI protein levels (Fig. 1, Ba and Bb). TGF-beta RI protein levels in cells exposed to DFMO for 4 and 6 days increased ~2 times over control values. The TGF-beta RI protein concentration was returned to normal levels when spermidine was given together with DFMO. Putrescine at 10 µM had an effect equal to that of spermidine on the expression of the TGF-beta RI gene when it was added to cultures that contained DFMO (data not shown). In contrast to TGF-beta RI, polyamine depletion did not induce expression of the TGF-beta RII gene in IEC-6 cells. There were no significant changes in the levels of TGF-beta RII mRNA and protein between control cells and cells exposed to DFMO with or without spermidine (Fig. 1, A and B).

To extend the finding of increased TGF-beta RI expression following polyamine depletion, we further explored the cellular distribution of TGF-beta RI protein in the cells grown in the presence or absence of DFMO for 4 days with the use of immunohistochemical staining technique. In control cells, a slight immunostaining for TGF-beta RI was observed in a loosely defined perinuclear area (Fig. 1Ca). Consistent with our data from Western blot analysis, these immunoreactivities for TGF-beta RI increased markedly after depletion of cellular polyamines by DFMO (Fig. 1Cb). Increased TGF-beta RI was scattered in punctate foci throughout the cytoplasm. Spermidine given together with DFMO prevented the increased immunostaining for TGF-beta RI, and the distribution of TGF-beta RI in the cells treated with DFMO plus spermidine was the same as that in control cells (Fig. 1Cc).

Ultrastructural Changes in Polyamine-Deficient IEC-6 Cells

Normal IEC-6 cells showed a simple monolayer of flat epithelial cells with sparse microvilli (Fig. 2A). In polyamine-deficient cells by treatment with DFMO for 4 days, there was appearance of "membranous lysosomal bodies," which were located throughout the cytoplasm and could be regularly identified in every experiment (Fig. 2, Ba and Bb). These ultrastructural changes were completely prevented by exogenous spermidine given together with DFMO. The ultrastructure in cells grown in the presence of DFMO plus spermidine was similar to that of control cells (data not shown).


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Fig. 2.   Electron microscopic analysis of IEC-6 cells grown in the absence (A) or presence (B) of 5 mM DFMO for 4 days. Cells were washed with Dulbecco's PBS and then fixed at room temperature in 2.5% glutaraldehyde-3.2% paraformaldehyde buffered with 0.1 M sodium cacodylate (pH 7.4). Three experiments were performed that showed similar results. In B, b shows boxed area of a.

Growth of Polyamine-Deficient Cells in Response to Exogenous TGF-beta

Exposure of normal IEC-6 cells (without DFMO) to exogenous TGF-beta significantly inhibited cell growth (Fig. 3Aa). When various doses of TGF-beta were tested, cell growth was inhibited linearly with concentrations of TGF-beta ranging from 2.5 to 10 ng/ml. Significant decreases in cell number occurred first at 10 ng/ml and were ~65% of normal values. As can be seen in Fig. 3Ab, polyamine depletion before the addition of TGF-beta increased the sensitivity of TGF-beta inhibition dramatically. In DFMO-treated cells, TGF-beta at a dose of 2.5 ng/ml significantly decreased cell number. When TGF-beta at a dose 5 or 10 ng/ml was given, cell numbers were ~25% of control values and were decreased by >70%. Consistent with the effect on the TGF-beta RI expression, spermidine given together with DFMO prevented the increased sensitivity of polyamine-deficient cells to growth inhibition caused by exogenous TGF-beta (Fig. 3Ac). The pattern of TGF-beta inhibition in cells treated with DFMO plus spermidine was identical to that of control cells.


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Fig. 3.   Effects of exogenous TGF-beta (A) and somatostatin (B) added to control cultures (without DFMO; a) or cultures containing either DFMO alone (b) or DFMO plus SPD (c) on cell growth. Cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO or DFMO + SPD for 4 days and then TGF-beta or somatostatin was given at different concentrations. Cell number was assayed 48 h after treatments. Values are means ± SE of data from 6 dishes. * P < 0.05 compared with groups treated with TGF-beta or somatostatin at a dose of 0.

Polyamine depletion-induced sensitivity to growth inhibition appears to be specific to exogenous TGF-beta because the pattern of growth inhibition induced by somatostatin in DFMO-treated cells was similar to that of control cells (Fig. 3B). Exposure to somatostatin at a dose of 250 ng/ml caused no significant inhibition of cell growth in control cells and DFMO-treated cells. When somatostatin at a dose of 500 ng/ml was given, a significant decrease in cell numbers occurred in both groups and was ~70% of normal values.

Effect of Laminin on TGF-beta Receptor Expression and Cell Growth

Our previous studies have demonstrated that IEC-6 cell growth decreased significantly when cells were cultured on laminin-coated dishes (44). To determine the specificity of polyamine depletion on TGF-beta RI expression, changes in the TGF-beta RI protein were investigated after cells were grown on laminin coated-dishes with or without DFMO for 4 and 6 days. As can be seen in Fig. 4, there were no significant differences in TGF-beta RI expression between control cells and cells grown on laminin for 4 and 6 days. Laminin also failed to prevent the activation of TGF-beta RI expression induced by depletion of cellular polyamines. In addition, expression of the TGF-beta RII protein was not altered when cells were grown on laminin with or without treatment with DFMO for 6 days (data not shown). Consistently, the patterns of TGF-beta inhibition in cells cultured on laminin in the presence or absence of DFMO were similar to those of cells grown on plastic dishes (Fig. 5), indicating that extracellular matrix laminin did not alter the cellular responsiveness to exogenous TGF-beta .


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Fig. 4.   Effect of extracellular matrix laminin (Lam) on TGF-beta RI protein expression in control cells and cells exposed to DFMO. A: representative autoradiograms of Western blot in cell extracts from cells grown in control (plastic) and laminin-coated dishes in the presence or absence of 5 mM DFMO for 4 and 6 days. Whole cell lysates (20 µg) from each group were subjected to electrophoresis on a 10% acrylamide gel. TGF-beta RI protein (~55 kDa) was identified by probing nitrocellulose with a specific anti-TGF-beta RI antibody. B: quantitative analysis of immunoblots by densitometry from cells described in A. Values are means ± SE of data from 3 separate experiments. * P < 0.05 compared with controls.



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Fig. 5.   Effect of exogenous TGF-beta on cell growth in IEC-6 cell grown in laminin-coated plates. Cells were cultured on plastic (A) or laminin-coated plates in the absence (B) or presence (C) of 5 mM DFMO for 4 days and then exposed to different concentrations of TGF-beta . Cell number was assayed 48 h after administration of TGF-beta . Values are means ± SE of data from 6 dishes. * P < 0.05 compared with groups treated with TGF-beta at a dose of 0.

Effect of RA on TGF-beta RI Expression and Cell Growth in IEC-6 Cells

To elucidate the significance of induced TGF-beta RI expression in the increased sensitivity to TGF-beta -mediated growth inhibition, we examined the effect of RA on TGF-beta RI expression and further determined whether observed reduction of TGF-beta RI protein by RA altered the growth response to exogenous TGF-beta . When IEC-6 cells were exposed to RA for 48 h, the levels of TGF-beta RI were reduced in a dose-dependent way, regardless of the presence or absence of DFMO (Fig. 6). In control cells, RA at a dose of 1 µM slightly inhibited TGF-beta RI expression, but a significant decrease in TGF-beta RI protein first occurred at doses of 2 µM and was ~55% of control values. In DFMO-treated cells, RA at doses of 1 and 2 µM significantly prevented increased TGF-beta RI expression, and the levels of TGF-beta RI protein were decreased by ~65% and ~80%, respectively. RA at a dose of 4 µM almost completely inhibited TGF-beta RI expression in both normal and polyamine-deficient cells. On the other hand, treatment with RA at various doses for 48 h did not decrease levels of TGF-beta RII protein in IEC-6 cells (data not shown).


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Fig. 6.   Effect of retinoic acid (RA) on TGF-beta RI protein expression in control cells (A) and cells treated with 5 mM DFMO (B). Cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO for 4 days and then exposed to different concentrations of RA for 48 h. TGF-beta RI protein in cell extracts was measured by Western immunoblotting analysis. Whole cell lysates (20 µg) from each group were subjected to electrophoresis on a 10% acrylamide gel, and TGF-beta RI protein (~55 kDa) was identified by probing nitrocellulose with a specific anti-TGF-beta RI antibody. C: quantitative analysis of immunoblots by densitometry from cells described in A and B. Values are means ± SE of data from 3 separate experiments. * P < 0.05 compared with controls.

To determine changes in growth response to exogenous TGF-beta in the presence of RA, cells were initially grown in DMEM medium with or without DFMO for 4 days and then incubated with RA and TGF-beta for an additional 48 h. Figure 7A clearly shows that TGF-beta at a dose of 10 ng/ml significantly inhibited normal cell growth (without DFMO), regardless of the presence or absence of RA. However, RA partially but significantly prevented the inhibitory effect of exogenous TGF-beta on cell growth. Consistent with the effect on TGF-beta RI expression, RA at a dose of 2 µM almost completely blocked the increased sensitivity of polyamine-deficient cells to growth inhibition induced by exogenous TGF-beta (Fig. 7B). When RA was given together with TGF-beta , the inhibitory effect of exogenous TGF-beta on cell growth was completely prevented in DFMO-treated cells.


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Fig. 7.   Effect of exogenous TGF-beta on cell growth in control cells and polyamine-deficient cells in the presence or absence of RA. Cells were grown in DMEM without (A) or with 5 mM DFMO (B) for 4 days, and 2 µM RA was added to cultures along with the TGF-beta at different concentrations. Cell number was assayed 48 h after treatments. Values are means ± SE of data from 6 dishes. * P < 0.05 compared with groups treated with TGF-beta at a dose of 0. + P < 0.05 compared with groups treated with the same dose of TGF-beta in the presence of RA.

Effect of Polyamine Depletion on TGF-beta RI and TGF-beta RII Expression in Other Intestinal Epithelial Cells

To determine whether the induction of TGF-beta RI expression by polyamine depletion is cell-type dependent, we examined changes in levels of TGF-beta RI and TGF-beta RII mRNAs and proteins following polyamine depletion in Caco-2 cells, a line of human colon carcinoma cells. Administration of 5 mM DFMO for 4 and 6 days completely inhibited ODC activity and depleted cellular polyamines in Caco-2 cells (data not shown), which is indicated in our previous publication (41). In contrast to IEC-6 cells, depletion of cellular polyamines by DFMO did not increase expression of the TGF-beta receptor genes in Caco-2 cells (Fig. 8). There were no significant differences in levels of TGF-beta RI and TGF-beta RII mRNAs and proteins between control cells and cells treated with DFMO in the presence or absence of exogenous spermidine. Polyamine depletion consistently failed to induce the sensitivity of TGF-beta -mediated growth inhibition in Caco-2 cells (data not shown). We also examined the effect of polyamine depletion on TGF-beta receptor expression in the HT-29 cell line. In general, the response of expression of the TGF-beta RI and TGF-beta RII genes to polyamine depletion in HT-29 cells was similar to that observed in Caco-2 cells exposed to DFMO. Treatment with DFMO for 4 and 6 days did not increase levels of TGF-beta RI and TGF-beta RII mRNA and protein in HT-29 cells (data not shown).


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Fig. 8.   Expression of TGF-beta RI and TGF-beta RII genes in Caco-2 cells treated with either DFMO (5 mM) or DFMO + spermidine (SPD, 5 µM) for 4 and 6 days. Caco-2 cells were purchased from American Type Culture Collection and grown in DMEM containing 5% dialyzed FBS, and medium was changed every 2nd day. A: TGF-beta receptor mRNA levels. PCR-amplified products were displayed in agarose gel for TGF-beta RI (177 bp) and TGF-beta RII (503 bp) when first-strand cDNAs, synthesized from total RNA extracted from Caco-2 cells, were amplified with the specific sense and antisense primers for TGF-beta RI and TGF-beta RII. B: TGF-beta receptor protein levels. Whole cell lysates (20 µg) were subjected to electrophoresis on a 10% acrylamide gel. TGF-beta RI and TGF-beta RII proteins were identified by probing nitrocellulose with a specific anti-TGF-beta RI or TGF-beta RII antibody. Molecular mass markers are indicated on left in kDa. Molecular masses of TGF-beta RI and TGF-beta RII proteins are ~55 and 70 kDa, respectively. Three experiments were performed that showed similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There is little doubt that intestinal epithelial growth is a complex process that is controlled at different levels and is modulated by numerous factors. An adequate supply of polyamines in the dividing cells within the crypts is absolutely required for small intestinal epithelial cell renewal (16, 17, 38, 40), but few specific molecular functions of polyamines in epithelial proliferation have been defined. The present study clearly shows that inhibition of polyamine synthesis by DFMO markedly increased expression of TGF-beta RI mRNA and protein in IEC-6 cells (Fig. 1). Increased expression of the TGF-beta RI gene was associated with an increase in sensitivity to growth inhibition induced by exogenous TGF-beta but not by somatostatin (Fig. 3). The increased TGF-beta RI expression and induced sensitivity to TGF-beta -mediated growth inhibition appear to be specific to polyamine depletion because laminin-induced growth inhibition did not increase TGF-beta RI expression (Fig. 4) and thus failed to affect cellular responsiveness to exogenous TGF-beta (Fig. 5). Furthermore, decreasing TGF-beta RI expression by treatment with RA not only decreased TGF-beta -mediated growth inhibition in normal cells but also prevented the increased sensitivity to exogenous TGF-beta in polyamine-deficient cells (Figs. 6 and 7). These findings suggest that expression of the TGF-beta RI gene is highly regulated by cellular polyamines and that increased TGF-beta RI expression after polyamine depletion may play an important role in the increased sensitivity to TGF-beta -mediated growth inhibition.

Although the importance of epithelial cell renewal in the maintenance of intestinal mucosal integrity is obvious, understanding the elements regulating intestinal epithelial growth and proliferation is far from clear. It has been shown that the TGF-beta signaling pathway makes a major contribution to these processes and that TGF-beta is a physiological regulator of normal intestinal epithelial growth (1, 29). Administration of TGF-beta inhibits proliferative activity and promotes the development of differentiated function in intestinal epithelial cells (1). Loss of TGF-beta sensitivity is frequently observed in tumors derived from cells that are normally sensitive, and the extent of TGF-beta resistance commonly correlates with malignancy (6, 15). The TGF-beta resistance in tumor cells may occur after inactivation of essential components of the TGF-beta signaling pathway (15, 28), through depletion of the p15INK4b locus (32), or by increased expression of the MDM2 gene (30).

In intestinal epithelial cells, resistance and sensitivity to growth inhibition by TGF-beta are mainly regulated by changes in TGF-beta receptor expression. Mulder et al. (19) reported that increased TGF-beta sensitivity in chemically mutagenized intestinal epithelial cell clones is associated with increases of 5- to 10-fold in both receptor numbers per cells and binding to signal-transducing (type I and II) receptors. The IEC-6, IPEC (porcine jejunal enterocytes), and RIE-1 (rat intestinal epithelial cells) cell lines, all of which are nontransformed, are growth inhibited by TGF-beta and express TGF-beta receptors. The ras-transformed RIE-1 and SW-620 transformed cells are not growth inhibited by TGF-beta and demonstrate a marked reduction of TGF-beta receptor levels (43). A similar correlation is also observed in rat hepatocytes (20). During liver regeneration and the condition of primary culture, increased expression of TGF-beta receptors is associated with an increased sensitivity to TGF-beta -mediated growth inhibition in hepatocytes. In many other cell types, the TGF-beta resistance has been found to relate to a decrease or absence of the expression of TGF-beta RI (11) and TGF-beta RII (39).

The results reported here indicate that expression of the TGF-beta RI is implicated in the process by which polyamine depletion increases the sensitivity to TGF-beta -mediated growth inhibition in intestinal epithelial cells. As can be seen in Fig. 1, exposure to DFMO for 4 and 6 days significantly increased levels of TGF-beta RI mRNA, which was paralleled by an increase in TGF-beta RI protein. In contrast, expression of the TGF-beta RII gene was not affected after exposure to DFMO in the presence or absence of exogenous spermidine. This result is not surprising, because the TGF-beta RII is a constitutive, active kinase (31, 45). Polyamines may regulate TGF-beta RII function through a mechanism rather than through its mRNA and protein synthesis. In addition, this increased expression of the TGF-beta RI gene in DFMO-treated cells is related to polyamine depletion rather than to the nonspecific effect of DFMO because the stimulatory effect of this compound on TGF-beta RI expression was completely prevented by the addition of exogenous spermidine.

Electron microscopic analysis shows that membranous lysosomal bodies appeared within the cytoplasm in DFMO-treated cells (Fig. 2). Although the significance and mechanisms responsible for these membranous lysosomal bodies are unknown, they are completely prevented when spermidine is given together with DFMO, indicating that these ultrastructural changes also result from polyamine depletion. These findings suggest that polyamines may be required for the maintenance of cellular distribution of membrane and nonmembrane organelles in intestinal epithelial cells. This possibility is supported by our previous findings (36), which indicated that inhibition of polyamine synthesis results in the appearance of many punctate foci of actin-myosin II in the cell interior.

It is interesting and of important biological consequences that the increased TGF-beta RI expression following polyamine depletion was associated with an increased sensitivity to growth inhibition induced by exogenous TGF-beta (Fig. 3). This increased sensitivity results from a specific correlation of TGF-beta ligand with its signaling receptors, because there is no change in the sensitivity of polyamine-deficient cells to somatostatin-mediated growth inhibition (Fig. 3B). In addition, the increased TGF-beta RI gene expression is related to polyamine depletion and is not a result of growth inhibition. Extracellular matrix laminin inhibited intestinal epithelial growth (44) but failed to induce TGF-beta RI expression (Fig. 4). Laminin consistently did not increase the sensitivity to TGF-beta -mediated growth inhibition in IEC-6 cells (Fig. 5). These results are consistent with our previous findings that indicate that cellular polyamines negatively regulate expression of growth-inhibited genes, including p53, junD, and TGF-beta in IEC-6 cells, and that polyamine depletion activates expression of these genes (13, 21, 23).

To further determine the significance of increased TGF-beta RI expression in the induced sensitivity to TGF-beta -mediated growth inhibition, RA was used to decrease TGF-beta RI levels in the presence or absence of cellular polyamines. Retinoids have been shown to modulate expression of TGF-beta receptors in a variety of tissues, and their effects are cell type dependent. In bovine endothelial cells, RA treatment upregulates the expression of TGF-beta RI and TGF-beta RII (46). On the other hand, TGF-beta receptors are markedly decreased after RA exposure in rat liver epithelial cells (18). Data presented in Fig. 6 show that administration of RA not only decreased basal levels of TGF-beta RI in normal IEC-6 cells but also prevented the increased TGF-beta RI expression in polyamine-deficient cells. RA treatment also diminished the inhibitory effect of TGF-beta on normal cell growth and prevented the increased sensitivity of polyamine-deficient cells to growth inhibition induced by TGF-beta (Fig. 7). Although it is possible that RA may have effects on other factors involved in the regulation of cell growth, these results support the possibility that the induced TGF-beta RI after polyamine depletion may be an important determinant of the increased sensitivity to TGF-beta action in intestinal epithelial cells.

Data presented in Fig. 8 clearly show that the increased TGF-beta RI expression by polyamine depletion is cell type dependent, because depletion of cellular polyamines by DFMO did not increase expression of TGF-beta receptor genes in Caco-2 cells. The reasons for the different responses to polyamine depletion in IEC-6 cells and Caco-2 cells remain obscure and may be related to the fact that the IEC-6 line is derived from the normal small intestine and Caco-2 line is from the colon carcinoma. Although we did not examine the effect of polyamine depletion on the expression of TGF-beta receptors in all types of cells available from small intestine and colon, the present studies also show that inhibition of polyamine synthesis did not induce the expression of TGF-beta receptor genes in the HT-29 cell line (data not shown).

The nature of molecular mechanisms that increase the expression of TGF-beta RI gene after polyamine depletion in IEC-6 cells remains to be demonstrated. Polyamines have been shown to play different roles in the expression of various genes in intestinal epithelial cells. For example, polyamines stimulate the transcription of protooncogene c-myc and c-jun but have no effect on these two gene posttranscriptions (22). In contrast, polyamines destabilize the TGF-beta mRNA without affecting its transcription (21). Data presented in Fig. 1A show that the increased TGF-beta RI protein level was paralleled by a significant increase in TGF-beta RI mRNA levels in IEC-6 cells exposed to DFMO for 4 and 6 days. It is not clear at present whether increased mRNA level for TGF-beta RI is due to an increase in gene transcription or results from the alteration of mRNA stabilization. Our previous studies have demonstrated that polyamines regulate growth-promoting genes such as c-myc and c-jun through the stimulation of gene transcription (22) but modulate growth-inhibited genes such as TGF-beta and p53 through the control of mRNA degradation (21). We postulate that the increased mRNA level for TGF-beta RI in polyamine-deficient cells may be regulated posttranscriptionally. This hypothesis awaits further study.

In summary, these results indicate that cellular polyamines are implicated in the regulation of TGF-beta RI gene expression in intestinal epithelial cells. Inhibition of polyamine synthesis by treatment with DFMO increases the TGF-beta RI expression, which associates with the increased sensitivity to growth inhibition induced by TGF-beta . Decreasing the TGF-beta RI level by RA prevents the increased sensitivity to exogenous TGF-beta in polyamine-deficient cells. These findings suggest that increased TGF-beta RI gene expression plays a critical role in the mechanism through which polyamine depletion increases the sensitivity of intestinal epithelial cells to TGF-beta -mediated growth inhibition.


    ACKNOWLEDGEMENTS

This work was supported by Merit Review Grants from the Department of Veterans Affairs to B. L. Bass and J.-Y. Wang and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45314 and DK-57819 to J.-Y. Wang.


    FOOTNOTES

Address for reprint requests and other correspondence: J.-Y. Wang. Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 N. Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 January 2000; accepted in final form 24 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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