Transforming growth factor-beta 1 signaling contributes to Caco-2 cell growth inhibition induced by 1,25(OH)2D3

Anping Chen, Bernard H. Davis, Michael D. Sitrin, Thomas A. Brasitus, and Marc Bissonnette

Gastroenterology Section, Department of Medicine, The University of Chicago, Chicago, Illinois 60637


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth of Caco-2 and many cancer cells is inhibited by 1,25(OH)2D3. Whereas TGF-beta 1 inhibits normal colonic epithelial cell growth, most human colon cancer-derived cells, including Caco-2 and SW480 cells, are resistant to it. The mechanisms underlying these antiproliferative actions and resistance to TGF-beta growth inhibition are largely unknown. We observed that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] sensitized Caco-2 and SW480 cells to TGF-beta 1 growth inhibitory effects. Versus 1,25(OH)2D3 alone, the combination of 1,25(OH)2D3 and TGF-beta 1 significantly reduced cell numbers. Also, the amount of active TGF-beta 1 was increased (~4-fold) by this secosteroid in conditioned media from Caco-2 cells. The 1,25(OH)2D3 increased the expression of IGF-II receptors (IGF-IIR), which facilitated activation of latent TGF-beta 1, and was found to activate TGF-beta signaling in Caco-2 cells. By using neutralizing antibodies to human TGF-beta 1, we showed that this cytokine contributes to secosteroid-induced inhibition of Caco-2 cell growth. Also, 1,25(OH)2D3 was found to enhance the type I TGF-beta receptor mRNA and protein abundance in Caco-2 cells. Whereas the 1,25(OH)2D3-induced sensitization of Caco-2 cells to TGF-beta 1 was IGF-IIR independent, the type I TGF-beta 1 receptor was required for this sensitization. Thus 1,25(OH)2D3 treatment of Caco-2 cells results in activation of latent TGF-beta 1, facilitated by the enhanced expression of IGF-IIR by this secosteroid. Also, 1,25(OH)2D3 sensitized Caco-2 cells to growth inhibitory effects of TGF-beta 1, contributing to the inhibition of Caco-2 cell growth by this secosteroid.

calcitriol; insulin-like growth factor-II receptor; colon cancer chemoprevention; antiproliferative; 1,25-dihydroxyvitamin D3


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

COLORECTAL CANCER IS A LEADING cause of cancer-related morbidity and mortality in the United States. Epidemiologic studies have found that the dietary intake of vitamin D and sunlight exposure are inversely associated with the risk of colon cancer (32). Studies from our laboratory and others have indicated that the active, hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and several of its analogs significantly inhibit colon cancer cell growth in vitro and in vivo (4, 16, 43). Prior studies from our group using Caco-2 cells have demonstrated that 1,25(OH)2D3 inhibits cell growth, enhances differentiation, and induces apoptosis (11, 16, 43), but many of the molecular mechanisms underlying these effects remain undefined.

In this regard, signaling pathways activated by 1,25(OH)2D3 and TGF-beta are known to have relevant biological interactions in several noncolonic cell types, including synergistic inhibitory effects on cell growth (17, 21, 50). The TGF-beta superfamily, like 1,25(OH)2D3, regulates a broad range of important cellular processes, including proliferation, differentiation, and apoptosis (38). Alterations in response to TGF-beta stimulation are thought to play important roles in colon cancer development. Whereas the growth of normal colonic epithelial cells is inhibited by TGF-beta 1, most human colon cancer cells, including Caco-2 and SW480 cells, are resistant to the growth inhibitory effects of TGF-beta 1 (6, 49). With the exception of several mutations in TGF-beta receptors and downstream signaling proteins (14, 31), which occur in a minority of colon tumors and colon cancer-derived cell lines, most sporadic colon cancers and cell lines have no identified molecular derangements responsible for their resistance to TGF-beta growth inhibition.

TGF-beta is synthesized and secreted in a latent, biologically inactive form, which must be activated before binding to TGF-beta receptors. The IGF-II receptor (IGF-IIR) is a multifunctional receptor with two distinct binding sites. One site binds to IGF-II, leading to its degradation and thereby regulating the bioavailability of extracellular IGF-II. The second site binds a variety of proteins bearing mannose-6-phosphate (Man-6-P) residues (28), including the inactive TGF-beta precursor. An established physiological function of IGF-IIR is to facilitate the proteolytic activation of the inactive TGF-beta precursor. TGF-beta signaling is mediated by a network of transmembrane serine/threonine kinase receptors and their downstream signal-transducing targets, the Smad proteins. This signaling is initiated by TGF-beta binding to the type II TGF-beta receptor (Tbeta -RII), which then phosphorylates and activates the type I TGF-beta receptor (Tbeta -RI). These two receptors then form a heteromeric complex (probably a heterotetramer), which, in turn, phosphorylates regulatory Smad2 and/or Smad3 proteins. The latter proteins subsequently form a heteromeric complex with Smad4 and migrate to the nucleus to regulate target gene expression. The Smad complexes often act in concert with other transcription factors, as well as with coactivators or corepressors.

We hypothesized that alterations in TGF-beta signaling may mediate, at least in part, Caco-2 cell growth inhibition by vitamin D secosteroids. In the present studies, 1,25(OH)2D3 was observed to increase the amount of active TGF-beta 1 in the conditioned media from Caco-2 cells. Furthermore, this secosteroid sensitized Caco-2 cells to TGF-beta 1 growth inhibitory effects. The mechanisms for these effects of 1,25(OH)2D3 on TGF-beta 1 activation and signaling, their influence on Caco-2 cell growth, and their potential importance in the chemoprevention of colon cancer form the basis of this study.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and reagents. We purchased 1,25(OH)2D3 from Steroids Laboratory (Chicago, IL). Active human TGF-beta 1 was obtained from Promega (Madison, WI). Man-6-P, glucose-6-phosphate (Glu-6-P), and other chemicals were of the highest purity available and were purchased from Sigma (St. Louis, MO) unless otherwise indicated.

Cell culture, transfection, and luciferase assay. Caco-2 and SW480 cells, each derived from human colonic carcinomas, were cultured at 37°C in 5% CO2 in DMEM, as previously described (11). Cells were treated with 1,25(OH)2D3 or vehicle (EtOH) for the indicated time and protected from fluorescent light. Sixty- to eighty-percent confluent cells (2-3 days after plating) in six-well cell culture plates were transfected by LipofectAMINE, following the protocol provided by the manufacturer (GIBCO Life Technologies, Grand Island, NY). Each transfection was performed in triplicate and repeated three to four times. The beta -galactosidase expression plasmid pSV-beta -galactosidase (Promega) was included to normalize for transfection efficiency. Luciferase assays were performed by using a kit from Promega and following the protocol provided by the manufacturer. The luciferase activities of each transfection were expressed as relative units after normalization for transfection efficiency by using beta -galactosidase activities.

Plasmid constructs. The plasmids, pT7-Tbeta RI and pT7-Tbeta RII, used for generating single-stranded RNA probes complimentary to Tbeta -RI and Tbeta -RII, respectively, for RNase protection assays (RPAs) were generous gifts from Dr. Michael Centrella (Yale University, New Haven, CT) (7). The plasmid p3TP-Luc is a TGF-beta -inducible luciferase reporter construct, containing the plasminogen activator inhibitor-1 (PAI-1) gene promoter, kindly provided by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY) (1, 5, 33).

RNA isolation and RPA. Total RNA was isolated by the Tri reagent, following the protocol recommended by the manufacturer (Sigma). A single-stranded RNA probe complimentary to IGF-IIR (411 bp) was generated as previously described (28, 48). To prepare RNA probes for Tbeta -RI and Tbeta -RII, pT7-Tbeta RI was linearized with SmaI and pT7-Tbeta RII was linearized with EcoRI, respectively (7). The 115 bp of 28S rRNA probe was used as an internal control (Ambion, Austin, TX). The antisense probes were synthesized and 32P-labeled by MAXIscript (Ambion). RPA was carried out with RPA II kits (Ambion) following the protocol provided by the manufacturer. The radioactivity in each band was measured by a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), as described previously (10, 11).

Western blotting analysis. Whole cell extracts were prepared from preconfluent Caco-2 cells, and membrane proteins were prepared as described (2). Human colonic specimens were obtained from the Department of Surgical Pathology of the University of Chicago Hospitals. These included paired colon cancer samples and normal colonic mucosa dissected from the underlying layers from the same patient. Specimens were flash frozen in liquid nitrogen within 1 h of resection. Tumor homogenates were prepared and stored at -70°C until use. Western blotting analyses were performed as described (10, 11). Anti-Tbeta -RI and -RII antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the anti-IGF-IIR serum was generously provided by Dr. Richard G. MacDonald (University of Nebraska) (29). Integrin-beta 1, a subunit of integrin receptors for the extracellular matrix (13), was used as an internal membrane protein control for the IGF-IIR Western blots.

Cell proliferation assays. Preconfluent Caco-2 or SW480 cells (2-3 days after plating) were incubated with the indicated concentrations of active human TGF-beta 1 (Promega) in the presence or absence of 1,25(OH)2D3 (100 nM) for 24 h in serum-free DMEM. In other experiments, neutralizing anti-active TGF-beta 1 antibodies (30 µg/ml; Promega), control normal rabbit IgG, Man-6-P (100 µM), or Glu-6-P (100 µM) was added to serum-free DMEM 30 min before the addition of 1,25(OH)2D3. Cell growth was determined by counting cell numbers and/or by MTS assays (Promega). For MTS assays, all experiments were carried out in 96-well plates. Cell growth was analyzed by using the CellTiter 96 AQueous non-radioactive cell proliferation assay kit following the protocol provided by the manufacturer.

TGF-beta 1 immunoassay. After cell treatment, conditioned medium was collected and centrifuged at 10,000 rpm for 10 min at 4°C. The supernatants were assayed for the active form of TGF-beta 1 by a TGF-beta 1 Emax ImmunoAssay system (ELISA) (Promega) following the protocol provided by the manufacturer. This immunoassay system was designed for the sensitive and specific detection of biologically active TGF-beta 1. The antibody in the system does not recognize the TGF-beta 1 precursor. To determine the amount of total TGF-beta 1 in the conditioned media, samples were pretreated with 1 N HCl for 15 min at room temperature before neutralization with 1 N NaOH, as suggested by the manufacturer. This procedure converts any latent TGF-beta 1 to the active form.

Suppression of IGF-IIR and Tbeta -RI protein expression by antisense oligonucleotides. These experiments were conducted as previously described (11). In brief, phosphorothioate-modified sense and antisense oligonucleotides were synthesized by GIBCO Life Technologies. The optimal concentrations to suppress the expression of these receptor proteins were determined by incubation of Caco-2 cells in DMEM with either antisense or sense oligonucleotides at concentrations between 0 and 100 µg/ml for 4 h before the addition of 1,25(OH)2D3. After 24 h incubation, the cells were harvested and the lysates were probed by Western blotting using anti-IGF-IIR or anti-Tbeta -RI. To study the roles of IGF-IIR and Tbeta -RI in the TGF-beta 1 sensitization induced by 1,25(OH)2D3, preconfluent cells were pretreated with 50 µg/ml of either antisense or sense oligonucleotides 4 h before the addition of 1,25(OH)2D3 (100 nM) and exogenous active TGF-beta 1. In preliminary experiments, we found that this concentration of antisense for Tbeta -R1 and antisense for IGF-IIR caused maximum inhibition of protein expression for each of these receptors. The incubation was then continued for an additional 24 h. Cell growth was determined by counting cell numbers and/or by MTS assays. The sequences for the antisense oligonucleotides used for the experiments were as follows: IGF-IIR, 5'-TCC TAG CTG AAC GGC CCG CAT-3' [the first 21 nucleotides from the start codon of the human IGF-IIR gene (37)] and Tbeta -RI, 5'-AGC AGC CGA CGC CGC CTC CAT-3' [the first 21 nucleotides from the start codon of the Tbeta -RI gene (20)].

Immunocytochemistry. Preconfluent Caco-2 cells in 12-well plates (2-3 days after plating) were pretreated with or without antisense IGF-IIR oligonucleotides (50 µg/ml) 4 h before the addition of 1,25(OH)2D3 (100 nM) for an additional 24 h. Cells treated with vehicle (0.08% EtOH) for 24 h were used as a control. Cells were fixed with 100% methanol. After being rinsed and blocked with PBS/1% BSA, cells were labeled with anti-IGF-IIR serum (1:100). As a negative control, cells were labeled with normal (nonimmune) rabbit IgG. The cells were immunostained by using biotinylated secondary antibodies and the ABC kit from Vector Laboratories (Burlingame, CA).

Statistical analysis. Differences between means were evaluated by using an unpaired Student's t-test (P < 0.05 was considered significant). Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by ANOVA with the Dunnett's test for post hoc analysis.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1,25(OH)2D3 sensitizes Caco-2 cells to the growth inhibitory effects of TGF-beta 1. Our previous studies demonstrated that 1,25(OH)2D3 significantly inhibited Caco-2 cell growth in a dose-dependent manner (10-10-10-7 M), with a maximal inhibition at 100 nM (16) and no toxicity up to 300 nM as assessed by lactate dehydrogenase release (41). On the basis of these observations, we chose to study preconfluent Caco-2 cells treated with 1,25(OH)2D3 at 100 nM for these experiments. Caco-2 cells were chosen to evaluate the potential ability of 1,25(OH)2D3 to sensitize colon cancer cells to the growth inhibitory effects of TGF-beta 1. Compared with cells without any treatment, 1,25(OH)2D3 alone (i.e., 0 ng/ml TGF-beta 1) significantly reduced Caco-2 cell numbers (Fig. 1). As shown in Fig. 1, in the absence of 1,25(OH)2D3, Caco-2 cells were resistant to the growth inhibitory effects of exogenous active TGF-beta 1 (0.1-4 ng/ml) alone. In contrast, in the presence of 1,25(OH)2D3 (100 nM), TGF-beta 1, in a dose-dependent manner, further significantly reduced Caco-2 cell numbers. The optimal TGF-beta 1 growth inhibitory dose was 2-2.5 ng/ml (Fig. 1). We also assessed whether another TGF-beta -resistant human colon cancer cell line, SW480, could also be sensitized by 1,25(OH)2D3 to TGF-beta growth inhibition (Fig. 1). In agreement with previous reports (6, 49), exogenous active TGF-beta 1 alone had no effect on SW480 cell growth (data not shown). In contrast, in the presence of 1,25(OH)2D3 (100 nM), exogenous active TGF-beta 1 caused a significant dose-dependent reduction in cell numbers of SW480 cells. As in the case of Caco-2 cells, maximal inhibition was seen at 2.5 ng/ml and was somewhat reduced at higher concentrations. These results indicated that SW480 cells, like Caco-2 cells, were sensitized by 1,25(OH)2D3 to the growth inhibitory effects of TGF-beta 1.


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Fig. 1.   1,25-dihydroxyvitamin D3 [1,25(OH)2D3] sensitizes Caco-2 and SW480 cells to the growth inhibitory effects of TGF-beta 1. Caco-2 and SW480 cells (2-3 days after plating) were treated for 24 h with the indicated doses of active TGF-beta 1 in the absence (TGF-beta alone) or presence of 1,25(OH)2D3 (100 nM). Cell growth was determined by counting cell numbers. Values are means ± SD (n = 6). *P < 0.05 vs. SW480 cells treated with 1,25(OH)2D3 alone. dagger P < 0.05 vs. Caco-2 cells treated with 1,25(OH)2D3 alone. Error bars, if not indicated, were contained within the data points.

1,25(OH)2D3 increases the amount of active TGF-beta 1 in Caco-2-conditioned media. To determine the potential ability of 1,25(OH)2D3 to alter the abundance of TGF-beta 1 in Caco-2 cells, conditioned media from these cells, treated with 1,25(OH)2D3 (100 nM) or vehicle for 24 h, were analyzed for both the total amount of TGF-beta 1 and the active form of this cytokine by immunoassays (ELISA) as described in MATERIALS AND METHODS. Our results indicated that, although 1,25(OH)2D3 did not change the total amount of TGF-beta 1, this secosteroid increased the amount of active TGF-beta 1 by approximately fourfold in Caco-2-conditioned media (Fig. 2). Prior studies demonstrated that Man-6-P prevented the activation of latent TGF-beta 1 by blocking the binding of the pro form of TGF-beta 1 to IGF-IIR (12). In agreement with these findings, we observed that the 1,25(OH)2D3-induced increase in active TGF-beta 1 in Caco-2-conditioned media could be blocked by Man-6-P (100 µM) (Fig. 2) but not by Glu-6-P (data not shown), suggesting the involvement of IGF-IIR in this process.


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Fig. 2.   1,25(OH)2D3 increases the active form of TGF-beta 1 in the conditioned media of Caco-2 cells. Aliquots of conditioned media from preconfluent Caco-2 cells, treated with 1,25(OH)2D3 (100 nM) or vehicle (EtOH) in the presence or absence of mannose-6-phosphate (M-6-P) for 24 h, were analyzed for total and active TGF-beta 1 by a TGF-beta 1 ELISA (see MATERIALS AND METHODS for details). Values are means ± SD (n = 6). *P < 0.05 vs. vehicle-treated cells. **P < 0.05 vs. 1,25(OH)2D3 alone.

1,25(OH)2D3 enhances the expression of IGF-IIR in Caco-2 cells. As noted earlier, the IGF-IIR facilitates the activation of TGF-beta 1 by binding the precursor of this cytokine and subsequently releasing the inhibitory Man-6-P-containing latency peptide (12, 15, 22). Prior studies have demonstrated that Caco-2 cells express functional IGF-IIR (18). Since 1,25(OH)2D3 significantly increased the active form of TGF-beta 1 in the conditioned media of Caco-2 cells and Man-6-P could prevent this increase (Fig. 2), it was plausible that 1,25(OH)2D3 might induce the expression of IGF-IIR in these cells. To study this possibility, we examined the effect of 1,25(OH)2D3 on the expression of IGF-IIR in Caco-2 cells. It significantly increased the steady-state levels of IGF-IIR mRNA by threefold in Caco-2 cells, as demonstrated by RNase protection assays (Fig. 3, A and B). Additional experiments were performed to assess the effects of 1,25(OH)2D3 on IGF-IIR membrane protein expression. Preconfluent Caco-2 cells were treated with 1,25(OH)2D3 (100 nM) or EtOH for 24 h, and 1,25(OH)2D3 significantly enhanced the expression of membrane IGF-IIR proteins by ~5.5-fold in Caco-2 cells (Fig. 3, C and D).


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Fig. 3.   1,25(OH)2D3 increases the IGF-II receptor (IGF-IIR) expression in Caco-2 cells. Caco-2 cells were exposed to 1,25(OH)2D3 (100 nM) for the indicated time. Total RNA or membrane proteins were prepared for the RNase protection assays (RPAs) or Western blotting analyses, respectively, as described in MATERIALS AND METHODS. A: representative IGF-IIR RPA. Fifteen micrograms of total RNA per sample were analyzed. Human 28S rRNA was used as the internal control to normalize for total RNA loading. The protected IGF-IIR and 28S rRNA are indicated at right. B: quantitation of IGF-IIR RPA with means ± SD from 4 independent experiments. *P < 0.05 vs. untreated control. C: representative Western blotting analyses of membrane IGF-IIR. Integrin-beta 1, a subunit of extracellular matrix receptors, was used as an internal membrane protein control (13). D: quantitation of membrane IGF-IIR protein, normalized to integrin-beta 1, with means ± SD from 3 independent experiments. *P < 0.05 vs. untreated control.

TGF-beta 1 contributes to the 1,25(OH)2D3-induced inhibition of Caco-2 cell growth. Having established that 1,25(OH)2D3 increased the active form of TGF-beta 1, we asked if immune neutralization of TGF-beta 1 would alter the ability of this secosteroid to inhibit Caco-2 cell growth. Caco-2 cells in serum-free DMEM were pretreated with anti-TGF-beta 1 antibodies (30 µg/ml) or nonimmune serum (normal rabbit IgG) for 30 min before the addition of this secosteroid. As shown in Fig. 4, pretreatment of cells with anti-TGF-beta 1 antibodies, but not nonimmune serum, significantly reduced the growth inhibition of Caco-2 cells by 1,25(OH)2D3 from 26% to 15%. These results thus suggested an autocrine/paracrine role for TGF-beta 1 and supported our hypothesis that TGF-beta 1 mediates, at least in part, the antiproliferative effects of this secosteroid in these cells. To further evaluate the role of the activation of latent TGF-beta 1 in the 1,25(OH)2D3-induced inhibition of cell growth, Caco-2 cells in serum-free DMEM were pretreated with Man-6-P (100 µM) or Glu-6-P (100 µM) before the addition of 1,25(OH)2D3. As shown in Fig. 4, Man-6-P, but not Glu-6-P, reduced the inhibition of Caco-2 cell growth induced by 1,25(OH)2D3 from 26 to 16%. These studies indicated that activation of latent TGF-beta 1, facilitated by IGF-IIR, played an important role in the inhibition of Caco-2 cell growth induced by this secosteroid. Increasing the concentrations of neutralizing anti-TGF-beta 1 antibodies or Man-6-P did not further limit this growth inhibition by 1,25(OH)2D3, suggesting that other TGF-beta -independent mechanisms also contribute to the inhibition of cell growth by this secosteroid. It should be emphasized, however, that additional (exogenous) active TGF-beta 1 alone failed to inhibit the growth of these cells in the absence of 1,25(OH)2D3. These results indicate, therefore, that TGF-beta 1 sensitization must involve more than an increased conversion of the latent to the active form of this cytokine.


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Fig. 4.   TGF-beta 1 contributes to the 1,25(OH)2D3-induced inhibition of Caco-2 cell growth. Caco-2 cells (2-3 days after plating) were treated with EtOH or active TGF-beta 1 (2 ng/ml) in the presence or absence of 1,25(OH)2D3 or with 1,25(OH)2D3 plus anti-TGF-beta 1 antibodies (alpha -TGF-beta 1; 30 µg/ml), normal rabbit IgG (NRIgG), mannose-6-phosphate (Man-6-P; 100 µM), or glucose-6-phosphate (Glu-6-P; 100 µM). alpha -TGF-beta 1, Man-6-P, or Glu-6-P was added before 1,25(OH)2D3. Cell growth was determined by counting cell numbers. Values are means ± SD (n = 6). *P < 0.05 vs. EtOH-treated cells. Dagger P < 0.05 vs. 1,25(OH)2D3 alone.

1,25(OH)2D3 activates TGF-beta signaling in Caco-2 cells. To assess the ability of 1,25(OH)2D3 to activate TGF-beta signaling in Caco-2, cells were transiently transfected with a TGF-beta 1-inducible luciferase reporter plasmid p3TP-Luc. This luciferase reporter plasmid contains the promoter for the PAI-1 gene, which includes TGF-beta response elements (1, 5, 33). After transfection, cells were treated with the indicated agents as described in Fig. 5 and luciferase activities in the treated cells were determined. As shown in Fig. 5, compared with EtOH as control, 1,25(OH)2D3 significantly increased the luciferase activity by 4.9-fold in cells transfected with p3TP-Luc. Man-6-P abrogated the ability of 1,25(OH)2D3 to increase the luciferase activity in these cells. Not surprisingly, TGF-beta 1 alone did not alter luciferase activity in Caco-2 cells. However, in the presence of 1,25(OH)2D3, TGF-beta 1 significantly increased luciferase activity by an additional 45% compared with 1,25(OH)2D3 treatment alone. Man-6-P pretreatment could not block the increase in luciferase activity in the cells treated with both 1,25(OH)2D3 and exogenous active TGF-beta 1. This result is not surprising since Man-6-P prevents the conversion of precursor TGF-beta 1 to the active form but does not inhibit the activated species. Together, these results indicate that 1,25(OH)2D3 confers competence to transcriptional activation of target genes by TGF-beta 1 in Caco-2 cells, which is likely involved in the ability of this secosteroid to sensitize Caco-2 cells to TGF-beta 1 growth inhibition.


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Fig. 5.   1,25(OH)2D3 activates TGF-beta 1 signaling. Preconfluent Caco-2 cells (2-3 days after plating) were transiently transfected with the TGF-beta -inducible luciferase reporter plasmid p3TP-Luc. The beta -galactosidase expression plasmid pSV-beta -galactosidase was cotransfected to normalize transfection efficiency. After transfection, cells were treated with 1,25(OH)2D3 or EtOH, plus or minus active TGF-beta 1 (2 ng/ml) for an additional 36 h. Where indicated, Man-6-P (100 µM) was added 3 h before 1,25(OH)2D3. Luciferase activities were determined by luciferase assay. The luciferase activities of each transfection were expressed as relative units after normalization for transfection efficiency from beta -galactosidase activity. Values are means ± SD (n = 6). *P < 0.05 vs. EtOH-treated cells. **P < 0.01 vs. cells treated with 1,25(OH)2D3 alone.

1,25(OH)2D3 increases the abundance of Tbeta -RI protein and mRNA in Caco-2 cells. As noted in the introduction, TGF-beta signaling is initiated by TGF-beta binding to Tbeta -RII, which phosphorylates and activates Tbeta -RI. The signaling subsequently passes to target genes through Smad proteins. As shown in Fig. 1, treatment with 1,25(OH)2D3 sensitized Caco-2 and SW480 cells to growth inhibition by TGF-beta 1. We postulated that this 1,25(OH)2D3-induced sensitization might arise, at least in part, by upregulation of TGF-beta receptor expression. To address this hypothesis, total mRNA and protein extracts were prepared from Caco-2 cells treated with 1,25(OH)2D3 for the indicated time. RPAs indicated that 1,25(OH)2D3 stimulated an increase in the steady-state levels of Tbeta -RI mRNA, but not Tbeta -RII mRNA, by 1.4-, 2.3-, 2.9-, and 2.8-fold after 3, 6, 16, and 24 h treatment, respectively, compared with control (0 h) (Fig. 6, A and B). As assessed by Western blotting analyses, 1,25(OH)2D3 treatment for 24 h significantly increased the protein abundance of Tbeta -RI by 2.5-fold (Fig. 6C) but did not increase Tbeta -RII (data not shown) in Caco-2 cells.


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Fig. 6.   1,25(OH)2D3 increases the steady-state levels of TGF-beta receptor (Tbeta -R) I mRNA and protein in Caco-2 Cells. Caco-2 cells in duplicate were treated with 1,25(OH)2D3 (100 nM) for the indicated time. Total proteins or RNA were prepared for Western blotting analyses or RPA, respectively. A: representative RPA for Tbeta -RI and Tbeta -RII and human 28S rRNA as the internal control to normalize for total RNA loading. B: quantitation of Tbeta -RI and Tbeta -RII mRNA with means ± SD from 3 independent experiments, each in duplicate. *P < 0.05 vs. control (0 h). C: representative Western blots of Tbeta -RI and an internal control of beta -actin.

Expression of both Tbeta -RI and RII are significantly reduced in human colonic carcinomas and Caco-2 cells. Since 1,25(OH)2D3 upregulated the expression of Tbeta -RI in Caco-2 cells and sensitized these cells to TGF-beta 1 inhibition, it was of interest to compare the expression of TGF-beta receptors in normal colon to that of colonic cancer and colon cancer-derived Caco-2 cells. To evaluate the expression levels of Tbeta -RI and -RII, protein extracts prepared from colonic carcinomas and corresponding normal colonic mucosa, as well as Caco-2 cells, were analyzed by Western blot analyses. Compared with matched normal colonic mucosa (Fig. 7A, lanes 1 and 2), expression levels of both Tbeta -RI and Tbeta -RII were significantly reduced by ~30% and 55%, respectively, in human colonic tumors (lanes 3 and 4). In addition, compared with normal colonic mucosa (lanes 1 and 2), expression of both Tbeta -RI and -RII in Caco-2 cells (lanes 5 and 6) were also markedly reduced. Tbeta -RI expression was induced by 1,25(OH)2D3 in Caco-2 cells by 2.3-fold (lanes 7 and 8). In contrast, Tbeta -RII was not altered by 1,25(OH)2D3 (lanes 7 and 8).


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Fig. 7.   Expression levels of both Tbeta -RI and -RII are reduced in human colonic cancers and Caco-2 cells. Protein lysates were prepared from 6 separate human colonic tumors and corresponding normal colonic mucosa, as well as from Caco-2 cells treated with or without 1,25(OH)2D3 (100 nM) for 24 h. A: Western blots of Tbeta -RI and -RII in two representative tumors and corresponding control mucosa as well as treated and untreated Caco-2 cells. beta -Actin was used as an internal control. B: quantitation of Tbeta -RI and -RII proteins with means ± SD (n = 6 human colonic tumors and matched normal colonic mucosa). Caco-2 cells were plated in duplicate (n = 3 independent experiments). * P < 0.05 vs. normal mucosa; **P < 0.02 vs. untreated Caco-2 cells (control).

Tbeta -RI is required for the TGF-beta 1 sensitization of Caco-2 cells induced by 1,25(OH)2D3. In initial experiments, we demonstrated that antisense Tbeta -RI or IGF-IIR oligonucleotides at 40-60 µg/ml significantly and specifically inhibited the expression of Tbeta -RI and IGF-IIR proteins, respectively, in Caco-2 cells (Fig. 8, A and B). In addition, we examined the effect of antisense IGF-IIR oligonucleotides on IGF-IIR protein expression detected by immunocytochemical staining (Fig. 9). Compared with the cells treated with 1,25(OH)2D3 alone, pretreatment of cells with antisense IGF-IIR oligonucleotides significantly reduced the level of IGF-IIR protein staining induced by 1,25(OH)2D3, demonstrating that IGF-IIR protein is significantly inhibited by antisense IGF-IIR oligonucleotides in Caco-2 cells. Using these antisense oligonucleotides, we next evaluated the roles of these receptors in the ability of 1,25(OH)2D3 to inhibit Caco-2 cell growth and to sensitize these cells to the antiproliferative effect of TGF-beta 1. Caco-2 cells were pretreated with either antisense Tbeta -RI or IGF-IIR oligonucleotides (50 µg/ml) 4 h before the addition of the indicated doses of active TGF-beta 1 and 1,25(OH)2D3 (100 nM) for an additional 24 h. As shown in Fig. 8C, antisense Tbeta -R1 oligonucleotides significantly reduced growth inhibition of Caco-2 cells by 1,25(OH)2D3 from 25 to 16% and abrogated the previously observed secosteroid-induced sensitization of these cells to TGF-beta 1. These results indicated that Tbeta -RI significantly contributes to the 1,25(OH)2D3-induced inhibition of Caco-2 cell growth. Furthermore, as expected, this receptor is required for 1,25(OH)2D3 to confer responsiveness of Caco-2 cells to the antiproliferative effect of TGF-beta 1. Downregulation of IGF-IIR in Caco-2 cells by antisense IGF-IIR oligonucleotides also significantly reduced the growth inhibition by 1,25(OH)2D3 from 25 to 14% (Fig. 8C). This is in agreement with the role of IGF-IIR in the activation of latent TGF-beta . Not surprisingly, however, antisense IGF-IIR oligonucleotides were unable to block the secosteroid-induced sensitization of Caco-2 cells to the growth inhibitory effects of exogenous active TGF-beta 1 (Fig. 8C).


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Fig. 8.   Antisense Tbeta -RI oligonucleotides reduced the 1,25(OH)2D3-induced inhibition of cell growth and completely abolished the sensitization of Caco-2 cells to TGF-beta 1 induced by this secosteroid. Caco-2 cells (2-3 days after plating) were incubated in DMEM with 1,25(OH)2D3 alone or containing the indicated antisense or sense oligonucleotides (see MATERIALS AND METHODS). A: representative Western blots of cells treated with 1,25(OH)2D3 alone or with either antisense or sense Tbeta -RI oligonucleotides at the indicated concentrations for 24 h. B: representative Western blots of cells treated with 1,25(OH)2D3 alone or with either antisense or sense IGF-IIR oligonucleotides at the indicated concentrations for 24 h. C: proliferation assays of cells pretreated with or without 50 µg/ml sense or antisense Tbeta -RI or antisense IGF-IIR oligonucleotides for 4 h. Cells were then incubated with the indicated concentrations of active TGF-beta 1 alone or with 1,25(OH)2D3 (100 nM) for 24 h. Cell growth was evaluated by MTS assays. Values are means ± SD (n = 6). *P < 0.05 vs. cells treated with 1,25(OH)2D3 alone (0 ng/ml TGF-beta 1).



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Fig. 9.   Antisense IGF-IIR oligonucleotides significantly reduce the protein expression of IGF-IIR induced by 1,25(OH)2D3 in Caco-2 cells. Caco-2 cells (2-3 days after plating) were pretreated with or without antisense IGF-IIR oligonucleotides (50 µg/ml) 4 h before the addition of 1,25(OH)2D3 (100 nM) for 24 h before fixation. Cells were labeled with anti-IGF-IIR antibodies (1:100) and immunostained by the biotinylated secondary antibodies and ABC kit from Vector Laboratories. Representative examples are shown here from 3 independent experiments (original magnification, ×100). Control samples, stained with nonimmune serum, were negative (data not shown). Left: cells treated with 0.08% EtOH as a negative control; Middle: cells treated with 1,25(OH)2D3; Right: cells pretreated with antisense IGF-IIR oligonucleotides (alpha -sense oligoes) before addition of 1,25(OH)2D3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this paper we report for the first time two important interactions of 1,25(OH)2D3 with the TGF-beta 1 signaling system that explain, at least in part, the antiproliferative effect of this secosteroid in Caco-2 cells. We found that 1,25(OH)2D3 (100 nM) increased the abundance of active TGF-beta 1 by stimulating the expression of IGF-IIR in Caco-2 cells. In addition, we observed that 1,25(OH)2D3 (100 nM) sensitized Caco-2 cells, as well as SW480 cells, to the growth inhibitory effects of TGF-beta 1.

Although the growth of normal epithelial cells, including colonocytes, is inhibited by TGF-beta 1 (39), primary colon neoplasms and most colon cancer cell lines are typically resistant to TGF-beta 1 (8). Progression from colonic adenoma to carcinoma is accompanied by increasing resistance to TGF-beta -induced growth inhibition (30). With the exception of several specific mutations in TGF-beta receptors or downstream Smad proteins that occur predominantly in tumors with DNA mismatch repair defects, the majority of sporadic colon cancers and colon cancer cell lines have no identified molecular derangements that explain their resistance to TGF-beta growth inhibition (19).

TGF-beta 1 is secreted by cells in a latent, biologically inactive form, which must be activated before binding to TGF-beta receptors. IGF-IIR binds and facilitates the proteolytic activation of the inactive TGF-beta precursor (12, 22). In the present study, total TGF-beta 1 secreted by Caco-2 cells was not affected by 1,25(OH)2D3; however, the abundance of active TGF-beta 1 was increased. In several noncolonic cell lines, 1,25(OH)2D3 was reported to increase total TGF-beta secretion (23, 51), suggesting that the effects of 1,25(OH)2D3 on TGF-beta 1 gene expression and activation are cell type specific. In Caco-2 cells, 1,25(OH)2D3 enhanced the protein and mRNA expression of IGF-IIR, as demonstrated by immunocytochemical and Western blotting studies and by RPAs, respectively. Moreover, treatment of Caco-2 cells with Man-6-P abolished the increase in active TGF-beta 1, which further supports our assumption that the upregulation of IGF-IIR expression was responsible for the increase in active TGF-beta 1 by 1,25(OH)2D3. We have demonstrated that induction of the IGF-IIR and subsequent TGF-beta 1 activation in Caco-2 cells is responsible, at least in part, for the 1,25(OH)2D3-induced growth inhibition of these cells. Downregulation of IGF-IIR by antisense oligonucleotides, blockage of latent TGF-beta binding to IGF-IIR by Man-6-P, or neutralization of TGF-beta 1 by antibodies diminished the antiproliferative effects of 1,25(OH)2D3 in Caco-2 cells, demonstrating that this secosteroid stimulated the activation of TGF-beta , which then acted by an autocrine/paracrine mechanism to inhibit growth. These results suggested that IGF-IIR indirectly contributes to the 1,25(OH)2D3-induced Caco-2 cell growth inhibition by facilitating activation of latent TGF-beta . We do not believe, however, that there is a direct requirement for IGF-IIR in the 1,25(OH)2D3-induced sensitization of Caco-2 cells to TGF-beta inhibitory effects. In this regard, antisense IGF-IIR oligonucleotides were unable to block the secosteroid-induced sensitization of Caco-2 cells to the growth inhibitory effects of exogenous active TGF-beta 1 (Fig. 8C)

In addition, the IGF-IIR may affect cell growth by regulating the bioavailability of extracellular IGF-II (12, 22). As a potent mitogenic polypeptide homologous to insulin, IGF-II, which is secreted by Caco-2 cells, binds to IGF-I receptors and stimulates cell growth (3). In contrast, binding to the IGF-IIR, which does not transduce a mitogenic signal, results in an accelerated degradation of IGF-II and thereby a reduction in cell growth (28, 34). Hence, the enhanced expression of the IGF-IIR by 1,25(OH)2D3 would be expected to reduce the bioavailability of mitogenic IGF-II and thereby contribute to the growth inhibition of Caco-2 cells caused by this secosteroid. Our results demonstrated that when IGF-IIR expression was blocked by IGF-IIR antisense oligonucleotides, large doses of exogenous TGF-beta 1 plus 1,25(OH)2D3 failed to inhibit growth to the same extent as cells without the antisense treatment. This observation suggests that the antisense oligonucleotides, which mediated downregulation of IGF-IIR, also positively influenced growth by a TGF-beta 1-independent mechanism, possibly by increasing the bioavailability of mitogenic IGF-II.

Signaling pathways induced by 1,25(OH)2D3 and TGF-beta 1 have been shown to interact in several noncolonic cell types, resulting in increased TGF-beta 1 release (17, 23), enhanced TGF-beta receptor expression (21, 50, 51), decreased cell growth (17, 23), and enhanced differentiation (35). In the present study, although TGF-beta 1 alone had no effect on cell growth, the combination of 1,25(OH)2D3 and TGF-beta 1 caused significantly more growth inhibition than 1,25(OH)2D3 alone. This indicated that 1,25(OH)2D3 sensitized these cells to the growth inhibitory effects of TGF-beta 1.

In this regard, our studies have demonstrated that the levels of both Tbeta -RI and -RII were significantly and comparably reduced in Caco-2 cells and in human colonic tumors compared with normal colonic mucosa. 1,25(OH)2D3 increased the expression of Tbeta -RI mRNA and protein by ~2.5-fold in Caco-2 cells but did not change the level of Tbeta -RII. Results from experiments with Tbeta -RI antisense oligonucleotides indicated that this receptor contributed to the 1,25(OH)2D3-induced inhibition of cell growth and, not surprisingly, was required for the sensitization of these cells to growth inhibition by exogenous TGF-beta 1. Interestingly, another human colon carcinoma-derived cell line, GEO cells, are also insensitive to TGF-beta 1 and express low levels of Tbeta -RI mRNA (47). Stable transfection of Tbeta -RI, but not Tbeta -RII, cDNA increased TGF-beta 1 binding and resulted in increased growth inhibition by exogenous TGF-beta 1 (47), indicating that the low level of Tbeta -RI was a limiting factor for the growth-inhibitory effects of TGF-beta 1 in those cells. Other studies have suggested a major role of Tbeta -RII in the regulation of gene expression, whereas Tbeta -RI appears to mainly transduce regulatory effects of TGF-beta 1 on cell growth (27, 40). Thus it is likely that the upregulation of Tbeta -RI by 1,25(OH)2D3 contributed, at least in part, to the sensitization of Caco-2 cells to the growth inhibitory effect of TGF-beta caused by this secosteroid. Maximal growth inhibition occurred at 2.5 ng/ml. The somewhat reduced inhibition at higher TGF-beta concentrations that others have also observed (36) may reflect a non-receptor-mediated effect.

In addition, vitamin D signaling has been found to interact with Smad proteins in other cell types. Transcriptionally active complexes containing the vitamin D receptor (VDR) and Smads have been identified (53). Smad proteins have been reported to activate or inhibit VDR transactivating ability (52). Studies are in progress in our laboratory to elucidate the roles of Smad proteins in the TGF-beta sensitization of Caco-2 cells by 1,25(OH)2D3.

The mechanisms by which 1,25(OH)2D3 upregulates IGF-IIR and Tbeta -RI expression remain unsolved. Our prior studies demonstrated that 1,25(OH)2D3 induced the activation of the transcription factor activator protein-1 (AP-1) in Caco-2 cells (11). It is possible that AP-1, activated by this secosteroid, might mediate the upregulation of IGF-IIR and Tbeta -RI gene expression, since putative AP-1 binding sites are present in the promoter regions of both genes (20, 26). Alternatively, these promoters may contain VDR response elements. Additionally, the c-Jun component of AP-1 may interact with Smad proteins in regulating gene expression as described by others (25, 46).

With regard to sensitization to TGF-beta 1 growth inhibition by other agents, previous studies have demonstrated that another differentiating agent, the short-chain fatty acid butyrate, like 1,25(OH)2D3, induced the maturation and inhibited the cell growth of several colon cancer cell lines, including Caco-2 cells (24, 42). Similar to our observation with 1,25(OH)2D3, butyrate was found to sensitize these cells to growth inhibition by TGF-beta 1 (24, 42). It was suggested that butyrate might alter the expression of proteins that mediate TGF-beta 1 responses in these cells, but the signal transduction elements involved in these processes were not identified. It bears emphasis that, although the additional inhibition of Caco-2 cell growth, induced by the combination of TGF-beta 1 and 1,25(OH)2D3 in this study, or by butyrate (24, 42) is relatively modest, its impact on tumor progression may be considerable. Mathematical models of tumorigenesis have suggested, for example, that even limited decreases in their proliferative rates may ultimately result in significant inhibition of tumor growth (44, 45).

Our previous studies demonstrated that 1,25(OH)2D3 causes a decrease in cell numbers without a specific change in the distribution of cells in G1, S, or G2/M. Specifically, we found that this secosteroid causes an increased doubling time (41). In the previous report, however, we did not examine the effects of TGF-beta antibodies on Caco-2 cell growth inhibition by 1,25(OH)2D3. We have also previously shown that 1,25(OH)2D3 increases apoptosis in Caco-2 cells (9). In this communication we now demonstrate that the antiproliferative effects of 1,25(OH)2D3 are mediated in part by sensitization of Caco-2 cells to growth inhibition by TGF-beta . It is likely that both apoptosis and increased doubling time contribute to the antiproliferative effects of 1,25(OH)2D3. The increased doubling time may reflect a generalized cell cycle slowing in addition to increased apoptosis. Additional studies, beyond the scope of this manuscript, will be required to characterize the selective contributions of these two antiproliferative pathways (i.e., apoptosis and generalized slowing of the cell cycle) to Caco-2 cell growth inhibition by the combination of TGF-beta and 25(OH)2D3.

In summary, our present studies have demonstrated that 1,25(OH)2D3 upregulated the expression of IGF-IIR in Caco-2 cells, which accounted for an increase in the amount of active TGF-beta 1 in the conditioned media of these cells. The increased active TGF-beta 1 mediated, at least in part, the 1,25(OH)2D3-induced inhibition of cell growth. In addition, our studies have shown that 1,25(OH)2D3 activated TGF-beta signaling in Caco-2 cells and induced the sensitization of Caco-2 cells to the growth inhibitory effects of TGF-beta 1. Furthermore, this secosteroid increased the expression of Tbeta -RI in Caco-2 cells, which played an important role in the 1,25(OH)2D3-induced inhibition of their growth, as well as the sensitization of these cells to TGF-beta growth suppression. These studies have, therefore, provided novel insights into the mechanisms by which 1,25(OH)2D3 inhibits colon cancer cell growth. Increased understanding of TGF-beta resistance in Caco-2 cells, which do not have identified mutations in TGF-beta signaling, may elucidate more general mechanisms by which sporadic colon cancers escape TGF-beta growth inhibition. It will be of interest to characterize the cis- and trans-activating elements involved in the upregulation of IGF-IIR and Tbeta -RI genes by 1,25(OH)2D3 in neoplastic colonic cells. Additional studies will be required to further characterize the interactions of TGF-beta and MAPK signaling pathways, both of which are activated by 1,25(OH)2D3 and are involved in the alterations in growth phenotype induced by this secosteroid (11). The pleiotropic actions of 1,25(OH)2D3 on critical signaling pathways that regulate the growth of colonic cells suggest that this secosteroid is a potentially potent and novel chemopreventive agent against colonic cancer.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Health grants, including DK-39573 (to T. A. Brasitus, M. D. Sitrin, and M. Bissonnette), CA-36745 (to T. A. Brasitus and M. Bissonnette), DK-47995 (to A. Chen and B. H. Davis), and P30-DK-42086 (to M. Bissonnette and T. A. Brasitus, Digestive Disease Research Core Center), as well as by the Samuel Freedman GI Cancer Laboratory at the University of Chicago.


    FOOTNOTES

Present address for A. Chen: Department of Pathology, Louisiana State Univ. Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130 (achen{at}lsuhsc.edu).

Address for reprint requests and other correspondence: M. Bissonnette, Gastroenterology Section, Dept. of Medicine, The Univ. of Chicago, MC4076, 5841 S. Maryland Ave., Chicago, IL 60637 (E-mail: mbissonn{at}medicine.bsd.uchicago.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.

June 12, 2002;10.1152/ajpgi.00524.2001

Received 18 December 2001; accepted in final form 6 June 2002.


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DISCUSSION
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