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Endocrinology Vol. 145, No. 7 3353-3362
Copyright © 2004 by The Endocrine Society

Growth Hormone Inhibits Apoptosis in Human Colonic Cancer Cell Lines: Antagonistic Effects of Peroxisome Proliferator Activated Receptor-{gamma} Ligands

Fausto Bogazzi, Federica Ultimieri, Francesco Raggi, Dania Russo, Renato Vanacore, Chiara Guida, Sandra Brogioni, Chiara Cosci, Maurizio Gasperi, Luigi Bartalena and Enio Martino

Department of Endocrinology and Metabolism (F.B., F.U., F.R., D.R., S.B., C.C., M.G., E.M.) and Transfusional Unit (R.V., C.G.), University of Pisa, 56124 Pisa, Italy; and Division of Endocrinology (L.B.), University of Insubria, 21100 Varese, Italy

Address all correspondence and requests for reprints to: Fausto Bogazzi, Dipartimento di Endocrinologia e Metabolismo, Università di Pisa, Ospedale di Cisanello, Via Paradisa 2, 56124 Pisa, Italy. E-mail: f.bogazzi{at}endoc.med.unipi.it or fbogazzi{at}hotmail.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GH has antiapoptotic effects on several cells. However, the antiapoptotic mechanisms of GH on colonic mucosa cells are not completely understood. Peroxisome proliferator activated receptor-{gamma} (PPAR{gamma}) activation enhances apoptosis, and a link between GH and PPAR{gamma} in the colonic epithelium of acromegalic patients has been suggested. We investigated the effects of GH and of PPAR{gamma} ligands on apoptosis in colonic cancer cell lines. Colonic cells showed specific binding sites for GH, and after exposure to 0.05–50 nM GH, their apoptosis reduced by 45%. The antiapoptotic effect was due to either GH directly or GH-dependent local production of IGF-1. A 55–85% reduction of PPAR{gamma} expression was observed in GH-treated cells, compared with controls (P < 0.05). However, treatment of the cells with 1–50 µM ciglitazone (cig), induced apoptosis and reverted the antiapoptotic effects of GH by increasing the programmed cell death up to 3.5-fold at 30 min and up to 1.7-fold at 24 h. Expression of Bcl-2 and TNF-related apoptosis-induced ligand was not affected by either GH or cig treatment, whereas GH reduced the expression of Bax, which was increased by cig treatment. In addition, GH increased the expression of signal transducer and activator of transcription 5b, which might be involved in the down-regulation of PPAR{gamma} expression. In conclusion, GH may exert a direct antiapoptotic effect on colonic cells, through an increased expression of signal transducer and activator of transcription 5b and a reduction of Bax and PPAR{gamma}. The reduced GH-dependent apoptosis can be overcome by PPAR{gamma} ligands, which might be useful chemopreventive agents in acromegalic patients, who have an increased colonic polyps prevalence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH AND OTHER growth factors have been shown to regulate the programmed cell death in several types of cells (1, 2, 3, 4, 5, 6). GH increases the expression of homeo box A1, which in turn is involved in the abrogation of the apoptotic response of mammary carcinoma cells to doxorubicin (1); in addition, rats treated with GH have a reduced apoptosis of intestinal cells during the early course of acute necrotizing pancreatitis (2). Autocrine GH-induced repression of apoptosis-promoting proteins, in mammary carcinoma cells, might be linked to tumor progression (3). GH promotes cell cycle progression of lymphoid cells and prevents their apoptosis, mainly through the phosphatidylinositol 3-kinase/Akt pathway and the transcription factor nuclear factor {kappa}B (NF-{kappa}B); by these molecular mechanisms, GH might play a role in regulating apoptosis, proliferation, and eventually neoplastic transformation of immune cells (4).

In bovine embryos GH did not affect expression of B-cell LL/lymphoma 2 (Bcl-2); as a matter of fact, GH treatment did decrease the levels of apoptosis-promoting Bcl-2-associated X protein (Bax) proteins (5). GH treatment did not modify the expression of Bcl-2 in human leukemic cells or chinese hamster ovary cell lines; in these cells the antiapoptotic effects of GH was mediated by induction of Akt phosphorylation (6). On the contrary, in human monocytes GH prevented Fas-induced apoptosis by up-regulating Bcl-2 protein expression (7). In IL-3-dependent Ba/F3 cell line, endogenous GH was shown to induce constant levels of Bcl-2 and Bag1, Bcl-2-associated athanogene 1 proteins, which might be involved in their short-term survival during cytokine deprivation (8). In humans, acromegaly, a disorder caused by GH excess usually due to a pituitary GH-secreting adenoma, is associated with an increased prevalence of colonic tumors due, at least in part, to excessive stimulation of colonic epithelial cells by GH and IGF-I (9). Transgenic mice overexpressing GH have an increased intestinal mass due to mucosal hyperplasia, which has been attributed to increased lifespan of mucosal cells (10). However, the effects of GH on apoptosis of colonic cells, as well as the molecular mechanisms underlying its effects, are not completely understood.

Peroxisome proliferator activated receptor (PPAR)-{gamma} is a ligand-dependent transcription factor belonging to the nuclear receptor superfamily (11); it is highly expressed in adipose tissue, in which it plays a key role in regulation of adipocyte differentiation and fat metabolism (12, 13), and in colonic mucosa in which it exerts key actions to differentiation (14, 15). It is a functional receptor for the thiazolidinedione antidiabetic drugs and may function as a tumor suppressor gene (16, 17); its activation has been reported to induce differentiation of liposarcoma (18), prostate cancer (19), or several transformed cells (14, 15, 16, 17, 18, 19, 20).

PPAR{gamma} expression increases during differentiation of colonic epithelial cells (21, 22), and its activation is associated with growth inhibition and increased levels of markers of cellular differentiation in cultured colon cancer cells (14).

In human and rat glioma cells, PPAR{gamma} activation induces apoptosis associated with a transient up-regulation of Bax and Bcl-2 antagonist of cell death protein levels (23). The troglitazone-induced apoptosis of human papillary thyroid carcinoma cells was associated with enhanced expression of c-myc, whereas expression of Bcl-2 and Bax proteins was unaffected (24). In colonic cancer HT-29 cell line, activation of PPAR{gamma} by troglitazone increased cell death associated with down-regulation of c-myc and up-regulation of c-jun and growth arrest and DNA damage-inducible gene 153 (25). In addition, in the colonic cancer cell line Caco2, PPAR{gamma} activation reduced cytokine gene expression inhibiting the NF-{kappa}B pathway (26).

The aim of the study was to examine the role of GH and PPAR{gamma} in the regulation of apoptosis in colonic epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and cell preparation
Caco2, HT29, and NIH3T3 cell lines were obtained from Data Bank ISDT (Genua, Italy) and cultured in DMEM, 20% (10% for NIH3T3 and HT29) fetal bovine serum, 1% nonessential amino acids (Caco2 cells only), and 2 mM glutamine and penicillin-streptomycin solution in a humidified 5% CO2 atmosphere at 37 C. Under starvation conditions, cells were extensively washed in PBS and then incubated for 12 h in serum-free medium.

To study the effect of GH or PPAR{gamma} activation on apoptosis, starved cells were cultured in the absence or presence of 0.05–50 nM human recombinant GH (Sigma-Aldrich, Milan, Italy), 0.1–100 µM ciglitazone (cig) (Alexis Biochemicals, San Diego, USA), alone or in combination. Some experiments were performed using 1–100 µM rosiglitazone (ros) (Alexis Biochemicals, San Diego, CA) or 10–100 ng/ml human recombinant IGF-I (Sigma-Aldrich, Milan, Italy). Ten micromoles deoxycholic acid (DCA, Sigma-Aldrich) were used as a positive control of the antiapoptotic effect of GH (27, 28). Assessment of apoptosis by annexin V, 7-actinomycin D (7-AAD), or DNA fragmentation assay was done after a 30-min to 24-h culture period (see below). Samples of normal skin were obtained during plastic surgery and were used as a negative control (together with nuclear extracts from NIH3T3 cells) of PPAR{gamma} expression (29). Patients gave their informed consent, and the procedure was approved by the institutional reviewer committee.

Radioligand binding assays
Recombinant radioiodinated human GH (specific activity of 3.33 MBq/µg) was purchased from PerkinElmer (Boston, MA). Competition binding assays were carried out as previously described (30). Scatchard analyses were performed using the GraphPad Prism software (San Diego, CA). Data represent the average of three independent experiments, each performed in triplicate.

Annexin V binding assay and 7-AAD assay
Expression of phosphatidylserine in the outer leaflet of the plasma membrane was detected by binding of fluorescein isothiocyanate (FITC)-conjugate annexin V using the annexin V-FITC apoptosis detection kit (Sigma-Aldrich); viable cells were distinguished from necrotic cells by simultaneous staining with propidium iodide (PI). Stained cells positive for annexin V and negative for PI were considered apoptotic. The samples were measured by FITC/PI flow cytometry on a FACS apparatus (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). For each sample 500,000 cells were analyzed. Data were analyzed using the CellQuest software (Becton Dickinson). For 7-AAD assay, cells were prepared as reported above, exposed to 7-AAD, and analyzed using a FACSCalibur apparatus according to the supplier’s manual (Beckman Coulter, Milan, Italy). Each experiment was performed in triplicate and repeated three times.

DNA fragmentation assay
Quantitative determination of fragmented DNA in cytoplasm was assessed using the cell death detection ELISA plus (Roche Diagnostics, Indianapolis, IN). This assay detects the amount of histone-associated DNA fragments. The assay was performed according to the supplier’s manual; briefly, 104 cells were grown in a 96-well culture plate for 24 h, starved for 12 h, and then incubated with GH, cig (or ros), or both for 30 min to 24 h as indicated in the cell culture and cell preparation section. Supernatant culture media were removed, lysis buffer added to each vial, and histone-associated DNA fragments colorimetrically quantified. Data represent the mean ± SD of five independent experiments, each performed in triplicate.

Antibodies
Human polyclonal anti-Bcl2, anti-TNF-related apoptosis-induced ligand (TRAIL), anti-BAX, anti-GH receptor (GHR) (clone SC-461), anti-IGF-I receptor (IGF-IR), anti-signal transducer and activator of transcription (STAT)5b, and anti-PPAR{gamma} antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phosphoserine antibody was purchased from Qiagen (Hilden, Germany).

Immunoblotting
Cells (9 x 106) were washed in PBS and lysed in lysis buffer [50 mM Tris-HCl (pH 6.8), 10% glycerol, 2.5% sodium dodecyl sulfate, 10 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride]. Protein concentration was measured by Bradford assay using the Bio-Rad reagent (Bio-Rad Laboratories, Hercules, CA). For detecting phosphorylated PPAR{gamma}, nuclear extracts were immunoprecipitated with an anti-PPAR{gamma} antibody (Ab) before resolving on SDS-PAGE. Proteins (25 µg) were resolved by 12% SDS-PAGE, transferred onto nitrocellulose membrane, and stained with red ponceau to verify the amount of protein per lane. The membranes were incubated overnight at 4 C in 50% Tris-buffered saline (TBS) [200 mM Tris-HCl (pH 7.6), 1.4 M NaCl] and 50% TTBS (TBS, 0.05% Tween 20), containing 5% nonfat dry milk, and subsequently incubated with the primary antibody for 1 h at room temperature. After extensive washing in TTBS, a horseradish peroxidase-conjugated antirabbit IgG was added for 1 h. After four washings of the membranes in TTBS and TBS, proteins were detected using enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ).

Transfections
The pBLCAT2PPRE vector containing the a single copy of the PPAR response element (PPRE) of the acylCoA-oxidase gene has been described (31). The pSG5-h-PPAR{gamma} plasmid containing the human cDNA of the human PPAR{gamma} gene was kindly provided by Dr. Beatrice Desvergne (Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland). 2 x 105 cells (Caco2 or NIH3T3, as specified) were transiently transfected with 10 µg pBLCAT2PPRE plasmid or the empty parental vector, 3 µg pSG5-h-PPAR{gamma} (as appropriate), and 1 µg PCH110 plasmid containing the ß-galactosidase gene, using the calcium phosphate method; the latter plasmid was used to account for the variability in transfection efficiency. Cell extracts were prepared 48 h later and chloramphenicol acetyl transferase (CAT) assay and B-gal assay performed as previously described (31). Results were expressed as arbitrary units considering 100% the value obtained in absence of cig. Data represent the mean of four independent experiments performed in triplicate.

Real-time PCR
Real-time PCR was performed as described (32). The primer set amplifies an 83-bp fragment, the identity of which was confirmed by DNA sequencing. A control in which reverse transcription was omitted before PCR amplification was always included to eliminate the possibility that any amplification was due to contaminating genomic DNA. Experiments were carried out in triplicate; data are expressed as number of copies of PPAR{gamma}.

Statistics
Data were expressed as mean ± SD. Comparison of parameters was performed by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH receptor expression
GH receptor expression on Caco2 cell membranes was evaluated by radioligand binding assay. High-affinity binding (1.53 x 10–10 M) of 125I human recombinant GH was detected (Fig. 1Go), similar to that described for GHR in rat gastrointestinal tract (33). Preincubation of cells with a monoclonal antibody against GHR resulted in 89% inhibition of specific binding (data not shown). The number of receptors (capacity) was in the range of 4–6 fmol/mg of proteins.



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  		FIG. 1. Scatchard analysis of 125I-GH binding to Caco2 cell membranes. Caco2 cell membranes were prepared as described in Materials and Methods and incubated with 10 µM cold GH and increasing concentrations of 125I-GH. High-affinity binding (Kd = 1.53 x 10–10) of 125I-GH was detected and a cell capacity ranging 4–6 fmol/mg protein. Results represent the average of three different experiments, each performed in triplicate.

 
Inhibition of apoptosis by GH
The level of apoptosis of starved Caco2 cells assessed by 7-AAD, histone-associated DNA fragments, or annexin V-PI, was 7.5 ± 0.9, 8.5 ± 1.1, and 10.3 ± 1.5%, respectively. The effects of GH on apoptosis of Caco2 cells was evaluated after a time range of 30 min to 24 h in serum-free medium. The fraction of apoptotic cells decreased after exposure to GH for up to 24 h by 45% (from 7.5 ± 0.9 to 3.5 ± 0.8%, as assessed by 7-AAD, 8.5 ± 1.1 to 4.8 ± 0.7% as assessed by histone-associated DNA, and 10.3 ± 1.5 to 5.2 ± 1.1% by annexin V) (Fig. 2Go, A and B) in a dose-dependent manner (P = 0.003) (Fig. 2CGo); similar results were obtained using a different human colonic cancer cell line (HT29) (Fig. 2Go, A and C). The inhibition of apoptosis in both cell lines (Caco2 or HT29) was comparable with that obtained with 10 µM DCA for 30 min (Fig. 2AGo). In addition, apoptosis was evaluated 24 h after incubation with either a single dose of GH or repeated administration (every 6 h for 24 h) of GH; the latter modality of GH exposure was done in an attempt to reproduce in vitro the pulsatility of GH secretion occurring in vivo. However, each modality of GH administration was associated with a similar apoptosis rate (Fig. 2DGo).



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  		FIG. 2. Inhibition of apoptosis by GH treatment. Cells were starved for 12 h and then exposed to 0.05–50 nM GH for up to 24 h. The rate of apoptosis was evaluated by annexin V PI (A) or 7-AAD methods (B) or by measuring the amount of fragmented DNA by the cell death kit as reported in Materials and Methods (C and D). A, Apoptotic cells, enclosed in the R2 box, decreased from 10.3 to 5.2% (19.0 to 7.&% in HT29 cells) after GH exposure as assessed by annexin V PI assay, similar to the antiapoptotic agent DCA (10 µM). B, Reduction of apoptosis from 7.5 to 3.5% in GH-treated Caco2 cells evaluated by the 7-AAD method (apoptotic cells are enclosed in the R2 box). C and D, The fraction of apoptotic cells [either Caco 2 (empty columns) or HT29 cells (filled columns)] decreased after exposure to 0.05–50 nM GH for either 30 min (C) or 24 h (D) by 30–40% (P = 0.003). Results are expressed as mean ± SD of five different experiments, each performed in triplicate.

 
GH reduces apoptosis through a IGF-I-dependent and IGF-I-independent mechanism
To assess whether the antiapoptotic effects of GH were mediated through local production of IGF-I, apoptosis in Caco2 cells was measured incubating the cells in the presence of GH alone or in combination with GHR-Ab or IGF-IR-Ab. The antiapoptotic effect of GH was reduced by blocking GH action using a specific anti-GHR-Ab (P < 0.0001); addition of IGF-IR-Ab to GH-containing cell medium reduced, albeit not abolishing, the antiapoptotic effect of GH (P < 0.03 vs. untreated cells, P < 0.006 vs. GH-treated cells), thus suggesting that IGF-I participated to the GH-dependent reduction of apoptosis. The antiapoptotic effect of IGF-I was confirmed by the reduction of apoptosis when it was added to the cell medium, and by the reversal of the effect by the simultaneous incubation with IGF-IR-Ab (Fig. 3Go). The fact that the level of apoptosis of cells treated with GH and IGF-IR-Ab and that of the cells treated with IGF-I is different (P < 0.05) lends further support to the notion that a quote of antiapoptotic effect is directly mediated by GH and does not require local IGF-I production (Fig. 3Go).



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  		FIG. 3. The GH-dependent inhibition of apoptosis occurs with an IGF-I-dependent and IGF-I-independent mechanism. Caco 2 cells were incubated with 50 nM GH alone or in combination with GHR-Ab or IGF-IR-Ab or with 10 ng/ml IGF-I for 1 h. The reduction of apoptosis observed with GH treatment was abolished by GHR-Ab (P < 0.0001) and reduced by IGF-IR-Ab (P < 0.006); the level of apoptosis of cells treated with GH and IGF-IR-Ab was different from that of cells treated with IGF-I (P < 0.05), thus lending support to the concept that part of apoptosis is mediated directly by GH. Data are expressed as percentage of apoptosis, considering 100% that found in Caco2 cells exposed to vehicle. Results represent the mean ± SD of seven independent results, each performed in duplicate.

 
Expression of PPAR{gamma}
The expression of PPAR{gamma} in Caco2 cells was studied by Western blot. Nuclear extracts were incubated with a specific anti-PPAR{gamma} antibody; extracts from normal colonic mucosa and in vitro synthesized PPAR{gamma} were used as positive controls, those from NIH3T3 cells and samples of normal human skin as negative controls (Fig. 4AGo). To evaluate the functional integrity of the receptor, a reporter plasmid containing a PPAR response element fused to a heterologous promoter and a reporter gene was transiently transfected into Caco2 cells. Addition of 1 µM cig increased the basal level of expression of a reporter plasmid containing a single copy of a PPRE by 2-fold; exposure to higher cig concentrations (10 µM) was not associated with increased levels of CAT (Fig. 4BGo). Exposure of NIH3T3 cells, which do not express detectable levels of PPAR{gamma}, to cig did not enhance the CAT activity of the reporter plasmid unless cells were cotransfected with a PPAR{gamma}-containing expression plasmid (Fig. 4BGo), thus suggesting the specificity of action of PPAR{gamma} activator.



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  		FIG. 4. PPAR{gamma} is expressed and functionally active in Caco2 cells. A, Nuclear extracts were prepared from Caco2 cells and normal human colonic mucosa; in vitro synthesized human PPAR{gamma} was used as a positive control, whereas nuclear extracts from NIH3T3 cells and normal human skin were used as negative control. Proteins were resolved by 12% SDS-PAGE, transferred onto nitrocellulose membrane, and incubated with a specific antibody against the NH2 terminus of PPAR{gamma}. After extensive washing a horseradish peroxidase-conjugated antirabbit IgG was added; proteins were detected by chemiluminescence. B, Caco2 or NIH3T3 cells were transiently transfected with the pBLCAT2PPRE vector containing a single copy of a PPRE, alone or cotransfected with psG5-PPAR{gamma} and exposed to 1–10 µM cig. Data were expressed as relative CAT activity, assuming that the CAT activity normalized by the ß-galactosidase activity obtained from the cells exposed to vehicle was considered 100%. Results represent the mean of four experiments carried out in triplicate.

 
Enhanced apoptosis by PPAR{gamma} activation
Caco2 cells exposed to 0.1–100 µM of either cig or ros for up to 24 h exhibited an increased apoptosis rate (Fig. 5Go, A and B). Apoptosis increased from 8 ± 0.5 to 15 ± 0.4% just after 30 min, as assessed by annexin V PI method (Fig. 5AGo), and in a dose-dependent manner up to 50 µM thiazolidinediones (TZDs) as assessed by measuring the amount of histone-associated DNA fragments (P < 0.001) (Fig. 5BGo). Because exposure of Caco2 cells to 100 µM TZDs increased necrosis but not apoptosis (Fig. 5AGo), the next experiments were performed using 50 µM TZDs (Fig. 5Go, A and B). The effect of cig on apoptosis was not due to the drug toxicity because it was ineffective in NIH3T3 cells not expressing PPAR{gamma} (Fig. 5BGo). Exposure to up 50 µM cig increased apoptosis leaving unaffected necrosis. Thus, 50 µM of either cig or ros was used in subsequent experiments (Fig. 5Go, A and B). Caco2 cells cultured in the presence of 0.5–50 nM GH exhibited an increased apoptosis up to 3.5-fold when exposed to 50 µM cig (P < 0.03) (Fig. 5Go C); at variance, cig increased apoptosis of GH-treated cells by only 80% at 24 h; this apparent discrepancy could be explained, taking into account that GH reduced the phosphorylation of PPAR{gamma}, which, in turn, might enhance its activity; this effect was present at 30 min and no longer detectable after 24 h (Fig. 5DGo); thus, cig was capable of overcoming the antiapoptotic effect of GH in Caco2 cells.



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  		FIG. 5. Activation of PPAR{gamma} increases Caco2 apoptosis and reverts GH-dependent increase of Caco2 survival. Cig or ros was added to the culture medium at 0.1–100 µM alone (A and B); because 50 µM cig determined the maximal increase in apoptosis leaving unaffected necrosis, subsequent experiments were performed with this dose of cig. Treatment with up to 50 µM cig significantly increased apoptosis of Caco2 cells from 8 ± 0.5 to 14 ± 0.4% but not necrosis, just after 30 min, as assessed by annexin V PI method; stained cells positive for annexin V and negative for PI were considered apoptotic. The samples were measured by FITC/PI flow cytometry on a FACS apparatus (A); cig or ros increased apoptosis up to 170% in untreated Caco2 cells in a dose-dependent manner (P < 0.03) (B) and in GH-treated cells up to 3.5-fold (P < 0.001) (C), as assessed by measuring the amount of histone-associated DNA fragments (see Materials and Methods). This paradoxical effect of GH was observed only after a short exposure of the cells to GH and probably was due to a reduction of phosphorylation of PPAR{gamma} (D): nuclear extracts were precipitated with an anti-PPAR{gamma} antibody, resolved on a 12% SDS-PAGE, blotted onto nitrocellulose membrane, and Western blot performed with an anti-phosphoserine antibody.

 
Effects of GH on PPAR{gamma} expression
Serum-deprived Caco2 cells treated with 50 nM GH for 6 or 24 h had a reduced number of PPAR{gamma} transcripts as assessed by either real-time PCR (Fig. 6AGo) or Western blot (Fig. 6BGo). After 6 and 24 h incubation with GH, the transcripts of PPAR{gamma} decreased from 5126 ± 798 to 914 ± 411 (P < 0.002) and from 14,691 ± 611 to 6767 ± 2090 (P < 0.05), respectively (Fig. 6AGo); the GH-dependent down-expression of PPAR{gamma} was confirmed in HT29 cells by either RT-PCR (data not shown) or Western blot (Fig. 6CGo). The mechanisms underlying the negative effects of GH on PPAR{gamma} expression was further investigated evaluating the expression of STAT5b protein; HT29 cells exposed to 5–50 nM GH for 6–24 h enhanced the expression of STAT5b (Fig. 7Go).



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  		FIG. 6. GH-treated cells have a reduced expression of PPAR{gamma}. Expression of PPAR{gamma} was measured by real-time PCR and Western blot analysis as described in Materials and Methods. A, After 6 and 24 h incubation with GH, the transcripts of PPAR{gamma} decreased from 5126 ± 798 to 914 ± 411 (P < 0.002) and from 14,691 ± 611 to 6767 ± 2090 (P < 0.05), respectively. Results represent the mean ± SD of three different experiments, each performed in triplicate. B and C, Western blot analysis showing the reduced expression of PPAR{gamma} protein in GH-treated Caco2 and HT29 cells.

 


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  		FIG. 7. GH enhances the expression of STAT5b. Cellular extracts of Caco2 cells were resolved on a 12% SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with a specific anti-STAT5b antibody After extensive washing a horseradish peroxidase-conjugated antirabbit IgG was added and proteins detected using enhanced chemiluminescence detection system. The level of expression of STAT5b increased in 50 nM GH-treated cells after 6 and 24 h.

 
Effects of GH or PPAR{gamma} activator on apoptosis-associated proteins
Caco2 cells were treated with 0.5–50 nM GH or 1–50 µM cig for 6–24 h. Cellular extracts were immunoblotted with antibody against Bcl-2, TRAIL, and Bax. Expression of Bcl-2 and TRAIL was not affected by either 50 nM GH or 50 µM cig treatment after 24 h; at variance, GH reduced the level of expression of Bax, which was increased by treatment with cig (Fig. 8AGo). The lower level of expression of Bax protein was revealed in 0.5–50 ng/ml GH-treated cells for 6–24 h (Fig. 8BGo). On the contrary, dose- and time-course experiments revealed the increased expression of Bax in 1–10 µM cig-treated Caco2 or HT29 cells (Fig. 8CGo).



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  		FIG. 8. Activation of PPAR{gamma} is associated with changes in the expression of apoptotic factors. Caco2 cell extracts were prepared as described in Materials and Methods. A, Cells were treated with 50 nM GH or 100 µM cig for 24 h. Western blot was done using anti-Bcl2-, anti-TRAIL-, and anti-Bax-specific antibody. Activation of PPAR{gamma} was associated with increased levels of expression of Bax, leaving unaffected Bcl-2 and TRAIL expression. GH did not modify the expression of TRAIL and Bcl-2 but reduced the expression of Bax. The down-regulation (B) and up-regulation (C) of Bax in cells treated with GH and cig, respectively, was confirmed by dose- and time-course experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Patients with acromegaly have an increased prevalence of colonic polyps (34, 35), and a link between colonic polyps and activity of the disease has been postulated on clinical grounds (36). Exposure of several cell lines to GH has been associated with reduced apoptosis (1, 2, 3, 4, 5, 6) and possibly with progression of neoplasms (3). GHR is widely expressed in several organs, including the grastrointestinal tract (30, 37, 38). Our study confirms that in Caco2 colonic-derived cells, high-affinity GH binding sites are present on the cytoplasmic membranes; albeit being expressed at a low density, GHRs were sufficient to drive GH action. GH actions depend on its binding to a membrane receptor that belongs to the cytokine receptor superfamily (38, 39). It was postulated that GH might cause prolonged survival of intestinal epithelial cells, based on studies of transgenic mice overexpressing bovine GH, in which the observed increased mucosal mass was not associated with an enhanced crypt cell production rate and DNA-specific activity (10); GH might also protect intestinal cells from apoptosis induced by radiation (40). GH seems to reduce apoptosis in many cell lines (3, 6, 15), but a direct involvement in colonic cells is largely speculative.

Using the pro-B murine Ba/F3 cell line expressing GH receptor, Baixeras et al. (41) reported that GH-dependent proliferation and survival of these cells were not mediated by IGF-I. The GH-dependent antiapoptotic effects were mediated by the NF-{kappa}B transcription factor. Our results demonstrated that the antiapoptotic effects of GH in Caco2 cells is not completely dependent on local production of IGF-I, albeit part of GH action might be counteracted by an anti-IGF-IR antibody. The molecular mechanisms underlying the antiapoptotic effects of GH might include a sustained production of Bcl-2 and NF-{kappa}B, as shown in Ba/F3 cells (8), or an increased homeo box A1-dependent expression of Bcl-2 as reported in human mammary carcinoma cells (1). At variance, in blastocysts GH treatment did not affect Bcl-2 expression but reduced the expression of the proapoptotic Bax proteins (5). In addition, GH treatment inhibited apoptosis in human leukemic cells and Chinese hamster ovary cells through activation of Akt phosphorylation, leaving unchanged the Bcl-2 levels (6). We herein reported that GH-dependent prolonged survival of Caco2 cells is mediated by a marked reduction of the proapoptotic Bax protein expression, without effect on Bcl-2; thus, the antiapoptotic effects of GH in this cell line may derive from a change in the Bax/Bcl-2 ratio. However, effects of GH on the regulation of pathways involving other proteins of the apoptotic process cannot be ruled out.

PPAR{gamma} is considered a tumor suppressor gene, the activation of which leads to cellular differentiation and apoptosis (14, 16, 17, 18, 19, 20, 23, 24, 25). Loss of heterozygosity and somatic mutations in the PPAR{gamma} gene might be an early event in colonic tumorigenesis (42, 43, 44); our recent findings of a reduced PPAR{gamma} expression in the colonic mucosa of patients with active acromegaly or colonic polyps lend support to this notion (32, 45). Thus, the lower levels of expression of PPAR{gamma} in Caco2 cells treated with GH might contribute to the antiapoptotic effects of GH in this cell line. Serum deprivation is associated with changes in the expression of several transcription factors, including PPAR{alpha} (46). Our findings suggest that a link between starvation and increased PPAR{gamma} levels might exist. However, the underlying mechanisms remain unknown. Furthermore, the different expression of PPAR{gamma} did not affect the GH-dependent down-regulation of this nuclear receptor. However, exposure of Caco2 cells, either GH treated or untreated, to cig increased apoptosis by about 3-fold; this suggests that even though reduced by GH treatment, PPAR{gamma} activation is able to overcome the antiapoptotic effects of GH. GH treatment was associated with a paradoxical increase of PPAR{gamma}-dependent apoptosis during the first half-hour; this might be due to the GH-dependent reduction of PPAR{gamma} phosphorylation, as shown by Western blot, which was no longer present after 24 h. In the colonic cancer HT-29 cell line, activation of PPAR{gamma} by troglitazone increased cell death associated with down-regulation of c-myc and up-regulation of c-jun and growth arrest and DNA damage-inducible gene 153 (31); at variance, in papillary thyroid cancer activation of PPAR{gamma} led to increased c-myc expression (30).

The levels of expression of Bcl-2, Bax, and p53 have been shown to be increased, unchanged, or decreased (47, 48, 49), likely depending on the cell lines. Activation of PPAR{gamma} in Caco2 cells increased the expression of Bax, leaving unaffected that of Bcl-2 and TRAIL, thus suggesting that, at least in this cell line, GH and PPAR{gamma} regulate apoptosis through a common pathway; alternatively, the reduced levels of expression of Bax found in GH-treated cells might be due to a down-regulation of PPAR{gamma} by GH; GH treatment was associated with a reduced level of PPAR{gamma} and an increased expression of STAT5b; albeit a direct link could not be proven from our studies, it is likely that STAT5b might be responsible for the GH-dependent down-regulation of PPAR{gamma} in colonic cells. Recent papers (50, 51) have demonstrated that the cross-talk between PPAR and STAT pathways is bidirectional, suggesting a mechanism whereby PPAR ligands or GH might interact to each other. Shipley et al. (51) reported that GH treatment of COS-1 cells transfected with PPAR{gamma}, STA5b and GHR was not associated with changes in the level of expression of STAT5b protein evaluated after 4 h. At variance with the report of Shipley and Waxman, our results show that GH increased STAT5b levels in Caco2 cells after 6 and 24 h. This discrepant result may be due to the different cell line, the exposure time to GH, and the fact that we did not transfect the cells with a STAT5b-containing plasmid.

In conclusion, we have reported a direct involvement of GH in prolonging survival of colonic cells, which might be opposed by activation of PPAR{gamma}. These data provide a molecular basis to the effects of GH on colonic epithelial cells and suggest a potential role of PPAR{gamma} agonists as chemopreventive agents in acromegalic patients at high risk of developing colonic neoplasms.


   Acknowledgments
 
We thank Professor Aldo Pinchera (Department of Endocrinology, University of Pisa, Italy) for his continuous encouragement and advice.


   Footnotes
 
This work was partially supported by grants from the University of Pisa (Fondi d’Ateneo) and Ministero dell’Istruzione, dell’Universitá e della Ricerca (M.I.U.R.), Rome (to E.M.), the University of Insubria at Varese (to L.B.), and M.I.U.R., Rome (to L.B.).

Abbreviations: 7-AAD, 7-Actinomycin D; Ab, antibody; Bax, Bcl-2-associated X protein; Bcl-2, B-cell LL/lymphoma 2; CAT, chloramphenicol acetyl transferase; cig, ciglitazone; DCA, deoxycholic acid; FITC, fluorescein isothiocyanate; GHR, GH receptor; IGF-IR, IGF-I receptor; NF-{kappa}B, nuclear factor {kappa}B, subunit 1; PI, propidium iodide; PPAR, peroxisome proliferator activated receptor; PPRE, PPAR response element; ros, rosiglitazone; STAT, signal transducer and activator of transcription; TBS, Tris-buffered saline; TRAIL, TNF-related apoptosis-induced ligand; TZD, thiazolidinedione.

Received February 20, 2004.

Accepted for publication March 31, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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