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Physiol. Genomics 30: 342-353, 2007. First published May 22, 2007; doi:10.1152/physiolgenomics.00042.2007
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Received 23 February 2007; accepted in final form 18 May 2007.
Physiological Genomics 30:342-353 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Differential expression, distribution, and function of PPAR-{gamma} in the proximal and distal colon

Weidong Su1, Craig R. Bush2, Brian M. Necela1, Shelly R. Calcagno1, Nicole R. Murray1, Alan P. Fields1 and E. Aubrey Thompson1

1 Department of Cancer Biology, Mayo Clinic College of Medicine, Jacksonville, Florida
2 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Suppression of colon carcinogenesis by peroxisome proliferator-activated receptor (PPAR)-{gamma} is likely due to some effect of PPAR-{gamma} on normal colonic epithelial cells. However, our understanding of the effects of PPAR-{gamma} in such cells is limited. We analyzed the abundance, distribution, and function of PPAR-{gamma} in epithelial cells isolated from the murine proximal and distal colon. Marked differences in PPAR-{gamma} abundance and distribution were observed, suggesting tissue-specific responses. Analysis of PPAR-{gamma} effects on DNA synthesis, formation of preneoplastic lesions, and activation of MAPK signaling in proximal and distal colonic epithelial cells in vivo indicates that PPAR-{gamma} regulates both tissue-specific and common responses within the proximal and distal colon. Three major functional cohorts of PPAR-{gamma} target genes were identified by genomic profiling of isolated colonic epithelial cells: genes that are involved in metabolism, in signaling, and in cellular adhesion and motility. Two subsets of PPAR-{gamma} target genes were differentially expressed in the proximal and distal epithelium. Proximal target genes were primarily involved in metabolic activities, whereas signal transduction, adhesion, and motility targets were more pronounced in the distal colon. Remarkably, those target genes that are differentially expressed in the proximal colon were all induced on activation of PPAR-{gamma}, whereas all target genes that are preferentially expressed in the distal colon were repressed. Our data indicate that PPAR-{gamma} exerts both common and tissue-specific effects in the colon and challenge the general conclusions that PPAR-{gamma} is induced on differentiation of colonic epithelial cells and that this receptor stimulates differentiated function in epithelial cells throughout the colon.

nuclear receptors; microarrays; colon cancer; gastrointestinal epithelium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
PEROXISOME PROLIFERATOR-ACTIVATED receptors (PPARs) are members of a subfamily of nuclear receptors (18, 35). Three related PPARs have been described, PPAR-{alpha}, PPAR-{delta}, and PPAR-{gamma} (11, 40); these differ in cellular distribution and function. PPAR-{alpha} is expressed at high levels in the liver, where it is believed to control fatty acid oxidation, and in the gastrointestinal epithelium (1, 25, 29). PPAR-{delta} is more widely distributed and has been implicated in regulation of differentiation and lipid metabolism (1, 2, 43, 53). PPAR-{gamma} has been studied in greatest detail in adipocytes, where it has been called a master switch in differentiation; however, PPAR-{gamma} is also very abundant in the colon (1, 31, 36), where its function is largely unknown.

All PPARs are believed to share a common mechanism of action. They appear to function as obligate heterodimers with retinoid X receptors (RXRs), and to bind as PPAR-RXR dimers to characteristic DNA sequences, the so-called PPAR response elements (PPREs), within the promoters of target genes (34, 51); such genes are regulated by both endogenous and pharmacological PPAR ligands. PPAR-{gamma} is activated in vitro by long-chain polyunsaturated fatty acids (27, 28) and by thiazolidinedione drugs such as rosiglitazone (Avandia), troglitazone (Rezulin), and piaglitazone (Actos) (3, 12, 32). We (6, 7) and others (8) have recently reported that PPAR-{gamma} is activated by RS5444, a high-affinity PPAR-{gamma} agonist that binds and activates PPAR-{gamma} in vitro with an EC50 of ~1 nM (6).

PPAR-{gamma} is expressed at high levels in some transformed colonic epithelial cell lines (9, 21, 26, 48). It was initially reported that very high concentrations of thiazolidinediones promote gastrointestinal tumor formation in the APC+/Min model of mouse colon carcinogenesis (30, 47). In contrast, PPAR-{gamma} agonists inhibit proliferation and induce differentiated markers in human colon cancer cells grown as xenografts in nude mice (26, 48), and thiazolidinediones inhibit colon tumor formation in the azoxymethane (AOM) mouse model of sporadic colon carcinogenesis (37, 44, 50). More recent studies by Niho et al. (42) failed to observe an increase in gastrointestinal tumorigenesis in APC+/Min mice treated with thiazolidinediones, consistent with the observation that hemizygous knockout of PPAR-{gamma} promotes tumorigenesis in such mice (16). Tissue-specific biallelic knockout of PPAR-{gamma} in gastrointestinal epithelial cells likewise enhances tumorigenesis in APC+/Min mice (39). Thus PPAR-{gamma} is abundant in the colon, and the preponderance of evidence indicates that this receptor affects a process or processes that block some early stage in the transformation of colonic epithelial cells in rodents. The role of PPAR-{gamma} in human colon carcinogenesis is less clear. Loss-of-function mutations to PPAR-{gamma} have been reported in human colon cancers (49), although the frequency with which such mutations occur is unclear (23). Moreover, decreased PPAR-{gamma} expression is associated with ulcerative colitis (10) and acromegaly (4), and these conditions are associated with increased risk of colon cancer. Thus the data are at least consistent with the hypothesis that PPAR-{gamma} is also a colon cancer suppressor in humans and that partial abrogation of PPAR-{gamma} activity may increase colon cancer risk.

It is plausible to assume that the tumor-suppressive effects of PPAR-{gamma} are related to the normal, physiological function of this receptor. However, little is known about the physiology of PPAR-{gamma} in the gastrointestinal epithelium. We have reported that PPAR-{gamma} regulates proliferation of nontransformed intestinal epithelial cells in culture (6) and regulates motility of such cells by a novel mechanism that involves activation of MAPKs by Rho family GTPases (7). It has been reported that PPAR-{gamma} inhibits proliferation of primary nontransformed colonic epithelial cells ex vivo (38). However, such cells have very poor survival in culture and have limited utility for mechanistic studies. It has not been shown, to our knowledge, that PPAR-{gamma} regulates proliferation or differentiated function of normal colonic epithelial cells in vivo.

Reports of PPAR-{gamma} expression and distribution in the normal colon are contradictory. Lefebvre et al. (31) reported that PPAR-{gamma} is induced during differentiation of colonic epithelial cells, whereas Saez et al. (47) reported that PPAR-{gamma} is expressed at the highest levels in the undifferentiated epithelial cells at the base of the colonic crypts, a conclusion that is obviously at variance with the conclusion that PPAR-{gamma} is induced during differentiation. Girnun et al. (16) reported that PPAR-{gamma} is expressed throughout the colonic epithelium. It is not clear whether these apparent discrepancies arise from technical issues or from region-specific differences in PPAR-{gamma} distribution. One of our first objectives was therefore to examine the expression and distribution of PPAR-{gamma} in the mouse colon. We also carried out genomic profiling of PPAR-{gamma} gene targets in isolated colonic epithelial cells to identify the potential roles of this receptor in normal epithelial cell physiology in the colon. Our data indicate that PPAR-{gamma} regulates a large number of genes that are involved in cellular metabolism, signal transduction, and cellular motility and adhesion. Analysis of PPAR-{gamma} target gene expression and cellular responses in the proximal and distal colon reveals that PPAR-{gamma} regulates both common and tissue-specific genomic and physiological processes in the proximal and distal colon.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
AOM-mediated colon carcinogenesis.
Female C57BL/6J mice, 6–8 wk of age, were used in all experiments. This strain was selected because of the availability of transgenic C57BL/6J strains that may be used in future experiments to study cross talk between PPAR-{gamma} and other pathways. Female mice were selected to minimize cage charges and because female mice exhibit significantly lower variance in aberrant crypt focus (ACF) formation compared with males, in our experience. Ten mice were enrolled in the control (saline injected) group, twenty mice in the AOM + diluent group, and twenty mice in the AOM + RS5444 group. Mice were anesthetized by CO2, followed by cervical dislocation. Mice were obtained from our breeding colonies, which are maintained as part of an American Association for Accreditation of Laboratory Animal Care facility. Animal experimentation was conducted in accordance with accepted standards of humane animal care according to protocols approved by the Mayo Clinic College of Medicine Institutional Animal Care and Use Committee. Quantification of ACF in methylene blue-stained colons from mice killed 12 wk after four weekly injections of 10 mg/kg AOM was carried out as previously described (17, 41).

The PPAR-{gamma} agonist RS5444 was obtained from Sankyo (6–8, 13), and 10 mg/kg was delivered by daily oral gavage. Control mice received an equivalent volume of diluent [0.5% carboxymethylcellulose (CMC)]. In those experiments in which ACF formation was measured, mice received oral gavage 5 days/wk beginning 1 wk before the first injection of AOM and continuing throughout the course of the experiment. Molecular analyses were carried out in mice that received 10 mg·kg–1·day–1 of RS5444 or diluent for 3 days before death.

Immunohistochemistry.
Immunohistochemical analysis of 5-µm sections from mouse colons was carried out as previously described (6) with antibodies against bromodeoxyuridine (BrdU; Oncogene Science), PPAR-{gamma} (Cell Signaling, 2492), RXR-{alpha} (Santa Cruz, SC-553), or phosphoERK1/2 (Cell Signaling, 9100). Only those longitudinally sectioned crypts that had an intact, complete, and well-orientated structure and that displayed lumen at the top and muscularis mucosa at the base were counted for the BrdU labeling index, as previously described (41). In most cases, the sections were counterstained with hematoxylin for 10 s. For peptide competition, the primary antibodies were preincubated with corresponding blocking peptides at 4°C for 8 h. The FITC-conjugated secondary antibody that was used for immunofluorescence analysis was obtained from Vector Laboratories (Vector M.O.M. Immunodetection Kit).

Isolation of proximal and distal colon crypts.
The colon was dissected from cecum to rectum, flushed with phosphate-buffered saline (4°C), and soaked in HBSS without Ca2+ or Mg2+. The cecum and rectum were removed, the colon was inverted, and the proximal and distal ends were tied and sealed. The inverted colon was washed three times for 5 min each in ice-cold HBSS containing 25 mM HEPES and 1% FBS. The colon was cut into proximal and distal segments, the open ends were tied, and the segments were suspended in HBBS-10 mM EDTA, shaken on ice for 30 min, and then shaken by hand vigorously for 5 min. The supernatant containing individual crypts was removed and stored at 4°C. The shaking process was repeated two more times or until no more crypts appeared in the solution. Supernatants from each wash were combined and centrifuged at 1,000 g for 5 min at 4°C. The pellets (pure crypt fraction) were stored at –80°C.

Protein preparation and Western blot analysis.
Lysis, protein extraction, electrophoresis, and blotting were carried out as previously described (6, 7) with aliquots of total protein (20 µg) and primary antibodies against PPAR-{gamma} (Santa Cruz, E8, SC-7273), RXR-{alpha} (Santa Cruz, SC-553), ERK1/2 (Cell Signaling, 9102), phosphoERK1/2 (Cell Signaling, 9100), or actin (Santa Cruz, SC-1616) and horseradish peroxidase-conjugated anti-mouse (Santa Cruz), anti-rabbit (Kirkegaard & Perry Labs), or anti-goat (Santa Cruz) secondary antibodies (depending on the primary antibody used). Antigen-antibody complexes were detected with the ECL Plus chemiluminescent system (Amersham Bioscience). Luminescence was measured with the Typhoon 9410 laser scanner (Amersham Bioscience).

Analysis of gene expression.
Four mice were treated with 10 mg·kg–1·day–1 RS5444 by oral gavage for 3 days. Controls were treated by daily oral gavage with diluent (CMC). Colonic crypts were prepared from total colon, and RNA was extracted with the RNAqueous Kit (Ambion). The integrity of the RNA was confirmed by microelectrophoresis on an Agilent 2100 analyzer. RNA was labeled and hybridized to Affymetrix MG430Ver2.0 chips, as previously described (6). The microarray data were analyzed along two parallel threads with Bioconductor v1.5 (14) or R v2.0 software (22). Preprocessing and scaling of microarrays were carried out as described by Pomeroy et al. (45), using threshold values of 100 and 20,000 for floor and ceiling, respectively. Fold change values (log base 2) were calculated with average values for four replicates of control and treated mice. For statistical analysis of differential expression, Espresso was run within the Bioconductor Affymetrix package with Irizarry's robust microarray analysis with correction for GC-rich regions (GCRMA; Ref. 24) for background correction, q-spline for normalization, and medianpolish for summarization routines. One-way ANOVA was calculated from the GCRMA data and probe sets were ranked by P value.

The abundance of individual mRNAs was determined with two-step quantitative reverse transcriptase-mediated real-time PCR (qPCR). Equal aliquots of total RNA from four control or four RS5444-treated animals were converted to cDNA with the High-Capacity cDNA Archive kit (Applied Biosystems), and qPCR reactions were performed in triplicate with 5 ng of cDNA and the TaqMan Universal PCR master mix (Applied Biosystems). Amplification data were collected with an Applied Biosystems Prism 7900 sequence detector and analyzed with Sequence Detection System software (Applied Biosystems). All primers and probes were purchased from Applied Biosystems. Data were normalized to GAPDH or 18S rRNA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
PPAR-{gamma} distribution in proximal and distal colon.
All previously published immunohistochemical analyses of PPAR-{gamma} expression in the colon have used histological sections containing crypts from different parts of the colon. Although such sections are useful for analyzing protein expression within a given group of crypts, it is difficult, for technical reasons, to compare staining intensities between independent sections, which may be subject to variation in fixation, antigen retrieval, antibody concentration, or development of the detection system. We utilized colonic "Swiss roll" preparations to circumvent such potential problems and thereby allow direct comparison of PPAR-{gamma} expression throughout the length of the mouse colon. The colon was excised, dissected from the cecum and rectum, split, and prepared as a Swiss roll, which was then fixed, sectioned, and immunostained. Representative data for PPAR-{gamma} and RXR-{alpha} expression are shown in Fig. 1A, in which the cecal (proximal) end of the colon is designated by the arrow on the upper left and the rectal (distal) end of the colon by the central arrow. PPAR-{gamma} staining was detected throughout the colon, with significantly more intense staining in the proximal one-third of the colon. High-resolution images of the proximal and distal colon stained with the PPAR-{gamma} antibody are shown in Fig. 1B. As previously reported by Lefebvre et al. (31), the most intense staining was observed in luminal cells of the proximal colon. Both nuclei and cytoplasm of luminal cells stained intensely. Weaker nuclear PPAR-{gamma} staining was observed in subluminal cells, indicating that PPAR-{gamma} is expressed at detectable levels in all cells of the proximal crypts (Fig. 1B1). Expression throughout the proximal epithelium was confirmed with a FITC-conjugated secondary antibody (Fig. 1B4).


Figure 1
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  		Fig. 1. Expression of peroxisome proliferator-activated receptor (PPAR)-{gamma} and retinoid X receptor (RXR)-{alpha} in the mouse colon. A: the colon from a C57BL/6J mouse was fixed as a "Swiss roll" and embedded in paraffin, and adjacent sections were cut and stained with antibodies against PPAR-{gamma} or RXR-{alpha} or without primary antibody (ab. Control). Arrow on top left indicates the cecal end of the proximal colon, whereas the central arrow indicates the rectal end of the distal colon. Sections cut from the proximal or distal colon were stained with antibodies against PPAR-{gamma} (B) or RXR-{alpha} (C). The secondary antibody used in B1-3 and C1-3 was conjugated with horseradish peroxidase, whereas the secondary antibody used in B4 and B5 was labeled with FITC. Sections shown in C were counterstained with hematoxylin, whereas sections shown in A and B were not counterstained because hematoxylin tended to obscure PPAR-{gamma} staining.

 
Staining in the distal epithelium was notably different from that observed in the proximal crypts. No pronounced basal-to-luminal PPAR-{gamma} staining gradient was observed in epithelial nuclei in the distal crypts (Fig. 1B, 3 and 5), although we consistently observed slightly higher staining in nuclei of epithelial cells at the base of distal colonic crypts. It is clear from these analyses that PPAR-{gamma} is primarily expressed in epithelial cells within the colon, although scattered cells that stain for PPAR-{gamma} are observed in the lamina propria of both the proximal and distal colon.

PPAR-{gamma} functions as an obligate heterodimer with RXR-{alpha}, which is the major RXR species in the gastrointestinal epithelium (33, 46, 52). Staining of a Swiss roll with RXR-{alpha} antibody revealed that this receptor is also expressed at significantly higher levels in the proximal one-third of the colon. High-resolution analysis of crypts from the proximal (Fig. 1C1) or distal (Fig. 1C3) colon revealed that all epithelial nuclei contain RXR-{alpha}. No significant basal-to-luminal gradient in nuclear staining was observed, although cytoplasmic staining was more intense in luminal cells from the proximal colon.

The immunohistochemical analyses indicated that PPAR-{gamma} is expressed at significantly higher levels in the proximal colon. To confirm this observation, we measured PPAR-{gamma} mRNA in the mouse small intestine and colon. The small intestine was removed, dissected from the colon, and cut into three sections of more or less equal length. Likewise, the colon was cut into three equal sections. Total RNA was extracted and PPAR-{gamma} mRNA abundance determined in reference to GAPDH mRNA. We had previously determined that GAPDH expression was constant throughout the length of the small and large intestines (data not shown). Representative data are shown in Fig. 2A.


Figure 2
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  		Fig. 2. Isolation and characterization of colonic epithelial crypts. Total RNA was extracted from segments cut from the small intestine (SB) and colon, and PPAR-{gamma} mRNA abundance was measured by quantitative reverse transcriptase-mediated real-time PCR (qPCR) in reference to GAPDH (A). A modified chelation procedure was used to isolate intact epithelial crypts (B), and qPCR was used to measure abundance of stromal markers, including smooth muscle action (SM actin) and vimentin, or epithelial markers, including epithelial membrane protein (EMP)1 and epithelial cadherin-1 (E-cadherin) in reference to GAPDH. C: expression of these markers in isolated epithelial crypt cells (black bars) was compared with that in scraped epithelial preparations (gray bars). D: protein and RNA were isolated from crypts of the proximal and distal colon, and PPAR-{gamma} mRNA and protein were measured by qPCR or Western blotting. All bars represent means of ≥3 independent preparations, and error bars represent 1 SD.

 
PPAR-{gamma} mRNA abundance was relatively constant throughout the small intestine (Fig. 2A), and immunoblotting of Swiss rolls prepared from the small intestine revealed relatively uniform staining of PPAR-{gamma} protein throughout the small intestine (not shown). However, we observed a gradient of PPAR-{gamma} expression in the colon. PPAR-{gamma} mRNA abundance was highest in the proximal colon (Fig. 2A), although PPAR-{gamma} mRNA in the distal colon was significantly more abundant than in the small intestine. However, PPAR-{gamma} expression is not limited to epithelial cells, and the data shown in Fig. 2A reflect the total PPAR-{gamma} mRNA abundance rather than the expression pattern in epithelial cells. We therefore prepared isolated epithelial crypts from the colon with a modified chelation procedure. These preparations retained their crypt morphology (Fig. 2B). We measured several epithelial and stromal marker genes to assess the relative enrichment of epithelial cells, compared with the scraping technique that we have used previously (17) to isolate colonic epithelial cells. Smooth muscle mRNA was undetectable in the isolated crypts (Fig. 2C), whereas vimentin was detected at very low levels relative to scraped preparations of colonic epithelial cells. Conversely, epithelial membrane protein (EMP)1 and epithelial cadherin-1 (E-cadherin) were expressed at high levels and were significantly enriched in isolated crypts relative to scraped epithelial preparations. These data indicate that the modified chelation procedure that we have developed yields very highly enriched preparations of epithelial cells, which were used to measure epithelial gene expression in proximal and distal colon.

We prepared crypts from proximal and distal colon and measured PPAR-{gamma} mRNA (relative to 18S) and protein. PPAR-{gamma} mRNA was significantly higher in the proximal epithelial cells (Fig. 2D). PPAR-{gamma} protein was likewise more abundant in the proximal epithelial cells, and RXR-{alpha} recapitulated this pattern. These data confirm the immunohistochemical analysis shown in Fig. 1 and are consistent with the hypothesis that PPAR-{gamma} activity and perhaps PPAR-{gamma} functions are likely to be different in the proximal and distal colonic epithelia.

PPAR-{gamma} functions in proximal and distal colon.
The physiological roles of PPAR-{gamma} in the colonic epithelium are largely unknown; however, PPAR-{gamma} suppresses preneoplastic ACF formation in AOM-treated mice (37, 44, 50). We therefore determined the effects of PPAR-{gamma} on ACF formation in the proximal and distal colons of AOM-treated mice that were treated by daily oval gavage with 10 mg·kg–1·day–1 of RS5444, a potent thiazolidinedione PPAR-{gamma} agonist (6–8), or diluent (CMC). AOM caused a significant increase in ACF in both proximal and distal colon compared with saline controls that did not receive AOM. The modal number of ACF per colon in CMC-treated mice was somewhat lower in the proximal colon than in the distal colon (AOM+CMC in Fig. 3A), consistent with our experience with C57BL/6J mice, in which the ratio of ACF in proximal and distal colon is about 2:3. RS5444 potently suppressed ACF in both proximal and distal colon (AOM+RS in Fig. 3A), indicating that PPAR-{gamma} is equally effective at suppressing preneoplastic events in both tissues.


Figure 3
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  		Fig. 3. Effects of PPAR-{gamma} on incidence of aberrant crypt focus (ACF) formation and proliferation in the proximal and distal colon. A: ACF were induced by azoxymethane (AOM) injection, as described in MATERIALS AND METHODS. Mice were killed 14 wk after the last injection of AOM, the colons were excised and stained with methylene blue, and ACF were counted in the proximal and distal colons of control [carboxymethylcellulose (CMC) treated] or RS5444-treated mice. Statistical analysis was carried out with Mann-Whitney rank sum analysis. B: control (CMC treated) and RS5444-treated mice were injected with bromodeoxyuridine (BrdU) before death. Sections from fixed colons were stained with anti-BrdU and counterstained with hematoxylin. C: proliferative index (BrdU-labeled cells/crypt) was counted and quantified as determined in MATERIALS AND METHODS. A minimum of 30 crypts for each of 5 control and RS5444-treated mice were counted, and statistical analysis was carried out with Mann-Whitney rank sum analysis.

 
The mechanism that accounts for colon cancer suppression by PPAR-{gamma} is unknown. However, it has been observed that PPAR-{gamma} inhibits proliferation of some colon cancer cell lines (5, 19, 20, 26, 48), suggesting that inhibition of proliferation might be involved in tumor suppression. PPAR-{gamma} inhibits proliferation of nontransformed intestinal epithelial cells in culture (6), and PPAR-{gamma} inhibits thymidine incorporation into colonic epithelial cells ex vivo (38), but inhibition of intestinal epithelial cell proliferation by PPAR-{gamma} had never been reported in vivo. We measured BrdU staining of proximal and distal colonic crypts from control (CMC treated) and RS5444-treated mice. As shown in Fig. 3B, PPAR-{gamma} inhibited BrdU labeling in both proximal and distal crypts. Quantification of BrdU-labeled cells per crypt indicated that a statistically significant decrease in BrdU labeling index occurs in mice that have been treated with the PPAR-{gamma} agonist. Furthermore, the extent of inhibition was similar in the proximal and distal colon (Fig. 3C). Thus activation of PPAR-{gamma} results in suppression of preneoplastic lesion formation and inhibition of proliferation in both proximal and distal colon.

We previously reported (7) that PPAR-{gamma} promotes epithelial to mesenchymal transformation and enhances motility of intestinal epithelial cells by a mechanism that involves activation of ERK1/2 both in culture and in vivo. Colonic crypts from control and RS5444-treated mice were stained with anti-phosphoERK1/2 to determine whether PPAR-{gamma} also activates ERK1/2 signaling in the colon. As shown in Fig. 4A, we observed no significant change in ERK1/2 phosphorylation in the proximal colon. However, nuclear staining for phosphoERK1/2 was significantly enhanced in epithelial cells from the distal colon. This result was confirmed by Western blotting of extracts from isolated proximal and distal crypts from control and RS5444-treated mice (Fig. 4B). Total ERK1/2 expression in proximal and distal epithelial cells was similar, although basal ERK1/2 activity (assessed by phosphorylation state) was significantly higher in the distal colon. Moreover, RS5444 induced phosphorylation of both ERK1 and ERK2 in the distal colon by about twofold (Fig. 4C), with no significant effect on ERK1/2 phosphorylation in the proximal colon. This observation demonstrates that some of the physiological responses to PPAR-{gamma} in the colon are region specific.


Figure 4
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  		Fig. 4. PPAR-{gamma}-dependent activation of MAPK signaling in colonic epithelial cells. A: sections from control (CMC treated) or RS5444-treated colons were stained with antibody against phospho (p)ERK1/2. B: protein extracts from isolated proximal and distal crypts from control and RS5444-treated animals were assayed for pERK1/2 or total ERK1/2 expression by Western blotting. C: fold changes in pERK1 relative to total ERK1 and pERK2 relative to total ERK2 were determined by Western blotting, followed by quantitative analysis of luminescence in extracts prepared from 3 independent pairs of control and RS5444-treated animals. Data represent means of 3 analyses, and error bars indicate 1 SD. Statistical analysis was carried out by t-test.

 
Identification of PPAR-{gamma} target genes in colonic epithelial cells.
Since PPAR-{gamma} is a transcription factor, we reasoned that PPAR-{gamma} function should be reflected by changes in gene expression after activation of the receptor in vivo, and that by profiling such changes we might be able to infer tissue-specific functional differences in the response to activation of PPAR-{gamma} in colonic epithelial cells. A genomic analysis was carried out to identify PPAR-{gamma}-regulated genes in colonic epithelial cells. Mice were treated by oral gavage with 10 mg RS5444·kg–1·day–1, and control mice received daily gavage of CMC. After 3 days, the mice were killed and epithelial crypts were prepared from total colon. Total RNA was prepared from four CMC-treated and four RS5444-treated mice, and Affymetrix microarrays were used to measure relative mRNA abundance. Data were processed as described in MATERIALS AND METHODS, and we identified 131 genes that were regulated, with P < 0.01 and a fold change of >1.99 or <0.51 (a list of these genes is provided in Supplemental Table I).1 The ontogeny of these genes was assigned by knowledge-based analysis using Ingenuity as well as literature searches. Three primary functional cohorts were identified by this approach (Fig. 5). The largest cohort of PPAR-{gamma} targets was associated with metabolic function. Among this cohort, the largest single group of genes was associated with lipid metabolism, consistent with the known functions of PPAR-{gamma} in other cell types. We also identified subcohorts involved in solute transport, protein modification and processing, and drug metabolism. A large cohort of genes was involved in other metabolic functions that could not be grouped into any single pathway.


Figure 5
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  		Fig. 5. Functional cohorts of PPAR-{gamma} target genes in isolated colonic epithelial cells. PPAR-{gamma} target genes were identified by statistical analysis of Affymetrix microarrays. Functional assignments were made based on Ingenuity pathway analysis and literature review of the published functions that have been assigned to these proteins: metabolism (A), signal transduction (B), and motility and adhesion (C). The reference lines indicate a fold change of 2-fold increase to –2 repression, equivalent to 50% inhibition.

 
The second largest functional cohort of PPAR-{gamma} target genes was comprised of genes with known roles in signal transduction. Among these genes were transcription factors, transcription regulatory proteins, receptors, genes that control second messenger activity, and a few genes that did not obviously fit into any of these categories but are thought to be involved in signaling. The third major cohort included genes that have known roles in controlling cellular morphology, motility, cell-cell adhesion, and interaction with the extracellular matrix. A small number of membrane proteins of unknown function were also included in this cohort because their localization suggested a potential role in cell/cell or cell/matrix interaction. Our genomic data indicate that activation of PPAR-{gamma} in the colonic epithelium has profound cellular consequences and regulates the metabolic activity of such cells, intracellular signal transduction activity, and cellular processes related to motility and adhesion. These responses conform broadly to those that we previously described (6) in nontransformed intestinal epithelial cells in culture.

Differential regulation of gene expression in proximal and distal colon.
We had previously carried out a genomic analysis of basal gene expression in isolated crypts from the proximal and distal colon of untreated female C57BL/6J mice (N. R. Murray, unpublished data). We therefore interrogated this data set to identify PPAR-{gamma} target genes that were differentially expressed in the proximal and distal colon. Proximal and distal target genes were assigned based on >2.49-fold (or <0.41-fold) difference in mRNA abundance in the proximal and distal epithelium (P < 0.01). PPAR-{gamma} target genes were then identified within these cohorts, using the criteria described above. The results were remarkable. The majority of the PPAR-{gamma} targets exhibited similar basal expression levels (<2.5-fold difference) in the proximal and distal colon, and among these there was an equal distribution of genes that were induced (37) and genes that were repressed (37) by PPAR-{gamma}. However, among those PPAR-{gamma} target genes that are predominantly expressed in the proximal colon (23 genes), all were induced by PPAR-{gamma} (Fig. 6). Conversely, all 34 of the PPAR-{gamma} target genes that were predominantly expressed in the distal colon were repressed by PPAR-{gamma}.


Figure 6
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  		Fig. 6. Regulation of proximal colon- and distal colon-specific PPAR-{gamma} target genes. Differential expression was defined as an expression difference of >2.49 or <0.41, proximal vs. distal, with P < 0.01 (n = 4). PPAR-{gamma} target genes were then segregated into 3 cohorts: those that were more or less equally abundant in proximal and distal colon (not shown), those that were predominantly expressed in proximal colon (A), and those that were predominantly expressed in distal colon (B). Data are expressed as mean log 2-fold change determined by comparison of 4 control and 4 RS5444-treated mice.

 
Regulation of a large number of these PPAR-{gamma} target genes was confirmed by using qPCR to measure mRNA abundance in RNA extracted from isolated colonic crypts from the total colon (see Supplemental Table I). In addition, we prepared epithelial cells from the proximal and distal colon and used qPCR to identify four distinct patterns of expression and response, as illustrated in Fig. 7. One group of genes (exemplified by SOCS3 and CKTM2) was expressed at similar abundance in untreated epithelial cells from the proximal and distal colon, and these genes were regulated by PPAR-{gamma} in both tissues. A second group of genes (e.g.,HSD17b4 and GJA1/connexin43) was expressed at similar abundance in both proximal and distal colon but was regulated by PPAR-{gamma} only in the proximal colon. The genes that were differentially expressed in the proximal and distal colon fell into two classes. One class of targets is illustrated by FABP1 and AQP3. These genes were expressed almost exclusively in the proximal colon and were all induced in that tissue. Conversely, target genes that were expressed predominantly in the distal colon (e.g., CCL28 and ATP12a) were all repressed by PPAR-{gamma}. The data indicate that PPAR-{gamma} response in the colonic epithelium is complex and likely to be regulated by a number of factors that may include differences in cellular distribution and tissue-specific expression, but are largely unknown at this time.


Figure 7
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  		Fig. 7. Analysis of PPAR-{gamma} target genes in isolated proximal and distal colonic epithelial cells. Expression of selected genes was measured by qPCR using pooled RNA samples from 4 preparations of isolated crypts from control proximal colon (PC), RS5444-treated proximal colon (PR), control distal colon (DC), or RS5444-treated distal colon (DR). Genes were selected to illustrate 4 distinct regulatory responses, as described in the text.

 

   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
Although the mechanism that accounts for the tumor-suppressive effects of PPAR-{gamma} is unknown, the data are generally consistent with the hypothesis that the critical tumor-suppressive effects of PPAR-{gamma} are exerted in normal, nontransformed colonic epithelial cells. However, our understanding of PPAR-{gamma} action in nontransformed colonocytes is limited by the fact that cell culture models do not exist. Consequently, one must focus on the effects of PPAR-{gamma} in the living colon, with all of the experimental limitations that are imposed by this approach. The experiments described in this article were undertaken to define the effects of PPAR-{gamma} on normal colonic epithelial cells, and, to our knowledge, these data represent the first report of global changes in gene expression in epithelial cells isolated from the proximal and distal colon of mice that have been treated with PPAR-{gamma} agonists.

Our initial objective was to resolve the controversy concerning PPAR-{gamma} distribution within colonic epithelial cells. We observed a strong basal-to-luminal expression gradient in the proximal colon, as reported by Lefebvre et al. (31). However, PPAR-{gamma} is expressed throughout the colonic epithelium, as reported by Girnun et al. (16), and in the distal colon PPAR-{gamma} expression is somewhat higher in those cells at the base of the crypts, as reported by Saez et al. (47). The highest level of PPAR-{gamma} expression occurs in the more highly differentiated luminal epithelial cells of the proximal colon, and PPAR-{gamma} is induced during differentiation of colon cancer cells in culture (31). Moreover, we have shown (6) that PPAR-{gamma} is induced at the crypt/villus junction in the small intestine, where intestinal epithelial cells differentiate from transit-amplifying to mature absorptive epithelial cells. Such observations have led to the general notion that PPAR-{gamma} is induced during differentiation of gastrointestinal epithelial cells. However, the cells at the base of the proximal crypts are also highly differentiated, and they express relatively low levels of PPAR-{gamma}. Likewise, the undifferentiated cells at the base of the distal crypts express relatively higher levels of PPAR-{gamma} compared with the more highly differentiated luminal epithelial cells of the distal colon. These data are inconsistent with the generalization that induction of PPAR-{gamma} in the colon epithelium occurs during differentiation throughout the colon, although this conclusion is correct in reference to luminal epithelial cells of the proximal colon.

These differences in the pattern of PPAR-{gamma} expression between the proximal and distal colon and within the epithelia of proximal and distal colon imply that PPAR-{gamma} functions in the colon are likely to be very complex and include both region-specific and cell-specific responses. We used a functional genomic approach to categorize the major responses that prevail in isolated colonic epithelial cells from mice treated with a potent PPAR-{gamma} agonist. We assume that the genomic responses that we have documented are directly due to activation of PPAR-{gamma} in epithelial cells. However, we cannot rigorously exclude the possibility that some of these responses may be due to systemic effects of PPAR-{gamma} agonists.

Genomic ontology was used to identify the major PPAR-{gamma}-regulated pathways in colonic epithelial cells. Pathway analysis of genomic data is complicated by the fact that many proteins function in more than one pathway, and rigorous assignment of gene function to discrete pathways is often ambiguous and debatable. Nevertheless, our ontological analysis indicates that PPAR-{gamma} regulates three broad patterns of response within the colon: metabolism, signaling, and motility/adhesion. We previously reported (6) that similar categories of response prevail in rat intestinal epithelial cells in which PPAR-{gamma} is activated in culture. It is not surprising that PPAR-{gamma} regulates a number of genes that are involved in energy metabolism, given the role of this receptor in regulating metabolic functions in other cell types. We observed that PPAR-{gamma} regulates a significant cohort of genes that are involved in drug metabolism, including several cytochrome P-450 genes and genes that encode glutathione and glutamyl transferases. The observation that PPAR-{gamma} regulates drug metabolism is provocative and suggests one possible mechanism to account for the ability of this receptor to suppress AOM-mediated colon carcinogenesis.

The observation that many signal transduction components are regulated by PPAR-{gamma} suggests that this receptor is likely to be involved in cross talk with other signaling pathways in colonic epithelial cells. Similar observations were reported in intestinal epithelial cells (6). Likewise, we have shown (6, 7) that PPAR-{gamma} plays an unexpected role in regulation of motility and adhesion of nontransformed intestinal epithelial cells in culture, and our genomic analysis indicates that a similar response is probable in the colonic epithelium. These data suggest that among the differentiated functions of PPAR-{gamma} is regulation of migration from the sites of proliferation to the luminal surface and perhaps exfoliation from the colonic epithelium.

We have carried out an ontological analysis to determine whether one can identify specific pathways that are characteristic of the PPAR-{gamma} response in the proximal and distal colon (Table 1). The data suggest that the metabolic functions of PPAR-{gamma} are more pronounced in the proximal colon, whereas responses related to signaling and motility are more pronounced in the distal colon. This analysis is consistent with the observation that MAPK activation is limited to the distal colon. However, this analysis is limited by the small number of tissue-specific PPAR-{gamma} target genes that we have identified, and the hypothesis that PPAR-{gamma} plays a different role in signal transduction in the proximal and distal colon requires a more detailed analysis.


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  		Table 1. Functional assignment of PPAR-{gamma} target genes in proximal and distal colon

 
It has been widely supposed that PPAR-{gamma} regulates proliferation of colonic epithelial cells in vivo, but our data represent, to our knowledge, the first direct demonstration of such a response in the intact colon. In contrast to our analysis of PPAR-{gamma} effects in nontransformed intestinal epithelial cells in culture (6), we did not observe a significant cohort of proliferation-related target genes (those involved in cell cycle progression, DNA replication, DNA repair, or genomic stability) in vivo. Failure to observe such a cohort in vivo probably reflects the fact that proliferative cells are a minor component of the intact epithelium, and genomic responses in such cells are likely to be diluted by mRNA from the major, nonproliferative component of the epithelium.

Perhaps the most striking result of our studies is the observation that PPAR-{gamma} has very different effects on expression of proximal and distal marker genes. If one assumes that a gene that is specifically expressed in a given tissue is a marker of the differentiated function of that tissue, then our data suggest that PPAR-{gamma} enhances differentiated function in the proximal epithelium, assessed by induction of differentiated markers. Aquaporin 3 (Aqp3) is one such example. This gene presumably functions in water recovery from the stool, a critical differentiated function, and Aqp3 is induced by PPAR-{gamma} in the proximal colon. However, a strikingly different response was observed in the distal colon. ATP12a is a well-known marker of differentiated function in the distal colon, involved in H+/K+ transport; and ATP12a expression is repressed by PPAR-{gamma}. Remarkably, all of the distal colon-specific PPAR-{gamma} target genes were repressed in distal epithelial cells. This observation raises the provocative notion that PPAR-{gamma} may repress differentiation in the distal colon.

Our data indicate that PPAR-{gamma} regulates some common functions in the proximal and distal colon. The majority of PPAR-{gamma} target genes that we have identified are expressed at similar levels in the proximal and distal colon, suggesting that a great many functions of PPAR-{gamma} are exerted to similar extents in both tissues. Among these common functions are inhibition of proliferation and suppression of early-stage transformation. However, there are some marked differences in response. For example, we did not observe activation of MAPK signaling in the proximal colon, whereas PPAR-{gamma} promotes ERK1/2 phosphorylation in the distal colon, as well as in the intestinal epithelium (7). It should be noted that ERK1/2 was activated in postmitotic cells, indicating that this response is unrelated to the proliferative response that is commonly associated with MAPK signaling. Instead, activation of MAPK signaling in postmitotic cells is probably related to motility of gut epithelial cells (7). If this conclusion is correct, then PPAR-{gamma} does not appear to regulate motility of proximal epithelial cells, or does so by a mechanism that does not depend on the MAPK pathway.

In conclusion, our data indicate that PPAR-{gamma} regulates important differentiated functions, including proliferation, metabolism, signal transduction, motility, and adhesion. It is equally clear that these responses are not manifest to the same extent in the proximal and distal colon. Regarding the tumor-suppressive effects of PPAR-{gamma}, our data indicate that this appears to be a common response in both the proximal and distal colon and is plausibly linked to the ability of PPAR-{gamma} to inhibit proliferation of transit-amplifying cells in both proximal and distal crypts. However, proliferative cells represent a very small subset within the colonic epithelium, and a more detailed analysis of PPAR-{gamma} response within this population will be required to more precisely define a testable hypothesis to account for the tumor-suppressive effects of PPAR-{gamma} in the colon. Our observations challenge several common generalizations with respect to PPAR-{gamma} action in the colon. PPAR-{gamma} is not invariably induced during differentiation of colonic epithelial cells, and PPAR-{gamma} clearly inhibits expression of differentiated markers in the distal colon. These observations therefore have very important implications for analysis of the role of PPAR-{gamma} in the physiology of the colonic epithelium.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
These experiments were supported in part by National Institutes of Health (NIH) Grants to E. A. Thompson (CA-121349 and CA-127996), N. R. Murray (CA-94122), and A. P. Fields (CA-81436). C. R. Bush was supported by a training fellowship from the Keck Center for Computational and Structural Biology of the Gulf Coast Consortia (NIH Grant 5-T15-LM-07093).


   DISCLOSURES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
These studies were funded in part by a grant from Sankyo Co., Ltd., which provided RS5444. This manuscript was reviewed by Sankyo before publication, but control of the decision to publish rested entirely with the authors.


   ACKNOWLEDGMENTS
 
We are grateful to Pamela Kreinest and the staff of the Mayo Clinic Cancer Histology laboratory for help with fixation, sectioning, and staining of tissues. Nancy Rutkoski and the staff of the Mayo Clinic Cancer Molecular Biology Core facility provided invaluable help in measuring gene expression by qPCR. Dr. Tom Wood and Michelle Guigneaux of the University of Texas Medical Branch Genomics Core facility are acknowledged for their excellent support in our microarray experiments.


   FOOTNOTES
 
Address for reprint requests and other correspondence: E. A. Thompson, Dept. of Cancer Biology, Griffin Cancer Research Bldg., Rm. 304, Mayo Clinic, 4500 San Pablo Rd., Jacksonville, FL 32225 (e-mail: thompson.aubrey{at}mayo.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 The online version of this article contains supplemental material. Back


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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