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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Veterinary Physiology, Vetsuisse Faculty, University of Bern, CH-3012 Bern, Switzerland
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.05) in the mRNA abundance among stomach (St), duodenum (Du), jejunum (Je), ileum (Il), and colon (Col) for NR [pregnane X receptor (Du, Je > Il, Col > St), peroxisome proliferator-associated receptor 
(St, Du, Col > Je, Il), constitutive androstane receptor (Je, Du > Il, Col), and retinoid x receptor 
(Du > Il)] and NR target genes [glutathione-S-transferase A3-3 (Du > Je > St, Il; St > Col), phenol-sulfating phenol sulfotransferase 1A1 (Du, Je > Il, St; Col > St), cytochrome P450 3A12 (Du, Je > St, Il, Col), multiple drug resistance gene 1 (Du, Je, Il, Col > St), multiple drug resistance-associated protein 2 (Je, Du > Il > St, Col), multiple drug resistance-associated protein 3 (Col > St > Il; Du > Je, Il; St > Il), NR corepressor 2 (St > Il, Col), and cytochrome P450 reductase (St, Du, Je > Il, Col)], but not for peroxisome proliferator-associated receptor . Differences (P > 0.05) in mRNA abundance in the liver relative to the GIT were also observed. In conclusion, the presence of numerous differences in expression of NR and NR target genes in different parts of the GIT and in liver of healthy dogs may be associated with location-specific functions and regulation of GIT regions.
Key Words: canine • gastrointestinal tract • liver • nuclear receptor • nuclear receptor target gene • reverse-transcription polymerase chain reaction
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Liddle and Goodwin, 2002).
Nuclear receptors (NR) are ligand-activated transcription factors that regulate the expression of target genes (Schoonjans et al., 1996). They are important for GIT and hepatic xenobiotic metabolism, transport, detoxification, and biotransformation reactions (de Bruin et al., 2000; Kast et al., 2002; Goodwin and Moore, 2004). Nuclear receptors contain several functional regions, including DNA- and ligand-binding domains (Schoonjans et al., 1996). After activation by various signaling molecules, they bind to intracellular receptors and cause receptor conformational changes (Honkakoski and Negishi, 2000). Activated NR bind to response elements within target gene promoters and control the transcription of specific genes (Mangelsdorf et al., 1995).
Few studies have investigated NR and NR target gene expression in domestic animals. Our previous studies in calves have shown that mRNA levels of NR and NR target genes are differentially expressed in GIT and liver and are influenced by nutrition, age, and hormones (Krüger et al., 2005; Greger et al., 2006a; Greger and Blum, 2006). Studies in canines with varying etiologies of chronic diarrhea have also shown differential expression, especially of NR target genes (Greger et al., 2006b).
The purpose of the current study was to investigate the expression profile of NR and NR target genes at different sites along the GIT and in the liver of healthy dogs.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals
Mature, healthy dogs (Beagles; 5 females and 6 males), kept at Novartis Animal Health (St. Aubin, Switzerland), Novartis Pharma (Muttenz, Switzerland), and MDS Pharma Services (Les Oncins, France) were studied. Mean age of the dogs was 14 mo (range 10 to 17 mo), and mean BW was 8.8 kg (range 6.6 to 11.4 kg).
Tissue Sampling
Within 5 min after euthanasia (with intravenous administration of pentobarbital, a barbiturate, at the place where kept), full-thickness sections of liver, stomach, duodenum, jejunum, ileum, and colon were taken. Tissue samples were rinsed once with PBS and then transferred into an RNA stabilization reagent (RNA-later, Qiagen, Hilden, Germany).
Isolation of Total RNA, Reverse Transcription of RNA, Primer Design, and PCR Analyses
Extraction of total RNA from the different tissues, measurement of RNA concentration, evaluation of the quality of RNA (by gel electrophoresis), reverse transcription of RNA into cDNA, primer design for the NR and NR target genes as well as for the housekeeping genes (HKG; Table 1), and conditions for real-time reverse-transcription PCR (performed with the LightCycler System; Roche Molecular Biochemicals, Rotkreuz, Switzerland) have been described in detail (Greger et al., 2006b). Ubiquitin, ß-actin, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were selected as HKG. The crossing-point values of GAPDH, ß-actin, and ubiquitin were positively correlated: Pearson’s rank coefficient of correlation (r) for ubiquitin vs. ß-actin was 0.44 (P < 0.001), for ubiquitin vs. GAPDH was 0.53 (P < 0.001), and for ß-actin vs. GAPDH was 0.72 (P < 0.001), indicating that these HKG were acceptable because of the consistent level of expression relative to the studied genes. Because they are involved in very different functions, it can be expected that they would not have been coordinately regulated and changed in a uniform manner. The PCR efficiency was established with the slope calculated by the LightCycler software 3.3 (Roche Applied Science, Rotkreuz, Switzerland); the precision of the assays was determined under the same conditions as described previously (Greger et al., 2006b). In PCR assays, interassay CV ranged from 0.14 to 1.60%, and intraassay CV ranged from 0.38 to 1.96%.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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.
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The abundance of pregnane X receptor (PXR) mRNA in duodenum was greater than in ileum (P < 0.001) and colon (P < 0.01) but not different from that in jejunum. Levels of PXR mRNA in ileum and colon were similar. In stomach, PXR was not detectable. It was the only gene that showed a sex effect across all tissues: mRNA levels were greater (P < 0.02) in females than in males. There were no sex x tissue interactions.
The abundance of peroxisome proliferator-activated receptor (PPAR) mRNA did not differ among stomach, duodenum, jejunum, ileum, and colon. The abundance of mRNA of PPAR in stomach, duodenum, and colon was similar but greater (P < 0.001) in stomach and colon than in jejunum and ileum, and the level in duodenum was greater (P < 0.05) than in jejunum and ileum (P < 0.001). Abundance of PPAR mRNA in jejunum was not different from ileum.
The abundance of retinoid receptor (RXR) mRNA was greater (P < 0.05) in duodenum than in ileum but not different from jejunum, colon, and stomach. The mRNA level was similar in jejunum, colon, stomach, and ileum. Additionally, RXR showed a tendency (P < 0.1) toward greater expression in females than in males.
Nuclear Receptor Target Gene mRNA Abundances in Stomach, Duodenum, Jejunum, Ileum, and Colon
The abundances of NR target genes in stomach, duodenum, jejunum, and colon relative to HKG are presented in Table 2
. The abundance of glutathione-S-transferase A3-3 (GST A3-3) mRNA was greater in duodenum than in jejunum (P < 0.01), stomach (P < 0.001), ileum (P < 0.001), and colon (P < 0.001). The mRNA level in jejunum was greater (P < 0.001) than in stomach, ileum, and colon. In stomach, GST A3-3 mRNA abundance was greater (P < 0.05) than in colon but not different (P > 0.05) from the mRNA level in ileum.
The abundance of phenol-sulfating phenol sulfotransferase (SULT1A1) mRNA was greater (P < 0.001) in duodenum than in ileum and stomach but not different from jejunum and colon. The mRNA level was greater (P < 0.01) in colon than in stomach but not different from that in ileum. Levels of SULT1A1 mRNA in ileum and stomach were not different.
The abundance of cytochrome P450-3A12 (CYP3A12) mRNA in duodenum and jejunum was similar but was greater (P < 0.001) in duodenum than in ileum, colon, and stomach. Similarly, CYP3A12 mRNA abundance was greater in jejunum than in colon (P < 0.01), ileum (P < 0.05), and stomach (P < 0.001). The mRNA levels in ileum, colon, and stomach were not different.
The abundance of multiple drug resistant gene 1 (MDR1) mRNA in ileum, jejunum, duodenum, and colon was similar, but in ileum, jejunum, and duodenum it was greater (P < 0.001) than in stomach. In colon, MDR1 mRNA abundance was also greater (P < 0.01) than in stomach.
The abundance of multiple drug resistance-associated protein (MRP) 2 mRNA was similar in duodenum and jejunum, but levels at both sites were greater (P < 0.05) than in ileum. No MRP2 mRNA was detectable in stomach and colon.
The abundance of MRP 3 mRNA was greater in colon than in ileum, jejunum (P < 0.001), and stomach (P < 0.01) but was not different from duodenum. The mRNA level in duodenum was greater than in jejunum (P < 0.01) and ileum (P < 0.001) but was not significantly different from stomach. The mRNA abundance in jejunum and ileum was similar.
The abundance of NR corepressor 2 (NCOR2) mRNA was greater (P < 0.05) in stomach than in ileum and colon but was not significantly different from duodenum and jejunum. Levels of NCOR2 mRNA levels in ileum and colon were similar.
The abundance of cytochrome P450 reductase (CPR) mRNA in duodenum was greater than in colon and ileum (P < 0.001). The mRNA levels in duodenum also tended to be greater than in stomach (P = 0.1) and jejunum (P = 0.1). Abundance of CPR mRNA was greater (P < 0.05) in stomach and jejunum than in ileum and colon. The mRNA levels in ileum and colon were similar.
Nuclear Receptor mRNA and Nuclear Receptor Target Gene mRNA Abundances in Liver
Abundances of mRNA for NR and NR target genes in liver were quite variable (Table 3). In addition, there were differences (P < 0.05) in the mRNA abundances in the liver relative to the GIT for PXR (liver > colon, ileum), PPAR(liver > jejunum, colon, duodenum, stomach, ileum), PPAR (liver < stomach, colon, duodenum), CAR (liver > jejunum, duodenum, stomach, ileum, colon), GST (liver > duodenum, jejunum > stomach, ileum, colon), SULT1A1 (liver > duodenum, jejunum, colon > ileum, stomach), CYP3A12 (liver > duodenum, jejunum, ileum, colon, stomach), MDR1 (liver > duodenum, colon > stomach), MRP2 (liver > ileum, colon, stomach), MRP3 (liver > jejunum, ileum), and CPR (liver > jejunum, stomach, ileum, colon).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Greger et al., 2006a). The data can be interpreted to suggest that these receptors and enzymes have variable functional roles at different GIT sites and in liver. Given that these organs have high metabolic activities and that they are regularly confronted with potentially harmful molecules that have to be metabolized, eliminated from the body, or both our results point to a role for the NR and their target genes in these processes. It is understood that mRNA levels are not necessarily mirrored by protein products of NR and NR target genes. Thus the present gene profiling study is the needed first step to substantiate the value in further investigating the functional differences of these variables across the different regions of the gut.
In the current study, mRNA levels of NR and NR target genes were measured in full-thickness GIT samples, whereas in the study of Greger et al. (2006b) analyses were performed in biopsies containing mainly the epithelium and parts of subepithelial layers. The differences (greater mRNA levels of all NR and NR target genes in samples from biopsies than in full-thickness samples) in the present and the previous study suggest that there exist differences within the different layers of the GIT.
In the current study the mRNA level of CAR was greater in jejunum than in ileum and colon, in accordance with studies in rats (Kanno et al., 2004). In contrast to that report, the mRNA level of CAR in stomach in the current study was not significantly different from other parts of the GIT. The greatest expression of CAR in liver has also been observed previously in a study in mice (Choi et al., 1997). Hepatic CAR may thus have a more important functional role than intestinal CAR. The role of CAR in the regulation of responses to metabolic and nutritional stress (Goodwin and Moore, 2004) is well known. Moreover, CAR influences the expression of numerous genes that are involved in oxidative metabolism, conjugation, and transport of different substances (Goodwin and Moore, 2004), such as in the metabolism and excretion of bilirubin (Maglich et al., 2002).
As already shown in studies in humans and mice (Lehmann et al., 1998) and calves (Krüger et al., 2005; Greger et al., 2006a; Greger and Blum, 2006), PXR was also expressed in the entire dog intestine. However, PXR expression was greater in females than in male dogs, although there were no sex x tissue interactions. We found that the abundance of PXR mRNA was greater in duodenum and jejunum than in ileum and colon. However, in contrast with mice (Lehmann et al., 1998), we could not detect mRNA for PXR in dog stomach. This indicates that there are species differences in the expression of this receptor. As shown previously in humans and calves (Lehmann et al., 1998; Krüger et al., 2005), hepatic PXR expression was greater than intestinal (ileum, colon) expression. After being activated by xenobiotics, PXR influences the expression of target genes which affect detoxification and elimination processes in intestine and liver (Goodwin and Moore, 2004). Additionally, the bilirubin metabolism and excretion is influenced by PXR and CAR (Maglich et al., 2002).
In accordance with a study in rats (Braissant et al., 1996), PPAR was evenly distributed in the entire GIT. In our study, PPAR was predominantly expressed in the liver, also in agreement with studies in rats (Braissant et al., 1996). The expression pattern of PPAR might reflect differences in the rate of mitochondrial and peroxisomal ß-oxidation activity (Braissant et al., 1996). This receptor regulates the ß-oxidation of fatty acids and thereby influences the metabolism of lipids (Schoonjans et al., 1996). Additionally, it has antiin-flammatory properties via stimulating the degradation of fatty acid metabolites (Chinetti et al., 2000).
We found a relatively high level of PPARmRNA in colon, similar to that reported in a study in humans (Fajas et al., 1997). Abundance in jejunum and ileum was lower than in colon, which was consistent with Fajas et al. (1997). In accordance with previous studies in humans, the abundance in liver was less than in colon (Fajas et al., 1997). This receptor might be necessary for physiological as well as pathological functions in the large intestine (Fajas et al., 1997) and liver, and in the regulation of inflammation by downregulation of the nuclear factor B and the mitogen-activated protein kinase cascades, which reduce the production of inflammatory cytokines (Dubuquoy et al., 2002). Perhaps because of those functions, PPARwas also highly expressed in stomach and duodenum, organs that are regularly exposed to high levels of ingested and potentially harmful nutrient components.
In agreement with a previous study in humans (Dubuquoy et al., 2002), we found no differences in the mRNA level of RXR between the small intestine and the colon. However, RXR showed a tendency to be more highly expressed in female than in male dogs. In the small intestine, however, a greater expression was found in duodenum than in ileum. The abundance of RXR mRNA was also very similar in the GIT and liver. This receptor forms heterodimers with different NR partners like PPAR, PXR, and CAR to regulate the transcription of target genes (Xu et al., 2005). Thereby RXR is involved in inflammation (Dubuquoy et al., 2002) and in numerous metabolic processes. The wide-ranging expression of RXR in the GIT and liver might be explained by its influence on the numerous NR and target genes.
We found that GST A3-3 expression is elevated in duodenum and jejunum and reduced in colon. In general, the GST are part of an important protective system of the body to inactivate toxic electrophiles of different compounds by conjugating glutathione to enhance their excretion (Johansson and Mannervik, 2001). Members of the GST alpha class participate in the metabolism of activated alkenes (such as GST A4-4; Hubatsch et al., 1998) and also possess fatty acid and phospholipid hydroperoxidase activity (GST A1-1 and GST A2-2; Zhao et al., 1999). The GST A3-3 may play an important role in the metabolism of steroid hormones (Johansson and Mannervik, 2001). However, in our study, GST A3-3 was highly expressed in tissues (duodenum and jejunum) that are not known to be particularly steroido-genic. Patel et al. (1997) also reported GST A3-3 expression, confined to the villus of the normal intestinal epithelium, and seems to be particularly involved in the regeneration of the small intestinal epithelium. We found greater expression in liver than in GIT, in contrast with a previous study in humans where no expression in liver was detectable (Johansson and Mannervik, 2001).
In the current study, the mRNA level of SULT1A1 was expressed in the entire intestine, in accordance with a previous study in dogs (Tsoi et al., 2002). The mRNA abundance in duodenum, jejunum, and colon was greater than in stomach and was particularly high in the liver. A relatively high mRNA expression in dogs of SULT1A1 in colon and a predominance of this enzyme in liver compared with other GIT sites has also been observed (Tsoi et al., 2002). In contrast with that study, we did not observe any differences in mRNA levels between ileum and colon. Sulfotransferases are phase II drug-metabolizing enzymes that promote sulfo-conjugation of endogenous substances and xenobiotics to produce polar and excretable substances and thus represent an important part in the chain of events leading to the detoxification of molecules (Glatt and Meinl, 2004).
Greater expression of CYP3A12 (the canine ortholog of human CYP3A4) in duodenum than in colon and stomach was also found in a study in humans, where CYP3A4 was also more highly expressed in duodenum (Thörn et al., 2005). Decreased CYP3A12 expression along the small intestine was also in line with previous studies that found less expression in ileum than in duodenum of humans (Wacher et al., 1995). Abundance of this gene was greater in liver than in GIT, which is indicative of the important role of this cytochrome in hepatic biotransformation reactions (Lehmann et al., 1998).
In our study, the mRNA levels of MDR1 were highly expressed in liver as well as in the small intestine and colon, in accordance with data obtained in tissues from humans (Farrell and Kelleher, 2003). This gene codes for an ATP-dependent P-glycoprotein that acts as an efflux pump to transport substances out of the cell into the intestinal lumen, which results in reduced intracellular concentrations (Farrell and Kelleher, 2003). From the low mRNA level of MDR1 in the stomach in dogs, it may be hypothesized that MDR1 has a small role in detoxification processes in the stomach compared with other parts of the GIT.
It was not surprising that MRP2, which acts as an ATP-binding cassette (ABC) transporter, was only detectable in the small intestine and not in stomach and colon. The small intestine is known for its high barrier functions (Langmann et al., 2003). A high expression of MRP2 is likely an important component of xenobiotic metabolism to protect the body against dietary harmful substances (Cherrington et al., 2002). Additionally, the expression of ABC-transporters in the different tissues is influenced by the direct and variable contact of those organs with xenobiotic and food components (Albermann et al., 2005). The mRNA levels of MRP2 in liver were greater than in duodenum and jejunum, which is in contrast with a study in rats (Cherrington et al., 2002). In liver and intestine, MRP2 enhances the excretion of various metabolites into bile and out of the body (Cherrington et al., 2002).
The mRNA of MRP3 was present in the entire GIT, with greatest levels in the colon, in agreement with studies in rats (Cherrington et al., 2002; Rost et al., 2002). In contrast to the report of Rost et al. (2002), we found a high mRNA level in duodenum and a low level in ileum. The high mRNA abundance in colon and liver may be indicative of an important involvement of this ABC-transporter in the absorption of bile acids into blood (Cherrington et al., 2002; Rost et al., 2002).
The mRNA of NCOR2 was present in the entire GIT, with greatest levels in stomach and a decline along the entire small intestine up to the colon. The expression level was only greater in stomach than in liver. This gene is able, in tandem with specific NR and different DNA binding transcription factors, to repress the transcription of other genes (Jepsen et al., 2000). Thus, it also has an important role in the regulation of vertebrate development (Tomita et al., 2004).
The mRNA levels of CPR were greatest in liver and tended to be greater in duodenum than in jejunum and stomach. The expression pattern of CPR was similar to the expression pattern of CYP3A12, where the expression in duodenum and jejunum was greater than in ileum and colon. The notable exception was the stomach, where the CPR expression was not lower than in duodenum and jejunum, and in the liver where the expression was similar to other tissues. This is possibly associated with differences in the function of CPR as an electron donor for cytochrome P450 (Vermilion et al., 1981).
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Footnotes |
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2 Present address: Department of Dairy and Animal Science, Penn State University, University Park, PA 16802-3503.
3 Present address: Berna Biotech Ltd., Rehhagstrasse 79, CH-3018 Bern, Switzerland.
4 Corresponding author: juerg.blum{at}itz.unibe.ch or juerg.blum{at}physio.unibe.ch
Received for publication March 23, 2006. Accepted for publication May 22, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, -ß, and -in the adult rat. Endocrinology 137:354–366.[Abstract], P1-1 and T1-1 in the human gastrointestinal tract. Jpn. J. Cancer Res. 91:310–316.[CrossRef]and retinoid X receptor heterodimer in hepatogas-troenterological diseases. Lancet 360:1410–1418.[CrossRef][Medline]gene. J. Biol. Chem. 272:18779–18789.
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