Differential regulation of cytochrome along rat intestinal crypt-villus axis PETER
G. TRABER,
WE1 WANG,
P-450 genes
AND LA1 YU
Division of Gastroenterology and the Department of Internal Medicine, University of Michigan Medical School and the Veterans Affairs Medical Center, Ann Arbor, Michigan 48109 Traber, Peter G., Wei Wang, and Lai Yu. Differential regulation of cytochrome P-450 genesalong rat intestinal cryptvillus axis. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G215-G223, 1992.-Mammalian small intestine contains cytochrome P-450-dependentmonooxygenaseenzymesthat are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cellslining the smallintestine are separatedinto a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expressionand induction by xenobiotics of genesthat encodecomponentsof the cytochrome P-450-dependentmonooxygenasesystem along the rat intestinal crypt-villus axis were investigated using isolated epithelial cellsand in situ hybridization. For each geneexamined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IAl mRNA (CYPlAl), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIBl mRNA (CYPZBl), the major phenobarbital-inducible P-450, wereconstitutively expressedin villus cellswith no detectablemRNA present in crypts. Treatment with severalchemical inducers resulted in a marked increasein CYPlAl mRNA in both crypt and villus cells. In contrast, although CYPZBl mRNA was inducible in villus cells,CYPZBl mRNA wasnot detectedin crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes,was expressed constitutively at low levels only in villus cells. Treatment with dexamethasoneinduced reductase mRNA in both crypt and villus cells.Taken together, theseresultsdemonstratethat there is a complex gene-specificpattern of expressionof the microsoma1monooxygenasesystem along the crypt-villus axis of rat small intestine. These findings have implications for transcriptional regulation of CYPlAl and CYP2Bl genesand for metabolism of xenobiotics and procarcinogensin crypt and villus cells. NADPH cytochrome P-450 reductase; cytochrome P-450dependent monooxygenase system; P-450IAl; CYPlAl; P-45011B1; CYPZB 1; in situ hybridization EPITHELIAL LINING of the small intestine is exposed to multiple ingested xenobiotics including plant toxins, pesticides, food additives, industrial chemicals, and pharmaceutical agents. The most intensively studied enzymes that are capable of metabolizing these xenobiotics are the cytochromes P-4501, essential constituents of the microsomal monooxygenase system (18). P-450 proteins are members of a superfamily of related gene products that provide the substrate specificity for the microsomal monooxygenase system (12, 19). The multiple forms of P-450 proteins that may be expressed THE
l Cytochrome P-450 genes in this paper are named according to the recent recommendations for standardization of P-450 gene nomenclature (19). Italicized CYP for cytochrome P-450 refers to the P-450 gene and nonitalicized CYP refers to the P-450 mRNA, cDNA, or protein. CYPlAl and CYP2Bl in rat were previously referred to as P-450IAl or P-450~ and P-450IIBl or P-450b, respectively. 0193-1857/92
in a given tissue allow this system to metabolize many substrates with diverse structures (12, 19). The best characterized P-450 enzymes that are expressed in the mucosa of rat small intestine include CYPlAl (28), the major aromatic hydrocarbon-inducible P-450, CYP2Bl (26, 29), the major phenobarbital-inducible P-450, and CYP3A (3 l), the major glucocorticoid-inducible P-450. These P-450s are capable of metabolizing multiple environmental chemicals and, depending on the form of P-450 and the type of xenobiotic, may either inactivate procarcinogens or convert them to their proximate mutagenic compounds. Furthermore, the synthesis of these P-450 enzymes is induced by a wide variety of compounds that are present in the environment. The cellular localization of P-450 enzymes in the small intestinal mucosa is not well defined. This is an important issue for the functional significance and regulation of P-450 expression, because the intestinal mucosa has a complex architecture with multiple cell types. Intestinal epithelial cells are continuously renewed by proliferation of stem cells located in crypts, migration of daughter cells onto villi, and, finally, extrusion of senescent cells into the intestinal lumen (3, 10). There are four epithelial phenotypes that arise from stem cells including absorptive enterocytes, goblet cells, enteroendocrine cells, and Paneth cells, which, in contrast to the others, reside in the base of crypts. Migration of cells from crypts to villi is coincident with cessation of proliferation and the appearance of the differentiated phenotype that equips the cells to perform specialized tasks. Therefore, the intestinal epithelium is spatially separated into a proliferating undifferentiated compartment (crypts) and a nonproliferating differentiated compartment (villi). The subepithelial tissue contains multiple cell types including fibroblasts, endothelial cells, macrophages, lymphocytes, and eosinophils; in addition, there are significant numbers of intraepithelial lymphocytes in normal mucosa. The cell type and compartment in which P-450 genes are expressed could potentially influence their metabolic role in intestinal function. Several investigators have shown that P-450 enzymatic activities are greater in villus epithelial cells than in crypt cells (6, 13). However, little information is available on the precise localization of specific gene products. Immunohistochemical studies in small intestine and colon have yielded divergent results possibly due to recognition of different epitopes by antibodies (1, 8, 24). The goal of this study was to determine the constitutive pattern of P-450 gene expression in the small intestinal mucosa of rats and to determine the cells in which these genes are induced after administration of known inducing agents. P-450 mRNA transcripts were localized using in situ RNA hybridization in combination
$2.00 Copyright 0 1992 the American Physiological
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with hybridization analysis of RNA extracted from isolated subpopulations of intestinal epithelial cells. We examined the constitutive expression and induction by xenobiotics of three major gene products of the P-450 enzyme system: CYP1A1, CYP2B1, and NADPH cytochrome P-450 reductase, which is essential for activity of all P-450 enzymes. The results indicate that there is a complex gene-specific pattern of expression in epithelial cells lining the crypt-villus axis of rat small intestine. These findings have implications for transcriptional regulation of CYPlAl and CYP2Bl genes in intestine and for metabolism of xenobiotics and procarcinogens in crypt and villus cells. METHODS
Isolation of intestinal epithelial cells. The method of cell isolation used in this study is a modification of the method describedby Weiser (33). We recently reported the validation of this method for the effective separation of villus and crypt populations of intestinal epithelial cells (27). Theseprocedureswere approved by the Animal Use Committee at The University of Michigan. The intestinal washsolution contained 0.15 M NaCl, 1 mM dithiothreitol (DTT), and 40 pg/ml phenylmethylsulfonyl fluoride (PMSF). Buffer A contained (in mM) 96 NaCl, 27 sodiumcitrate, 1.5 KCl, 8 KH2POd, 5.6 Na2HP04, aswell as 40 pg/ml of PMSF (pH 7.4). Buffer B contained (in mM) 109 NaCl, 2.4 KCl, 1.5 KH2P04, 4.3 Na2HP04, 1.5 EDTA, 10 glucose,5 glutamine, 0.5 DTT, aswell as 40 pg/ml PMSF (pH 7.4). Buffers A and B were preoxygenated with 100%0, and warmed to 37°C. Male F344 rats (ZOO-250g), fed standard rat chow and housedin wire-floored cages,were anesthetized with ether and a 20 cm length of jejunum wasremoved starting 10 cm from the pylorus. The length of intestine was rinsed thoroughly with intestinal wash solution and then filled with buffer A; the ends wereclampedand the intestine wasfilled to a pressureof 50 cm of water. The filled intestine wassubmergedin oxygenated 0.15 M NaCl at 37°C for 15 min and then drained and the solution discarded.The intestine was then filled with solution B, incubated at 37”C, and drained. This was repeatedfor 10 cycles at the time intervals of 4, 2, 2, 3, 4, 5, 7, 10, 10, and 10 min; after the 9th and 10th fractions the intestine was flushed with phosphate-buffered saline,pH 7.4. The cellsfrom eachfraction were collectedby pelleting at 100g for 5 min at 4°C and washedonce with phosphate-bufferedsaline. RNA extraction and analysis. Total RNA wasextracted from isolated epithelial cells using the method of Chomczynski and Sacchi (5) as previously described(29). RNA was separatedin 2.2 M formaldehyde, 1% agarosegels,transfered to nylon membranes (Hybond, Amersham) and hybridized and washed as previously described(25,29). The efficiency of RNA transfer to the nylon membraneswas examined by illuminating the membrane with ultraviolet light to visualize the transferred RNA and by restaining the gel after transfer. The Posiblot method of transfer (Stratagene, La Jolla, CA) hasconsistently yielded uniform transfer of RNA (27, 29). Probesusedfor hybridization analysisincluded I) pSBF-1, a CYP2Bl cDNA that was cloned from rat small intestine in our laboratory (29); 2) ~210, a cDNA of the 3’-untranslated portion of the CYPlAl mRNA that was a gift from Dr. J. Fagan [Maharishi Univ., Fairfield, Iowa (7)]: the 762 basepair (bp) insert was excisedwith Pst I and subclonedinto pGEM 42 for usein generating RNA probes (p2lOST); and 3) pSP65-OR, a fulllength P-450 reductase cDNA that was cloned from rat liver, that was a gift from Dr. C. Kasper [Univ. of Wisconsin, Madison, WI (9)]. Digestion of this plasmid with BamH I and Hind
GENE
EXPRESSION
III releasesa 2.4-kb cDNA containing the full coding region. The cDNA inserts were isolated from gelsusing diethylaminoethyl membranesand radiolabeledwith 32P-labeleddeoxycytidine triphosphate using a random-primedoligonucleotidelabeling method as previously described (25, 26). Unincorporated nucleotideswere removed from the labeled DNA using Sephadex G-50 chromatography (Nick columns,Pharmacia, Uppsala, Sweden). In situ hybridization. In situ hybridization wasperformed as previously described(25). Tissue was embeddedin OCT compound (Miles Laboratories) and frozen by immersion in a dry ice-acetone bath. Sections (6-8 pm) were cut using a cryostat and placed on polylysine-treated slides.The sectionswere fixed by sequential immersion in 4% paraformaldehyde (2 min) and ice-cold 70% ethanol (10 min), hydrated through decreasing concentrations of ethanol, treated with 0.2% acetic anhydride in 0.1 M triethanolamine, pH 8.0, and dehydrated through increasing concentrations of ethanol. The sectionswere air dried and immediately hybridized with 1 x lo5 cpm/pl of 35S-labeledRNA probe in a solution containing 50% formamide, 2 x standard sodiumcitrate (SSC), 10 mM DTT, 1 mg/ml nuclease-freeEscherichia coli transfer RNA, 1 mg/ml salmonsperm DNA, and 2 mg/ml nuclease-freebovine serum albumin. The single-strandedRNA probe was synthesized from the DNA template using the protocol recommended by PromegaBiotech (Madison, WI) aspreviously reported (25). To yield an antisenseCYPlAl RNA probe, p2lOST waslinearized with Eco RI and a 762-bp 35S-labeledsingle-strandedRNA probe was synthesized using T7 polymerase.To yield a sense CYPlAl RNA probe, p2lOST was linearized with Hind III and a 762-bp 35S-labeledsingle-strandedRNA probe was synthesized using SP6 polymerase. To yield an antisense CYP2Bl RNA probe, pSBF-1 was linearized with Sea I, and a 310-bp 35S-labeledsingle-strandedRNA probe was synthesized using T7 polymerase.To make the senseCYPBBl, RNA probe pSBF-1 waslinearized with Kpn I and a 380-bp 35S-labeledsinglestranded RNA probe was synthesized using SP6 polymerase. After synthesis, the DNA template was removed by treatment with ribonuclease (RNase)-free deoxyribonuclease (RQl DNase, Promega), extracted with phenol:chloroform, and precipitated with ethanol. The slideswere incubated at 65°C for 16 h. Washeswere performed as follows: I) 50% formamide, 2 x SSC at room temperature, 10 min; 2) 2 X SSC at room temperature, 15 min; 3) RNase A (10 mg/ml) in 0.5 M NaCl, 1 mM EDTA at 37°C for 30 min; 4) 0.5 M NaCl, 1 mM EDTA at 37” C for 30 min; 5) 2 x SSC, 10 mM ,8-mercaptoethanol,55°C for 30 min; and 6) 0.1 x SSC, 10 mM ,&mercaptoethanol,55°C for 30 min, twice. After washing, slides were dehydrated through increasing concentrations of ethanol, dipped in liquid emulsion (Kodak NTB2, diluted 1:l with water), and exposedin light-tight boxes at 4°C. The slideswere developedat intervals and stained with hematoxylin and eosin. RESULTS
Mucosal localization of CYPlAl expression. CYPlAl is an aromatic hydrocarbon-inducible P-450 that is responsible for metabolic activation of a number of important procarcinogens including benz[a]pyrene. The localization of CYPlAl transcripts within the intestinal mucosa was investigated initially using isolated subpopulations of epithelial cells. Rats were treated with a single intraperitoneal dose of 3-methylcholanthrene (20 mg/kg body wt, in corn oil vehicle) or corn oil alone (control), and the animals were killed 16 h later. The intraperitoneal route was chosen to ensure that cells in intestinal
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INTESTINAL
P-450 GENE EXPRESSION
crypts would be exposed to the inducer. Intestinal epithelial cells were isolated from individual rats and extracted RNA analyzed for expression of CYPlAl. The results of three control and two 3-methylcholanthrene-treated rats were the same, and, therefore, a representative Northern blot from one animal is presented (Fig. 1). In control animals, CYPlAl mRNA was expressed predominantly in villus cell fractions with very little found in crypt fractions (Fig. 1). In contrast, 3-methylcholanthrene-treated animals showed induction of CYPlAl mRNA in both villus and crypt cell fractions, which appeared to equalize the levels of mRNA in each subpopulation of cells. When Aroclor 1254, a mixture of polychlorinated biphenyls, was used as the inducer similar results were obtained (Fig. 5, described below). These results suggested that CYPlAl mRNA was expressed constitutively in villus cells but was inducible in both villus and crypt cells. In situ hybridization was performed on jejunal tissue sections for comparison to the results obtained from isoCONTROL FRACl NUMB
3-MC /213/415/617/81
9 I10
1
o/P1 Al
Fig. 1. CYPlAl mRNA in isolated subpopulations of intestinal epithelial cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells as described in METHODS. Fraction l/2 represents cells predominantly from villus tips and fraction 10 is last fraction and represents primarily crypt cells. Controls were administered corn oil (1 ml ip), and treated rats were given 3-methylcholanthrene (20 mg/kg in 1 ml of corn oil ip) 16 h before death. Analysis of 1 representative control and 1 treated animal is shown. Top: ethidium bromide-stained agarose gel. Ultraviolet illumination of nylon membrane and restaining of gel confirmed adequacy of RNA transfer to membrane. Middle: autoradiogram of Northern blot that was hybridized with 32P-labeled cDNA isolated from p21OST (8 h exposure). Bottom: graphic representation of densitometric analysis of Northern blot. Densitometry of both the 28s ribosomal band on photograph of ethidium-stained gel and the autoradiograph was performed using a Zeiss digital image analysis system. Ratio of the density of the band from the autoradiograph to that of the 28s band was used to compare the amount of mRNA between samples.
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lated epithelial cells. This was necessary for two reasons. First, we recently observed for sucrase-isomaltase mRNA that fine detail of mRNA expression along the cryptvillus axis may be obscured because of overlap between isolated cell fractions (25, 30). Second, the effect of inducer treatment on the isolation of intestinal epithelial cells was unknown. Multiple tissue sections from animals treated with either 3-methylcholanthrene or Aroclor 1254 were studied. In control animals, CYPlAl mRNA was expressed in epithelial cells located exclusively on the villus (Fig. 2, A-C). Figure 2, A and B, shows low-power light and dark field views, respectively, of a cross-section of rat jejunum. Autoradiographic grains were concentrated over epithelial cells located on the villus. A higher power view of one region of the section demonstrated that there were no grains over background in crypt cells with appearance of grains at the crypt-villus junction (Fig. 2C). Hybridization of serial sections with a sense-strand probe did not show grains over epithelial cells, which confirmed the specificity of results obtained with the antisense probe (Fig. 20). In addition, prehybridization with loo-fold excess of unlabeled antisense RNA effectively blocked hybridization of the labeled antisense probe to epithelial cells (data not shown). Taken together with the findings in isolated epithelial cells, these results suggest that constitutive CYPlAl gene transcription is activated as cells move from the crypt to the villus. This is similar to the pattern observed for other genes expressed in the small intestine of rats (15, 20, 25). After treatment with inducers there was marked induction of CYPlAl mRNA in crypt cells (Fig. 2, E-G). The low-power views demonstrate the dramatic induction in crypts compared with control sections (Fig. 2, E and F). A high-power view of the crypt region demonstrates the intense pattern of grains overlying the crypt cells (Fig. 2G); cells at the very base of the crypts, which may represent Paneth cells, had fewer grains. As for the sections from control animals, use of the sense probe did not result in grains over epithelial cells demonstrating specificity of hybridization with the antisense probe (Fig. 2H). Several crypt-villus units of a treated animal are shown in Fig. 2, I and J. These photomicrographs demonstrate the expression of CYPlAl mRNA in both crypt and villus cells and allow direct comparison of the intensity of grain density in both compartments. Although not quantified, the density of grains over crypts appears to be greater than that over villi, which is somewhat of a different impression than was obtained from Northern blot analysis of isolated subpopulations of epithelial cells, which suggested that the levels were similar in crypt and villus compartments (Fig. 1). Previous studies from our laboratory have shown that fine detail of mRNA localization determined by in situ hybridization is lost when analyzing isolated subpopulations of cells (25, 27, 30). This is most likely due to intermixing of villus and crypt cells in the middle cell fractions (27). However, the overall pattern of expression in crypt and villus compartments as determined by in situ hybridization confirms the pattern of CYPlAl gene expression along the crypt-villus axis that was suggested by Northern blot analysis of isolated
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Fig. 2. In situ hybridization analysis of rat jejunum for CYPlAl mRNA. Jejunal sections were prepared from control (corn oil 1 ml ip) and Aroclor 1254-treated rate (300 mg/kg in corn oil ip) and in situ hybridization performed as described in METHODS. Each slide was developed 2 days after application of radiographic emulsion and then stained with hematoxylin and eosin. The photographs shown represent examples taken from multiple sections of 2 control and 2 Aroclortreated animals. A: jejunal section from control rat hybridized with antisense CYPlAl RNA-probe. B: dark field microscopic view of field seen in A. C: higher vower view of a vortion of section shown in A. D: jejunalsection from control rat hybridized with sense CYPlAl RNA probe. E: ieiunal section from rat treated with Aroclor 1254 hybridized with antisense CYPlAl RNA probe. F: dark field microscopic view of field seen in E. G: higher power view of a portion of section shown in E. H: jejunal section from rat treated with Aroclor 1254 hvbridized with sense CYPlAl RNA probe.“l: jejunal section from a rat treated with Aroclor 1254 hybridized with antisense CYPlAl RNA probe. Bright field microscopic view. J: dark field microscopic view of field seen in I.
epithelial cells. Cellular localization of CYP2Bl expression. The pattern of CYP2Bl mRNA expression was examined using both isolated subpopulations of cells and in situ hybridization as described above for CYPlAl. Rats were treated with a single intraperitoneal dose of phenobarbital (80 mg/kg body wt), a potent inducer of CYP2Bl gene transcription in small intestine (29), and controls were given an intraperitoneal injection of 0.15 M NaCl; the animals were killed 16 h later. Northern analysis showed that CYP2Bl mRNA was constitutively expressed in villus epithelial cells with an apparent linear decrease in mRNA levels in successive cell fractions (Fig. 3). Treatment with phenobarbital produced induction of CYP2Bl mRNA, but the relative pattern of expression along the crypt-villus axis appeared to be maintained (Fig. 3). Identical results were obtained in two separate control and two phenobarbital-treated animals. In situ hybridization analysis demonstrated that control animals expressed CYP2Bl mRNA in villus tip cells with little mRNA in lower villus cells and none in crypt cells (Fig. 4, A and B). After phenobarbital treatment, CYP2Bl mRNA was induced in villus cells and was now detectable in lower villus cells; however, there remained no detectable mRNA in crypt cells (Fig. 4, C and D). Therefore, the results of isolated epithelial cells and in situ hybridization suggest that treatment with phenobarbital induces CYP2Bl mRNA only in villus enterocytes with little effect on crypt cells. Induction of both CYPlAl and CYP2Bl with a single agent. One possible explanation for differential induction of CYPlAl and CYP2Bl along the crypt-villus axis would be differential exposure of crypt cells to 3-methyl-
cholanthrene and phenobarbital, respectively. Therefore, animals were treated with Aroclor 1254, a mixture of polychlorinated biphenyls, which is a known inducer of both CYPlAl and CYP2Bl in small intestine (26). A Northern blot was sequentially hybridized with cDNA probes for both P-450s (Fig. 5). This analysis demonstrated that the pattern of expression in control rats was identical for CYPlAl and CYP2Bl mRNA. However, treatment with Aroclor 1254 induced expression of CYPlAl mRNA in crypt cell fractions, whereas there was no induction of CYP2Bl mRNA in the same cell fractions. These results suggest that the differential induction of P-450s in crypt cells is due to intrinsic differences in the ability of the cells to respond to inducers rather than different accessibility to the inducing chemicals. Expression of NADPH cytochrome P-450 reductase. The enzymatic activity of each P-450 enzyme is dependent on the presence of NADPH cytochrome P-450 reductase. Therefore, we examined the expression of NADPH cytochrome P-450 reductase mRNA in cell fractions of control animals and those treated with dexamethasone (300 mg/kg). Very low levels of mRNA were found in control intestine with the majority expressed in villus tip cells (Fig. 6). After treatment with dexamethasone, there was marked induction of NADPH cytochrome P-450 reductase mRNA in both villus and crypt cell compartments (Fig. 6). DISCUSSION
These studies reveal a complex pattern of expression along the rat intestinal crypt-villus axis for genes that encode components of the microsomal monooxygenase
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PB
NUMBER
CYP2Bl
Fig. 3. CYPZBl mRNA in isolated subpopulations of intestinal epitheha1 cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells that were obtained as described in METHODS. Control animals were administered saline and treated animals were given phenobarbital (80 mg/kg) 16 h before death. Analysis of 1 representative control and 1 treated animal is shown. Top: ethidium bromide-stained agarose gel. Ultraviolet illumination of nylon membrane and restaining of gel confirmed adequacy of RNA transfer to membrane. Middle: autoradiogram of Northern blot that was hybridized with 32P-labeled cDNA isolated from pSBF-1 (6 h exposure). Bottom: graphic representation of densitometric analysis of Northern blot. Autoradiographs were quantified as described in Fig. 1, legend.
system. We chose to examine levels of mRNA along the crypt-villus axis as a measure of gene expression for two reasons. First, molecular hybridization methods represent the most definitive way to identify which P-450 genes are expressed in a given tissue. Enzymatic activities for P-450 enzymes have overlapping substrate specificities (ll), and there may be differences in specificity for the same enzyme in different tissues (22). Identification of P-450 proteins using antibodies has yielded conflicting results possibly because of cross-reactivity to homologous forms of P-450s. Second, we have previously shown that CYPlAl and CYP2Bl genes are regulated at the level of mRNA abundance in small intestine after treatment with inducers, predominantly by changes in gene transcription (28, 29). Therefore, analysis of mRNA levels provides information on the mechanisms for regulation along the crypt-villus axis. The molecular probes used in this study are specific for identification of CYPlAl and CYP2Bl mRNA in small intestine. We previously showed that CYP2Bl is the only member of this family expressed in small intestine by amplifying cDNA using the polymerase chain reaction and sequencing of the product (29). The probe used for CYPlAl was shown by Fagan et al. (7) to differentiate between CYPlAl and the only other mem-
c
i
Fig. 4. In situ hybridization analysis of rat jejunum for CYPBBl mRNA. Jejunal sections were prepared from control (1 ml 0.15 M NaCl ip) and phenobarbital-treated rats (80 mg/kg ip) and in situ hybridization performed as described in METHODS. Photographs shown represent examples taken from multiple sections of 2 control and 2 phenobarbitaltreated animals. Each slide was developed 2 days after application of radiographic emulsion and then stained with hematoxylin and eosin. Jejunal sections from control and treated rats hybridized with sense CYPBBl RNA probe did not show detectable signal over background (data not shown). A: jejunal section from control rat hybridized with antisense CYPBBl RNA probe. B: dark field view of field shown in A. C: jejunal section from a phenobarbital-treated rat hybridized with antisense CYP2Bl RNA probe. D: dark field view of field shown in C.
ber of this subfamily, CYPlA2. CYPlAl was constitutively expressed in epithelial cells located on the villus and was absent in crypt and submucosal cells. The epithelial cells that express the mRNA appear to be predominantly absorptive enterocytes although the resolution of the in situ hybridization method did not allow the definitive identification of goblet and enteroendocrine cells. CYPlAl mRNA was induced in both villus and crypt cells. Because these experiments
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AROCLOR
CONTROL
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1Al 2.0 -
FRACTION NUMBER
Fig. 5. CYPlAl and CYP2Bl mRNA in isolated intestinal epithelial cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells as described in METHODS. Control animal was administered corn oil (1 ml ip), and treated animal was given Aroclor 1254 (300 mg/kg ip) 16 h before death. Top: ethidium bromide-stained agarose gel. Middle: autoradiograms of Northern blots that were hybridized sequentially with labeled cDNAs isolated from pSBF-1 (3 h exposure) and p21OST (12 h exposure), respectively. Bottom: graphic representation of densitometric analysis of Northern blot. Autoradiographs were quantified as described in Fig. 1, legend.
evaluated steady-state levels of CYPlAl mRNA, induction could represent either increased gene transcription, diminished mRNA degradation, or both. We have previously shown that induction of CYPlAl mRNA in small intestine occurs predominantly by an increase in gene transcription (28). Because those studies used nuclei isolated from scraped mucosa, which only partially removes crypts (26), this was felt to be primarily a measure of transcription in villus cells. The results of in situ hybridization revealing absence of CYPlAl mRNA in the crypts of control intestine and abundant mRNA in treated intestine suggests that activation of transcription also occurs in crypt cells. Induction of CYPlAl is considered a marker for the presence of the aromatic hydrocarbon (Ah) receptor (16, 21). The Ah receptor is a cytosolic protein that is translocated to the nucleus on binding of a ligand where it activates transcription of the CYPlAl gene via binding to DNA regulatory elements (34). Therefore, our data
Fig. 6. NADPH cytochrome P-450 reductase mRNA in isolated intestinal epithelial cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells as described in METHODS. Control animal was administered corn oil (1 ml ip) and treated animal was given dexamethasone (300 mg/kg ip) 16 h before death. Top: ethidium bromide-stained agarose gel. Middle: autoradiogram of Northern blot that was hybridized with 32P-labeled cDNA isolated from pSP450-OR (14 h exposure). Bottom: graphic representation of densitometric analysis of Northern blot. Autoradiographs were quantified as described in Fig. 1, legend.
suggest that the factors required for transcriptional activation of the CYPlAl gene including the Ah receptor (34) and the recently described arnt protein (14, 16) are present in all cells along the crypt-villus axis. Whether these factors are constitutively expressed in both crypt and villus compartments or are inducible in response to xenobiotics will require examination of the proteins or their respective mRNAs once the reagents necessary for these experiments become available. Differences in the degree of induction between crypt and villus compartments may be related to multiple factors including different levels of inducers, transcriptional regulatory proteins, or rates of mRNA degradation. The presence of the Ah receptor in intestinal epithelial cells may have implications beyond those of xenobiotic metabolism. The Ah receptor is responsible for the induction of a battery of genes (18) and may play a role in control of cellular growth and differentiation (17). It was recently shown that 2,3,4,8-tetrachlorodibenzo-p-dioxin, a compound that binds avidly to the Ah receptor, causes proliferation of keratinocytes via synthesis and secretion of transforming growth factor (TGF)-a which acts as an autocrine growth factor (4). Furthermore, TGF-a! and TGF-/3 may be involved in the regulation of intestinal
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growth and differentiation (2, 23). The role of Ah receptor and xenobiotics in the regulation of intestinal growth requires investigation. CYP2Bl mRNA was expressed constitutively and was inducible only in cells located on villi. As for CYPl Al, our laboratory has previously shown that transcriptional activation of the CYP2Bl gene is responsible for induction of mRNA in small intestine (29). These data suggest several possibilities for the regulation of CYP2Bl gene transcription in the small intestine including I) the transcription factors that mediate induction by xenobiotics are not present in crypt cells; 2) the factors necessary for basal transcription of the gene are not present in crypt cells and, therefore, factors involved in induction are ineffective; or 3) there are repressors in proliferating crypt cells that prevent transcription of the gene. There is presently no information on the factors that regulate basal or induced transcription of the CYP2Bl gene. Differences in crypt and villus cells may be exploited to investigate these mechanisms. An intriguing finding of this study was the pattern of expression of NADPH cytochrome P-450 reductase mRNA along the crypt-villus axis. Undetectable levels of reductase in the crypt cells raise the possibility that CYPlAl expressed in the crypt after induction may not be enzymatically active. Furthermore, if levels of reductase are rate-limiting for enzymatic activity in control animals, it is possible that induction of the reductase alone may increase P-450 enzyme activity. This would be a novel situation, because NADPH cytochrome P-450 reductase in liver is felt to be in excess of P-450 proteins such that the level of P-450 protein determines enzymatic activity. Quantitative histochemical measurements must be developed to accurately identify cells that contain monooxygenase activity in order to investigate this possibility and to determine if differential induction of P-450 genes in crypt and villus compartments lead to differences in metabolic capacity. Previous histochemical localization of benz [ a] pyrene hydroxylase activity in small intestine (32) suggests that quantitative analysis of this type may be possible. Finally, it will be important to investigate the effect of inducers of P-450 genes on the expression of reductase mRNA and enzymatic activity. Taken together, our data demonstrate that the CYPlAl, CYP2B1, and NADPH cytochrome P-450 reductase genes are differentially regulated along the intestinal crypt-villus axis. This results in a gene-specific pattern of induction in crypt and villus cell compartments. Therefore, transcription factors required for induction of certain members of this gene family are present in undifferentiated crypt cells, and certain other factors are expressed only in more differentiated cells located on villi. Investigation of the transcriptional regulation of CYHAl and CYP2Bl genes in crypt and villus cells may allow elucidation of the molecular mechanisms that control basal and induced transcription in intestine. Differences in the complement of P-450 enzymes or the activity of the monooxygenase system in crypt and villus cell compartments may have profound effects on the fate of ingested xenobiotics and procarcinogens. This studv was supnorted bv National
Institute
of Diabetes and
GENE EXPRESSION Digestive and Kidney Diseases Grant DK-41393 and a Veteran’s Affairs Research Associate Award and Merit Review (P. G. Traber). This study was presented in part as an abstract (28) and at the annual meeting of The American Gastroenterological Association, San Antonio, TX, 1990. Address for reprint requests: P. G. Traber, 6520a MSRB-1, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0682. Received 4 December 1991; accepted in final form 31 March 1992. REFERENCES 1. Anderson, Junker,
L. M., J. M. Ward, S. S. Park, A. B. Jones, J. L. H. V. Gelboin, and J. M Rice. Immunohistochemical
determination of inducibility phenotype with a monoclonal antibody to a methylcholanthrene-inducible isozyme of cytochrome P-450. Cancer Res. 47: 6079-6085, 1987. 2. Barnard, Moses.
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Regulation of intestinal epithelial cell growth by transforming growth factor type B. Proc. Natl. Acad. Sci. USA 86: 1578-1582, 1989. 3. Cheng, H., and C. P. Leblond. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat. 141: 537-562, 1974. 4. Choi, E. J., D. G. Toscano, J. A. Ryan, N. Riedel, and W. A. Toscano. Dioxin induces transforming growth factor-a in human keratinocytes. J. Biol. Chem. 266: 9591-9597, 1991. 5. Chomczynski, P., and N. Sacchi. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform traction. Anal. Biochem. 162: 156-159, 1987.
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6. Dawson, J. R., and M. Schwenk. Isolated epithelial cells. In: Progress in Pharmacology and Clinical Pharmacology: Intestinal Metabolism of Xenobiotics, edited by A. Sj. Koster, E. Richter,
F. Lauterbach, and F. Hartmann. lag, 1989, p. 21-41.
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7. Fagan,
J. B., J. V. Pastewka, S. C. Chalberg, E. Gozukara, F. P. Guengerich, and H. V. Gelboin. Noncoordinate regula-
tion of the mRNAs encoding cytochromes P-450BNF/MC-B P-450ISF/BNF-G. Arch. Biochem. Biophys. 244: 261-272, 8. Foster, Sesardic, A. Lock.
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J. R., C. R. Elcombe, A. R. Boobis, D. S. Davies, J. McQuade, R. T. Robson, C. Hayward, and
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Immunocytochemical localization of cytochrome P450 in hepatic and extra-hepatic tissues of the rat with a monoclonal antibody against cytochrome P45Oc. Biochem. Pharmacol. 35: 4543-4554, 1986. Gonzalez, F. J., and C. B. Kasper. Cloning of DNA complementary to rat liver NADPH-cytochrome c(P-450) oxidoreductase and cytochrome P-450b mRNAs. Evidence that phenobarbital augments transcription of specific genes. J. Biol. Chem. 257: 59625968, 1982. Gordon, J. I. Intestinal epithelial differentiation: new insights from chimeric and transgenic mice. J CeZLBiol 108: 1187-1194, 1989. Guengerich, F. P. Enzymology of rat liver cytochromes P-450. In: Critical Reviews in Biochemistry, The Mammalian Cytochromes P450, edited by F. P. Guengerich. Boca Raton, FL: CRC, 1987, p. l-54. Guengerich, F. P. Reactions and significance of cytochrome P-450 enzymes. J. Biol. Chem. 266: 10019-10022, 1991.
H., C. H. Woo, S. B. Raffin, and R. Schmid. Ox13. Hoensch, idative metabolism of foreign compounds in rat small intestine: cellular localization and dependence in dietary iron. Gastroenterology
70:1063-1070,1976. E. C., H. Conley, B. A. Brooks,
14. Hoffman,
Reyes, F. OF. Chu, and 0. Hankinson.
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Cloning of a factor receptor. Science Wash.
required for activity of the Ah (dioxin) DC 252: 954-958, 1991. S., and H. Kondo. Light microscopic localization of he15. Iseki, patic fatty acid binding protein mRNA in jejunal epithelia of rats using in situ hybridization, immunohistochemical, and autoradiographic techniques. J. Hktochem. Cytochem. 38: 111-115, 1990. E. F. A partnership between the dioxin receptor and a 16. Johnson, basic helix-loop-helix proteins. Science Wash. DC 252: 924-925, 1991.
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17. Nebert, D. W. Growth Signal Pathways. Nature Lond. 347: 709710, 1990. 18. Nebert, D. W., and F. J. Gonzalez. P45O genes: structure, evolution, and regulation. Annu. Rev. Biochem. 56: 945-993, 1987. 19. Nebert, D. W., D. R. Nelson, M. J. Coon, R. W. Estabrook, R. Feyereisen, Y. Fujii-Kuriyama, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, J. C. Loper, R. Sato, M. R. Waterman, and D. J. Waxman. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 10: 1-14, 1991. 20. Noren, O., E. Dabelsteen, P. E. Hoyer, J. Olsen, H. Sjostrom, and G. H. Hansen. Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation. FEBS L&t. 259: 107-112, 1989. 21. Poland, A., and J. C. Knutson. 2,3,7,8-tetrachlorodibenzo-pdioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22: 517-554, 1982. 22. Sesardic, D., K. J. Cole, R. J. Edwards, D. S. Davies, P. E. Thomas, W. Levin, and A. R. Boobis. The inducibility and catalytic activity of cytochromes P45Oc (P450IAl) and P450d (P450IA2) in rat tissues. Biochem. Pharmacol. 39: 499-506, 1990. 23. Suemori, S., C. Ciacci, and D. K. Podolsky. Regulation of transforming growth factor expression in rat intestinal epithelial celllines. J. Clin. Invest. 87: 2216-2221, 1991. 24. Tamura, S., S. Kawata, M. Okamoto, and S. Tarui. Localization of cytochrome P-450 in the colonic mucosa of S-methylcholanthrene-pretreated and untreated rats. Cell Tissue Res. 252: 397-401, 1988. 25. Traber, P. G. Regulation of sucrase-isomaltase gene expression along the crypt-villus axis of rat small intestine. Biochem. Biophys. Res. Commun. 173: 765-773, 1990. 26. Traber, P. G., J. Chianale, R. Florence, K. Kim, E. Wojcik, and J. J. Gumucio. Expression of cytochrome P45Ob and P450e
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genes in small intestinal mucosa of rats following treatment with phenobarbital, polyhalogenated biphenyls, and organochlorine pesticides. J. Biol. Chem. 263: 9449-9455, 1988. 27. Traber, P. G., D. L. Gumucio, and W. Wang. Isolation of intestinal epithelial cells for the study of differential gene expression along the crypt-villus axis. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G895-G903, 1991. 28. Traber, P. G., W. M. McDonnell, W. Wang, and R. Florence. Expression and transcriptional regulation of P4501 genes in the rat alimentary tract (Abstract). Gastroenterology 98: A316, 1990. 29. Traber, P. G., W. Wang, M. McDonnell, and J. Gumucio. P450IIB gene expression in rat small intestine: cloning of intestinal P450IIBl mRNA using the polymerase chain reaction and transcriptional regulation of induction. Mol. Pharmacol. 37: 810819, 1990.
30 Traber, P. G., L. Yu, G. Wu, and T. Judge. Sucrase-isomaltase gene expression along the crypt-villus axis of human small intestine is regulated at the level of mRNA abundance. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G123-GMO, 1992. 31. Watkins, P. B., S. A. Wrighton, E. G. Schuetz, D. T. Molowa, and P. S. Guzelian. Identification of glucocorticoidinducible cytochromes P-450 in the intestinal mucosa of rats and man. J. Clin. Invest. 80: 1029-1036, 1987. 32. Wattenberg, L. W., J. L. Leong, and P. J. Strand. Benzpyrene hydroxylase activity in the gastrointestinal tract. Cancer Res. 22: 1120-1125, 1962. 33. Weiser, M. M. Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J. Biol. Chem. 248: 2536-2541, 1973. 34. Whitlock, J. P. Genetic and molecular aspects of 2,3 7,8-tetrachlorodibenzo-p-dioxin action. Annu. Rev. Pharmacol. Toxicol. 30: 251-277, 1990.
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G. TRABER,
WE1 WANG,
P-450 genes
AND LA1 YU
Division of Gastroenterology and the Department of Internal Medicine, University of Michigan Medical School and the Veterans Affairs Medical Center, Ann Arbor, Michigan 48109 Traber, Peter G., Wei Wang, and Lai Yu. Differential regulation of cytochrome P-450 genesalong rat intestinal cryptvillus axis. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G215-G223, 1992.-Mammalian small intestine contains cytochrome P-450-dependentmonooxygenaseenzymesthat are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cellslining the smallintestine are separatedinto a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expressionand induction by xenobiotics of genesthat encodecomponentsof the cytochrome P-450-dependentmonooxygenasesystem along the rat intestinal crypt-villus axis were investigated using isolated epithelial cellsand in situ hybridization. For each geneexamined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IAl mRNA (CYPlAl), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIBl mRNA (CYPZBl), the major phenobarbital-inducible P-450, wereconstitutively expressedin villus cellswith no detectablemRNA present in crypts. Treatment with severalchemical inducers resulted in a marked increasein CYPlAl mRNA in both crypt and villus cells. In contrast, although CYPZBl mRNA was inducible in villus cells,CYPZBl mRNA wasnot detectedin crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes,was expressed constitutively at low levels only in villus cells. Treatment with dexamethasoneinduced reductase mRNA in both crypt and villus cells.Taken together, theseresultsdemonstratethat there is a complex gene-specificpattern of expressionof the microsoma1monooxygenasesystem along the crypt-villus axis of rat small intestine. These findings have implications for transcriptional regulation of CYPlAl and CYP2Bl genesand for metabolism of xenobiotics and procarcinogensin crypt and villus cells. NADPH cytochrome P-450 reductase; cytochrome P-450dependent monooxygenase system; P-450IAl; CYPlAl; P-45011B1; CYPZB 1; in situ hybridization EPITHELIAL LINING of the small intestine is exposed to multiple ingested xenobiotics including plant toxins, pesticides, food additives, industrial chemicals, and pharmaceutical agents. The most intensively studied enzymes that are capable of metabolizing these xenobiotics are the cytochromes P-4501, essential constituents of the microsomal monooxygenase system (18). P-450 proteins are members of a superfamily of related gene products that provide the substrate specificity for the microsomal monooxygenase system (12, 19). The multiple forms of P-450 proteins that may be expressed THE
l Cytochrome P-450 genes in this paper are named according to the recent recommendations for standardization of P-450 gene nomenclature (19). Italicized CYP for cytochrome P-450 refers to the P-450 gene and nonitalicized CYP refers to the P-450 mRNA, cDNA, or protein. CYPlAl and CYP2Bl in rat were previously referred to as P-450IAl or P-450~ and P-450IIBl or P-450b, respectively. 0193-1857/92
in a given tissue allow this system to metabolize many substrates with diverse structures (12, 19). The best characterized P-450 enzymes that are expressed in the mucosa of rat small intestine include CYPlAl (28), the major aromatic hydrocarbon-inducible P-450, CYP2Bl (26, 29), the major phenobarbital-inducible P-450, and CYP3A (3 l), the major glucocorticoid-inducible P-450. These P-450s are capable of metabolizing multiple environmental chemicals and, depending on the form of P-450 and the type of xenobiotic, may either inactivate procarcinogens or convert them to their proximate mutagenic compounds. Furthermore, the synthesis of these P-450 enzymes is induced by a wide variety of compounds that are present in the environment. The cellular localization of P-450 enzymes in the small intestinal mucosa is not well defined. This is an important issue for the functional significance and regulation of P-450 expression, because the intestinal mucosa has a complex architecture with multiple cell types. Intestinal epithelial cells are continuously renewed by proliferation of stem cells located in crypts, migration of daughter cells onto villi, and, finally, extrusion of senescent cells into the intestinal lumen (3, 10). There are four epithelial phenotypes that arise from stem cells including absorptive enterocytes, goblet cells, enteroendocrine cells, and Paneth cells, which, in contrast to the others, reside in the base of crypts. Migration of cells from crypts to villi is coincident with cessation of proliferation and the appearance of the differentiated phenotype that equips the cells to perform specialized tasks. Therefore, the intestinal epithelium is spatially separated into a proliferating undifferentiated compartment (crypts) and a nonproliferating differentiated compartment (villi). The subepithelial tissue contains multiple cell types including fibroblasts, endothelial cells, macrophages, lymphocytes, and eosinophils; in addition, there are significant numbers of intraepithelial lymphocytes in normal mucosa. The cell type and compartment in which P-450 genes are expressed could potentially influence their metabolic role in intestinal function. Several investigators have shown that P-450 enzymatic activities are greater in villus epithelial cells than in crypt cells (6, 13). However, little information is available on the precise localization of specific gene products. Immunohistochemical studies in small intestine and colon have yielded divergent results possibly due to recognition of different epitopes by antibodies (1, 8, 24). The goal of this study was to determine the constitutive pattern of P-450 gene expression in the small intestinal mucosa of rats and to determine the cells in which these genes are induced after administration of known inducing agents. P-450 mRNA transcripts were localized using in situ RNA hybridization in combination
$2.00 Copyright 0 1992 the American Physiological
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with hybridization analysis of RNA extracted from isolated subpopulations of intestinal epithelial cells. We examined the constitutive expression and induction by xenobiotics of three major gene products of the P-450 enzyme system: CYP1A1, CYP2B1, and NADPH cytochrome P-450 reductase, which is essential for activity of all P-450 enzymes. The results indicate that there is a complex gene-specific pattern of expression in epithelial cells lining the crypt-villus axis of rat small intestine. These findings have implications for transcriptional regulation of CYPlAl and CYP2Bl genes in intestine and for metabolism of xenobiotics and procarcinogens in crypt and villus cells. METHODS
Isolation of intestinal epithelial cells. The method of cell isolation used in this study is a modification of the method describedby Weiser (33). We recently reported the validation of this method for the effective separation of villus and crypt populations of intestinal epithelial cells (27). Theseprocedureswere approved by the Animal Use Committee at The University of Michigan. The intestinal washsolution contained 0.15 M NaCl, 1 mM dithiothreitol (DTT), and 40 pg/ml phenylmethylsulfonyl fluoride (PMSF). Buffer A contained (in mM) 96 NaCl, 27 sodiumcitrate, 1.5 KCl, 8 KH2POd, 5.6 Na2HP04, aswell as 40 pg/ml of PMSF (pH 7.4). Buffer B contained (in mM) 109 NaCl, 2.4 KCl, 1.5 KH2P04, 4.3 Na2HP04, 1.5 EDTA, 10 glucose,5 glutamine, 0.5 DTT, aswell as 40 pg/ml PMSF (pH 7.4). Buffers A and B were preoxygenated with 100%0, and warmed to 37°C. Male F344 rats (ZOO-250g), fed standard rat chow and housedin wire-floored cages,were anesthetized with ether and a 20 cm length of jejunum wasremoved starting 10 cm from the pylorus. The length of intestine was rinsed thoroughly with intestinal wash solution and then filled with buffer A; the ends wereclampedand the intestine wasfilled to a pressureof 50 cm of water. The filled intestine wassubmergedin oxygenated 0.15 M NaCl at 37°C for 15 min and then drained and the solution discarded.The intestine was then filled with solution B, incubated at 37”C, and drained. This was repeatedfor 10 cycles at the time intervals of 4, 2, 2, 3, 4, 5, 7, 10, 10, and 10 min; after the 9th and 10th fractions the intestine was flushed with phosphate-buffered saline,pH 7.4. The cellsfrom eachfraction were collectedby pelleting at 100g for 5 min at 4°C and washedonce with phosphate-bufferedsaline. RNA extraction and analysis. Total RNA wasextracted from isolated epithelial cells using the method of Chomczynski and Sacchi (5) as previously described(29). RNA was separatedin 2.2 M formaldehyde, 1% agarosegels,transfered to nylon membranes (Hybond, Amersham) and hybridized and washed as previously described(25,29). The efficiency of RNA transfer to the nylon membraneswas examined by illuminating the membrane with ultraviolet light to visualize the transferred RNA and by restaining the gel after transfer. The Posiblot method of transfer (Stratagene, La Jolla, CA) hasconsistently yielded uniform transfer of RNA (27, 29). Probesusedfor hybridization analysisincluded I) pSBF-1, a CYP2Bl cDNA that was cloned from rat small intestine in our laboratory (29); 2) ~210, a cDNA of the 3’-untranslated portion of the CYPlAl mRNA that was a gift from Dr. J. Fagan [Maharishi Univ., Fairfield, Iowa (7)]: the 762 basepair (bp) insert was excisedwith Pst I and subclonedinto pGEM 42 for usein generating RNA probes (p2lOST); and 3) pSP65-OR, a fulllength P-450 reductase cDNA that was cloned from rat liver, that was a gift from Dr. C. Kasper [Univ. of Wisconsin, Madison, WI (9)]. Digestion of this plasmid with BamH I and Hind
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EXPRESSION
III releasesa 2.4-kb cDNA containing the full coding region. The cDNA inserts were isolated from gelsusing diethylaminoethyl membranesand radiolabeledwith 32P-labeleddeoxycytidine triphosphate using a random-primedoligonucleotidelabeling method as previously described (25, 26). Unincorporated nucleotideswere removed from the labeled DNA using Sephadex G-50 chromatography (Nick columns,Pharmacia, Uppsala, Sweden). In situ hybridization. In situ hybridization wasperformed as previously described(25). Tissue was embeddedin OCT compound (Miles Laboratories) and frozen by immersion in a dry ice-acetone bath. Sections (6-8 pm) were cut using a cryostat and placed on polylysine-treated slides.The sectionswere fixed by sequential immersion in 4% paraformaldehyde (2 min) and ice-cold 70% ethanol (10 min), hydrated through decreasing concentrations of ethanol, treated with 0.2% acetic anhydride in 0.1 M triethanolamine, pH 8.0, and dehydrated through increasing concentrations of ethanol. The sectionswere air dried and immediately hybridized with 1 x lo5 cpm/pl of 35S-labeledRNA probe in a solution containing 50% formamide, 2 x standard sodiumcitrate (SSC), 10 mM DTT, 1 mg/ml nuclease-freeEscherichia coli transfer RNA, 1 mg/ml salmonsperm DNA, and 2 mg/ml nuclease-freebovine serum albumin. The single-strandedRNA probe was synthesized from the DNA template using the protocol recommended by PromegaBiotech (Madison, WI) aspreviously reported (25). To yield an antisenseCYPlAl RNA probe, p2lOST waslinearized with Eco RI and a 762-bp 35S-labeledsingle-strandedRNA probe was synthesized using T7 polymerase.To yield a sense CYPlAl RNA probe, p2lOST was linearized with Hind III and a 762-bp 35S-labeledsingle-strandedRNA probe was synthesized using SP6 polymerase. To yield an antisense CYP2Bl RNA probe, pSBF-1 was linearized with Sea I, and a 310-bp 35S-labeledsingle-strandedRNA probe was synthesized using T7 polymerase.To make the senseCYPBBl, RNA probe pSBF-1 waslinearized with Kpn I and a 380-bp 35S-labeledsinglestranded RNA probe was synthesized using SP6 polymerase. After synthesis, the DNA template was removed by treatment with ribonuclease (RNase)-free deoxyribonuclease (RQl DNase, Promega), extracted with phenol:chloroform, and precipitated with ethanol. The slideswere incubated at 65°C for 16 h. Washeswere performed as follows: I) 50% formamide, 2 x SSC at room temperature, 10 min; 2) 2 X SSC at room temperature, 15 min; 3) RNase A (10 mg/ml) in 0.5 M NaCl, 1 mM EDTA at 37°C for 30 min; 4) 0.5 M NaCl, 1 mM EDTA at 37” C for 30 min; 5) 2 x SSC, 10 mM ,8-mercaptoethanol,55°C for 30 min; and 6) 0.1 x SSC, 10 mM ,&mercaptoethanol,55°C for 30 min, twice. After washing, slides were dehydrated through increasing concentrations of ethanol, dipped in liquid emulsion (Kodak NTB2, diluted 1:l with water), and exposedin light-tight boxes at 4°C. The slideswere developedat intervals and stained with hematoxylin and eosin. RESULTS
Mucosal localization of CYPlAl expression. CYPlAl is an aromatic hydrocarbon-inducible P-450 that is responsible for metabolic activation of a number of important procarcinogens including benz[a]pyrene. The localization of CYPlAl transcripts within the intestinal mucosa was investigated initially using isolated subpopulations of epithelial cells. Rats were treated with a single intraperitoneal dose of 3-methylcholanthrene (20 mg/kg body wt, in corn oil vehicle) or corn oil alone (control), and the animals were killed 16 h later. The intraperitoneal route was chosen to ensure that cells in intestinal
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crypts would be exposed to the inducer. Intestinal epithelial cells were isolated from individual rats and extracted RNA analyzed for expression of CYPlAl. The results of three control and two 3-methylcholanthrene-treated rats were the same, and, therefore, a representative Northern blot from one animal is presented (Fig. 1). In control animals, CYPlAl mRNA was expressed predominantly in villus cell fractions with very little found in crypt fractions (Fig. 1). In contrast, 3-methylcholanthrene-treated animals showed induction of CYPlAl mRNA in both villus and crypt cell fractions, which appeared to equalize the levels of mRNA in each subpopulation of cells. When Aroclor 1254, a mixture of polychlorinated biphenyls, was used as the inducer similar results were obtained (Fig. 5, described below). These results suggested that CYPlAl mRNA was expressed constitutively in villus cells but was inducible in both villus and crypt cells. In situ hybridization was performed on jejunal tissue sections for comparison to the results obtained from isoCONTROL FRACl NUMB
3-MC /213/415/617/81
9 I10
1
o/P1 Al
Fig. 1. CYPlAl mRNA in isolated subpopulations of intestinal epithelial cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells as described in METHODS. Fraction l/2 represents cells predominantly from villus tips and fraction 10 is last fraction and represents primarily crypt cells. Controls were administered corn oil (1 ml ip), and treated rats were given 3-methylcholanthrene (20 mg/kg in 1 ml of corn oil ip) 16 h before death. Analysis of 1 representative control and 1 treated animal is shown. Top: ethidium bromide-stained agarose gel. Ultraviolet illumination of nylon membrane and restaining of gel confirmed adequacy of RNA transfer to membrane. Middle: autoradiogram of Northern blot that was hybridized with 32P-labeled cDNA isolated from p21OST (8 h exposure). Bottom: graphic representation of densitometric analysis of Northern blot. Densitometry of both the 28s ribosomal band on photograph of ethidium-stained gel and the autoradiograph was performed using a Zeiss digital image analysis system. Ratio of the density of the band from the autoradiograph to that of the 28s band was used to compare the amount of mRNA between samples.
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lated epithelial cells. This was necessary for two reasons. First, we recently observed for sucrase-isomaltase mRNA that fine detail of mRNA expression along the cryptvillus axis may be obscured because of overlap between isolated cell fractions (25, 30). Second, the effect of inducer treatment on the isolation of intestinal epithelial cells was unknown. Multiple tissue sections from animals treated with either 3-methylcholanthrene or Aroclor 1254 were studied. In control animals, CYPlAl mRNA was expressed in epithelial cells located exclusively on the villus (Fig. 2, A-C). Figure 2, A and B, shows low-power light and dark field views, respectively, of a cross-section of rat jejunum. Autoradiographic grains were concentrated over epithelial cells located on the villus. A higher power view of one region of the section demonstrated that there were no grains over background in crypt cells with appearance of grains at the crypt-villus junction (Fig. 2C). Hybridization of serial sections with a sense-strand probe did not show grains over epithelial cells, which confirmed the specificity of results obtained with the antisense probe (Fig. 20). In addition, prehybridization with loo-fold excess of unlabeled antisense RNA effectively blocked hybridization of the labeled antisense probe to epithelial cells (data not shown). Taken together with the findings in isolated epithelial cells, these results suggest that constitutive CYPlAl gene transcription is activated as cells move from the crypt to the villus. This is similar to the pattern observed for other genes expressed in the small intestine of rats (15, 20, 25). After treatment with inducers there was marked induction of CYPlAl mRNA in crypt cells (Fig. 2, E-G). The low-power views demonstrate the dramatic induction in crypts compared with control sections (Fig. 2, E and F). A high-power view of the crypt region demonstrates the intense pattern of grains overlying the crypt cells (Fig. 2G); cells at the very base of the crypts, which may represent Paneth cells, had fewer grains. As for the sections from control animals, use of the sense probe did not result in grains over epithelial cells demonstrating specificity of hybridization with the antisense probe (Fig. 2H). Several crypt-villus units of a treated animal are shown in Fig. 2, I and J. These photomicrographs demonstrate the expression of CYPlAl mRNA in both crypt and villus cells and allow direct comparison of the intensity of grain density in both compartments. Although not quantified, the density of grains over crypts appears to be greater than that over villi, which is somewhat of a different impression than was obtained from Northern blot analysis of isolated subpopulations of epithelial cells, which suggested that the levels were similar in crypt and villus compartments (Fig. 1). Previous studies from our laboratory have shown that fine detail of mRNA localization determined by in situ hybridization is lost when analyzing isolated subpopulations of cells (25, 27, 30). This is most likely due to intermixing of villus and crypt cells in the middle cell fractions (27). However, the overall pattern of expression in crypt and villus compartments as determined by in situ hybridization confirms the pattern of CYPlAl gene expression along the crypt-villus axis that was suggested by Northern blot analysis of isolated
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Fig. 2. In situ hybridization analysis of rat jejunum for CYPlAl mRNA. Jejunal sections were prepared from control (corn oil 1 ml ip) and Aroclor 1254-treated rate (300 mg/kg in corn oil ip) and in situ hybridization performed as described in METHODS. Each slide was developed 2 days after application of radiographic emulsion and then stained with hematoxylin and eosin. The photographs shown represent examples taken from multiple sections of 2 control and 2 Aroclortreated animals. A: jejunal section from control rat hybridized with antisense CYPlAl RNA-probe. B: dark field microscopic view of field seen in A. C: higher vower view of a vortion of section shown in A. D: jejunalsection from control rat hybridized with sense CYPlAl RNA probe. E: ieiunal section from rat treated with Aroclor 1254 hybridized with antisense CYPlAl RNA probe. F: dark field microscopic view of field seen in E. G: higher power view of a portion of section shown in E. H: jejunal section from rat treated with Aroclor 1254 hvbridized with sense CYPlAl RNA probe.“l: jejunal section from a rat treated with Aroclor 1254 hybridized with antisense CYPlAl RNA probe. Bright field microscopic view. J: dark field microscopic view of field seen in I.
epithelial cells. Cellular localization of CYP2Bl expression. The pattern of CYP2Bl mRNA expression was examined using both isolated subpopulations of cells and in situ hybridization as described above for CYPlAl. Rats were treated with a single intraperitoneal dose of phenobarbital (80 mg/kg body wt), a potent inducer of CYP2Bl gene transcription in small intestine (29), and controls were given an intraperitoneal injection of 0.15 M NaCl; the animals were killed 16 h later. Northern analysis showed that CYP2Bl mRNA was constitutively expressed in villus epithelial cells with an apparent linear decrease in mRNA levels in successive cell fractions (Fig. 3). Treatment with phenobarbital produced induction of CYP2Bl mRNA, but the relative pattern of expression along the crypt-villus axis appeared to be maintained (Fig. 3). Identical results were obtained in two separate control and two phenobarbital-treated animals. In situ hybridization analysis demonstrated that control animals expressed CYP2Bl mRNA in villus tip cells with little mRNA in lower villus cells and none in crypt cells (Fig. 4, A and B). After phenobarbital treatment, CYP2Bl mRNA was induced in villus cells and was now detectable in lower villus cells; however, there remained no detectable mRNA in crypt cells (Fig. 4, C and D). Therefore, the results of isolated epithelial cells and in situ hybridization suggest that treatment with phenobarbital induces CYP2Bl mRNA only in villus enterocytes with little effect on crypt cells. Induction of both CYPlAl and CYP2Bl with a single agent. One possible explanation for differential induction of CYPlAl and CYP2Bl along the crypt-villus axis would be differential exposure of crypt cells to 3-methyl-
cholanthrene and phenobarbital, respectively. Therefore, animals were treated with Aroclor 1254, a mixture of polychlorinated biphenyls, which is a known inducer of both CYPlAl and CYP2Bl in small intestine (26). A Northern blot was sequentially hybridized with cDNA probes for both P-450s (Fig. 5). This analysis demonstrated that the pattern of expression in control rats was identical for CYPlAl and CYP2Bl mRNA. However, treatment with Aroclor 1254 induced expression of CYPlAl mRNA in crypt cell fractions, whereas there was no induction of CYP2Bl mRNA in the same cell fractions. These results suggest that the differential induction of P-450s in crypt cells is due to intrinsic differences in the ability of the cells to respond to inducers rather than different accessibility to the inducing chemicals. Expression of NADPH cytochrome P-450 reductase. The enzymatic activity of each P-450 enzyme is dependent on the presence of NADPH cytochrome P-450 reductase. Therefore, we examined the expression of NADPH cytochrome P-450 reductase mRNA in cell fractions of control animals and those treated with dexamethasone (300 mg/kg). Very low levels of mRNA were found in control intestine with the majority expressed in villus tip cells (Fig. 6). After treatment with dexamethasone, there was marked induction of NADPH cytochrome P-450 reductase mRNA in both villus and crypt cell compartments (Fig. 6). DISCUSSION
These studies reveal a complex pattern of expression along the rat intestinal crypt-villus axis for genes that encode components of the microsomal monooxygenase
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PB
NUMBER
CYP2Bl
Fig. 3. CYPZBl mRNA in isolated subpopulations of intestinal epitheha1 cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells that were obtained as described in METHODS. Control animals were administered saline and treated animals were given phenobarbital (80 mg/kg) 16 h before death. Analysis of 1 representative control and 1 treated animal is shown. Top: ethidium bromide-stained agarose gel. Ultraviolet illumination of nylon membrane and restaining of gel confirmed adequacy of RNA transfer to membrane. Middle: autoradiogram of Northern blot that was hybridized with 32P-labeled cDNA isolated from pSBF-1 (6 h exposure). Bottom: graphic representation of densitometric analysis of Northern blot. Autoradiographs were quantified as described in Fig. 1, legend.
system. We chose to examine levels of mRNA along the crypt-villus axis as a measure of gene expression for two reasons. First, molecular hybridization methods represent the most definitive way to identify which P-450 genes are expressed in a given tissue. Enzymatic activities for P-450 enzymes have overlapping substrate specificities (ll), and there may be differences in specificity for the same enzyme in different tissues (22). Identification of P-450 proteins using antibodies has yielded conflicting results possibly because of cross-reactivity to homologous forms of P-450s. Second, we have previously shown that CYPlAl and CYP2Bl genes are regulated at the level of mRNA abundance in small intestine after treatment with inducers, predominantly by changes in gene transcription (28, 29). Therefore, analysis of mRNA levels provides information on the mechanisms for regulation along the crypt-villus axis. The molecular probes used in this study are specific for identification of CYPlAl and CYP2Bl mRNA in small intestine. We previously showed that CYP2Bl is the only member of this family expressed in small intestine by amplifying cDNA using the polymerase chain reaction and sequencing of the product (29). The probe used for CYPlAl was shown by Fagan et al. (7) to differentiate between CYPlAl and the only other mem-
c
i
Fig. 4. In situ hybridization analysis of rat jejunum for CYPBBl mRNA. Jejunal sections were prepared from control (1 ml 0.15 M NaCl ip) and phenobarbital-treated rats (80 mg/kg ip) and in situ hybridization performed as described in METHODS. Photographs shown represent examples taken from multiple sections of 2 control and 2 phenobarbitaltreated animals. Each slide was developed 2 days after application of radiographic emulsion and then stained with hematoxylin and eosin. Jejunal sections from control and treated rats hybridized with sense CYPBBl RNA probe did not show detectable signal over background (data not shown). A: jejunal section from control rat hybridized with antisense CYPBBl RNA probe. B: dark field view of field shown in A. C: jejunal section from a phenobarbital-treated rat hybridized with antisense CYP2Bl RNA probe. D: dark field view of field shown in C.
ber of this subfamily, CYPlA2. CYPlAl was constitutively expressed in epithelial cells located on the villus and was absent in crypt and submucosal cells. The epithelial cells that express the mRNA appear to be predominantly absorptive enterocytes although the resolution of the in situ hybridization method did not allow the definitive identification of goblet and enteroendocrine cells. CYPlAl mRNA was induced in both villus and crypt cells. Because these experiments
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1Al 2.0 -
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Fig. 5. CYPlAl and CYP2Bl mRNA in isolated intestinal epithelial cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells as described in METHODS. Control animal was administered corn oil (1 ml ip), and treated animal was given Aroclor 1254 (300 mg/kg ip) 16 h before death. Top: ethidium bromide-stained agarose gel. Middle: autoradiograms of Northern blots that were hybridized sequentially with labeled cDNAs isolated from pSBF-1 (3 h exposure) and p21OST (12 h exposure), respectively. Bottom: graphic representation of densitometric analysis of Northern blot. Autoradiographs were quantified as described in Fig. 1, legend.
evaluated steady-state levels of CYPlAl mRNA, induction could represent either increased gene transcription, diminished mRNA degradation, or both. We have previously shown that induction of CYPlAl mRNA in small intestine occurs predominantly by an increase in gene transcription (28). Because those studies used nuclei isolated from scraped mucosa, which only partially removes crypts (26), this was felt to be primarily a measure of transcription in villus cells. The results of in situ hybridization revealing absence of CYPlAl mRNA in the crypts of control intestine and abundant mRNA in treated intestine suggests that activation of transcription also occurs in crypt cells. Induction of CYPlAl is considered a marker for the presence of the aromatic hydrocarbon (Ah) receptor (16, 21). The Ah receptor is a cytosolic protein that is translocated to the nucleus on binding of a ligand where it activates transcription of the CYPlAl gene via binding to DNA regulatory elements (34). Therefore, our data
Fig. 6. NADPH cytochrome P-450 reductase mRNA in isolated intestinal epithelial cells. RNA was isolated in 6 fractions from subpopulations of epithelial cells as described in METHODS. Control animal was administered corn oil (1 ml ip) and treated animal was given dexamethasone (300 mg/kg ip) 16 h before death. Top: ethidium bromide-stained agarose gel. Middle: autoradiogram of Northern blot that was hybridized with 32P-labeled cDNA isolated from pSP450-OR (14 h exposure). Bottom: graphic representation of densitometric analysis of Northern blot. Autoradiographs were quantified as described in Fig. 1, legend.
suggest that the factors required for transcriptional activation of the CYPlAl gene including the Ah receptor (34) and the recently described arnt protein (14, 16) are present in all cells along the crypt-villus axis. Whether these factors are constitutively expressed in both crypt and villus compartments or are inducible in response to xenobiotics will require examination of the proteins or their respective mRNAs once the reagents necessary for these experiments become available. Differences in the degree of induction between crypt and villus compartments may be related to multiple factors including different levels of inducers, transcriptional regulatory proteins, or rates of mRNA degradation. The presence of the Ah receptor in intestinal epithelial cells may have implications beyond those of xenobiotic metabolism. The Ah receptor is responsible for the induction of a battery of genes (18) and may play a role in control of cellular growth and differentiation (17). It was recently shown that 2,3,4,8-tetrachlorodibenzo-p-dioxin, a compound that binds avidly to the Ah receptor, causes proliferation of keratinocytes via synthesis and secretion of transforming growth factor (TGF)-a which acts as an autocrine growth factor (4). Furthermore, TGF-a! and TGF-/3 may be involved in the regulation of intestinal
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growth and differentiation (2, 23). The role of Ah receptor and xenobiotics in the regulation of intestinal growth requires investigation. CYP2Bl mRNA was expressed constitutively and was inducible only in cells located on villi. As for CYPl Al, our laboratory has previously shown that transcriptional activation of the CYP2Bl gene is responsible for induction of mRNA in small intestine (29). These data suggest several possibilities for the regulation of CYP2Bl gene transcription in the small intestine including I) the transcription factors that mediate induction by xenobiotics are not present in crypt cells; 2) the factors necessary for basal transcription of the gene are not present in crypt cells and, therefore, factors involved in induction are ineffective; or 3) there are repressors in proliferating crypt cells that prevent transcription of the gene. There is presently no information on the factors that regulate basal or induced transcription of the CYP2Bl gene. Differences in crypt and villus cells may be exploited to investigate these mechanisms. An intriguing finding of this study was the pattern of expression of NADPH cytochrome P-450 reductase mRNA along the crypt-villus axis. Undetectable levels of reductase in the crypt cells raise the possibility that CYPlAl expressed in the crypt after induction may not be enzymatically active. Furthermore, if levels of reductase are rate-limiting for enzymatic activity in control animals, it is possible that induction of the reductase alone may increase P-450 enzyme activity. This would be a novel situation, because NADPH cytochrome P-450 reductase in liver is felt to be in excess of P-450 proteins such that the level of P-450 protein determines enzymatic activity. Quantitative histochemical measurements must be developed to accurately identify cells that contain monooxygenase activity in order to investigate this possibility and to determine if differential induction of P-450 genes in crypt and villus compartments lead to differences in metabolic capacity. Previous histochemical localization of benz [ a] pyrene hydroxylase activity in small intestine (32) suggests that quantitative analysis of this type may be possible. Finally, it will be important to investigate the effect of inducers of P-450 genes on the expression of reductase mRNA and enzymatic activity. Taken together, our data demonstrate that the CYPlAl, CYP2B1, and NADPH cytochrome P-450 reductase genes are differentially regulated along the intestinal crypt-villus axis. This results in a gene-specific pattern of induction in crypt and villus cell compartments. Therefore, transcription factors required for induction of certain members of this gene family are present in undifferentiated crypt cells, and certain other factors are expressed only in more differentiated cells located on villi. Investigation of the transcriptional regulation of CYHAl and CYP2Bl genes in crypt and villus cells may allow elucidation of the molecular mechanisms that control basal and induced transcription in intestine. Differences in the complement of P-450 enzymes or the activity of the monooxygenase system in crypt and villus cell compartments may have profound effects on the fate of ingested xenobiotics and procarcinogens. This studv was supnorted bv National
Institute
of Diabetes and
GENE EXPRESSION Digestive and Kidney Diseases Grant DK-41393 and a Veteran’s Affairs Research Associate Award and Merit Review (P. G. Traber). This study was presented in part as an abstract (28) and at the annual meeting of The American Gastroenterological Association, San Antonio, TX, 1990. Address for reprint requests: P. G. Traber, 6520a MSRB-1, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0682. Received 4 December 1991; accepted in final form 31 March 1992. REFERENCES 1. Anderson, Junker,
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