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Acylase 1 expression in rat intestinal crypt–villus axis
Cell Biology International 31 (2007) 966e973 www.elsevier.com/locate/cellbi
Acylase 1 expression in rat intestinal cryptevillus axis Natacha Cigna a,c, Cendrine Nicoletti a, Anne Durand b, Jean Claude Chaix a, Thierry Giardina a, Josette Perrier a,* a
BiosCiences FRE CNRS 3005, Universite´ Paul Ce´zanne e Aix-Marseille III, Faculte´ des Sciences, 13397 Marseille Cedex 20, France b Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK c Institut Curie section de recherche, UMR 146, Bt. 110, Centre Universitaire, 91405 Orsay, France Received 11 December 2006; revised 31 January 2007; accepted 11 March 2007
Abstract Acylase 1 was investigated at the cellular level in the rat small intestine along the enterocyteedifferentiation axis. As confirmed by microscopic analysis, villus tip cells and crypt cells of rat jejunal mucosa were successfully separated using the Weiser method. The proliferating undifferentiated crypt cells showed much higher ACY 1 activity levels than the villus cells, with a 6.4-fold decrease as the cells migrated and differentiated along the cryptevillus axis. RT-PCR studies on mRNA extracted from isolated cells showed that ACY 1 mRNA was mainly expressed in crypt cells, reaching levels that were 12-fold higher than those recorded in other cell types along the whole enterocyte differentiation axis. It was concluded that the expression of ACY 1 in the intestinal crypt cells is regulated at the mRNA level. Immunohistochemistry revealed the expression of ACY 1 in the absorbing lineage cells from the ileal and colonic crypts and the absence of ACY 1 in the mucus producing goblet cells. These findings proposed ACY 1 as a new marker transcript for absorbing cells of intestinal crypt, which can be used to monitor the process of intestinal N-a-acetylated protein metabolism. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Acylase 1; N-terminal acetylated protein; Enterocyte differentiation; Weiser method
1. Introduction Enterocytes constitute the bulk of the epithelial population, which is mainly responsible for the absorptive process occurring after the action of specialized brush border hydrolases has been completed. Mammalian small intestinal epithelial cell production begins with crypt cells dividing and subsequently differentiating along the cryptevillus structural axis. Highly regulated mechanisms control the rate of proliferation and differentiation of enterocytes as they move along the cryptevillus axis during
Abbreviations: Ac, acetyl; ACY 1, acylase 1. * Corresponding author. BiosCiences FRE CNRS 3005, Laboratoire de Biochimie de la Nutrition, Universite´ Paul Ce´zanne e Aix-Marseille III, Service 342, Faculte´ des Sciences et Techniques, Avenue Escadrille Normandie Niemen, F-13397 Marseille Cedex 20, France. Tel.: þ33 4 91 28 84 44; fax: þ33 4 91 28 84 40. E-mail address: [email protected] (J. Perrier).
their relatively short lifetime (48e72 h in the rat) (De Santa et al., 2003; Vidrich et al., 2003). The process of mitosis ends and that of differentiation starts in the crypt. Differentiation is completed in the lower half of the villus, and there exists histological evidence that apoptosis occurs at the villus tip (Potten and Loeffler, 1987; Madara and Trier, 1987; Weiser, 1973; Altmann and Leblond, 1982; Gravieli et al., 1992). During differentiation of the mouse small intestinal epithelium, a general decrease in transcriptome complexity, using filter-based cDNA arrays, was demonstrated (Tadjali et al., 2002). The marked polarization of this cell, as well as the expression of hydrolytic brush border enzymes, provide specific markers which can be used to monitor the process of enterocyte differentiation (Weiser, 1973; Tadjali et al., 2002; Traber, 1990; Noren et al., 1989; Darmoul et al., 1991). Several studies have also shown that a few xenobiotic-metabolizing enzymes are present in the fully differentiated enterocytes (Traber et al., 1992; Cornell and Meister, 1976; Ware et al., 1998).
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.03.006
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An interesting online intestinal crypt/villus in situ hybridization database holds information for relative gene expression patterns in the mammalian intestine (Olsen et al., 2004). In mouse small intestine, a high expression of cell cycle and DNA synthesis genes occurs in the crypt, and cytoskeletal and lipid uptake genes are present in the villus, in respect with the enterocyte differentiation (Mariadson et al., 2005). In mammalian cells, as much as 70% of the cytosolic proteins can be a-N-acetylated, and the N-protected amino acids which are released during proteolysis must be free of amino acids before they can be used for protein synthesis (Driessen et al., 1985), since it seems hardly likely that they would be wasted in the cells. Post-translational acetylation of intracellular proteins and peptides is generally thought to be an efficient mechanism protecting them from proteolytic degradation in eukaryotic cells (Persson et al., 1985; Tsunasawa and Sakiyama, 1984). The NH2-a-acetylated amino acid released from the protein or the resulting peptide is catalyzed by the sequential action of the acylpeptide hydrolase (APH or acylamino acid releasing enzyme, EC 3.4.19.1) and acylases (ACY, EC 3.5.1.x) generating a free amino acid and an acetate, yielding the corresponding free amino acid during the process of intracellular protein catabolism. Acylase 1 (ACY 1 or N-a-acyl-L-amino acidamidohydrolase, EC 3.5.1.14) is by far the best characterized form, which hydrolyses aliphatic N-acyl-a-amino acids in tissue extracts (Gade and Brown, 1981; Perrier et al., 2005). It is worth noting here that the a-N-acetylamino acids which are usually found at the N-terminal position of the acylated protein, namely Acmethionine, Ac-alanine and Ac-serine (Driessen et al., 1985), correspond to the substrate specificity of ACY 1. The free amino acid released, especially the essential amino acid methionine (Met), is required for initiating protein synthesis by MettRNA formation, in relation to the polyamine synthesis or the trans-methylation pathway. Alternatively, polyamine synthesis abets normal cell proliferation (Ogier et al., 1993). However, until now, the exact physiological function of ACY 1 has not yet been completely elucidated. The fact that high levels of this enzyme have been detected in various tissues, including intestinal epithelia (Giardina et al., 1997), strongly suggested that this enzyme might have some additional physiological functions. For example, the expression of ACY 1 has been reported to be correlated with the existence and activity of acetylated growth factors in carcinoma cells and to be down-regulated in these cells (Scaloni et al., 1992; Hwa et al., 2005). ACY 1 might also regulate the activity of type 1 sphingosine kinase, which has been found to promote cell growth and to inhibit the apoptosis of tumor cells (Maceyka et al., 2004). ACY 1 deficiency was recently detected in lymphoblast cells from a young encephalopathic patient (Van Coster et al., 2005; Sass et al., 2006). Acylases also play an important role in the metabolism of the prodrug N-acetyl-L-cysteine (NAC), used in clinical practice to treat lung disorders (De Flora et al., 1995), and thus promote glutathione synthesis (Lauterburg et al., 1983). ACY 1 has been reported to be involved in xenobiotic bioactivation as well as in the interorgan processing of
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N-acetyl-amino acid-conjugated xenobiotic derivatives (Ssubstituted-N-acetyl-L-cysteine) (Uttamsingh et al., 1998). The transport of acetylated amino acids found to occur across the rabbit intestinal epithelium has suggested the involvement of ACY 1 in protein metabolism (Brachet et al., 1991; Wallace et al., 1998). It was also recently suggested that ACY 1 may play a protective role by degrading bacterial and mitochondrial N-formylated peptides in the rat intestine and may constitute the first line of defense against constant exposure to commensal bacteria (Nguyen and Pei, 2005). Certain features related to protein metabolism in the intestine are relatively unique to that tissue. Intestinal protein metabolism will be considered as occurring by two major mechanisms: (1) the formation, migration, differentiation and loss of whole cells, and (2) intracellular synthesis and degradation. In keeping with these two major mechanisms, the rat intestinal cryptevillus axis represents a useful model for study of the involvement of ACY 1 in N-acetylated protein/peptide metabolism, which represents a major event in the cell. Therefore, in the present study, ACY 1 enzyme activity was investigated at the cellular level in the rat small intestine along the enterocyteedifferentiation axis. We showed that ACY 1 is expressed at a high level in the undifferentiated crypt cells, from the absorptive lineage. In addition, we showed that ACY 1 expression in the rat intestinal cryptevillus axis is controlled at the mRNA level. Understanding ACY 1 cellular localization and expression in the intestine could give us indications on function(s) associated with this enzyme in this tissue. 2. Materials and methods 2.1. Materials N-Acetyl-L-methionine, p-nitrophenyl phosphate, L-leucine-p-nitroalanine, acid oxidase, horseradish peroxidase (type II), o-dianisidine were obtained from Sigma (St. Louis, MO, USA). L-amino
2.2. Preparation of intestinal epithelial cells Adult male Wistar rats weighing 260e300 g were used in this study. A 30cm segment of small intestine was removed, and villus and crypt epithelial cells were prepared directly by shaking the intestinal segment for successive periods in a solution of EDTA as previously described in detail (Weiser, 1973). Briefly, after removal, the intestine segment was rinsed thoroughly with ice-cold phosphate-buffered saline (PBS), and flushed twice with icecold PBS containing 1 mM DTT. The segment was incubated at 37 C for 15 min in a citrate buffer, pH 7.3, containing 96 mM NaCl, 1.5 mM KCl, 27 mM Na citrate, 8 mM KH2PO4 and 5.6 mM Na2HPO4 (Buffer A), then everted, filled and incubated at 37 C in Buffer B (PBS buffer containing 1.5 mM EDTA, 0.5 mM DTT). The intestinal segment was gently shaken at 37 C to remove adhering enterocytes from the villusecrypt axis, for nine incubation steps (F1eF9) after 4, 2, 2, 3, 4, 5, 7, 10 and 15 min, respectively. The cells from each fraction were collected by pelleting, washed twice in 10 ml fresh ice-cold PBS.
2.3. Enzyme activities Immediately after isolating the cells, the enzyme activities were measured. The cells were lysed with an Ultraturax for 10 s in ice-cold PBS buffer, the resulting homogenate was centrifuged at 100,000 g for 30 min and the supernatant was directly used for enzyme activities determinations.
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Aminopeptidase N (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and ACY 1 (EC 3.5.1.14.) activities were determined spectrophotometrically by using L-leucine-p-nitroalanine, p-nitrophenyl phosphate, N-acetyl-L-methionine, respectively, as the substrate (Weiser, 1973; Raul et al., 1987; Perrier et al., 2002). Specific activities were expressed as milliunits per milligram of protein. One unit was defined as the activity hydrolyzing 1 mmol of substrate/min at 37 C. Protein concentrations were determined using Bradford’s method (Bradford, 1976), with bovine serum albumin as the standard, to calculate the specific activities.
2.4. Immunofluorescence and electron microscopy After harvesting cells, the cells from fractions 1 (villus), 5 (middle crypte villus axis) and 9 (crypt) were fixed with 4% paraformaldehyde in PBS for 30 min at 4 C, and 200 ml were then adsorbed onto polylysine-coated slides for 30 min at room temperature, before being post fixed with 4% PFA for 10 min at room temperature and permeabilized for 10 min with 0.1% saponin in PBS. After several washes with PBS, the cells were saturated for 30 min in 10% fetal calf serum in PBS and incubated for an additional 30-min period at room temperature with a 1:100 dilution of the commercial Texas Red phalloidin derivative anti-actin. Lastly, after undergoing several washes, the cells were mounted with Vectashield (Vecto Laboratories, Burlingame, USA). Epifluorescence was detected and visualized with an Axiovert 200M Zeiss microscope equipped with a Hamamatsu camera. For the electron microscopy procedure, the tissue and the cell fractions (F1, F5, F9) were fixed for 1 h in 2.5% glutaraldehyde in cold PBS. After being washed with PBS, they were post-fixed in 2% OSO4 in PBS for 1 h at room temperature, and then dehydrated in a graded ethanol series and embedded in Epon 812. Ultrathin sections (50e60 nm) were layered on Maxtaform HR 24Cu/Rh (200 mesh), stained with uranyl acetate for 20 min and lead citrate for 10 min, and examined with a transmission electron microscope Zeiss EM109, at 80 kV. The tissue immunofluorescence reaction was performed on semithin frozen sections (1 mm), by using the anti-ACY 1 polyserum (1:200) (Abnova Corporation Taiwan) or the normal mouse serum (1:200) and a commercial fluorescein derivative of anti-mouse Igs diluted 200 times as secondary antibody (both from Sigma Corporation, France).
2.5. Total RNA isolation and RT-PCR Cells were washed once with cold PBS, and 200 ml of RNAwizÔ isolation solution (Ambion, Inc. Austin, USA) were added to each sample. Total RNA was immediately extracted in line with the manufacturer’s instructions and stored at 20 C until used. Total RNA (101 mg) was used for RT and the cDNA synthesis reaction was performed in keeping with the instructions given by Invitrogen, using their SuperScriptÔ III/PlatiniumÒ Taq polymerase Onestep RT-PCR System (Invitrogen Life Technologies) at 50 C for 20 min, ending in a 5-min heating step at 95 C to inactivate the RT enzyme inactivation. PCR primers were designed for use in this study, based on the EMBL/GenBank (accession no.: Rattus b actin, NM031144; APN, Y18765; ACY 1, NM001005383): the primer sequences are shown in Table 1. Each PCR cycle included 30 s of denaturation at 94 C. The annealing step was carried out at an appropriate temperature, as indicated in Table 1, using 0.4 mmol of primer pairs, for 2 min, whereas the extension step was performed at 72 C for 3 min, using an overall volume of 50 ml and a total number of 30 PCR cycles in a MastercyclerÒ gradient 5331 thermocycler (Eppendorf). Lastly, aliquots of the PCR products were separated on a 1.5% Triseacetate EDTA agarose gel, stained with ethidium bromide, and photographed under UV light. The signal intensity of each band was normalized by comparing it with the intensity of b-actin, using the PhotoCapt software.
2.6. Immunoblotting Cells were washed with PBS buffer. The resulting pellets were homogenized in buffer containing 10 mM sodium phosphate, 150 mM sodium chloride (pH 7.4), and centrifuged at 12,000 g. Protein concentration in the supernatant was determined according to Bradford (1976). An equal loading of proteins (5 mg) were separated in a 10% SDS-PAGE gel as described by
Laemmli (1970). Electroblotting was performed according to Towbin et al. (1979). As primary antibody the anti-aminoacylase 1 polyclonal serum was used (Abnova, Taiwan) (dilution 1:1000). A horseradish peroxidase-conjugated goat anti mouse antibody (dilution 1:5000) was used as secondary antibody (Pierce, Perbio Science Company) and the super signal West Pico Chemiluminescent substrate (Pierce) was used to detect the ACY 1 protein.
2.7. Statistical analysis All the enzyme activity data recorded were assessed using the Student’s t-test for paired data. Differences were taken to be significant at p < 0.05.
3. Results 3.1. Actin network and cellular profile changes in rat intestinal cryptevillus axis Under the experimental conditions used to freshly isolate enterocytes from adult rats, the morphology of the cells obtained was found to be normal. As was to be expected (Fig. 1A), the highly differentiated villus cells (fraction 1, F1) were found to have a polarized morphology, closely packed apical microvilli or brush borders (BB) and its terminal web (TW); the cells in the intermediate stage of differentiation observed in the middle of the villusecrypt axis (fraction 5, F5) were found to be characterized by their very small size and their very slight apical microvilli (A), which contrasts strongly with the undifferentiated, non-polarized crypt cells from F9 showing no microvilli. A photomicrograph of villus cells (F1), middle cryptevillus axis cells (F5) and crypt cells (F9) from isolated enterocytes showed the presence of a normal peripheral actin network after interactions had been performed with an anti-actin phalloidin-Texas Red-conjugated moiety (Fig. 1B). Cellular sections were taken from the cell fractions of F1, F5, and F9 collected. Electron microscopy examination of cell fractions (Fig. 1C) showed that the closely adjacent enterocytes had a highly polarized pattern of organization and numerous microvilli were observed in the brush border (BB) in F1; the cells and the microvilli network, were much smaller in F5eF9. In the last fraction (F9), the cells were found to be small and round, without any microvilli; these characteristics are typical of undifferentiated cells in the deep crypts. Microscope analysis of electron micrographs showed that a 3-fold increase in the absolute size of these cellular fractions occurred as the cells differentiated (28 mm, 18 mm, 9 mm from F1 to F9, respectively). These findings show that epithelial cells were successfully isolated here from along the cryptevillus axis with minimum damage and contamination of cells from the lamina propria, with respect to the normal enterocyte differentiation process. 3.2. Enzyme expression changes along the rat intestinal cryptevillus axis and immunoblotting As expected, the specific activities of all villus marker enzymes used (alkaline phosphatase (AP), aminopeptidase N (APN) increased sharply from F9 (the fraction from the bottom
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Table 1 Primer sequences corresponding to the rat cDNA sequences used for RT-PCR analysis of each enzyme investigated in this study (accession no. Rattus b actin, NM031144; aminopeptidase N (APN), Y18765; aminoacylase 1 (ACY 1), NM001005383) Gene
Primer
Nt
Annealing temperature ( C)
% GC
Nucleotide sequence (50 to 30 )
ADNc size expected (bp)
ACY 1
d81 S c294 AS 5’ primer 3’primer S2 AS3
34 28 32 32 19 20
59 65 60 60 48 45
47 71 53 53 57 45
GCT AAA CTC CCA CAC AGA TGT GGT ACC TGT CTT C TCC CGC TTC CTG GGA CCA GCT CTG CAG C ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC AAC AAG GAG CGG GTG GTC A TCT GAA TCC TTC CTC CAG TT
666
b-Actin APN
838 770
S, sense primer (foward primer); AS, antisense primer (reverse primer); Nt, nucleotide primer length.
crypt cells) to F1 (the villus tip cell fraction) (Fig. 2A). The specific activity of AP increased 19-fold from 28 24.7 to 518 100 (U/mg), and the specific activity of APN increased 11-fold from 13 3.5 to 140 55 (U/mg). Inversely, our data also showed that the specific activity of acylase 1 (ACY 1)
decreased 6.4-fold from 16 3.1 to 2.5 1.8 (U/mg) from F9 (undifferentiated cells) to F1 (differentiated cells). Immunoblotting blotting (Fig. 2B) with an anti-aminoacylase 1 polyclonal antibody revealed a decrease of the band corresponding to ACY 1 in Weiser’s fraction from F9 to F1. It
Fig. 1. Actin network and cellular profile changes in rat intestinal crypt- villus axis. (A) The Normaski view and (B) its corresponding specific phalloidin-Texas Red complex against the actin network. (C) The electron microscope image. The actin network is well represented in the brush border (BB), basolateral membrane (BL), apical membrane (A), terminal web (TW). Note: nucleus (N), gobelet cell (GC). Fraction 1 (F1), villus cells; fraction 5 (F5), middle villusecrypt axis cells; fraction 9 (F9), crypt cells. Bar 2 and 10 mm.
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A
A
F1
Cell fractions F5
F7
F9
1000
β-Act. AP
ACY 1
APN
APN 100
Relative mRNA levels
SA (U/mg)
ACY 1
10
40 30
B
20 10 0 F9
(F1-F9)fold
1
0
22
14
24
12
F1
F5
F7
APN
22
18
ACY1
2
13
1 F1 Villus
F2
F3
F4
F5
F6
F7
F8
F9 crypts
Villus-crypts cells fractions
B ACY 1
Fig. 2. Enzyme activities profile in rat intestinal cryptevillus axis homogenates. (A) The specific activities (U/mg protein) of AP, APN and ACY 1 were determined as indicated in Section 2. Values are means obtained from duplicate samples, and comparable results were obtained in four independent experiments. (B) The corresponding immunoblotting profile with the anti-aminoacylase 1 antibody.
therefore seems likely that ACY 1 enzyme may gradually decrease as the cells migrate and differentiate from the crypt to the villus. 3.3. Changes in mRNA expression by ACY 1 along the rat intestinal cryptevillus axis Fig. 3A shows the RT-PCR products separated on 1.5% agarose gel. The size of b-actin, APN and ACY 1 mRNAs was consistent with the predicted values (respectively 838 bp, 770 bp and 667 bp as shown in Table 1). As was to be expected, b-actin mRNA expression occurs in the epithelial cells located along the whole cryptevillus axis, and the levels of expression were found to increase slightly from F9 to F1 (Fig. 3A), reaching a 1.3-fold increase in mRNA level in the completely differentiated cells. These results are in good agreement with the pattern of immunofluorescence labeling shown in Fig. 1B. In addition, APN mRNA levels were higher in cells F1 and F5 than in fractions isolated from the lower part of the villus (F7 and F9, Fig. 3A), corresponding to a 22-fold relative mRNA increase from F9 to F1 (Fig. 3B). These results matched the 11-fold increase in the APN enzyme activity observed from F9 to F1 (Fig. 2A). In marked contrast, the ACY 1 mRNA levels were higher in F7 and F9 than in F1 and F5 (Fig. 3A), and a 12-fold relative decrease in the mRNA levels
Cell fractions Fig. 3. RT-PCR products in intestinal epithelial cells along the cryptevillus axis. Cells were isolated separately from rat jejunum (F1 villus, F5 middle villuse crypt axis and F7, F9 crypt), and the mRNA levels were determined using semi-quantitative RT-PCR methods, as described in Section 2. Typical ethidium bromide (EtBr) staining of RT-PCR products is shown in (A), for aminopeptidase N (APN), b-actin (Act), aminoacylase 1 (ACY 1). In the graph above (B), the RT-PCR signal intensity was normalized by comparing each value with that obtained on b-actin, used as the internal PCR control values. Results are means SE of values obtained on five rats.
was found to occur from F9 to F1 (Fig. 3B). These results matched the 6.4-fold decrease in ACY 1 enzyme activity observed from F9 to F1 (Fig. 2A). The distribution pattern of ACY 1 mRNA correlates well with the enzyme activity pattern reported above. A translational regulation of ACY 1 expression may occur along the cryptevillus axis of the small intestine, which would account for the proliferative status and/ or for the protein metabolism status of the cells. 3.4. Cellular localization of acylase 1 along the rat intestinal cryptevillus axis Fig. 4A shows the patterns of immunolabeling observed with the anti acylase 1 antibody on sections from ileal and colonic tissue segments along the digestive tract in the rat, also confirmed by Western blotting experiments (data not shown). The ACY 1 labeling is restricted to the cytosol of the crypt cells, between the nucleus and the terminal web, with a tendency for apical staining, in the ileum and in the proximal colon. The ACY 1 labeling is evident in both lower and upper crypt areas. The absence of acylase 1 labeling in the differentiated cells from ileal villi and colonic villi (the flat surface epithelium or plateau) is well observed. The acylase 1 labeling is also absent from the goblet cells (GC) from the secretory lineage, numerous in the colon. The immunolabeling study revealed also that ACY 1 is
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Fig. 4. Immunolocalization of acylase 1 in the digestive tract. Sections from villi and crypt regions of the rat ileum and proximal colon are revealed with the antiACY 1 plyclonal serum (A) or normal mouse serum (B). The Normaski view (left) and immunofluorescence labeling of the same field (right) are given side by side. Note the presence of the cryptevillus gradient. Bar 10 mm; BB, brush border; LP, lamina propria; N, nucleus; GC, goblet cell; L, gut lumen; PC, Paneth cell.
not expressed in other cell types among the lamina propria epithelial cells and in Paneth cells (PC) (data not shown). The specificity of staining was checked using a normal mouse serum as a control. Results clearly show the absence of staining (Fig. 4B). Here again, it therefore seems likely that ACY 1 enzyme immunofluorescence labeling may gradually decrease as the cells migrate and differentiate from the crypt to the villus, in the digestive tract. The distribution pattern of ACY 1 immunolabeling correlates well with the enzyme activity and the mRNA patterns. We proposed ACY 1 as a new important marker enzyme, for crypt absorbing cells in rat intestine. 4. Discussion ACY 1 is found to be expressed in the mucosa throughout the gastrointestinal tract where is thought to play a role in Nacetylated protein metabolism (Giardina et al., 1997). Also, the activities of alkaline phosphatase, aminopeptidase N, and ACY 1 were studied here in normal rat intestine, after crypt cell proliferation. As was to be expected, AP and APN were found to be low from normal rat intestinal crypt cells (Weiser, 1973; Noren et al., 1989), but to reach measurable levels after the cells had migrated and matured, reaching the non-proliferative state and the functional villus stage. Measurable levels of the cytoplasmic enzyme ACY 1 were detected in the rat
intestine. In strong contrast with these other enzymes, ACY 1 enzyme activity showed a 6.4-fold decrease during crypt cell differentiation. In this model, mucosal crypt epithelial cells divide in the zone of cell proliferation towards the bottom of the crypt. Also, our results clearly show that the ACY 1 mRNA is down-regulated during crypt cell differentiation indicating that the ACY 1 enzyme activity reflected well the mRNA expression level of the protein. We proposed that ACY 1 expression is controlled at mRNA level in rat intestinal cells. Also, our method of tissues (ileon and colon) immunolabeling appears to give the same patterns of ACY 1 enzyme activity, protein and mRNA expression. No acylase 1 labeling is observed in the goblets cells (secreting protecting mucins), in Paneth cells and in other epithelial cell types. Based on these data, the activity of ACY 1 seems to be coordinated in specific ways depending on the cell type (absorptive/secretory lineage), the functional state of the cell, and on whether the crypt cells were in the proliferative undifferentiated state. These data supported well the idea that ACY 1 may contribute to the process of N-acetylated proteins catabolism, which, logically occurs preferentially in the proliferative cells as crypts cells. ACY 1 has also been recently described as a SphK1-interacting protein, which may affect the physiological functions of the SphK1 enzyme, promoting cell growth and
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inhibiting the apoptosis of mammalian kidney HEK 293 tumor cells (Maceyka et al., 2004). The fact that high levels of ACY1 were detected here in the proliferative crypt cells (lower and upper area) is consistent with the idea that this enzyme may be involved in cell growth via interactions with SphK1. It seemed to be worth investigating whether the biological utilization of intrinsic and food N-acetylated proteins, as well as that of acetylated amino acids, and N-acetyl-amino acid-conjugated xenobiotic derivatives (S-substituted-N-acetyl-L-cysteine) (Uttamsingh et al., 1998), might affect the activity of ACY 1 enzyme in an undifferentiated/differentiated polarized living enterocyte cells. ACY 1 is activated by its substrates (N-acetyl-amino acid) in rat and pig primary hepatocyte cell cultures (Perrier et al., 2002). Also, the presence of ACY 1 which catalyses the deacetylation of the haloalkene-derived mercapturates in several tissues studied may contribute to the organ-selective toxicity of these mercapturates (Cooper et al., 2002). The strong activity of ACY 1 in the crypt area correlates well with an active amino acid turnover found in the proliferating undifferentiated crypt cells from the small intestine and may also explain the intestine toxicity of acetylated-xenobiotics. It would be interesting for us to analyze the ACY 1 expression along the cryptevillus axis under N-acetylated substrates and under Nacetylated xenobiotic conditions. Therefore, we have described recently a natural phenolic compound as a powerful competitive inhibitor of porcine kidney ACY 1 (Stocker et al., 2005). For all the conditions discussed above, the rat intestinal cryptevillus axis could represent a good alternative approach to the in vitro colon cancer cell line Caco-2 and could give us indications on in vivo function(s) associated with ACY 1 in this tissue. Studies to elucidate these points are currently in progress. Acknowledgements We are grateful to Dr Jessica Blanc for revising the English manuscript. References Altmann GG, Leblond CP. Changes in the size and structure of the nucleolus of columnar cells during their migration from crypt base to villus top in rat jejunum. J Cell Sci 1982;56:83e99. Brachet P, Gaertner H, Tome D, Dumontier AM, Guidoni A, Puigserver A. Transport of N-a (or N-e)-L-methionyl-L-lysine and acetylated derivatives across the rabbit intestinal epithelium. J Nutr Biochem 1991;2:387e94. Bradford M. A rapid and sensitive method for the quantification of microgram quantity for protein utilizing the principle of dye binding. Anal Biochem 1976;72:248e54. Cornell JM, Meister A. Glutathione and g-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa. Proc Natl Acad Sci U S A 1976;73:420e2. Cooper AJL, Bruschi AS, Anders MW. Toxic halogenated cysteine S-conjugates and targeting of mitochondrial enzymes of energy metabolism. Biochem Pharmacol 2002;64:553e64. Darmoul D, Rouyer C, Blais A, Trugnan G. Dipeptidyl peptidase IV expression in rat jejunal crypt-villus axis is controlled at RNA levels. Am J Physiol 1991;261:G763e9. De Flora S, Cesarone CF, Balansky RM, Izzotti A. Chemopreventive properties and mechanisms of N-acetylcysteine. J Cell Biochem 1995;22:33e41.
De Santa BP, Van den Brick GR, Robert DJ. Development and differentiation of the intestinal epithelium. Cell Mol Life Sci 2003;60:1322e32. Driessen HP, de Jong WW, Tesser GI, Bloemendal H. The mechanism of N-terminal acetylation of proteins. CRC Crit Rev Biochem 1985;18:281e325. Gade W, Brown JL. Purification, characterization and possible function of aN-acylamino acid hydrolase from bovine liver. Biochim Biophys Acta 1981;66:286e93. Giardina T, Biagini A, Dalle Ore F, Ferre E, Reynier M, Puigserver A. The hog intestinal mucosa acylase I: subcellular localization, isolation, kinetic studies and biological function. Biochimie 1997;79:265e73. Gravieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493e501. Hwa JS, Park HJ, Jung JH, Kam SC, Park HC, Kim CW, et al. Identification of proteins differentially expressed in the conventional renal cell carcinoma by proteomic analysis. J Korean Med Sci 2005;20:450e5. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680e5. Lauterburg BH, Corcoran GB, Mitchell JR. Mechanism of action of N-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo. J Clin Invest 1983;71:980e91. Maceyka M, Nava VE, Milstien S, Spiegel S. Aminoacylase is a sphingosine kinase 1-interacting protein. FEBS Lett 2004;568:30e4. Madara JL, Trier R. Functional morphology of the mucosa of the small intestine. In: Johnson LR, editor. Physiology of the gastrointestinal tract. New York: Raven Press; 1987. p. 1209e49. Mariadson JM, Nicholas C, L’Italien KE, Zhuang M, Smartt HJM, Heerdt BG, et al. Gene expression profiling of intestinal cell maturation along the crypt-villus axis? Gastroenterology 2005;128:1081e8. Nguyen KT, Pei D. Purification and characterization of enzymes involved in the degradation of chemotactic N-formyl peptides. Biochemistry 2005; 44:8514e22. Noren O, Dabelsteen E, Hoyer PE, Olsen J, Sjo¨stro¨m H, Hansen GH. Onset of transcription of the aminopeptidase N gene at the crypt/villus transition zone during rabbit enterocytic differentiation. FEBS Lett 1989;259:107e12. Ogier G, Chantepie J, Deshayes C, Chantegrel B, Charlot C, Doutheau A, et al. Contribution of 4-methylthio-2-oxobutanoate and its transaminase to the growth of methionine-dependent cells in culture. Effect of transaminase inhibitors. Biochem Pharmacol 1993;22:1631e44. Olsen L, Hansen M, Ekstrom CT, Troelsen JT, Olsen J. CVD: the intestinal crypt/villus in situ hybridization database. Bioinformatics 2004;20: 1327e8. Perrier J, Giardina T, Durand A, Puigserver A. Specific enhancement of acylase I and acylpeptide hydrolase activities by the corresponding N-acetylated substrates in primary rat hepatocyte cultures. Biol Cell 2002;94: 45e54. Perrier J, Durand A, Giardina T, Puigserver A. Catabolism of intracellular Nterminal acetylated proteins: involvement of acylpeptide hydrolase and acylase. Biochimie 2005;87:673e85. Persson B, Flinta C, von Heijne G, Jornvall H. Structures of N-terminally acetylated proteins. Eur J Biochem 1985;152:523e7. Potten CS, Loeffler M. A comprehensive model of the crypts of the small intestine in the mouse provides insight into the mechanisms of cell migration and the proliferation hierarchy. J Theor Biol 1987;127:381e91. Raul F, Goda T, Gosse F, Koldowsky O. Short term effect of high-protein/low carbohydrate diet on aminopeptidase N in adult rat jejunum. Biochimie 1987;247:401e5. Sass JO, Mohr V, Olbrich H, Engelke U, Horvath J, Fliegauf M, et al. Mutations in ACY 1, the gene encoding aminoacylase 1, cause a novel inborn error of metabolism. Am J Hum Genet 2006;78:401e9. Scaloni A, Jones W, Pospischil M, Sassa S, Schneewind O, Popowicz AM, et al. Deficiency of acylpeptide hydrolase in small cell lung carcinoma cells lines. J Lab Clin Med 1992;120:546e52. Stocker P, Yousfi M, Salmi C, Perrier J, Brunel JM, Moulin A. Maackian 3-O-(60 -O-malonyl-b-D-glucopyranoside) from Oudneya africana, a powerful inhibitor of porcine kidney acylase I. Biochimie 2005;87:507e12. Tadjali M, Seidelin J, Troelsen JT. Transcriptome changes during intestinal cell differentiation. Biochim Biophys Acta 2002;1589:160e7.
N. Cigna et al. / Cell Biology International 31 (2007) 966e973 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad U S A 1979;76:4350e4. Traber PG. Regulation of sucrase-isomaltase gene expression along the crypte illus axis of rat small intestine. Biochem Biophys Res Commun 1990; 173:765e73. Traber PG, Wang W, Yu L. Differential regulation of cytochrome P-450 genes expression along rat intestinal crypt-villus axis. Am J Physiol 1992;263: G215e23. Tsunasawa S, Sakiyama F. Amino-terminal acetylation of proteins: an overview. Methods Enzymol 1984;106:165e70. Uttamsingh V, Keller DA, Anders MW. Acylase I catalyzed deacetylation of N-acetyl-L-cysteines and S-Alkyl-N-L-cysteines. Chem Res Toxicol 1998;11:800e9.
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Van Coster RN, Gerlo EA, Giardina TG, Engelke UF, Smet JE, De Praeter CM, et al. Aminoacylase I deficiency: a novel inborn error of metabolism. Biochem Biophys Res Commun 2005;338:1322e6. Vidrich A, Buzan JM, Cohn SM. Intestinal stem cells and mucosal gut development. Curr Opin Gastroenterol 2003;19:583e90. Wallace RJ, Min WK, Witt MW, Grant G, MacRae JC, Maeng WJ, et al. Uptake of acetylated peptides from the small intestine in sheep and their nutritive value in rats. Br J Nutr 1998;80:101e8. Ware JA, Reilly TP, Svensson CK. Cellular distribution of N-acetyltransferase activity in the rat small intestine. Biochem Pharmacol 1998;55: 1475e9. Weiser MM. Intestinal epithelial cell surface membrane glycoprotein synthesis I. An indicator of cellular differentiation. J Biol Chem 1973;248: 2536e41.
Acylase 1 expression in rat intestinal cryptevillus axis Natacha Cigna a,c, Cendrine Nicoletti a, Anne Durand b, Jean Claude Chaix a, Thierry Giardina a, Josette Perrier a,* a
BiosCiences FRE CNRS 3005, Universite´ Paul Ce´zanne e Aix-Marseille III, Faculte´ des Sciences, 13397 Marseille Cedex 20, France b Department of Molecular Microbiology, John Innes Centre, Norwich, NR4 7UH, UK c Institut Curie section de recherche, UMR 146, Bt. 110, Centre Universitaire, 91405 Orsay, France Received 11 December 2006; revised 31 January 2007; accepted 11 March 2007
Abstract Acylase 1 was investigated at the cellular level in the rat small intestine along the enterocyteedifferentiation axis. As confirmed by microscopic analysis, villus tip cells and crypt cells of rat jejunal mucosa were successfully separated using the Weiser method. The proliferating undifferentiated crypt cells showed much higher ACY 1 activity levels than the villus cells, with a 6.4-fold decrease as the cells migrated and differentiated along the cryptevillus axis. RT-PCR studies on mRNA extracted from isolated cells showed that ACY 1 mRNA was mainly expressed in crypt cells, reaching levels that were 12-fold higher than those recorded in other cell types along the whole enterocyte differentiation axis. It was concluded that the expression of ACY 1 in the intestinal crypt cells is regulated at the mRNA level. Immunohistochemistry revealed the expression of ACY 1 in the absorbing lineage cells from the ileal and colonic crypts and the absence of ACY 1 in the mucus producing goblet cells. These findings proposed ACY 1 as a new marker transcript for absorbing cells of intestinal crypt, which can be used to monitor the process of intestinal N-a-acetylated protein metabolism. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Acylase 1; N-terminal acetylated protein; Enterocyte differentiation; Weiser method
1. Introduction Enterocytes constitute the bulk of the epithelial population, which is mainly responsible for the absorptive process occurring after the action of specialized brush border hydrolases has been completed. Mammalian small intestinal epithelial cell production begins with crypt cells dividing and subsequently differentiating along the cryptevillus structural axis. Highly regulated mechanisms control the rate of proliferation and differentiation of enterocytes as they move along the cryptevillus axis during
Abbreviations: Ac, acetyl; ACY 1, acylase 1. * Corresponding author. BiosCiences FRE CNRS 3005, Laboratoire de Biochimie de la Nutrition, Universite´ Paul Ce´zanne e Aix-Marseille III, Service 342, Faculte´ des Sciences et Techniques, Avenue Escadrille Normandie Niemen, F-13397 Marseille Cedex 20, France. Tel.: þ33 4 91 28 84 44; fax: þ33 4 91 28 84 40. E-mail address: [email protected] (J. Perrier).
their relatively short lifetime (48e72 h in the rat) (De Santa et al., 2003; Vidrich et al., 2003). The process of mitosis ends and that of differentiation starts in the crypt. Differentiation is completed in the lower half of the villus, and there exists histological evidence that apoptosis occurs at the villus tip (Potten and Loeffler, 1987; Madara and Trier, 1987; Weiser, 1973; Altmann and Leblond, 1982; Gravieli et al., 1992). During differentiation of the mouse small intestinal epithelium, a general decrease in transcriptome complexity, using filter-based cDNA arrays, was demonstrated (Tadjali et al., 2002). The marked polarization of this cell, as well as the expression of hydrolytic brush border enzymes, provide specific markers which can be used to monitor the process of enterocyte differentiation (Weiser, 1973; Tadjali et al., 2002; Traber, 1990; Noren et al., 1989; Darmoul et al., 1991). Several studies have also shown that a few xenobiotic-metabolizing enzymes are present in the fully differentiated enterocytes (Traber et al., 1992; Cornell and Meister, 1976; Ware et al., 1998).
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.03.006
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An interesting online intestinal crypt/villus in situ hybridization database holds information for relative gene expression patterns in the mammalian intestine (Olsen et al., 2004). In mouse small intestine, a high expression of cell cycle and DNA synthesis genes occurs in the crypt, and cytoskeletal and lipid uptake genes are present in the villus, in respect with the enterocyte differentiation (Mariadson et al., 2005). In mammalian cells, as much as 70% of the cytosolic proteins can be a-N-acetylated, and the N-protected amino acids which are released during proteolysis must be free of amino acids before they can be used for protein synthesis (Driessen et al., 1985), since it seems hardly likely that they would be wasted in the cells. Post-translational acetylation of intracellular proteins and peptides is generally thought to be an efficient mechanism protecting them from proteolytic degradation in eukaryotic cells (Persson et al., 1985; Tsunasawa and Sakiyama, 1984). The NH2-a-acetylated amino acid released from the protein or the resulting peptide is catalyzed by the sequential action of the acylpeptide hydrolase (APH or acylamino acid releasing enzyme, EC 3.4.19.1) and acylases (ACY, EC 3.5.1.x) generating a free amino acid and an acetate, yielding the corresponding free amino acid during the process of intracellular protein catabolism. Acylase 1 (ACY 1 or N-a-acyl-L-amino acidamidohydrolase, EC 3.5.1.14) is by far the best characterized form, which hydrolyses aliphatic N-acyl-a-amino acids in tissue extracts (Gade and Brown, 1981; Perrier et al., 2005). It is worth noting here that the a-N-acetylamino acids which are usually found at the N-terminal position of the acylated protein, namely Acmethionine, Ac-alanine and Ac-serine (Driessen et al., 1985), correspond to the substrate specificity of ACY 1. The free amino acid released, especially the essential amino acid methionine (Met), is required for initiating protein synthesis by MettRNA formation, in relation to the polyamine synthesis or the trans-methylation pathway. Alternatively, polyamine synthesis abets normal cell proliferation (Ogier et al., 1993). However, until now, the exact physiological function of ACY 1 has not yet been completely elucidated. The fact that high levels of this enzyme have been detected in various tissues, including intestinal epithelia (Giardina et al., 1997), strongly suggested that this enzyme might have some additional physiological functions. For example, the expression of ACY 1 has been reported to be correlated with the existence and activity of acetylated growth factors in carcinoma cells and to be down-regulated in these cells (Scaloni et al., 1992; Hwa et al., 2005). ACY 1 might also regulate the activity of type 1 sphingosine kinase, which has been found to promote cell growth and to inhibit the apoptosis of tumor cells (Maceyka et al., 2004). ACY 1 deficiency was recently detected in lymphoblast cells from a young encephalopathic patient (Van Coster et al., 2005; Sass et al., 2006). Acylases also play an important role in the metabolism of the prodrug N-acetyl-L-cysteine (NAC), used in clinical practice to treat lung disorders (De Flora et al., 1995), and thus promote glutathione synthesis (Lauterburg et al., 1983). ACY 1 has been reported to be involved in xenobiotic bioactivation as well as in the interorgan processing of
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N-acetyl-amino acid-conjugated xenobiotic derivatives (Ssubstituted-N-acetyl-L-cysteine) (Uttamsingh et al., 1998). The transport of acetylated amino acids found to occur across the rabbit intestinal epithelium has suggested the involvement of ACY 1 in protein metabolism (Brachet et al., 1991; Wallace et al., 1998). It was also recently suggested that ACY 1 may play a protective role by degrading bacterial and mitochondrial N-formylated peptides in the rat intestine and may constitute the first line of defense against constant exposure to commensal bacteria (Nguyen and Pei, 2005). Certain features related to protein metabolism in the intestine are relatively unique to that tissue. Intestinal protein metabolism will be considered as occurring by two major mechanisms: (1) the formation, migration, differentiation and loss of whole cells, and (2) intracellular synthesis and degradation. In keeping with these two major mechanisms, the rat intestinal cryptevillus axis represents a useful model for study of the involvement of ACY 1 in N-acetylated protein/peptide metabolism, which represents a major event in the cell. Therefore, in the present study, ACY 1 enzyme activity was investigated at the cellular level in the rat small intestine along the enterocyteedifferentiation axis. We showed that ACY 1 is expressed at a high level in the undifferentiated crypt cells, from the absorptive lineage. In addition, we showed that ACY 1 expression in the rat intestinal cryptevillus axis is controlled at the mRNA level. Understanding ACY 1 cellular localization and expression in the intestine could give us indications on function(s) associated with this enzyme in this tissue. 2. Materials and methods 2.1. Materials N-Acetyl-L-methionine, p-nitrophenyl phosphate, L-leucine-p-nitroalanine, acid oxidase, horseradish peroxidase (type II), o-dianisidine were obtained from Sigma (St. Louis, MO, USA). L-amino
2.2. Preparation of intestinal epithelial cells Adult male Wistar rats weighing 260e300 g were used in this study. A 30cm segment of small intestine was removed, and villus and crypt epithelial cells were prepared directly by shaking the intestinal segment for successive periods in a solution of EDTA as previously described in detail (Weiser, 1973). Briefly, after removal, the intestine segment was rinsed thoroughly with ice-cold phosphate-buffered saline (PBS), and flushed twice with icecold PBS containing 1 mM DTT. The segment was incubated at 37 C for 15 min in a citrate buffer, pH 7.3, containing 96 mM NaCl, 1.5 mM KCl, 27 mM Na citrate, 8 mM KH2PO4 and 5.6 mM Na2HPO4 (Buffer A), then everted, filled and incubated at 37 C in Buffer B (PBS buffer containing 1.5 mM EDTA, 0.5 mM DTT). The intestinal segment was gently shaken at 37 C to remove adhering enterocytes from the villusecrypt axis, for nine incubation steps (F1eF9) after 4, 2, 2, 3, 4, 5, 7, 10 and 15 min, respectively. The cells from each fraction were collected by pelleting, washed twice in 10 ml fresh ice-cold PBS.
2.3. Enzyme activities Immediately after isolating the cells, the enzyme activities were measured. The cells were lysed with an Ultraturax for 10 s in ice-cold PBS buffer, the resulting homogenate was centrifuged at 100,000 g for 30 min and the supernatant was directly used for enzyme activities determinations.
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Aminopeptidase N (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and ACY 1 (EC 3.5.1.14.) activities were determined spectrophotometrically by using L-leucine-p-nitroalanine, p-nitrophenyl phosphate, N-acetyl-L-methionine, respectively, as the substrate (Weiser, 1973; Raul et al., 1987; Perrier et al., 2002). Specific activities were expressed as milliunits per milligram of protein. One unit was defined as the activity hydrolyzing 1 mmol of substrate/min at 37 C. Protein concentrations were determined using Bradford’s method (Bradford, 1976), with bovine serum albumin as the standard, to calculate the specific activities.
2.4. Immunofluorescence and electron microscopy After harvesting cells, the cells from fractions 1 (villus), 5 (middle crypte villus axis) and 9 (crypt) were fixed with 4% paraformaldehyde in PBS for 30 min at 4 C, and 200 ml were then adsorbed onto polylysine-coated slides for 30 min at room temperature, before being post fixed with 4% PFA for 10 min at room temperature and permeabilized for 10 min with 0.1% saponin in PBS. After several washes with PBS, the cells were saturated for 30 min in 10% fetal calf serum in PBS and incubated for an additional 30-min period at room temperature with a 1:100 dilution of the commercial Texas Red phalloidin derivative anti-actin. Lastly, after undergoing several washes, the cells were mounted with Vectashield (Vecto Laboratories, Burlingame, USA). Epifluorescence was detected and visualized with an Axiovert 200M Zeiss microscope equipped with a Hamamatsu camera. For the electron microscopy procedure, the tissue and the cell fractions (F1, F5, F9) were fixed for 1 h in 2.5% glutaraldehyde in cold PBS. After being washed with PBS, they were post-fixed in 2% OSO4 in PBS for 1 h at room temperature, and then dehydrated in a graded ethanol series and embedded in Epon 812. Ultrathin sections (50e60 nm) were layered on Maxtaform HR 24Cu/Rh (200 mesh), stained with uranyl acetate for 20 min and lead citrate for 10 min, and examined with a transmission electron microscope Zeiss EM109, at 80 kV. The tissue immunofluorescence reaction was performed on semithin frozen sections (1 mm), by using the anti-ACY 1 polyserum (1:200) (Abnova Corporation Taiwan) or the normal mouse serum (1:200) and a commercial fluorescein derivative of anti-mouse Igs diluted 200 times as secondary antibody (both from Sigma Corporation, France).
2.5. Total RNA isolation and RT-PCR Cells were washed once with cold PBS, and 200 ml of RNAwizÔ isolation solution (Ambion, Inc. Austin, USA) were added to each sample. Total RNA was immediately extracted in line with the manufacturer’s instructions and stored at 20 C until used. Total RNA (101 mg) was used for RT and the cDNA synthesis reaction was performed in keeping with the instructions given by Invitrogen, using their SuperScriptÔ III/PlatiniumÒ Taq polymerase Onestep RT-PCR System (Invitrogen Life Technologies) at 50 C for 20 min, ending in a 5-min heating step at 95 C to inactivate the RT enzyme inactivation. PCR primers were designed for use in this study, based on the EMBL/GenBank (accession no.: Rattus b actin, NM031144; APN, Y18765; ACY 1, NM001005383): the primer sequences are shown in Table 1. Each PCR cycle included 30 s of denaturation at 94 C. The annealing step was carried out at an appropriate temperature, as indicated in Table 1, using 0.4 mmol of primer pairs, for 2 min, whereas the extension step was performed at 72 C for 3 min, using an overall volume of 50 ml and a total number of 30 PCR cycles in a MastercyclerÒ gradient 5331 thermocycler (Eppendorf). Lastly, aliquots of the PCR products were separated on a 1.5% Triseacetate EDTA agarose gel, stained with ethidium bromide, and photographed under UV light. The signal intensity of each band was normalized by comparing it with the intensity of b-actin, using the PhotoCapt software.
2.6. Immunoblotting Cells were washed with PBS buffer. The resulting pellets were homogenized in buffer containing 10 mM sodium phosphate, 150 mM sodium chloride (pH 7.4), and centrifuged at 12,000 g. Protein concentration in the supernatant was determined according to Bradford (1976). An equal loading of proteins (5 mg) were separated in a 10% SDS-PAGE gel as described by
Laemmli (1970). Electroblotting was performed according to Towbin et al. (1979). As primary antibody the anti-aminoacylase 1 polyclonal serum was used (Abnova, Taiwan) (dilution 1:1000). A horseradish peroxidase-conjugated goat anti mouse antibody (dilution 1:5000) was used as secondary antibody (Pierce, Perbio Science Company) and the super signal West Pico Chemiluminescent substrate (Pierce) was used to detect the ACY 1 protein.
2.7. Statistical analysis All the enzyme activity data recorded were assessed using the Student’s t-test for paired data. Differences were taken to be significant at p < 0.05.
3. Results 3.1. Actin network and cellular profile changes in rat intestinal cryptevillus axis Under the experimental conditions used to freshly isolate enterocytes from adult rats, the morphology of the cells obtained was found to be normal. As was to be expected (Fig. 1A), the highly differentiated villus cells (fraction 1, F1) were found to have a polarized morphology, closely packed apical microvilli or brush borders (BB) and its terminal web (TW); the cells in the intermediate stage of differentiation observed in the middle of the villusecrypt axis (fraction 5, F5) were found to be characterized by their very small size and their very slight apical microvilli (A), which contrasts strongly with the undifferentiated, non-polarized crypt cells from F9 showing no microvilli. A photomicrograph of villus cells (F1), middle cryptevillus axis cells (F5) and crypt cells (F9) from isolated enterocytes showed the presence of a normal peripheral actin network after interactions had been performed with an anti-actin phalloidin-Texas Red-conjugated moiety (Fig. 1B). Cellular sections were taken from the cell fractions of F1, F5, and F9 collected. Electron microscopy examination of cell fractions (Fig. 1C) showed that the closely adjacent enterocytes had a highly polarized pattern of organization and numerous microvilli were observed in the brush border (BB) in F1; the cells and the microvilli network, were much smaller in F5eF9. In the last fraction (F9), the cells were found to be small and round, without any microvilli; these characteristics are typical of undifferentiated cells in the deep crypts. Microscope analysis of electron micrographs showed that a 3-fold increase in the absolute size of these cellular fractions occurred as the cells differentiated (28 mm, 18 mm, 9 mm from F1 to F9, respectively). These findings show that epithelial cells were successfully isolated here from along the cryptevillus axis with minimum damage and contamination of cells from the lamina propria, with respect to the normal enterocyte differentiation process. 3.2. Enzyme expression changes along the rat intestinal cryptevillus axis and immunoblotting As expected, the specific activities of all villus marker enzymes used (alkaline phosphatase (AP), aminopeptidase N (APN) increased sharply from F9 (the fraction from the bottom
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Table 1 Primer sequences corresponding to the rat cDNA sequences used for RT-PCR analysis of each enzyme investigated in this study (accession no. Rattus b actin, NM031144; aminopeptidase N (APN), Y18765; aminoacylase 1 (ACY 1), NM001005383) Gene
Primer
Nt
Annealing temperature ( C)
% GC
Nucleotide sequence (50 to 30 )
ADNc size expected (bp)
ACY 1
d81 S c294 AS 5’ primer 3’primer S2 AS3
34 28 32 32 19 20
59 65 60 60 48 45
47 71 53 53 57 45
GCT AAA CTC CCA CAC AGA TGT GGT ACC TGT CTT C TCC CGC TTC CTG GGA CCA GCT CTG CAG C ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC AAC AAG GAG CGG GTG GTC A TCT GAA TCC TTC CTC CAG TT
666
b-Actin APN
838 770
S, sense primer (foward primer); AS, antisense primer (reverse primer); Nt, nucleotide primer length.
crypt cells) to F1 (the villus tip cell fraction) (Fig. 2A). The specific activity of AP increased 19-fold from 28 24.7 to 518 100 (U/mg), and the specific activity of APN increased 11-fold from 13 3.5 to 140 55 (U/mg). Inversely, our data also showed that the specific activity of acylase 1 (ACY 1)
decreased 6.4-fold from 16 3.1 to 2.5 1.8 (U/mg) from F9 (undifferentiated cells) to F1 (differentiated cells). Immunoblotting blotting (Fig. 2B) with an anti-aminoacylase 1 polyclonal antibody revealed a decrease of the band corresponding to ACY 1 in Weiser’s fraction from F9 to F1. It
Fig. 1. Actin network and cellular profile changes in rat intestinal crypt- villus axis. (A) The Normaski view and (B) its corresponding specific phalloidin-Texas Red complex against the actin network. (C) The electron microscope image. The actin network is well represented in the brush border (BB), basolateral membrane (BL), apical membrane (A), terminal web (TW). Note: nucleus (N), gobelet cell (GC). Fraction 1 (F1), villus cells; fraction 5 (F5), middle villusecrypt axis cells; fraction 9 (F9), crypt cells. Bar 2 and 10 mm.
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A
A
F1
Cell fractions F5
F7
F9
1000
β-Act. AP
ACY 1
APN
APN 100
Relative mRNA levels
SA (U/mg)
ACY 1
10
40 30
B
20 10 0 F9
(F1-F9)fold
1
0
22
14
24
12
F1
F5
F7
APN
22
18
ACY1
2
13
1 F1 Villus
F2
F3
F4
F5
F6
F7
F8
F9 crypts
Villus-crypts cells fractions
B ACY 1
Fig. 2. Enzyme activities profile in rat intestinal cryptevillus axis homogenates. (A) The specific activities (U/mg protein) of AP, APN and ACY 1 were determined as indicated in Section 2. Values are means obtained from duplicate samples, and comparable results were obtained in four independent experiments. (B) The corresponding immunoblotting profile with the anti-aminoacylase 1 antibody.
therefore seems likely that ACY 1 enzyme may gradually decrease as the cells migrate and differentiate from the crypt to the villus. 3.3. Changes in mRNA expression by ACY 1 along the rat intestinal cryptevillus axis Fig. 3A shows the RT-PCR products separated on 1.5% agarose gel. The size of b-actin, APN and ACY 1 mRNAs was consistent with the predicted values (respectively 838 bp, 770 bp and 667 bp as shown in Table 1). As was to be expected, b-actin mRNA expression occurs in the epithelial cells located along the whole cryptevillus axis, and the levels of expression were found to increase slightly from F9 to F1 (Fig. 3A), reaching a 1.3-fold increase in mRNA level in the completely differentiated cells. These results are in good agreement with the pattern of immunofluorescence labeling shown in Fig. 1B. In addition, APN mRNA levels were higher in cells F1 and F5 than in fractions isolated from the lower part of the villus (F7 and F9, Fig. 3A), corresponding to a 22-fold relative mRNA increase from F9 to F1 (Fig. 3B). These results matched the 11-fold increase in the APN enzyme activity observed from F9 to F1 (Fig. 2A). In marked contrast, the ACY 1 mRNA levels were higher in F7 and F9 than in F1 and F5 (Fig. 3A), and a 12-fold relative decrease in the mRNA levels
Cell fractions Fig. 3. RT-PCR products in intestinal epithelial cells along the cryptevillus axis. Cells were isolated separately from rat jejunum (F1 villus, F5 middle villuse crypt axis and F7, F9 crypt), and the mRNA levels were determined using semi-quantitative RT-PCR methods, as described in Section 2. Typical ethidium bromide (EtBr) staining of RT-PCR products is shown in (A), for aminopeptidase N (APN), b-actin (Act), aminoacylase 1 (ACY 1). In the graph above (B), the RT-PCR signal intensity was normalized by comparing each value with that obtained on b-actin, used as the internal PCR control values. Results are means SE of values obtained on five rats.
was found to occur from F9 to F1 (Fig. 3B). These results matched the 6.4-fold decrease in ACY 1 enzyme activity observed from F9 to F1 (Fig. 2A). The distribution pattern of ACY 1 mRNA correlates well with the enzyme activity pattern reported above. A translational regulation of ACY 1 expression may occur along the cryptevillus axis of the small intestine, which would account for the proliferative status and/ or for the protein metabolism status of the cells. 3.4. Cellular localization of acylase 1 along the rat intestinal cryptevillus axis Fig. 4A shows the patterns of immunolabeling observed with the anti acylase 1 antibody on sections from ileal and colonic tissue segments along the digestive tract in the rat, also confirmed by Western blotting experiments (data not shown). The ACY 1 labeling is restricted to the cytosol of the crypt cells, between the nucleus and the terminal web, with a tendency for apical staining, in the ileum and in the proximal colon. The ACY 1 labeling is evident in both lower and upper crypt areas. The absence of acylase 1 labeling in the differentiated cells from ileal villi and colonic villi (the flat surface epithelium or plateau) is well observed. The acylase 1 labeling is also absent from the goblet cells (GC) from the secretory lineage, numerous in the colon. The immunolabeling study revealed also that ACY 1 is
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Fig. 4. Immunolocalization of acylase 1 in the digestive tract. Sections from villi and crypt regions of the rat ileum and proximal colon are revealed with the antiACY 1 plyclonal serum (A) or normal mouse serum (B). The Normaski view (left) and immunofluorescence labeling of the same field (right) are given side by side. Note the presence of the cryptevillus gradient. Bar 10 mm; BB, brush border; LP, lamina propria; N, nucleus; GC, goblet cell; L, gut lumen; PC, Paneth cell.
not expressed in other cell types among the lamina propria epithelial cells and in Paneth cells (PC) (data not shown). The specificity of staining was checked using a normal mouse serum as a control. Results clearly show the absence of staining (Fig. 4B). Here again, it therefore seems likely that ACY 1 enzyme immunofluorescence labeling may gradually decrease as the cells migrate and differentiate from the crypt to the villus, in the digestive tract. The distribution pattern of ACY 1 immunolabeling correlates well with the enzyme activity and the mRNA patterns. We proposed ACY 1 as a new important marker enzyme, for crypt absorbing cells in rat intestine. 4. Discussion ACY 1 is found to be expressed in the mucosa throughout the gastrointestinal tract where is thought to play a role in Nacetylated protein metabolism (Giardina et al., 1997). Also, the activities of alkaline phosphatase, aminopeptidase N, and ACY 1 were studied here in normal rat intestine, after crypt cell proliferation. As was to be expected, AP and APN were found to be low from normal rat intestinal crypt cells (Weiser, 1973; Noren et al., 1989), but to reach measurable levels after the cells had migrated and matured, reaching the non-proliferative state and the functional villus stage. Measurable levels of the cytoplasmic enzyme ACY 1 were detected in the rat
intestine. In strong contrast with these other enzymes, ACY 1 enzyme activity showed a 6.4-fold decrease during crypt cell differentiation. In this model, mucosal crypt epithelial cells divide in the zone of cell proliferation towards the bottom of the crypt. Also, our results clearly show that the ACY 1 mRNA is down-regulated during crypt cell differentiation indicating that the ACY 1 enzyme activity reflected well the mRNA expression level of the protein. We proposed that ACY 1 expression is controlled at mRNA level in rat intestinal cells. Also, our method of tissues (ileon and colon) immunolabeling appears to give the same patterns of ACY 1 enzyme activity, protein and mRNA expression. No acylase 1 labeling is observed in the goblets cells (secreting protecting mucins), in Paneth cells and in other epithelial cell types. Based on these data, the activity of ACY 1 seems to be coordinated in specific ways depending on the cell type (absorptive/secretory lineage), the functional state of the cell, and on whether the crypt cells were in the proliferative undifferentiated state. These data supported well the idea that ACY 1 may contribute to the process of N-acetylated proteins catabolism, which, logically occurs preferentially in the proliferative cells as crypts cells. ACY 1 has also been recently described as a SphK1-interacting protein, which may affect the physiological functions of the SphK1 enzyme, promoting cell growth and
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inhibiting the apoptosis of mammalian kidney HEK 293 tumor cells (Maceyka et al., 2004). The fact that high levels of ACY1 were detected here in the proliferative crypt cells (lower and upper area) is consistent with the idea that this enzyme may be involved in cell growth via interactions with SphK1. It seemed to be worth investigating whether the biological utilization of intrinsic and food N-acetylated proteins, as well as that of acetylated amino acids, and N-acetyl-amino acid-conjugated xenobiotic derivatives (S-substituted-N-acetyl-L-cysteine) (Uttamsingh et al., 1998), might affect the activity of ACY 1 enzyme in an undifferentiated/differentiated polarized living enterocyte cells. ACY 1 is activated by its substrates (N-acetyl-amino acid) in rat and pig primary hepatocyte cell cultures (Perrier et al., 2002). Also, the presence of ACY 1 which catalyses the deacetylation of the haloalkene-derived mercapturates in several tissues studied may contribute to the organ-selective toxicity of these mercapturates (Cooper et al., 2002). The strong activity of ACY 1 in the crypt area correlates well with an active amino acid turnover found in the proliferating undifferentiated crypt cells from the small intestine and may also explain the intestine toxicity of acetylated-xenobiotics. It would be interesting for us to analyze the ACY 1 expression along the cryptevillus axis under N-acetylated substrates and under Nacetylated xenobiotic conditions. Therefore, we have described recently a natural phenolic compound as a powerful competitive inhibitor of porcine kidney ACY 1 (Stocker et al., 2005). For all the conditions discussed above, the rat intestinal cryptevillus axis could represent a good alternative approach to the in vitro colon cancer cell line Caco-2 and could give us indications on in vivo function(s) associated with ACY 1 in this tissue. Studies to elucidate these points are currently in progress. Acknowledgements We are grateful to Dr Jessica Blanc for revising the English manuscript. References Altmann GG, Leblond CP. Changes in the size and structure of the nucleolus of columnar cells during their migration from crypt base to villus top in rat jejunum. J Cell Sci 1982;56:83e99. Brachet P, Gaertner H, Tome D, Dumontier AM, Guidoni A, Puigserver A. Transport of N-a (or N-e)-L-methionyl-L-lysine and acetylated derivatives across the rabbit intestinal epithelium. J Nutr Biochem 1991;2:387e94. Bradford M. A rapid and sensitive method for the quantification of microgram quantity for protein utilizing the principle of dye binding. Anal Biochem 1976;72:248e54. Cornell JM, Meister A. Glutathione and g-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa. Proc Natl Acad Sci U S A 1976;73:420e2. Cooper AJL, Bruschi AS, Anders MW. Toxic halogenated cysteine S-conjugates and targeting of mitochondrial enzymes of energy metabolism. Biochem Pharmacol 2002;64:553e64. Darmoul D, Rouyer C, Blais A, Trugnan G. Dipeptidyl peptidase IV expression in rat jejunal crypt-villus axis is controlled at RNA levels. Am J Physiol 1991;261:G763e9. De Flora S, Cesarone CF, Balansky RM, Izzotti A. Chemopreventive properties and mechanisms of N-acetylcysteine. J Cell Biochem 1995;22:33e41.
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