1Developmental Gastroenterology Laboratory, Massachusetts General Hospital, Boston, Massachusetts; 2The Aga Khan University Hospital, Karachi, Pakistan; and 3Combined Program in Pediatric Gastroenterology and Nutrition, Children's Hospital, Boston, Massachusetts
Submitted 19 November 2004 ; accepted in final form 20 March 2005
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ABSTRACT |
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clathrin; caveolae; endocytosis; intestinal development
Pathological response to CT is dependent on a complex, highly coordinated sequence of events. First, it is released from the bacterium onto the intestinal surface. Then, it binds to enterocyte receptors and is endocytosed as a toxin-receptor complex leading to the accumulation of intracellular cAMP. Finally, stimulation of chloride channels actively secretes Cl (32).
CT is a pentavalent protein comprised of one active (A) and five binding (B) subunits that interact with an enterocyte microvillus glycolipid ganglioside (GM1). In polarized epithelial cells, activation of signal transduction by CT requires endocytosis of toxin-receptor complexes into the apical endosome, translocation by retrograde transport into the Golgi cisternae or endoplasmic reticulum, and finally movement of the toxin to its site of action in the basolateral membrane (reviewed in Refs. 18, 23, and 32). In most cell types, a large fraction of the CT receptor GM1 clusters in caveolae, and therefore GM1 has been used as a marker for caveolae (glycolipid/raft domains). Previous studies by Lencer and colleagues (45, 46) and others (30, 38) have demonstrated that attachment to GM1 partitions CT into detergent-insoluble glycosphingolipid-rich (DIG) membranes that are critical to the toxin's function. Glycolipid rafts are DIG membranes that can be isolated from most mammalian cell types by virtue of their insolubility in a nonionic detergent (e.g., Triton X-100) and their floatation characteristics on low-density sucrose gradients (46).
Endocytosis of CT can occur in dynamin- and caveolae-mediated pathways in multiple cell types including enterocytes (17). However, CT can also be taken up by a clathrin-dependent pathway (10, 13). There has been compelling evidence that in some cell types, CT can be taken via clathrin- and caveolae-independent pathways (26, 41), and these different endocytic pathways can function simultaneously. In studies using mature enterocytes (e.g., T84 cells), CT has been shown to interact and be endocytosed by caveolae/lipid raft domains (45). T84, a crypt-like adult epithelial cell line traditionally used for these studies, was selected because other intestinal epithelial cell lines (Caco-2, HT-29, etc.) have less well-defined secretory and endocytic pathways (11, 48). The difference has also been shown in studies in this laboratory (data not shown). To distinguish endocytosis by the caveolae pathway from other endocytic pathways, particularly from the clathrin-dependent pathway, agents [filipin and methyl--cyclodextrin (M
CD)] that gradually deplete membrane cholesterol have been previously used as caveolae/lipid raft endocytosis inhibitors (37, 45).
During development, the immature intestine passes through a cellular phase in which enterocytes are highly endocytic (43). The existence of an enhanced endocytic capacity by the developing intestine may play a critical role in growth and differentiation due to an increased capacity to engulf intact hormones and growth factors (16, 42). However, the immature intestine is not able to distinguish between substances necessary for growth and development from those that can be potentially harmful. This enhanced capacity to endocytose microorganisms and protein antigens may help to explain the increased susceptibility of preterm as well as term infants to enteric infection and gastrointestinal allergic disorders (15).
In previous animal studies (5, 6, 36) and in a recent human study (24) from this laboratory, we have shown that at lower doses of CT, the immature intestine responds more actively than the mature intestine by increasing cAMP accumulation and an excessive chloride secretion. To further explain this increased response to CT by the immature gut and with the realization that the immature enterocyte has an increased endocytic capacity (7, 8, 42, 43), we have begun to examine endocytosis of CT as a potential contributing factor in this process.
Accordingly, we hypothesize that an age-dependent difference in CT uptake in immature vs. mature enterocytes may, in part, be due to differences in binding to the surface GM1 receptor and enhanced CT endocytosis leading to a subsequent excessive elevation of cAMP and enhanced secretion.
In this study, we utilized H4 and T84 cell lines as models for the study of the developmental response to CT in humans. H4 cells are a nontransformed human fetal small intestinal cell line and have morphologic and functional characteristics typical of intestinal crypt-like enterocytes. They are poorly differentiated (34) and therefore represent an appropriate cellular model for defining the CT response in the immature intestine.
T84 is a well-characterized and highly differentiated human colonic carcinoma cell line that functions as a mature crypt-like cell and is a useful model for studies of Cl secretion as well as endo- and transcytosis of CT (20, 22, 44). When grown on collagen-coated permeable supports, T84 cells form monolayers with increased high transepithelial resistance and consist of highly polarized columnar cells with structural similarity to adult intestinal crypt cells (25). It has been demonstrated that T84 cells have all the necessary cellular components to produce pathophysiological components to produce a pathophysiological response to CT (19, 20, 22).
By carefully examining the different endocytic processes in immature vs. mature intestinal epithelia, we provide evidence to support a developmental difference in endocytosis of CT as a partial explanation for its accentuated secretory response in the immature gut.
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MATERIALS AND METHODS |
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Cell cultures. T84 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown on collagen-coated Transwell inserts (Costar, Cambridge, MA) or tissue culture plates as previously described (22). Cell passages 5163 were used for all experiments. H4 cells are nontransformed human fetal small intestinal cells generated in our laboratory (obtained from a 16-wk-old therapeutically aborted fetus) (34) with the approval from the committee for human studies in Massachusetts General Hospital, which are comparable to the intestinal epithelial cell line (IEC)-6 rodent crypt cell line developed by Quaroni et al. (31). The cells were maintained in DMEM supplemented with 10% FBS, 20 mM L-glutamine, human recombinant insulin (0.5 U/ml), and standard antibiotics. Cell passages 1022 were used for these experiments. Cells were plated on six-well tissue culture plates and grown to confluence before they were used in experiments.
Endocytic inhibitors.
Cells were treated with various endocytic inhibitors to distinguish clathrin-dependent from -independent endocytosis using the dose previously shown to have an optimal effect in cell lines (29, 38, 41, 45). For clathrin-dependent endocytosis, cells were preincubated with 20 µg/ml chlorpromazine (CPZ). Other cells were pretreated with 1 µg/ml filipin, or 1 mM MCD for disruption inhibition of caveolar endocytosis. BFA (1 µg/ml) was used to block CT activity. Specificity of each treatment modality was evaluated by incubating cells with CT (20 nM) at 4°C to let binding take place or 37°C for 2 h, at which temperature the endocytosis of CT occurs, and by monitoring the internalization of CT by immunoblotting and fluorescence staining.
Trophic factors. Hydrocortisone was selected as a known maturation factor for this study (13, 28). H4 cells, and in some cases T84 cells, were grown to near confluence and then incubated with 1 µM hydrocortisone for 1 wk. After incubation, corticosteroids were washed away, and cells were then subjected to various stimuli and further analysis.
Animals. Four-week-old homozygous SCID mice (Jackson Laboratory, Bar Harbor, ME) were housed in a specific pathogen-free facility and fed with rodent chow and water ad libitum. Female Sprague-Dawley rats were purchased from Taconic (Germantown, NY). The animals were kept in an institutional animal facility with a 12:12-h light-dark cycle. They were fed rat chow and water ad libitum. This study has been approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. Experiments were performed when the litters were 2 wk of age for the preweaned group and 5 wk of age for the postweaned group. Adult animals were killed at 10 wk or later.
CT-response in human fetal small intestine xenografts. Human fetal small intestine xenografts were supplied by the Xenograft Core Facility in our laboratory. Acquisition of the fetal tissue complied with the American Physiological Society publications policy on the publication of research on human fetuses, fetal tissue, embryos, and embryonic cells. Fetal tissue was obtained with the approval from the Partners Human Study Committee and with signed permission. Briefly, fetal small intestines from 10- to 14-wk-old fetuses were transplanted subcutaneously into SCID mice and kept in a pathogen-free environment for 3 mo as previously reported (35). We have shown in several recent publications that fetal tissue transplanted into SCID mice reepithelializes and matures in a manner similar to that which occurs in the intrauterine environment as documented by expression of microvillous disaccharides and other enzymes (35). Mice were injected subcutaneously with hydrocortisone (50 mg/100 g body wt) or an equal volume of normal saline 1 wk before CT exposure. Tissue was obtained by carefully removing all the serosal and muscle tissues surrounding the intestinal mucosa and cutting them into small pieces using a surgical blade while immersed in HBSS containing 0.1% BSA. After exposure to CT or media under conditions previously used for cells at 4°C, tissues were washed with ice-cold HBSS to remove unbound CT, half of the samples were placed in media at 4°C to measure the surface binding of CT, and the other half were placed in media at 37°C to determine internalization. After incubation, tissues were washed with either HBSS or acidified HBSS in some samples to remove surface CT before being snap frozen in liquid nitrogen and stored at 70°C for further analysis.
Isolation of enterocytes from rat small intestine. Pre- and postweaned and adult rat small intestinal enterocytes were isolated by a method previously described (3). Briefly, a 3040 cm length of small intestine (jejunum and ileum) was excised and washed with ice-cold HBSS (in mM: 140 NaCl, 5 KCl, 10 HEPES, 1.3 CaCl2, 0.5 MgCl2, 0.36 K2PO4, 5.5 D-glucose, and 4.2 NaCO3, pH 7.2, with 2.5 M Tris) and inverted. The preparation was then immersed in ice-cold Ca2-free isolation buffer (in mM: 30 EDTA, 52 NaCl, 5 KCl, 10 HEPES, 2 DTT, and 60 HCl, pH 7.1, with 2.5 M Tris) and shaken for 5 min to disaggregate mucus. After discarding the first wash, intestine was washed twice in the same buffer and shaken for 10 min each time. The successive fractions were collected after 5 min centrifugation at 100 g. Pellets were rinsed once with HBSS and resuspended in DMEM containing 0.1% BSA. Cells were maintained at 4°C and used within 6 h of enterocyte isolation. The CT internalization in these cells was carried out as described in the previous section.
Cell lysis and fractionation. H4 and T84 cells were grown to confluence, washed with HBSS, and incubated with 20 nM CT for 1 h at 4°C. After reaching a steady state of CT binding, cells were washed with HBSS to remove unbound CT and incubated either at 4 or 37°C for 1 h. Surface-bound toxin was washed away with acidified HBSS (pH 2.5) in specific additional experiments as detailed elsewhere (46). Cells were then scraped into Tris-buffered solution (4°C) containing 1% Triton X-100 (1% TTBS, 10 mM Tris, 150 mM NaCl, 1% Triton X-100), 1 mM PMSF and a complete protease inhibitor tablet. Triton-soluble proteins were collected and membrane-associated proteins were solublized in 1% TTBS containing 0.5% SDS. Isolated mucosal tissues from xenografts were homogenized in 1% TTBS. Triton-soluble and insoluble fractions were collected as described above. Both fractions were analyzed by SDS-PAGE and Western blotting.
Sucrose equilibrium density centrifugation. Confluent monolayers of polarized or nonpolarized T84 and H4 cells were used for isolation of DIGs. All steps were completed at 4°C as described in detail elsewhere (1, 46). Briefly, cells were scraped into 2 ml of 1% TTBS and homogenized with seven strokes in a tight-fitting Dounce homogenizer on ice. The subsequent homogenate was adjusted to 40% sucrose with equal volume of 80% sucrose in 1% TTBS, layered under a linear 530% sucrose gradient, and centrifuged at 39,000 rpm for 1620 h in a swinging bucket rotor (model SW 41; Beckman Instruments, Palo Alto, CA). Sequential 0.5- or 1-ml fractions were collected from the top of the gradient and a 20-µl sample from each fraction was analyzed by SDS-PAGE and Western blotting. Sucrose density was monitored by refractometry.
Measurement of cAMP. The content of cAMP in the cells treated with various agents and CT was measured by a cAMP EIA system from Amersham, as previously described (24).
Fluorescent microscopy of CT binding and internalization. H4 and T84 cells were plated on glass coverslips until confluence. After incubation with CTB-B at 4 or 37°C, cells were washed three times with HBSS at 4°C, and in some experiments, surface-bound CTB was disassociated and washed away with an acid wash. Cells were fixed with 4% ice-cold paraformaldehyde in PBS for 10 min at 4°C, blocked with 3% BSA in TBS (10 mM Tris, 150 mM NaCl pH 8.0) for 1 h before incubation with a Cy3-streptavidin conjugate for 1 h, and examined using a Zeiss Axiophot photomicroscope (Germany).
Immunoblotting. H4 and T84 cells were grown to confluence and subjected to various treatments. Cell lysates were collected and added onto 1020% denaturing Tris·HCl polyacrylamide gels (Bio-Rad, Hercules, CA). Following electrophoresis, proteins were transferred to nitrocellulose membranes. After blocking with a 5% nonfat milk solution, membranes were probed with primary and secondary antibodies and visualized by enhanced chemiluminesence (Pierce, Rockford, IL). Quantification of the protein was measured by densitometry and expressed as the relative density unit ratio between the protein of interest and GAPDH (as a normal control for loading).
Statistics. Data were analyzed using StatView 4.1 software (Brainpower, Calabasas, CA).
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RESULTS |
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Effect of endocytic inhibitors on the internalization of CT in H4 compared with T84 cells.
We next investigated the pathways of CT internalization in immature (H4 cells) vs. mature human enterocytes (T84 cells). Using endocytic pathway-specific pharmacological inhibitors, we examined differences in endocytosis between these two cell lines. We first examined the effect of two classes of drugs on the uptake of CT by these cells. Cationic amphiphilic drugs, such as CPZ, act on the clathrin-dependent endocytic pathway and inhibit receptor-mediated endocytosis by reducing the number of coated pit-associated receptors. In contrast, cholesterol-binding agents, such as filipin or MCD, disrupt caveolae structure and function, thus blocking caveolae-mediated endocytosis. Cells were treated with CPZ, filipin, or M
CD for 30 min and were exposed to CT at 4 or 37°C for 2 h. They were then washed and surface-bound toxin was removed by an acid wash (2 times) in selective experiments. CT internalization was monitored using anti-CTB antiserum and immunoblotting. We demonstrated that CT internalization in H4 cells was markedly inhibited by CPZ but was not significantly affected by filipin and M
CD (Fig. 2A). In contrast, internalization of CT was significantly inhibited by filipin and M
CD in T84 cells, whereas CPZ only partially inhibited CT uptake (Fig. 2B).
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Age-related differences of CT uptake in pre- and postweaned and adult rat small intestinal epithelia.
To further validate the differences found in CT uptake between immature and mature IECs, we investigated whether there was a difference in CT internalization in enterocytes from pre- and postweaned and adult rat small intestinal epithelia. Western blot analysis from pre- and postweaned and adult rat enterocytes demonstrated an age-dependent decrease in CT uptake, and preweaned rat enterocytes exhibited the highest rate of internalization of CT compared with postweaned and adult rats (Fig. 7A). Since the investigations of H4 and T84 cells with pathway-specific endocytic inhibitors suggested different CT-uptake processes in these cells, we further investigated the endocytosis of CT in pre- and postweaned rat enterocytes. The isolated small intestinal enterocytes from pre- and postweaned rats were pretreated with CPZ, filipin, and MCD for 45 min before incubation with CT. Our preliminary study suggested a parallel pattern of endocytosis of CT presented in pre- and postweaned rat enterocytes to that in H4 vs. T84 cells. As depicted in Fig. 7B, CPZ (20 µg/ml) demonstrated a greater inhibition of CT internalization in preweaned rat enterocytes, whereas filipin (1 µg/ml) and M
CD (1 mM) showed greater inhibition of CT internalization in the postweaned rat intestine.
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DISCUSSION |
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The endocytosis of CT in T84 cells has been previously well characterized to be mainly a caveolae/raft-mediated process (46). To compare the caveolae-mediated endocytosis for CT with other endocytic pathways, we first had to define the endocytic pathways of CT in H4 cells. H4 cells are nontransformed human fetal small intestinal cells and comparable in structure and function to the primary IEC-6 rodent crypt cell line developed by Quaroni et al. (31). H4 cells have morphologic and functional characteristics typical of intestinal enterocytes, but they are poorly differentiated (34) and represent an appropriate cellular model for defining the CT response in the immature intestine. These studies demonstrate that CT binds to the H4 cellular surface where a small fraction of surface-bound CT is associated with sphingolipid/cholesterol-enriched detergent-insoluble domain/raft, e.g., the caveolae-mediated endocytic pathway. However, the initial step of CT internalization cannot be blocked by drugs that deplete cholesterol, such as MCD or filipin, which inhibits caveolae-mediated endocytosis but can be blocked by CPZ, which causes aberrant endosomal accumulation of clathrin on internalized vesicles (e.g., inhibits the clathrin endocytic pathway). These findings are indistinguishable from internalization of the transferrin receptor, previously reported by Shogomori and Futerman (38), which is taken up by enterocytes via an exclusively clathrin-dependent mechanism, suggesting that in H4 cells, the endocytosis of CT is mediated principally by a clathrin-dependent mechanism. This endocytic pathway results in a more rapid and efficient internalization of the toxin-receptor complex compared with the caveolae-mediated pathway operational in adult enterocytes.
T84 cells, an established model for the adult enterocyte, have also been used as a model for the study of CT endocytosis and action on intestinal epithelia (1, 21). T84 cells when grown on collagen-coated permeable supports form morphologically well-differentiated monolayers and express the necessary ion channel symporters and pumps in the appropriate membrane domains for vectorial transport (22, 29). It has been demonstrated that T84 cells have all the necessary cellular components to produce a pathophysiologic response to CT. We confirmed that T84 cells exhibit a caveolae-mediated endocytosis of CT as previously reported by other studies (1, 46). In contrast to the internalization of CT in H4 cells, this process can be blocked by the cholesterol depletion agents filipin and MCD, confirming its caveolae-associated nature.
A central dogma of the caveolae/glycolipid raft endocytosis has been that the "flask shaped" invagination of caveolae is a specific consequence of the association of caveolin-1 with selected raft domains (reviewed in Ref. 30). Caveolin-1 is essential for the formation and stability of caveolae. Few to no caveolae invaginations are present in cells in which caveolin-1 expression levels are reduced or absent. Since the levels of caveolin-1 present in H4 and T84 cells are comparable (unpublished data), we have reasoned that there are caveolae on the H4 cell plasma membrane and the surface-bound CT-GM1 complex is at least partially caveolae/raft-associated as suggested by its presence in DIGs. While the different endocytic mechanisms used by T84 and H4 cells may simply reflect different cell types, we propose the possibility that endocytosis of CT by human enterocytes is developmentally regulated, and the sensitivity of different membrane components such as lipids, glycolipid-binding toxins, and their intracellular signal responses to intracellular trafficking machinery depend on the maturation of these cells.
To support this hypothesis, experiments using glucocorticoids as a maturational stimulus show that CT internalization in H4 cells shifts from a clathrin-dependent to a clathrin-independent mechanism, and a significantly larger amount of surface-bound CT becomes lipid raft-associated. In addition, studies by Badizadegan et al. (1) comparing isolated primary mature human enterocytes to T84 cells have shown a similar capacity for CT signal transduction via specific association of the toxin-GM1 complex with DIGs. In these studies, the internalization of CT was indistinguishable in human small intestinal epithelia from that in T84 cells, suggesting that T84 cells are an appropriate model for studying the cellular mechanism of the CT response.
In like manner, among different age groups using isolated rat enterocytes from pre- and postweaned and adult small intestine, we showed an age-dependent decrease in the internalization of CT but no significant difference in CT binding to its surface receptor. The animal model further validated our findings in cell lines and strengthened our argument that the difference in the endocytosis of CT between H4 and T84 cells is not merely a cell-type difference but represents a developmentally regulated process. Our preliminary data suggest that a clathrin-dependent endocytosis of CT exists in preweaned rat enterocytes compared with a cholesterol-dependent endocytosis of CT in postweaned rat enterocytes (Fig. 7B). However, further studies are needed to characterize this change in endocytosis during gut development.
The developing intestine expresses an enhanced capacity for endocytosis of macromolecules present in the luminal compartment. During development of the intestinal epithelium, a transition occurs from a stratified to a simple columnar epithelium (9, 12). After the conversion, enterocytes assemble an extensive endocytic complex in the apical cytoplasm. This apical endocytic complex is located just beneath the microvillus membrane and is comprised of an elaborate array of membrane tubules and vesicles (40). In the human fetus, this complex is present by the 10th wk and persists at least until the 22nd wk of gestational age (7). Clathrin-coated vesicles have long been considered the most important pathway for internalization of ligand-receptor complexes during intestinal development and are thought to provide a rapid clearance of proteins from fetal amniotic fluid that contains abundant amounts of growth factors and hormones (42). Thus it is presumed that this developmental endocytotic transfer of biologically active molecules may play an important role in the pre- and postnatal maturation of the intestine.
Unfortunately premature infants are also more susceptible to intestinal infection by a variety of infectious agents and their toxins. This may also be due to the same enhanced endocytic capacity and/or increased transepithelial transport capacity in the developing intestine. Our results suggest a role for the clathrin-dependent process in immature human enterocytes leading to a more rapid and efficient uptake of CT, which subsequently results in increased activation of Gs and adenylyl cyclase leading to a CT-stimulated cAMP accumulation and an excessive secretory response. In animal studies (14), cortisone administration could alter the diffuse pattern of anionic sites on the microvillus membrane of newborn rats. This change parallels a premature reduction in the endocytic apparatus of the cell. In our cell line studies, we demonstrate that corticosteroid treatment induces a conversion from a clathrin-dependent pathway to favor the more mature caveolae/raft-mediated pathway. To support this conclusion, we have shown that corticosteroid treatment also inhibits the excessive CT-induced cAMP-mediated secretory response noted in immature human enterocytes. We also provide additional experiments using hydrocortisone treatment in human fetal small intestinal xenografts and noted that cortisone treatment decreased uptake of CT receptor complex and caused a shift in endocytosis to a caveolae/raft-mediated pathway as well as a decreased activation of cAMP. These studies in cell lines confirmed by pre- and postweaned and adult rats as well as human xenograft studies suggest that the excessive secretory response to CT by immature enterocytes appears to be due to an immature, excessive endocytosis of the CT-receptor complex.
In summary, this study represents a preliminary observation that in human fetal cells CT-GM1-associated endocytosis is developmentally regulated and involves a clathrin-dependent mechanism. The enhanced internalization of CT plays a crucial role in CT-induced Gs activation and cAMP elevation and may help to explain our previous observation that at lower doses, CT is more active in the immature gut. The use of CT as a probe to examine developmental regulation of endocytic processes in human fetal enterocytes may present a powerful tool for our understanding of the association between signal transduction and intracellular trafficking that is crucial to cell function. Moreover, these studies may lead to a better understanding of the development of mucosal barrier function and provide new insight into reducing the excessive CT response in the immature intestine leading to more frequent and severe age-related secretory diarrhea.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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