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CELLULAR |
Division of Gastroenterology and Hepatology, Departments of
1 Medicine
2 Biochemistry, Medical College of Wisconsin and Veterans Administration Medical Center, Milwaukee, Wisconsin 53295, USA
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Abstract |
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(Received 25 January 2007;
accepted after revision 5 March 2007;
first published online 8 March 2007)
Corresponding author B. Seetharam: Zablocki VA Medical Center, 5000 West National Avenue, Research 151, Bldg 70C, Milwaukee, WI 53295, USA. Email: seethara{at}mcw.edu
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Introduction |
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Both renal and intestinal epithelial cells have developed elaborate systems to transport Cbl across their plasma membrane domains (Seetharam, 1999). While Cbl bound to gastric intrinsic factor is internalized apically via a multipurpose receptor, cubilin (Seetharam & Yammani, 2003), Cbl bound to plasma TC is internalized via basolaterally expressed TC-R (Seetharam & Li, 2000). One remarkable property of intestinal epithelial Caco-2 cells is that they mediate transcytosis of apically internalized Cbl to allow it to enter circulation (Ramanujam et al. 1991) to provide Cbl to other tissues/cells. However, Cbl internalized basolaterally bound to TC via TC-R is retained in the cell and utilized as Cbl coenzymes (Bose et al. 1997). Although the mechanism by which Caco-2 cells are able to sort Cbl differentially depending on the side of entry or the protein ligand to which Cbl is bound is not fully understood, it does reflect in vivo Cbl transport across the mucosal barrier. In addition, both intact intestinal mucosal epithelial and Caco-2 cells are also able to internalize Cbl bound to TC from the apical side. Interestingly, like the situation with intrinsic factor (IF)-mediated apical entry of Cbl (Ramanujam et al. 1991), Cbl internalized bound to TC is also transcytosed to the basolateral side (Bose et al. 1997). The in vivo functional significance of TC-R expressed in BBM is not apparent due to absence of ligand TC in the intestinal lumen. However TC-R expressed in the BLM appears to play a crucial role in plasma Cbl transport to the intestinal epithelial cells which are under constant regeneration.
Although Caco-2 cells are able to mediate endocytosis of TC–Cbl from both its plasma membrane domains, very little is known about the trafficking pathways and intracellular vesicles involved in the plasma membrane delivery of de novo synthesized TC-R. To address these issues we have used post-confluent Caco-2 cells grown for 12 days as they express mature enterocyte-like functional and morphological properties, and are in a stationary growth phase (Rousset et al. 1985). Results of the current study show that newly synthesized TC-R is initially delivered rapidly to BLM and following endocytosis, it is degraded by leupeptin-sensitive proteases. However, a small percentage (15%) of TC-R escapes degradation and is transcytosed to BBM and during this process it associates with megalin.
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Methods |
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Caco-2 cells (passages 76–80) were routinely grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA) as described earlier (Bose et al. 1996b, 1997). Domain-specific biotinylation studies were carried out using cells grown on 30 mm diameter, 0.45 µm pore size filter inserts Millicell-HA (Millipore, Bedford, MA, USA). Formation and integrity of monolayers were assessed by measuring transepithelial resistance and cells with resistance over 250–300 
cm–2 were used.
Isolation of BBM and BLM from Caco-2 cells
Post-confluent Caco-2 cells grown on culture inserts were used for simultaneous isolation of BBM and BLM using the sucrose density gradient and differential precipitation method (Ellis et al. 1992). Alkaline phosphatase, which is chiefly located in BBM, was enriched 15-fold and BLM was enriched with K+-stimulated p-nitrophenyl phosphatase by 18-fold. Sucrase–isomaltase, an exclusive marker for BBM, was present less than 0.1% in BLM.
Immunoblotting studies
Isolated BBM and BLM (75–100 µg protein) were subjected to non-reducing SDS-PAGE (5%) and separated proteins were transferred onto Immobilin-P membrane (Millipore) at 4°C for 1 h at 90 V for TC-R or 20 h at 30 V for megalin. The membranes were probed with diluted (1: 5000) antiserum to human TC-R or rat renal megalin and the immunoreactive bands were visualized by enhanced chemiluminescence (ECL) (Amersham-Pharmacia, NJ, USA). Antiserum to human placental TC-R was raised in rabbits as described earlier (Bose et al. 1995). Megalin was purified from rat kidney according to Kanalas & Makker (1990) and its antibody was raised in rabbits as described earlier (Yammani et al. 2001).
BBM association of TC-R and megalin in Caco-2 cells
To examine the possible association of megalin and TC-R in the BBM, we carried out sequential immunoprecipitation and immunoblotting experiments. Isolated BBM (150 µg protein) was solubilized in 1 ml of 10 mM Tris-HCl, pH 7.4 containing 140 mM NaCl (TBS buffer) containing 1% Triton X-100. The Triton X-100-solubilized fraction was subjected to immunoprecipitation with undiluted (5 µl) TC-R or megalin antiserum and 50 µl of a 1: 1 suspension of protein A coupled to Sepharose (Sigma-Aldrich, St Louis, MO, USA). The immunopellets were subjected to non-reducing SDS-PAGE and immunoblotting as above.
Preparation of endosomes from Caco-2 cells
An endosomal-rich fraction was prepared from post-nuclear supernatants of Caco-2 cell monolayers as described by Matter et al. (1990b). Briefly, Caco-2 cell monolayers were allowed to take up horseradish peroxidase (HRP) for 10 min at 37°C. The cells were washed exhaustively and subjected to a Percoll gradient (initial density 1.061). 2 ml collected from the bottom of the percoll gradient was overlayed with metrizamide gradient (14.5% –21.5%) and centrifuged for 5h at 75 000 x g. Fifteen fractions (0.3 ml each) were collected from the bottom. Fractions 1–5 were enriched 15-fold for glucosaminidase (lysosomes), fractions 6–10 in the middle of the gradient with the first peak of HRP activity, and fractions 11–15 were enriched 12-fold for alkaline phosphatase, a marker for BBM. Our initial dot blot immunoanalysis revealed that fractions 8 and 9 containing HRP also contained TC-R and megalin and thus, we focused on these two fractions. Fractions 8 and 9 were precipitated with trichloroacetic acid (5%) and the pellet was washed with acetone to remove acid, resuspended in 10 mM Tris-HCl buffer 7.4 and subjected to SDS-PAGE (7.5%). The strips of gels were immunoblotted for TC-R and Rab5 at 90 V for 60 min, Tfr at 90 V for 90 min and megalin at 30 V for 20 h at 4°C. Antibodies for Rab5 and Tfr were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Zymed laboratories Inc. (Carlsbad, CA, USA), respectively.
Effect of processing and trafficking inhibitors
Caco-2 cells grown on culture inserts were treated with colchicine (20 µg ml–1), tunicamycin (2 µg ml–1), wortmannin (20 nmol ml–1), BFA (2 µg ml–1), or leupeptin (1 mg ml–1) for 12 h. All these inhibitors were purchased from Sigma-Aldrich. Due to its metabolism over time, wortmannin was added every 4 h. The medium was removed from both apical and basolateral chambers after 12 h, and then fresh medium containing the same amount of inhibitors was added along with 25 µCi ml–1 [35S]-methionine (7 mCi, Perkin Elmer Life Sciences, Boston, MA, USA) and further incubated for 2 h. After 2 h, the medium was removed, cells were washed with ice-cold DMEM and fresh ice-cold medium containing TC-R antiserum (5 µl) was added to the basolateral side in one set of cells and to the apical side in the other. Following 30 min incubation at 4°C, the cells were lysed and incubated with 50 µl of a 1: 1 suspension of protein A–Sepharose. The immunoprecipitated radioactivity was washed 10 times with 1.5 ml of TBS containing 0.1 mM phenylmethylsulphonylfluoride and counted in a beta-counter: a part of the radioactivity was subjected to non-reducing SDS-PAGE (7.5%).
Pulse-chase labelling of Caco-2 cells
In order to determine the half-life of TC-R, post-confluent cells grown in T-75 flasks were treated with or without leupeptin (1 mg ml–1) for 1 h at 37°C and then incubated with methionine-free DMEM for 30 min. The cells were pulsed for 1 h with [35S]-methionine (200 µCi (10 ml)–1). The medium was removed; cells were washed with DMEM and chased for 0–16 h with DMEM containing non-radioactive methionine (10 mM) with or without leupeptin (1 mg ml–1). At each time interval, the cells were harvested, washed in DMEM and homogenized in 1 ml of TBS containing 0.1 mM phenylmethylsulphonylfluoride, extracted with Triton X-100 (1% v/v) for 12 h at 4°C. The Triton X-100 extract containing [35S]-labelled TC-R was immunoprecipitated with 5 µl of TC-R antiserum and 50 µl of a 1: 1 suspension of protein A–Sepharose. The immunoprecipitate was washed 10 times with 3 ml of TBS and the radioactivity released was processed for non-reducing SDS-PAGE (7.5%).
Plasma membrane delivery of TC-R
Post-confluent Caco-2 cells grown on filters were initially incubated with methionine-free DMEM for 30 min. The medium was removed and fresh DMEM containing [35S]-methionine (100 µCi ml–1) was added and incubated for 10 min. The medium was removed; cells were washed in DMEM and were chased for 0–60 min at 37°C with DMEM containing non-radioactive methionine (10 mM). At each time interval, the medium was replaced with ice-cold medium alone on the apical side and that containing 5 µl TC-R antiserum on the basolateral side followed by incubation for 60 min at 4°C. In some filters, antiserum (5 µl) was added to the apical instead of the basolateral side. The cells were then harvested, washed 3 times with 2 ml of TBS to remove unbound TC-R antiserum, extracted with 1 ml of TBS containing Triton X-100 (1% v/v), and treated with protein A–sepharose. The intracellular pool of [35S]-TC-R was determined using the supernatant fraction obtained following removal of [35S]-TC-R from the BLM. Radioactivity immunoprecipitated from the supernatant fraction at each time interval of chase was corrected for [35S]-TC-R radioactivity due to apical [35S]-TC-R. The apical [35S]-TC-R present in the supernatant fraction varied depending on the chase time, but was a maximum 6–7% of BLM levels at 60 min of chase. The immunoprecipitated radioactivity obtained from BLM and the intracellular pool was processed for non-reducing SDS-PAGE.
Cell surface biotinylation
In order to study the endocytic pattern of TC-R from the two surface domains of Caco-2 cells and to study transcytosis of TC-R from BLM to BBM, a combination of metabolic labelling and domain-specific biotinylation techniques was utilized. Initially, separate sets of filter-grown cells were pulsed with 25 µCi of 35[S]-methionine per filter for 60 min. After labelling, the medium was replaced by ice-cold DMEM, followed by cell surface biotinylation either at the apical or basolateral side using sulfosuccinimido biotin (S-NHS-biotin; Pierce Chemical Company, Rockford, IL, USA) as described before (Bose et al. 1997). The filters were warmed to 37°C with addition of serum-free DMEM containing non-radioactive methionine (10 mM) and chased for 0–120 min. At each time interval, cells were washed with ice-cold medium and incubated at 4°C with TC-R antiserum (5 µl) added either to apical or basolateral medium. The immunoprecipitated radioactivity containing both biotinylated and non-biotinylated [35S]-TC-R was released by boiling with SDS (1% v/v) and precipitated with ice-cold acetone (80%). The precipitated radioactivity was washed with TBS, extracted with Triton X-100 (1%) and treated with 25 µl of a 1: 1 suspension of streptavidin–agarose (Sigma-Aldrich) in TBS. Following 1 h incubation, the precipitated radioactivity was released in SDS-sample buffer [Tris-HCL buff pH 6.8 containing glycerol (20%)] and subjected to non-reducing SDS-PAGE (7.5%). In some experiments, the protocol was modified by adding ligand, human TC-[57Co]Cbl (500 fmol per filter) to the basolateral medium (serum free) to examine the effect of ligand on the endocytic pattern of TC-R.
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Results |
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Immunoblot analysis (Fig. 1) for TC-R (panel A) revealed that it was expressed in both BLM and BBM with about 6-fold enrichment in BLM. In contrast, megalin was expressed only in the BBM (panel B) and could not be detected in BLM. We have shown earlier (Yammani et al. 2003) that TC-R is associated with megalin in the renal BBM. Thus, we wanted to examine whether a similar type of association exists between the two in Caco-2 cells. Immunoprecipitation of Triton X-100 extract of isolated BBM with either antiserum to TC-R (Fig. 1C) or megalin (Fig. 1D) followed by SDS-PAGE and immunoblotting with megalin (panel C) or TC-R antiserum (panel D) revealed that the two proteins are associated in native BBM of Caco-2 cells. Due to extraction of BBM with Triton X-100, molecular mass of TC-R immunoprecipitated is 62 kDa. Previously (Bose et al. 1996a), we have shown that TC-R exists as a dimer of mass 124 kDa in native plasma membranes and as monomer of mass 62 kDa upon its treatment with Triton X-100.
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Following synthesis, [35S]-TC-R was delivered rapidly to BLM (Fig. 3A, top) with a T1/2 of 15 min. At 30 min of chase, nearly 100% of [35S]-TC-R synthesized was delivered to BLM. Incubation for an additional 30 min resulted in nearly 70% loss of TC-R from BLM due to endocytosis. During the same time period of chase, intracellular [35S]-TC-R levels declined in the first 15 min, reaching to undetectable levels at 30 min, followed by an increase during 30–60 min of additional chase (Fig. 3B, top). Immunoprecipitated radioactivity from both BLM (Fig. 3A, bottom) and the intracellular pool (Fig. 3B, bottom) when analysed on SDS-PAGE revealed a single band. In a parallel pulse–chase experiment using a different set of filters, TC-R antiserum added to the apical side failed to immunoprecipitate any radioactivity for up to 30 min of chase, but about 5–7% of radioactivity present in BLM was immunoprecipitated from the BBM between 30 and 60 min of chase (data not shown). These observations indicated that delivery of newly synthesized TC-R to BLM is rapid (T1/2, 15 min) reaching completion by 30 min and after that TC-R was endocytosed.
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Effect of processing and trafficking inhibitors on BLM and BBM delivery
TC-R contains one N-linked oligosaccharide (Bose et al. 1995) and we wanted to test whether N-glycosylation at this single site is important for its BLM delivery. Following a 12 h incubation with tunicamycin, an inhibitor of N-glycosylation, there was a 50% reduction in [35S]-TC-R levels in BLM (Fig. 4, bar 3, top) relative to cells that were not exposed to any inhibitors (bar 1) suggesting that a single N-linked sugar chain on TC-R may play a role in its BLM delivery. As a consequence of decreased levels in BLM, tunicamycin treatment also substantially decreased TC-R expression in BBM (bar 3). In contrast, treatment of cells with colchicine, a microtubule disruption agent, had no effect on BLM delivery (bar 2), but it inhibited the delivery of TC-R to BBM by nearly 90%. Brefeldin A, a fungal antibiotic which is known to disrupt Golgi organization and endosomal fusion, resulted in inhibition of TC-R delivery to both BLM and BBM (lanes 5), indicating that intact Golgi and vesicular fusion events are important in TC-R delivery to both surface domains. Wortmannin, a PI-3 kinase inhibitor had no effect on TC-R delivery to both BLM and BBM (lanes 4). Finally, treatment of cells with leupeptin, an inhibitor of lysosomal proteolysis, resulted in increased (20%) levels in both BLM and BBM, indicating that inhibition of intracellular turnover might indeed result in reverse delivery of undegraded TC-R back to the membrane surfaces (lanes 6). SDS-PAGE of immunoprecipitated [35S]-TC-R from these experiments (Fig. 4, bottom panels) revealed a single band, and its relative intensity corresponded fairly well to the amount of radioactivity associated with TC-R. Since treatment of cells with leupeptin had small, but significant, effect on the surface membrane expression of TC-R in both BBM and BLM, it implied that lysosomes play a role in its degradation and turnover. In order to verify the role of lysosomes in turnover of TC-R, pulse–chase labelling experiments were carried out in the presence and absence of leupeptin.
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Pulse–chase studies revealed that in Caco-2 cells, newly synthesized [35S]-TC-R radioactivity decayed linearly (Fig. 5A) during the chase period (0–16 h). Total cellular labelled receptor decayed to very low levels in 16 h of chase and the T1/2 of decay was 8 h. When pulse–chase experiments were carried out in the presence of leupeptin (Fig. 5A), the rate of decay of [35S]-TC-R slowed down, and nearly 77% of [35S]-TC-R was still present in the cell extracts at 16 h of chase. SDS-PAGE followed by fluorography of immunoprecipitated [35S]-TC-R (Fig. 5B) revealed a single band of TC-R monomer whose intensity declined to very low levels at 16 h of chase in leupeptin-untreated, but not in treated cells. Densitometric analyses (data not shown) of the bands in fluorography revealed an identical decay pattern as the decay of actual radioactivity associated with TC-R (Fig. 5A) in these cells. These results indicated that TC-R is degraded by leupeptin-sensitive lysosomal proteases.
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Endocytosis of TC-R
Relative to labelled TC-R levels at 0 min chase, nearly 66% and 74% disappeared from BLM following 15 and 30 min of chase, respectively (Fig. 6, left panel). At 60–240 min of chase, TC-R levels in BLM decreased further to about 20% and 0% indicating that TC-R does not recycle to the BLM during this period of chase. TC-R levels in BBM also decreased during a chase for up to 2 h (Fig. 6, left panel). SDS-PAGE analysis (Fig. 6, right panels) of [35S]-TC-R extracted from the BLM (top) and BBM (bottom) revealed a single band. The image intensity pattern of these bands obtained from SDS-PAGE was identical to the amount of immunoprecipitated radioactivity. The time-dependent pattern of endocytosis from BLM did not change when these experiments were carried out with the ligand, TC–Cbl (data not shown).
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Discussion |
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Expression of TC-R in the apical membranes associated with megalin and identification of the two in an intracellular compartment containing Rab5 and Tfr (Figs 1 and 2) have the following implications: first, apical TC-R is derived from BLM by transcytosis since labelled TC-R containing a biotin tag present in BLM appeared in BBM. This is not too surprising since the apical delivery of proteins occurs both by a direct as well as an indirect (transcytosis) pathway in most polarized epithelial cells (Caplan, 1997), including Caco-2 cells (LeBivic et al. 1990). Second, inhibition of apical delivery of TC-R by colchicine is consistent with the involvement of microtubules whose disruption is known to affect apical delivery of proteins by both direct and indirect pathways (Gilbert et al. 1991). Third, BBM expression of TC-R was not altered significantly when cells were incubated with leupeptin, an inhibitor of lysosomal proteases indicating that lysosomes are unlikely to be an intermediate vesicle in the transcytotic pathway of TC-R. Fourth, lack of recycling of apically endocytosed TC-R may be due to its association with megalin, since most ligands internalized bound to megalin are transported to lysosomes for their degradation (Kounnas et al. 1994; Hussain et al. 1999; Marino et al. 2001). Finally, association of TC-R and megalin is an intracellular event and does not occur in situ following their apical delivery. Since Rab5 has been implicated in both apical and basolateral endocytosis (Mostov et al. 2000), it is likely that TC-R endocytosed from the BLM and megalin endocytosed from BBM associate in a common or a mixed endosomal fraction which has been shown to exist in Caco-2 cells (Hughson & Hopkins, 1990).
The delivery of newly synthesized TC-R to BLM is rapid (Fig. 3) with the bulk of the endocytosed receptor being degraded by leupeptin-sensitive proteases. Another interesting feature of TC-R expression in Caco-2 cells is that, unlike most cell surface receptors, it is not retained in an intracellular pool and most of the newly synthesized receptor is expressed on the membrane surface. This is rather unusual for a cell surface receptor, and lack of retention in an intracellular pool or its endocytosis in the absence of ligand TC-Cbl suggest strongly that TC-R expression on the cell surface is a constitutive process.
Our studies using trafficking inhibitors also provide some important clues regarding the trafficking pathways of TC-R. Unlike its delivery to BBM, TC-R delivery to BLM is not affected by colchicine, consistent with a lack of any role of microtubules in this process. In contrast, both tunicamycin and BFA seem to delay BLM delivery of TC-R suggesting that both intact Golgi and post-Trans-Golgi Network (TGN) fusion events are important for its trafficking to BLM. While tunicamycin treatment may affect trafficking of TC-R from endoplasmic reticulum to Golgi, resulting in degradation (Elbein, 1987), we have shown previously (Bose et al. 1998) that BFA treatment results in accumulation of TC-R in a non-sialylated form. Treatment with wortmannin is known to delay trafficking of some cell surface receptors such as cation-indepenedent mannose 6-phosphate (Kundra & Kornfeld, 1998) and platelet-derived growth factor receptor (Joly et al. 1995). However, lack of effect of wortmannin on BLM expression of TC-R suggests that BLM delivery of TC-R is independent of phosphatidyl inositol-3-kinase activity. Finally, leupeptin treatment resulted in higher levels of TC-R in BLM suggesting that inhibition of degradation of endocytosed TC-R might enable it to recycle.
Although the absence of recycling of TC-R from BBM can be explained based on its association with megalin at this location, the reason for the absence of recycling of TC-R from BLM is not clear. It is possible that following basolateral endocytosis, TC-R is initially sorted into a compartment from which it can only recycle if it is not degraded by the lysosomes. This suggestion is supported by the observation that basolateral TC-R levels are increased when the metabolic labelling was carried out in the presence of leupeptin. This may also explain why, in leupeptin-treated cells, there was also a small increase in the apical levels of TC-R, suggesting that some of the undegraded TC-R may once again enter the transcytotic pathway. In contrast to TC-R, its ligand TC is degraded by the lysosomes when taken up as TC–Cbl from the BLM and it undergoes transcytosis bound to Cbl when taken up from the BBM. Inhibition of lysosomal degradation of TC–Cbl taken up from the BLM results in its secretion to the basolateral medium (Bose et al. 1997). TC–Cbl entering the cell from the apical side is transcytosed to the basolateral side both in the presence and absence of leupeptin and other lysosomotropic agents. This indicates that lysosomes are involved in both TC–Cbl and TC-R processing when the ligand enters the cell from the BLM, while it is involved in only receptor processing when the ligand enters the cell from the BBM.
A relatively short half-life of TC-R and its efficient endocytosis from both plasma membrane domains is consistent with the generally accepted idea (Hare, 1990) that the half-life of membrane proteins undergoing efficient endocytosis is less than 22 h. The identical half-lives of 8 h of both total cellular TC-R (Fig. 5A) and that endocytosed from BLM (data not shown) suggest strongly that BLM enrichment (85%) of TC-R is not due to a preferential retention at this site, but rather to its preferential delivery to this location.
Data presented in the current studies, rapid and complete delivery of de novo synthesized TC-R to plasma membranes, its lack of recycling, constitutive endocytosis and lysosomal degradation have been obtained using Caco-2 cells which are derived from human colon adenocarcinoma. This raises the question whether the data obtained represent trafficking events in a normal cell or in a cancer cell. In addition, earlier studies (Hall, 1984, 1987; Lindemans et al. 1989) using ligand binding to cell surface have shown that TC-R is up-regulated in cancer cells or in cells that are methionine dependent for growth and proliferation (Fiskerstrand et al. 1998). While it is possible that TC-R is up-regulated in many cancer cells, trafficking events noted in Caco-2 cells probably represent those occurring in a normal mature enterocyte. It is well known that Caco-2 cells grown under culture conditions used in these studies do differentiate as mature entrocytes and have all the absorptive, transport and defensive functions, and structural properties as a normal enterocyte (Sambuy et al. 2005).
Expression of TC-R is important to maintain Cbl balance, and cells accomplish this by regulating its expression on the cell surface. In addition to the trafficking pathways outlined in this study, additional regulatory factors such as intracellular signals and events may also modulate its cell surface expression. Recent studies using immunoblot (Bauer et al. 2002) and radioimaging (Collins & Hogenkamp, 1997; Collins et al. 2000) have shown that TC-R is indeed up-regulated in various types of cancer. In addition, recent studies (Kalra S, Ahuja R, Mutti E, Veber D, Seetharam S, Scalabrino G & Seetharam B, unpublished data) from our laboratory have shown that TC-R levels are up-regulated in inflammatory disorders such as intestinal inflammation and by proinflammatory cytokines such as tumour necrosis factor-alpha when added to cells. The molecular mechanisms and signalling pathways involved in the cell surface expression of TC-R and import of Cbl in these disorders are not known. It is very likely that import of high levels of Cbl, and hence up-regulation of TC-R, may be a fundamental property of cells that are under stress/shock. It has been proposed (Wheatley, 2006) that high levels of Cbl may have very beneficial effect in many human diseases such as systematic inflammatory response syndrome, sepsis, septic and traumatic shock.
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) or without (
) leupeptin (1 mg ml–1) for 1 h prior to pulse labelling with [35S]-methionine (200 µCi per flask). The cells were then chased for 0–16 h with DMEM with or without leupeptin (1 mg ml–1). B, the immunoprecipitated radioactivity (4 500–88 000 c.p.m. in the absence of leupeptin and 68 000–88 000 c.p.m. in the presence of leupeptin) was liberated and subjected to non-reducing SDS-PAGE and the bands were visualized by fluorography. Other details of labelling, cell extraction and immunoprecipitation are provided in the Methods. The data shown are mean ±
S.D. of 4 pulse–chase experiments.
) are shown and are the mean ±
S.D. of 5 separate labelling experiments (P
0.01). Right panel, SDS-PAGE of biotinylated [35S]-TC-R (1 000–60 000 c.p.m.) in the BLM (top) and 200–10 000 c.p.m. in the BBM (bottom) were used during the indicated chase time intervals.