ARTICLE |
Correspondence to: Joseph L. Dixon, Dalton Cardiovascular Research Center, University of MissouriColumbia, Columbia, MO 65211. E-mail: DixonJ@missouri.edu
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Summary |
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We studied apolipoprotein B100 (apoB) metabolism in a series of non-hepatic cell lines (HT29 colon adenocarcinoma, HeLa cervical epithelioid carcinoma, and 1321N1J astrocytoma human cell lines) and in the human hepatoma cell line HepG2. ApoB mRNA was detected by reverse transcription polymerase chain reaction in each non-hepatic cell line. ApoB was detected in HepG2 cells by immunoprecipitation, Western blotting, and immunocytochemistry using a polyclonal anti-human low-density lipoprotein (LDL) antibody, an anti-human apoB peptide antibody, and several monoclonal anti-apoB antibodies. ApoB was identified in the three non-hepatic cell lines by each method using the anti-apoB peptide and monoclonal antibodies, but not with the anti-LDL antibody. Immunocytochemistry indicated that epitopes of apoB were evident throughout the endoplasmic reticulum, and gel mobility of newly labeled apoB and immunoblot with anti-ubiquitin showed that apoB was highly ubiquinated in non-hepatic cells. The observations that apoB is synthesized in non-hepatic cell lines but never recognized by the anti-LDL antibody suggests that apoB is not processed into a nascent lipoprotein in these cells. Immunocytochemical localization of apoB epitopes at many locations throughout non-hepatic cells raises the exciting possibility that apoB can be used for other purposes in these cells.
(J Histochem Cytochem 50:629639, 2002)
Key Words: apolipoprotein B, HeLa, HepG2, HT29, 1321N1J, colon, cervix, astrocytes, LDL, proteasome
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Introduction |
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APOLIPOPROTEIN B (apoB) is the primary structural protein present in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and chylomicron particles of human blood (
It is generally recognized that apoB is produced primarily in two human tissues: liver, where a very large version of apoB (apoB100, 4536 amino acids, 540 kD) is secreted in the form of a VLDL particle, and small intestine, where mRNA editing leads to a truncated version of apoB (apoB48, the N-terminal 2152 amino acids, 259 kD) that is secreted in the form of a chylomicron particle (
In the course of our immunocytochemical studies of the intracellular metabolism of apoB in HepG2 cells, we observed that epitopes of apoB were present in three human non-hepatic cell lines (HT29, HeLa, and 1321N1J cells). The current studies were performed to investigate the intracellular metabolism of apoB in non-hepatic cell lines compared to HepG2 cells. The results provide evidence that endogenous apoB mRNA is expressed and full-size apoB is synthesized in the non-hepatic cells examined, but apoB is processed differently than in HepG2 cells and eventually degraded. Observations of apoB metabolism in non-hepatic cell lines provide insights into the early events involved in nascent apoB metabolism.
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Materials and Methods |
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Chemicals
L-[4,5-3H]-leucine (135 Ci/mmol) was purchased from Nycomed/Amersham (Princeton, NJ). Protein ASepharose CL-4B was obtained from Pharmacia LKB Biotechnology (Piscataway, NJ). Minimal essential medium, non-essential amino acids, sodium pyruvate, MEM select amine kit, and penicillin/streptomycin were from GIBCO/BRL (Gaithersburg, MD). Leupeptin and pepstatin A were from Peninsula Laboratories (Belmont, CA). Bovine serum albumin (BSA) (essentially fatty acid-free) and fetal bovine serum were purchased from Sigma Chemical (St Louis, MO). ALLN was from Boehringer Mannheim (Indianapolis, IN). Lactacystin was from Calbiochem Novabiochem (San Diego, CA).
Antibodies
The domains recognized by the anti-apoB antibodies are described in Table 1. Sheep anti-human LDL was purchased from Serotec (Raleigh, NC). The epitopes recognized by the anti-LDL antibodies are not defined. The B4 peptide (apoB amino acids 32213240) was synthesized on a multiple antigenic peptide (MAP) resin on an Applied Biosystems model 432A synthesizer using Fmoc chemistry, cleaved, and purified according to the manufacturer's protocol (Applied Biosystems; Foster City, CA). A polyclonal antibody to the B4 peptide was prepared in New Zealand White rabbits. Before use in immunocytochemistry, the B4 anti-peptide antibody and the sheep polyclonal anti-human LDL antibodies were affinity-purified on columns containing either purified B4 peptide or human LDL, respectively, as previously described (
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Cell Culture
HT29 (human colon adenocarcinoma), HeLa (human cervical epithelioid carcinoma), and NCI-H292 (mucoepidermoid pulmonary carcinoma) cells were obtained from the ATCC (Manassas, VA). Human astrocytoma cells (1321N1J) were obtained from Dr. Gary Weisman of the University of MissouriColumbia. NCI-H292 cells were used only as a negative control. HepG2 cells were grown in collagen-coated tissue culture dishes as previously described (
RNA Isolation and RT-PCR
Total RNA from confluent monolayers of cultured cells was isolated using total RNA isolation reagent (TRIzol Reagent; Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. PCR primers based on the human apoB sequence were synthesized by CyberSyn (Lenni, PA). The downstream primer was apoB6786F (67866807), 5'CACGGATATGATAGTGCTCAT; the upstream primer was apoB7276R (72547276), 5'CTTGTTGTAGGACATTGCTTAG. Reverse transcription polymerase chain reaction (RT-PCR) was performed using the Access RT-PCR system from Promega (Madison, WI). Amplification was carried out for 32 cycles and the PCR products were analyzed on 1.5% agarose gels.
Treatment and Labeling of Cells
At the start of an experiment, medium was removed from culture dishes and the cells were washed twice with PBS and preincubated for 1 hr in a serum-free medium containing 0.4 mM oleate complexed to 1.5% BSA. For labeling, the medium was removed, the cells were again washed once with PBS, and leucine-free medium containing [3H]-leucine (75 µCi/ml) was added to the cells. After 2 hr, labeling medium was removed, the cells washed twice with PBS, and harvested with ice-cold lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 62.5 mM sucrose, 0.5% Triton X-100, and 0.5% sodium deoxycholate) containing protease inhibitors (final concentrations: 1 mM benzamidine, 5 mM EDTA, 0.86 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units aprotinin/ml, 50 µg leupeptin/ml, and 50 µg pepstatin A/ml) to protect against further proteolysis. Protease inhibitors were also added to the medium.
Immunoprecipitation of 3H-labeled protein was carried out at 4C by the method described previously (
Culture of Cells for Immunocytochemistry
HepG2, HT29, HeLa, and 1321N1J cells were seeded onto collagen-coated coverslips and grown for 48 hr to 2030% confluence in complete growth medium in a CO2 incubator at 37C. For certain controls, cells were grown in serum-free medium for 4 hr before fixing. Cells were washed three times with intracellular buffer BB II (75 mM potassium acetate, 25 mM HEPES, pH 7.2;
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Immunoblotting
Cells were grown to 90% confluence and washed with PBS. Proteins were extracted with 2 ml of lysis buffer containing protease inhibitors. The proteins were concentrated in a Centricon-10 concentrator (Amicon), boiled in electrophoresis sample buffer, separated by SDS-PAGE (315% gel), and transferred overnight from gels to polyvinyl difluoride (Immobilon P) membranes (
Cellular Protein
Protein was determined by the Lowry method (
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Results |
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ApoB mRNA is expressed in HepG2 and in selected non-hepatic cell lines. The presence of apoB mRNA in HepG2 and the three non-hepatic cell lines was investigated by RT-PCR. The primers used are complementary to nucleotides 67866807 in exon 27 and nucleotides 72547276 in exon 28 of the human apoB gene. The PCR product generated from apoB mRNA was expected to be 209 bp and the product generated from DNA contamination was expected to be 492 bp. Only the smaller 209-bp PCR product was observed in total RNA derived from each of the four cell lines (Fig 1, right four lanes), indicating that apoB mRNA is synthesized in all four cell lines. Although the signal from the 1321N1J and HT29 cells is more intense than that from HepG2 and HeLa cells, the qualitative nature of RT-PCR precludes comparison of the relative concentration of message between the cell lines.
Immunoprecipitation of apoB
ApoB synthesis and secretion were investigated in HepG2, HT29, HeLa, and 1321N1J cells that were grown to 90% confluency. Cells were labeled for 2 hr in medium containing [3H]-leucine to detect immunoprecipitable apoB using polyclonal anti-LDL or anti-B4 peptide antibodies in non-hepatic cell lines in comparison to HepG2 cells. Cells were preincubated in medium containing 0.4 mM oleate complexed to 1.5% BSA to enhance neutral lipid synthesis and apoBlipoprotein assembly (
The anti-LDL antiserum effectively precipitated 2.6% of the pulse-radiolabeled proteins from cell extracts of control HepG2 cells (Table 2). ALLN and lactacystin treatment substantially increased the amount of pulse-radiolabeled apoB precipitated from HepG2 cellular extracts by 114% and 99%, respectively. The anti-LDL antisera was able to detect the equivalent of 14.6% of the total TCA-precipitable radiolabeled protein in the conditioned medium collected from the HepG2 cells. ALLN and lactacystin increased the amount of protein that could be precipitated by the anti-LDL antisera by 86% and 57%, respectively. The rabbit anti-B4 peptide antiserum was almost as effective (7287%) as the anti-LDL antiserum in precipitating radiolabeled protein from HepG2 cell extracts but was only 2534% as effective in precipitating secreted radiolabeled proteins from the culture media of these cells. When both anti-LDL and anti-B4 precipitates of HepG2 cell and media extracts were analyzed on gradient electrophoresis gels, prominent bands with a mobility corresponding to full-length apoB were observed (Fig 2).
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The anti-LDL antiserum precipitated less than 0.25% of pulse-radiolabeled proteins from extracts of the non-hepatic cells (Table 2). This antiserum was also ineffective (<1%) in precipitating radiolabeled proteins from the medium of HT29 or HeLa cells but was able to precipitate up to 9.5% of the radiolabel in the 1321N1J medium. When the cell extract and media immunoprecipitates were analyzed by gel electrophoresis, no bands corresponding to full-length apoB were found in the HT29 or HeLa cell lanes (Fig 2). The anti-LDL immunoprecipitates from both the cell extracts and media of 1321N1J cells resulted in a prominent band with a slightly greater mobility than full-length apoB (Fig 2).
The anti-B4 peptide antiserum, on the other hand, was more effective in precipitating pulse-radiolabeled proteins from cellular extracts of the non-hepatic cell lines (Table 2). This antiserum pulled down between 0.81.0% of the pulse-radiolabeled protein in the cell extracts of non-hepatic cell lines. ALLN and lactacystin treatment caused a modest increase (1137%) in the fraction of total cell protein that could be detected by the anti-B4 antibody from HeLa and 1321N1J cells but not from HT29 cells. Gel electrophoresis of the immunoprecipitates of all three non-hepatic cell lines produced clear bands running with a mobility similar to full-length apoB100 (Fig 2B). In addition, each of the lanes had a prominent band at the top of the gel consistent with the limited mobility of polyubiquinated apoB (Fig 2B). Both the full-size apoB100 band and the band with slower mobility were increased by ALLN or lactacystin treatment in 1321N1J and HeLa cells (Fig 2B). The anti-B4 antiserum brought down only 0.50.6% of the radiolabeled proteins present in the conditioned medium collected from HT29 and HeLa cells. Like the anti-LDL antisera, the anti-B4 antiserum was more effective (up to 3.4%) in precipitating radiolabeled proteins from the 1321N1J cell line's medium. Gel electrophoresis of the anti-B4 immunoprecipitates from the medium of either the HT29 or the HeLa cell line resulted in no high molecular weight bands (Fig 2). The anti-B4 immunoprecipitates of the 1321N1J cell line's medium resulted in a prominent band running with a mobility slightly greater than full length apoB, similar to that seen in the anti-LDL immunoprecipitates from this cell line's medium. To confirm that the bands with slower mobility in Fig 2 were ubiquitinated apoB, extracts from HepG2 and 1321N1J cells were immunoprecipitated with anti-B4, separated on an SDS 315% polyacrylamide gel, and probed with anti-ubiquitin. The results (Fig 3) show that in both HepG2 and 1321N1J cells the bands with slower mobility contained ubiquitin but that apoB that migrated with the apoB100 standard was not ubiquitinated. Cell extracts that were brought through the identical immunoprecipitation procedure but without added anti-B4 (Fig 3, lanes with "-" at the top) did not stain with anti-ubiquitin, indicating that the high molecular weight band was not due to nonspecific insoluble material in the extract.
Immunoblotting Analysis
Cells were incubated with ALLN for 2 hr and then harvested in lysis buffer, and total extracted proteins were probed by immunoblotting with antibodies to apoB. ApoB100 was identified in all of the cell lines (Fig 4).
In HepG2 cells, all of the antibodies produced a strong signal for full-size apoB100 at the same mobility as standard apoB (arrow) isolated from human plasma (Fig 4A). The anti-B4 peptide antiserum and monoclonal antibody B1B6 stained a few smaller peptides, but these were of very weak signal intensity. The anti-LDL also identified a peptide in the 6070-kD range in HepG2 cells (Fig 4A). These data confirm the observation that relatively few degradation products of apoB are observed in HepG2 cells even though most newly synthesized apoB is degraded intracellularly in this cell line (
In non-hepatic cells, the anti-LDL did not produce a convincing band for full-size apoB, but identified many smaller bands, including peptides at approximately 228, 125, 6070 kD, and smaller (Fig 4B4D). In contrast to anti-LDL, in all three non-hepatic cell lines the rabbit anti-B4 antiserum and 3 monoclonal antibodies stained a protein with a mobility consistent with full-size apoB. In HT-29 cells (Fig 4B), the bands in the 4050-kD range stained by anti-B4 and D7.2 cannot be the same fragment because of the distance between these epitopes. In some cases, especially in 1321N1J cells, the monoclonal antibodies also stained bands at the top of the gel, consistent with the presence of polyubiquinated apoB. In 1321N1J cells (Fig 4D), the anti-B4 antiserum stained with equal intensity both apoB and a peptide with a slightly greater mobility.
The many smaller bands stained by the polyclonal anti-LDL antiserum in the non-hepatic cells may be breakdown peptides of apoB or other proteins that are recognized nonspecifically by the polyclonal antiserum. When exposure times to films were increased, more breakdown bands could be seen in non-hepatic cell extracts probed with the monoclonal antibodies. The observations that four different antibodies recognized apoB100 (Fig 4B4D) is strong evidence that full-size apoB is present in the three non-hepatic cell lines but that the polyclonal anti-LDL antibody has only limited affinity for it.
Immunocytochemistry of apoB
HepG2, HT29, HeLa, and 1321N1J cells were cultured to 2030% confluency on collagen-coated coverslips, permeabilized with saponin, probed with antibodies to apoB, and analyzed with a laser scanning confocal microscope. Saponin permeabilizes both plasma and internal membranes, giving antibodies access to both cytosolic and luminal sides of the ER membrane. The locations of cell compartments were determined using a series of antibodies to marker proteins (
HT29 cells (Fig 5F5J) were probed with the same series of antibodies as HepG2 cells. The polyclonal anti-LDL antibody did not produce significant staining in HT29 cells (Fig 5F). The slight diffuse signal observed was not different from that observed when non-immune preparations were used. Taking into consideration the different morphology of HT29 cells, the staining patterns produced by the anti-B4 peptide antiserum and the anti-apoB monoclonals CC3.4, D7.2, and B1B6 (Fig 5G5J) were similar to those observed in HepG2 cells. There was a thin bright line of staining surrounding the nuclei of HT29 cells probed with the B4 antiserum (Fig 5G), but the line was not as continuous or defined as the signal observed in HepG2 cells (Fig 5B).
Both HeLa and 1321N1J cells were stained differently by the series of antibodies to apoB than were HepG2 cells. Similar to HT29 cells, the polyclonal anti-LDL antiserum (Fig 5K and Fig 5P) did not stain HeLa and 1321N1J cells. The B4 antiserum produced a very light continuous line of staining around the nuclei of HeLa and 1321N1J cells, but the reticular staining was much reduced compared to HepG2 cells and HT29 cells. The CC3.4 and D7.2 antibodies produced bright reticular staining in HeLa and 1321N1J cells, similar to that seen in HepG2 and HT29 cells. The staining pattern produced by the B1B6 antibody was diffuse without punctate signals in HeLa cells (Fig 5O), and was mostly diffuse with a few punctate signals per cell in 1321N1J cells (Fig 5T). These observations suggest that either a difference in the epitope's protein conformation or the presence of a chaperone binding to this site prevents the B1B6 antibody from binding to apoB in HepG2 and HT-29 cells but not in HeLa and 1321N1J cells.
As a control for the above studies, we identified a human cell line (NCI-H292 mucoepidermal pulmonary carcinoma cells) that was negative for apoB protein expression by either immunocytochemistry or immunoprecipitation followed by SDS-PAGE analysis, utilizing anti-B4 and D7.2 antibodies. Compared to HepG2 cells run in the same experiment, the signals generated in NCI-H292 cells by immunocytochemistry with anti-B4 and D7.2 antibodies were of very low background intensity (data not shown). These observations indicate that apoB expression does not universally occur in cultured human cell lines.
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Discussion |
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Full-length apoB mRNA and protein were found to be expressed in HepG2 cells and the three non-hepatic cell lines using RT-PCR, immunoprecipitation, immunoblotting, and immunocytochemical techniques. We believe that our demonstration of apoB expression in HeLa and 1321N1J cells is the first report of any cell line derived from cervical epithelia or astrocytes expressing this protein. Although this is the first demonstration that HT29 cells, a widely used intestinal model cell line, produce apoB100, it is consistent with earlier reports that the human colon-derived CaCo2 cell line also expresses it (
Our observation that antisera against plasma LDL are relatively ineffective in recognizing apoB expression in non-hepatic cells was not unexpected. The conformation of apoB is sensitive to the lipid environment, and many antibodies against apoB epitopes have been found to be dependent on the lipid composition or size of the lipoprotein particle with which apoB is associated (
What is the biological significance of apoB 100 production in non-hepatic tissues? Our observation that apoB is synthesized in cell lines derived from non-hepatic tissues but is not processed correctly or recognized by a polyclonal anti-LDL antibody is similar to an observation made by
The differences in metabolism of apoB in the four cell lines as detected by immunocytochemistry give insights into the cellular machinery required for lipoprotein assembly and secretion. In HepG2 cells, apoB is synthesized and is present throughout the endoplasmic reticulum (Fig 5B and Fig 5C; B4 and CC3.4 antibodies), is assembled into a nascent lipoprotein (recognized by the polyclonal anti-LDL antibody), traverses the secretory pathway, and is secreted. If lipid substrates are limiting, the newly synthesized apoB is immediately targeted for degradation. Although the proteasome is involved in apoB degradation (
The staining pattern for apoB in HT29 cells was similar to that of HepG2 cells except that the polyclonal anti-LDL antiserum did not produce a signal in HT29 cells. This is consistent with the observation that full-sized apoB was not immunoprecipitated from HT29 cells or convincingly identified by immunoblotting analysis using the polyclonal anti-LDL antibody. ApoB is synthesized in HT29 cells but is never assembled into a nascent lipoprotein, at least not under the culture conditions we employed. The B4 antibody and monoclonals CC3.4 and D7.2 recognized their epitopes in a reticular pattern throughout the cytoplasm of HT29 cells, indicating that intact apoB or fragments of apoB are present throughout the ER. The B1B6 monoclonal antibody gave a punctate staining pattern in HT29 cells, suggesting that, as in HepG2 cells, fragments of apoB reach the lysosomes. Therefore, apoB is not totally degraded by the proteasome in the ER of HT29 cells. The differences in response to ALLN and lactacystin between HepG2 cells and the HT29 and other non-hepatic cell lines may be due to differences in transport or metabolism of the protease inhibitors (
Slightly different pictures can be painted for apoB metabolism in HeLa and 1321N1J cells, which are not of hepatic or intestinal origin. ApoB is synthesized in HeLa and 1321N1J cells but, as in HT29 cells, is differently processed and is not assembled into a lipoprotein. The reticular staining produced in HeLa and 1321N1J cells by CC3.4 and D7.2 indicated that some intact apoB or fragments of apoB with these epitopes were found throughout ER. The absence of a punctate staining pattern in HeLa cells and the observance of a very minor punctate pattern in 1321N1J cells probed with the B1B6 antibody indicated that the region of apoB that includes the B1B6 epitope is processed differently in these cells than in HepG2 cells.
A mechanistic explanation for extensive reticular staining in non-hepatic cells is that a large portion of synthesized apoB cannot be lipidated and remains in the ER, as proposed by
The present findings join the growing number of reports of apoB expression in non-hepatic, non-intestinal cells. In addition, the current report reinforces the hypothesis that the mechanism of apoB degradation is multi-step, multi-compartmental, and may differ among cell types. Future studies will need to focus on the physiological role of apoB expression in these other cell types and the impact of mechanism of degradation on its functional role.
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Acknowledgments |
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Supported by grant HL-47586 to JLD from the National Heart, Lung, and Blood Institute, National Institutes of Health.
We thank Sean Johnston for technical assistance, Jill Cunningham for culturing cells, Elizabeth Norton for preparing figures, and JoAnn Lewis for preparing the manuscript.
Received for publication June 12, 2001; accepted November 30, 2001.
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