Khosrow Adeli

Apolipoprotein B Is Intracellularly Associated with an ER-60 Protease Homologue in HepG2 Cells

Two ALLN (N-acetyl-leucyl-leucyl-norleucinal)-sensitive endoplasmic reticulum (ER)-localized proteases (ER-60 and ER-72) were recently purified from rat liver. We used an antibody to rat ER-60 to investigate the possible role of this protease in apolipoprotein B (apoB) degradation. First, immunoprecipitation and immunoblotting experiments with the anti-rat ER-60 antibody suggested that HepG2 cells contain a homologue of ER-60 with an approximate molecular mass of 58–60 kDa. The ER-60 homologue was mostly associated with the luminal contents of HepG2 microsomes. Evidence from co-immunoprecipitation and cross-linking experiments appear to suggest that the ER-60 homologue in HepG2 cells is associated with apoB intracellularly. A small pool of apoB was recovered when HepG2 lysates were subjected to immunoprecipitation with anti-rat ER-60 antibody followed by a second immunoprecipitation with anti-apoB antibody. Furthermore, cross-linking of permeabilized cells with dithiobis(succinimidylpropionate) further demonstrated association of apoB with the ER-60 homologue in HepG2 cells. Three polypeptides with molecular masses of 78, 66, and 50 kDa were consistently found to be associated with apoB as well as the 58-kDa ER-60 homologue. The 78-kDa protein associated with both apoB and ER-60 appeared to represent immunoglobulin heavy chain-binding protein (BiP) based on immunoprecipitation with a monoclonal antibody. Cross-linking and immunoblotting experiments suggested the association of the 78-kDa BiP with both the 58-kDa ER-60 homologue as well as the 550-kDa apoB. In summary, the data suggests that HepG2 cells contain a 58-kDa protein which is homologous to the rat liver ER-60 in size, antigenecity, and intracellular localization. The ER-60 homologue in HepG2 cells appears to be closely associated with apoB, as well as other proteins possibly representing ER chaperones such as BiP. We hypothesize that the ER-60 homologue may be involved in the degradation of apoB in the ER lumen of HepG2 cells.

Studies on Intracellular Translocation of Apolipoprotein B in a Permeabilized HepG2 System

Recent evidence suggests that the rate of apolipoprotein B-100 (apoB) translocation may be a key regulatory point in the production of apoB-containing lipoproteins. We have developed an in vitro system to measure the translocation rate of apoB in HepG2 cells. Intact cells were initially pretreated with oleate and N-acetyl-Leu-Leu-norleucinal to maximize the translocation rate while minimizing degradation. Cells were pulsed with [35S]methionine, chased (5-30 min), and then permeabilized with digitonin (75 μg/ml). Permeabilized cells were incubated with or without trypsin (200 μg/ml) for 10 min, and digestion was halted with soybean trypsin inhibitor (2 mg/ml). The rate of translocation was determined by comparing the amount of immunoprecipitable intact apoB in trypsin-treated cells with that in control cells at each time point. Under these conditions, two control proteins, α1-antitrypsin and transferrin, were fully protected from trypsin digestion, confirming the integrity of the secretory pathway in permeabilized cells. The percentage of apoB translocated steadily increased from 36% after 5 min to 71% after a 30-min chase (mean percentage, n = 3). A characteristic apoB fragmentation pattern resulted from trypsin digestion, and protected fragments of various size including N-terminal 60-70-kDa fragments were identified. Subcellular fractionation of the cells confirmed that the apoB pool protected from trypsin digestion was luminal in nature, confirming its translocation. ApoB translocation was significantly increased in oleate-treated cells compared with untreated cells. Inhibition of peptidylprolyl isomerase through the use of cyclosporin A and disruption of disulfide bond formation using dithiothreitol reduced the percentage of translocated apoB by 37 and 63%, respectively. Dithiothreitol induced specific changes in the pattern of protected apoB fragments, suggesting a conformational change in apoB that may hinder its translocation. Inhibition of N-linked glycosylation with tunicamycin did not significantly alter the rate of apoB translocation but appeared to stimulate its degradation. Together, the data suggest that the rate of apoB translocation across the membrane of the ER is determined by both lipid availability as well as the correct conformation of nascent apoB molecules.

Secretion of apolipoprotein B in serum-free cultures of human hepatoma cell line, HepG2

We have developed a defined medium which can maintain efficient growth of HepG2 cells sustaining the synthesis of a variety of plasma proteins including apolipoprotein B. This defined system was used to investigate long‐term effects of insulin, estrogen, triiodothyronine, cholesterol, and oleate on the growth pattern of HepG2 cells and secretion rate of apolipoprotein B. Oleate and triiodothyronine caused significant increases in secretion of apolipoprotein B. The stimulatory effect of triiodothyronine was only observed after long (6 days) exposure of cells to the hormone. In contrast, insulin caused up to a 4‐fold decrease in the secretion rate of apolipoprotein B during the early growth periods. This inhibitory effect appeared to be partially abolished after 6 days. Our data suggest that some important questions on regulation of apolipoprotein B expression can be addressed by the long‐term culture of HepG2 cells in defined medium.

Thyroid hormone modulates apolipoprotein B gene expression in HepG2 cells

We have investigated the modulation of apolipoprotein B gene expression in HepG2 cells by thyroid hormone. ApoB secretion rate in serum-free media was found to be significantly increased in the presence of the hormone in long-term cultures (48 h, 37%). This stimulatory effect was dose-dependent. The mechanisms underlying the stimulatory effect of triiodothyronine on apoB production were investigated. Triiodothyronine increased apoB mRNA levels by about 25-36% as determined by slot- and Northern-blot analysis of total RNA. ApoB synthesis rate was also found to be increased both in in vivo pulse-chase experiments (61%) and in in vitro translation studies (54.5%). Despite the 54.5-61% increase in apoB synthesis with triiodothyronine, only a 30% increase in apoB secretion was noted suggesting that part of the increase in the intracellular apoB pool may be lost by degradation. Overall, apoB gene expression appears to be modulated by thyroid hormone at both transcriptional and posttranscriptional levels.

Intracellular Assembly and Degradation of Apolipoprotein B-100-containing Lipoproteins in Digitonin-permeabilized HEP G2 Cells

Permeabilized Hep G2 cells have been used to investigate the turnover of apolipoprotein B-100 (apoB-100). When such cells were chased in the presence of buffer, there was no biosynthesis of apoB-100, nor was the protein secreted from the cells. Thus the turnover of apoB-100 in these cells reflected the posttranslational degradation of the protein. Pulse-chase studies indicated that apoB-100 was degraded both when associated with the membrane and when present as lipoproteins in the secretory pathway. Neither albumin nor α1-antitrypsin showed any significant posttranslational intracellular degradation under the same condition. The kinetics for the turnover of apoB-100 in the luminal content differed from that of apoB-100 that was associated with the microsomal membrane. Moreover, while the degradation of the luminal apoB-100 was inhibited by N-acetyl-leucyl-leucyl-norleucinal (ALLN), this was not the case for the membrane-associated protein. Together these results suggest the existence of different pathways for the degradation of luminal apoB-100 and membrane-associated apoB-100. This was further supported by results from pulse-chase studies in intact cells, showing that ALLN increased the amount of radioactive apoB-100 that associated with the microsomal membrane during the pulse-labeling of the cells. However, ALLN did not influence the rate of turnover of the membrane-associated apoB-100. The presence of an ATP-generating system during the chase of the permeabilized cells prevented the disappearance of pulse-labeled apoB-100 from the luminal lipoprotein-associated pool. The ATP-generating system combined with cytosol protected the total apoB-100 in the system from being degraded. The cells cultured in the presence of oleic acid and chased after permeabilization in the presence of cytosol and the ATP-generating system showed an increase in the amount of apoB-100 present on dense (“high density lipoprotein-like”) particles. This increase was linear during the time investigated (i.e. from 0 to 2 h chase) and independent of protein biosynthesis. Our results indicate that the dense particle was generated by a redistribution of apoB-100 within the secretory pathway and that it most likely was assembled from the membrane- associated form of apoB-100. These results indicate that the release of apoB-100 from this membrane-associated form to the microsomal lumen is dependent on cytosolic factors and a source of metabolic energy.

Human bioassays to assess environmental genotoxicity: development of a DNA break bioassay in HepG2 cells.

OBJECTIVES Increasing interest in environmental health issues has created a demand for improved methods for the assessment of pollutant effects on humans. Our laboratory has developed an in vitro assay for the quantification of genotoxicity, monitored as DNA single strand breaks (SSB), in the HepG2 human hepatoma cell line. DESIGN AND METHODS This assay procedure, which is based upon alkaline unwinding and hydroxylapatite DNA chromatography, is both rapid and simple to perform. RESULTS HepG2 cells responded to the standard mutagen, 4-nitroquinoline N-oxide, demonstrating SSB formation at concentrations above 0.1 mumol/L. Phenanthrene-9,10-quinone, a component of diesel exhaust, mediated SSB formation at concentrations above 250 nmol/L. Finally, an extract of contaminated sediment from the Great Lakes Basin mediated SSB formation in a dose-dependent manner. CONCLUSIONS These results illustrate the utility of this human genotoxicity assay for future use in screening of environmental pollutants.

Intracellular degradation of apolipoprotein B generates an N-terminal 70 kDa fragment in the endoplasmic reticulum

Regulated apoB degradation in HepG2 cells occurs in the endoplasmic reticulum (ER), is catalyzed by an N-acetylleucylleucylnorleucinal (ALLN)-sensitive proteinase, and generates a specific 70 kDa fragment (Adeli, K., 1994, J. Biol. Chem. 269, 9166-9175) [corrected]. In the present report, we have characterized the 70 kDa fragment by immunoprecipitation of permeabilized HepG2 cells with a battery of monoclonal antibodies against various sites on the apoB molecule. N-Terminal monoclonal antibodies (1D1 and 2D8) were capable of binding to the 70 kDa fragment suggesting that this polypeptide is an N-terminal fragment of the intact apoB. Subcellular fractionation of permeabilized cells and carbonate extraction resulted in the detection of the 70 kDa fragment in the ER lumen. Endoglycosidase H treatment confirmed that the fragment is N-linked glycosylated. We hypothesize that the ALLN-sensitive proteinase which may be located on the luminal side of the ER membrane, catalyzes an initial cleavage of apoB near the N-terminus generating a 70 kDa fragment, which is then released into the ER lumen.

Regulation of apolipoprotein B biogenesis in human hepatocytes: Posttranscriptional control mechanisms that determine the hepatic production of apolipoprotein B-containing lipoproteins

OBJECTIVES Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins appears to be a common cause of hyperlipoproteinemia in humans. Patients with overproduction states secrete denser cholesterol ester-rich lipoprotein particles which are highly atherogenic. The formation of apoB particles involves a very complex process that requires the coordinated synthesis and assembly of apoB, triglycerides, cholesterol esters, phospholipids, and other components. ApoB expression is an important prerequisite for the assembly and secretion of apoB particles. Evidence to date appears to suggest that apoB expression is regulated posttranscriptionally. ApoB secretion rate is determined at the levels of apoB translocation into the endoplasmic reticulum (ER) as well as degradation within the ER. RESULTS AND HYPOTHESIS Based on available data, we postulate that the rate of apoB particle secretion is determined at the critical point where newly-synthesized apoB interacts with core lipids, particularly triglycerides. The supply of these lipids determines the rate of translocation of the apoB molecule across the ER membrane and into the ER lumen. Lipidation of apoB facilitates its proper folding, its assembly into a lipoprotein particle, and its extracellular secretion. In the absence of lipids, apoB is misfolded resulting in the abortion of ER translocation and subsequent degradation by an apoB specific protease. CONCLUSIONS The balance between intracellular degradation and extracellular secretion determines the rate at which the human liver secretes apoB particles.

Expression of cathepsin B and aryl hydrocarbon hydroxylase activities, and of apolipoprotein B in human hepatoma cells maintained long-term in a serum-free medium

SummaryWe have established the human hepatoma cell line, HepG2, in a defined, serum-free medium. These cells were maintained and studied over a 100-generation period (i.e. 10 serial transfers). Cells maintained in serum-free medium exhibited growth parameters (i.e. saturation density, efficiency of plating, and population doubling time) similar to those obtained with HepG2 cells maintained in serum-supplemented medium. Serum-free cells were also similar to their serum-supplemented counterparts with respect to the expression of cathepsin B activity and the induction of aryl hydrocarbon hydroxylase by 2,3,7,8-tetra-chlorodibenzo-p-dioxin. Significantly, HepG2 cells maintained in serum-free conditions also retained the ability to synthesize and secrete proteins, including the liver plasma protein, apo-lipoprotein B. These results indicate that the serum-free medium used in this study supports the long-term growth and maintenance of human hepatoma, HepG2, cells in culture. Inasmuch as these cells retain phenotypes, including differentiated markers previously reported for their serum-supplemented counterparts, they may provide a more reliable, standardized culture system to study the expression, secretion, and regulation of proteins during biological and pathologic processes.

ROLES OF DT DIAPHORASE IN THE GENOTOXICITY OF NITROAROMATIC COMPOUNDS IN HUMAN AND FISH CELL LINES

The genotoxicity of nitroaromatic compounds was examined in two cultured cell lines, namely, a human hepatoma cell line, HepG2, and a brown bullhead fibroblast cell line, BB. Furthermore, the role of the quinone-reducing enzyme DT diaphorase [NAD(P)H:(quinone acceptor) oxidoreductase] was examined with respect to its influence on the genotoxic effects of model nitroaromatic pollutants. The nitroreductive characteristics of these two cell lines were examined using an acetylated cytochrome c reduction assay for enzymatic nitroreductase activity. Subsequently, the influence of DT diaphorase on the genotoxicity of two model nitroaromatics, 4-nitroquinoline 1-oxide (4NQ) and nitrofurantoin (NF), revealed that DT diaphorase was the predominant 4NQ reductase in cytosols of both cell lines, but played a lesser role in NF reduction in both species. Despite these interspecific similarities, results revealed marked qualitative differences between the two species in terms of the influence of DT diaphorase on quinone-mediated genotoxicity. When pretreated with the DT diaphorase inhibitor dicoumarol, HepG2 cells exhibited an exacerbation of genotoxicity in the presence of 4NQ, indicating a protective influence of the enzyme. In contrast, 4NQ genotoxicity in BB cells was reduced in the presence of dicoumarol, indicating a deleterious effect of DT diaphorase activity. Conversely, dicoumarol pretreatment was moderately protective against NF-mediated genotoxicity in HepG2 cells but exacerbated NF toxicity in BB cells. This study illustrates the manner in which functionally analogous enzymes may have markedly distinct influences on xenobiotic toxicity in different cellular systems.