Effects of estrogen on apolipoprotein secretion by the human hepatocarcinoma cell line, HepG2

We have examined the effect of estrogen on the rate of accumulation of apolipoproteins secreted by the human hepatocarcinoma cell line, HepG2. Prior to exposure to hormone, we detected less than 300 high-affinity, nuclear, estrogen-binding sites/cell. Within 48 h of growth in the presence of 20 nM 17 beta-estradiol this number rose to 3000-3500 sites/cell. Rates of accumulation of two of the major apolipoproteins, apo-C-II and apo-A-I increased 2.5- and 2.0-fold, respectively, in response to estrogen treatment. Other major apolipoproteins were not affected at this concentration of hormone. Induction of both proteins was completely antagonized by 20 nM testosterone. The density distribution of apolipoproteins secreted by the hepatocytes was similar to that reported using perfused liver systems. The consequences of estrogen treatment were to increase the apo-C-II/apo-C-III ratio in very low density lipoproteins as well as to decrease the overall very low density lipoprotein:high density lipoprotein ratio.     

Apolipoprotein C-II mRNA levels in primate liver. Induction by estrogen in the human hepatocarcinoma cell line, HepG2

We have demonstrated that physiological concentrations of 17 beta-estradiol increase nuclear estrogen-specific binding sites in the human hepatoma cell line HepG2 7- to 10-fold and the rate of accumulation of secreted apolipoprotein C-II (apo-C-II), 2.5-fold (Tam, S-P., Archer, T. K., and Deeley, R. G. (1985) J. Biol. Chem. 260, 1670-1675). Apo-C-II is the major activator of lipoprotein lipase, an enzyme which plays a key role in lipoprotein catabolism. In order to define more precisely the mechanism by which estrogen influences apo-C-II production, we have synthesized a triacontanucleotide DNA probe that is complementary to apo-C-II mRNA. We have used the probe both in Northern hybridization experiments and in DNA excess titrations to quantify apo-C-II mRNA in hormonally treated HepG2 cells and various primate tissues. These studies revealed that: 1) the concentration of apo-C-II mRNA in HepG2 cells is comparable with that present in human liver; 2) treatment of the cells with low levels of estrogen results in a doubling of the apo-C-II mRNA concentration; 3) the apo-C-II mRNA concentration in monkey liver is 60- to 70-fold greater than in the intestine and 2.5-fold higher than in human liver.     

Induction of rat E and chicken A-I apolipoproteins and mRNAs during optic nerve degeneration

Apolipoprotein synthesis was measured in control optic nerves and optic nerves undergoing Wallerian degeneration. After short term organ culture with radiolabeled amino acid, optic nerve extracts were reacted with antiserum to rat or chicken apolipoproteins. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the degenerating rat optic nerve, apo-E synthesis increased from 0.30 to 0.90% of newly synthesized protein and from 0.45 to 1.4% of secreted protein. A DNA-excess solution hybridization assay was constructed to measure the absolute amount of apo-E mRNA in control and degenerating optic nerves. Paralleling the increase in apo-E protein synthesis, the absolute amount of apo-E mRNA was elevated 3- to 4-fold after enucleation. Similar to rat apo-E, apo-A-I synthesis was increased in degenerating chicken optic nerve. Chicken apo-A-I represented 0.65 and 3.5% of newly synthesized protein from control and enucleated optic nerves, respectively. Apo-A-I increased from 0.85 to 5.5% of secreted protein following enucleation. Using in vitro translation to quantitate relative amounts of chicken apo-A-I mRNA, enucleated optic nerve apo-A-I mRNA content was increased 5-fold. These results suggest that local apolipoprotein synthesis may be involved in the mobilization of myelin cholesterol which occurs during Wallerian degeneration. The similar response of the rat and chicken to increase optic nerve apolipoprotein synthesis during degeneration supports the idea that avian peripheral apo-A-I and mammalian peripheral apo-E may be performing functions common to both classes of animals.     

Regulation of apo-A-I processing in cultured hepatocytes

Apo-A-I, the major protein component of high density lipoproteins, appears intracellularly as an intermediate precursor (pro-apo-A-I) with a hexapeptide extension (RHFWQQ) at its amino terminus. Proteolytic processing of pro-apo-A-I to apo-A-I has been shown to occur extracellularly in cell and organ cultures from rat and human tissues. Recently, however, intracellular conversion has been detected in chickens. To determine what distinguishes and regulates these two processing methods, the proteolytic processing and secretion of apo-A-I was studied by metabolic labeling in chick hepatocytes and in Hep-G2 cells (derived from a human hepatocellular carcinoma). The proportions of intracellular and secreted pro-apo-A-I and apo-A-I were measured by sequencing NH2-terminal portions of the proteins and determining the location of radio-labeled amino acids. Chick hepatocytes cultured in the absence of hormones or fetal bovine serum secreted primarily processed apo-A-I (83%). In the presence of serum these cells secreted only pro-apo-A-I, whereas incubation with a combination of hormones (insulin, triiodothyronine, dexamethasone) resulted in secretion of a nearly equal mixture of the pro- and processed forms of the protein. In contrast, Hep-G2 cells, maintained in the absence of serum, secreted only pro-apo-A-I; when grown in the presence of serum these cells secreted a mixture of pro- and processed apo-A-I. Under conditions in which chick hepatocytes and Hep-G2 cells secreted both forms of the protein, a mixture of pro- and processed apo-A-I was also found intracellularly; when only the pro-form was secreted, the cells likewise contained only pro-apo-A-I. Under all the above conditions, the secreted apo-A-I exhibited similar isoform patterns in two-dimensional gel electrophoresis. These data show that both chick hepatocytes and human hepatoma cells are capable of intracellularly processing pro-apo-A-I to apo-A-I, and that the extent of intracellular processing is controlled by the cell's hormonal environment.     

Tissue-specific expression of genes encoding apolipoprotein E and apolipoprotein A-I in rabbits

We determined the site of synthesis of apolipoprotein (apo) E and apo-A-I in rabbit by measuring in vitro translational activity of their mRNAs from the liver and from the intestine. Poly(A+) RNA isolated from liver and intestinal epithelium of rabbits fed either a chow diet or a cholesterol-rich diet was translated in vitro in the rabbit reticulocyte lysate system using [35S] methionine as the labeled precursor. Newly synthesized apolipoproteins were immunoprecipitated with specific antisera and quantitated after electrophoresed on 10% polyacrylamide slab gels in the presence of 0.2% sodium dodecyl sulfate. The levels of liver apo-E and apo-A-I mRNAs from chow-fed rabbits are 0.41 and 0.002% of total translatable mRNA, respectively. The level of liver apo-A-I mRNA in the rabbit is approximately 500-fold lower than the reported level of apo-A-I mRNA in rat and human livers. Rabbit intestinal apo-E and apo-A-I mRNAs levels are 0.0036 and 0.67%, respectively. Our results indicate that in rabbits apo-E is synthesized primarily in the liver and that apo-A-I is synthesized primarily in the intestine. When rabbits are fed a cholesterol-rich diet, liver and intestinal apo-E in mRNA levels and intestinal apo-A-I mRNA levels are not changed. In contrast, the liver apo-A-I mRNA level increases 5-fold in response to the cholesterol-rich diet. However, because the intestinal liver apo-A-I mRNA level is so low, the 5-fold induction only increases liver mRNA levels to 2.7% of the corresponding intestinal apo-A-I mRNA level.     

Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells

The effects of androgen withdrawal and replacement on the concentrations of androgen receptor (AR) protein and AR mRNA were investigated in rat ventral prostate and seminal vesicles and in cultured human hepatoma (HepG2) cells. AR mRNA concentrations were determined by Northern blotting with single stranded AR cRNA as the hybridization probe, whereas antibodies raised against two synthetic 17-amino acid long peptides corresponding to the N-terminal and steroid-binding regions of the AR were employed in immunological receptor assays. AR mRNA levels in both prostate and seminal vesicles increased about 2-fold within 24 h after castration and continued to rise within the next 48 h to values that were 9- to 11-fold higher than those in intact controls. Administration of pharmacological doses of testosterone (400 micrograms steroid/day) to 1-day castrated animals for 24-48 h brought about a decrease in AR mRNA levels in accessory sex organs to levels in intact controls. Similar results were obtained in cultured HepG2 cells where a switch to serum- and steroid-free medium elicited a rapid increase (approximately 4-fold in 10 h) in the AR mRNA level, which was prevented by inclusion of 10(-7) M testosterone in culture medium. Similar, but quantitatively less marked, changes occurred in the AR protein concentration in prostate, seminal vesicles, and HepG2 cells, as determined by immunoblotting using antibodies against AR peptides. In addition, immunohistochemical studies showed that AR is a nuclear protein of the prostatic epithelial cells in both intact and castrated rats, and suggested that short term castration increases the concentration of nuclear AR in the prostate. Taken together, these data indicate that androgens down-regulate the concentration of AR protein and AR mRNA in a variety of target tissues.     

Cytokine production by cholesterol-loaded human peripheral monocyte-macrophages: the effect on fibrinogen mRNA levels in a hepatoma cell-line (HepG2)

Conditioned medium from human monocyte-macrophages incubated under various conditions was tested for its ability to stimulate fibrinogen mRNA levels in the hepatoma cell line HepG2. Recombinant human interleukin-6 (IL-6) stimulated fibrinogen mRNA levels 4.4-fold over control levels; this response was blocked by an anti-IL-6 antibody. Conditioned medium from 3-day-cultured monocyte-macrophages produced a slight stimulation of fibrinogen synthesis in HepG2 cells which was enhanced when the monocyte-macrophages had been treated with lipopolysaccharide (LPS). This stimulation was blocked by the anti IL-6 antibody. The cytokines, interleukin-1 (IL-1) and tumour necrosis factor (TNF) were also detected in the conditioned medium from the 3-day-cultured monocyte-macrophages. Monocyte-macrophages were cultured for 17 days and then incubated with acetylated low density lipoprotein (AcLDL) for 48 h. Such cells were 'foamy' in appearance and showed a 4-fold increase in apoE mRNA and a 10 to 50-fold increase in apoE secretion. This increase in apoE production was suppressed by almost a third when cells were coincubated with AcLDL and LPS. Conditioned medium from these 17-day-cultured AcLDL-treated human monocyte-macrophages did not stimulate fibrinogen mRNA synthesis in HepG2 cells, nor did the conditioned medium contain detectable levels of cytokines. These results suggest that cytokine production from foam cells in the atherosclerotic lesion is unlikely to be a major contributing factor in determining the elevated fibrinogen levels seen in the plasma of patients with IHD.     

Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells.

The objective of this study was to examine effects of interleukin-6 (IL-6) on the expression and activity of the drug resistance transporters (MDR1 and MRP) in human hepatoma cell lines. Expression and activity of MDR1 and MRP transporters were examined in IL-6-treated and control HuH 7 and HepG2 cells using semi-quantitative RT-PCR analysis and by rhodamine 123 and 5-carboxyfluorescin efflux assays. Results from RT-PCR demonstrated expression of MRP3, MRP6, and MDR1 in HuH 7 cells and expression of MRP1, MRP2, MRP3, MRP6, and MDR1 in HepG2 cells. Compared with controls, treatment of HuH 7 cells with IL-6 (10 ng/mL, 24 h) resulted in a 1.8-fold increase in MRP-mediated efflux of 5-CF with a corresponding 1.5-fold induction of MRP3 mRNA levels (p < 0.05). Similarly, in HepG2 cells, a 2-fold increase in MRP functional activity and a 1.8-fold induction of MRP1 mRNA levels were seen in the IL-6 treated cells (p < 0.05). Treatment of cells with IL-6 was also found to cause significant reductions in the expression and activity of MDR1 in HuH 7 cells, but not in HepG2 cells. Our data suggest that IL-6 induces MRP expression and activity in human hepatoma cell lines. Suppressive effects of IL-6 on MDR1 expression and activity were also observed in HuH 7 cells. This underscores the importance of examining the regulation of multiple drug resistance proteins as these proteins may have opposing regulatory mechanisms in malignant cells.     

Regulation of angiotensinogen gene expression in a human hepatoma cell line

Angiotensinogen is the precursor of biologically active peptide angiotensin II and its synthesis is increased in the liver during acute inflammation. We have used radiolabeled human angiotensinogen cDNA to study the effect of hepatocyte stimulating factor (HSF), a protein synthesized in differentiating monocytes which increases the synthesis of various hepatic proteins during inflammation, on angiotensinogen mRNA levels in human hepatoma cells (HepG2). Our results indicate that angiotensinogen mRNA is present in human hepatoma (HepG2) cells and its levels are decreased when treated with hepatocyte stimulating factor. Although dexamethasone elevated angiotensinogen mRNA levels, HSF reduced this increase. These results suggest that a factor other than HSF may be involved in elevating the angiotensinogen mRNA levels in the liver during inflammation.     

Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis

Two alternative splice variants of the interleukin-1 receptor accessory protein (IL-1RAcP) mRNA are known. Membrane-bound IL-1RAcP (mIL-1RAcP) promotes intracellular interleukin-1 (IL-1) signalling whereas soluble IL-1RAcP (sIL-1RAcP) is probably an inhibitor of IL-1 signalling. Here we establish that sIL-1RAcP mRNA levels increase 16-fold in response to phorbol esters in the human hepatoma cell line HepG2 via a mechanism that depends on de novo protein synthesis. Following exposure of cells to UV light, a potent inducer of apoptosis, mIL-1RAcP mRNA is rapidly down-regulated and a new steady-state level established briefly before a gradual return to pretreatment levels. Following treatment with staurosporine, also an inducer of apoptosis, mIL-1RAcP mRNA levels steadily decrease through 72 h, with little change in sIL-1RAcP mRNA levels. A novel alternative splice variant, sIL-1RAcP-beta, was identified. Its sequence indicates that sIL-1RAcP-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of IL-1RAcP mRNAs suggest important functions in regulating sensitivity to IL-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.     

Uterine type II estrogen-binding sites are not of eosinophil origin

A recent report by Lyttle et al. (Lyttle, C. R., Medlock, R. L., and Sheehan, D. M. (1984) J. Biol. Chem. 259, 2697-2700) suggested that nuclear type II sites in the rat uterus are of eosinophil origin and may represent [3H]estradiol binding to eosinophil peroxidase. To further evaluate this hypothesis we examined the response of nuclear type II sites to estrogen under conditions where eosinophils are not present. Results of our experiments show that physiological levels of estradiol-17 beta (10 nM for 72 h) will stimulate nuclear type II sites in highly purified cultures (21-25 days; 4 passages) of rat uterine stromal and myometrial cells. The magnitude of the response of type II sites to estradiol in these stromal (4-fold) and myometrial (80-fold) cell cultures was essentially identical to that observed in the uterine cell types following in vivo estrogen treatment. Since these highly purified cultures of uterine cells were prepared from the uterus of a 21-day ovariectomized rat which is devoid of eosinophils, we conclude that estradiol stimulation of nuclear type II sites is a direct intracellular response to estrogen which occurs independent of eosinophil accumulation. Furthermore, we have found that type II sites in the rat uterus are not peroxidase. This was demonstrated by experiments which show type II sites are present in the 39,000 X g supernatant fraction of uterine cytosol, whereas peroxidase activity is quantitatively recovered in the crude mitochondrial (39,000 X g) pellet. Likewise, the small amount of peroxidase activity (approximately 10%) in the total homogenate which contaminates our nuclear pellet preparations was extracted (98-100%) with 0.5 M CaCl2. Type II estrogen-binding sites (95-100%) remained associated with the nuclear pellet fraction after peroxidase extraction. Therefore, stimulation of cytosol and nuclear type II sites by estrogen in the rat uterus is a direct intracellular response to the hormone unrelated to eosinophil accumulation and/or peroxidase activity.     

Insulin action on activity and cell surface disposition of human HepG2 glucose transporters expressed in Chinese hamster ovary cells

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, delta, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this delta-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using delta-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar-uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.