Ethanolamine modulates DNA synthesis through epidermal growth factor receptor in rat primary hepatocytes

Summary Ethanolamine (Etn) stimulates hepatocyte proliferation in vivo and in vitro; however, the physiological function of Etn in hepatocytes has yet to be elucidated. In the present study, we examined the effect of Etn using a primary culture of rat hepatocytes. The level of membrane phosphatidylethanolamine (PE) significantly decreased when the hepatocytes were cultured without Etn but increased to the level found in the liver when the culture medium was supplemented with 20–50 μM Etn. Moreover, Etn stimulated DNA synthesis in a dose-dependent manner and had a synergistic effect with epidermal growth factor (EGF). A binding assay and Western blotting showed that the number of EGF receptors was 22–30% lower in cells grown in the absence of Etn compared to those grown in its presence, but the respective Kd values were almost the same. Furthermore, tyrosine phosphorylation of the EGF receptor was significantly lower in cells grown without Etn. Phosphatidylcholine (PC) synthesis in the liver is unique in that it occurs via stepwise methylation of PE. We found that without Etn supplementation, bezafibrate-induced inhibition of PE methylation increased the level of PE by decreasing its conversion to PC and stimulated DNA synthesis. Moreover, the function of EGF in stimulating DNA synthesis was significantly enhanced under Etn-sufficient conditions. These data suggest that Etn is a nutritional factor required for synthesis of adequate PE, levels of which are important for hepatocyte proliferation.     

Formation of bile canaliculi in long-term primary cultures of adult rat hepatocytes on permeable membrane: an ultrastructural study

Adult rat hepatocytes were cultured for 15 days on type I collagen-coated permeable membranes in a hormonally defined Waxman's modified medium supplemented with very low concentrations of insulin, glucagon and dexamethasone. Phase contrast examination showed that 15-day-old cultures still formed a regular monolayer of polygonal cells. In similarly aged cultures, intracellular glycogen was abundant and evenly distributed, while steatosis remained very limited. Scanning and transmission electron microscopy showed that well developed bile canaliculi could be observed on the lateral side of the hepatocyte membrane after 4 days of incubation and persisted for 2 weeks. These canalicular structures probably originated from coalescence of membrane invaginations observed in 1-day-old cultures. Transmission electron microscopy showed that the ultrastructure of the cells was very close to that of normal rat hepatocytes in the intact liver. These results suggest that rat hepatocytes cultured under these experimental conditions are able to develop and maintain tissue-specific cytochemical and morphological properties for at least 15 days.     

The effect of amino acid composition of serum-free medium on DNA synthesis in primary hepatocyte cultures in the presence of epidermal growth factor

Summary The presence of optimal nutritional elements in cell culture medium is very important in studies of cultured cells. For this reason, several researchers have experimented with adding or increasing the concentration of one or more amino acids to the medium they were using to determine “essential” amino acids and optimal concentrations. We studied how leaving out one amino acid at a time from Dulbecco’s modified Eagle’s medium would affect epidermal growth factor-induced DNA synthesis in primary hepatocytes of the rat. Our “modified” DMEM contained only eight amino acids: arginine, cysteine, isoleucine, leucine, lysine, phenylalanine, tryptophan, and valine. Proline was found to be an essential amino acid in normal DMEM but not in the modified DMEM, and some other amino acids reduced DNA synthesis in this medium. This study showed that perhaps no single amino acid such as proline can be called “essential,” but rather an optimal balance of amino acids is required for each major function of each cell type cultured.     

Effect of calf and rat serum on the induction of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes by cyproterone acetate and epidermal growth factor

Summary Induction of hepatocyte DNA synthesis in culture by cyproterone acetate (CPA), a potent hepatomitogen in vivo, was studied. Adult rat hepatocytes were grown on collagen gels in primary culture for 3 to 10 d. Epidermal growth factor (EGF) was used as a model inducer to establish appropriate culture conditions. (a) In serum-free medium EGF stimulated a wave of DNA synthesis in 10 to 30% of the hepatocytes. CPA had only a weak effect. (b) Increasing concentrations of newborn bovine serum (NBS) at 5 to 95% progressively inhibited the stimulatory effect of EGF. A similar inhibition was obtained by adding bovine serum albumin; 20% NBS, however, had a slightly stimulatory effect on the induction of DNA synthesis by CPA. (c) Portal rat serum (RS) at concentration of 5 to 95% markedly stimulated DNA synthesis, a plateau being reached between 20 and 95%. EGF had a distinct enhancing effect on DNA synthesis in the presence of 5 and 20% RS but not at 50 and 95%. CPA stimulated DNA synthesis in the presence of 20, 50, and 95% RS in a synergistic way. (d) Mitoses were found after treatment with EGF or with CPA. These results show that CPA can induce DNA synthesis in cultured hepatocytes and that RS contains factors facilitating the response to CPA. This study was supported by Gesellschaft für Strahlen-und Umweltforschung mbH, München, Germany.     

Activation and glucagon regulation of mitogen-activated protein kinases (MAPK) by insulin and epidermal growth factor in cultured rat and human hepatocytes

Many hepatocellular activities may be proximally regulated by intracellular signalling proteins including mitogen-activated protein kinases (MAPK). In this study, signalling events from epidermal growth factor (EGF) and insulin were examined in primary cultured human and rat hepatocytes. Using Western immunoblots, rat and human hepatocytes were found to produce a rapid tyrosine phosphorylation of the EGF receptor and MAPK following 0·5–1 min exposure to EGF. Phosphorylation of p42 and p44 MAPK was observed following 2·5 min exposure to EGF. Insulin treatment produced phosphorylation of the insulin receptor β subunit; shc phosphorylation was not observed. MAPK phosphorylation corresponded with a shift in molecular weight and an increase in kinase activity. Insulin-dependent activation of MAPK was unequivocally observed only in human hepatocytes, though a slight activation was detected in rat. Co-treatment with insulin and EGF produced phosphorylation and complete electrophoretic shift in molecular weight of MAPK, with an additive or synergistic increase in enzyme activity in rat but not human hepatocytes; human hepatocyte MAPK was maximally stimulated by EGF alone. Glucagon pretreatment blocked phosphorylation, gel mobility shift and kinase activity of MAPK induced by insulin but only partially blocked EGF-induced MAPK activation in human hepatocytes. Glucagon also reduced the activation of MAPK by EGF in rat hepatocytes. Pre-treatments with forskolin or cyclic AMP analogues diminished in the insulin-, EGF- and insulin plus EGF-dependent activation of MAPK in rat hepatocytes without effecting phosphorylation of receptors or MAPK. These results indicate that although EGF and insulin may both signal through the MAPK/ras/raf/MAPK pathway, the response for MAPK differs between these ligands and between species. Further, in both rat and human, glucagon exerts its effects through a cyclic AMP-dependent mechanism at a level in the insulin and EGF signal transduction pathways downstream of MAPK but promixal to MAPK. The partial inhibition of EGF-induced MAPK phosphorylation by glucagon in human hepatocytes provides further evidence for a raf-1-independent pathway for activation of MAPK. © 1998 John Wiley & Sons, Ltd.     

Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes

Summary Because the metabolic environment can alter gene expression in cultured cells, we examined the effects of change of medium on the levels of several cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on Matrigel. The amounts of P450 1A2, 2B1/2, or 3A1 mRNA were unaffected by changing the medium. In contrast, P450 1A1 mRNA levels were increased 1 to 2 h after media change, reached maximum levels by 6 h, and declined to baseline by 24 h. Supplementing day-old media with components of the medium revealed that only addition of amino acids resulted in 1A1 mRNA induction. From the results of direct additions and omissions, we showed that tryptophan, but not histidine, was largely responsible for the 1A1 mRNA induction. Moreover, mild photoactivation of the tryptophan resulted in a substantially increased magnitude of 1A1 mRNA induction. The time course for 1A1 mRNA induction by treatment with photoactivated tryptophan was identical to that observed after medium change. Treatment of hepatocyte cultures with β-naphthoflavone, which is metabolized by 1A1, also resulted in a transient 1A1 mRNA induction time-course profile over a 24-h period, whereas treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which is relatively stable to metabolic transformation, produced sustained elevations of 1A1 mRNA, suggesting that the transient response to tryptophan also may involve metabolism of the inducer. Our results extend previous data showing that oxidized products of tryptophan induce 1A1, and suggest that the transient nature of the induction may be due to elimination of the activated tryptophan molecule.     

Phenolsulfonphthalein (phenol red) metabolism in primary monolayer cultures of adult rat hepatocytes

Summary The sulfonic acid dye, phenolsulfonphthalein (PSP or phenol red), has been incorporated as a pH indicator in many tissue culture media formulations since the emergence of tissue culture methodologies. The present study was designed to examine the pathway, time course, and degree of metabolism of this anionic dye in monolayer cultures of adult rat hepatocytes. Thin layer chromatographic studies coupled with β-glucuronidase studies show that glucuronidation is the major metabolic pathway for PSP in vitro. About 20% of the dye is metabolized in the first 24 h, but this functional activity is decreased by approximately half at 48 h, and even further at 72 h of culture. This metabolic activity was not affected by continuous exposure to the dye. The effect of PSP concentration on its rate of metabolism by the adult rat hepatocyte in culture seemed to be biphasic, and at concentrations of less than 100μM there was indication of a saturable process. Although PSP seemed not to be toxic to hepatocyte cultures, it is partially metabolized by these cells (as opposed to no observed metabolism in human fibroblasts or HeLa cells). Therefore, its incorporation into tissue culture media formulations for use in hepatocyte cultures should be avoided, especially when studying the mechanism(s) of glucuronidation or metabolic pathways thought to be affected by this anionic dye. This study was supported in part by NIH Grants HL-11945-11 and 1 R01 AM 26520-01A1.     

Isolation of rat hepatocytes with EDTA and their metabolic functions in primary culture

Summary Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance, viability (94.5%) and yield (1.76.10⁷ cells/g liver) compare well with those of previously described methods using collagenase. Differentiated functions of the hepatocytes in primary culture such as albumin secretion (10.9 μg/mg cell protein/d) and triglyceride synthesis and secretion are maintained. Induction of triglyceride synthesis and secretion by oleic acid takes place to an extent similar to that observed in vivo and liver perfusion. Particles with a lipid composition resembling circulating very low density lipoproteins are secreted into the medium. These characteristics demonstrate the ability of hepatocytes isolated with EDTA and subsequently used in primary culture to retain complex and highly differentiated functions of the intact liver.     

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.     

Factors controlling epidermal growth factor (EGF) gene expression in the endometrium of the mare

Previous studies showed a dramatic increase in EGF gene expression in the endometrial glands of pregnant mares around day 40 after ovulation. To investigate how the steroid hormones of pregnancy might regulate this expression, in situ hybridization was used to monitor the levels of EGF mRNA in endometrial biopsies obtained from seasonally anoestrous or ovariectomised mares given exogenous progesterone and oestrogen, alone or in combination, for up to 46 days. Biopsies were also taken from mares during the non‐pregnant cycle, during normal pregnancies and pregnancies compromised by endometrial pathology (endometrosis) or because of incompatible extraspecific embryo transfers (donkey‐in‐horse pregnancies). Only a few samples showed weak EGF expression during the late luteal phase of the oestrous cycle. During normal pregnancy, the previously observed dramatic increase of expression after day 40 of gestation was confirmed. Although aged mares suffering from endometrosis and mares carrying an extraspecific donkey conceptus showed the same increase of EGF mRNA in normal glands, this was virtually absent from gland cross‐sections compromised due to inflammatory or fibrotic changes. Administration of various doses and combinations of progesterone and oestrogen for <35 days yielded negative or only weakly positive hybridization results, whereas progesterone alone for ≥40 days upregulated EGF expression strongly irrespective of additional treatment with oestrogen. This is the first experimental evidence that EGF expression in the endometrium can be induced by progesterone alone. The requirement for prolonged progesterone priming is of considerable interest in the context of the unusually late stage of gestation at which placental attachment commences in equids. Mol. Reprod. Dev. 53:255–265, 1999. © 1999 Wiley‐Liss, Inc.     

Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium

We have analyzed the receptors for epidermal growth factor (urogastrone) (EGF-URO) and insulin in primary cultures of adult rat hepatocytes maintained for up to 3 weeks on human placental cell matrix in serum-free defined medium. Cross-link labeling experiments revealed that the insulin receptor, partially damaged by the collagenase isolation procedure, was rapidly regenerated to yield an intact receptor. In contrast, cross-link labeling of the EGF-URO receptor revealed that, upon prolonged culture, there was a progressive disappearance of the high molecular mass (175 kilodaltons (kDa)) receptor form, and an appearance of low molecular mass receptor species (130 and 105 kDa). After 3 weeks of culture, the low molecular mass receptor forms accounted for all of the labeled EGF-URO receptor present in the cells. Measurements of EGF-URO binding indicated that the number of EGF-URO binding sites per cell (2.0 x 10(5) +/- 0.3 x 10(5)) did not change during the 3 weeks of culture. However, there was a decrease in EGF-URO binding affinity, reflected by an increase in the KD from 0.6 to 3.0 nM. At zero time and after 3 weeks in culture, Scatchard plots of the binding data were linear; at intermediate time points, the plots were curvilinear. Despite the changes in the EGF-URO receptor that occurred, cells were still responsive to EGF-URO in terms of the inhibition of acetate incorporation into lipid. The ED50 for EGF-URO (about 0.2 nM) was the same for short-term cultures (48 h) as for cells maintained in culture for 3 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the human hepatocyte growth factor cDNA in primary cultures of rat hepatocytes

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are primary mitogens for hepatocytes in culture. hepatocytes express the HGF-receptor MET but not HGF itself. To investigate the influence of autocrine HGF expression on the proliferative potential of hepatocytes, primary cultures were submitted to retrovirus-mediated transduction of the human hgf (huHGF) cDNA. Expression of the transduced cDNA revealed a minimum 2-fold increase in HGF-mRNA, whereas expression of the Escherichia coli beta-galactosidase gene remained even. Estimation of huHGF copy numbers showed there was a minimum 4-fold increase, suggesting an increase in the population of transduced cells. Immunoprecipitation of excreted huHGF and growth bioassays proofed that HGF was present and functional. HGF is excreted into the medium and therefore, by diffusion, available to transduced and non-transduced cells. The increase in huHGF-transduced cells suggests that the autocrine pathway as opposed to the paracrine pathway, which are both present at the same time, confers a growth advantage to these cells.     

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.     

DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: effects of epidermal growth factor, insulin, glucagon, and cyclic-AMP

Epidermal growth factor (EGF) especially in combination with insulin and glucagon, has been shown to stimulate DNA synthesis in liver cells, both in the whole animal and in cell cultures. As a further development we have found that in primary monolayer cultures of freshly isolated adult rat liver parenchymal cells, in which contamination with nonparenchymal cells was negligible, DNA synthesis was substantially stimulated by these substances. In control cultures, incorporation of [3H]thymidine into DNA and labeling of nuclei in autoradiographs was low. The stimulation by EGF was enhanced by insulin and glucagon, whereas these hormones by themselves exhibited only limited activity. These observations were made in cultures of hepatocytes that were never exposed to serum, even during cell isolation and plating. Hence for stimulation of DNA synthesis under these conditions neither serum factors nor interactions with other types of cells or their products were required. The effects of glucagon were reproduced by substances that elevate intracellular concentration of cyclic-AMP, including cholera toxin, isoproterenol, and methylisobutylxanthine. These various substances, especially EGF, glucagon, or cyclic-AMP, altered the morphological characteristics of the cultures during early stages, promoting cellular spreading and aggregation.     

Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF

Summary To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco’s modified Eagle’s medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6×10⁵ cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo. but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleoid and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentiate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.     

Stimulation by epidermal growth factor of phospholipid methyltransferase in isolated rat hepatocytes

Epidermal growth factor produces a time- and dose-dependent activation of phospholipid methyltransferase activity in hepatocytes isolated from juvenile and mature hepatectomized rats. This treatment however has no effect with hepatocytes isolated from mature or laparotomized rats. Dansylcadaverine (50μM), an inhibitor of receptor-mediated internalization of epidermal growth factor, has no effect on basal phospholipid methyltransferase but inhibits the stimulation of this enzyme by epidermal growth factor.

These results indicate a possible role of phospholipid methylation during liver proliferation.