Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.

A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined.     

Modulation by oxygen of zonal gene expression in liver studied in primary rat hepatocyte cultures

The different endowment with key enzymes and thus different metabolic capacities of periportal and perivenous cell types led to the model of "metabolic zonation." The periportal and perivenous hepatocytes receive different signals owing to the decrease of substrate concentrations including O2 and hormone levels during passage of blood through the liver sinusoids. These different signal patterns should be important for the short-term regulation of metabolism and also for the long-term induction and maintenance of the different enzyme pathways by control of gene expression. The periportal to perivenous drop in oxygen tension was considered to be a key regulator in the zonated expression of carbohydrate-metabolizing enzymes. In primary hepatocyte cultures, glucagon activated the phosphoenolpyruvate carboxykinase (PCK) gene to higher levels under arterial than under venous oxygen. The insulin-dependent activation of the glucokinase (GK) gene was reciprocally modulated by oxygen. Exogenously added hydrogen peroxide mimicked the effects of arterial oxygen on both the glucagon-dependent PCK gene and the insulin-dependent GK activation. Therefore, the oxygen sensor could be a hydrogen peroxide-producing oxidase which could contain a heme group for "measuring" the O2 tension. This notion was corroborated by the finding that CO mimicked the positive effect of O2 on PCK gene activation. Transfection of PCK promoter-CAT gene constructs into primary hepatocytes showed that the oxygen modulation of the PCK gene activation occurred in the region -281/+69. The modulation by O2 was not mediated by isolated cAMP-responsive elements. Nuclear protein extracts prepared from hepatocytes cultured under venous Po2 as compared to arterial Po2 showed an enhanced binding activity to the promoter fragment -149/-43. Oxidative conditions such as H2O2 reduced the DNA-binding activity, thus supporting the role of H2O2 as a mediator in the O2 response of the PCK and GK genes.     

Regulation of malic enzyme gene expression by nutrients,hormones, and growth factors in fetal hepatocyte primary cultures

The culture of fetal hepatocytes for 64 h in medium supplemented with 5 mM glucose, T3, insulin, and dexamethasone resulted in the coordinate precocious expression of malic enzyme mRNA, protein, and specific activity. T3 was the main inducer; meanwhile, insulin exerted a small synergistic effect when added with T3. Dexamethasone had a potentiation effect on the T3 response of malic enzyme mRNA expression regardless of the presence of insulin. This effect of dexamethasone on T3 response of malic enzyme mRNA expression was time (64 h) and glucose dependent. Glucagon, and to a greater degree dibutyryl-cAMP, repressed malic enzyme mRNA as well as protein expression by T3 and dexamethasone, in the absence of insulin. Glucose and other carbon sources such as lactate-pyruvate or dihydroxyacetone induced the abundance of malic enzyme mRNA in the absence of hormones. Insulin and T3 produced a high accumulation of malic enzyme mRNA in lactate-pyruvate medium, this effect being decreased by dexamethasone. EGF supressed the induction produced by T3 and dexamethasone on malic enzyme mRNA, while the expression of β-actin mRNA remained essentially unmodified. © 1993 Wiley-Liss, Inc.     

Post-transcriptional regulation of P-glycoprotein expression in cancer cell lines

The present study of inhibitors shows that the histone deacetylase-induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5' end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5' end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5' untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA.     

Regulation of collagenase-3 gene expression in osteoblastic and non-osteoblastic cell lines

Collagenase-3 expression in osteoblastic (UMR 106-01, ROS 17/2.8) and non-osteoblastic cell lines (BC1, NIH3T3) was examined. We observed that parathyroid hormone (PTH) induces collagenase-3 expression only in UMR cells but not in BC1 (which express collagenase-3 constitutively) or ROS and NIH3T3 cells. Since we know from UMR cells that the AP-1 factors and Cbfa1 are required for collagenase-3 expression, we analyzed the expression and PTH regulation of these factors by gel shift and Northern blot analysis in all cell lines. Gel mobility shift with a [(32)P]-labeled collagenase-3 AP-1 site probe indicated the induction of c-Fos in osteoblastic cells upon PTH treatment. While c-fos was induced in UMR cells, both c-fos and jun B were induced in ROS cells. Since Jun B is inhibitory of Fos and Jun in the regulation of the rat collagenase-3 gene in UMR cells, it is likely that high levels of Jun B prevent PTH stimulation of collagenase-3 in ROS cells. When we carried out gel shift analysis with a [(32)P]-labeled collagenase-3 RD (runt domain) site probe and Northern blot analysis with a Cbfa1 specific probe, we have observed the presence of Cbfa1 in both osteoblastic and non-osteoblastic cell lines, but there was no change in the levels of Cbfa1 RNA or protein in these cells under either control conditions or PTH treatment. From our studies above, it is evident that the expression of collagenase-3 and its regulation by PTH in osteoblastic and non-osteoblastic cells may be influenced by differential temporal stimulation of the AP-1 family members, especially c-Fos and Jun B along with the potential for posttranslational modification(s) of Cbfa1.     

Ethanol-responsive gene expression in neural cell cultures.

We have studied the molecular mechanisms underlying neuronal adaptation to chronic ethanol exposure. NG108-15 neuroblastoma cells were used to perform a detailed analysis of ethanol-induced changes in neuronal gene expression. High resolution, quantitative two-dimensional (2-D) gel electrophoresis of in vitro translation products showed both dose-dependent increases and decreases in specific mRNA abundance following treatment with ethanol at concentrations seen in actively drinking alcoholics (50-200 mM). Dose response curves for representative members of the increasing or decreasing response groups had very similar profiles, suggesting that similar mechanisms may regulate members of a response group. Some mRNAs that increased with ethanol treatment appeared identical to species induced by heat shock while other mRNAs were only induced by ethanol. We conclude that chronic ethanol exposure can produce specific coordinate changes in expression of neuronal mRNAs, including some members of the stress protein response. However, the overall pattern of ethanol-responsive gene expression is distinct from the classical heat shock subgroup of stress proteins response. Changes in gene expression and specifically, mechanisms regulating a subset of stress protein expression, could be an important aspect of neuronal adaptation to chronic ethanol seen in alcoholics.     

Regulation of cell growth and motility by hepatocyte growth factor and receptor expression in various cell species.

Hepatocyte growth factor (HGF), a humoral mediator for regeneration of liver and kidney, possesses multiple biological activities. To investigate target cell specificity and to examine whether multiple actions of HGF are related to properties of the HGF receptor on target cells, we examined the effects of HGF on cell growth and motility and analyzed the HGF receptor in various species of cells. HGF stimulated growth and DNA synthesis of PAM212 (naturally immortalized mouse keratinocytes), Mv1Lu (mink lung epithelia), and A431 (human epidermoid carcinoma) cells, as well as mature hepatocytes, but inhibited those of IM-9 (human B-lymphoblasts). Conversely, HGF had a marked stimulatory effect on cell motility of MDCK (Mardin-Darby canine kidney epithelia) cells, but not on their growth. Also, HGF enhanced the motility of various species of cells, including A431, PAM212, HepG2 (human hepatoma), KB (human epidermoid carcinoma), and J-111 (human monocytes) cells. Scatchard analysis of 125I-HGF binding to hepatocytes indicated that the cells expressed both high- and low-affinity binding sites for HGF with Kd values of 23 and 260 pM, respectively. High-affinity HGF receptor with Kd values of 20-25 pM was detected at 40-720 sites/cell in MDCK, A431, PAM212, Lu99, and IM-9 cells, but not in fibroblasts and hematopoietic cells. In contrast, low-affinity binding sites were detected in all cell lines examined, even in those not responsive to HGF. Northern blots revealed that cells possessing a high-affinity HGF receptor expressed c-MET/HGF receptor mRNA. Therefore, HGF probably regulates both cell growth and motility of various types of epithelial cells and some types of mesenchymal cells. The multiple biological activities of HGF may be exerted through a high-affinity HGF receptor linked to multiple distinct intracellular signaling pathways.     

Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures

Mammalian liver exhibits expression of members of the family of multidrug resistance (mdr) transporters (P-glycoproteins). P-glycoprotein isoforms encoded by mdr1 genes participate in extrusion of an array of xenobiotics into the bile. Induction of mdr1b mRNA expression has been shown to occur in rat hepatocytes in response to hepatotrophic growth factors. As the cytokine tumor necrosis factor alpha (TNF-α) is known to exert a direct mitogenic effect on hepatocytes, its influence on mdr1b expression was investigated. In primary rat hepatocytes cultured in the absence of TNF-α, a time-dependent increase in basal expression of mdr1b mRNA and in immunodetectable P-glycoprotein was observed. In cells treated with TNF-α (4,000 U/ml) for 3 days, expression of mdr1b mRNA and of immunodetectable P-glycoprotein was induced approximately twofold. Moreover, intracellular steady-state levels of the mdr1 substrate rhodamine 123 were decreased in cells pretreated with TNF-α in comparison to controls, indicating an increase in functional transporter(s) mediating dye extrusion. Treatment of hepatocytes with antioxidants (1 mM ascorbic acid and 2% dimethyl sulfoxide) for 3 days markedly suppressed mdr1b mRNA and P-glycoprotein expression both in cells cultured in the presence of TNF-α and in the absence of the cytokine, but did not fully abolish mdr1b mRNA induction by TNF-α, supporting the notion that reactive oxygen species participate in regulation of basal mdr1b gene expression during hepatocyte culture. In conclusion, the present data indicate that by inducing mdr1b expression in hepatocytes, TNF-α may affect the capacity of the liver for extrusion or detoxification of endogenous or xenobiotic mdr1 substrates. J. Cell. Physiol. 176:506–515, 1998. © 1998 Wiley-Liss, Inc.