Nucleotide sequences required for the regulation of a rat alpha 2u-globulin gene by glucocorticoids.

alpha 2u-Globulin is a rat protein of as yet unknown function whose synthesis can be induced by glucocorticoids and several other hormones. Induction by glucocorticoids is a secondary response to the hormone: protein synthesis is required before the hormone can exert its stimulatory effect on alpha 2u-globulin transcription. We have used the linker-scanning mutagenesis procedure, followed by transfer of the mutant genes into mouse L-cells for analysis of their phenotype, to determine sequences within a cloned alpha 2u-globulin promoter that are required for its regulation by glucocorticoids. Mutations between positions -115 and -160 abolish or greatly reduce the inducibility of alpha 2u-globulin by the hormone. Mutations just upstream from this region, between positions -177 and -220, have an opposite effect; they increase induction two- to fourfold.

Tissue-specific expression of the rat alpha 2u globulin gene family.

The rat alpha 2u globulin gene family encodes approximately 20 low-molecular-weight (20,000) proteins with pIs ranging from 4.5 to 7.9. alpha 2u globulin protein isoforms were detected in the liver and in the submaxillary, lachrymal, preputial, and mammary glands of Sprague-Dawley rats. The hormonal and developmental regulation of alpha 2u globulin synthesis in each of these tissues was unique, and it appears that different alpha 2u gene sets were transcribed in the various tissues.

C/EBPalpha inhibits cell growth via direct repression of E2F-DP-mediated transcription.

Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPalpha, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPalpha in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPalpha is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPalpha has no affinity for these E2F sites, but when recombinant C/EBPalpha is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPalpha protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPalpha directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPalpha can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPalpha-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

Comparison of in vivo translation rates and messenger RNA levels of alpha2U-globulin in rat liver and Morris hepatoma 5123D.

The synthesis of the male rat hepatic protein alpha2U-globulin has been examined in Morris hepatoma 5123D and male host liver using pulse incorporation of labeled amino acids in vivo, followed by immunoprecipitation of the newly synthesized alpha2U-globulin from the soluble protein fraction of liver and hepatoma tissue. It was found that no alpha2U-globulin synthesizes alpha2U-globulin at a normal level (0.9 to 1.0% of total hepatic protein synthesis). A variety of liver-derived cell culture lines also did not have alpha2U-globulin synthesis. The level of the specific mRNA coding for alpha2U-globulin can be quantitated using in vitro translation of polyadenylate-containing RNA in a Krebs II ascites cell-free translational system, followed by immunoprecipitation of the alpha2U-globulin synthesized in vitro. Using this technique, it was found that host liver contained alpha2U-globulin mRNA at normal levels, whereas hepatoma tissue contained no detectable mRNA coding for this protein. Thus, alpha2U-globulin synthesis is deleted in the minimal-deviation hepatoma 5123D as a consequence of the inability of that tissue to produce functional mRNA coding for alpha2U-globulin. The implications for the regulation of gene expression in malignant cells are discussed.

Cloning of alpha 2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs.

An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200--500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA of alpha 2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of alpha 2u globulin mRNA in male and female rats.

The N54 mutant of Galphas has a conditional dominant negative phenotype which suppresses hormone-stimulated but not basal cAMP levels.

The phenotype of a Ser to Asn mutation at position 54 of the alpha subunit of G(s)(N54-alpha(s)) was characterized in transient transfection experiments in COS and HEK293 cells. Expression of either wild type or N54-alpha(s) increased basal cAMP levels. In contrast, expression of wild type alpha(s), potentiated agonist-stimulated cAMP levels, while expression of N54-alpha(s)caused a decrease. Thus, the N54-alpha(s) mutant possesses a conditional dominant negative phenotype, suppressing preferentially hormone-stimulated effects.

Extraction of nuclear proteins with increased DNA binding activity.

We have developed a method for the rapid extraction of nuclear proteins from cultured cells. The ammonium sulfate method described here extracts larger quantities of proteins that retain DNA binding activity than the modified Dignam method and another popular method used for the extraction of transcription factors. The ammonium sulfate method is rapid and can be used to process a large number of samples for gel shift analysis.

Maitotoxin-elevated cytosolic free calcium in GH4C1 rat pituitary cells nimodipine-sensitive and -insensitive mechanisms.

Maitotoxin includes an extracellular Ca2+-dependent membrane depolarization predominantly via activation of L-type voltage-dependent Ca2+ channels (L-VDCC) in GH4C1 rat pituitary cells. In contract to studies employing intracellular dyes, electrophysiological studies have indicated that maitotoxin activates voltage-independent conductances. In the present study, we used fura-2 calcium digital analysis to investigate the actions of very low concentrations of maitotoxin on cytosolic free calcium ([Ca2+]i) in GH4C1 cells in an effort to distinguish different calcium entry mechanisms. Maitotoxin at concentrations as low as 0.01 ng/mL elevated [Ca2+]i 35 +/- 3% and induced membrane depolarization. The concentration dependency for maitotoxin-elevated [Ca2+]i was biphasic with the first phase maximal at 0.05 to 0.5 ng/mL and the minimum EC50 of the second phase about 2.0 ng/mL. Nimodipine (100 nM), a dihydropyridine antagonist of L-VDCC, prevented the [Ca+2]i increase and depolarization induced by up to 0.1 ng/mL maitotoxin, but not at higher concentration (0.5 ng/mL) of maitotoxin. This indicates that lower concentrations (0.1 ng/mL) of maitotoxin require L-VDCC, whereas higher concentrations (>-0.5 ng/mL) of maitotoxin may require additional ionic mechanisms. Maitotoxin (0.5 ng/mL) induced 45Ca2+ uptake and depolarization in Ltk-cells which lack VDCC. Reducing extracellular Cl- from 123 to 5.8 microM increased the magnitude of membrane depolarization by maitotoxin (0.5 ng/mL), which suggests that a Cl- conductance participated in depolarization induced by higher maitotoxin concentrations. Taken together, our results indicate that maitotoxin activates at least two ionic mechanisms. At lower concentrations of maitotoxin, the primary ionic mechanism requires the activation of L-VDCC; however, at higher maitotoxin concentrations, additional ionic mechanisms are involve in the entry of extracellular Ca2+. This latter mechanism may represent the voltage-independent pathway evident under voltage clamp conditions.

Tyrosine phosphorylation of phosphatidylinositol 3-kinase and of the thromboxane A2 (TXA2) receptor by the TXA2 mimetic I-BOP in A7r5 cells.

Thromboxane A2 (TXA2) interacts with its G-protein coupled receptor, the TP receptor, to produce contraction and proliferation of vascular smooth muscle cells. We have shown previously that proliferation of primary cultures of vascular smooth muscle cells initiated by [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (I-BOP), a stable TXA2 mimetic, is mediated by activation of mitogen-activated protein (MAP) kinase. In the present study, we examined further the intracellular mediators involved in TXA2 activation of vascular smooth muscle cells. Transient transfection of the cDNA for the TP receptor into A7r5 vascular smooth muscle cells resulted in expression of TP receptors with a receptor density, Bmax, of 0.7 +/- 0.2 pmol/mg protein and a receptor affinity, Kd, of 0.6 +/- 0.1 nM (N = 7). Mock transfected cells lacked significant receptor expression. In TP receptor transfected cells, I-BOP increased the activation of MAP kinase 2-fold, stimulated tyrosine phosphorylation of cellular proteins of relative molecular mass (Mr) of 140, 85, 60, 56, and 45 kDa, and increased the message for c-jun, a nuclear transcription factor involved in mitogenesis, 2.6-fold. Immunoblot analysis indicated that the 85-kDa protein represented phosphoinositide 3-kinase (PI3-K), while the 60 kDa protein was the TP receptor. The activity of PI3-K was increased 3.5-fold by the addition of I-BOP (0.1 microM). In summary, the present study demonstrated that stimulation of the TP receptor results in tyrosine phosphorylation of the receptor and of PI3-K.

Sequence requirements for secondary glucocorticoid inducibility of rat alpha 2u globulin genes.

The transcription of the rat alpha 2u globulin gene family is under complex hormonal control, involving the participation of glucocorticoids, estrogens, insulin, and growth hormone. The glucocorticoid induction of alpha 2u globulin is a secondary response; that is, ongoing protein synthesis is necessary for induction of alpha 2u globulin mRNA by the hormone. This secondary response is maintained when alpha 2u globulin genes are transfected into tissue culture cells which contain the glucocorticoid receptor. We have found that the glucocorticoid induction of alpha 2u globulin occurs only in permanent cell lines, in which the alpha 2u globulin genes are integrated into the host cell DNA; induction in transient transfections is minimal. Further, the DNA sequences required for alpha 2u globulin secondary response lie entirely in the 5' proximal promoter region; no intragenic sequence elements are required for, or participate in, the secondary response to glucocorticoids.

Enhanced activity of the cardiac endocrine system during right ventricular hypertrophy.

In a model of pulmonary hypertension induced by a single injection of monocrotaline (MCT), we observed a time-dependent right ventricular hypertrophy, which became apparent in treated rats 21 days after administration of MCT and progressed through day 45. Associated with this right ventricular hypertrophy were time-dependent increases in ventricular levels of immunoreactive atrial natriuretic peptide (iANP). Forty-five days after MCT treatment, treated rats exhibited a 72-fold increase in right ventricular iANP levels and a 7-fold increase in left ventricular iANP levels. Hybridization analysis of total RNA extracted from cardiac tissue indicated that both atrial and ventricular ANP mRNA levels were elevated in treated rats. These data suggest that during pulmonary hypertension and cardiac hypertrophy the endocrine activity of the heart expands to include ventricular tissue. ANP binding site autoradiography revealed decreased binding site density in the kidney and hearts of treated rats at 49 days, consistent with the occurrence of desensitization/down-regulation. Enhanced ventricular ANP production may serve as a compensatory response to sustained elevation of pulmonary arterial pressure or may function as an autocrine/paracrine system regulating cardiac function. In either case, the effects of augmented ANP production may be subject to modulation by the status of ANP receptors in target organs and cells.

Rat alpha 2u globulin is encoded by a multigene family.

The hormonally responsive rat liver protein designated alpha 2u globulin is encoded by a multigene family. Southern blot hybridization analysis, and solution hybridization using a cloned alpha 2u globulin cDNA, indicate the presence of 18-20 alpha 2u globulin genes per haploid genome. In situ hybridization to rat metaphase chromosomes, using 125I-labeled alpha 2u globulin cDNA, indicates that most, and perhaps all, of the alpha 2u globulin genes are clustered on chromosome 5. Several alpha 2u globulin genes have been isolated from a rat library cloned in lambda phage. At least six of these clones appear to contain the entire alpha 2u globulin mRNA coding region. These genes are contained on different-sized restriction fragments, and are surrounded by distinct flanking sequences. Two of the six clones containing the alpha 2u globulin genes were found to have diverged greatly from the other four in the DNA sequence corresponding to the 3' untranslated region of mRNA. The implications for the hormonal control of alpha 2u globulin synthesis are discussed.

Hormonal inducibility of rat alpha 2u globulin genes in transfected mouse cells.

Two different genes coding for the hormonally regulated rat liver protein alpha 2u globulin were introduced into mouse Ltk- cells through co-transfection with the HSV-1 thymidine kinase gene. Three to ten copies of the alpha 2u globulin genes were detected several tk+ clones, over 50% of which could be induced with dexamethasone, the produce alha 2u globulin mRNA and protein. This suggests that the information necessary for hormonal response is contained in the DNA fragment used for transfer.

Identification of nuclear proteins that bind to the glucocorticoid regulatory region of a rat alpha 2u-globulin gene.

The induction of rat alpha 2u-globulin by glucocorticoids is a secondary response to the hormone, that is protein synthesis is absolutely required for induction. Using the DNase I protection assay, we have identified three proteins present in rat liver nuclei that bind in or near the regulatory region of a cloned alpha 2u-globulin gene. One protein which we term alpha 2u-globulin nuclear factor 1 (alpha 2uNF1), binds to precisely the sequence we have previously shown to be required for hormonal induction. Genes containing linker-scanning mutations in this region show diminished binding to this nuclear factor and display greatly reduced or abolished glucocorticoid response. alpha 2uNF1 was detected in nuclei from several sources, and its level is apparently unaffected by glucocorticoids. Its recognition sequence is unlike those of previously reported transcription factors. We detect two other proteins, alpha 2uNF2 and alpha 2uNF3, that bind near the alpha 2u-globulin regulatory region. A mutant alpha 2u-globulin promoter which does not bind alpha 2uNF2 shows increased inducibility by glucocorticoids in transfected mouse L-cells. The binding of alpha 2uNF3 is not required for alpha 2u-globulin induction by the hormone.

Multihormonal induction of hepatic alpha2u-globulin mRNA as measured by hybridization to complementary DNA.

A procedure is presented for the preparation of a (3)H-labeled complementary DNA (cDNA) specific for the mRNA coding for alpha(2u)-globulin, a male rat liver protein under multihormonal control that represents approximately 1% of hepatic protein synthesis. Rat liver polysomes are incubated with monospecific rabbit antiserum to alpha(2u)-globulin, which binds to the nascent alpha(2u)-globulin chains on the polysomes. These antibody-polysome complexes are then adsorbed to goat antiserum to rabbit IgG that is covalently linked to p-aminobenzylcellulose. mRNA preparations are thus obtained that contain 30-40% alpha(2u)-globulin mRNA. A labeled cDNA is made to this alpha(2u)-globulin-enriched mRNA preparation by using RNA-dependent DNA polymerase (reverse transcriptase). To remove the non-alpha(2u)-globulin sequences, this cDNA preparation is hybridized to an RNA concentration x incubation time (R(0)t) of 1000 mol of ribonucleotide per liter x sec with female rat liver mRNA, which, though it shares the vast majority of mRNA sequences with male liver, contains no alpha(2u)-globulin mRNA sequences. The cDNA remaining single-stranded is isolated by hydroxylapatite chromatography and is shown to be specific for alpha(2u)-globulin mRNA by several criteria. Good correlation was found in all endocrine states studied between the hepatic level of alpha(2u)-globulin, the level of functional alpha(2u)-globulin mRNA as assayed in a wheat germ cell-free translational system, and the level of alpha(2u)-globulin mRNA sequences as measured by hybridization to the alpha(2u)-globulin cDNA. Thus, the hormonal control of hepatic alpha(2u)-globulin synthesis by sex steroids and thyroid hormone occurs through modulation of the cellular level of alpha(2u)-globulin mRNA sequences, presumably by hormonal control of transcriptive synthesis.

The effect of the trichloroethylene metabolites trichloroacetate and dichloroacetate on peroxisome proliferation and DNA synthesis in cultured human hepatocytes.

Dichloroacetate (DCA) and trichloroacetate (TCA) are metabolites of the environmental contaminant trichloroethylene (TCE) that are thought to be responsible for its hepatocarcinogenicity in B6C3F1 mice. TCA and DCA induce peroxisomal proliferation and are mitogenic in rodent liver. The susceptibility of humans to TCA- and DCA-induced hepatocarcinogenesis is unknown. The current studies were aimed at using both primary and long-term human hepatocyte cultures to study the effects of TCA, DCA, and a potent peroxisome, proliferator, WY-14,643, on peroxisomal activity and DNA synthesis in human hepatocytes. Peroxisome proliferation, as assessed by palmitoyl-CoA oxidation activity, was below the limit of detection in all human cell lines tested. However, the human cell lines did display small but significant increases in CYP450 4A1 1 levels following treatment with WY-14,643 (0.1 mmol/L), indicating that the CYP 4A11 gene may be regulated by peroxisome proliferator-activated receptor alpha in humans. Similarly to their effect in rodent hepatocyte cultures, TCA and DCA were not complete mitogens in human hepatocyte cultures. In fact, DNA synthesis tended to be significantly decreased following treatment of the cells with WY-14,643, TCA, or DCA. In contrast to rodent hepatocyte responses, TCA and DCA did not increase palmitoyl-CoA oxidation and caused a decrease in DNA synthesis in human hepatocyte cultures, suggesting that humans may not be susceptible to TCA- and DCA-induced hepatocarcinogenesis.

Expression of PPAR(alpha) in human hepatocytes and activation by trichloroacetate and dichloroacetate.

Dichloroacetate (DCA) and trichloroacetate (TCA) are carcinogenic metabolites of trichloroethylene (TCE), a known hepatocarcinogen in B6C3F1 mice. This hepatocarcinogenesis is believed to result from peroxisome proliferation via PPAR(alpha) and/or stimulation of hepatocyte replication. In this study hPPAR(alpha) levels in six human liver tissues and in a long-term human hepatocyte cell line are compared. PPAR(alpha) levels varied significantly between individual tissues and are generally lower than PPAR(alpha) levels detected in mouse liver. Long-term cultured human hepatocytes display PPAR(alpha) levels only slightly lower than cultured mouse hepatocytes. Transfection studies examining the endogenous hPPAR(alpha) activity revealed little or no receptor activation, even following treatment with high concentrations of peroxisome proliferators. In contrast human hepatocytes transfected with mPPAR(alpha) and mRXR(alpha) display increased expression of PPAR(alpha), and increased PPRE-reporter activity when treated with WY-14,643, TCA, and DCA. This human hepatocyte transfection system is a promising tool for examinin the regulation of genes by PPAR(alpha) from different species.

Profound elevation of ventricular and pulmonary atriopeptin in a model of heart failure.

Recently, the concept of an atrial endocrine system has expanded to that of a cardiac endocrine system. In support of this expanded view, the cardiac ventricles have been demonstrated to be a source of the atrial hormone (atriopeptin). Markedly enhanced ventricular expression of atriopeptin has been shown to be associated with cardiac hypertrophy. In this study, we measured the levels of atriopeptin in atrial and extra-atrial tissues of the BIO 14.6 hamster, a genetic model of cardiomyopathy and congestive heart failure. The BIO 14.6 hamsters (approximately 1 year of age) weighed 7.4% more than their age-matched controls, an indication of edema, and showed overt cardiac hypertrophy (control vs. BIO 14.6 heart weight: .556 +/- .045 g vs. .990 +/- .043 g). A survey of extra-atrial tissues indicated that pulmonary and ventricular tissue from both control and BIO 14.6 hamsters possessed measurable levels of immunoreactive atriopeptin. However, a comparison of atriopeptin levels in the lungs and cardiac ventricles, respectively, of control and BIO 14.6 hamsters revealed profound differences. Pulmonary atriopeptin levels were 30-fold greater, and ventricular atriopeptin levels were 13.3-fold greater, in the BIO 14.6 hamsters. In addition, the total content of atriopeptin was 2.2-fold greater in the atria of BIO 14.6 hamsters. Dot blot analysis indicated that atriopeptin mRNA levels were greater in the atria (3.4-fold) and ventricles (17.9-fold) of BIO 14.6 hamsters. A similar analysis of atriopeptin mRNA in pulmonary tissue proved inconclusive. The function of the marked increase of pulmonary and ventricular atriopeptin is unknown; however, it is plausible that the peptide hormone serves to regulate the formation of pulmonary and peripheral edema.

Transcription of the alpha2u-globulin gene in male rat liver nuclei in vitro.

Alpha2u-globulin is a male rat liver protein under multihormonal control which represents approximately 1% of hepatic protein synthesis. We have measured the rate of transcription of the alpha2u-globulin gene using a nuclear cell-free transcriptional system with mercurated CTP as substrate for the endogenous RNA polymerases. The newly synthesized, mercurated RNA was purified free of endogenous RNA by chromatography on sulfhydryl agarose and hybridized to 3H-labeled alpha2u-globulin cDNA. It was found that, in male rat liver nuclei, alpha2u-globulin RNA sequences represent 0.005% of the total newly synthesized RNA. Actinomycin D or alpha-amanitin completely blocks the synthesis of alpha2u-globulin RNA in these nuclei. No alpha2u-globulin RNA synthesis was detectable in female rat liver nuclei. Thus, within the limits of our detection the absence of hepatic alpha2u-globulin mRNA in female rats appears to be due to a lack of transcription of the alpha2u-globulin gene in these animals.

Glucocorticoid induction of hepatic alpha2u-globulin synthesis and messenger RNA level in castrated male rats in vivo.

alpha2u-Globulin is a male rat liver protein of Mr = 20,000 which is synthesized in the liver of adult male rats, secreted into the serum, and excreted in the urine. Its function is unknown. The hepatic synthesis of this protein is under complex hormonal control. We had previously shown that castration of male rats diminishes hepatic alpha2u-globulin synthesis and the level of its mRNA, and that administration of androgen to these castrated animals results in the reinduction of the synthesis of this protein and the level of its mRNA. We now report that alpha2u-globulin synthesis and the level of its mRNA can be fully reinduced in castrated males by administration of glucocorticoid alone. This induction is much more rapid than the androgenic induction and is inhibited by the glucocorticoid antagonist progesterone. Administration of glucocorticoid to intact male animals does not induce alpha2u-globulin synthesis above normal levels; however, if alpha2u-globulin synthesis has been depressed in intact male rats by pretreatment with estrogen or cyproterone acetate, the level of this protein can be reinduced by administration of glucocorticoids. The implications for the control of alpha2u-globulin gene expression are discussed.