Nuclear Run-off Transcription Assays Research Articles

Hyaluronan Mixed Esters of Butyric and Retinoic Acid Drive Cardiac and Endothelial Fate in Term Placenta Human Mesenchymal Stem Cells and Enhance Cardiac Repair in Infarcted Rat Hearts

We have developed a mixed ester of hyaluronan with butyric and retinoic acid (HBR) that acted as a novel cardiogenic/vasculogenic agent in human mesenchymal stem cells isolated from bone marrow, dental pulp, and fetal membranes of term placenta (FMhMSCs). HBR remarkably enhanced vascular endothelial growth factor (VEGF), KDR, and hepatocyte growth factor (HGF) gene expression and the secretion of the angiogenic, mitogenic, and antiapoptotic factors VEGF and HGF, priming stem cell differentiation into endothelial cells. HBR also increased the transcription of the cardiac lineage-promoting genes GATA-4 and Nkx-2.5 and the yield of cardiac markerexpressing cells. These responses were notably more pronounced in FMhMSCs. FMhMSC transplantation into infarcted rat hearts was associated with increased capillary density, normalization of left ventricular function, and significant decrease in scar tissue. Transplantation of HBR-preconditioned FMhM-SCs further enhanced capillary density and the yield of human vWF-expressing cells, additionally decreasing the infarct size. Some engrafted, HBR-pretreated FMhMSCs were also positive for connexin 43 and cardiac troponin I. Thus, the beneficial effects of HBR-exposed FMhMSCs may be mediated by a large supply of angiogenic and antiapoptotic factors, and FMhMSC differentiation into vascular cells. These findings may contribute to further development in cell therapy of heart failure.

Identification of Newly Transcribed RNA

Newly transcribed RNA can be identified using the nuclear runoff transcription assay. In this assay, isolated nuclei, free of membranes and cytoplasmic debris, are used in an in vitro transcription reaction in the presence of (32)P-labeled UTP. The labeled RNA can then be hybridized to cDNAs immobilized on nitrocellulose. This unit describes three methods for isolating suitable nuclei by detergent lysis, Dounce homogenization, and centrifugation on a sucrose gradient. Two Support Protocols describe the preparation of nitrocellulose filter strips containing double-stranded and single-stranded DNAs, which are used to detect the presence of specific transcripts in the nuclear runoff transcription assay.

Clinical predictors of cognitive function change in schizophrenia

Serine dehydratase mRNA in the livers of rats maintained on laboratory chow (containing 22% protein) under a 12-h light (7:00-19:00)/12-h dark (19:00-7:00) cycle showed a daily oscillation that was maximal at 19:00 and minimal at 7:00 with an amplitude of more than 20-fold. The feeding of high-protein diets (60 and 91% casein) did not affect the oscillation phase. Virtually identical oscillatory phases for the mature serine dehydratase mRNA and its precursor RNAs, and the results of nuclear run-off transcription assays indicated that the oscillation was generated at the level of transcription. Similar oscillation patters were seen in adrenalectomized, starved, or diabetic rats. However, the mRNA oscillation was not precisely reflected in the oscillation of enzyme activity or enzyme protein in both the phase and the amplitude. Rats maintained on laboratory chow under constant light for 2 weeks still showed mRNA oscillation with a peak at 3:00, whereas rats exposed to constant light and fed between 7:00 and 9:00 exhibited an oscillation peak at 3:00-7:00. Under the conditions of a 12-h light/dark cycle and a 2-h restricted feeding between 7:00 and 9:00, a broad peak was seen at 23:00-3:00. These results indicate that the rhythmic expression of serine dehydratase gene is controlled by both meal-responsive and photoresponsive regulators.

Transcriptional activation by the human Hsp70-associating protein Hap50.

We investigated human Hap50, the large isoform of the previously characterized Hsp70/Hsc70-associating protein Hap46, also called BAG-1, for effects on transcriptional activities. Overproduction by transient transfection led to enhanced expression of reporter gene constructs in various cell types using different promoters, suggesting independence of promoter type. Similarly, overexpression of Hap50 resulted in increased levels of poly(A)(+ )mRNAs in HeLa, COS-7, 3T3 and HTC cells. Concomitantly, the expression of some selected endogenous genes, such as those coding for c-Jun and the glucocorticoid receptor, was enhanced significantly relative to actin. Nuclear runoff transcription assays using HeLa cells showed that the effect is caused by increased transcription rates rather than mRNA stabilization. Activation of transcription by Hap50 occurred at 37 degrees C and did not require prior thermal stress, as is the case for Hap46. In accordance with these biological effects, Hap50 is localized exclusively in the nuclear compartment of different cell types, whereas Hap46 is mostly cytoplasmic in unstressed cells, as revealed by use of fusion constructs with green fluorescent protein. High cellular levels of Hap50 were found to make cells less susceptible to adverse environmental effects such as heat stress. Our data suggest that Hap50 is a nuclear protein that acts in cells to increase the transcription of various genes.

Elf-pulsed magnetic fields modulate opioid peptide gene expression in myocardial cells.

Magnetic fields have been shown to affect cell proliferation and growth factor expression in cultured cells. Although the activation of endorphin systems is a recurring motif among the biological events elicited by magnetic fields, compelling evidence indicating that magnetic fields may modulate opioid gene expression is still lacking. We therefore investigated whether extremely low frequency (ELF) pulsed magnetic fields (PMF) may affect opioid peptide gene expression and the signaling pathways controlling opioid peptide gene transcription in the adult ventricular myocyte, a cell type behaving both as a target and as a source for opioid peptides. Prodynorphin gene expression was investigated in adult rat myocytes exposed to PMF by the aid of RNase protection and nuclear run-off transcription assays. In PMF-exposed nuclei, nuclear protein kinase C (PKC) activity was followed by measuring the phosphorylation rate of the acrylodan-labeled MARCKS peptide. The effect of PMF on the subcellular distribution of different PKC isozymes was assessed by immunoblotting. A radioimmunoassay procedure coupled to reversed-phase high performance liquid chromatography was used to monitor the expression of dynorphin B. Here, we show that PMF enhanced myocardial opioid gene expression and that a direct exposure of isolated myocyte nuclei to PMF markedly enhanced prodynorphin gene transcription, as in the intact cell. The PMF action was mediated by nuclear PKC activation but occurred independently from changes in PKC isozyme expression and enzyme translocation. PMF also led to a marked increase in the synthesis and secretion of dynorphin B. The present findings demonstrate that an opioid gene is activated by myocyte exposure to PMF and that the cell nucleus and nuclear embedded PKC are a crucial target for the PMF action. Due to the wide ranging importance of opioid peptides in myocardial cell homeostasis, the current data may suggest consideration for potential biological effects of PMF in the cardiovascular system.

Transcriptional regulation of the human Na/K ATPase via the human mineralocorticoid receptor.

The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) are members of the steroid/thyroid hormone receptor superfamily of ligand inducible transcription factors and have been shown to bind the glucocorticoid response element (GRE). Sodium-potassium ATPase (Na/KATPase) is a major target of mineralocorticoids. Both aldosterone and glucocorticoids activate the human Na/K ATPase alpha1 subunit and beta1 subunit genes transcriptionally. However, the mechanisms of corticosteroid regulation of mammalian Na/K ATPase subunit gene expression are not known. In this investigation, we report for the first time that cell lines (T-84 and 293) express endogenous MR by RT-PCR message expression. However, the protein product was not expressed as determined by western blot analyses. In transactivation studies of MR with GRE31, we detected MR expression at low concentrations of aldosterone. We also performed Northern blot and nuclear run-off transcription assays to further confirm that the regulation is transcriptional. We conclude that the transcriptional regulation of the human Na/K ATPase alpha1 and beta1 subunits by aldosterone occurs via the involvement of the MR.

Regulation of brain glucose transporters by glucose and oxygen deprivation

Brain cells are dependent on glucose and oxygen for energy. We investigated the effects of hypoxia, glucose deprivation, and hypoxia plus glucose deprivation on mRNA and protein levels of glucose transporter (GLUT1) and GLUT3 and 2-deoxyglucose (2-DG) uptake in primary cultures of rat neurons and astroglia. Hypoxia for 24 hours did not significantly affect cell viability but increased neuronal GLUT1 and GLUT3 mRNA up to 40-fold and fivefold, respectively, above control levels. Similar changes in GLUT1 mRNA were measured in glia. The effects of hypoxia on GLUT1 and GLUT3 mRNA were reversible. The increase in GLUT1 mRNA could be detected within 20 minutes of hypoxia and was blocked by actinomycin D. Nuclear runoff transcription assays showed that hypoxia did not alter the transcription rate of GLUT1. However, hypoxia enhanced the stability of GLUT1 mRNA in neurons ( half-life [t 1 2 ] > 12 hours ) compared with normoxic conditions ( t 1 2 ∼ 10.4 hours ), suggesting the existence of a posttranscriptional mechanism for the regulation of GLUT1 transcript levels. Twenty-four hours of normoxia and 1.0 mmol/L glucose increased neuronal GLUT1 mRNA less than threefold above basal, but 24 hours of glucose and oxygen deprivation increased GLUT1 over 111-fold above basal. Induction of neuronal GLUT1 mRNA was temporally associated with increased levels of GLUT1 protein and with stimulation of intracellular 2-DG accumulation. We conclude that hypoxia reversibly increases the transcript levels of GLUT1 and GLUT3 in rat brain cells and stimulates GLUT1 transcript levels by posttranscriptional mechanisms. Although glucose deprivation alone produces minimal effects on GLUT mRNA levels, hypoxia plus glucose deprivation synergize to markedly increase GLUT gene expression.

Epigenetic inhibition of lysyl oxidase transcription after transformation by ras oncogene.

Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts as a phenotypic suppressor of the ras oncogene. To understand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays indicated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcription. Interferon-mediated phenotypic reversion of ras transformed cells, in which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transfected mouse lysyl oxidase promoter indicated that it was active in the transformed background, despite the silencing of the endogenous lysyl oxidase promoter. The detection of comparable amounts of mRNA for transcription factors IRF-1 and IRF-2 in normal and ras-transformed cell lines suggests that the differential transcription of lysyl oxidase was not due to regulation of IRFs. Lysyl oxidase promoter activity was localized to a 126 bp region that includes two consensus TATA boxes with associated confirmed cap signals. Analysis of a human lysyl oxidase promoter sequence indicated similar promoter elements and extensive sequence identity with the mouse promoter. The binding of transcription factor AP2 to sites predicted in the control region was confirmed by DNase footprinting. Lysyl oxidase transcription was stimulated by dexamethasone treatment of cells, but this effect could not be assigned within the approximately 3 kb region tested in reporter gene constructs. The promoter activity of the lysyl oxidase reporter gene construct was completely abolished by in vitro DNA methylation, suggesting that the transcriptional suppression after transformation by the ras oncogene may involve DNA methylation.

Interferon Inhibits the Replication of HIV-1, SIV, and SHIV Chimeric Viruses by Distinct Mechanisms

Interferon (IFN) treatment of lentivirus-infected cells substantially reduces virus replicationin vitro. Although the replication of both HIV-1 and simian immunodeficiency virus (SIV) is inhibited, IFN blocks the replication of these viruses at different stages of the viral life cycle. We previously demonstrated that in HIV-1–infected cells, IFN blocks a late step in viral replication, leading to a decrease in viral protein stability and a deregulation of polyprotein processing. In contrast, in SIV-infected cells, IFN blocks an early step in viral replication, between virus binding and reverse transcription. Thus, the viral gene products targeted by IFN may be different for each of these viruses. To attempt to define which viral proteins are targeted by the IFN response, we examined the effects of IFN on the replication of two SIV/HIV-1 (SHIV) chimeric viruses, SHIV-4(vpu+) and SHIV-4(vpu−) in 174 × CEM cells. These viruses were grown from constructs in which the SIVmac239env,tat, andrevgenes have been replaced with those of HIV-1. The use of SHIV-4(vpu+) allowed us to examine whethervpu, which is unique to HIV-1, might contribute to the differential effects of IFN on HIV-1 and SIV replication. Surprisingly, we found that IFN inhibited SHIV replication differently than the replication of either HIV-1 or SIV. IFN treatment of SHIV-infected cells resulted in a decrease in the level of viral RNA expression but had no apparent effect on the integration of proviral DNA. Nuclear runoff transcription assays indicated that the reduction in SHIV RNA expression in IFN-treated cells was not due to alterations in RNA polymerase II-mediated transcription, suggesting that IFN may block SHIV replication by promoting the increased degradation of viral RNA. The presence or absence of thevpugene did not alter the effects of IFN on SHIV replication, indicating that Vpu is not responsible for the differential effect of IFN on HIV-1 and SIV replication. Thus the response of SHIVs to antiviral agents such as IFN may be unique from either HIV-1 or SIV. This may be an important consideration when using SHIVs to evaluate anti-HIV-1 therapies in animal models of AIDS.

Agonist-induced down-regulation of α1B-adrenergic receptor in HEK293 cells transfected with α1B cDNA

HEK293 cells stably expressing hamster alpha(1B)-adrenergic receptor (alpha(1B)-AR) were used to observe the effect of nonepinephrine (NE) on alpha(1B)-AR gene expression. Radioligand binding assys and RNase protection assays were used to determine alpha(1B)-AR number and the mRNA level, respectively. Exposure (2-24 h) of HEK293 cella to NE (10 mumol) caused a decrease in alpha(1B)-AR mRNA with maximum change found at the 4th hour. and in alpha(1B)-AR density at the 24th hour. NE-induced decrease in alpha(1B)-AR mRNA was inhibited by protein kinase C (PKC) inhibitor calphostin C (0.1. mumol) and mimicked by PKC activator PMA (1 mumol). Nuclear run-off transcription assay showed that treatment of the cells with NE (10 mumol) exerted no effect on the transcription rate of alpha(1B)-AR, After the synthesis of new RNAs was inhibited by actinomycin D, NE muld not accelerate the degradation of alpha(1B)-AR mRNA. The results suggested that in the HEK293 cells NE muld induce the down-regulation of alpha(1B)-AR, and the effects were mediated by PKC pathway. NE could not alter the transcription rate of alpha(1B)-AR mRNA, but it might induce the synthesis of some factors and indirectly accelerate the degradation.

Epidermal growth factor induces cyclin D1 in a human prostate cancer cell line

BACKGROUND The human prostate carcinoma cell line, LNCaP, proliferates under stimulation by a limited number of mitogenic signals, which include members of the growth factor and steroid hormone families. Androgens and epidermal growth factor (EGF) are among the LNCaP cell mitogens. We tested the hypothesis that these mitogens stimulate LNCaP cell proliferation at least in part through the induction of cyclin D1, a protein requisite for cell cycle progression, which is expressed in the G1 phase of the cell cycle. METHODS LNCaP cells were grown in serum-free medium with 10 ng/ml or 100 ng/ml EGF, 0.1 nM or 1.0 nM mibolerone (a potent androgen agonist), or vehicle (distilled water or 0.01% ethanol). Expression of cyclin D, mRNA, and protein were assessed by Northern and Western blot analyses. Transcription regulation was assessed by nuclear runoff assay. RESULTS Western analyses demonstrated that EGF stimulated cyclin D1 protein expression 4-fold over 12 hr. Northern analyses showed a 4-fold increase in mRNA expression, peaking within 4 hr of EGF stimulation. There were no effects on cyclin D1 protein or mRNA expression with mibolerone treatments. We further explored the mechanism of cyclin D1 induction. LNCaP cells stimulated for 1 hr with EGF demonstrated a 2-fold increase in cyclin D1 message, as assayed by nuclear runoff transcription assay. In addition, we demonstrated the involvement of the protein kinase C pathway in mediating the EGF induction of cyclin D1. CONCLUSIONS We conclude that one of the mechanisms by which growth factors such as EGF may stimulate prostate cell proliferation is through the direct induction of cyclin proteins, which are necessary for entry of cells into mitosis. Prostate 35:117–124, 1998. © 1998 Wiley-Liss, Inc.

Epidermal growth factor induces cyclin D1 in a human prostate cancer cell line.

The human prostate carcinoma cell line, LNCaP, proliferates under stimulation by a limited number of mitogenic signals, which include members of the growth factor and steroid hormone families. Androgens and epidermal growth factor (EGF) are among the LNCaP cell mitogens. We tested the hypothesis that these mitogens stimulate LNCaP cell proliferation at least in part through the induction of cyclin D1, a protein requisite for cell cycle progression, which is expressed in the G1 phase of the cell cycle. LNCaP cells were grown in serum-free medium with 10 ng/ml or 100 ng/ml EGF, 0.1 nM or 1.0 nM mibolerone (a potent androgen agonist), or vehicle (distilled water or 0.01% ethanol). Expression of cyclin D, mRNA, and protein were assessed by Northern and Western blot analyses. Transcription regulation was assessed by nuclear runoff assay. Western analyses demonstrated that EGF stimulated cyclin D1 protein expression 4-fold over 12 hr. Northern analyses showed a 4-fold increase in mRNA expression, peaking within 4 hr of EGF stimulation. There were no effects on cyclin D1 protein or mRNA expression with mibolerone treatments. We further explored the mechanism of cyclin D1 induction. LNCaP cells stimulated for 1 hr with EGF demonstrated a 2-fold increase in cyclin D1 message, as assayed by nuclear runoff transcription assay. In addition, we demonstrated the involvement of the protein kinase C pathway in mediating the EGF induction of cyclin D1. We conclude that one of the mechanisms by which growth factors such as EGF may stimulate prostate cell proliferation is through the direct induction of cyclin proteins, which are necessary for entry of cells into mitosis.

Up-regulation of Transforming Growth Factor (TGF)-β Receptors by TGF-β1 in COLO-357 Cells

In the present study we investigated the actions of transforming growth factor (TGF)-beta1 on gene induction and cyclin-dependent kinase inhibitors in relation to TGF-beta receptor modulation in COLO-357 pancreatic cancer cells. TGF-beta1 inhibited the growth of COLO-357 cells in a time- and dose-dependent manner and caused a rapid but transient increase in plasminogen activator inhibitor-I and insulin-like growth factor binding protein-3 mRNA levels. TGF-beta1 caused a delayed but sustained increase in the protein levels of the cyclin-dependent kinase inhibitors p15(Ink4B), p21(Cip1), and p27(Kip1) and a sustained increase in type I and II TGF-beta receptors (TbetaRI and TbetaRII) mRNA and protein levels. The protein synthesis inhibitor cycloheximide (10 microg/ml) completely blocked the TGF-beta1-mediated increase in TbetaRI and TbetaRII expression. Furthermore, a nuclear runoff transcription assay revealed that the increase in receptor mRNA levels was due to newly transcribed RNA. There was a significant increase in TbetaRI and TbetaRII mRNA levels in confluent cells in comparison to subconfluent (</=80% confluent) controls, as well as in serum- starved cells when compared with cells incubated in medium containing 10% fetal bovine serum. COLO-357 cells expressed a normal SMAD4 gene as determined by Northern blot analysis and sequencing. These results indicate that TGF-beta1 modulates a variety of functions in COLO-357 cells and up-regulates TGF-beta receptor expression via a transcriptional mechanism, which has the potential to maximize TGF-beta1-dependent antiproliferative responses.

Regulation of PDGF-B in endothelial cells exposed to cyclic strain.

The present study was designed to examine the regulation by cyclic strain of endothelial cell (EC) platelet-derived growth factor-B chain (PDGF-B) expression. We demonstrate in this study that bovine aortic ECs subjected to 10% (but not 6%) average strain resulted in a 2.6-fold increase in PDGF-B steady state mRNA and immunoreactive protein. Nuclear runoff transcription assays confirmed the induction of PDGF-B transcripts. To address the regulation of PDGF-B gene expression by cyclic strain, we transfected bovine aortic ECs with a construct containing 450 bp of human PDGF-B promoter sequence coupled to chloramphenicol acetyltransferase (CAT), and found that subjecting these cells to 10% average strain resulted in a twofold increase in CAT activity by 4 hours. Analysis of nested 5' deletions of the promoter transfected into ECs demonstrated a 55% drop-off in activity between position -313 and -153, with no induction of activity with the - 101-bp minimal promoter. Since a shear stress response element (SSRE) is located at position -125, we tested the hypothesis that the SSRE site was necessary and/or sufficient for induction of PDGF-B activity with strain. Electromobility shift assays revealed that nuclear proteins from ECs exposed to strain for short intervals (30 minutes) bound to the PDGF-B SSRE. However, transfection of ECs with hybrid promoter constructs containing the SV40 sequence promoter downstream of the SSRE or the -153 PDGF-B promoter sequence bearing a mutation in the SSRE demonstrated that the SSRE was not necessary for inducible reporter gene expression in ECs exposed to cyclic strain.

Regulation of Tyrosinase mRNA in Mouse Melanoma Cells by α-Melanocyte–Stimulating Hormone

Cloudman S-91 mouse melanoma cells respond to alpha-melanocyte-stimulating hormone) by demonstrating a marked increase in tyrosinase activity (O-diphenol-O2 oxidoreductase, EC 1.14.18.1). This increase is the result of increased levels of tyrosinase mRNA with a subsequent increase in tyrosinase abundance. Our studies were carried out to determine the effect of melanocyte-stimulating hormone on tyrosinase gene transcription and to measure the kinetics of the hormone-induced increase in tyrosinase mRNA. When melanoma cells were exposed continuously to melanocyte-stimulating hormone for 6 d, a large but transient increase in both tyrosinase mRNA abundance and enzyme activity were observed. The maximum increase in tyrosinase mRNA occurred 60 h after melanocyte-stimulating hormone stimulation and was followed by a decline in message levels even though cells were continuously exposed to hormone. Results of nuclear run-off transcription assays showed that melanocyte-stimulating hormone caused a slow increase in the rate of transcription of the tyrosinase gene with a maximal 6-fold stimulation occurring at 48 h. In cells treated with the ribonucleic acid synthesis inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, tyrosinase mRNA levels decayed with a half-life of 4-5 h. This decay rate was unaffected by treatment of cells with melanocyte-stimulating hormone, indicating that the hormone does not act to stabilize tyrosinase ribonucleic acid. Inhibition of protein synthesis by treatment with cycloheximide had no effect on the melanocyte-stimulating hormone-induced increase in tyrosinase messenger ribonucleic acid levels suggesting that ongoing protein synthesis is not required for, at least, the initial stimulation of tyrosinase gene transcription by melanocyte-stimulating hormone.

Synthesis and Accumulation of αB Crystallin in C6 Glioma Cells Is Induced by Agents That Promote the Disassembly of Microtubules

When C6 cells in culture were exposed at 37 degrees C to 1 microM colchicine or to 1 microM colcemid, a tubulin-binding antimitotic alkaloid, levels of alphaB crystallin in cells began to increase after about 10 h, reaching a maximum of more than 1 microg/mg protein after 24 h. The level of alphaB crystallin returned to near the control level within two subsequent days of culture in the normal medium. Northern blot analysis showed that the accumulation of alphaB crystallin was preceded by an increase in the level of the mRNA for alphaB crystallin. Nuclear run-off transcription assays showed that colchicine induced new synthesis of mRNA for alphaB crystallin. Immunofluorescence staining revealed that alphaB crystallin accumulated in the peripheral areas of cells, as did the depolymerized tubulin, after several hours of treatment with colcemid, and then it gradually became more conspicuous in the cytoplasm. Vinblastine and nocodazole, which also promote the disassembly of microtubules by binding to tubulins, also induced the synthesis of alphaB crystallin. Furthermore, induction of alphaB crystallin by these drugs was observed in quiescent cells that had been cultured in serum-free medium. However, taxol, a microtubule-stabilizing antimitotic agent, did not stimulate the synthesis of alphaB crystallin, but rather, it suppressed the induction of synthesis of alphaB crystallin by the microtubule-disrupting drugs. Induction of alphaB crystallin by colchicine or by other drugs that promote the disassembly of microtubules was sensitive to staurosporine, an inhibitor of protein kinases, and the induction was completely suppressed in the presence of 10 nM staurosporine. These results suggest that the expression of alphaB crystallin is stimulated, via phosphorylation reactions that are sensitive to staurosporine, when the depolymerization of microtubules is enhanced.

Protein-DNA Interactions at a Drug-responsive Element of the Human Apolipoprotein A-I Gene

Previously, we demonstrated that when two human hepatoma cell lines, Hep3B and HepG2, were exposed to gemfibrozil, a hypolipidemic drug, a 2-fold induction in apolipoprotein A-I (apoA-I) mRNA levels resulted. To determine if mRNA stabilization was responsible for the changes in apoA-I mRNA levels, the half-lives for apoA-I mRNA were measured in the presence of actinomycin D with and without gemfibrozil. These experiments revealed no differences in stability. However, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was increased 2-fold in gemfibrozil-treated cells. Transient transfection experiments also indicated that the induction of apoA-I mRNA level in response to gemfibrozil is mediated at the transcriptional level. We have identified two copies of the "drug-responsive element" (DRE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gemfibrozil. Using gel mobility shift assays with a synthetic DRE oligonucleotide, we have demonstrated that exposure of Hep3B and HepG2 cells to gemfibrozil resulted in strong induction of a protein-DNA complex. The formation of this complex is highly sequence-specific as indicated by the DNA competition experiments. The drug-inducible nuclear proteins bind to the DRE of the human apoA-I gene with an apparent Kd of 4.1 nM. Methylation interference experiments have localized the contact sites of nuclear factors to the DRE region. Southwestern blot analyses have identified two groups of drug-inducible nuclear proteins with molecular masses of approximately 30 and 15 kDa. When a copy of synthetic DRE oligonucleotide was inserted upstream of the thymidine kinase promoter and luciferase reporter construct, a significant 2-fold induction in luciferase activity was observed in the presence of gemfibrozil following transient transfection of two human hepatoma cell lines, HepG2 and Hep3B. However, a plasmid containing one copy of mutated apoA-I-DRE oligomer did not confer responsiveness to gemfibrozil treatment. Furthermore, pGL2 (apoA-I -250 mutant DRE), which carried an internal mutation of the DRE in the human apoA-I proximal promoter region, showed no increase in luciferase activity in response to gemfibrozil. These results implicate protein-DNA interactions at the DRE region in the transcriptional induction of human apoA-I gene expression by gemfibrozil.

Transcriptional and posttranscriptional mechanisms regulate human renin gene expression in Calu-6 cells.

We have recently identified a human pulmonary carcinoma cell line (Calu-6) that expresses human renin (hREN) mRNA endogenously, and we use it herein as a model to examine the regulation of the hREN gene. Transfection analysis of a deletion series (-2750 to -149) of hREN promoter-luciferase fusion constructs revealed the presence of multiple weak regulatory elements within the first 1,301 bp of the 5'-flanking region and a classic silencer element within the first intron (intron A) of the gene. The 5'-flanking regulatory domain consisted of three closely linked elements, two negative and one positive, each contributing a cell-specific threefold modulation of transcriptional activity. Treating Calu-6 cells with forskolin caused a 100-fold increase in steady-state endogenous hREN mRNA but no increase in hREN promoter activity in transient transfections or in nuclear runoff transcription assays. Nevertheless, de novo transcription and translation were necessary for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated induction. Our results suggest that multiple regulatory elements regulate basal transcriptional activity of the hREN gene and the increase in hREN mRNA by cAMP may be mediated by posttranscriptional mechanisms.

Hormonal modulation of c-fos expression in isolated hepatocytes. Effects of angiotensin II and phorbol myristate acetate on transcription and mRNA degradation.

It has been shown that angiotensin II and PMA increase the expression of proto-oncogenes (c-fos, c-myc and c-mos) in liver cells. In this study the effects of angiotensin II and PMA on c-fos transcription and mRNA stability were investigated. Using nuclear run-off transcription assays, it was observed that PMA and angiotensin II induced a rapid increase in c-fos transcription. The transcription rate of the GAPDH gene did not change, indicating that the effects were not general on gene transcription. The ability of these agents to modulate proto-oncogene mRNA stability was tested by measuring c-fos mRNA half-life. It was observed that c-fos mRNA half-life was relatively short (approximately 14-18 min) and that angiotensin II and PMA markedly stabilized mRNA, increasing its half-life (approximately 4-fold and approximately 2-fold, respectively). The protein synthesis inhibitor cycloheximide increased mRNA stability to a much greater extent. Our results clearly demonstrate that angiotensin II and PMA increased c-fos mRNA accumulation in liver cells through two actions: induction of c-fos gene transcription and increase in mRNA stability.

Beta-Amyloid regulates gene expression of glial trophic substance S100 beta in C6 glioma and primary astrocyte cultures.

S100 beta, a calcium-binding protein synthesized by CNS astrocytes, has trophic effects in vitro (neurite extension and glial proliferation). In Alzheimer's disease and Down's syndrome, severely afflicted brain regions exhibit up to 20-fold higher levels of S100 beta protein, and astrocytes surrounding neuritic plaques exhibit highly elevated levels of S100 beta immunostaining. A major constituent of plaques, beta-amyloid, has been reported to have neurotoxic and neurotrophic effects in vitro. In our study we examined the responses of CNS glia to beta-amyloid. C6 glioma cells and primary rat astrocyte cultures were treated with beta A(1-40) peptide at doses up to 1 microM. Weak mitogenic activity, measured by [3H]thymidine incorporation, was observed. Northern blot analysis revealed increases of S100 beta mRNA within 24 h in a dose-dependent manner. Nuclear run-off transcription assays showed that beta A(1-40) specifically induced new synthesis of S100 beta mRNA in cells maintained in serum, but under serum-free conditions, there was a general elevation of several mRNA species. Corresponding increases of S100 beta protein synthesis were observed by immunoprecipitation of 35S-labeled cellular proteins. To evaluate whether this effect of beta-amyloid was mediated via neurokinin receptors or by calcium fluxes, various agonists and antagonists were tested and found to be ineffective at stimulating S100 beta synthesis. In sum, these in vitro data suggest that in neuropathological conditions, beta-amyloid itself is an agent which may provoke chronic gliosis and the production of trophic substances by astrocytes.