Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: involvement of both the glucocorticoid and pregnane X receptors.

Glucocorticoids regulate the rat glutathione S-transferase A2 (GSTA2) gene in a biphasic manner in cultured hepatocytes that repress gene expression at low concentration (10--100 nM) but induce gene expression at high concentration (>1 microM). High concentrations of the glucocorticoid receptor (GR) antagonist RU38486 (5--10 microM) also induced the expression of GSTA2. These effects were reproduced in HepG2 cells transfected with a luciferase reporter containing 1.6 kilobase pairs of 5'-flanking sequence of GSTA2 and expression plasmids for either GR, pregnane X receptor (PXR) or a combination of both. Dexamethasone t-butylacetate (1 microM t-Bu-DEX) repressed gene expression between 60 to 75% when only GR was expressed. When PXR was expressed, both basal and t-Bu-DEX-dependent gene expression was increased over 2-fold, respectively. Biphasic regulation of gene expression was observed over a broad range of t-Bu-DEX concentrations when expression plasmids for both receptors were cotransfected. Other steroids of the pregnane class induced GSTA2 expression as expected for a PXR-dependent process. Because no canonical responsive element for the PXR-RXR alpha heterodimer was observed in the 5'-flanking region of the construct, deletion analysis was used to identify a pregnane responsive region between base pairs -700 and -683; this 20-bp region contains the antioxidant response element (ARE). When the ARE sequence was mutated, basal, t-butylhydroquinone- and 17 alpha-hydroxypregnenolone-inducible expression were all lost. These results suggest that PXR interacts with factors binding to the ARE to elicit the pregnane inductive response for GSTA2.

Expression of prostaglandin receptors EP4 and FP in human lens epithelial cells.

PURPOSE: To examine the expression of prostaglandin (PG) receptors EP2, EP4, and FP in a human lens epithelial cell line (HLE-B3) at molecular and pharmacologic levels. METHODS: Reverse transcription-polymerase chain reactions (RT-PCR) were performed with total RNA preparations from HLE-B3 cells using sense and antisense primers for each of the three prostaglandin receptors. The PCR products were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. At the pharmacologic level, the expression of EP4 receptors was determined by measuring intracellular cyclic adenosine monophosphate (cAMP) formation in response to PGE2 (EP1, EP2, EP3, and EP4 agonist) and the EP4 receptor-selective antagonist AH23848. The expression of FP receptors in HLE-B3 cells was explored by measuring intracellular [Ca2+]i mobilization. RESULTS: RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors. Hybridization of the PCR products with specific 32P-labeled probes and restriction digestion of the PCR products further confirmed that they were generated from the respective EP2, EP4, and FP mRNAs. The EP receptor agonist PGE2 significantly increased the cAMP level in HLE-B3 cells. The formation of cAMP by PGE2 was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. Stimulation of HLE-B3 cells by the FP receptor agonist fluprostenol increased [Ca2+]i in a time-dependent manner. CONCLUSIONS: The results of the molecular biologic and pharmacologic experiments showed conclusively the presence of EP4 and FP receptor messenger RNAs and proteins, respectively, in HLE-B3 cells.

Cytochalasin D-induced actin gene expression in murine erythroleukemia cells.

There is a dynamic equilibrium between monomeric G-actin and polymeric F-actin microfilaments (MFs) in eucaryotic cells. We have previously shown that disruption of MFs with cytochalasin D (CD) induced beta-actin gene transcription, resulting in elevated levels of beta-actin mRNA and protein synthesis. CD also inhibited cell growth by arresting progression through the S phase of the cell cycle. These CD-induced responses were reversible since recovering cells progressed through the G2 phase and resumed normal growth while beta-actin mRNA and protein synthesis rapidly returned to control levels. In the present study, we show that the response of beta- and gamma-actin genes is due to the synthesis of a protein(s) acting at a 5' regulatory element that may be independent of or require sequences in addition to the serum response element (SRE). CD induces beta- and gamma-actin mRNA in a dose-dependent manner, reaching a maximum of 20-fold over control mRNA levels at 30 microM. beta- and gamma-Actin gene expression was also induced 5-fold by serum stimulation of quiescent murine erythroleukemia (MEL) cells, while combined treatment with serum and CD had an additive effect. Two protein synthesis inhibitors, cycloheximide and puromycin, blocked the CD-induced increase in beta-actin mRNA, in contrast to the serum-induced increase which is insensitive to inhibitors of protein synthesis. The rapid return of beta-actin mRNA to basal levels following CD removal did not require protein synthesis nor did it require progression through the G2 phase of the cell cycle. A vector containing the 5' end of the beta-actin gene linked to a CAT reporter responded to CD when transfected into MEL cells, localizing the responsive element to the 5' portion of the beta-actin gene. By contrast, a minimal 99-bp actin promoter-CAT construct containing a functional SRE did not respond to CD.

Actin gene expression in murine erythroleukemia cells treated with cytochalasin D.

The expression of cytoskeletal protein genes may be linked to both cell growth and the status of the cytoskeleton. Actin gene expression was examined in murine erythroleukemia cells treated with the microfilament disrupting agent, cytochalasin D (CD), at a concentration which was determined to inhibit cell growth and arrest cells in the S and G1 phase of the cell cycle. Levels of actin mRNA and protein synthesis were elevated eight- and sixfold, respectively, after 9 h in CD. This increase was reflected in levels of nuclear run-on actin transcripts and prevented by actinomycin D, suggesting that enhanced transcription of the actin gene was responsible for the increase. Removal of CD resulted in immediate resumption of cell cycle progression with the accumulation of a G2-phase-enriched population and a rapid return of actin mRNA and protein synthesis to control levels (half-life 4.8 h). These results are consistent with a model linking actin gene expression to cell growth by regulating transcription during the G1 and mRNA decay during the G2 phase of the cell cycle.

Transforming growth factor alpha and its receptor in neural retina.

Transforming growth factor alpha (TGF-alpha) stimulates mitosis of many ectodermal cells but has not previously been studied for its role in neural tissues such as retina. We examined bovine retina for the presence of TGF-alpha mRNA, TGF-alpha protein and for the presence and location of the TGF-alpha/EGF receptor. Biochemical studies demonstrated a high level (770 fmol/mg protein) of specific, high affinity (Kd = 2 nM) TGF-alpha/EGF receptors in membrane homogenates of neural retina, but undetectable binding to homogenates of retinal pigment epithelium. Light microscopic autoradiograms of sections of neural retinal tissue incubated with 125I-EGF indicated that specific TGF-alpha/EGF receptors were present on one or more cell types of the retina with the exception of the outer segments of the photoreceptor cells. In addition, retinal cells appear to synthesize TGF-alpha since both mRNA for TGF-alpha and TGF-alpha protein (4.2 ng/mg protein) were detected in retinal extracts using cDNA hybridization and TGF-alpha RIA techniques. The role(s) of TGF-alpha and its receptor in retina is unknown, but it is possible that they interact via an autocrine/paracrine mechanism to influence retinal regeneration, proliferative retinopathies or neural transmission.

Altered cardiac tissue gene expression during acute hypoxic exposure.

Anoxia has been shown to induce the expression of one or more "stress proteins' in mammalian cells and tissues. A less severe form of oxygen depletion, hypoxic hypoxia, occurs in response to hypobaric decompression which simulates high altitude conditions. Under these conditions mouse hearts accumulate mRNAs for at least two polypeptides at substantially elevated levels. The molecular weights of these proteins, 85 kDa and 95 kDa, are similar to those reported for other mammalian stress proteins or glucose-regulated proteins. Time course experiments suggest that mRNAs for these species increase continuously for up to 16 hours of treatment, while mRNA for 71 kDa and 79 kDa polypeptides are elevated early in the treatment, but later decrease to control values. Total heart mRNA template activity is also increased by the hypobaric treatment. These results demonstrate that mouse cardiac tissue is capable of mounting a cellular stress-like response when exposed to moderately stressful conditions. It also provides a model for studying the direct effects of acute hypoxic stress on cellular gene expression, and its relationship to physiological adaptation.

Effects of starvation on rat liver mRNA translation products.

The synthesis of rat liver protein and RNA decreases with starvation. It is not yet known whether such decreases are regulated strictly at a transcriptional level, or if post-transcriptional controls are also involved. In this study we investigate the effects of 0, 2, or 4 days starvation on the levels of specific, abundant mRNAs in total and polysomal RNA populations. The mRNAs were analyzed by translation in vitro in mRNA-dependent, cell-free, protein synthesizing systems. The resulting polypeptide products were separated by gel electrophoresis and visualized with fluorography. The amount of albumin translated from both polysomal and total cellular mRNA decreased 20-40% with fasting. In contrast, a specific peptide having a molecular mass of approximately 30 kDa increased two- to three-fold in total cellular RNA with a smaller increase observed in polysomal RNA. These changes were maximal at 2 days of starvation. Since starvation is known to cause alterations in liver metabolism the 30-kDa polypeptide may be related to enzymes or other proteins involved in this homeostatic response.

Purification of transferrin and albumin from mouse ascites fluid.

Mouse ascites fluid, which is readily obtained when cell lines and hybridomas are maintained in host mice, is a convenient source of several plasma proteins. This paper describes procedures for the purification of albumin and transferrin from mouse ascites fluid. Mouse transferrin was prepared from a 50-75% ammonium sulfate fraction of mouse ascites fluid by CM- and DEAE-cellulose chromatography. Mouse albumin was obtained by the same purification route, but required an additional chromatography step on Cibacron Blue F3GA-agarose. Both proteins were shown to be homogeneous by polyacrylamide gel electrophoresis and immunoelectrophoresis. Characterization, which included a determination of amino acid composition, partial N-terminal sequence, molecular weight and extinction coefficient, correlated well with known values reported for human transferrin and albumin. The purified mouse proteins may be useful for biochemical studies, antibody preparation, and as growth factors for hybridomas or other mouse cell lines maintained in culture.

Decrease in the levels of specific non-polysomal messenger ribonucleoproteins during the mouse sarcoma-180 ascites cell cycle.

A few mRNAs in S-180 ascites cells are found as non-polysomal messenger ribonucleoprotein particles, whose translation is restricted both in vivo and in vitro. To determine if these mRNPs remain untranslated throughout the cell cycle we obtained S-180 ascites cells enriched in cell cycle stages by centrifugation through ficoll gradients. The "inactive" mRNA species were clearly present in the pre-polysomal fraction from S and pre-S phase cells, however they were not detected in the mRNP fraction from post S phase cells. Hybridization of a cloned cDNA probe for one of these species (P-40) to polysomal RNA from pre- or post-S phase cells demonstrated it was present in substantially higher concentrations in polysomes from post-S phase cells, consistent with its translation after S phase. We also observed that a poly (A)-minus form of the mRNA coding for actin was restricted to post S phase cells.

A major species of mammalian messenger RNA lacking a polyadenylate segment.

Translation of total polysomal RNA from sarcoma 180 ascites cells in a wheat germ cell-free system produces two major polypeptides, A and B, with molecular weights of 50,000 and 45,000, respectively. Fractionation on Millipore filters or on oligo(dT)-cellulose leads to retention of the mRNA specific for protein A in the poly(A)-containing fraction and to accumulation of the B mRNA in the unadsorbed poly(A)-deficient fraction. The mRNA for B sediments at approximately 18 S; it is released as a 50S ribonucleorprotein upon EDTA treatment of polysomes. Its translation is particularly sensitive to an inhibitor present in the polysomal RNA. The poly(A)-deficient mRNA for the 45,000 dalton polypeptide is also present in mouse myeloma MPC-11 cells, where it seems to be localized in membrane-bound polysomes.