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.

Cytochrome P-450 mRNAs are modulated by dehydroepiandrosterone, nafenopin, and triiodothyronine.

Dehydroepiandrosterone (DHEA) is the only known naturally occurring compound that promotes peroxisome proliferation in rodent liver, and stimulates transcriptional induction of genes involved in lipid metabolism and peroxisomal beta-oxidation. Therefore, we examined mRNA for several such genes in rat liver, specifically acyl-CoA oxidase and the cytochromes P-450 (CYP4A1, CYP4A3, and CYP3A23), after 5 to 6 day treatments with either DHEA, or nafenopin, a known peroxisome proliferator. Acyl-CoA oxidase and CYP4A1 were induced nearly identically by DHEA and nafenopin, with induction being more pronounced in female rats. However, CYP3A23 was induced only by DHEA, suggesting an induction mechanism independent of the peroxisome proliferator activated receptor. Previously, we observed triiodothyronine (T3) suppression of peroxisome proliferator induced CYP4As and we sought to determine whether CYP3A23 might be regulated in a different manner. T3 was found to also suppress DHEA-dependent induction of CYP3A23. CYP4A2 expression in kidney was also negatively regulated by T3. To characterize a putative negative thyroid hormone response element (nTRE) in the 5' flanking region of this gene, a luciferase reporter gene containing a rat CYP4A2 flanking sequence extending to -1865 bp was transfected into HepG2 cells along with human thryroid hormone receptor expression vector. Expression of luciferase activity was unaffected by T3, suggesting the absence of a functional nTRE within this portion of CYP4A2. These data demonstrate gene regulatory activity by DHEA different from that of nafenopin, and a suppressive effect of T3, consistent with indirect regulatory mechanisms not involving an nTRE.

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.

Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells.

We have examined the expression of three prostaglandin (PG) receptors, EP2, EP4, and FP, in a nonpigmented ciliary epithelial cell line (ODMCl-2) and in human ciliary muscle (HCM) cells. Total RNA preparations from either ODMCl-2 or HCM cells were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with sense and antisense primers for each of the three PG receptors. The RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors in both ODMCl-2 and HCM cells. PCR products corresponding to each receptor were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. Pharmacological studies with the EP2 receptor-selective agonist butaprost resulted in a significant increase in the cyclic AMP level in ODMCl-2 cells. The stimulation of cyclic AMP in ODMCl-2 cells by PGE2 and 11-deoxy PGE1, the respective EP1/EP2/EP3/EP4 and EP2/EP3/EP4 receptor agonists, was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. These results conclusively demonstrate the presence of both mRNA and protein for EP2, EP4, and FP receptors in ODMCl-2 and HCM cells.

Regulation of CYP4A expression in rat by dehydroepiandrosterone and thyroid hormone.

Dehydroepiandrosterone (DHEA) is a peroxisome proliferating agent when administered in pharmacological dosages, but it has not been shown to function through the peroxisome proliferator-activated receptor in cell-based assays. Because members of the thyroid hormone/vitamins A and D nuclear receptor subfamily, including PPAR, are known to modulate each other's function in gene expression by heterodimerization, we sought to establish whether DHEA and thyroid hormone interact to regulate several of the hepatic and renal enzymes associated with peroxisome proliferation, i.e., peroxisomal beta-oxidation and microsomal NADPH:cytochrome P450 oxidoreductase and the cytochromes P450 4A. In rats administered exogenous T3 to attain a hyperthyroid state, induction of the three isozymes of CYP4A (4A1, 4A2, and 4A3) by DHEA was suppressed > 60-80% at the mRNA level, with induction of CYP4A2 mRNA being completely inhibited. Nuclear run-on transcription assays indicated that this inhibitory effect was regulated at the level of transcription. Induction of hepatic peroxisomal beta-oxidation by DHEA or the peroxisome proliferator nafenopin was in large part unaffected by treatment of animals with T3 under any condition tested. Microsomal NADPH:cytochrome P450 oxidoreductase activity was induced by either DHEA or T3; cotreatment resulted in an additive induction. When animals were treated with a lower dose of exogenous T3 that rendered the animals slightly hyperthyroid, only induction of hepatic CYP4A2 mRNA by DHEA or nafenopin was significantly inhibited (> 80%) compared with euthyroid control animals. Animals that had been rendered hypothyroid through removal of the thyroid gland showed normal induction of CYP4A genes by DHEA in liver, suggesting that their induction by DHEA was not dependent on the presence of thyroid hormone. The administration of exogenous T3 to thyroidectomized rats in the presence of DHEA potently suppressed hepatic induction of all three genes at the mRNA and protein level. In experiments with cultured rat hepatocytes, physiological concentrations of T3 potently inhibited the induction of CYP4A2 mRNA levels by nafenopin but had little effect on induction of CYP4A1 or 4A3 mRNA. At higher T3 concentrations, the induction of CYP4A1/4A3 mRNA and protein was also inhibited. These results suggest that T3 modulates the expression of CYP4A2 at the level of transcription in physiologically relevant concentrations but that hyperthyroid conditions are required to suppress expression of CYP4A1/4A3 genes. In euthyroid rodent kidney, which only expresses CYP4A2 under either basal or DHEA-induced conditions, near-physiological levels of T3 caused potent suppression of peroxisome proliferator-dependent induction of CYP4A2 mRNA levels by either DHEA or nafenopin. In thyroidectomized rats, basal expression of CYP4A2 mRNA was decreased relative to euthyroid controls, but DHEA was as effective an inducer of this mRNA as it is in euthyroid rats. As seen in euthyroid rats, T3 administration potently suppressed DHEA induction of CYP4A2 mRNA levels under either basal or induced conditions. Although CYP4A expression was not derepressed in liver or kidneys of hypothyroid animals, our results indicated that the thyroid status of the animal did affect basal expression of CYP4A2, suggesting involvement of thyroid hormone or some other factor regulated by the thyroid gland on its constitutive expression.

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.

Age effects on rat hindlimb muscle atrophy during suspension unloading.

Disuse can induce numerous adaptive alterations in skeletal muscle. In the present study the effects of hindlimb unloading on muscle mass and biochemical responses were examined and compared in adult (450 g) and juvenile (200 g) rats after 1, 7, or 14 days of whole body suspension. Quantitatively and qualitatively the soleus (S), gastrocnemius (G), plantaris (P), and extensor digitorum longus (EDL) muscles of the hindlimb exhibited a differential sensitivity to suspension and weightlessness unloading in both adults and juveniles. The red slow-twitch soleus exhibited the most pronounced atrophy under both conditions, with juvenile responses being greater than adult. In contrast, the fast-twitch EDL hypertrophied during suspension and atrophied during weightlessness, with no significant difference between adults and juveniles. Determination of biochemical parameters (total protein, RNA, and DNA) indicated a less rapid rate of response in adult muscles. This was corroborated by assessment of muscle alpha-actin mRNA levels, which indicated a rapid (within 1 day) and significant (P less than 0.05) effect in juveniles but not in adults. The results of this investigation indicate 1) a qualitatively similar differential effect of unloading on muscles of adults and juveniles, 2) a quantitatively reduced and less rapid effect of suspension on adult muscles, and 3) a close similarity of adult and juvenile muscle responses during suspension and spaceflight, suggesting that this ground-based model simulates many of the unloading effects of weightlessness.

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.

Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading.

Muscle atrophy resulting from disuse is associated with marked decrements in protein synthesis. The objective of the present investigation was to determine levels of total muscle RNA and the content and composition of the mRNA fraction as a qualitative assessment of the potential regulatory role of transcriptional alterations in unloaded skeletal muscles. Hindlimb muscle unloading was produced by whole-body suspension of rats for up to 7 days. The soleus, gastrocnemius, and extensor digitorum longus (EDL) were excised from 1-, 3-, and 7-day suspended and pair-fed controls, and RNA was extracted by homogenization in 5 M guanidinium thiocyanate. Total RNA and mRNA contents were lower in soleus and gastrocnemius after 7 days of suspension compared with pair-fed controls, but total RNA and mRNA concentrations (per g muscle and per microgram total RNA, respectively) were unaltered. alpha-Actin mRNA, assessed by dot blot hybridization, was significantly reduced in soleus after 1 (37%), 3 (28%), and 7 (59%) days of suspension and in gastrocnemius after 3 (44%) and 7 (41%) days. However, alpha-actin mRNA was unchanged in the EDL after suspension. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked (30-400%) alterations in mRNAs coding for several small (15- to 25-kDa) proteins. The results of this study suggest that altered transcription and availability of specific mRNAs could contribute significantly to the regulation of protein synthesis during unloading of skeletal muscle.

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.

Biogenesis and cell cycle relationship of poly(A)- actin mRNA in mouse ascites cells.

A variety of rapidly growing mammalian cells contain a substantial portion of their actin mRNA in a poly(A)- form. We have used DNA-driven hybridization of a cloned actin cDNA-containing plasmid with pulse-labeled RNA from mouse S-180 ascites cells to examine newly synthesized actin mRNA. Our results indicate that the same proportion of newly synthesized and steady-state actin mRNA (approx. 40%) exists in a poly(A)- deficient form. This suggests that the poly(A)- form arises by some process other than slow cytoplasmic de-adenylation of a poly(A)+ precursor. We have also examined cell cycle-enriched populations of S-180 ascites cells for the presence of poly(A)- actin mRNA. Results from these experiments indicate that cells in G1 phase of the cell cycle contain predominantly poly(A)+ actin mRNA, while the poly(A)- form is restricted to late-S and post-S phase cells.

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.

Histone interactions in solution and susceptibility to denaturation.

Histone interactions in solution may depend upon treatments used for purification. Optical rotatory dispersion and sedimentation-velocity measurements have been made in a reference solvent, before and after exposure to various treatments, to investigate histone susceptibility to irreversible denaturation. Some acid conditions and urea and guanidine solutions may denature. Interaction studies performed on nondenatured histones indicate that the dimer, (H4)(H3), and tetramer, (H4)2(H3)2, dissociate to monomers at low ionic strength. Sedimentation-velocity experiments suggest a model for the (H4)2(H3)2 tetramer, with a compact semispherical center and four protruding amino-terminal regions. Fractions H2a and H2b interact to form the mixed dimer in equilibrium with monomers. Fraction H2a self-associates readily to dimers, tetramers, and octamers, while fraction H1 associates only weakly to form dimers.