Regulation of tyrosine aminotransferase messenger ribonucleic acid in rat liver. effect of cycloheximide on messenger ribonucleic acid turnover

Tyrosine aminotransferase messenger ribonucleic acid (mRNA) activity in rat liver was rapidly increased 3-6-fold following in vivo administration of hydrocortisone acetate, dibutyryladenosine cyclic 3',5'-phosphate, or the protein synthesis inhibitor cycloheximide. Treatment with the steroid hormone or cyclic nucleotide in combination with cycloheximide resulted in levels of tyrosine aminotransferase mRNA 10-20-fold greater than control values. These changes in mRNA activity were not accompanied by changes in albumin mRNA or total liver template activity. The rapid decline in tyrosine aminotransferase mRNA activity following cordycepin inhibition of de novo RNA synthesis was prevented by cycloheximide treatment. This protection was not observed when pactamycin was substituted for cycloheximide, demonstrating that the inhibition of protein synthesis per se was not responsible for the stabilization of tyrosine aminotransferase mRNA. Based upon the effects of cycloheximide and pactamycin on rat liver polysome structure, it is concluded that the cycloheximide-mediated increase in tyrosine aminotransferase mRNA activity is the result of stabilization of the mRNA molecule which renders the message less susceptible to inactivation and degradation in the cytoplasm. The action of cycloheximide is very specific for tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and probably several other mRNAs that code for minor liver proteins that turn over rapidly in response to hormonal or metabolic stimuli.     

Effect of thyroid hormone on the abundance of Na,K-adenosine triphosphatase alpha-subunit messenger ribonucleic acid

The effects of thyroid hormone on Na,K-ATPase alpha-subunit mRNA (mRNA alpha) content and Na,K-ATPase activity were measured in renal cortex, heart, and cerebrum of hypothyroid rats 24 and 72 h after injection of diluent or T3. Use of a cDNA probe complementary to rat brain mRNA alpha in Northern blot analysis revealed a single 26-27 S band in RNA isolated from these three tissues regardless of thyroid status. Tissue mRNA alpha content was estimated by dot blot analysis of whole cell extracts and isolated total RNA. Injection of T3 augmented mRNA alpha content by 2.1- to 2.5-fold in kidney cortex and myocardium at 24 h. After three daily injections of T3, the increases in mRNA alpha were evident despite a global increase in RNA content associated with hypertrophy of these target tissues. Furthermore, the increases in abundance of mRNA alpha after 72 h of T3 treatment correlated with enhancement of Na,K-ATPase activity. In contrast, both mRNA alpha and enzyme activity were invariant in the cerebrum. These data suggest that T3-induced augmentation of Na,K-ATPase activity is mediated, at least in part, by increased mRNA alpha content in target tissues.     

Regulation of two c-erbA messenger ribonucleic acids in rat GH3 cells by thyroid hormone

There is recent evidence suggesting that c-erbA is the thyroid hormone nuclear receptor, and that there may be multiple c-erbA genes. We investigated the effect of T3 on two c-erbA mRNAs present in GH3 cells. A partial cDNA was isolated from rat GH3 cells which is nearly identical (99.6% nucleotide identity) to rat c-erbA alpha, except for a unique 3'-region corresponding to the carboxyl terminal region of the predicted protein sequence. This cDNA (c-erbA alpha-2), like rat c-erbA alpha, hybridizes to a 2.6 kilobase (kb) mRNA which is distinct from a 6.2 kb species that hybridizes to c-erbA beta. Since nuclear T3-binding is down-regulated by T3, we hypothesized that one or both c-erbA mRNAs might be regulated by T3. GH3 cells were treated with 10 nM T3 for up to 24 h, a manipulation known to decrease nuclear T3 binding by approximately 2-fold in GH cells. Both the 6.2 kb and 2.6 kb mRNA species decreased to nearly 50% of control values at 24 h. These data indicate that these two c-erbA mRNAs are regulated by T3 and suggest that the T3 effect on T3 binding-activity in GH cells may be mediated, in part, by down-regulation of c-erbA mRNA levels.     

Thyrotropin increases malic enzyme messenger ribonucleic acid levels in rat FRTL-5 thyroid cells

The addition of TSH to FRTL-5 thyroid cells induces a 7- to 8-fold increase in the steady state level of malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] mRNA, but does not alter beta-actin mRNA levels. Insulin alone or together with TSH has no effect on malic enzyme mRNA. The effect of TSH is not the result of thyroid hormone formation, since the addition of T3 in the presence or in the absence of TSH and the addition of 5% serum (which includes T3 and T4) have no effect. Forskolin (10(-6) M) reproduces the TSH effect, suggesting that cAMP is involved.     

Additive effects of thyroid hormone,growth hormone and testosterone on deoxyribonucleic acid-dependent ribonucleic acid polymerase in rat-liver nuclei

1. The stimulations of DNA-dependent RNA polymerase in isolated rat-liver nuclei by thyroid hormone, human growth hormone and testosterone are compared. 2. Single or multiple administrations of growth-promoting doses of tri-iodo-l-thyronine, human growth hormone and testosterone stimulate the Mg²⁺-activated RNA-polymerase reaction in nuclei from thyroidectomized, hypophysectomized and castrated rats respectively. The magnitude of stimulation was proportional to the degree of enhancement of liver growth by each hormone. After a single injection, the latent period preceding the stimulation was 1, 2 and 10hr. for growth hormone, testosterone and tri-iodothyronine respectively. The time-course of stimulation of enzyme activity and the synthesis of rapidly labelled nuclear RNA in vivo were also different for each hormone. 3. Growth hormone administration failed to stimulate the Mn²⁺/ammonium sulphate-activated RNA-polymerase reaction. Thyroid hormone and testosterone, however, stimulated it but the effect was less pronounced and occurred several hours later than that observed for the Mg²⁺-activated RNA-polymerase reaction. 4. In combination experiments, hypophysectomized or the thyroidectomized rats were given growth hormone or tri-iodothyronine in a single or repeated doses at levels that produced the maximum stimulation of Mg²⁺-activated RNA-polymerase activity. Taking into account the different latent period for each hormone, a single administration of the second hormone caused an additional stimulation of the enzyme activity. Similar additive effects were observed in thyroidectomized–castrated rats after treatment with tri-iodothyronine and testosterone. The magnitude of the additional stimulation caused by the administration of the second hormone was compatible with the capacity of that hormone to promote liver growth in rats deprived of it. 5. It is concluded that, although these hormones have some similar effects, the regulation of nuclear RNA synthesis may be mediated via different routes for each hormone.     

Cell-free synthesis of metallothionein directed by rat liver polyadenylated messenger ribonucleic acid.

Total RNA was isolated from rat liver polyribosomes and fractionated by oligo(dT)-cellulose chromatography to obtain polyadenylated mRNA. The mRNA was translated in a wheat-germ cell-free protein-synthesizing system containing [3H]glycine, [3H]lysine and [3H]serine. Most of the newly synthesized 3H-labelled polypeptides were removed from the cell-free products by precipitation at pH 4.0. 3H-labelled thionein chains, which were soluble at pH 4.0, were purified by activated-thiol-Sepharose 4B chromatography or by gel-filtration chromatography. Polyribosomal thionein mRNA was found to increase by at least 3-fold after parenteral administration and by 20 h thereafter the ratio of thionein mRNA to total mRNA approached that found in controls. Actinomycin D administration in vivo blocked the Zn2+-induced increase in polyribosomal thionein mRNA content. These data strongly suggest that metallothionein is an inducible protein. The mechanism of regulation appears to involve changes in the synthesis de novo of thionein mRNA and hence the pool of thionein mRNA available for translation.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.     

Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues

Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.