Studies on the mechanism by which prolactin regulates protein, RNA, and DNA synthesis in Nb2 node lymphoma cells

Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C.     

Mitochondrial RNA and protein synthesis in enucleated African green monkey cells

The behavior of extramitochondrial protein synthesis and of mitochondrial RNA and protein synthesis was examined in the cytoplasts of African green monkey kidney cells (TC-7 subline) at different times following enucleation by cytochalasin B. The rate of incorporation of [³H]isoleucine into protein of the soluble cytoplasmic fraction decreased in an approximately exponential fashion, with a half-life of about five hours, during the first 26 hours after enucleation. Discrete mitochondrial 16 S, 12 S and 4 S RNA components were identified among the products of cytoplast RNA synthesis. The rates of [³H]uridine incorporation into the 16 and 12 S RNA components as well as into total RNA declined progressively after enucleation to a barely detectable level by the 20th hour. By contrast, the rate of chloramphenicol-sensitive [³H]isoleucine incorporation into protein (due to mitochondrial protein synthesis) did not undergo a substantial decline for at least 20 hours in TC-7 cytoplasts; instead, a reproducible transient stimulation occurred in the first hours following enucleation. The products of mitochondrial protein synthesis pulse-labeled in nucleated cells and in cytoplasts 24 hours after enucleation exhibited similar electrophoretic profiles.     

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.     

Polyamine stimulation of ribosomal synthesis and activity in a polyamine-dependent mutant of Escherichia coli

A polyamine-dependent mutant of Escherichia coli KK101 was isolated by treatment of E. coli MA261 with N-methyl-N'-nitro-N-nitrosoguanidine. In the absence of putrescine, doubling time of the mutant was 496 min. The mutation was accompanied by a change in the nature of the 30 S ribosomal subunits. Addition of putrescine to the mutant stimulated the synthesis of proteins and subsequently, this led to stimulation of RNA and DNA synthesis. Under these conditions, we determined which proteins were preferentially synthesized. Putrescine stimulated the synthesis of ribosomal protein S1 markedly, but stimulated ribosomal proteins S4, L20, and X1, and RNA polymerase slightly. The amounts of initiation factors 2 and 3 synthesized were not influenced significantly by putrescine. The preferential stimulation of the synthesis of ribosomal protein S1 occurred as early as 20 min after the addition of putrescine, while stimulation of the synthesis of the other ribosomal proteins and RNA polymerase appeared at 40 min. The stimulation of the synthesis of ribosomal RNA also occurred at 40 min after addition of putrescine. Our results indicate that putrescine can stimulate both the synthesis and the activity of ribosomes. The increase in the activity of ribosomes was achieved by the association of S1 protein to S1-depleted ribosomes. The early stimulation of ribosomal protein S1 synthesis after addition of putrescine may be important for stimulation of cell growth by polyamines.     

Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 microU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added to the cells two hours after the hormone. Synthesis of RNA measured by the incorporation of [3H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.     

Planarian regeneration: quantitative differences in RNA and protein synthesis related to age

A comparative study of RNA and protein synthesis during regeneration of immature and adult planarians reveals fundamental differences in the regeneration process. Young planarians, which contain about 20 times more RNA/protein in their tissues than adults, actively synthesize RNA prior to any wound. A single stimulation of RNA synthesis is observed after 24 h following sectioning. The electrophoretic pattern of labelled RNA extracted either from intact or regenerating young planarians does not change significantly and shows, besides ribosomal RNA, an important fraction of RNA of heterogeneous molecular weights. This pattern is similar to that observed with extracts of RNA from regenerating adults but only after 24 h following sectioning (Martelly, I. and Le Moigne, A., Reprod. Nutr. Dev., 20 (1980) 1527–1537). Indeed, in adults, a preliminary phase of RNA metabolism is observed during the first day of regeneration. Young and adult planarians differ also in their time course of stimulation of protein synthesis after sectioning. While a lag time of more than 6 h is necessary in adults, protein synthesis is stimulated immediately after sectioning in the young. These differences in the pattern of macromolecular synthesis related to age are discussed in relation with the idea of cellular activation during the regeneration process.     

UAP56 is an important regulator of protein synthesis and growth in cardiomyocytes

UAP56, an ATP dependent RNA helicase that also has ATPase activity, is a DExD/H box protein that is phylogenetically grouped with the eukaryotic initiation factor eIF4A, the prototypical member of the DExD/H box family of helicases. UAP56, also known as BAT1, is an essential RNA splicing factor required for spliceosome assembly and mRNA export but its role in protein synthesis is not known. Here we demonstrate that UAP56 regulates protein synthesis and growth in cardiomyocytes. We found that wild-type (WT) UAP56 increased serum induced protein synthesis in HeLa cells. UAP56 mutants lacking ATPase and/or helicase activity inhibited protein synthesis compared with WT UAP56, suggesting that the ATPase and RNA helicase activity of UAP56 is important for protein synthesis. UAP56 siRNA inhibited phenylephrine (PE) induced protein synthesis in cardiomyocytes and inhibited PE induced cardiomyocyte hypertrophy. Our data demonstrate that UAP56 is an important regulator of protein synthesis and plays an important role in the regulation of cardiomyocyte growth.     

Senescence: action of auxin and kinetin in control of RNA and protein synthesis in subcellular fractions of bean endocarp

A comparative study was made of the effects of auxin (α-naphthalene acetic acid), kinetin (6-furfurylaminopurine) and a mixture of auxin and kinetin applied in vivo on synthesis of RNA and protein and the distribution of such synthesis amongst the subcellular fractions of sections of endocarp from Kentucky Wonder pole beans (Phaseolus vulgaris, L.). Auxin caused considerable enhancement of incorporation of labeled precursors into RNA and protein of all subcellular fractions, and induced net synthesis of RNA and protein. That auxin-induced net synthesis of protein is repressed by actinomycin D indicates that auxin acts primarily to stimulate synthesis of RNA, as a result of which synthesis of protein is enhanced. The effect of kinetin alone on synthesis of RNA, or of kinetin on auxin-induced synthesis of RNA was variable, with either stimulation or inhibition observed in different experiments. Kinetin-enhancement of synthesis of both RNA and protein in subcellular fractions also varied, with enhancement of synthesis in 1 or all subcellular fractions among different experiments. The variable effect of kinetin did not seem to be related to the amount of endogenous or added auxin. The mode of action of kinetin is discussed.     

Myofibrillar protein synthesis in myostatin-deficient mice

Either increased protein synthesis or prolonged protein half-life is necessary to support the excessive muscle growth and maintenance of enlarged muscles in myostatin-deficient mice. This issue was addressed by determining in vivo rates of myofibrillar protein synthesis in mice with constitutive myostatin deficiency (Mstn(DeltaE3/DeltaE3)) or normal myostatin expression (Mstn(+/+)) by measuring tracer incorporation after a systemic flooding dose of l-[ring-(2)H(5)]phenylalanine. At 5-6 wk of age, Mstn(DeltaE3/DeltaE3) mice had increased muscle mass (40%), fractional rates of myofibrillar synthesis (14%), and protein synthesis per whole muscle (60%) relative to Mstn(+/+) mice. With maturation, fractional rates of synthesis declined >50% in parallel with decreased DNA and RNA [total, 28S rRNA, and poly(A) RNA] concentrations in muscle. At 6 mo of age, Mstn(DeltaE3/DeltaE3) mice had even greater increases in muscle mass (90%) and myofibrillar synthesis per muscle (85%) relative to Mstn(+/+) mice, but the fractional rate of synthesis was normal. Estimated myofibrillar protein half-life was not affected by myostatin deficiency. Muscle DNA concentrations were reduced in both young and mature Mstn(DeltaE3/DeltaE3) mice, whereas RNA concentrations were normal, so the ratio of RNA to DNA was approximately 30% greater than normal in Mstn(DeltaE3/DeltaE3) mice. Thus the increased protein synthesis and RNA content per muscle in myostatin-deficient mice cannot be explained entirely by an increased number of myonuclei.     

Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 μU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+ 15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added by the incorporation of [³H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

Preferential stimulation of the in vivo synthesis of a protein by polyamines in Escherichia coli: purification and properties of the specific protein

The possibility that polyamines can stimulate the synthesis of special kinds of proteins has been examined by using a polyamine-requiring mutant of Escherichia coli. It was found that the synthesis of some proteins, particularly one with a molecular weight (Mr) of 62K, was significantly stimulated following polyamine supplementation of polyamine-starved cells. The preferential stimulation of the synthesis of this polyamine-induced protein of Mr 62K (PI protein) was followed by the stimulation of overall protein synthesis by polyamines. PI protein was purified to homogeneity and some of its properties were examined. From studies on the effect of PI protein on MS2 RNA directed protein synthesis, it was shown that this protein stimulated the synthesis of RNA replicase by 2.2-fold in the presence of 1 mM spermidine.     

Effect of polyamines on the fidelity of macromolecular synthesis

Addition of polyamines to in vitro systems containing suboptimal concentrations of Mg2+ markedly stimulated protein and RNA synthesis. This stimulation is observed only within a marrow range of polyamine concentration. The extend of stimulation of RNA synthesis was dependent on assay conditions. Addition of spermidine to the wheat germ system not only stimulated poly(U) directed polyphenylalanine synthesis, but also reduced the misincorporation of leucine. MS2-coat protein synthesis, studied in an E.coli cell-free system using either one of the two glutamyl-tRNAs as the only source of glutamine, suggested that in the presence of spermidine, codon recognition by these two isoacceptor tRNA molecules was more stringent. From these results it is concluded that polyamines contribute to the specificity of codon/anticodon interactions and thereby increase the fidelity of protein synthesis.     

Prostaglandin synthesis regulation in human amnion tissue: involvement of protein kinase C and dependence on ribonucleic acid and protein synthesis.

The role of protein kinase C (PKC) in the control of prostaglandin production by the human amnion was studied. Amnion membranes delivered spontaneously at term were minced and treated with phorbol esters, protein kinase inhibitors, cycloheximide, and actinomycin D; prostaglandin E2 (PGE2) output then was determined. Untreated tissue produced 3.97 +/- 1.13 ng PGE2/micrograms DNA/14 h (mean +/- SEM, n = 19). Phorbol dibutyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated PGE2 output up to 20-fold in a concentration-dependent manner with potencies corresponding to their efficacy as PKC activators. Four-beta-phorbol and 4-methoxy-TPA, which do not stimulate PKC, did not affect PGE2 output. Stimulation by TPA was blocked by staurosporine (IC50 = 57 nM) and H7; however, these PKC inhibitors did not decrease basal prostaglandin production. Cycloheximide inhibited basal and TPA-promoted PGE2 production and amino acid incorporation. Actinomycin D abolished TPA stimulation without decreasing unstimulated prostaglandin synthesis. These results show that amnion PGE2 production after labor is not maintained by PKC action, but PKC activation in this tissue causes a protein synthesis-dependent and RNA synthesis-dependent increase of PGE2 output. However, basal PGE2 production is dependent upon protein synthesis which, presumably, utilizes pre-existing mRNAs.     

Sequential stimulation of cellular RNA synthesis in polyoma-infected mouse kidney cell cultures.

Lytic infection with polyoma virus leads in Go-arrested primary mouse kidney cell cultures to a mitotic host response. In the present work we focused our attention on cellular RNA synthesis shortly after onset of polyoma T-antigen synthesis. Onset of polyoma-induced stimulation of 45S pre-rRNA synthesis was determined by hybridization of total cellular RNA with a plasmid (pMrSalB) containing the 5'-end of the mouse ribosomal gene and of the other cellular RNA species by standard biochemical analysis of cellular fractions. The results showed that polyoma-induced stimulation of cellular hnRNA (hnRNP) synthesis, the earliest presently known host cell reaction, preceded onset of stimulated 45S pre-rRNA synthesis and that the latter was paralleled by polyoma-induced stimulation of 5S RNA, tRNA and overall protein synthesis. The polyoma-induced mitotic response is similar to that triggered by simian virus 40 and by certain nonviral mitogens.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.