Translational regulation of mammalian ornithine decarboxylase by polyamines

Ornithine decarboxylase, which catalyses the formation of putrescine, is the first and rate-limiting enzyme in the biosynthesis of polyamines in mammalian cells. The enzyme is highly regulated, as indicated by rapid changes in its mRNA and protein during cell growth. Here we report that ornithine decarboxylase is regulated at the translational level by polyamines in difluoromethylornithine-resistant mouse myeloma cells that overproduce the enzyme due to amplification of an ornithine decarboxylase gene. When such cells are exposed to putrescine or other polyamines, there is a rapid and specific decrease in the rate of synthesis of ornithine decarboxylase, assayed by pulse-labeling. Neither the cellular content of ornithine decarboxylase mRNA nor the half-life of ornithine decarboxylase protein is affected. Our results indicate that polyamines negatively regulate the translation of ornithine decarboxylase mRNA, thereby controlling their own synthesis.     

Interrelation between the charge isoforms of mammalian ornithine decarboxylase

Ornithine decarboxylase (ODC) isolated from a variety of tissues has been separated, using DEAE ion-exchange chromatography, into multiple peaks of activity that appear to be related to control of this enzyme stability. Reports of these charge isoforms in current literature are generally unclear as to whether these represent a covalent posttranslational modification or merely an alteration in structural conformation or association. In this study we investigated the relationship of this form separation to the degree of enzyme polymerization, interaction with other proteins and buffer components, and the multiple isoelectric forms of this enzyme noted in denaturing concentrations of urea. High-performance chromatography techniques were used to demonstrate that two of the major enzyme forms, ODC I and II, are really monomers of the enzyme, while minor peaks of activity frequently observed to elute after ODC II contain various dimeric enzyme states. Pyridoxal 5'-phosphate (0.05 mM) added to isolated enzyme preparations composed of I and II monomers induced the formation of I and II dimers as well as a mixed I-II dimer. All three dimer forms were observed to be natural components of freshly isolated crude cell homogenates. The charge distinction between the monomer forms I and II was found to be maintained during ion-exchange chromatography in the presence of 8 M urea, and the enzyme isoforms demonstrated distinct bands on isoelectric focusing gels run in the presence of 9 M urea. Thus, although some of the multiple ornithine decarboxylase forms identified by ion-exchange chromatography of crude mammalian cell homogenates are related to enzyme conformation, the two major forms are distinctly charged protein states that can be visualized using two-dimensional gel electrophoresis of highly purified samples.     

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney

An immunoblotting technique was used to study the forms of ornithine decarboxylase present in androgen-induced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (approximately 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive alpha-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr approximately 53 000) without substantial loss of enzyme activity. The synthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S]methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fraction of total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1,3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity did not greatly selectively inhibit the synthesis of the enzyme. However, 1,3-diaminopropane did produce an increase in the rate of degradation of ornithine decarboxylase and a general reduction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1,3-diaminopropane.     

Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine, and spermine

We have recently isolated, without using any inhibitors, a mutant of Chinese hamster ovary cell line which greatly overproduces ornithine decarboxylase in serum-free culture. Addition of polyamines (putrescine, spermidine, or spermine, 10 microM) or ornithine (1 mM), the precursor of polyamines, to the culture medium of these cells caused a rapid and extensive decay of ornithine decarboxylase activity. At the same time the activity of S-adenosylmethionine decarboxylase showed a less pronounced decrease. Notably, the polyamine concentrations used were optimal for growth of the cells and caused no perturbation of general protein synthesis. Spermidine and spermine appeared to be the principal regulatory amines for both enzymes, but also putrescine, if accumulated at high levels in the cells, was capable of suppressing ornithine decarboxylase activity. The amount of ornithine decarboxylase protein (as measured by radioimmunoassay) declined somewhat more slowly than the enzyme activity, but no more than 10% of the loss of activity could be ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation through ornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeled ornithine decarboxylase revealed a rapid reduction in the synthesis and acceleration in the degradation of the enzyme after polyamine additions. No decrease in the amounts of the two ornithine decarboxylase-mRNA species, hybridizable to a specific cDNA, was detected, suggesting that polyamines depressed ornithine decarboxylase synthesis by selectively inhibiting translation of the message.     

Proposed model of major sequential biochemical events of a trophic response.

It appears that the induction of ornithine decarboxylase regulates the rate of ribosomal RNA synthesis as well as regulating the rate of synthesis of polyamines. Further, ornithine decarboxylase, in most cases, is induced after a significant activation of cAMP-dependent protein kinase. We propose a model for the process of hypertrophy based on studies of a considerable number of mammalian growth systems. The mechanism of parallel regulation of polyamines and RNA appears to be initiated by the direct effect of ornithine decarboxylase on RNA polymerase I.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

Polyamines appear to be second messengers in mediating Ca2+ fluxes and neurotransmitter release in potassium-depolarized synaptosomes

High potassium (50 mM) depolarization induces a rapid (less than 15 sec) increase in the levels of the polyamines putrescine, spermidine and spermine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine blocked the K+-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca2+ influx and efflux and the Ca2+-dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of alpha-difluoromethylornithine. These results suggest that enhanced polyamine synthesis is required for potassium depolarized stimulation of synaptic function.     

Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.     

Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines—such as putrescine, spermidine and spermine—are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion—by means of α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase—abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.     

Relation of polyamine biosynthesis to the initiation of sprouting in potato tubers

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase are present in all parts of dormant potato (Solanum tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally.     

Effect of inhibitors of protein synthesis on rat liver spermidine N-acetyltransferase

The increase in spermidine N-acetyltransferase activity in rat liver produced by carbon tetrachloride was completely prevented by simultaneous treatment with inhibitors of protein and nucleic acid synthesis suggesting that the increase results from the synthesis of new protein rather than the release of the enzyme from a cryptic inactive form. Treatment with cycloheximide 2 h after carbon tetrachloride also completely blocked the rise in spermidine N-acetyltransferase seen 4 h later. Such treatment completely prevented the fall in spermidine and rise in putrescine in the liver 6 h after carbon tetrachloride confirming the importance of the induction of spermidine N-acetyltransferase in the conversion of spermidine into putrescine. When cycloheximide was administered to rats in which spermidine N-acetyltransferase activity had been stimulated by prior treatment with carbon tetrachloride or thioacetamide, the activity was lost rapidly showing that the enzyme protein has a rapid rate of turnover. The half-life for the enzyme in thioacetamide-treated rats was 40 min, whereas the half-life for ornithine decarboxylase (which is well known to turn over very rapidly) was 27 min. In carbon tetrachloride-treated rats the rate or protein degradation was reduced and the half-life of spermidine N-acetyltransferase was 155 min and that for ornithine decarboxylase was 65 min. It appears that three of the enzymes involved in the synthesis and interconversion of putrescine and spermidine namely, ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine N-acetyltransferase have rapid rates of turnover and that polyamine levels are regulated by changes in the amount of these enzymes.     

Studies of mammalian ornithine decarboxylase using a monoclonal antibody.

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.     

Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. Implications for enzyme turnover and selective difluoromethylornithine inhibition

Ornithine decarboxylase of the African trypanosome Trypanosoma brucei brucei had an estimated native molecular weight of 100,000 by gel filtration and a subunit molecular weight of 45,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding this enzyme, present in a single copy in T. brucei, was identified by mouse ornithine decarboxylase cDNA under relatively stringent conditions of hybridization and subcloned in a 5.9-kilobase (kb) SstI fragment from a cosmid clone into the plasmid pUC 19. This clone encompassed a 2.8-kb SstII fragment that contained the entire T. brucei ornithine decarboxylase gene. The 2.8-kb SstII fragment hybridized to a 2.4-kb mRNA that presumably encodes the parasite enzyme. The 2.8-kb SstII fragment was partially sequenced and found to contain an open reading frame of 445 amino acids that has 61.5% homology with the corresponding sequence of the mouse enzyme. The only major discrepancies between the two enzymes are the addition of a 20-amino acid N-terminal peptide and the deletion of a 36-amino acid C-terminal peptide and the T. brucei ornithine decarboxylase. The C terminus has been postulated to be one of the structural factors associated with rapid in vivo turnover of mammalian ornithine decarboxylase. The absence of this C-terminal peptide in T. brucei ornithine decarboxylase predicts a slow turnover for the parasite enzyme in vivo, and this is supported by our experimental data. The lack of turnover of ornithine decarboxylase in trypanosomes may constitute the basis of selective antitrypanosomal action of the irreversible enzyme inhibitor DL-alpha-difluoromethylornithine.     

Association of diamine oxidase and ornithine decarboxylase with maturing cells in rapidly proliferating epithelium.

Diamine oxidase and ornithine decarboxylase activities are shown to have a parallel distribution across rat small intestine mucosa; levels of both enzyme activities are sharply higher in mature cells in the villus tip region than in proliferating cells in the crypt areas. Histidine decarboxylase levels were not measurable in the same cell preparations and aromatic-L-amino-acid decarboxylase activity was distributed in an opposite pattern to diamine oxidase and ornithine decarboxylase. The results suggest that intestinal diamine oxidase could be involved with polyamine metabolism. The new findings for ornithine decarboxylase suggest an in vivo role for polyamines in non-proliferative cells; rat small intestinal mucosa may be an excellent model for investigating the function of polyamines in regenerating cells.     

Association of diamine oxidase and ornithine decarboxylase with maturing cells in rapidly proliferating epithelium

Diamine oxidase and ornithine decarboxylase activities are shown to have a parallel distribution across rat small intestine mucosa; levels of both enzyme activities are sharply higher in mature cells in the villus tip region than in proliferating cells in the crypt areas. Histidine decarboxylase levels were not measurable in the same cell preparations and aromatic-L-amino-acid decarboxylase activity was distributed in an opposite pattern to diamine oxidase and ornithine decarboxylase. The results suggest that intestinal diamine oxidase could be involved with polyamine metabolism. The new findings for ornithine decarboxylase suggest an in vivo role for polyamines in non-proliferative cells; rat small intestinal mucosa may be an excellent model for investigating the function of polyamines in regenerating cells.     

Expression of arginase II and related enzymes in the rat small intestine and kidney

Arginase, which catalyzes the conversion of arginine to urea and ornithine, and consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Arginine is also used for the synthesis of nitric oxide and creatine phosphate, while ornithine is used for the synthesis of polyamines and proline, and thus collagen. Arginase II mRNA and protein are abundant in the intestine (most abundant in the jejunum and less abundant in the ileum, duodenum, and colon) and kidney of the rat. In the kidney, the levels of arginase II mRNA do not change appreciably from 0 to 8 weeks of age. In contrast, arginase II mRNA and protein in the small intestine are not detectable at birth, appear at 3 weeks of age, the weaning period, and their levels increase up to 8 weeks. On the other hand, mRNAs for ornithine aminotransferase (OAT), ornithine decarboxylase, and ornithine carbamoyltransferase (OCT) are present at birth and their levels do not change much during development. Arginase II is elevated in response to a combination of bacterial lipopolysaccharide, dibutyryl cAMP, and dexamethasone in the kidney, but is not affected by these treatments in the small intestine. Immunohistochemical analysis of arginase II, OAT, and OCT in the jejunum revealed their co-localization in absorptive epithelial cells. These results show that the arginase II gene is regulated differentially in the small intestine and kidney, and suggest different roles of the enzyme in these two tissues. The co-localization of arginase II and the three ornithine-utilizing enzymes in the small intestine suggests that the enzyme is involved in the synthesis of proline, polyamines, and/or citrulline in this tissue.     

Regulation of polyamine synthesis in physarum polycephalum during growth and differentiation

The levels and synthesis of polyamines were investigated in Physarum polycephalum to obtain information about their regulation during growth and differentiation in a lower eukaryote. Putrescine pools rapidly increased 4–5 fold during the change from dormant spherules to growing plasmodia. The activity of ornithine decarboxylase (EC 4.1.1.17), which converts ornithine to putrescine, reflected this rapid change in the level of putrescine. Spermidine levels were closely correlated with protein concentrations during differentiation due to variations in the activity of S-adenosyl-l-methionine decarboxylase which is involved in the conversion of putrescine to spermidine This enzyme was not stimulated by putrescine, unlike the similar enzyme in other eukaryotes, thereby permitting independent regulation of putrescine and spermidine levels. The high levels of both putrescine and spermidine suggest separate functions for these polyamines in Physarum.The half-lives of ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase were 14 and 21.5 min, respectively. These short half-lives keep the polyamine metabolism under a very tight control as illustrated by the rapid fluctuations in enzyme activity during differentiation and the synchronous mitotic cycle. The step patterns of these unstable enzymes during the mitotic cycle suggest that these enzyme levels are limited by gene dosage.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Putrescine and spermidine control degradation and synthesis of ornithine decarboxylase in Neurospora crassa

Neurospora crassa mycelia, when starved for polyamines, have 50-70-fold more ornithine decarboxylase activity and enzyme protein than unstarved mycelia. Using isotopic labeling and immunoprecipitation, we determined the half-life and the synthetic rate of the enzyme in mycelia differing in the rates of synthesis of putrescine, the product of ornithine decarboxylase, and spermidine, the main end-product of the polyamine pathway. When the pathway was blocked between putrescine and spermidine, ornithine decarboxylase synthesis rose 4-5-fold, regardless of the accumulation of putrescine. This indicates that spermidine is a specific signal for the repression of enzyme synthesis. When both putrescine and spermidine synthesis were reduced, the half-life of the enzyme rapidly increased 10-fold. The presence of either putrescine or spermidine restored the normal enzyme half-life of 55 min. Tests for an ornithine decarboxylase inhibitory protein ("antizyme") were negative. The regulatory mechanisms activated by putrescine and spermidine account for most or all of the regulatory amplitude of this enzyme in N. crassa.