Down-regulation of ornithine decarboxylase by an increased degradation of the enzyme during gastrulation of Xenopus laevis

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Biochi ~mie~a et BiophysicaActa Biochimica et BiophysicaActa 1264 (1995) 121- 128

Down-regulation of ornithine decarboxylase by an increased degradation of the enzyme during gastrulation of Xenopus laevis Ulla Rosander, Ingvar Holm *, Birgitta Grahn, Huguette LOvtrup-Rein, Mats-Olof Mattsson, Olle Heby Department ~( Cellular and Det'elopmental Biology. Uni~'ersityOf"Ume~. S-901 87 Ume~, Sweden

Received 19 December 1994; revised 9 June 1995:accepted 12 June 1995

Abstract

The present study was designed to analyze the regulation of the levels of the polyamines and their biosynthetic enzymes during embryonic development of Xenopus laet:is. The activity of omithine decarboxylase (ODC), a rate-controlling enzyme in polyamine biosynthesis, is elevated until, during gastrulation, there is a precipitous drop in activity. This is not attributable to a decrease in ODC mRNA content and polysome profiles reveal no apparent decrease in ODC message associated with polysomes. ODC synthesis seems to be maintained at a low, relatively constant rate until neurulation whereupon ribosome loading of ODC mRNA increases. During gastrulation the rate of ODC degradation increases dramatically, which can account for the decrease in ODC. S-Adenosylmethionine decarboxylase (AdoMetDC), another rate-controlling enzyme in polyamine biosynthesis, shows a low and constant activity from cleavage to neurulation. Subsequently, the AdoMetDC activity increases dramatically. The changes in AdoMetDC activity parallel the changes in AdoMetDC mRNA levels, suggesting a transcriptional control of AdoMetDC expression during this developmental period. The activities of ODC and AdoMetDC produce a steady increase in putrescine and spermidine content of the embryo. The spermine content also increases until gastrulation, but then decreases until the tailbud stage. Keywords: maternal mRNA; Ornithine decarboxylasemRNA; S-Adenosylmethioninedecarboxylase mRNA: Polyamine:Protein degradation: Post-translational control; ( X. lael'is)

1. Introduction

Ornithine decarboxylase ( O D C ) and S-adenosylmethionine decarboxylase (AdoMetDC) are rate-controlling enzymes in the polyamine biosynthetic pathway [1,2]. In mammalian cells their expression is feedback-regulated by the polyamines. Thus, ODC is regulated mainly at the translational [3] and post-translational levels [4], whereas AdoMetDC is regulated at the transcriptional, translational and post-translational levels [2]. When polyamine synthesis is blocked, most cell types cease to grow and divide, but upon addition of polyamines they rapidly regain their normal rate of growth [5]. Terato-

Abbreviations: ODC, ornithine decarboxylase; AdoMetDC, S-adenosylmethionine decarboxylase; MBT, mid-blastula transition; hCG, human chorionic gonadotropin;HPLC, high-performanceliquid chromatography; SDS, sodium dodecyl sulfate; UTR, untranslated region. * Corresponding author. E-mail: [email protected]. Fax: + 46 90 16669I. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67-4781 (95)001 36-0

carcinoma stem cells do not only stop growing, but are induced to differentiate terminally when subjected to polyamine depletion [6]. With the exception of multiple cell lines of human small cell lung carcinoma and a human promyelocytic leukemia cell line (HL-60), cells do not die as a result of polyamine depletion [7]. Most embryos are able to develop through cleavage in the absence of polyamine synthesis, but at about the time of gastrulation there is a critical period for which polyamine synthesis is absolutely essential for continued embryonal growth and differentiation [8]. Highly specific polyamine synthesis inhibitors are known to interfere with hormoneinduced ODC activity in Xenopus oocytes and, as a consequence, inhibit their meiotic maturation as well as their ovulation [9]. However, the consequences of polyamine synthesis inhibition for the developing Xenopus embryo have not yet been studied. The present study addresses the question whether ODC and AdoMetDC expression change during early Xenopus development. Since the activities of these enzymes are cell cycle dependent [5] as well as developmentally regulated

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[8], one would expect changes to occur during the progression of the fertilized egg to the tailbud stage. In early Xenopus embryos, where virtually no new transcripts are formed until mid-blastula transition (MBT = stage 8.5) [10,11], changes in enzyme expression would have to be regulated at post-transcriptional, translational a n d / o r post-translational levels. We find a major down-regulation of ODC expression during gastrulation attributable to an increase in ODC degradation. AdoMetDC activity remains at a rather low and constant level from fertilization through gastrulation, but from neurulation on the activity increases. Similarly, the ODC activity increases from neurulation and until the tailbud stage. These increases in ODC and AdoMetDC activities are, at least partly, attributable to increases in mRNA content.

2. Materials and methods

2.1. Experimental animals Female South African clawed frogs (Xenopus laet,is) were injected with 180 IU of pregnant mares' serum gonadotropin (3800 IU/mg; Sigma). Two days later they were induced to lay eggs by injection with 350 IU of human chorionic gonadotropin (hCG; Sigma). Males were simultaneously injected with 150 IU of hCG to induce spawning, and after about 15 h fertilized eggs were collected, Eggs and embryos were dejellied by treatment with 0.75% thioglycolate in Holtfreter's solution (60 mM NaCI, 0.67 mM KCI, 0.91 mM CaC12, 2.4 mM N a H C Q ) (pH 8.0), and were then incubated at room temperature in Hoitfreter's solution (pH 7.2). The developmental stages were determined according to Nieuwkoop and Faber [12]. Embryos were stored at - 7 0 ° C until analyzed.

2.2. Analysis of ODC actici O, The activity of ODC was determined by measuring the release of 14C02 from L-[l-~4C]ornithine (Amersham) essentially as described by Jfinne and Williams-Ashman [13]. Embryos were disrupted by sonication in a small volume of 0.1 M Tris-HC1 buffer (pH 7.5) containing 0.1 mM EDTA and 2.5 mM dithiothreitoh The samples were centrifuged at 20000 × g (20 rain; 4°C). Pyridoxal 5'-phosphate was added to the supernatant to a final concentration of 2 mM (saturating). The ODC activity was assayed for in the presence of a saturating level (5 mM) of L-ornithine (final specific activity, 2 mCi/mmol). The 14CO2 released was trapped in hyamine hydroxide and the radioactivity was measured in a scintillation counter.

2.3. Analysis qf AdoMetDC acticity The embryos were processed as for the ODC assay and the activity of AdoMetDC was determined by measuring

the release of HCO2 from 2 mM S-adenosyl-L-[carboxvl~4C]methionine (Amersham; final specific activity, 2 mCi/mmol) in the presence of 2.5 mM putrescine [14]. The ~4CO~ released was trapped and quantified as described above.

2.4. Analysis q[ polyamine content Embryos were sonicated in 0.2 M perchloric acid and centrifuged at 20000 × g (10 min; 4°C). The polyamines contained in the supernatant were analyzed quantitatively using the reversed-phase high performance liquid chromatography (HPLC) method described by Seiler et al. [15]. In principle the polyamines were determined by separation of the ion pairs formed with l-octanesulfonic acid on a reversed-phase column (Kromasil KR 100-5C18; Eka Nobel; 15 cm × 4.6 mm ID). For these analyses we used a Varian Vista 5500 Liquid Chromatography System equipped with a Model 9090 AutoSampler, a Model 2050 UV Variable Wavelength Detector, a Model 2010 HPLC Pump and a Fluorichrom Fluorescence Detector. A Dynamax HPLC Method Manager and MacIntegrator (Rainin Instrument Company) was used together with a Macintosh S E / 3 0 for method editing and HPLC control, and for data collection and analysis (peak identification and quantification).

2.5. Analysis of ODC mRNA and AdoMetDC mRNA content Four dejellied embryos were homogenized manually in 400 /zl of 10 mM Tris-HC1 buffer (pH 8.6) containing 0.14 M NaC1, 1.5 mM MgC12, 0.15% Nonidet P-40, I mM dithiothreitol, 500 units/ml RNasin ~ ribonuclease inhibitor (Promega), using a Pellet pestle homogenizer (Kontes). The homogenate was kept on ice for 5 min and then centrifuged at 12000 × g (2 min; 4°C). Equal volumes of the supernatant and a 0.2 M Tris-HC1 buffer (pH 8.0) containing 25 mM EDTA, 0.3 M NaCI, 2% sodium dodecyl sulfate (SDS), proteinase K (100 /xg/ml), were mixed by vortexing and then incubated for 30 min at 37°C. Protein was removed by phenol-chloroform extraction. One volume of ice-cold isopropanol was added to the aqueous phase, and the solution was mixed and chilled at - 2 0 ° C for at least 30 min. RNA was collected by centrifugation at 12000 × g (10 min) and washed once with 70% ethanol at room temperature. RNA samples were fractionated by electrophoresis in 1.2% agarose gels containing 0.6 M formaldehyde using l0 mM sodium phosphate (pH 6.5) as the running buffer. The RNA was transferred to nylon membranes (Hybond-N, Amersham) using a Vacu-Blot XL (Pharmacia). Hybridization to 3_~P-labelled probes was carried out in 0.25 M sodium phosphate buffer (pH 7.2) containing 50% formamide, 7% SDS, 0.25 M NaC1, I mM EDTA, 100 /xg/ml tRNA (Gibco-BRL), at 60°C (ODCprobe) or 55°C (AdoMetDC probe) for 15 h. The mere-

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branes were then washed three times 20 min in 1 X PES (0.04 M sodium phosphate buffer (pH 7.2) containing 1 mM EDTA and 1% SDS) at 65°C for the ODC probe and at 60°C for the AdoMetDC probe, and were finally autora-

diographed (Hyperfilm-MP, Amersham) at --70°C for 3 days. Less stringent hybridization conditions were used for the AdoMetDC probe because of its human origin (see below).

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Fig. 2. AdoMetDC activity during embryonic development of Xenopus laet'is. The data points are means (___S.D.) of three different series of experiments. AdoMetDC activity was determined by measuring the release of 14CO2 from S-adenosyl-L-[carboxyl-14C]methionine.

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The ODC and AdoMetDC mRNA levels were quantified by means of densitometric analysis of the autoradiograms using a Pharmacia/LKB Imagemaster DTS.

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Embryo extracts were prepared at 4°C by homogenizing 12 embryos in 400 /zl of hypotonic buffer (10 mM Hepes (pH 7.5), 10 mM KCI, 1.5 mM magnesium acetate, 7 mM /3-mercaptoethanol and 500 units/ml RNasin ~ (Promega) or 140 units/ml heparin (Sigma)) using a Pellet pestle homogenizer (Kontes). Then 0.25% Nonidet P-40 was added. The cytosolic extracts obtained by 10 min centrif-

U. Rosander et al. / Biochimica et Biophysica Acta 1264 (1995) 12 I - 128

3. Results

ugation at 10000 × g were layered onto 12 ml 15-50% ( w / v ) linear sucrose gradients prepared in 20 mM Tris-HCl (pH 7.6), 0.1 M KCI, 3 mM magnesium acetate and 140 units/ml heparin. Free ribosomal subunits, monosomes and polysomes were resolved by centrifugation at 40000 rpm (260000 × g) for 2.5 h at 4°C in a Beckman SW 41 rotor, and fractionation into 1.2 ml aliquots using an ISCO Density Gradient Fractionator (Model 640) equipped with a UA-5 absorbance (254 nm) detector. The RNA was purified by one extraction with phenol/chloroform. Equal volumes of all fractions were then subjected to Northern blot analysis as described above.

In the present study we have analyzed the regulation of the polyamines and their biosynthetic enzymes during early Xenopus laevis development. We found the ODC activity to increase slightly during the cleavage period of the embryos (Fig. 1). A greater increase in ODC activity from fertilization to MBT was reported by Russell [19] and Osborne et al. [20] The present study revealed maximum ODC activity in late blastulae (stage 9), and a dramatic decrease from stage 9 to 10. The ODC activity remained at a low, almost nondetectable, level during gastrulation (stage 10 to 13), but from neurulation on, the activity increased progressively (Fig. 1). The disappearance of ODC activity during gastrulation is supported by the study of Osborne et al. [20], but at variance with that of Russell [19], who found a continuous rise in ODC activity from fertilization to late gastrulation (stage 12.5). The AdoMetDC activity was approximately one order of magnitude lower than the ODC activity during early cleavage, and remained relatively constant until the tailbud stage (Fig. 2). Accordingly, there was no analogy to the precipitous drop in ODC activity during gastrulation. Later during development, however, the activities of ODC and

2.8. Analv.ris ~5~"ODC half l(/~" The half-life of ODC was determined for dejellied embryos at stage 2, 9, 12 and 20. The embryos were incubated in the presence of cycloheximide (1 m g / m l in Holtfreter's solution, pH 7.2) for 5-150 rain at room temperature. ODC activity was measured at various time points, using the method described above. Embryos incubated in the absence of cycloheximide (in Holtfreter's solution, pH 7.2, at room temperature) served as controls.

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Fig. 5. Distribution of ODC mRNA in polysome gradient profiles of cytosolic extracts from Xenopus laevis embryos at various stages of development. Free ribosomal subunits and polysomes were separated on a 15-50% sucrose gradient. Serial fractions across the gradient were analyzed for their content of ODC mRNA using an antisense RNA probe. Embryo extracts were prepared and analyzed in the presence of heparin in order to prevent RNA degradation. Peaks representing 40S and 60S ribosomal subunits, and 80S monosomes appear to the left of the polysomes containing 2-6 ribosomes. The vertical arrow indicates the position of the disomes in each sucrose velocity gradient.

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AdoMetDC changed in parallel, with ODC increasing somewhat ahead of AdoMetDC, beginning during early neurulation and progressing steadily until the tailbud stage (Figs. 1 and 2). These increases in enzyme activity coincided with growth of the embryo. Despite the dramatic decrease in ODC activity from stage 9 to 13 (Fig. 1), there were no significant changes in the putrescine, spermidine or spermine content of the embryo during this period (Fig. 3). During the subsequent growth phase, the spermidine content showed a tendency to increase while the spermine content decreased slightly (23%; P < 0.05). Similar reciprocal changes in spermidine and spermine content also characterize rapidly proliferating mammalian cells [21]. The putrescine content was approx. 2-fold higher than the spermidine content during the development of the embryo from zygote to tailbud (Fig. 3). There is one major discrepancy between the present data on polyamines and those published by Osborne et al. [20]. Although these investigators detected spermine in Xenopus oocytes [22], they were unable to detect spermine in early embryos, implying that each embryo contained less than 10 pmol, which is the detection limit of their method [20]. Notably, the spermine content recorded in the present study was at least 200-fold greater. This is largely in agreement with a study by Russell [19], who also found spermine to be present throughout development, albeit at a somewhat lower level than that observed in the present study. The relatively small changes in ODC mRNA content (Fig. 4A), established by densitometric scanning (results not shown), can only partly explain the changes in ODC activity (Fig. 1). Thus, the increase in ODC activity taking place from neurulation on, may be a consequence of the increase in ODC mRNA, but there is no change in ODC message content to explain the precipitous drop in ODC activity during gastrulation. Duval et al. [23] also found the ODC mRNA content to be constant during early development. In agreement with the suggestions made above regarding the regulation of ODC expression, the increase in AdoMetDC activity taking place from neurulation on, may be a consequence of an increase in AdoMetDC mRNA transcription (Fig. 4B). Since there is virtually no transcription prior to MBT and since AdoMetDC has a short half-life, at least in mammalian cells [2], the AdoMetDC activity is likely to be maintained by continuous translation of a stable message during this period. Fig. 5 shows the distribution of ODC mRNA in polysome gradient profiles at various stages of development. From fertilization to MBT there was essentially no ODC mRNA associated with polysomes containing more than 2 ribosomes. Slowly, however, the ODC message was found to be associated with polysomes containing 3 and 4 ribosomes, but even at neurulation (stage 20) there was little ODC mRNA in polysome fractions containing more than 3 ribosomes.

In view of the fact that there were no changes at the transcriptional or translational levels to explain the decrease in ODC activity during gastrulation, it seemed probable that this down-regulation would be due to increased degradation of the enzyme. Using cycloheximide to inhibit protein synthesis we showed that this was indeed the case. Thus, the half-life of ODC was relatively long Slage

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U. Rosander et al. / Biochimica et Biophysica Acta 1264 (1995) 121-128

(tl/2

= 74 to 122 min) during the cleavage period (stages 2 and 9), but decreased dramatically (tl/2 = 5 min) during gastrulation (stage 12) (Fig. 6). The same, short half-life has been observed for ODC in high density, stationary phase rat hepatoma cells [24], implying that ODC is among the most labile of cellular proteins. At the end of neurulation (stage 20) the half-life of ODC was again relatively long (t~/2 = 45 min).

4. Discussion

Development of Xenopus laeL'is occurs in the virtual absence of gene transcription from the time of fertilization and until MBT [10,11]. Thus, gene expression is controlled at post-transcriptional levels during this period, and the first 12 rapid and synchronous cleavage divisions depend exclusively on maternal mRNAs and proteins that have been produced during oogenesis [25]. ODC mRNA does not belong to the class of maternal transcripts that are specifically deadenylated and released from polysomes after fertilization, and then degraded after MBT [26]. The 3' UTR of ODC mRNA is unable to promote the deadenylation of chloramphenicol acetyltransferase-coding chimeric mRNA in Xenopus embryos, and the two ODC mRNA-binding proteins (p35 and p40) that are present in extracts made from 4-h embryos (pre-MBT) apparently have another function than to promote deadenylation [26]. The present study shows that the amounts of ODC mRNA and AdoMetDC mRNA in the Xenopus embryo remain constant until transcription is reactivated at MBT and then increase gradually. This implies that these maternal mRNAs are entirely stable for at least 8 h. In proliferating mammalian cells, the half-life of ODC mRNA is shorter, varying between 3 and 9 h [27,28]. The poly(A) tail of ODC mRNA remains unchanged or increases slightly between fertilization and MBT [20], suggesting that the ODC mRNA is translatable during this period. This is also supported by our finding that the message is present on polysomes, and that the ODC activity increases despite the fact that the enzyme has a short half-life (t~/2 < 2 h). Osborne et al. [20] interpreted their data to indicate that more than 90% of the ODC mRNA sedimented with the polysomes present in extracts from eggs, 4-h embryos and gastrulae. Moreover, they claimed that most of the ODC message was present in large polysomes. These conclusions are at variance with the data obtained in the present study, where most of the ODC mRNA is found in fractions containing ribosomal subunits and monosomes, a finding that is consistent with studies on mammalian cells [29,30]. Nevertheless, some ODC mRNA is always present in polysome fractions - during early cleavage almost exclusively in fractions containing disomes, but subsequently also in fractions containing 3 - 4 ribosomes. In agreement with the study of Woodland [31] we find very few polysomes during the early developmental stages.

127

The present study indicates that Xenopus ODC mRNA is poorly translated during early development. In fact, all ODC mRNAs isolated from vertebrates have been found to be poorly translated, apparently because of stable secondary structures in their 5' UTRs [32,33]. Since the 5' UTR of Xenopus ODC mRNA has not been fully sequenced [16], it remains to be determined whether stable secondary structures in this region also can explain the poor translatability of the ODC message in early Xenopus embryos. Even though ODC mRNA is poorly translated during early Xenopus development, the rate of translation is sufficient to account for a slight increase in the ODC level - despite the fact that the enzyme has a relatively short half-life (t~/2 < 2 h). After MBT and during gastrulation, however, we find the half-life of ODC to decrease dramatically. Inasmuch as there appears to be no major change in ODC gene transcription or message translation, the ODC activity drops precipitously. Surprisingly, however, the decrease in ODC activity does not cause any major changes in the levels of putrescine, spermidine or spennine (Fig. 3), nor does the amount of substrate (L-ornithine) change significantly [20]. There may be a temporary halt in polyamine accumulation, but in view of studies where polyamine synthesis inhibitors have been used to reduce polyamine levels, these small changes should have no biological effect, unless large changes in compartments (cellular as well as subcellular) are obscured by analyzing whole embryos. Therefore, a question that remains unanswered is why ODC expression is strongly down-regulated during gastrulation. An interesting coincidence is that embryo development in many species is blocked at gastrulation. following exposure to ODC inhibitors from fertilization on [8]. The conclusion arrived at by Osborne et al. [20], that the decrease in ODC activity may be due to feedback regulation by polyamines, is not supported by our data, not even by their own, because the polyamine content does not change sufficiently to elicit such a response, unless subcellular and cellular compartments differ markedly in their polyamine content. The rapid degradation of certain mammalian proteins, including ODC, has been attributed at least partly to the presence of PEST regions, i.e., regions rich in proline, glutamic acid, serine and threonine [34]. Since Xenopus ODC does not possess any PEST region [20], other mechanisms have to be involved in the rapid degradation of the enzyme. Baby and Hayashi [35] have shown that antizyme, a protein that exhibits high-affinity binding to ODC [36], is present in Xenopus cells. Recent findings in mammalian systems indicate that antizyme binding causes a conlormational change in ODC, thus exposing its C-terminus and reversibly inactivating the enzyme [37,38]. The C-terminus of ODC together with a portion of the N-terminal half of antizyme direct the enzyme for degradation by the 26S proteasome in a ubiquitination-independent but ATP-de-

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pendent manner. In v i e w o f the p r e s e n c e in X e n o p u s o f both a n t i z y m e and a d e v e l o p m e n t a l l y regulated proteas o m e [39], it is c o n c e i v a b l e that a m e c h a n i s m , similar to that described for m a m m a l i a n O D C degradation, is responsible for the rapid degradation o f O D C during gastrulation of the X e n o p u s e m b r y o . T a k e n together, the present data s h o w that O D C expression is regulated mainly at the translational level from fertilization to M B T . A gradual increase in translation bringing about an increase in the O D C activity and the p o l y a m i n e content. D u r i n g gastrulation, h o w e v e r , there is a switch to post-translational control, causing a dramatic increase in O D C degradation, and c o n s e q u e n t l y a rapid decrease in O D C activity, but not in p o l y a m i n e content. At variance with the activity o f O D C , that o f A d o M e t D C remains at a low and constant level from fertilization through gastrulation. F r o m neurulation on, h o w e v e r , the activities o f both e n z y m e s increase, p r o d u c i n g an increase o f m a i n l y spermidine. The increase in O D C and A d o M e t D C e x p r e s s i o n during this d e v e l o p m e n t a l period are, at least partly, attributable to increases in their m R N A contents, but effects at translational and post-translational levels are also evident.

Acknowledgements This w o r k was supported by the S w e d i s h Natural Science R e s e a r c h Council ( B - B U - 4 0 8 6 and B - B U - 2 6 5 7 ) , the S w e d i s h Council for Planning and Coordination o f Research, the Knut and A l i c e W a l l e n b e r g F o u n d a t i o n and the J.C. K e m p e Foundation.

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