Ornithine decar☐ylase activity and the accumulation of its mRNA during early stages of liver regeneration

Biochimica et Biophysica Actao 1007 (1989) 120-123 Elsevier

120 uu~ 90127

BBA Report

Ornithine decarboxylase activity and the accumulation of its mRNA during early stages of liver regeneration Ari H i r v o n e n Delut,tment of Biochemistry. University of Kuopio, Kuopio (Finland) (Received 3 October 1988)

Key words: Ofnithine decarboxylase; mRNA accumulation: Liver regeneration; (Rat); (Mouse)

The maAed and well documented stimulation of hepatic omithine dec~rboxylase (ODC; EC 4.1.1.17} in response to partial hepetectomy is at lemt to some extent attributable to an enhanced accumulation of the enzyme's mRNA, The stimulation of ODC activity was associated with an increased accumulation of two ODC.related mRNA species (2.1 and 2.6 ~ kb) as revealed by Northern blot hybridization analyses. The levels of the above-mentioned messages elevated for 6 h after partial hepatectomy, at which time the enzyme activity had returned to #]most control I~ Fm'thennm~ ODC protein levels remained relatively stable after the first peak of ODC activity, suggesting that pmttrm~tional activity was responsible for the changes in ODE activity after the initial burst, in addition to the two ~A species typical of mouse cells, rat tissues contained a third bybridizable messaSe (I.6 kb). This smaller poly(A) + IINA was never seen in samples obtained from mouse or human cells, but was always present in samples obtained from rat tissues. Interestingly this rat.specific message appeared to be expressed in somewhat opposite manner to the other two mRNA species.

Many of the mammalian enzymes which regulate the flow of substrates through metabofic pathways by rapidly changing their activity in response to variety of stimuli are labile proteins with short molecular half-lives [1]. ODC, the rate-controlling enzyme of the biosyntheti¢ pathway of the natural polyamines (putrescine, s~ine and spem~ne), is probably the most inducible mammalian enzyme, having a half-life about 10 rain in rat fiver [2|. Its activity is s,,rikingly stimulated in response to various anabolic ~fimuli, such as hormones in their appropriate target tissues [3-6]. Partial hepatectomy of the rat causes a dra.matic enhancement of ODC activity in the regenerating fiver remnant. Rapid stimulation of the enzyme activity occurs in two phases, reaching its first peak invariably at 4 h postoperatively [7], and evidently requires the synthesis of new enzyme protein and/or new mRNA for the enzyme [2,8-10]. In this paper I have extended the studies on the regulation of ODC in regenerating rat fiver by measur-

Abbreviatioas: ODC. omithine decarboxylase (EC 4.1.1.17). ~ : A. Hirvonen, Department of Biochemistry, University of Kuopio, P.O. Box 6, SF-70211 Kuopio, Finland.

ing the accumulation of the mRNA and protein of the enzyme during early stages of liver regeneration. It appears that the primary stimulation of ODC activity is to some extent attributable to an enhanced accumulation of enzyme's message, yet the subsequent fluctuations of the enzyme activity may be based on post-translational regulation. Partial hepatectomy resulted in an striking stimulation of ODC activity 3-4 h after the operation (Fig. 1). Fig. 2 shows a Northern blot analysis of poly(A) + RNA isolated from liver after different times postoperatively. As shown, rat liver appears to contain three ODC mRNA species hybridizable to mouse kidney eDNA. This is in contrast to mouse cells, which only contain two ODC mRNA species arising from size heterogeneity at their Y-t~rmini [11]. The accumulation of 2.1 kb mRNA, which was the main hybridizable message at 4 h postoperatively, already began at 1 h and remained at elevated level at 6 h after partial hepatectomy (Fig. 2 and Table I). On the other hand, the accumulation of 1.6 kb mRNA, which was the main hybridizable message in the control fiver, decreased 1 h after the operation and returned to almost basal level at 6 h postoperatively (Fig, 2 and Table I). The nature and significance of this rat-specific hybridization signal is not known at present.

0167-4781/89/~03.50 © 1989 Elsevier Science Pubfishers B.V. (Biomedical Division)

121

T|ME{hours~ 0 I 2 3 4 6 Size kb) Z 0 e'l

-2.6 =2.1

E a. =g am

-1.6

lU g ¢g

:IOO.

Fig. 2. Northern blot analyses of ODC mRNA from partially hepat~tomize rat livers. Tissue samples (pooled groupwise) were removed, placed in liquid nitrogen and processed for RNA i,~lation. Total RNA was isolated by guanidine isothiocyanate method [21]. Polyadenylated RNA was isolated with the aid of an mRNA affinity paper (polyuridylated) (Hybond-mAP, Amersham) by employing the loading and washing conditions described by Werner et al. [22]. Poly(A)' RNA (14 t~g) was fracfionated by electrophoresis in 1.4~6 agarose gels in the presence of I M glyoxai, transferred [23] onto HybondoN filters (Amersham), hybridized to nick.translated [24] mouse pODC16 It 3,18] and autoradiographed. The hybridizations were essentially performed as described by the manufacturer of the hybridization filters (Amersham: Membrane transfer and detection methods). The molecular sizes of mRNA species are shown to the right,

~OO.

tOO,

HOURS AFTER PARTIAL HEPATECTOM¥

Fig. 1. Omithine decarbo,tylase activity of partially hepatectomized rat liver. Female Sprague.Dawley rats (body wt. about 100 g) were used in all experiments. Partial hepatectomy was performed under aether aneasthesia [17] and the animals were killed by cervical dislocation at different times after the operation. The tissue extracts were prepared and ODC activity assayed essentially as described in Ref. 19. Protein was determined by the method of Lowry et al. [20]. The vertical bars represent standard deviations of the means (three animals in each group).

bridized with nick-translated mouse cDNA probe pODC16 [13,18]. Using a mouse probe instead of rat should nol cause any problems because mouse-rat eDNA homology is known to be about 94~ in the coding region [26]. Mouse liver contained mainly 2.1 kb mRNA species and there was no sign of the 1.6 kb mRNA species which was always present in rat samples (Fig. 3). To exclude the possibility of a plasmid-derived artifact, TABLE !

When the time period studied was extended over the full period of stimulation of ODC activity, it appeared that not only the amount of ODC mRNA but the amount of immunoreactive ODC protein, too, remained at a relatively stable level after the first peak of ODC activity (Table II). Northern blot analyses of the total RNA showed similar fluctuations in ODC mRNA levels compared to protein amounts. This might indicate that the early (up to 4 h) stimulation of ODC activity in regenerating rat liver is due mainly to enhanced accumulation of its mRNA. However, at later stage it is possible that posttranslational rather than posttranscriptional activity plays a role in ODC regulation° To exclude possible artifacts, I isolated rat (R) and mouse (M) poly(A) + RNA side by side from rat and mouse liver. Poly(A) + RNA was electrophoresed and blotted to hybridization filters and the filters were by-

Relative amounts of ODC mRNA species and enzyme activity during first peak of ODC activity in rat livers after partial hepatecwmy For treatment of animals, see legend to Fig. i and for Northern blot analysis see legend to Fig. 2. Autoradiographs of Northern blot analysis shown in Fig. 2 were scanned with a Shimadzu DualoW~,,cle,gth Chromato Scanner. The relative amount of 2.1 and 2.6 kb ODC mRNA species from livers of untreated rats was set to 1.0. Relative ODC activities were obtained from Fig. 1. Time (h)

Relative amount of ODC mRNA species

Relative ODC activity

2.1 + 2.6 kb

1.6 kb

0

1.0

4.5

1.0

1

1.6

4.1

0.6

2 3

2.1 2.8 3.7

2.7 2.4 2.1

2.9 17.0

2.9

2.5

4 6

16.0 2.8

122 TABLE il Relative ODC mRIVA amounts, enzyme activity and the amount of immunoreactive protein at different times in regenerating rat livers

For treatments of animals see legend to Fig. ! and for ODC activity measurements see ref. 19. Tissue extracts for radioimmunoassay were prepared and analysis carried out essentially as described in Ref. 25. Total RNA was isolated and Northern blot analysis carried out as described in legend to Fig. 2. Autoradiographs were scanned as ~bed in Table !. Relative mRNA leveh and ODC protein were assayed from pooled samples (three animals in each group). Time (h)

4 8 10 12 24

Relative amounts of ODe MRNA species 2A+ 2,6 kb 1,6 kb

ODC activity (pmol of CO2 / n~ protein per 30 rain :I:S,D.)

ODC (ng/mg protein

1,0 2,s !,2 2,3 0,5 1,9

57.2 :t: 16.4 6 ,2 ± 7s.4 111.2:1:19,6 351,6 :t: 46.2 87,2:1:24,2 259,4:1:82,8

0.2:~ 0.63

.....

12",S ~

7,7 8,7 9,2 10,5 4,9

0.40

0,51 0,40 0,51

the filters were washed and reprobed with nick-translated pBR322. After autoradiography there were never any signs of 2.6, 2.1 or 1.6 kb hybridizable poly(A) + RNA, rul/ng out the possibility of plasmid-derived signals. The possibili'y of antisense RNA [14,15] was excluded by hybridi:,.ation analyses with single-stranded probes (Jltnne, O.A., personal communication). Indirect studies have suggested that partial hepatectomy of the cat results in transcriptional activation of ODC followed by regulation at some post-transcriptional level ll0]. However, no one has directly shown the time-course of ODC mRNA accumulation and its

LIVER

R M Size (kb)

relation to ODC activity at the early stages of rat liver regeneration. The present results support the notion that the enhanced accumulation of ODC mRNA is in all likelihood mainly responsible for the primary stimulation of the enzyme activity (up to 4 h) after partial hepatectomy and that after the first peak of enzyme activity, the mRNA of ODC remained relatively stable, although the enzyme activity rapidly declined. The comparison of ODC activity and the accumulation of its mRNA (Table I) reveals that the marked decrease in the enzyme activity between 4 and 6 h was not accompanied by similar reduction of the amount of mRNA, suggesting post-transcriptional regulation. It is likewise worth noting that the enzyme activity and the amount of mRNA increased in parallel only up to 2 h, whereafter the enzyme activity sharply rose, accompanied by a modest increase in the amount of mRNA and protein (Table 1 and I!). This phenomenon suggests that the expression of the enzyme after the initial stimulation might primarily be controlled post-translationally at relatively stable mRNA levels. An interesting finding concerning an extra hybridizable message is also included in the present results. Unlike human [12] and mouse [11,13] cells, rat liver contained a smaller 1.6 kb ODC-related transcript which appeared to accumulate in opposite manner in comparison to the 2.1 kb and 2.6 kb messages typical of mouse cells. Whether this transcript represents a truncated (3' or 5' terminus) ODC mRNA or not is currently under investigation. A similar, smaller mRNA species has recently been reported in samples obtained from rat hepatoma cells [16]. The skilful technical assistance of Ms. Tiina Iramourn is gratefully acknowledged. This investigation was financially supported by the University of Helsinki, by grants from the Academy of Finland, from the Farmos Research Foundation and from the National Institutes of Health, U.S.A. (grant No. R01 CA3769503). Referenees

- 2.6

-2,1 - 1.6

Fig, 3. Northern blot analyses of ODC mRNA from rat and mouse fiver. Rat and mouse fiver mRNAs were isolated side by side, electro(10 Fig), blotted, hybridized to nick-translated pODCI6 and autogadiqgaphed. For treatments of animals see legend to Fig. 1, and Northern blot analyses see legend to Fig. 2. R,rat liver; M, mouse liver. The molecular sizes of mRNA species are shown to the right.

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