Cell Type-Specific Mechanisms of Regulating Expression of the ...

Vol. 10, No. 10

MOLECULAR AND CELLULAR BIOLOGY, Oct. 1990, p. 5525-5528 0270-7306/90/105525-04$02.00/0 Copyright C) 1990, American Society for Microbiology

Cell Type-Specific Mechanisms of Regulating Expression of the Ornithine Decarboxylase Gene after Growth Stimulation MITCHELL S. ABRAHAMSEN AND DAVID R. MORRIS* Department of Biochemistry, University of Washington, Seattle, Washington 98195 Received 2 November 1989/Accepted 1 July 1990

Ornithine decarboxylase (ODC) mRNA is strongly induced by mitogenic activation of resting Swiss 3T3 fibroblasts and T lymphocytes. Nuclear run-on analysis revealed a low level of nascent transcripts in resting fibroblasts that was elevated upon activation. In contrast, there was a high level of transcription across the entire ODC gene in resting T ceUls, which remained unchanged upon activation. The stability of the mature ODC message was found to be unaffected by mitogenic stimulation. These results indicate that ODC mRNA levels are regulated transcriptionaHly in Swiss 3T3 ceUs and posttranscriptionally within the nucleus of T lymphocytes in response to mitogenic stimuli. In this unique situation, the mitogenic induction of a single gene, ODC, is regulated by two very distinct, cell-specific mechanisms. cells by ConA were very similar to those seen in the serum-activated fibroblasts (Fig. 1B). To evaluate the mechanisms of regulation of ODC mRNA levels in fibroblasts and lymphocytes, the relative numbers of nascent ODC transcripts in nuclei isolated from quiescent and activated cells were measured by nuclear run-on transcription. Nuclei were isolated from cells according to McKnight and Palmiter (21), and nascent RNA transcripts were labeled with [a-32P]UTP (6). Total RNA was extracted (4), and the RNA pellet was suspended in water and treated with 0.2 M NaOH for 10 min on ice (to partially hydrolyze the transcripts). Equal radioactivity from each RNA preparation (107 cpm/ml) was hybridized (16) to nitrocellulose filters containing 5 ,ug of single-stranded DNA with specific inserts, cloned in opposite orientations, which will detect sense (+) and antisense (-) transcription. ODC 5' is a 386-base-pair (bp) SalI-PstI subclone, covering exons 2 through 5, and ODC 3' is a 340-bp PstI subclone, covering exons 10, 11, and 12, of pOD 20.7 (7). The c-myc probe is a 350-bp PstI fragment from exon 2 (3). No measurable transcription above a control using wild-type M13 bacteriophage DNA has ever been detected in nuclei from resting 3T3 cells (not shown), but increased ODC transcription was readily observed after treatment of the fibroblasts with serum, TPA, or forskolin (Fig. 2A). In the same experiments, the increase in transcription of the c-myc gene was consistent with that previously published (6) and served as an internal control for the competence of the nuclei to demonstrate transcriptional regulation. The use of 5'- and 3'-specific ODC probes allowed measurement of the distribution of nascent transcripts across the gene. Since the nascent transcripts are labeled with radioactive uridine, the anticipated ratio of hybridization to the 5' and 3' ODC probes is dependent on the number of adenine bases present in each probe. ODC 5' contains 97 adenine bases and the 3' probe contains 74, yielding an anticipated ratio of 1.3. Densitometric scanning of the autoradiogram gave a ratio of approximately 1.7, in reasonable agreement with the anticipated result. Thus, we find no evidence of significant transcriptional attenuation within the ODC gene, as seen in some other genes that are up regulated during mitogenesis (14, 15). In striking contrast to the fibroblasts, run-on transcription with nuclei isolated from lymphocytes before and after ConA treatment, using the same probes as were used with

Ornithine decarboxylase (ODC) is a member of the immediate-early family of growth-regulated genes (5, 9-11, 19) and catalyzes the key regulated step in the biosynthesis of the polyamines spermidine and spermine (25, 27). The rapid and dramatic induction of ODC activity in response to growth stimuli, which has been found in all cells and tissues in which it has been studied (25, 26, 32), strongly suggests an important role for ODC and the generated polyamines in the growth of mammalian cells (18). In these growth-regulated systems, ODC activity seems to be controlled mainly through changes in the rate of de novo synthesis, which in turn is regulated in most cases through modulation of the level of its mRNA (23). We have examined the mechanism of regulation of ODC mRNA level in two growth-stimulated cell systems: fibroblasts activated by serum mitogens and T lymphocytes stimulated through interaction of the antigen receptor with the mitogenic lectin concanavalin A (ConA). Responses of this mRNA to various stimuli in these two cell types are shown in Fig. 1. Cells were harvested at the times shown after stimulation, and total RNA was isolated (4). Equal amounts of total RNA were separated on formaldehydeagarose gels, transferred to nitrocellulose, and hybridized (17) to the insert from the mouse cDNA clone pOD48 (20). Activation of quiescent Swiss 3T3 cells by addition of serum led to a rapid and transient increase in the relative level of ODC mRNA (Fig. 1A). Message level began to increase within 30 min after the addition of serum and reached a maximal induction of 18-fold at 3 h. Similar results were obtained by treatment of these cells with epidermal, fibroblast, or platelet-derived growth factor (not shown). Activation of protein kinase C by 12-O-tetradecanoylphorbol-13acetate (TPA) or elevation of intracellular cyclic AMP with forskolin also led to increased levels of ODC mRNA (Fig. 1A), but with different kinetics and lower magnitudes than observed with serum. The effects of TPA and forskolin added in combination were synergistic (not shown). In T lymphocytes, the induction of ODC mRNA by ConA is through a protein kinase C-dependent pathway (22). The kinetics and magnitude of induction after activation of T

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Corresponding author. 5525

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FIG. 1. Induction of ODC mRNA by various stimuli in fibroblasts and T lymphocytes. (A) Swiss 3T3 cells were arrested by incubating subconfluent cultures for 3 days in 0.5% calf serum. The cells were activated for times indicated by the addition of 10% calf serum (U), 30 nM TPA (l), or 10 ,uM forskolin (A). (B) Resting bovine T lymphocytes (30) were activated for various times with ConA (18 ,ug/ml). The relative levels of ODC mRNA were determined by densitometric scanning of autoradiograms produced by Northern (RNA) blot analysis.

the 3T3 cells, did not detect any change in the rate of transcription of the ODC gene (Fig. 2B), despite the large increases seen in mRNA level (Fig. 1B). The data are representative of three independent experiments, and similar results were seen using nuclei isolated from cells activated for 1 and 2 h (data not shown). The high level of hybridization to the ODC 3' antisense probe (Fig. 2B) was repeatedly seen with transcripts from bovine T-cell nuclei but not with those from 3T3 cells (Fig. 2A). This high level of antisense transcription appeared to be bovine specific, since it was also seen in bovine fibroblasts (not shown) but not in human (Fig. 3) or mouse (Fig. 2A) fibroblasts. This signal most likely resulted from cross-hybridization to a transcript from an unrelated bovine gene or from a transcriptional unit oriented opposite to, but overlapping, the 3' end of the bovine ODC gene. The increase in c-myc transcnrption upon activation (Fig. 2B) demonstrated that the nuclei did indeed reflect the functional state of the cells from which they were isolated. In addition, the ratio of hybridization of radioactive A ODC

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transcripts to the 5' and 3' ODC probes did not change upon activation of the cells with ConA. Thus, increased ODC mRNA in mitogen-activated T lymphocytes was due neither to elevated transcriptional initiation nor to release of an intragenic block to transcriptional elongation. The level of ODC mRNA in T lymphocytes must therefore be regulated primarily through a posttranscriptional mechanism. The results presented above were obtained with primary T-cell cultures. Primary human foreskin fibroblasts were examined to determine whether posttranscriptional regulation of the ODC gene is a characteristic associated with primary cell cultures. Run-on transcription with nuclei isolated from resting foreskin fibroblasts before and after treatment with TPA demonstrated that the ODC gene, as well as the c-myc gene, is regulated transcriptionally in these cells (Fig. 3). A common mechanism of posttranscriptional regulation is through modulating the rates of decay of the mature message in response to external stimuli (28, 29, 31). Posttranscriptional regulation of ODC synthesis in lymphocytes could be achieved through increased stability of the mature message after mitogenic activation, as has been found for a variety of genes expressed in this cell type (13). The rate of decay of the mature ODC message was measured in resting and 3-h-activated lymphocytes after treatment with an inhibitor of transcriptional initiation, 5,6-dichloro-1-p-D-ribofuranosylbenzimidazole (24) (Fig. 4). In both resting and activated cells, ODC mRNA decayed with a half-life of approximately 1.5 h. Therefore, the increase in ODC mRNA upon activation could not be due to an increase in the stability of mature message. Similarly, experiments in Swiss 3T3 cells detected no change in message stability upon activation, although the message was more stable than that seen in lymphocytes (half-life of 5 h; not shown). FORSKOLIN

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FIG. 2. Nuclear run-on transcription in nuclei isolated from (A) Swiss 3T3 fibroblasts activated for 0 and 1 h as for Fig. 1 and (B) T cells activated for 0 and 3 h with ConA. Nuclei were isolated at the indicated times and labeled with [32P]UTP. Total RNA was isolated, and equal radioactivity from each sample (107 cpm/ml) was hybridized to nitrocellulose filters containing 5 ,ug of the described single-stranded DNAs. CS, Calf serum.

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transcripts of adenosine deaminase (2), T-cell receptor alpha and beta subunits (33), and c-myc (8) that seem to be involved in regulating the levels of the mature mRNA species. The intranuclear event leading to elevation of ODC mRNA upon activation of T cells could be stabilization of a pre-mRNA species that is unstable in the resting cells, a change in pre-mRNA processing from an unproductive pathway to one which leads to mature message, or an enhancement of the transport of processed ODC mRNA out of the nucleus. It is possible that posttranscriptional regulation of the ODC gene also occurs in 3T3 cells but, in contrast to the T-cells, is not apparent experimentally because of the large element of transcriptional control in the fibroblasts.

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FIG. 4. Stability of ODC mRNA in resting (O) and activated (X) T lymphocytes. Resting and 3-h ConA-activated T cells were treated with 5,6-dichloro-1-,-D-ribofuranosylbenzimidazole (63 p.M). Total RNA was isolated at the times indicated and subjected to Northern analysis as before. The relative levels of ODC mRNA were determined by densitometric scanning of autoradiograms exposed in the linear range for each set of samples; the autoradiogram of the unactivated samples was exposed approximately 10 times longer than that of the activated samples. The data are presented as arbitrary units on a semilog graph.

The difference in regulation of the ODC gene between the seen in the basal level of transcription in the resting cells (Fig. 2). No significant transcription above background could be detected in nuclei from resting fibroblasts, in clear contrast to the high level of transcription seen in resting T cells. In Swiss 3T3 cells, activation of two separate signaling pathways, protein kinase C with TPA and protein kinase A with forskolin, and multiple signaling pathways with serum, resulted in increased ODC transcription (Fig. 2A). In addition, activation of protein kinase C in foreskin fibroblasts also resulted in increase ODC transcription (Fig. 3), demonstrating the ability of primary cell cultures to transcriptionally regulate the ODC gene. Although we cannot rule out the possibility of some posttranscriptional control in the fibroblasts, it is apparent that increased transcription of the ODC gene is a major element of regulation in this particular cell type. It seems likely, given undetectable transcription in resting cells, that any stimulus given to fibroblasts that results in increased levels of ODC mRNA would be accompanied by an increase in transcription. In contrast to the situation in fibroblasts, ODC transcription is already elevated in resting T lymphocytes and is not altered on activation of the cells. Since the strong elevation of ODC mRNA was not due to stabilization of the mature message, we suggest that T cells must be regulating this gene posttranscriptionally within the nucleus. Relatively few examples of nuclear posttranscriptional mechanisms have been reported to date in cells not infected with viruses. Evidence suggesting modulation of intranuclear stability or transport of transcripts was found with cell cycle regulation of histone H3 (1) and induction of dihydrofolate reductase after growth stimulation (12). More recent studies have demonstrated qualitative and quantitative changes in nuclear pre-mRNA two cell types studied here is striking and is best

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