CAMP Stimulation of Dictyostelium discoideum Destabilizes the mRNA for 117 Antigen*

destabilization synthesis of The data suggest that a CAMP-induced protein(s) be involved in the destabilization of selective mRNAs.

In a number of cases, changes in intracellular cAMP levels are associated with altered gene expression in eukaryotes, but the mechanism(s) by which CAMP affects gene activity is poorly understood. Promoter regions of an increasing number of CAMP-regulated genes have been isolated and characterized to define CAMP-regulatory elements (1). Although the possibility exists that CAMP controls mRNA stability (2), this has not been extensively documented experimentally. The phosphoenolpyruvate carboxykinase (GTP) mRNA has been reported to be stabilized by CAMP (3). More generally, CAMP appears to alter the rate of gene transcription. Some genes are rapidly regulated by CAMP in a manner that seems to be insensitive to cycloheximide (4). Other classes of genes are induced only after several hours of CAMP treatment, a period that probably suggests the need for continued protein synthesis (1). The distinction of genes transcriptionally activated by CAMP by the requirement for protein synthesis would be similar to that made in the case of steroid hormone induced primary and secondary responses (5).
For Dictyostelium discoideum, extracellular CAMP has been shown to play an important role in development, while little is known about its possible intracellular effects. Extracellular CAMP pulses function as a chemoattractant during cell aggregation (6) as well as an effector of cell differentiation to aggregation competence (7). The effects of extracellular CAMP are mediated by a cell surface CAMP receptor which shows a number of similarities to mammalian hormone receptors including ligand-induced phosphorylation (8, 9) and down-regulation (10). During aggregation, CAMP pulses effect the mRNA levels of certain developmentally regulated genes and pharmacological studies suggest that these effects are mediated by the cell surface CAMP receptor (11)(12)(13)(14). In the cases studied, CAMP alters the rate of gene transcription (15)(16)(17) and acts additionally to stabilize developmentally regulated mRNAs (18,19). Upon disaggregation of post-aggregative cells, many of the developmentally increased mRNAs disappear. The addition of CAMP to cells at the time of disaggregation restores many of those mRNAs, primarily by restoring their rates of transcription (18), but also by preventing the destabilization of labile mRNAs (19).
We have previously demonstrated that 117 antigen is expressed cyclically during the development of D. discoideum. Both cell surface protein and mRNA levels are nondetectable in vegetative cells, maximal in aggregating cells, and then return to nondetectable levels as cells form slugs. Latter in development, as slugs begin to culminate, 117 protein and mRNA reappear as prespore cells differentiate into spores. Both are then lost as cells complete their differentiation into mature spores (20,21). To begin to understand the factors that regulate the expression of 117 antigen, we have examined the effects of CAMP stimulation on 117 mRNA levels. We show here that CAMP pulses can induce 117 mRNA in cells, while high concentrations of CAMP result in a rapid loss of that message. Using pulse-chase and nuclear run-off assays, combined with inhibitors of RNA and protein synthesis, we examined the basis for the effects of high concentrations of CAMP. The data indicate that stimulation of cells with high concentrations of CAMP destabilizes the 117 mRNA and does so by a mechanism requiring protein synthesis. Nuclear Run-off Assay-Nuclei were isolated as described by Sol1 and Sussman (30) using their alternative method. Run-off transcription was also performed as they describe with a few modifications. A 100~~1 suspension of 6 X 10' nuclei in 50 mM Hepes, pH 7.5, 40 mM MgCI,, 20 mM KCl, and 13.5% sucrose, was added to 800 &I of 40 mM Tris, pH 7.9, 2 mM NaF, 5 mM KCl, 1 mM B-mercaptoethanol, 100 mM (NH&SO+ 10 mM .MgCl?, 3.4% sucrose, 30 units of RNasin (Promesa). 0.3 mM CTP. GTP. and ATP. and 200 uCi of ln-32P1UTP (600 Ci/mmol).
The mixture was incubated at 25 "C for 60 mih and the RNA extracted by the addition of 0.5 ml of 20% SDS, 10 ml of phenol/CHC&, and 10 ml of 50 mM Tris, pH 8.  Fig. 3, when 6-h starved cells were treated with 1 mM CAMP, the level of 117 mRNA decreased such that by 30 min of such treatment, cells contained approximately 10% of the message present in untreated control cells (also see Fig. 8). This decrease in 117 mRNA levels did not reflect a general cellular change since the levels of actin mRNA were not affected under these conditions (data not shown).
To assess if the effects of high concentrations of CAMP on 117 mRNA levels were the result of receptor down-regulation, we performed similar experiments using a range of concentrations of two CAMP analogues. 2'-Deoxy-CAMP is a potent agonist for the CAMP cell surface receptor, but has a very weak ability to activate the CAMP-dependent protein kinase. Dibutyryl CAMP shows the opposite specificity (34, 12). As Ax-2 cells were starved for 6 h at which time CAMP, 2'-deoxy-CAMP, or dibutyryl CAMP was added. Cells were starved for an additional 30 min and their levels of 117 mRNA determined by Northern analysis. Lanes l-3. cells were treated with 1. 0.1. and 0.01 mM dibutvrvl CAMP, respectively. Lanes 4-6, cells were treated with the same eoncentrations of CAMP, while in lanes 7-9, 2'-deoxy-CAMP was used. Lane 10 shows the level of 117 mRNA in cells incubated for the additional 30 min in the absence of any treatment. The picture represents one autoradiogram from which individual lanes were cut and repositioned for ease of discussion. A significant gradation in the background across the filter occurred and is accentuated by the repositioning of the lanes. The data are representative of three experiments.
shown in Fig. 4, dibutyryl CAMP treatment of cells, at any of the concentrations tested, had little or no effect on the level of 117 mRNA. In contrast, 2'-deoxy-CAMP treatment resulted in a dramatic decrease in 117 mRNA levels and its effects exhibited a dose dependence similar to that of CAMP treatment. The results indicate that the effects of high concentrations of CAMP on mRNA 117 are also mediated by the cell surface CAMP receptor.
Nuclear run-off transcription assays were employed to determine if the decrease in 117 mRNA levels elicited by high doses of CAMP was due to an arrest of gene transcription. In these experiments, transcription from nuclei isolated from 6h starved cells that had been incubated for 30 min in the absence or presence of 1 mM CAMP were compared. As shown in Fig. 5, no obvious decrease in the level of 117 mRNA transcription was observed in nuclei from cells that had, or had not, been treated with high concentrations of CAMP. As expected, such nuclei synthesized equivalent amounts of actin mRNA, a gene unaffected by CAMP (35). No detectable 117 mRNA was synthesized by nuclei isolated from vegetative cells, consistent with the fact that vegetative cells do not accumulate detectable 117 mRNA. Thus, the developmental increase in this mRNA reflects changes in gene transcription. The data indicate that treatment of cells with high concentrations of CAMP alters 117 mRNA levels, not by arresting in response to CAMP stimulation. 6-h starved Ax-2 cells were incubated in the absence (-) or presence (+) of 1 mM CAMP for 30 min. At that time, nuclei were isolated and run-off transcription assayed as described under "Experimental Procedures." Nuclei from vegetative amoebae (VI were also analyzed. Transcripts were hybridized to varied amounts of either the Bluescribe plasmid without any insert (plasmid) or to the plasmid containing a cDNA insert for actin or 117, respectively. From top to bottom, the amounts of DNA immobilized onto the filter were 5, 2. Pulse-chase analysis of 117 mRNA. Ax-2 amoebae were starved for 5 h, pulse-labeled with "PO, for 1.5 h, and placed in the chase medium for 45 min. 1 mM CAMP was then added to onehalf of the population. At the indicated times after CAMP addition, RNA was extracted and hybridized to 5,2.5, and 1 pg of immobilized plasmid containing the 117 cDNA (top filters). Alternatively, samples were hybridized to the B-l actin plasmid (time 0 and 45 min) or to plasmid alone (times 15, 30, and 60 min) (bottom filter). The autoradiogram showing hybridization to the B-l plasmid was exposed for a shorter period to provide signal intensities similar to that observed with the plasmid containing the 117 cDNA. The data are representative of five experiments.
its synthesis but more likely by enhancing its degradation. This hypothesis was substantiated by measuring 117 mRNA stability using pulse-chase analysis. Cells were labeled with 32P04 for 1.5 h and then incubated in phosphate buffer for 45 min. At that time, one-half of the cells were stimulated with 1 mM CAMP and after varied times, were examined for their levels of radioactive 117 mRNA. As seen in Fig. 6, cells that had been treated with CAMP showed an accelerated degradation of 117 mRNA compared to untreated cells. In the absence of CAMP treatment, only a slight decrease in radiolabeled 117 mRNA was observed during the 60-min chase period. In contrast, that level was dramatically reduced in CAMP-stimulated cells. In the first 15min incubation of cells with CAMP, the decrease in radioactive 117 mRNA was slight. During the subsequent 15 min, the major loss in radioactive 117 mRNA occurred. After this time only slight decreases in radioactive 117 mRNA were observed. The effect of cell incubation with CAMP on 117 mRNA stability was not a general one. The level of radiolabeled actin mRNA remained relatively constant during this chase period, as previously reported (31) and was not affected by CAMP stimulation of cells.

Destabilization of 117 mRNA Requires RNA and Protein
Synthesis-The above experiments indicate that CAMP stimulation of cells results in an enhanced degradation of 117 mRNA. To determine if this is a primary effect, or one that requires new protein synthesis (secondary effect), we examined 117 mRNA stability in cells treated with inhibitors of RNA or protein synthesis. Inhibition of RNA synthesis was accomplished using a mixture of actinomycin D and daunomycin (18). Fig. 7A shows that the decrease in 117 mRNA in the presence of 1 mM CAMP(+) was largely prevented when RNA synthesis was inhibited. To eliminate the possibility that the inhibitors themselves were stabilizing the message to the effects of CAMP, we also performed these experiments using nogalamycin. This RNA synthesis inhibitor has been used in D. discoideum to determine mRNA half-lives in germinating spores (36). The same results were obtained (Fig.   7B).
The inability of CAMP stimulation to alter 117 mRNA stability in the above experiments suggests that RNA synthesis is required for that to occur. A newly synthesized RNA may itself act to destabilize 117 mRNA or it may encode a protein that does so. In accordance with the latter interpretation, the addition of protein synthesis inhibitors prevented the CAMP-induced loss of 117 mRNA (Fig. 8). In the experiment shown, pactamycin was added to cells and, after a 30min incubation, CAMP was added to one half the culture. Also shown are the results obtained when cells which had not been incubated with pactamycin were stimulated with CAMP. In the presence of pactamycin, CAMP treatment did not alter the levels of 117 mRNA. Similar results were obtained when the experiment was performed using cycloheximide to inhibit protein synthesis (data not shown). It would appear that Ax-2 cells were starved for 6 h at which time 125 pg/ml actinomycin D and 250 rg/ml daunomycin (A) or 300 kg/ml nogalamycin (B) were added. After an additional 15 min, 1 mM CAMP was added to one-half of the population (+). RNA levels were determined after 15, 30, 45, and 60 min of incubation with CAMP (lanes 7-10) or without CAMP (lanes 3-6). Lane I shows the level of 117 mRNA prior to the addition of the inhibitors, and lane 2 shows the level after the first 15 min incubation with the drugs. The data are representative of three experiments. Ax-2 amebae were starved for 6 h after which time 300 pg/ml pactamycin was added. After an additional 30 min, 1 mM CAMP was added to one-half of the population. Levels of 117 mRNA were determined by Northern analysis. Shown are the densitometer scans of linear-range autoradiograms. Controls were incubated in the absence of any additions (0); or with CAMP alone (0). Pactamycin (PM) (A); pactamycin + CAMP (A). The data are representative of three experiments. protein synthesis is required for CAMP stimulation to destabilize 117 mRNA. It should be noted that although CAMP stimulation no longer altered the level of 117 mRNA, a decrease in that level was observed over the course of the experiment when cells were incubated with the inhibitors. This may indicate that protein synthesis is also necessary for maintaining 117 mRNA levels.

DISCUSSION
Our previous experiments have shown that cell stimulation with high concentrations of CAMP results in a loss of cell surface CAMP receptor sites (10). Incubation of cells with 0.1 mM CAMP for 30 min results in an approximate 70% loss of receptor binding activity on the cell surface while 1 mM CAMP can induce an almost 90% loss of receptors. The need for such high concentrations of CAMP reflects the activity of the extracellular phosphodiesterase which severely restricts the lifetime of the added CAMP. Much lower concentrations of CAMP effectively induce receptor down-regulation in the phosphodiesterase mutant (37). The mechanism of this downregulation is not clear but may involve the phosphorylation of the CAMP receptor (8,9,38) and the formation of a slowly dissociating form of the receptor (37). Here we have demonstrated that conditions which lead to receptor down-regulation result in decreased levels of 117 mRNA. Stimulation of cells with dibutyryl CAMP, which is a poor agonist for the CAMP receptor but which can permeate cells and activate the intracellular CAMP-dependent protein kinase (12, 34), was not an efficient effector of 117 mRNA levels. In contrast, 2deoxy-CAMP, which has a high affinity for the cell surface receptor and elicits its down-regulation, but binds poorly to the CAMP-dependent protein kinase (34), showed effects similar to those of CAMP.
The above observations would also argue that changes in intracellular CAMP are not necessary for the decrease in 117 mRNA that occurs when cells are incubated with high concentrations of CAMP. Similarly, the increase in 117 mRNA levels by pulsing cells with low concentrations of CAMP was shown not to require changes in intracellular CAMP. Thus, although both of these phenomena are mediated by the CAMP surface receptor, it would appear that receptor coupling to, and subsequent activation of, adenylate cyclase is not the mechanism by which these events occur. Other second messengers like Ca*+, diacylglycerol, or cGMP may be involved in increasing 117 mRNA levels in response to CAMP pulses since such receptor activation appears to be linked also to inositol triphosphate production, changes in Ca*+ concentrations, and guanylate cyclase activation (39, 40). It is not yet known if these changes also accompany the receptor downregulation response that is associated with the decrease of 117 mRNA when cells are incubated with high CAMP concentrations.
Predominantly, those studies have documented changes in gene transcription (15)(16)(17)(18) or enhanced mRNA stability (19). The data presented in this paper describe a different effect of CAMP. They show that events mediated by the CAMP surface receptor result in a destabilization of an mRNA. Three lines of evidence were obtained to support this conclusion: Cells that were incubated with CAMP showed a relatively rapid decrease in their level of 117 mRNA, but retained normal levels of the actin mRNA. The rate of transcription of the 117 gene, as determined from nuclear run-off assays, was not reduced by treating cells with this concentration of CAMP. Also, pulse-chase experiments indi-cated that such treatment lead to an enhanced rate of degradation of the 117 message. It should also be noted that this destabilization of 117 mRNA was observed after approximately 15 min of cell exposure to CAMP. This was observed in both the experiments measuring changes in cellular 117 mRNA levels in response to CAMP stimulation and in the pulse-chase analyses. This period could reflect the time necessary to synthesize a protein(s) that influences 117 mRNA stability. Such an hypothesis is supported by the observation that inhibitors of either RNA or protein synthesis blocked the effect of CAMP stimulation of 117 mRNA levels. Each of the two inhibitors of protein synthesis used in this investigation has a distinct mode of action. Cycloheximide inhibits the elongation step of protein synthesis while pactamycin inhibits the formation of the initiation complex. Consequently, polysomes accumulate or break-down under these respective treatments. Kelly et al. (41) have shown that some mRNAs may be stabilized by cycloheximide but not affected by pactamycin treatment. Since both of these drugs prevented the CAMP-induced decrease in 117 mRNA levels, it is unlikely that such levels reflect the state of polysomes after inhibition of protein synthesis. Taken together, the data support the hypothesis that CAMP stimulation induces the expression of an mRNA-destabilizing protein.
A similar hypothesis has been presented to explain the glucocorticoid-enhanced destabilization of interleukin-lb mRNA in human cell lines and the requirements for protein synthesis for this to occur (42). We have also observed that although CAMP could no longer stimulate 117 mRNA decay in cells incubated with cycloheximide or pactamycin, the rate of loss of the mRNA was faster when these drugs were present (but never as great as with CAMP treatment alone). The meaning of this is as yet unclear but may suggest that ongoing protein synthesis is also required for the stabilization of 117 mRNA. It is a rather unusual finding that the same compound (CAMP) can elicit two opposing processes, depending upon the manner the stimulus is presented to the cells. Low doses, in the form of pulses, result in increased levels of 117 mRNA, a probable reflection of enhanced transcription.
Higher doses of CAMP elicit a rapid loss in 117 mRNA, a result of mRNA destabilization.
This dual regulation of 117 mRNA by CAMP, however, correlates well with the events that accompany the development of the amoebae. During the initial phase of the developmental cycle, 117 mRNA increases as cells produce CAMP pulses and develop aggregation competence. Nuclear run-off experiments indicated that this rise in mRNA levels reflects changes in gene transcription.
When cells terminate their aggregation program and begin to form slugs, 117 mRNA is not detectable (21, 28). During this transition stage, the cell surface levels of the CAMP receptor also decrease (43, 10). It is possible that this decrease reflects the down-regulation of receptors induced by the high external CAMP concentrations in the micro-environment of the receptor (10). Thus, the events we have associated with the destabilization of 117 mRNA, receptor down-regulation and new protein synthesis leading to 117 mRNA degradation, may underlie the loss of 117 mRNA seen at this developmental period. As mentioned earlier, 117 mRNA reaccumulates later in development, during culmination, and then disappears as mature fruiting bodies are formed (20, 21). Although a role for CAMP has not been established for cells at this stage in development, it is intriguing to imagine that 117 mRNA is regulated in a manner similar to that seen during aggregation. Continued investigations should identify the mechanism by which CAMP elicits mRNA degradation, the intermediate gene products involved,