Differential Regulation and Transcriptional Control of Immediate Early Gene Expression in Forskolin-Treated WEHI7.2 Thymoma Cells

Dailing Mao, Elizabeth A. Warner, Susan A. Gurwitch and Diane R. Dowd

E. A. Doisy Department of Biochemistry and Molecular Biology Saint Louis University Health Sciences Center St. Louis, Missouri 63104


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Agents that increase intracellular cAMP are frequently growth inhibitory for lymphocytes and induce apoptosis in cortical thymocytes by regulating gene expression. In the present study, immediate early gene expression was examined in WEHI7.2 thymoma cells undergoing cAMP-mediated apoptosis. Temporal differences in c-fos, junB, and inducible cAMP early repressor (ICER) steady-state mRNA levels were observed after forskolin exposure. Maximal induction of c-fos and junB occurred within 1 h, returning to basal levels by 3.5 h. In contrast, a 1.5-h time lag was observed before ICER transcript levels increased, reaching maximal levels after 3.5 h. This rise in expression, correlating with the decrease in c-fos and junB levels, preceded apoptotic DNA fragmentation by 1.5 h. Transient expression of ICER promoter constructs demonstrated that cAMP responsiveness occurred through cAMP-autoregulatory response element (CARE)3/4, two of the four proposed response elements in the ICER promoter. In contrast to the cAMP-responsive cell line JEG-3, CARE1/2 was not functional for cAMP-activated transcription in WEHI7.2 cells. An observed differential binding pattern of WEHI and JEG nuclear extracts to these elements may account for the cell-specific differences in expression patterns. To determine the role of endogenous ICER in regulating gene expression, cells were treated with two sequential doses of forskolin after which ICER and c-fos mRNA levels were measured. The high levels of cAMP-induced ICER expression dramatically reduced a second induction of c-fos. These data suggest that ICER expression may function as an antioncogene to attenuate the expression of certain protooncogenes, thereby preventing transformation and oncogenesis due to continuous overexpression. Moreover, inhibition of growth-stimulatory genes may be required for the activation of the cell death machinery in specific cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The balance between cellular differentiation, proliferation, and cell death is controlled by complex signaling pathways. These pathways include such diverse molecules as receptors, kinases, and transcription factors interacting in concert to exert an effect on growth and viability. A variety of pathways act in the differentiating thymocyte as the cell undergoes positive and negative immunological selection. Mitogenic agents trigger maturation and differentiation, whereas exposure to glucocorticoids and the E series prostaglandins lead to apoptotic cell death in certain populations of thymocytes. PGE1 and PGE2 signal through a receptor/G protein/adenylate cyclase-coupled pathway. As cAMP levels in the cell rise, the subsequent activation of protein kinase A (PKA) results in the phosphorylation of cytoplasmic and nuclear proteins. One of the nuclear targets is the transcription factor cAMP response element-binding protein (CREB). In the thymocyte cell line WEHI7.2, we have shown that forskolin treatment results in the phosphorylation of the CREB homodimer and subsequent cAMP response element (CRE)-dependent transcription (1). While the activation of cAMP-mediated transcription requires the phosphorylation of CREB, transcriptional attenuation may be due in part to its dephosphorylation (2, 3, 4). A refractory period begins 6–8 h after stimulation of adenylate cyclase, lasts for 3–5 days, and represents a time during which additional stimulation of adenylate cyclase does not result in the activation of transcription. The down-regulation of the c subunit of PKA appears to be responsible for the observed refractory period (5). Thus, three phases have been observed for cAMP-mediated transcription: the activation phase, the attenuation phase, and the refractory phase.

The recent discovery of the cAMP response element modulator (CREM) proteins suggests that the activity of the functional CREB dimer is regulated not only by its phosphorylation state, but also by protein/protein interactions (6, 7). CREM{alpha}, CREMß, CREM{gamma}, and inducible cAMP early repressor (ICER) are alternatively spliced isoforms of the CREM gene, whose protein products function to repress CREB activity either by the formation of a nonfunctional CREB:CREM heterodimer or by competition between CREM and CREB homodimers for binding to a CRE. The ICER isoform utilizes a distinct promoter located in an intron and is itself induced by cAMP (6). The induction of ICER was originally identified in neuroendocrine cells but more recently has been shown to occur during some stages of lymphocyte development (8). The cAMP inducibility of the ICER promoter may be due to the presence of four cAMP-autoregulatory response elements (CAREs) located in two clusters of two elements each (6). Each cluster was shown to exert cAMP inducibility in JEG-3 choriocarcinoma cells and was able to bind the ICER protein in vitro. Moreover, ICER is proposed to down-regulate its own expression and to down-regulate the expression of cAMP-inducible genes. Thus, CREM/ICER may function to attenuate the transcriptional response to cAMP.

The cellular effects of agents that increase cAMP are varied. For example, cAMP stimulates proliferative pathways in some endocrine cells and antiproliferative pathways in many immune cells. Increases in cAMP levels in developing thymocytes block differentiation by blocking cell cycle progression and by inducing apoptosis in a subset of cells (9, 10). Furthermore, cAMP-induced apoptosis in thymocytes is preceded by a G1 block in the cell cycle (11) and proceeds through a mechanism requiring activated gene expression (12, 13). The molecular bases of cAMP cell cycle arrest and apoptosis are poorly understood.

In this study, we examined the effect of forskolin, an activator of adenylate cyclase, on the expression of the immediate early genes c-fos, junB, and ICER in the apoptotic thymocyte. The c-fos and junB protooncogenes have previously been shown to be cAMP-responsive, and each have been implicated in apoptosis induced by a variety of stimuli (14, 15, 16, 17, 18). Moreover, junB expression has been implicated as a negative regulator of cell growth in neuronal cells, fibroblasts, and mammary carcinoma cells (19, 20). ICER regulation by cAMP was detected recently in developing and mature human T lymphocytes, and it was suggested that ICER may function in the circadian control of lymphocyte development and activity (8) similar to its proposed role in the pineal gland (21, 22). Because of the importance of cAMP signaling in the developing thymocyte, it was of considerable interest to determine whether these immediate early genes were induced during cAMP-induced apoptosis. We observed a rapid and transient induction of the c-fos and junB protooncogenes and a delayed induction of ICER expression. The kinetics of ICER expression was similar for the thymoma cells and a neuroendocrine cell line; however, there were qualitative differences that may be explained by thymoma-specific complexes bound to the ICER promoter. Moreover, ICER induction precedes DNA fragmentation in the WEHI7.2 cells and thus may represent an early step in the apoptotic cascade. The induced endogenous ICER expression correlated with the attenuated expression of c-fos and a reduced responsiveness to a second treatment of forskolin. Thus, ICER may function in the attenuation of the cAMP response, in an early transcriptional refractory period, as well as in apoptosis of thymocytes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Rapid Induction of c-fos, junB, and ICER in Forskolin-Treated WEHI7.2 Precedes DNA Fragmentation
The WEHI7.2 murine thymoma cell line was used in this study as a model system for cAMP-mediated activation of gene expression and apoptosis (1, 23, 24). To determine whether the expression of c-fos, junB, and CREM genes is induced by cAMP in the WEHI7.2 cell line, cells were treated with forskolin for various times, and mRNA was analyzed by Northern blot protocols (Fig. 1AGo). An increase in steady-state CREM mRNA levels was detected within 1.5 h. A maximal 10-fold increase in CREM RNA levels was observed at 3.5 h, after which time expression was reduced essentially to basal levels by 24 h. This gradual decline after CREM induction is consistent with negative autoregulation by CREM/ICER on ICER gene transcription (6). c-fos and junB were also induced by forskolin, although the kinetics of induction are significantly different than CREM. c-fos and junB steady-state mRNA levels increased within 20 min and were maximal at 1.5 h. Transcript levels were attenuated to basal levels by 3.5 h and were reduced to below basal levels by 6 h. Thus, the increase in CREM expression correlated with the decrease in c-fos and junB.



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Figure 1. Effect of Forskolin on Expression of Immediate Early Genes and Apoptosis

A, WEHI7.2 cells were incubated with 10 µM forskolin for the indicated time periods. mRNA was isolated and 3 µg were subjected to Northern blot analysis. The resulting bloti was sequentially hybridized with probes for CREM, c-fos, junB, and {gamma}-actin (for normalization). B, WEHI7.2 cells were preincubated with 10 µg/ml cycloheximide for 20 min to block protein synthesis. This was effective at blocking 96% of protein synthesis as analyzed by incorporation of [35S]methionine in newly synthesized proteins (40). Forskolin (10 µM) then was added to the cells and incubated for 20 min or 3 h. mRNA was isolated and analyzed as in panel A. f, Forskolin; c, cycloheximide; c/f, cycloheximide pretreatment followed by forskolin treatment. C, WEHI7.2 cells were incubated with 10 µM forskolin for the indicated time periods. Soluble DNA fragments were extracted and separated by electrophoresis on a 1.5% agarose gel (see Materials and Methods for details).

 
The cAMP-mediated induction of Fos and Jun family members has been shown to be a primary response, independent of protein synthesis. Indeed, the rapid induction of c-fos and junB in the WEHI7.2 cells was not blocked by cycloheximide at levels that inhibited protein synthesis by 96% (Fig. 1BGo). Moreover, in the cycloheximide-treated cells, the return of c-fos and junB mRNA to basal levels was inhibited. Similarly, cycloheximide was ineffective at blocking the induction of CREM transcripts. Three hours of cycloheximide and forskolin cotreatment led to a 26-fold induction of CREM whereas forskolin alone induced CREM expression 10-fold. These data are consistent with the hypothesis that the ICER protein represses its own expression in a negative autoregulatory loop and suggests that CREM/ICER may also function to attenuate the cAMP-induced expression of c-fos and junB.

The relative kinetics of induced gene expression and cell death were compared. Because WEHI7.2 cells excluded vital dyes for up to 16 h after treatment with forskolin (D. Dowd, unpublished observations), we used DNA fragmentation as a marker of apoptosis. During apoptosis, the DNA is cleaved into double-stranded fragments whose lengths are multiples of 180 bp, the length associated with the nucleosome (25, 26). DNA cleavage was observed after 3 h of forskolin treatment, and the concentration of fragments increased over time (Fig. 1CGo). Thus, the induction of CREM/ICER gene expression precedes DNA fragmentation by approximately 1.5 h.

ICER Is the CREM Isoform Induced by Forskolin
The CREM gene is spliced to yield multiple isoforms of the protein, which can differ by the presence or absence of the N terminus and by the DNA-binding domain used (6, 7). Using a full-length CREM{alpha} cDNA probe, we detected induction of four transcripts: 2.3, 1.6, 1.4, and 1.1 kb. To specifically identify which of the CREM products were regulated, we used probes specific for the N terminus and C terminus of CREM (Fig. 2AGo). The 3'-CREM probe, which encodes C-terminal sequences common to all CREM and ICER isoforms, demonstrated an induction pattern virtually identical to the full- length probe with an approximately 10-fold induction in each of the four transcripts. In contrast, the 5'-probe, specific for CREM{alpha}, -ß, -{gamma}, and -{tau} but absent in the ICER isoforms, demonstrated multiple transcripts with no induction of expression of the 2.3-kb band and a 1.6-fold induction of the smaller 1.4- and 1.1-kb species. JEG-3 choriocarcinoma cells treated with forskolin demonstrated only a single transcript hybridizing to the 5'-sequence that was not regulated by cAMP. A pattern of induced transcripts similar to WEHI7.2 was observed when the 3'-ICER-specific probe was used (Fig. 2BGo), consistent with that previously reported (6). It is interesting to note that although the kinetics of induction appear similar in WEHI7.2 and JEG-3, the pattern of transcripts was slightly different. Whereas the level of the 2.3-kb transcript increased 10-fold in WEHI7.2 cells, it represented only a small fraction of the regulated transcripts in the JEG-3 cells.



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Figure 2. The ICER Isoform Is Regulated in WEHI7.2 and JEG-3 Cells

A, WEHI7.2 cells were incubated with 10 µM forskolin for the indicated time periods, mRNA was isolated, and 3 µg were subjected to Northern blot analysis. The blot was probed sequentially with full-length CREM, CREM (3'), and CREM (5'). CREM (3') encodes sequences of CREM corresponding to nucleotides 389–745 of CREM{alpha} cDNA and common to CREM/ICER; the CREM (5') probe corresponds to nucleotides -1 to 353 of CREM{alpha} cDNA, which do not hybridize to ICER isoforms; {gamma}-actin was used for normalization. B, JEG-3 cells were incubated with 10 µM forskolin for the indicated time periods, and mRNA was isolated and analyzed as in panel A. C, RNase protection to determine which DNA-binding domain is encoded by the induced ICER isoform. RNA from WEHI7.2 cells treated with ethanol vehicle (-f) or 10 µM forskolin (+f) for 3 h was hybridized to probe 1 (specific for DBD I, lanes 1–5), probe 2 (to detect DBD II of ICER II or intron/DBD II encoded by ICER I, lanes 6–11), and probe 3 (actin control, lanes 12–15). Lanes 3 and 8 ({alpha}) contain sense RNA produced using CREM{alpha} cDNA and is a control for the presence of DBD I and intron/DBD II. Lane 9 (ß) contains sense RNA produced using CREMß cDNA as a control for DBD II (ICER II) in the absence of DBD I and intronic sequences. Lanes 2, 7, and 13 (tRNA) represent complete digestion of probe in the absence of WEHI7.2 RNA and presence of tRNA. The lengths of the probes and protected fragments are given in nucleotides (nt). The presence of multiple bands with each of the CREM probes corresponds to partial RNase cleavage of protected probe fragments or incomplete digestion in the presence of RNA. Shown are representative experiments.

 
The 3'-probe used for Northern blot analysis encoded both DNA-binding domain (DBD) I and DBD II and thus is capable of detecting isoforms that utilize either of the DBDs. We employed ribonuclease (RNase) protection assays to determine whether the ICER isoform(s) induced in WEHI7.2 cells used DBD I or II, (ICER I and II, respectively; Fig. 2CGo). Probe I was used to detect DBD I, specifically. A DBD I-specific fragment was protected in ethanol-treated cells (lane 4), which comigrated with the protected band resulting from hybridization with CREM{alpha}. This band was induced in cells treated with forskolin (lane 5). These observations were confirmed using probe II, a probe that contained a portion of DBD II and noncoding sequence between DBD I and DBD II. This noncoding sequence is spliced from ICER II, and thus this probe generates two products of different sizes when hybridized to CREM/ICER isoforms encoding DBD I, DBD II, and the intervening sequence (e.g. ICER I, CREM {alpha}; lane 8) or those encoding only DBD II (e.g. ICER II, CREM ß; lane 9). We observed a forskolin-induced increase in the protected band comigrating with DBD I, and no detectable band corresponding to the exclusive use of DBD II (compare lanes 10 and 11). Thus, these data indicate that the ICER isoform induced by forskolin in WEHI7.2 cells utilizes DBD I.

CARE3/4 Directs cAMP-Induced Expression in WEHI7.2 Cells
Because of the observed differences in transcript pattern between WEHI7.2 thymocytes and JEG-3 choriocarcinoma cells (Fig. 2Go, A and B), ICER promoter activity was examined in transient expression studies in these two cell lines. The ICER native promoter, containing approximately 400 bp of 5'- untranslated sequence, was isolated by PCR amplification of genomic DNA and cloned into a CAT reporter vector. This ICER-CAT construct conveyed cAMP- inducible CAT activity in WEHI7.2 and JEG-3 cells (Fig. 3Go). The ICER promoter encodes a cluster of four CRE-like elements (termed cAMP-autoregulatory elements, CAREs) that direct cAMP inducibility in JEG-3 choriocarcinoma cells (6). The 62-bp cluster of four CAREs conferred cAMP inducibility to the thymidine kinase (tk) promoter (27), as did two copies of the CREs from the {alpha}-glycoprotein hormone promoter (1, 27, 28). This CARE1–4 construct was positively responsive to forskolin in both cell lines. The CAREs were further divided into two segments, CARE1/2 and CARE3/4, and tested for cAMP inducibility in WEHI7.2 and JEG-3 cells. CARE1/2 demonstrated cAMP inducibility in JEG-3 cells, in agreement with the observations of Molina et al. (6), but it was ineffective in WEHI7.2 cells. In contrast, CARE3/4 was responsive to forskolin in both JEG-3 and WEHI7.2 cells. However in WEHI7.2 cells, the level of CAT activity measured utilizing the CARE3/4tkCAT construct was 3-fold less than that obtained with CARE1–4tkCAT. This suggests that although CARE1/2 was unresponsive to cAMP, it augments the activation of CARE3/4. CARE3 and CARE4 were examined separately for the ability to induce expression from a CAREtkCAT expression vector. Both CARE3 and CARE4 were capable of sustaining forskolin-activated transcription in WEHI7.2 and JEG-3 cells (data not shown).



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Figure 3. Transcriptional Activation by cAMP in WEHI7.2 and JEG-3 Cells

WEHI7.2 cells or JEG-3 cells were transfected by lipofection with the pCRE(2)tkCAT control plasmid or with ICER-CAT, CARE1–4tkCAT, CARE1/2tkCAT, or CARE3/4tkCAT reporter constructs. (Plasmid construction is detailed in Materials and Methods). Cells were treated with 5 µM forskolin for 16 h and extracts analyzed for CAT activity. Fold induction is reported relative to vehicle-treated cells ± SEM, n >= 2.

 
The sequences of CARE1 (TGAGCTGCA) and CARE2 (TGATGGCA) each are 62% identical to the consensus palindromic CRE, TGACGTCA. In comparison, CARE4 (TGATGTCA) differs by only 1 bp from the palindrome, and CARE3 is a perfect CRE. Thus, we reasoned that the inability of forskolin to activate the CARE1/2tkCAT construct in WEHI7.2 cells may be due to a reduced affinity for binding to CREBs. Gel mobility shift analyses were employed to determine the relative affinity of CREM and CREB for the CARE1/2 and CARE3/4 DNA fragments (Fig. 4Go). Whereas CREB{alpha} and CREM{alpha} bound avidly to the CARE3/4 oligonucleotide (Fig. 4AGo, lanes 9–14), binding of equivalent amounts of extracts to CARE1/2 was greatly reduced (lanes 2–7). Note that at the highest levels of CREB or CREM protein extracts, virtually all of the CARE3/4 probe is bound, whereas only a small fraction of the CARE1/2 probe is bound by these proteins. When CARE3/4 was used as the probe, both the CREB and CREM extracts resulted in two major bands of retarded mobility. These complexes may correspond to the occupancy of one CARE (band I) and two CAREs (band II). CARE1/2 demonstrated only a single major band with the CREB and CREM extracts comigrating with CREB band I and CREM band I, respectively. These data suggest that only one of the two CARE sites is occupied at these protein concentrations.



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Figure 4. Mobility Shift Analysis of ICER Fragments Bound to CREB{alpha} or CREM{alpha}

A, CREB and CREM have higher affinity for binding to CARE3/4 than to CARE1/2. Lanes 1–7, Extracts were incubated with 32P-labeled CARE1/2 probe. Lanes 8–14, Extracts were incubated with 32P-labeled CARE3/4 probe. Lanes 1 and 8, Free probe; lanes 2–4 and 9–11, increasing amounts of CREB{alpha} extract (0.16, 0.32, and 0.64 µg); lanes 5–7 and 12–14, increasing amounts of CREM{alpha} extract (0.2, 0.6, and 1.2 µg). The protein-DNA complexes were resolved from free probe with a nondenaturing polyacrylamide gel and visualized by autoradiography of the dried gel. The filled arrows labeled I and II represent CREB occupancy of one or two CARE sites, respectively; the open arrows labeled I and II represent CREM occupancy of one or two CARE sites, respectively. B, DNA competition for binding to CREB and CREM. CREB extract (0.06 µg; lanes 1–7), or 0.16 µg CREM extracts (lanes 9–15) were preincubated in the absence of DNA-radiolabled probe, with increasing amounts of unlabeled CARE1/2 oligonucleotide (5-, 20-, and 100-fold excess; lanes 2–4 and 10–12) or with increasing amounts of unlabeled CARE3/4 oligonucleotide (5, 20, and 100-fold excess; lanes 5–7 and 13–15). 32P-labeled CARE3/4 probe was then added and incubated for 20 min, and the complexes were analyzed as in panel A. Filled arrows labeled I and II represent CREB occupancy of one or two CARE sites, respectively; open arrow labeled I represents CREM occupancy of one CARE site. At this concentration of extract we observed minimal occupancy of both sites. Lane 8 contains 0.16 µg control COS7 extract. Note that there are no protein-DNA complexes at this concentration of extract when CREM is not expressed.

 
The lower affinity of CARE1/2 compared with CARE3/4 was confirmed in competition assays (Fig. 4BGo). As before, the level of CREB protein used resulted in the appearance of two slowly migrating bands (lane 1), presumably due to the occupancy of one or both CAREs. The amount of CREM extract used in this experiment produced primarily one band, corresponding to the occupancy of one CARE (lane 9, Fig. 4BGo; and lane 12, Fig. 4AGo). A 20-fold excess of unlabeled CARE3/4 was effective at eliminating CREB-specific band II (lane 6) and reducing the CREM-specific band by 50% (lane 14), whereas a 100-fold excess completely eliminated binding of labeled CARE3/4 to extracts containing recombinant CREB or CREM (lanes 7 and 15). A 100-fold excess of CARE1/2 was an ineffective competitor when CREB or CREM protein was bound to CARE3/4 (lanes 4 and 12). When CREB was bound to the CARE3/4 probe, there did appear to be slight reduction in band II with 100-fold excess of CARE1/2. This suggests that there was a small competition effect of the CARE1/2 oligonucleotide reducing the amount of probe in which both CARE sites were occupied.

Both CARE3 and CARE4 were efficient at binding CREB and CREM. Indeed, the binding affinities of CREB and CREM to each of these sequences were similar. Gel mobility shift DNA-binding assays demonstrated that both CARE3 and CARE4 were effective competitors for CREB and CREM binding to a 32P-labeled CARE3 probe (data not shown). The ability of CREB to bind to both CARE3 and CARE4 is consistent with the ability of both of these elements to confer cAMP responsiveness on a heterologous reporter system (Ref. 6 and data not shown).

To further address the question of differential behavior of CARE-driven transcription in WEHI7.2 and JEG-3 cells, we examined the binding of nuclear proteins to the CARE1/2 and CARE3/4 oligonucleotides (Fig. 5Go). Increasing concentrations of nuclear extracts were incubated with the CARE3/4 oligonucleotide and examined by gel mobility shift analysis (Fig. 5AGo). Extracts from both cell lines formed a protein/DNA complex that comigrated with the baculoviral-expressed CREB protein. Its identity as CREB was confirmed with the use of an anti-CREB antibody, which resulted in a supershifted protein/antibody/DNA complex. The WEHI7.2 extract also formed a complex of slower mobility (denoted by the open arrow, lanes 3–5), which was absent in the JEG-3 extract (lanes 7–9). This complex was not supershifted by the anti-CREB antibody. However, the supershifted CREB band comigrated with this unique band, making it difficult to determine whether the antibody adversely affected it.



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Figure 5. Mobility Shift Analysis of CAREs Bound to WEHI7.2 and JEG-3 Nuclear Extracts

A, Differential binding to CARE3/4. Lane 1, 0.03 µg baculoviral-expressed CREB{alpha}; lane 2, 0.03 µg CREB{alpha} incubated with an anti-CREB antibody. Note the supershifted complex formed in the presence of antibody. Lanes 3–5, Increasing concentrations of WEHI7.2 nuclear extract (0.2, 0.6, and 1.0 µg protein, respectively); lane 6, 1.0 µg WEHI7.2 nuclear extract incubated with an anti-CREB antibody; lanes 7–9, increasing concentrations of JEG-3 nuclear extract (0.2, 0.6, and 1.0 µg protein, respectively); lane 6, 1.0 µg JEG-3 nuclear extract incubated with an anti-CREB antibody. The WEHI7.2-specific complex is denoted by an open arrow; CREB is denoted by a filled arrow. B, Differential binding to CARE1/2. Lanes are as in panel A with the exception that lanes 1 and 2 contain 0.15 µg baculoviral-expressed CREB{alpha}. C, DNA competition for binding to nuclear protein complexes. WEHI7.2 nuclear extract (1.0 µg) was preincubated in the absence of DNA-radiolabeled probe, with increasing amounts of unlabeled CARE1/2 oligonucleotide (2.5, 10, and 50-fold excess; lanes 1–3) or with increasing amounts of unlabeled CARE3/4 oligonucleotide (2.5, 10, and 50-fold excess; lanes 5–7). Lane 4 contains WEHI7.2 extract in the absence of competitor DNA. 32P-Labeled CARE1/2 probe was then added and incubated for 20 min and the complexes were separated. The filled arrow represents the complex common between WEHI7.2 and JEG-3 extracts; open arrows designate complexes specific for WEHI7.2, and the asterisk denotes the only complex with which CARE3/4 competes.

 
In addition, there were observed differences in the pattern of complex formation when CARE1/2 was used as the probe (Fig. 5BGo). Both WEHI7.2 and JEG-3 extracts formed complexes that comigrated with baculoviral-expressed CREB (filled arrow), however, these complexes did not interact with the anti-CREB antibody, suggesting that the complexes did not contain CREB. As with the CARE3/4 probe, WEHI7.2 extracts formed two additional protein/DNA complexes, which were absent in the JEG-3 extracts (open arrows). The three complexes formed by the WEHI extracts were competed with unlabeled CARE1/2 oligonucleotide (Fig. 5CGo), but only the fastest migrating complex (denoted by the asterisk) was competed by the CARE3/4 oligonucleotide, suggesting that this protein complex can bind to both CARE1/2 and CARE3/4. In contrast, the remaining two slower migrating bands were unaffected by CARE3/4, suggesting that they are specific for the CARE1/2 oligonucleotide and are not capable of binding to a typical CRE. These data support the conclusion that the protein/CARE1/2 complex that is seen in both WEHI and JEG extracts is not CREB, as CREB binding the CARE1/2 would be competed by the CARE3/4 oligonucleotide.

Induction of c-fos Is Reduced after a Second Treatment of Forskolin
The overexpression of ICER in transient expression studies blocks the cAMP-induced expression of a number of cAMP-responsive promoter constructs (8, 29, 30, 31). Moreover, ectopic expression of the CREM{alpha} transcriptional repressor in WEHI7.2 cells significantly inhibits the responsiveness of a c-fos promoter to cAMP in agreement with data reported earlier by Foulkes et al. (Ref. 32; and data not shown). Although these experiments provide functional information on CREM and ICER bioactivity, they do not address whether such an inhibitory activity occurs in the context of native cellular levels of endogenous ICER. We addressed this question by examining whether the cAMP-induced expression of native ICER altered gene regulation after a second stimulation of adenylate cyclase. Because activation of adenylate cyclase by forskolin does not desensitize the enzyme with respect to subsequent treatments (33, 34), we were able to reactivate adenylate cyclase by removing the first treatment and stimulating a second time with forskolin (see Materials and Methods for details). Forskolin was added to WEHI7.2 cells for 3.5 h to achieve maximal levels of ICER expression. Then, cells were harvested and resuspended in fresh media, and a second treatment of forskolin was added. Gene induction in the presence of increased ICER was then monitored (Fig. 6Go; quantification in Table 1Go). As shown previously, c-fos expression was induced approximately 3-fold at 0.5 h after the initial incubation with forskolin, and the levels of c-fos dropped below basal levels by 3.5 h. In contrast, maximal levels of ICER message were observed at 3.5 h. At 3.5 h, cells were harvested and resuspended in fresh media, and a second treatment with forskolin was immediately initiated. After 0.5 h of additional incubation, c-fos levels were only modestly induced and were not significantly higher than basal levels. This observation could not be explained by a reduction in CREB activation as the CREB protein was phosphorylated after the second treatment to levels similar to the initial treatment (Fig. 6Go and Table 1Go). Thus, induction of endogenous ICER severely impaired c-fos expression.



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Figure 6. Secondary Induction of cAMP-Mediated Gene Expression

WEHI7.2 cells were treated with 10 µM forskolin. After 3.5 h incubation, media was replaced with fresh media and 10 µM forskolin was immediately added. Cells were incubated for an additional 0.5 h. When indicated, a sample of cells was harvested and divided for mRNA isolation or Western blot analysis. An antiphospho-CREB antibody was used to examine CREB phosphorylation. Lane 1, Control; lane 2, forskolin, 0.5 h; lane 3, forskolin 3.5 h; lane 4, forskolin 4 h; lane 5, forskolin 3.5 h + forskolin 0.5 h. Experimental detail is described in Materials and Methods. Quantification of the data is included in Table 1Go. Shown is a representative experiment.

 

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Table 1. Secondary Induction of cAMP-Mediated Gene Expression

 
It is worth noting that the first treatment of forskolin had to be removed before the second incubation. If the second addition of forskolin was placed directly in the initial forskolin-containing media, an effect was not seen, i.e. CREB was not phosphorylated and c-fos mRNA levels were unaffected (data not shown). This is in agreement with earlier observations whereby forskolin-treated membranes were washed before subsequent reactivation of adenylate cyclase by this agent (33, 34). Thus, removal of the initial forskolin treatment was absolutely required to reactivate adenylate cyclase and subsequently rephosphorylate CREB.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We and others have shown that cAMP induces gene expression in thymocytes, and the regulation of gene expression is critical for induction of the apoptotic response. In the present study, we examine the expression of immediate early genes in WEHI7.2 thymoma cells in response to adenylate cyclase stimulation. Forskolin led to the rapid, transient induction of c-fos and junB gene expression. Expression of both genes was maximal within 20 min of stimulation. In contrast, induction of ICER was not apparent for 1.5 h and was maximal at 3.5 h. Although slightly delayed in comparison to c-fos and junB, additional protein synthesis was not required, suggesting a primary transcriptional response typical of immediate early genes. The kinetics of cAMP-mediated induction of ICER were similar for an unrelated cell line, JEG-3, suggesting similar pathways to up-regulation of transcription of the ICER gene.

The ICER protein exists as multiple isoforms. RNase protection assays revealed that the ICER protein(s) induced in WEHI7.2 cells encoded DBD I. Isoforms that lacked DBD I and that used DBD II exclusively were not detected. However, the Northern blot analysis suggests the presence of multiple transcripts; therefore we cannot rule out the possibility that there are additional isoforms that we were unable to distinguish by these experiments.

Although ICER was induced in both WEHI7.2 and JEG-3 cells, there were three differences in the expression and regulation of CREM/ICER between the cell lines. First, 5'-sequences of CREM{alpha} hybridized to multiple transcripts in WEHI7.2 cells whereas only a single transcript was observed for JEG-3 cells. These transcripts were not regulated by forskolin and may represent cell-specific differences in the basal expression of various CREM isoforms. The significance of this finding remains to be elucidated. Second, the levels of the 2.3-kb transcript increased more in the WEHI7.2 cells relative to the JEG-3 cells. The 2.3-kb transcript represented only a minor species in JEG-3 cells treated with forskolin, whereas it was a major isoform in the WEHI cells. Third, regulation in JEG-3 cells occurred through both CARE1/2 and CARE3/4. Moreover, CARE3/4 was sufficient in JEG-3 cells for maximal induction equivalent to CARE1–4. In WEHI7.2 cells, CARE3/4 was able to support cAMP-induced transcription whereas CARE1/2 was nonfunctional in transient transfection assays. Although CARE3/4 conferred cAMP sensitivity to the tk promoter, it was not sufficient for full activity. Other sequences appear to be required for maximal induction. In this regard, CARE1/2 augmented the activity of CARE3/4 to produce the maximal effect. One possible explanation for this observation is that these sequences serve as binding sites for other proteins that are required for maximal activation. Gel mobility shift experiments using nuclear extracts demonstrate the presence of multiple complexes associated with CARE1/2 that are unique to WEHI7.2. Although CARE1/2 does not support cAMP-mediated gene expression in WEHI7.2 cells, it may recruit specific proteins to the area, which leads to the synergistic effect on transcription observed with CARE1–4 in WEHI7.2 cells. The absence of these complexes in JEG-3 cells may explain the lack of synergy of CARE1/2 and CARE3/4 in these cells. This would suggest the presence of cell type-specific factors in thymocytes that function in the regulation of ICER transcription in a manner different from choriocarcinoma cells.

How CARE1/2 functions in cAMP-mediated transcription in JEG-3 cells is currently unclear. CREB from nuclear extracts did not bind to these elements, in agreement with our results demonstrating a low affinity of CREB for these sequences. However, a nuclear protein/DNA complex was formed that was common to both JEG-3 and WEHI extracts. This protein(s) may be responsible for the transcriptional activation observed. The absence of a response in WEHI7.2 cells may be due to the presence of other complexes not observed in JEG-3 extracts that may be inhibiting the response to cAMP. There are more than 12 different CREB/activating transcription factor (ATF) family members that bind to similar CRE-like sequences and may have a higher affinity for these sequences than does CREB. Thus the presence or absence of these may represent an additional level of transcriptional control and specificity. We are currently attempting to identify these factors to determine their potential role in the response to cAMP.

The kinetics of cAMP-mediated induction and attenuation of c-fos transcription in WEHI7.2 cells are typical of immediate early genes: rapid induction followed by transcriptional attenuation and down-regulation. Subsequent to the rapid induction of c-fos, we detected a reduction in mRNA levels to basal levels within 3 h, decreasing below basal levels within 6 h after forskolin treatment. It is proposed that transcriptional attenuation after cAMP induction of the somatostatin gene requires phosphatase activation and dephosphorylation of CREB (2, 3, 4). Although this mechanism is certainly of importance in the attenuation of expression of genes such as somatostatin, its role in the attenuation of c-Fos expression in WEHI7.2 cells is unclear. Significant CREB phosphorylation is detected after 3 h of forskolin treatment (1), a time when c-fos and junB mRNA levels have returned to basal. CREM transcript levels also are increased at 3.5 h after forskolin treatment, suggesting that CREB remains functional at this time point. Thus, it is unlikely that dephosphorylation of CREB plays a predominant role in the attenuation of c-fos expression in WEHI7.2 cells. It has been suggested that c-Fos may act to feedback and negatively regulate its own expression after stimulation with growth factors (35, 36, 37). This attenuation occurs through the palindromic sequence known as the dyad symmetry element, which is also the element that confers phorbol ester and serum responsiveness. Thus, the activation and repression of fos expression by phorbol ester and serum occur through the same DNA element. In comparison, the dyad symmetry element is not responsive to cAMP, whereas the CREs located in the promoter are responsive. Although it is possible that Fos may negatively regulate cAMP-induced transcription, it is unlikely, since these transcription factors act through separate response elements.

Another potential candidate for the transcriptional attenuation is ICER. It is suggested that ICER functions to attenuate the transcriptional response to cAMP (6, 8, 22, 29, 30, 31). In the WEHI7.2 cells, the induction of ICER correlates with the return of c-fos and junB expression to basal levels, suggesting that ICER may be involved in the attenuation phase of the response. Cotreatment of cells with forskolin and cycloheximide prevents the transcriptional attenuation of c-fos and junB and indicates that new protein synthesis is required for the rapid return of these oncogene transcripts to basal levels (reviewed in Ref.38). This is consistent with the idea that one component of the superinduction is the lack of ICER repression.

In addition to transcriptional attenuation, overexpression of ICER in transient reporter assays prevents the induction of a variety of cAMP-responsive genes (8, 29, 30, 31), and expression of the CREM {alpha}, ß and {gamma} transcriptional repressors can inhibit cAMP induction of c-fos in transient transfection assays (Ref. 32 and data not shown). While these studies are paramount in understanding the role for this family of transcriptional repressors, the levels of CREM/ICER in the cells are superphysiological and thus do not represent levels of protein actually occurring in the cell under normal physiological conditions. Thus, we asked whether induced levels of native ICER were sufficient to prevent the regulation of cAMP-induced gene expression at time periods subsequent to transcriptional attenuation of the c-fos gene. WEHI cells were treated with forskolin for 3.5 h, a time period required for induction of ICER and attenuation of c-fos expression. Cells were then treated a second time with forskolin and c-fos levels were measured. Although c-fos levels increased, the relative levels increased only to slightly above basal levels. The reduced induction of c-fos could not be explained by the down-regulation of the cAMP signaling machinery because the CREB protein was phosphorylated after the second treatment to levels similar to the initial treatment. These data indicate that: 1) adenylate cyclase was activated; 2) the phosphatases responsible for attenuating the effects of CREB activation did not prevent its reactivation; and 3) PKA was active; thus the refractory period due to PKA down-regulation had not begun. Thus, other factors appear to be responsible for preventing the relative levels of the c-fos protooncogene from rising above the basal levels upon subsequent activation.

In this regard, the production of transcriptional attenuators/repressors such as ICER may be required for early stages in the rapid down-regulation of cAMP-induced genes and the reduced responsiveness to subsequent activation. The dephosphorylation of CREB may prevent further activation of gene expression when cAMP levels remain elevated. At later time periods, down-regulation of the catalytic subunit of PKA functions to make the cell refractory to stimulation by the same agent. This concerted effort to reduce Fos in the cells after stimulation and keep levels low may be required to prevent oncogenesis. Deregulated expression of c-fos results in neoplastic transformation of rat fibroblasts (reviewed in Ref.38). Thus, ICER may function as an antioncogene by preventing protooncogenes such as c-fos from being expressed at high levels for an extended period of time, a condition supporting neoplasia.

In WEHI7.2 thymoma cells, the induction of ICER immediately precedes the detection of apoptosis-associated DNA cleavage. Thus, ICER may function in the early steps of apoptosis in cells that have an intact death cascade. The growth-promoting effects of protooncogenes might be dominant to death factors and block apoptosis. By down-regulating protooncogenes such as c-fos, ICER may be allowing for the death program to proceed. Moreover, ICER may function in part by inhibiting cell growth by down-regulating cell cycle genes. For example, cyclin A expression and cell cycle progression are induced by cAMP. ICER may function to return the level of cyclin A to its noninduced state and return the cells to the G0/G1 stage of the cell cycle (39). Clearly, the stable expression of ICER sense and antisense cDNA in WEHI7.2 cells will help to address the issue surrounding the possible involvement of ICER in both transcriptional attenuation and the apoptotic cascade, and such studies will be instrumental in enhancing our understanding of cAMP-induced gene expression leading to apoptosis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Forskolin was purchased from Sigma (St. Louis, MO), dissolved in ethanol, and stored at -20 C in the dark.

Cell Culture
WEHI7.2 thymoma cells were grown in DMEM containing 10% calf bovine serum, 0.063 g/liter penicillin, 0.1 g/liter streptomycin. JEG-3 cells were grown in MEM containing 10% calf bovine serum, 0.063 g/liter penicillin, 0.1 g/liter streptomycin. All cells were grown at 37 C in an atmosphere of 6% CO2 and 90% humidity.

Northern Blot Analysis
Northern analysis was performed as described previously (40) with the following modification: mRNA was purified using a PolyATract mRNA isolation system (Promega, Madison, WI), according to manufacturer’s instructions, and 3 µg mRNA were subjected to electrophoresis. Quantification of Northern blots was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

The analysis of gene expression after two treatments with forskolin was performed as follows. Five aliquots of WEHI7.2 cells were pooled and one aliquot (control, 0 h) was removed for Northern and Western analyses. The remaining cells were treated with 10 µM forskolin and aliquots removed for analysis after 0.5 h (+forskolin, 0.5 h) and 3.5 h (+forskolin, 3.5 h). The final two samples were divided, and one sample continued to incubate for an additional 0.5 h (+forskolin, 4 h). The other sample was harvested, and the cells were resuspended in fresh media, 10 µM forskolin was added, and the cells were incubated 0.5 h (+forskolin, 3.5 h; +forskolin, 0.5 h). Individual pools were divided for the analysis of phospho-CREB by Western blot and for mRNA isolation and Northern blot analysis.

DNA Fragmentation
DNA fragmentation was assessed by the methods of Wyllie et al. (26). Briefly, cells were treated with 10 µM forskolin for the indicated time points, harvested by centrifugation at 900 g, and lysed on ice in an excess volume of hypotonic buffer consisting of 5 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 0.5% Triton X-100. The lysates were centrifuged at 13,000 x g, and the supernatant fraction containing soluble DNA fragments were extracted two times with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1). The DNA in the supernatants was precipitated in 66% ethanol and 300 mM sodium acetate, pH 5.3. Precipitates were resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and incubated for 30 min at 25 C with 25 µg/ml RNase. Soluble DNA fragments from equivalent number of cells were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.

RNase Protection
RNA was analyzed according to manufacturer’s directions using the RPA II Ribonuclease Protection Assay Kit (Ambion, Austin, TX). The cDNAs corresponding to CREM probe I [nucleotides 554–653 of the CREM cDNA sequence (7)] and probe II (nucleotides 841-1158) were constructed in pBluescriptKS+ and 32P-labeled probes were made using MAXIscript (Ambion). The mouse ß-actin probe was supplied by Ambion and was used as a control for normalization of RNA concentration. CREM{alpha} and CREMß cDNAs were cloned into pSG5 and unlabeled RNA made using MAXIscript. Briefly 2 µg total RNA were hybridized to a 32P-labeled RNA probe, single-stranded RNA was digested, and the product was separated on a standard urea gel. The gel was dried and products were visualized by autoradiography.

Gel Mobility Shift Analysis
Gel mobility shift analysis used procedures and conditions described previously (41). The CARE1/2 probe consists of the following sense-strand sequence corresponding to positions -154 to -121 in the ICER promoter (6): TTTCAGTGAGCTGCACATTGATGGCAGTGATAGG. The CARE3/4 probe consists of the following sense strand sequence corresponding to positions -120 to -92: CTGGTGACGTCACTGTGATGTCAGTGCTC. CREM{alpha}-containing extracts were obtained by transfecting COS-7 cells with pSG5-CREM{alpha} (7) and preparing nuclear extracts by the method of Shapiro et al. (42). Control COS-7 extracts were obtained in a similar manner from cells transfected with the pSG5 plasmid lacking the cDNA insert. Murine CREB{alpha} cDNA was subcloned into the pVL1392 polyhedrin transfer plasmid for expression in a baculovirus system. Recombinant baculovirus was isolated and plaque purified by standard procedures (43). Sf-9 cells were infected for 48 h with CREB{alpha} recombinant baculovirus. Whole cell extracts were prepared by sonication of infected cell pellets in 10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 0.3 mM zinc acetate, 5 mM dithiothreitol, 0.3 M KCl, followed by centrifugation at 200,000 x g for 30 min. WEHI7.2 and JEG-3 nuclear extracts were prepared by the method of Shapiro et al. (42). Supershift experiments were performed using an anti-CREB antibody (New England Biolabs, Beverly, MA).

Construction of Reporter Plasmids
The ICER promoter from -378 to +1 from the translational start site was isolated by PCR amplification of genomic DNA. DNA primers were designed corresponding to position -378 to -365 and +1 to -13, and standard PCR conditions were employed for amplification of genomic DNA (44). The amplified sequence was subcloned into the ptkCAT plasmid (27) in which the tk promoter was removed. CARE1/2tkCAT and CARE3/4tkCAT were constructed using complimentary DNA oligonucleotides corresponding to base pairs -154 to -121 and -120 to -92, respectively, and subcloned into ptkCAT. The sequence of all constructs was confirmed using a Sequenase kit (USB Corp., Cleveland, OH) according to manufacturer’s recommendation.

Transient Transfection Assay
Logarithmically growing cells were harvested and resuspended in DMEM, 0.5% calf bovine serum at a concentration of 3 x 106 cells/ml. Cells were transfected with the indicated reporter plasmid (2 µg) for 5 h using Lipofectamine reagent (Life Technologies, Gaithersburg, MD), according to manufacturer’s recommendations. Then cells were diluted with DMEM, 10% calf bovine serum to 6 x 105 cells/ml and treated with forskolin or ethanol vehicle for 16 h. Cell extracts were prepared and assayed for CAT activity as described (45).

Western Blot (Immunoblot) Analysis
Western blot analysis with antiphosphorylated CREB was performed as previously described (1). Phosphorylated CREB was quantitated by densitometric analysis of the resulting autoradiogram.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the generosity of Pamela Mellon for the tkCAT and pCRE(2)tk CAT plasmids, Günther Schütz for the CREB{alpha} cDNA, Paolo Sassone-Corsi for the CREM{alpha} and CREMß cDNA vectors, and M. Greenberg for the antiphosphorylated CREB antibody. We would like to thank Paul MacDonald for help with baculovirus-mediated expression of CREB and for helpful discussions. Special thanks go to Darcy Krueger for critical reading of the manuscript and to Monique Heitmeier and Michelle Benoit for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Diane R. Dowd, Ph.D., E.A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, 1402 South Grand Boulevard, St. Louis, Missouri 63104. E-mail: dowddr{at}wpogate.slu.edu

This work was supported by NIH Grant AI-35910 (to D.R.D.).

Received for publication September 9, 1997. Revision received December 12, 1997. Accepted for publication December 30, 1997.


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