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Molecular Endocrinology, doi:10.1210/me.2005-0386
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Molecular Endocrinology 20 (5): 1112-1120
Copyright © 2006 by The Endocrine Society

Interference with 3',5'-Cyclic Adenosine Monophosphate Response Element Binding Protein Stimulates Apoptosis through Aberrant Cell Cycle Progression and Checkpoint Activation

Jessica H. Dworet and Judy L. Meinkoth

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6061

Address all correspondence and requests for reprints to: Judy L. Meinkoth, Department of Pharmacology, University of Pennsylvania School of Medicine, 420 Curie Boulevard, Philadelphia, Pennsylvania 19104-6061.E-mail: meinkoth{at}pharm.med.upenn.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We previously reported that protein kinase A activity is an important determinant of thyroid cell survival. Given the important role of cAMP response element binding protein (CREB) in mediating the transcriptional effects of protein kinase A, we explored whether interference with CREB family members impaired thyroid cell survival. Expression of A-CREB, a dominant-negative CREB mutant that inhibits CREB DNA binding activity, induced apoptosis in rat thyroid cells. A-CREB inhibited CRE-regulated gene expression but failed to alter the expression of bcl-2 family members or of well-characterized inhibitors of apoptosis. To elucidate the mechanism through which impaired CREB function triggered apoptosis, its effects on cell proliferation were examined. Expression of A-CREB inhibited cell number increases, in part due to delayed cell cycle transit. Protracted S-phase progression in A-CREB-expressing cells was sufficient to activate a checkpoint response characterized by Chk-1, histone H2A.X, and p53 phosphorylation. To determine whether cell cycle progression was required for apoptosis, the effects of p27 overexpression were investigated. Overexpression of p27 prevented cell cycle progression, checkpoint activation, and apoptosis in A-CREB-expressing cells. These data reveal a novel mechanism through which interference with CREB abrogates cell survival, through checkpoint activation secondary to cell cycle delay. This study may explain how interference with CREB induces apoptosis in cells where alterations in the expression of pro- and anti-survival genes are not detected.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
AS AN IMPORTANT regulator of the transcriptional effects of cAMP and growth factors, CREB [cAMP response element (CRE) binding protein] plays critical roles in metabolic regulation, differentiation, and cell survival. CREB activity is essential for neurotropin-mediated survival of sympathetic and cerebellar neurons (1, 2). Mice nullizygous for CREB exhibit excess sensory neuron apoptosis and degeneration (3). Inducible cAMP early repressor (ICER), a dominant-negative CRE modulator (CREM) isoform that potently inhibits CREB function, induces apoptosis in neuronal cells (4). CREB regulates the survival of B (5) and T cells (6). The ability of B cells to survive and proliferate was dramatically reduced in mice expressing dominant-negative M1-CREB (5). CREB also plays an important role in the survival of endocrine cells including ovarian granulosa cells (7) and pancreatic ß-cells (8, 9).

Apoptosis is often accompanied by decreases in CREB activity and/or expression. Neuronal cells dying in response to hypoxic-ischemic episodes showed dramatic losses of CREB expression (10). Reactive oxygen species decreased CREB content in hippocampal neurons (11). The HIV TAT protein down-regulated CREB expression and induced apoptosis in PC12 cells (12). Staurosporine (13) and iron chelators (14) induced caspase-mediated CREB cleavage, whereas hypoxia stimulated the proteasomal turnover of CREB (15). The mechanisms that underlie apoptosis in these cases remain to be determined, although it seems reasonable to assume that apoptosis results, at least in part, from the inactivation of gene expression related to survival secondary to CREB depletion. In support of this idea, expression of CREB-sequestering CRE oligonucleotides was sufficient to induce apoptosis in ovarian cancer cells (16).

One way in which CREB contributes to cell survival is through the regulation of bcl-2 expression. Overexpression of bcl-2 rescued apoptosis induced by impaired CREB function in neurons (2) and IGF-I prevented apoptosis through CREB-dependent up-regulation of bcl-2 expression in PC12 cells (17, 18). Overexpression of CREB increased bcl-2 levels and rescued cytokine-induced apoptosis in ß-cells (8) as well as calcium-mediated apoptosis in immature B cells (19). However, there are a number of instances where interference with CREB induced apoptosis without detectable alterations in bcl-2 family gene expression. No alterations in bcl-2, bcl-x, bax, or bad expression were observed in human melanoma cells expressing K-CREB (a dominant-negative CREB mutant that fails to bind DNA) (20). K-CREB induced apoptosis in 3T3-L1 adipocytes without altering bcl-2 or bax expression (21). Similarly, expression of A-CREB (a dominant-negative CREB mutant that inhibits CREB DNA binding activity) failed to alter bcl-2 expression in ß-cells (9). The mechanism through which impaired CREB function induces apoptosis in these instances is presently unknown.

Protein kinase A (PKA) plays a critical role in thyroid cell survival (22). Given the integral role of CREB as a PKA substrate, we examined whether CREB regulated thyroid cell viability by expressing A-CREB (23, 24), a well-characterized dominant-negative mutant that inhibits CREB DNA binding activity. A-CREB inhibits multiple CREB family members, including CREB, CREM, and ATF-1. Although we refer to A-CREB as an inhibitor of CREB function, its effects could be mediated through any of the CREB family members that serve as positive regulators of CRE-dependent transcription. Expression of A-CREB induced apoptosis in thyroid cells. Although A-CREB inhibited CRE-dependent gene expression, the expression of bcl-2 family members, as well as that of inhibitors of apoptosis, was not affected. The mechanism through which A-CREB-induced apoptosis was explored. These experiments revealed a novel role for CREB in the regulation of cell cycle progression and checkpoint activation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A-CREB Inhibits CRE-Dependent Gene Expression
To investigate the contribution of CREB family members to thyroid cell survival, an adenovirus (Adv) was used to express dominant-negative A-CREB in Wistar rat thyroid (WRT) cells. A-CREB contains the CREB leucine zipper dimerization domain but lacks the basic domain required for DNA binding, which is replaced by an acidic extension. Hence, A-CREB heterodimerizes with CREB family members and prevents their binding to DNA (23, 24). Because the A-CREB Adv expresses green fluorescent protein (GFP) from an internal ribosome entry site, an Adv expressing GFP was used as a control for these experiments. Conditions were derived where more than 90% of the cells were infected and where GFP was expressed at similar levels after infection with A-CREB or GFP Advs (data not shown). These conditions were used for most of the experiments described below. Similar results were obtained using A-CREB and GFP Advs at equal multiplicity of infection (MOI) (data not shown).

Because A-CREB does not react with CREB-directed antibodies, functional assays were employed to derive conditions where A-CREB selectively impaired CRE-regulated gene expression. WRT cells that express integrated lacZ genes under the control of CRE- (CRE cells) or activator protein-1 (AP-1)- (AP-1 cells) regulated promoters (25) were used to titrate the amount of A-CREB required to selectively inhibit CRE-regulated expression. As shown in Fig. 1AGo, infection of A-CREB at MOI 80 markedly impaired CRE-regulated ß-galactosidase expression. Importantly, A-CREB did not inhibit phorbol ester-stimulated ß-galactosidase expression when used at this MOI (Fig. 1BGo), indicating that its effects are not due to squelching. As expected, expression of GFP alone had no effect on ß-galactosidase expression.


Figure 1
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  		Fig. 1. A-CREB Selectively Impairs CRE-Regulated Gene Expression

A, Starved CRE cells (see Materials and Methods) were infected with A-CREB (A/C) or GFP Advs at the MOIs indicated overnight in basal medium, allowed to recover in 3H medium for 6 h, and starved overnight in basal medium. CRE cells were stimulated with TSH (1 mU/ml), IBMX (100 µM) for 6 h. Total cell lysates were prepared and analyzed by Western blotting for ß-galactosidase (ßgal) and GFP expression. The blots were reprobed with actin to confirm equal protein loading. B, Starved AP-1 cells (see Materials and Methods) were infected as described for the CRE cells, above. Expression of ß-galactosidase, GFP, and actin in A-CREB- and GFP-infected AP-1 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) for 6 h is shown. C, WRT cells were infected overnight in basal medium and stimulated with 3H growth medium for 24 h. Total cell lysates were prepared and ICER expression analyzed by Western blotting. Western blotting for ERK2 confirmed equal protein loading.

 
To confirm that A-CREB repressed the expression of endogenous CRE-regulated genes, its effects on ICER expression (26) were analyzed. Stimulation of starved WRT cells with TSH-containing growth medium (3H) increased ICER expression, and this was blocked by A-CREB (Fig. 1CGo). These results demonstrate that A-CREB selectively impairs CRE-dependent gene expression under the conditions used in these studies.

CREB Function Is Required for Thyroid Cell Survival
Inhibition of basal PKA activity induced apoptosis in starved thyroid cells (22). Because CREB is an important PKA substrate, we analyzed whether CREB function was required for thyroid cell survival. Cells were infected with A-CREB or GFP Advs, and stimulated with 3H growth medium for 1, 3, or 4 d. Floating and adherent cells were collected and analyzed by flow cytometry for hypodiploid (sub-G1) DNA content. A-CREB-expressing cells exhibited a time-dependent increase in the proportion of cells with hypodiploid DNA (Fig. 2AGo). To confirm that hypodiploid DNA content reflected apoptotic cell death, effects on procaspase 3 cleavage and DNA laddering, hallmarks of apoptosis, were examined. A-CREB, but not GFP, induced cleavage of procaspase 3 (33 kDa) to its active form (17 kDa) (Fig. 2BGo) and stimulated DNA laddering (Fig. 2CGo). As expected, procaspase 3 cleavage (Fig. 2BGo) and DNA fragmentation (Fig. 2AGo, inset) were blocked by the pan-caspase inhibitor, QVD-OPH (QVD). To ensure that apoptosis induced by A-CREB was not limited to WRT cells, similar experiments were performed in rat thyroid PC-Cl3 cells. All of the effects of A-CREB were reproduced in both thyroid cell lines (data not shown). Collectively, these data demonstrate that expression of A-CREB induces caspase-mediated apoptosis in rat thyroid cells.


Figure 2
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  		Fig. 2. CREB Is Required for Thyroid Cell Survival

A, WRT cells were infected overnight with A-CREB (A/C, MOI 60), GFP (MOI 3), or mock-infected (m) and the cells stimulated with 3H growth medium for the times indicated (days). Cells were harvested and analyzed for DNA content by FACS analysis. Inset shows that pretreatment with QVD (20 µM, 1 h before infection with A-CREB) blocked hypodiploid DNA content in cells harvested at d 4. B, m, GFP (MOI 3) and A/C (MOI 60)-infected cells were stimulated with 3H growth medium, harvested at d 3 after infection and lysates subjected to Western blotting for pro- (procasp3) and cleaved-caspase 3 (cl casp3). QVD (20 µM, added 1 h before infection and upon stimulation with 3H growth medium) blocked procaspase 3 cleavage in A-CREB-infected cells. C, m, GFP (MOI 3), and A/C, MOI 60-infected cells were stimulated with 3H growth medium, harvested at d 2 after infection, and low molecular weight DNA isolated and analyzed for fragmentation by electrophoresis on agarose gels.

 
Interference with CREB Function Stimulates Aberrant Cell Cycle Regulation
In many cells, CREB promotes cell survival through the regulation of bcl-2 expression (1, 2, 8, 11, 17, 18, 19, 27). Unexpectedly, A-CREB did not alter the expression of pro- or antiapoptotic genes, including bcl-2, bax, bcl-XL, bad, bid, xIAP, and c-IAP2 (28, 29) in thyroid cells (data not shown). This suggested that interference with CREB induced apoptosis through another mechanism. CREB plays an important role in the growth and development of the thyroid gland (30) and in thyroid cell proliferation in vitro (31, 32). To determine whether A-CREB impaired cell proliferation in WRT cells, growth curve experiments were performed. A-CREB blocked the increase in cell number stimulated by 3H growth medium (Fig. 3AGo). The effects of A-CREB on cell cycle progression were also analyzed. Preliminary experiments performed in starved cells revealed that A-CREB delayed transit through S phase after stimulation with 3H growth medium (data not shown). To confirm these findings, experiments were performed in synchronized cells. Cells were arrested at the G1/S boundary using the DNA polymerase inhibitor, aphidicolin, and subsequently released into 3H growth medium for different times. Figure 3BGo demonstrates that cells incubated for 24 h in aphidicolin-containing 3H medium were arrested primarily in G1 phase (left panel, aphid), although some cells arrested in G2/M phase (right panel, aphid). Upon removal of aphidicolin and release into 3H medium for 4 h, GFP- and A-CREB-infected cells exited G1 phase (left panel) and entered S phase (middle panel) in a similar manner. At 10 h after aphidicolin release, a greater proportion of GFP-infected vs. A-CREB-infected cells had entered G2/M (right panel). Conversely, more A-CREB-expressing cells remained in S phase at this time (middle panel). These data suggested that A-CREB-expressing cells are delayed in S phase. In support of this idea, when starved cells were stimulated with 3H for 23 h, the proportion of cells undergoing DNA synthesis was consistently higher in A-CREB-infected cells compared with mock- or GFP-infected cells (Fig. 3CGo). Finally, expression of cyclins A and B1, markers of S- and G2/M-phase cells, respectively (33), was prolonged in A-CREB-expressing cells, consistent with the notion that these cells encounter a delay in cell cycle progression (Fig. 3DGo).


Figure 3
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  		Fig. 3. CREB Is Required for Cell Cycle Progression

A, Starved WRT cells were infected overnight [mock (m), GFP (MOI 3) and A-CREB (A/C, MOI 60)], trypsinized and replicate dishes plated at 2 x 105 cells/dish in 3H growth medium. Duplicate dishes were harvested at each time point (d 1–7) and cell number determined using a hemocytometer. Two counts were performed for each dish. Fold-increase over cell number at d 1 is shown. Inset shows GFP expression at d 5 in mock, GFP and A-CREB-infected cells. B, m, GFP (MOI 3), and A/C, MOI 60-infected cells were arrested at the G1/S boundary by incubation in 3H growth medium containing aphidicolin (10 µM) for 24 h (aphid). Cells were washed and released into 3H growth medium in the absence of aphidicolin for 4 and 10 h, harvested and analyzed by flow cytometry for DNA content. % cells in G1, S, and G2/M phases are shown. C, Starved cells were infected overnight and stimulated with 3H growth medium for 23 h. BrdU was added for 3 h (at 20–23 h after stimulation). Percent BrdU-positive cells in GFP-positive (for GFP- and A/C-infected cells) or DAPI-positive (mock-infected cells) cells was determined. D, m, GFP and A/C-infected cells were stimulated with 3H growth medium for 24, 48, and 72 h. Total cell lysates were prepared and subjected to Western blotting for cyclin A, cyclin B1, and GFP. ERK2 Western blotting confirmed equal protein loading. Expression of cyclin B1 was analyzed in two experiments.

 
Stalled replication activates an S-phase checkpoint that is recognized by the checkpoint kinases, ATM (ataxia-telangiectasia mutated protein), ATR (ATM and Rad-3-related protein kinase), and DNA-dependent protein kinase (34, 35). Activation of the S-phase checkpoint serves to inhibit DNA synthesis and to facilitate DNA repair. When DNA damage is extensive, checkpoint activation culminates in apoptosis. Because stalled replication is a robust stimulator of ATR activity, we investigated whether the delay in cell cycle progression in A-CREB-infected cells was sufficient to activate an S-phase checkpoint. Activating phosphorylation of Chk-1 at serine 345, a marker of ATR activity, was increased in A-CREB-expressing cells (Fig. 4AGo). ATM and ATR phosphorylate p53 at serine 15, and phosphorylation at this site was also increased in A-CREB-expressing cells. Although an established marker of DNA double-strand breaks, phosphorylation of histone H2A.X (generating {gamma}-H2A.X) has been observed as a consequence of replication stress in precancerous lesions (36, 37). Similarly, aberrant DNA replication in yeast causes double-strand breaks (38, 39). {gamma}-H2A.X was increased in a time-dependent manner in A-CREB-expressing cells (Fig. 4Go, B and C). To exclude the possibility that checkpoint activation was secondary to the DNA fragmentation that accompanies apoptosis, phosphorylation of Chk-1, p53, and H2A.X was examined in cells pretreated with the caspase inhibitor, QVD. QVD had no effect on Chk-1, p53, or histone H2A.X phosphorylation, documenting that checkpoint activation was not secondary to apoptosis-induced DNA fragmentation. These data indicate that impaired CREB function induces three markers of replication stress, resulting in checkpoint activation.


Figure 4
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  		Fig. 4. Interference with CREB Activates an S Phase Checkpoint

A, Cells were infected overnight and stimulated with 3H growth medium for 1–3 d. Lysates prepared from mock (m), GFP (MOI 3) and A-CREB (A/C, MOI 60)-infected cells were subjected to Western blotting with phospho-Chk-1 serine 345 (Chk-1-S345P) and phospho-p53 serine 15 (p53-S15P) antibodies as described previously (56 ). B, Cells plated on coverslips were infected as described above, stimulated with 3H growth medium for 48 h (d 2) and subjected to immunostaining for serine 139-phosphorylated H2A.X ({gamma}-H2A.X). Where indicated, QVD (20 µM) was added 1 h before infection, and again when the cells were stimulated with 3H growth medium. C, Summary of three time course experiments that examined H2A.X phosphorylation in GFP- and A/C-infected cells. Cells were infected overnight, stimulated with 3H growth medium and analyzed at d 1, 2, and 3 after stimulation. At least 300 cells were scored for each condition.

 
Blockade of S-Phase Entry Prevents Checkpoint Activation and Apoptosis
We reasoned that if impaired CREB function-induced apoptosis through its ability to prolong transit through S phase, then blocking cell cycle progression before S-phase entry should prevent checkpoint activation and apoptosis. To test this hypothesis, an Adv was used to overexpress the cyclin-dependent kinase inhibitor p27, a potent inhibitor of G1/S-phase cell cycle progression. As expected, overexpression of p27 blocked S-phase entry as assessed by DNA content (Fig. 5AGo) and DNA synthesis measured by bromodeoxyuridine (BrdU) incorporation (data not shown). Moreover, phosphorylation of Chk-1, p53, and H2A.X induced by A-CREB was markedly reduced by p27 (Fig. 5BGo; H2A.X data not shown). Strikingly, overexpression of p27 inhibited A-CREB-induced DNA laddering (Fig. 5CGo) and procaspase 3 cleavage (Fig. 5DGo). These data strongly suggest that impaired CREB function induces apoptosis through the induction of replication stress.


Figure 5
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  		Fig. 5. Blockade of Cell Cycle Progression Rescues Apoptosis

A, Starved cells (basal, b) were infected overnight with A-CREB (A/C, MOI 60) in the absence (–) and presence (+) of coinfection with p27 (MOI 300). The cells were stimulated with 3H growth medium for 16 h and harvested for FACS analysis. B, Mock (m, +/– p27, MOI 300), GFP (MOI 3) and A-CREB (A/C, MOI 60, +/– p27 MOI 800)-infected cells were stimulated with 3H growth medium for 48 h, cell lysates prepared and subjected to Western blotting for serine 345-phosphorylated Chk-1, serine 15-phosphorylated p53, and GFP. ERK2 expression was used to document equal protein loading. C. m (+/– p27 MOI 300), GFP (MOI 3), and A/C, MOI 60 +/– p27 MOI 300)-infected cells were stimulated with 3H growth medium for 48 h, DNA isolated and analyzed for laddering. D, Cells infected and treated as in B above were analyzed for the presence of cleaved caspase 3 by immunostaining. Results from two independent experiments are summarized.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
A dominant-negative CREB mutant, A-CREB, was used to inactivate CREB DNA binding activity in rat thyroid epithelial cells. Interference with CREB function stimulated apoptosis, implicating a critical role for one or more CREB family members in the regulation of thyroid cell survival. Thyroid-specific expression of dominant-negative M1-CREB impaired thyroid development (30), an effect believed to reflect the inhibition of follicular cell expansion secondary to effects on proliferation. Our data suggest that apoptosis may contribute to the decrease in follicular cell number seen in these animals.

A-CREB inhibited the expression of an integrated CRE-regulated reporter gene as well as that of ICER, an endogenous CREB-regulated gene. Surprisingly, A-CREB failed to alter the expression of a variety of pro- and antiapoptotic gene products, including members of the bcl-2 family. cAMP induces the expression of only a subset of the large number of genes that are regulated by CREB. Clearly, factors in addition to CREB dictate whether a particular CRE-regulated gene is induced by cAMP in a particular cell type, for example, bcl-2 in thyroid cells. Moreover, although it has been widely held that CREB is constitutively bound to CRE-regulated promoters, more recent data challenge this notion and suggest that CREB binding is regulated (40). Intriguingly, occupancy of the bcl-2 promoter by CREB may be subject to regulation (41). Finally, the expression of CREM {tau} was up-regulated in A-CREB-expressing thyroid cells (data not shown), reminiscent of the situation in CREB-null mice where CREM {tau}, {alpha}, and ß are overexpressed (42, 43). CREM {tau} is a transcriptional activator that binds to the promoters of CREB-responsive genes. This raises the possibility that CREM {tau} restores the expression of at least some CREB-regulated genes, perhaps including bcl-2 in A-CREB-expressing thyroid cells.

In considering mechanisms through which impaired CREB function could abrogate cell survival, we were reminded of the important role ascribed to CREB in the regulation of thyroid cell proliferation (31, 32). Although A-CREB inhibited increases in thyroid cell number measured over days, it failed to induce G1-phase cell cycle arrest after its acute introduction into thyroid cells. Paradoxically, A-CREB increased the number of S-phase cells. Experiments performed in synchronized cells revealed that A-CREB-expressing cells exited G1 phase similar to mock- and GFP-infected cells but were delayed in reaching G2/M, resulting in prolonged transit through S phase. Cycling cells, and in particular S-phase cells, are especially prone to apoptosis (34, 35, 44). Based on this, we assessed whether cell cycle progression was required for apoptosis induced by A-CREB. Several lines of evidence support that this is the case. First, the ability of A-CREB to stimulate apoptosis was correlated with its ability to delay S-phase cell cycle progression. A-CREB prolonged S-phase transit in two lines of rat thyroid cells (WRT and PC-Cl3) but not in a continuous line of rat embryonic fibroblasts, REF52 cells. Unlike thyroid cells, A-CREB failed to stimulate apoptosis in REF52 cells (data not shown). Second, expression of A-CREB resulted in the activation of an S-phase checkpoint. Two downstream markers of ATR activation, phosphorylation of Chk-1 on serine 345 and of p53 on serine 15, were increased by A-CREB. Moreover, Chk-1 and p53 phosphorylation were temporally correlated with the delay in S phase and preceded the appearance of large numbers of dying cells. Inclusion of the caspase inhibitor QVD did not prevent Chk-1 or p53 phosphorylation, results that indicate that checkpoint activation is not secondary to DNA fragmentation in apoptotic cells. Third, and perhaps the most telling, blockade of S-phase entry by overexpression of the CDK inhibitor p27 prevented both checkpoint activation and apoptosis. We interpret this to indicate that p27 rescues apoptosis through its ability to prevent entry into S phase. In support of that hypothesis, we confirmed that p27 did not rescue thyroid cells from cell cycle-independent apoptosis induced by sodium nitroprusside, and that p27 did not affect the ability of A-CREB to impair CRE-regulated gene expression (data not shown). Based on these data, we suggest that impaired CREB function delays progression through S phase, and that this delay is sufficient to activate ATR and induce apoptosis (summarized schematically in Fig. 6Go). Phosphorylation of H2A.X was increased in A-CREB-expressing cells. Although typically a marker of DNA damage associated with double-strand breaks, replication stress stimulated H2A.X phosphorylation in precancerous lesions (36, 37).


Figure 6
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  		Fig. 6. Induction of Apoptosis by CREB Inhibition

Expression of A-CREB delayed transit through S phase. The delay in S phase was sufficient to stimulate ATR activity, as indicated by phosphorylation of Chk-1 at serine 345 and p53 at serine 15. Phosphorylation of H2A.X, a marker of double-strand breaks and replication stress (36 37 57 ), was also increased in A-CREB-expressing cells. Overexpression of p27 blocked entry into S phase, Chk-1, p53, and H2A.X phosphorylation and prevented apoptosis. These results indicate that A-CREB-induced apoptosis is dependent upon S-phase entry, supporting the notion that apoptosis is induced by replication stress.

 
These are not the first data to link impaired CREB function with aberrant effects on the cell cycle. Overexpression of ICER, an endogenous CREB inhibitor, in forskolin-treated pituitary corticotrophs induced S- and G2/M-phase arrest (45). Expression of K-CREB increased the number of 3T3-L1 adipocytes in S and G2/M phases (21). In vascular smooth muscle cells, M1-CREB inhibited the up-regulation of proliferating cell nuclear antigen (PCNA) (46), an S-phase gene whose expression is CREB dependent (47, 48, 49). Expression of dominant-negative M1-CREB or K-CREB decreased the basal activity of the PCNA promoter, reduced PCNA protein levels, and impaired the repair of double-strand breaks, thereby enhancing apoptosis induced by {gamma}-irradiation (50). We were unable to demonstrate effects of A-CREB on PCNA expression in thyroid cells; therefore, the delay in S phase that we observe does not appear to be due to impaired PCNA levels. In addition to PCNA, CREB regulates the expression of several other genes that function in DNA repair, including DNA polymerase ß (51) and BRCA1 (52). Moreover, CREB itself is a target of the DNA damage response. CREB is phosphorylated by ATM in response to {gamma}-irradiation or hydrogen peroxide treatment, stresses that culminate in apoptosis (53). Phosphorylation decreased the interaction of CREB with CREB-binding protein, resulting in impaired transcription of CREB-dependent genes. The observation that CREB levels are decreased in apoptotic cells, together with our finding that impaired CREB function triggers an S-phase checkpoint, suggests the existence of a feed-forward mechanism through which stress further decreases CREB activity, reinforcing the decision of a cell to undergo apoptosis.

In summary, we report that one or more members of the CREB family play critical roles in the regulation of thyroid cell proliferation and survival, processes that are intimately linked. Interference with CREB function prolonged the duration of S phase, activated cell cycle checkpoints, and induced apoptosis (Fig. 6Go). This represents a novel mechanism through which impaired CREB function sensitizes cells to apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents
Insulin, transferrin, crude bovine TSH, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (St. Louis, MO). QVD was from Calbiochem (EMD Biosciences, La Jolla, CA). The A-CREB Adv (CMV-FLAG-A-CREB-GFP) was kindly provided by Dr. M. Montminy (The Salk Institute, La Jolla, CA), the GFP Adv (CMV-GFP) was a generous gift from Dr. A. Zeleznik (University of Pittsburgh, Pittsburgh, PA) and the p27 Adv was generously provided by Dr. P. Seth (Northwestern University, Chicago, IL). Sheep anti-BrdU antibody was from Biodesign International (Saco, ME). Antibodies to procaspase 3, cleaved caspase 3, ERK2, phospho-Chk1 serine 345, phospho-p53 serine 15, and cyclin B1 were from Cell Signaling Technology (Beverly, MA). The ß-galactosidase antibody was from Organon Teknika Corp. (Durham, NC). Actin, GFP, bcl-2, bax, and cyclin A (C-19) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). p27 monoclonal antibody was from BD Biosciences Pharmingen (San Diego, CA). The H2A.X serine 139 antibody was from Upstate (Lake Placid, NY) and the ICER antibody was kindly provided by Dr. C. Molina (New Jersey Medical School, Newark, NJ).

Cell Culture and Viral Infection
WRT and PCCL3 rat thyroid cells were grown in Coon’s modified Ham’s F12 medium supplemented with crude bovine TSH (1 mU/ml), insulin (10 µg/ml), transferrin (5 µg/ml), and 5% calf serum (3H medium) as described previously (54). Cells were infected overnight in Coon’s modified Ham’s F12 medium devoid of growth factors and serum (basal medium). Virus was removed and the cells stimulated with 3H medium for 1–7 d, depending upon the assay. Multiplicities of infection (MOI, expressed as TCID50 per cell) were determined by limiting dilution in 293T cells. Unless otherwise indicated, A-CREB and GFP Advs were used at MOIs where they directed similar levels of GFP expression. Infection with the A-CREB and GFP Advs at equal MOIs yielded identical results. Coinfection of A-CREB with the p27 Adv decreased its expression because adenoviral p27 is expressed from a CRE-regulated promoter. Therefore, higher MOIs of the p27 Adv were often used to drive similar levels of p27 expression when coinfected with A-CREB.

Effects of A-CREB on Gene Expression
WRT cells stably expressing CRE- or AP-1-regulated lacZ genes (25) were propagated in 3H growth medium containing geneticin (150 µg/ml). CRE and AP-1 cells were infected overnight in basal medium, allowed to recover in 3H medium for 6 h to allow transgene expression, and starved in basal medium overnight. CRE cells were stimulated with TSH (1 mU/ml) and IBMX (100 µM) and AP-1 cells with 12-O-tetradecanoylphorbol-13-acetate (100 nM) for 6 h.

DNA Synthesis
DNA synthesis was assessed by BrdU incorporation as previously described (54) except that cells were pulse labeled with BrdU from 20–23 h after stimulation with 3H growth medium.

Flow Cytometry for DNA Content
Floating and adherent cells were collected, fixed and processed for flow cytometry as described previously (22). Cells were analyzed on a Becton Dickinson (Franklin Lakes, NJ) FACScan using CELLquestpro software (Becton Dickinson).

Western Blotting
Cells were lysed in RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate] supplemented with pepstatin, aprotinin, leupeptin, pefablock, and sodium orhovanadate as described previously (55). Antibody incubation was performed as recommended by manufacturer.

DNA Laddering
Cells were disrupted in 10 mM Tris/0.2% Triton-X and incubated on ice for 10 min. An aliquot of total DNA was removed and low molecular weight DNA isolated as described previously (56).

Immunostaining
Immunostaining for cleaved caspase 3 and {gamma}-H2A.X was performed as described in Ref. 56 . Images were captured using a Zeiss axiphot fluorescence microscope (Carl Zeiss, Jena, Germany) fitted with a Hamamatsu ORCA-ER digital camera using axiovision software. Photomicrographs to be compared were imaged for the same times.

Statistics
The data shown in all figures reflect experiments that were performed at least three times with the same results unless otherwise indicated. Data are presented as mean ± SEM.


   FOOTNOTES
 
This work was supported by United States Public Health Service Grants DK45696 and DK55757 (to J.L.M).

J.H.D. and J.L.M have nothing to declare.

First Published Online January 12, 2005

Abbreviations: A-CREB, Dominant-negative CREB mutant that inhibits CREB DNA binding activity; Adv, adenovirus; AP-1, activator protein-1; ATM, ataxia-telangiectasia mutated protein; ATR, ATM and Rad3-related protein kinase; BrdU, bromodeoxyuridine; CRE, cAMP response element; CREB, CRE binding protein; CREM, CRE modulator; GFP, green fluorescent protein; IBMX, 3-isobutyl-1-methylxanthine; ICER, inducible cAMP early repressor; K-CREB, dominant-negative CREB mutant that fails to bind DNA; MOI, multiplicity of infection; PCNA, proliferating cell nuclear antigen; PKA, protein kinase A; QVD, pan-caspase inhibitor, QVD-OPH; WRT, Wistar rat thyroid.

Received for publication September 20, 2005. Accepted for publication January 5, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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