Human Endometrial Stromal Cells Express Novel Isoforms of the Transcriptional Modulator CREM and Up-Regulate ICER in the Course of Decidualization

Birgit Gellersen, Rita Kempf and Ralph Telgmann

Institute for Hormone and Fertility Research University of Hamburg 22529 Hamburg, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Decidualization of human endometrial stromal (ES) cells in culture can be triggered by a sustained elevation of intracellular cAMP for several days and is characterized by activation of the cAMP-responsive decidual PRL (dPRL) gene promoter. We investigated the expression of the cAMP response element (CRE) binding protein CREB, and the modulators CREM (cAMP response element modulator) and ICER (inducible cAMP early repressor), in relation to decidualization of ES cells. We isolated all four known ICER isoforms from ES cells, which differ by the presence or absence of the small exon {gamma} and the presence of either DNA-binding domain (DBD) I or II. Of the various CREM isoforms, we cloned six transcript species, all containing DBD I. These were the known repressor CREM-{alpha}, the potential activator CREM-{tau}2{alpha}, and four novel forms whose reading frames were blocked upstream of the DBD. Two of these forms contained a novel exon {Psi}, which is 100 bp in length, resides downstream of the first protein-coding exon of the CREM gene, and introduces an early in-frame stop codon. Surprisingly, in cotransfection assays, all four novel CREM isoforms were potent inhibitors of protein kinase A-stimulated transcription of a reporter gene construct driven by a CRE. By in vitro transcription/translation of all six CREM cDNAs, we demonstrated internal translation initiation at three different methionine residues, giving rise to novel short and very short C-terminal proteins comprising DBD I. These proteins bound to a cAMP response element as homodimers or as heterodimers with each other or with CREB. Immunofluorescence showed nuclear localization of C-terminal CREM proteins expressed from all six CREM cDNAs. Comparison of undifferentiated and decidualized ES cells showed no difference in the level of expression of any of the CREM transcript species. Likewise, CREB was evenly expressed between the two populations. In contrast, ICER transcripts were strongly up-regulated in decidualized ES cells in parallel with the induction of dPRL expression. It appears paradoxical that in vivo, in response to a permanent cAMP stimulus, ICER is up-regulated without displaying negative autoregulation of its own gene or suppression of the dPRL promoter. Elevated ICER levels in decidualized ES cells may be indicative of the presence of overriding amounts of transcriptional activators such as full length CREM-{tau} or CREB which, in turn, upon cAMP-induced phosphorylation, contribute to the induction of the dPRL gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human endometrial stromal (ES) cells undergo a profound transformation into decidualized cells in the late luteal phase of the menstrual cycle. This differentiation process occurs independently of the presence of a blastocyst and prepares the endometrial lining for implantation and pregnancy. The establishment of in vitro decidualization models has allowed identification of hormonal signals that induce the morphological and biochemical alterations leading to the differentiated phenotype (1, 2, 3, 4). Among the typical products of decidualized endometrial cells are laminin, fibronectin, insulin-like growth factor-binding protein-1, and PRL (5, 6, 7). It has become increasingly evident that progesterone alone is a very weak inducer of decidualization in vitro, which needs to be present for at least 6 days (1, 8). A much faster and more pronounced differentiation is achieved by ligands that activate the protein kinase A (PKA)-dependent signal transduction pathway, such as gonadotropins, PGE2, CRF, and relaxin (RLX), or cAMP analogs (9, 10, 11, 12, 13, 14). The relevance of progesterone in vitro lies in a synergistic enhancement of the action of these agents (2, 13). The onset of PRL production serves as a marker of decidualization.

The PRL gene in the human endometrium is transcribed from an alternative promoter located 6 kb upstream of the pituitary PRL promoter (15). This decidual-type PRL (dPRL) promoter is not responsive to progesterone but is cAMP-inducible (16). We have shown previously that ES cells, decidualized by long-term treatment with RLX and progestin, do not desensitize to the persistent cAMP stimulus. The protein for the regulatory subunit of PKA, RI{alpha}, is down-regulated whereas the level of catalytic subunit remains unaltered, resulting in an increase in free catalytic subunit (17). Here we investigated the involvement of downstream mediators of the cAMP signal, i.e. the transcriptional modulators CREM (cAMP response element modulator) and ICER (inducible cAMP early repressor). CREM was identified as a modulator of cAMP response element binding protein (CREB) action (18). CREB binds to cAMP response element (CRE) sequences and activates gene transcription upon phosphorylation (19). CREM can form heterodimers with CREB or compete for the binding site as a homodimer (20, 21, 22). The CREM gene shares regions of high homology with the CREB gene and probably arouse by gene duplication (19, 23). CREM expression is regulated at multiple levels to give rise to both activators and inhibitors of PKA-stimulated transcription. First, numerous isoforms are generated by alternative splicing of exons encoding functional modules (20). These modules include two alternative DNA binding and dimerization domains (DBD I and II), a phosphorylation domain (P box), and two glutamine-rich regions conveying activator function (Q1, Q2). CREM isoforms lacking Q domains are transcriptional repressors, e.g. CREM-{alpha} and CREM-{gamma}, which differs from the former by the absence of the small {gamma}-exon (18). The various CREM-{tau} forms, including either one or both Q domains, are transcriptional activators and are significantly up-regulated during spermatogenesis (22, 24). At a second level, alternative translation initiation on a CREM-{tau} transcript can give rise to the shorter inhibitory form S-CREM, as has been demonstrated in the mouse brain (25). Finally, complexity is added to CREM expression by the use of an alternative cAMP-inducible intronic promoter (P2) upstream of the DBDs to generate the inducible early repressor ICER (26) (Fig. 1Go). P2 carries a cluster of four CREs that mediate cAMP induction of ICER expression. ICER protein consists of no other functional domain than a DBD and acts as a repressor on cAMP-responsive promoters including its own promoter, thereby establishing a negative autoregulatory loop (27). This mechanism has been implicated in the circadian rhythmicity of cAMP responsiveness in the neuroendocrine system (26).



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Figure 1. Schematic Representation of Modules Composing CREM and ICER Transcripts

Transcripts are generated by alternative splicing of modules B to J (CREM) or A to J (ICER). Module E is composed of two exons, whereas I and J are located on one exon. ICER is transcribed from an alternative intronic promoter. Functional domains are two glutamine-rich transactivating regions (Q1, Q2), the phosphorylation domain (P box), and two alternative dimerization and DNA binding domains (DBD I and DBD II). The major translational start codons (ATG) are indicated by broken arrows; termination codons (STOP) are indicated by vertical arrows. An internal start site downstream of the P box has been described for murine CREM (20, 25, 26). Positions and orientations of module-specific oligonucleotide primers are shown by horizontal arrows.

 
We used an in vitro model of decidualization in which human ES cells are either maintained undifferentiated in the presence of 17ß-estradiol (E2) alone, induced to decidualize by the addition of medroxyprogesterone acetate (MPA) to achieve a low level of dPRL expression, or by the addition of MPA and RLX to obtain a high level of dPRL expression. The expression of CREB, CREM, and ICER isoforms was compared under these different conditions. Novel CREM transcripts were identified and found to encode previously undescribed C-terminal proteins that bind DNA and inhibit PKA-stimulated gene transcription. CREB and CREM transcript levels did not change in the course of decidualization whereas ICER was strongly up-regulated in decidualized cells. The persistent stimulation of the cells by RLX, leading to a permanently elevated intracellular cAMP level, apparently elicits events that override the negative autoregulation of the ICER promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RT-PCR Analysis of dPRL, CREB, CREM, and ICER Expression in Human ES Cells
To determine the level of dPRL and CREB expression, RT-PCR was performed on RNA isolated from undifferentiated ES cells (-/-) and cells maintained in the presence of MPA (+/-) or in the presence of MPA and RLX (+/+) (Fig. 2Go). Decidual-type PRL transcripts were strongly up-regulated in decidualized cells in response to MPA plus RLX, weakly expressed in +/- cells in response to MPA alone, and barely detectable in undifferentiated cells (Fig. 2AGo). In contrast, CREB was evenly expressed under all three culture conditions. Amplification of CREB mRNA using primers anchored in exons B and H, upstream of the DBD sequence, yielded a single product of the predicted size for CREB-327 (28) (Fig. 2BGo).



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Figure 2. RT-PCR Analysis of dPRL, CREB, CREM, and ICER Expression in Human ES Cells

ES cells were cultured for 12 days in the absence of MPA and RLX (-/-), in the presence of MPA (+/-) or in the presence of MPA and RLX (+/+) before preparation of cDNA. PCR products were subjected to Southern hybridization. A, dPRL transcripts were amplified using primers located in the 5'- and 3'-UTRs and hybridized with an internal oligonucleotide. B, CREB transcripts were amplified with primers located in exons B and H upstream of the DBD and hybridized with an exon C-specific probe (28). C and D, Amplification of ICER (C) and CREM (D) transcripts was performed with the indicated primer pairs (for location of primers, see Fig. 1Go). The resultant Southern blots were successively hybridized with the indicated module-specific probes (see Fig. 1Go). Positions of markers are indicated in kilobases (kb) on the left.

 
When we amplified ICER transcripts using the primer pairs A-H and A-J (see Fig. 1Go), various isoforms were detected. In contrast to the even expression of CREB, ICER transcripts were strongly up-regulated in decidualized +/+ cells, which also displayed the highest level of dPRL expression (Fig. 2CGo).

The function of CREM isoforms is dictated by their modular composition as a consequence of alternative splicing. We wanted to determine which isoforms were expressed in human ES cells. Amplification of the 5'-portion of CREM, upstream of the two alternative DBDs, with primer pair C-H (see Fig. 1Go) yielded multiple products, indicating that alternative splicing involving modules D to G takes place (Fig. 2DGo). Amplification of the entire sequences using primer pair C-J indicated that both DBDs are used, because isoforms expressing DBD II are 399 bp shorter (lacking module I) than those expressing DBD I. The latter forms include module I, leading to translation termination at the stop codon immediately after DBD I. Hybridizations with module-specific probes revealed no change in the level of expression of any of the CREM isoforms between undifferentiated and differentiated cells (Fig. 2DGo). Probe H visualized all isoforms. Probe G detected a subpopulation that includes exon {gamma} and therefore is 36 bp larger than the corresponding forms lacking this exon. Hybridization with the module-specific probes E and F revealed the presence of the P box or the Q2 domain in some, but not all, isoforms. The Q1 domain was not present in any of the transcripts inasmuch as probe D yielded no signal (not shown).

Cloning of CREM- and ICER-Isoforms from Human ES Cells
The size of some CREM isoforms was not compatible with the sum of any of the combinations of the known modules. We therefore performed PCR with the primers B and J anchored in the 5'- and 3'-untranslated region (UTR) of CREM, respectively, to obtain full-length coding regions (Fig. 3Go). The resultant pattern was similar to that shown for primer pair C-J in Fig. 2DGo. Products between 0.8 and 1.3 kb in size were isolated and cloned. Six different populations of clones were obtained based on size and restriction mapping, all including DBD I but none containing exon {gamma} (Fig. 3Go). Clone CREM-22 represented the known inhibitor CREM-{alpha} whereas all other forms have not yet been described. CREM-10 is a potential activator as it includes P box and Q2 domain but, in contrast to the established CREM-{tau} forms, it contains the first of the two DBDs and was therefore termed CREM-{tau}2{alpha}. CREM-17 lacks Q domains and P box (CREM-{Delta}1,P, 2). In general, the absence or presence of the Q domains or of exon {gamma} does not affect the open reading frame (ORF), since their nucleotide numbers represent multiples of 3. However, the presence of the P box (module E; 241 nucleotides) is required because it contributes one additional nucleotide to complement the incomplete codon -CT at the 5'-end of module H. The alternative splicing, leading to omission of the P box (module E) in CREM-17, was therefore expected to interrupt the ORF. Sequencing of the splice junction revealed an additional irregularity due to a missing nucleotide at the 3'-end of module C (Fig. 4Go). However, the ORF remains disrupted because an in-frame stop codon is encountered soon after entry into module H. Likewise, in the isoform CREM-20, which is lacking Q1 and P box (CREM-{Delta}1,P), a stop codon appears immediately after entry into Q2 (module F) (Fig. 3Go).



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Figure 3. CREM Transcripts Cloned from Human ES Cells

Top, All known modules composing CREM transcripts are shown. Primers B and J were used to amplify full-length CREM cDNAs from human ES cells by RT-PCR. Below, Six different isoforms were isolated, which will be referred to by their clone numbers (given in brackets; CREM-22, -10, -17, -20, -23, -24). Clone CREM-22 represents the known inhibitor CREM-{alpha} originally isolated from mouse (18); the remaining clones are novel isoforms. All clones contained module I. This leads to inclusion of DBD I in the ORF of CREM-22 and CREM-10 and utilization of the stop codon immediately downstream of DBD I in module I. A novel module {Psi} was identified in CREM-23 and CREM-24, introducing an early in-frame stop codon (see also Fig. 5AGo). Clones CREM-17 and CREM-20 also contain in-frame stop codons due to out-of-frame splicing of modules H or F, respectively, onto module C (see also Fig. 4Go). Locations of primers used for further analysis of module {Psi} are indicated at the bottom.

 


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Figure 4. Sequence of Splice Junction between Modules C and H in CREM-17

Top, In CREM isoforms with a full-length ORF (such as CREM-{alpha}, -ß, -{gamma}, -{tau}) (18, 24, 41), one or several exons are inserted between modules C and H such that a multiple of 3 plus one extra nucleotide [(XXX)nX] maintain the downstream ORF. Bottom, In clone CREM-17 (CREM-{Delta}1,P, 2), module H, lacking one nucleotide, is spliced directly to module C, the proper spacing is disrupted, and an in-frame stop codon is encountered soon after entry into module H.

 
Finally, two isoforms were found that contained a novel sequence, module {Psi}, inserted between modules C and F (clone CREM-23) or between C and E (clone CREM-24). Module {Psi} is 100 bp in length and also introduces an in-frame stop codon (Fig. 5AGo). Hybridization of a Southern blot containing CREM cDNAs amplified with primer pairs C-H or C-J (as shown in Fig. 2DGo) with a {Psi}-specific probe revealed the presence of this module in at least three different CREM isoforms in human ES cells (Fig. 5BGo). Expression of module {Psi} is, however, not restricted to ES cells; RT-PCR screening of a variety of human tissues revealed its presence in CREM transcripts from decidua, placenta, HeLa cells, B- and T-cell lines (DAUDI, Jurkat), peripheral blood lymphocytes, and thymus. It was not detected, however, in pituitary (not shown). The position of module {Psi} in CREM-23 and -24 indicated that it might reside either between modules C and D or D and E on the CREM gene. We performed genomic PCR with module-specific primers to span the genomic region between C and E, followed by hybridization with a {Psi}-specific primer. This analysis located module {Psi} approximately 4 kb downstream of module C (Fig. 5CGo). Its position between the first protein-coding exon and the first Q domain resembles that of exon {Psi} of the CREB gene (29). We termed these novel CREM isoforms CREM-{Psi}{Delta}1,P and CREM-{Psi}{Delta}1,2. Exon {Psi} of the CREB gene also introduces an in-frame stop codon. It is longer (175 bp), however, and has no sequence homology with the exon {Psi} identified by us in the human CREM gene.



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Figure 5. Sequence and Localization of the Novel Exon {Psi} in the CREM Gene

A, Exon {Psi} is 100 bp in length and carries an early in-frame stop codon (TAA). Additional stop codons are present in the other reading frames. B, The same Southern blot as shown in Fig. 2DGo was hybridized with oligonucleotide {Psi}' to demonstrate presence of module {Psi} in CREM transcripts from human ES cells. Cells had been cultured for 12 days in the absence of MPA and RLX (-/-), in the presence of MPA (+/-), or in the presence of MPA and RLX (+/+). C, Genomic PCR was performed to localize the novel sequence in the CREM gene. Primer pairs B-D' or C-E were employed for long range PCR on human genomic DNA, followed by nested PCR (indicated by the curved arrows) using primer pairs C-{Psi}" or D-{Psi}", respectively. The Southern blot of the PCR products was hybridized with the oligonucleotide {Psi}' (for location of primers, see Fig. 3Go). Positions of markers (in kilobases) are indicated on the left. The minor band at about 1.5 kb in lane 2 was not visible on the ethidium bromide-stained gel and probably represents a single-stranded product whereas the major product of 4 kb was clearly visible on the gel.

 
Cloning of ICER cDNAs, amplified with primer pair A-J, revealed the presence of all four known isoforms, ICER-I, -I{gamma}, -II, and II-{gamma}, which differ by the absence or presence of exon {gamma} and by the presence of either DBD I or II (27) (Fig. 6Go).



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Figure 6. Cloning of ICER Isoforms from Human ES Cells

Top, Modules composing ICER transcript isoforms and positions of primers used for RT-PCR amplification of ICER transcripts from cultured human ES cells are shown. Below, All four known ICER isoforms were isolated (27, 44). The {gamma}-isoforms are lacking the small exon {gamma}. In the ICER-II forms module I is absent, leading to expression of DBD II and utilization of the second stop codon.

 
Transcriptional Modulation by CREM- and ICER-Isoforms
Four of the six CREM isoforms isolated from ES cells included early in-frame stop codons. We wished to determine whether these isoforms encoded functional protein. Internal translation initiation has been described for the transcriptional activator CREM-{tau} in mouse brain (25). An alternative start codon 3' to the P box (module E in our scheme, see Fig. 1Go) is used in addition to the first AUG to give rise to the short form S-CREM, which is a transcriptional repressor. Internal translation initiation downstream of the short 5'-ORF would also have to operate on the human isoforms CREM-17, -20, -23, and -24 to give rise to proteins that include DBD I and would be predicted to act as repressors. We generated eukaryotic expression vectors of all CREM- and ICER-isoforms isolated from ES cells to assess their effect on PKA-stimulated gene expression. Transient transfection assays were performed in primary cultures of undifferentiated ES cells and in the uterine sarcoma cell line SKUT-1B (Fig. 7Go). Reporter gene constructs were either a classical CRE element, linked to a minimal promoter and the luciferase reporter gene (pCRE/-36rPRL/luc3), or various portions of the dPRL promoter that we have shown to be cAMP-responsive in a delayed fashion (17).



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Figure 7. Transcriptional Modulatory Function of CREM and ICER Isoforms

Transient transfection assays were performed in primary cultures of human ES cells (A, B, and C) and the SKUT-1B cell line (D, E, and F). The expression vectors pRc/CREM-10, pRc/CREM-17, pRc/CREM-20, pRc/CREM-23, pRc/CREM-24, pRc/ICER-I, pRc/ICER-II, or empty vector pRc/CMV (-) were cotransfected with PKA expression vector pRSV-Cß (crosshatched bars) or the inactive mutant pRSV-Cßmutant (controls, open bars). Cyclic AMP-responsive reporter gene constructs were: pCRE/-36rPRL/luc3 (A and D), dPRL-332/luc3 (B and E) or dPRL-3000/luc (C and F).

 
ICER-I and -II inhibited PKA-stimulated activity of both the CRE control construct and the dPRL promoter. Expression vectors for all CREM isoforms with the exception of CREM-10 were equally efficient in repressing PKA-induced promoter activity. CREM-10 displayed variable function depending on promoter context and cell type. It repressed the CRE construct in SKUT-1B cells while having no effect on this construct in ES cells. The short dPRL promoter fusion (dPRL-332/luc3) was stimulated in both cell types whereas the long dPRL fragment (dPRL-3000/luc) was inhibited (Fig. 7Go). The weak or absent induction of dPRL-332/luc3 by PKA alone within the time frame of this experiment is due to the delayed kinetics of cAMP responsiveness of this region of the dPRL promoter (17).

None of the constructs affected basal activity of the reporter constructs in the absence of PKA expression vector (not shown), suggesting a function linked to PKA-induced nuclear events. To further exclude that the CREM expression vectors, particularly those carrying in-frame stop codons upstream of the DBD (pRc/CREM-17, -20, -23, -24), inhibited production of reporter protein nonspecifically, we tested these constructs in a different, PKA-independent system. SKUT-1B cells were transfected with the Pit-1 responsive reporter gene construct mPit-0.3/luc, which carries 0.3 kb 5'-flanking DNA of the mouse Pit-1 gene linked to the luciferase reporter gene (30). Cotransfection of a Pit-1 expression vector, pCMV-hPit1 (30), caused a 4-fold induction of reporter gene activity, which was not altered by introduction of any of the CREM or ICER expression vectors (data not shown).

Novel CREM Protein Isoforms Are Generated by Internal Translation Initiation
The ambiguity in the function of overexpressed CREM-10 indicated that both negative and positive modulators were translated from the transcript, which could be explained by translation initiation both at the first (or closely adjacent second) AUG in module C (see Fig. 8CGo, methionine residues Met 1 and Met 2) and at a downstream AUG. The same alternative start codon that gives rise to S-CREM from the murine CREM-{tau} mRNA is conserved in the human CREM sequence in module E (adjacent to the P box within a Kozak context, ACC AUG G) (31) (Fig. 8CGo, Met 3). The fact that CREM-17, -20, -23, and -24 were efficient inhibitors of PKA-stimulated gene transcription suggested that functional protein was translated from these transcripts despite the short 5'-ORF. Several in-frame AUG codons are present in these transcripts downstream of the stop codons indicated in Fig. 3Go. The AUGs shown in Fig. 8CGo in module F (Met 4) and at the 5'- and 3'-ends of module H (Met 5, Met 7) are in a rather poor initiation context, whereas the Met 6 in module H (GUG AUG G) complies with the Kozak rules.



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Figure 8. CREM Transcripts Encode Multiple Proteins Arising from Alternative Translation Initiation

A, The constructs CREM-10/FLAG, CREM-17/FLAG, CREM-20/FLAG, CREM-22/FLAG, CREM-23/FLAG, CREM-24/FLAG, and the empty vector pRc/CMV were subjected to in vitro transcription/translation in the presence of [35S]methionine. Products were fractionated on a 3–27% SDS polyacrylamide gel and autoradiographed. Sizes of major products are indicated in kilodaltons. A potential 9-kDa product translated from CREM-17 (marked in brackets) is not visible as it comigrated with the reticulocyte lysate pigment. B, An aliquot of the reaction shown in panel A was immunoprecipitated with FLAG antiserum and resolved on a 10–20% SDS polyacrylamide gel. The 9-kDa band resulting from translation of CREM-17/FLAG is now clearly visible. C (top), The modules of CREM-{alpha} transcript isoforms (those encoding DBD I) are depicted with AUG codons indicated by arrowheads. Methionine codons embedded in a sequence complying with the rules of Kozak (31) are shown above the modules (Met 2, Met 3, Met 6); methionine codons in a less favorable context are shown below the modules (Met 1, Met 4, Met 5, Met 7). C (bottom), Deduced modular compositions of the C-terminal proteins obtained by in vitro transcription/translation of the CREM/FLAG constructs are shown. Approximate sizes of proteins are given in kilodaltons and are derived from the migration of the isoforms on the gels shown in panels A and B and from the number of protein-coding nucleotides indicated above the modules. The flag symbol represents the FLAG sequence incorporated at the 3'-end of the DBD I for immunological detection. The apparent size of SS-CREM-{tau}2{alpha} translated from CREM-23/FLAG is approximately 2.3 kDa larger than the 16 kDa SS-CREM-{tau}2{alpha} obtained with CREM-10/FLAG and CREM-20/FLAG (see also panels A and B) due to an artefactual shift of the termination codon from the 3'-end of the FLAG sequence into the vector polylinker.

 
To determine whether shorter, carboxy-terminal proteins were translated from downstream AUGs, we inserted the FLAG peptide sequence C-terminally to DBD I in all CREM expression vectors including the known inhibitor CREM-22 (CREM-{alpha}). In vitro transcription/translation of these constructs yielded protein products ranging from 13–32.5 kDa in size (Fig. 8Go). A potential 9-kDa translation product generated by initiation at Met 6 was not visible, however, because the reticulocyte lysate pigment migrated in this region. Subsequent immunoprecipitation with FLAG antiserum showed that proteins encompassing the DBD I were translated from all constructs. CREM-10 yielded the full-length 32.5-kDa CREM-{tau}2{alpha} and two N-terminally truncated forms, a short (20 kDa) S-CREM-{tau}2{alpha} originating from Met 3 in module E and a yet shorter (16 kDa) SS-CREM-{tau}2{alpha} starting at Met 4 in module F. This same SS-CREM-{tau}2{alpha} was also generated in reticulocyte lysates programmed with CREM-20 and CREM-23. (SS-CREM-{tau}2{alpha} generated from CREM-23 migrated at a higher molecular mass compared to CREM-20 because during PCR-based construction of the CREM-23/FLAG fusion, the stop codon immediately 3' to the FLAG sequence was lost. This leads to read-through into the vector until a stop codon is encountered 20 triplets further downstream, resulting in additional 2.3 kDa of protein). CREM-22 yielded the known CREM-{alpha} protein of approximately 25 kDa but also a prominent shorter form S-CREM-{alpha} (13 kDa) again due to translation initiation at Met 3. S-CREM-{alpha} was also produced from CREM-24. The shortest form, a 9-kDa SS-CREM-{alpha} protein consisting of merely DBD I, was encoded by CREM-17 and likely results from initiation at Met 6 in module H. A trace of SS-CREM-{alpha} was also detectable in lysates programmed with CREM-20 (Fig. 8Go). In summary, all constructs gave rise to CREM isoforms, which are devoid of the P box but include the DBD I and are presumably transcriptional inhibitors.

CREM, S-CREM, and SS-CREM Isoforms Homo- and Heterodimerize and Bind to a CRE
The FLAG-tagged proteins obtained by in vitro transcription/translation as described above were used for electrophoretic mobility shift assays (EMSA) on a consensus CRE probe (Fig. 9Go). Two retarded complexes, which were competed by excess unlabeled CRE but were not affected by the addition of FLAG antiserum, were seen with translation product from the empty vector (pRc/CMV) and represent CREBs endogenous to the transcription/translation cocktail. All preparations resulting from CREM/FLAG cDNAs yielded additional complexes that were quantitatively supershifted by FLAG antiserum. CREM-10 yielded CREM-{tau}2{alpha} and S-CREM-{tau}2{alpha} homodimers and an intermediate heterodimer. CREM-17 generated SS-CREM-{alpha} homodimer. CREM-20 gave rise to a prominent SS-CREM-{tau}2{alpha} homodimer and a second smaller complex probably resulting from heterodimerization with a limited amount of SS-CREM-{alpha}. CREM-22 generated CREM-{alpha} and S-CREM-{alpha} homodimers and an intermediate heterodimer. SS-CREM-{tau}2{alpha} homodimer was obtained with CREM-23; this complex caused a higher retardation than that of CREM-20-based SS-CREM-{tau}2{alpha} homodimer, again due to the C-terminally shifted stop codon in CREM-23/FLAG. Finally, with CREM-24, only S-CREM-{alpha} homodimer was formed.



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Figure 9. Novel CREM Protein Isoforms Bind to a CRE

In vitro transcribed/translated products from the constructs CREM-10/FLAG, CREM-17/FLAG, CREM-20/FLAG, CREM-22/FLAG, CREM-23/FLAG, CREM-24/FLAG, and empty vector (pRc/CMV) were incubated with a 32P-labeled CRE consensus probe. Complexes were supershifted by the addition of FLAG antiserum ({alpha}-FLAG). A 100-fold molar excess of unlabeled CRE was used as competitor. The large asterisk indicates migration of free probe; the small asterisks indicate two faintly visible bands resulting from CRE-binding proteins endogenous to the reticulocyte lysate cocktail. The large arrow marks supershifted complexes. The intermediate complexes in lanes 4 and 13 represent heterodimers between CREM-{tau}2{alpha} and S-CREM-{tau}2{alpha} (obtained with CREM-10/FLAG) or between CREM-{alpha} and S-CREM-{alpha} (obtained with CREM-22/FLAG), respectively. The faster migrating complex in lane 10 (from lysates programmed with CREM-20/FLAG) is most likely due to heterodimerization between the major product SS-CREM-{tau}2{alpha} and a limited amount of the minor product SS-CREM-{alpha} (see also Fig. 8Go), which is not visible as a homodimeric complex. As already pointed out in the legend to Fig. 8Go, CREM-23/FLAG encodes a protein with additional 2.3 kDa C-terminal sequence derived from the cloning vector (SS-CREM-{tau}2{alpha}*). The complex due to binding of this protein (lane 16) therefore migrates differently from the SS-CREM-{tau}2{alpha} complex obtained with CREM-20/FLAG (lane 10).

 
The relative electrophoretic mobilities of all the homodimer/DNA adducts are in agreement with the relative apparent sizes of the six CREM protein isoforms depicted in Fig. 8Go, and heterodimeric adducts are observed in those cases in which several proteins were translated from one cDNA template. Therefore, CREM, S-CREM, and SS-CREM proteins are able to homodimerize, heterodimerize, and bind to DNA.

To examine whether short CREM isoforms also heterodimerized with CREB, we produced FLAG-tagged S-CREM-{alpha} protein by in vitro transcription/translation of CREM-24/FLAG. Upon EMSA, this protein forms a single complex with labeled CRE, as already shown in Fig. 9Go. We then added the DBD of CREB (human CREB bZIP, amino acids 254–327, ~10 kDa), which results in a faster migrating complex. Coincubation of S-CREM-{alpha} and CREB led to the formation of an intermediate complex that represents a CREM/CREB heterodimer because this complex and the S-CREM-{alpha} homodimeric complex were supershifted by FLAG antibody, whereas CREB homodimer remained unaffected (Fig. 10Go).



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Figure 10. S-CREM-{alpha} Heterodimerizes with CREB

The same in vitro translation product generated from CREM-24/FLAG as shown in Fig. 9Go was used for heterodimerization studies with the DBD of CREB (CREB-bZIP) on a 32P-labeled CRE consensus probe. When CREB bZIP protein was added in the absence of CREM-24/FLAG-programmed lysate (lanes 3, 4, 7, 8), an equivalent volume of lysate from empty vector was added to maintain equal protein concentrations. Therefore, the two lysate-derived complexes, indicated by the small asterisks, are also present in those lanes. Supershifts were performed with FLAG antiserum ({alpha}-FLAG) and resulted in specific retardation of homodimeric S-CREM-{alpha} and heterodimeric S-CREM-{alpha}/CREB bZIP complexes but not of CREB homodimer.

 
S-CREM and SS-CREM Proteins Are Translated in Vivo and Translocate to the Nucleus
We wanted to determine next whether S-CREM and SS-CREM forms were also translated in vivo. COS-7 cells were transfected with expression vectors for CREM/FLAG cDNAs or with empty vector, and nuclear extracts were prepared and subjected to the same EMSA protocol as described in Fig. 9Go (Fig. 11AGo). In mock-transfected cells, several protein/DNA complexes resulting from endogenous CREBs were observed. The expression of all CREM/FLAG constructs with the exception of CREM-23/FLAG (not shown) resulted in the formation of additional complexes that were supershifted by FLAG antiserum. CREM-10/FLAG and CREM-22/FLAG produced larger adducts (lanes 3 and 12), which might result from the predominant translation of full-length CREM-{tau}2{alpha} and CREM-{alpha} proteins, respectively. Transfection of CREM-17/FLAG and CREM-24/FLAG led to the formation of distinct faster migrating complexes (lanes 6 and 15) indicative of the predicted SS-CREM-{alpha} and S-CREM-{alpha} isoforms (see also Fig. 8CGo). The expected small SS-CREM-{tau}2{alpha} protein, encoded by CREM-20/FLAG cDNA, was not discernible; however, a larger complex appeared, possibly resulting from heterodimerization of a low amount of SS-CREM-{tau}2{alpha} protein with endogenous CREM/CREB (lane 9). This complex was removed by addition of FLAG antiserum. No additional complexes were formed with nuclear extracts from COS-7 cells transfected with CREM-23/FLAG; equivalent extracts from SKUT-1B cells, however, allowed the detection of adducts resulting from expression of this construct (data not shown).



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Figure 11. Downstream Translation Initiation Occurs in Vivo

Nuclear extracts were prepared from COS-7 cells transfected with CREM-10/FLAG, CREM-17/FLAG, CREM-20/FLAG, CREM-22/FLAG, CREM-23/FLAG, CREM-24/FLAG, or empty vector (pRc/CMV). A, The nuclear extracts were used for EMSA on a 32P-labeled CRE consensus probe. Migration of the free probe is indicated by the asterisk. Competition was performed with a 100-fold molar excess of unlabeled CRE. Complexes were supershifted with FLAG antiserum D-8 ({alpha}-FLAG). Lane 1 shows the CREB/CREM complexes endogenous to COS-7 cells. B, Nuclear extracts were immunoprecipitated with FLAG antiserum, fractionated on a 16.5% SDS polyacrylamide gel, and subjected to Western immunoblotting using FLAG antiserum. Upper panel, A short exposure (5 min) of the blot is shown. The portion of the membrane containing proteins larger than 50 kDa was removed because it contained the primary antibody, which produced a strong signal with the secondary anti-rabbit antiserum. The asterisks denote proteins that were nonspecifically precipitated by the FLAG antibody. Apparent molecular masses of CREM isoforms containing the C-terminal FLAG epitope are indicated in kilodaltons. The isoforms are: CREM-{tau}2{alpha} (32.5 kDa), CREM-{alpha} (25 kDa), S-CREM-{tau}2{alpha} (20 kDa), S-CREM-{alpha} (13 kDa), and SS-CREM-{alpha} (9 kDa) (for structure of these isoforms, see also Fig. 8CGo). Lower panel, Upon longer exposure (2 h) additional low molecular mass proteins became detectable: SS-CREM-{alpha} (9 kDa) in COS-7 cells transfected with CREM-20/FLAG and CREM-22/FLAG, and SS-CREM-{tau}2{alpha} (16 kDa) in cells transfected with CREM-20/FLAG (the higher molecular mass bands were overexposed and are therefore not shown). C, The Western blot shown in panel B was stripped and immunodecorated with antibody against human CREM-1.

 
To precisely identify which isoforms were being produced in vivo, we analyzed the above nuclear extracts from transfected COS-7 cells by immunoprecipitation with FLAG antiserum followed by Western blotting. Precipitated proteins were visualized with antibodies directed against the FLAG epitope (Fig. 11BGo) or human CREM-1 (Fig. 11CGo). No CREM/FLAG fusion protein was detectable in cells transfected with CREM-23/FLAG, consistent with the lack of specific DNA/protein complexes in the EMSA. Identical products were detected with both antibodies albeit with different efficiencies. Most notably, all isoforms obtained by in vitro transcription/translation (see Fig. 8Go) are also produced in vivo. From each cDNA, every possible isoform resulting from internal translation initiation at the AUG codons Met 3, 4, or 6 was produced. As predicted from the less favorable context of Met 4, this residue is used somewhat less efficiently for initiation than are Met 3 and 6. As a result, isoform SS-CREM-{tau}2{alpha} (16 kDa) is less abundant compared with the prominent smaller isoforms S-CREM-{alpha} and SS-CREM-{alpha} (13 and 9 kDa, respectively). The nature of the two proteins migrating just below full-length CREM-{tau}2{alpha} and CREM-{alpha} translated from CREM-10 and CREM-22, respectively, is unclear at present. They were not visible in other nuclear protein preparations and may be due to proteolytic cleavage or to altered electrophoretic mobility caused by posttranscriptional modification.

To assess the subcellular localization of the CREM protein isoforms as summarized in Fig. 8Go, we applied immunofluorescent staining to SKUT-1B cells transfected with the CREM/FLAG constructs. FLAG antibody detected FLAG-tagged proteins produced from all constructs and localized these proteins almost exclusively to the nuclei of the transfected cells (Fig. 12Go). Equivalent results were obtained using either polyclonal FLAG antiserum or M2 monoclonal FLAG antibody, which detects C-terminally inserted FLAG epitope.



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Figure 12. Nuclear Localization of C-Terminal CREM Isoforms

SKUT-1B cells were transfected with empty vector pRc/CMV (panel A), CREM-10/FLAG (panel B), CREM-17/FLAG (panel C), CREM-20/FLAG (panel D), CREM-22/FLAG (panel E), CREM-23/FLAG (panel F), or CREM-24/FLAG (panel G). Immunofluorescence was performed with FLAG antiserum D-8 followed by Cy3-conjugated anti-rabbit secondary antibody (panels A-D, F, and G) or with monoclonal FLAG antibody M2 in conjunction with Cy3-conjugated anti-mouse secondary antibody (E). To show the spindle-like shape of the cells, a well of mock-transfected cells with high nonspecific staining due to inappropriately high antibody concentration is included (panel A). This clarifies the almost exclusive nuclear staining obtained with all CREM/FLAG expression vectors (panels B-G). Equivalent results were obtained using either D-8 or M2 antibody.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Multitude of Inhibitory CREM Isoforms Is Generated by Alternative Splicing and Alternative Translation Initiation
Our understanding of the transduction of cAMP-mediated signaling to a CRE is becoming increasingly complex. The first CRE-binding protein identified was CREB (32), which was characterized as a transcriptional activator regulated by phosphorylation (33). Phosphorylation allows interaction with the CREB-binding protein CBP, which is thought to act as a link between CREB and the transcription preinitiation complex (34). Later, a group of transcription factors modulating CREB function was identified (18). These are encoded by the CREM gene, which is highly related to the CREB gene and probably arouse by gene duplication (23). Multiple isoforms are generated by alternative splicing; CREM-{alpha}, -ß, and -{gamma} comprise the P box but lack transactivation domains and are transcriptional repressors whereas the CREM-{tau} variants possess either one or both transactivation domains and are transcriptional activators (22, 24). From an intronic second promoter (P2) in the CREM gene, the inhibitor ICER is transcribed in a cAMP-regulated fashion due to a cluster of four CREs in the P2 control region. ICER proteins have a molecular mass of about 13 kDa, consist almost exclusively of either DBD I or DBD II sequences, and are among the smallest transcription factors ever described (35).

All alternatively spliced forms of CREM described so far retain the coding sequence in frame (19). In contrast, numerous exons have been identified in the CREB gene (reviewed in Refs. 19 and 20): {gamma}, {Omega}, {Psi} (29), W (36), Y (37), and Z (38) which, when inserted into the CREB transcript, introduce in-frame stop codons upstream of the DBD. The function of such transcripts remained elusive, as carboxy-terminally truncated proteins would be lacking the DBD and the nuclear translocation signal that resides within the basic region of the CREB DBD. Only recently the polycistronic nature of CREB transcripts containing exon W was demonstrated in that internal translation initiation in exons W and H could lead to the formation of short transcriptional repressors, I-CREB(l) and I-CREB(s), expressing the DBD, in testes (38, 39). Expression of these repressors is potentially involved in cAMP-dependent regulatory pathways during spermatogenesis.

Here we report for the first time a similar multilevel complexity in the expression of transcriptional repressors from the CREM gene, involving both alternative splicing and alternative translation initiation. Upon cloning of CREM transcripts from human ES cells, we identified a novel exon {Psi} residing about 4 kb downstream of the first protein-coding exon and 5' to the sequence encoding the transactivation domain Q1. CREM exon {Psi} resembles CREB exon {Psi} both by its genomic location (exon {Psi} is separated from the first protein coding exon of the CREB gene by a 5.7-kb intron) and by the presence of in-frame stop codons (29); exons {Psi} of the CREB and of the CREM gene, however, share no sequence homology. Exon {Psi} was found in several transcript isoforms; in the cDNA clones CREM-23 and CREM-24 isolated from human ES cells it was either spliced to the Q2 domain (CREM-{Psi}{Delta}1,P) or to the first exon coding for the P box (CREM-{Psi}{Delta}1,2). In addition to these CREM-{Psi} isoforms, we isolated previously undescribed splice variants lacking the P box in which the first protein-coding exon was either spliced to the Q2 domain (clone CREM-20, termed CREM-{Delta}1,P) or directly to the exon encoding the 5'-portion of DBD I (clone CREM-17, termed CREM-{Delta}1,P, 2). All these novel isoforms appeared nonfunctional at first glance because their ORF was blocked upstream of the DBD. In cotransfection experiments, however, they proved to be potent inhibitors of PKA-stimulated activity of a reporter gene construct driven by a consensus CRE. This prompted us to investigate whether C-terminal peptides were synthesized from these cDNAs. To allow immunological detection of such peptides, we tagged the cDNAs by inserting the FLAG sequence between DBD I and the 3'-UTR. Using these constructs we were able to demonstrate that, 1) C-terminal proteins were translated in vitro from CREM-17, -20, -23, and -24 templates by internal translation initiation downstream of the in-frame stop codons, and that 2) these proteins bound to a CRE as homo- and heterodimers.

The methionine residues predicted to be used as start sites for downstream translation initiation in the above constructs are Met 3 (adjacent to the P box), Met 4 in Q2, and Met 6 in module H, 19 amino acids upstream of the DBD I. All three of these residues are also present in CREM-{tau}2{alpha} (clone CREM-10), and Met 3 and Met 6 are present in CREM-{alpha} (our clone CREM-22), both transcript isoforms possessing a full-length ORF. We assessed whether internal translation initiation would also take place on these templates. By in vitro transcription/translation, SDS-PAGE analysis, and EMSA, we could show that shorter carboxy-terminal peptides were synthesized from CREM-10 and CREM-22 in addition to the full-length proteins and that long and short isoforms were cotranslated at comparable levels. A corresponding observation has been made for the CREM-{tau} transcript of the mouse brain, where translation can also initiate at Met 3 to yield the short repressor S-CREM (25). S-CREM, however, differs from S-CREM-{tau}2{alpha} described by us in that it includes the second rather than the first DBD. We have thus identified four novel CREM protein isoforms, S-CREM-{tau}2{alpha} (20 kDa), SS-CREM-{tau}2{alpha} (16 kDa), S-CREM-{alpha} (13 kDa), and SS-CREM-{alpha} (9 kDa).

We have demonstrated that these novel short isoforms are produced in vivo and act as transcriptional repressors: 1) Expression vectors for CREM-23, CREM-24, and CREM-17 cDNAs served as powerful inhibitors of PKA-stimulated promoter activity; due to the upstream in-frame stop codons, SS-CREM-{tau}2{alpha}, S-CREM-{alpha}, and SS-CREM-{alpha} are the only carboxy-terminal products that can be translated from these templates. 2) Protein translated from these templates in vivo was efficiently translocated to the nucleus. 3) The AUG codons Met 3, 4, and 6, allowing translational initiation in vitro, are also used in vivo, giving rise to all predicted isoforms as visualized by Western analysis. These data indicate that CREM transcripts are polycistronic and that short inhibitory isoforms are translated from CREM-10, -17, -20, -22, and -24 in vivo. We were not able to identify protein translated from CREM-23 in COS-7 cells by immunoblotting. However, transfected CREM-23 cDNA resulted in transcriptional repression in primary ES and in SKUT-1B cells, and C-terminal FLAG-tagged protein appeared in the nucleus, indicating that CREM-23 is expressed either in the form of SS-CREM-{tau}2{alpha} or of SS-CREM-{alpha}, at least in these uterine cell types.

The start codon for production of the shortest isoform, SS-CREM-{alpha}, is most likely Met 6, although we have no final proof for this assumption. The other two methionine codons (Met 5 and 7) in module H are in an unfavorable context whereas Met 6 has a purine residue at position -3 and a guanosine at +4 (GTG ATG G). This region is almost identical to the corresponding site in exon H of the CREB gene (GTT ATG G), which has recently been described to give rise to the short transcriptional repressor I-CREB(s). This protein of about 8 kDa also consists of the DBD only and is expressed in human, mouse, and rat testis (38, 39). The nuclear translocation signal of CREB has been defined in the first half of the DBD (RRKKKEYVK) (40). This amino acid sequence is conserved in DBD I of CREM and is located downstream of the last methionine residue. Therefore, even in the unlikely case of translation initiation at Met 7, rather than Met 6, the resultant CREM protein would contain this signal sequence.

The AUG codon in module H (Met 6) is common to all CREM transcript isoforms. It is most efficiently used for translational initiation in CREM-17 where no alternative more upstream AUG start codon leads to elongational occlusion by ribosomes initiating upstream. It has been shown that the internal AUG start codon in exon H of CREB mRNA, which is embedded in a very similar sequence as Met 6 and gives rise to the short repressor I-CREB(s) (8 kDa), serves as an internal ribosomal entry site (39). Albeit less efficiently, Met 6 is also used in the other CREM cDNAs as demonstrated by Western analysis of COS-7 cells transfected with CREM-10, -20, -22, and -24. The SS-CREM-{alpha} isoform therefore appears to be a translational byproduct of CREM transcripts in general. The presence of CREM transcripts in vivo, whether or not they include transactivation domains, is therefore expected to lead to an accumulation of SS-CREM-{alpha} repressor protein.

Our findings not only demonstrate that one transcript can encode several proteins (CREM-10 yields CREM-{tau}2{alpha}, S-CREM-{tau}2{alpha}, SS-CREM-{tau}2{alpha}; CREM-22 yields CREM-{alpha} and S-CREM-{alpha}), but also, conversely, that one protein can be encoded by numerous transcripts, e.g. SS-CREM-{tau}2{alpha} by CREM-10 and CREM-20, S-CREM-{alpha} by CREM-22 and CREM-24, and SS-CREM-{alpha} potentially by every CREM transcript isoform.

The expression of a CREM transcript similar to CREM-17 has been reported in rat spermatids. This form, CREM{Delta}C-G, also lacks both transactivation domains and the P box and includes DBD I (21). It contains, however, the small exon X (also termed exon {gamma}), which adds 36 nucleotides 5' to the DBD I. In striking contrast to CREM-17, however, CREM{Delta}C-G displays a full-length ORF and encodes a protein of about 17 kDa. This is due to the insertion of one additional guanosine in the splice junction between the first protein-coding exon (module C, see Figs. 1Go and 3Go) and exon X (exon {gamma}, see Figs. 1Go and 3Go), which restores the ORF leading into the DBD. As depicted in Fig. 4Go, regular splicing of module C to exons downstream of the P box leads to disruption of the ORF. Interestingly, the junction between modules C and H in CREM-17 is also irregular; it does not, however, restore the ORF. Two highly related transcripts, rat isoform CREM{Delta}C-G and human isoform CREM-17 (CREM-{Delta}1,P, 2), therefore encode dissimilar proteins of 17- and 9-kDa molecular mass, respectively. Functionally, these isoforms are analogs, both acting as transcriptional repressors.

We were not able to detect the upstream transactivation domain, Q1, in CREM transcripts amplified from human ES cells. Likewise, others have reported the lack of Q1 sequence in transcripts from human sources, and the sequence of the human Q1 segment has only been deduced by genomic cloning of this region using a mouse Q1 probe (41). It is uncertain whether Q1 sequence of the human CREM gene is expressed at all. Species-specific splicing has been reported for exon Z of the human CREB gene, an exon that is not spliced into rat and mouse transcripts due to mutations in the flanking splice signals (38).

Our data demonstrate that expression of the human CREM gene at the posttranscriptional level is highly complex and gains versatility both by alternative splicing and by alternative translation initiation. The majority of CREM protein isoforms represent transcriptional repressors, some of which are remarkably small.

Human ES Cells Override the Presence of Multiple Inhibitory CREM Isoforms and of Up-Regulated ICER in the Course of Decidualization
Human ES cells decidualize in response to a sustained cAMP stimulus. In the course of decidualization, the regulatory subunit isoform of PKA, RI{alpha}, is significantly decreased at the protein level, while all other regulatory and catalytic subunits remain unaltered. This leads as a net effect to an increase in free catalytic subunit, which can traffic to the nucleus and phosphorylate target proteins such as CREB and CREM (17). A marker of decidualization is the activation of the dPRL promoter. This promoter is induced by cAMP via an indirect mechanism. While induction of promoter constructs with a classical CRE becomes detectable in transfected ES cells within 4 h of cAMP stimulation, induction of the dPRL promoter appears with a delay of at least 20 h. This induction can be abolished by the transfection of an expression vector for protein kinase inhibitor (17, 42). It was of interest to us, therefore, to investigate the expression of transducers of the PKA-signaling pathway in ES cells undergoing decidualization. Amplification of CREB mRNA using primers anchored in exons B and H, upstream of the DBD sequence, yielded a single product of the predicted size for CREB-327 (28), indicating that no alternative splicing involving exons W or Z takes place in ES cells. These exons have been described in human testis; their inclusion in CREB transcripts introduces in-frame stop codons upstream of the DBD and leads to the generation of carboxy-terminal repressors I-CREB(l) and I-CREB(s) due to alternative translation initiation (38). No difference in the level of CREB expression was detected when we compared undifferentiated to differentiated ES cells. A constant level of CREB-327 is maintained in the cells, which will be activated by phosphorylation in response to a suitable stimulus, while no inhibitory CREB isoforms are present or induced.

In contrast, we found a variety of novel CREM isoforms in ES cells. While we have shown that these mRNAs are polycistronic and encode small repressor proteins, we have not been able to investigate to what extent these S-CREM and SS-CREM proteins are present in ES cells. We provide evidence, however, that the downstream AUG codons are used for translation initiation in ES cells, because transfection of polycistronic constructs had an antagonistic effect on PKA-mediated promoter activation. In addition, transfection of the CREM-{tau}2{alpha} expression vector pRc/CREM-10 led to either an enhancement or an inhibition of PKA-stimulated promoter activity depending on the promoter/reporter construct. This indicates that not only full-length activator protein CREM-{tau}2{alpha}, but also shorter repressor forms (S-CREM-{tau}2{alpha} and/or SS-CREM-{tau}2{alpha}) are translated from this template. It will be of great interest to determine whether the ratio between translation of activator and repressor forms is hormonally regulated and linked to the differentiation status of the cells. In general, ES cells seem to contain more CREM transcripts potentially encoding repressors than CREM transcripts encoding activators. The abundance of all these transcripts does not change in the course of decidualization, but it is unknown whether expression is regulated at the level of translation.

In contrast to the unaltered levels of CREM transcripts, we observed a dramatic up-regulation of ICER transcripts in decidualized cells. This up-regulation was dependent upon the presence of RLX in the medium and did not occur in the presence of MPA alone. Even though the RLX receptor has not been cloned to date and its signal transduction pathway has not been fully elucidated, RLX action is clearly linked to the PKA system in ES cells. Not only does RLX cause an enormous acute increase in intracellular cAMP in cultured human ES cells (43), but we have also shown that the elevated cAMP levels are maintained for at least 6 days with continued presence of RLX (17). We observed up-regulation of ICER in three independent ES cell preparations that had been induced to decidualize and activate the dPRL promoter by RLX treatment for 12, 16, or 28 days. The internal P2 promoter of the CREM gene that gives rise to ICER is cAMP-responsive. The induction of ICER has been described as transient and cell-specific. Expression in neuroendocrine cells peaks after 2–4 h and drops 4–6 h later. This is due to an autoregulatory negative feedback loop by which newly synthesized ICER protein antagonizes PKA-induced activation of the CREs in its own promoter (27). Fluctuating expression of ICER in the pineal gland in response to adrenergic signals plays an important role in circadian rhythmicity (26). In addition to neuroendocrine cells, cAMP inducibility of ICER has also been reported in human medullary thymocytes, peripheral blood T lymphocytes, and T cell lines. Again, ICER expression peaked within 3 h of stimulation (by forskolin or PGE2) and ceased soon after (44). The authors noted a relatively high level of ICER in untreated freshly prepared peripheral blood T cells, which they attributed to either an activation of cAMP signaling during preparation of the cells or to a normal physiological property of this cell type. We also observed an unexpectedly high level of ICER transcripts in decidualized ES cells treated with RLX+MPA. This was not due to the preparation procedure because 1) the cells were harvested directly into denaturing solution and 2) no up-regulation occurred in cultures harvested in parallel that had been maintained without hormone or in the presence of MPA alone. Elevated ICER was therefore caused by the long-term exposure to RLX.

Our finding of high ICER expression together with induction of the dPRL promoter in decidualized cells subjected to a persistent cAMP stimulus exerted by RLX seems paradoxical at first glance: In cotransfection experiments ICER expression vectors inhibited PKA-stimulated activity of the dPRL promoter construct dPRL-3000/luc. With regard to the cotransfection experiments, it must be remembered, however, that ICER was overexpressed in undifferentiated cells without being complemented or opposed by as yet unknown mechanisms that may evolve in differentiating cells.

What we see in decidualized cells is the apparently paradoxical activity of two promoters in the presence of ICER, even though these promoters are known to be repressed by isolated ICER: the dPRL promoter and the ICER promoter itself. We suggest the following explanation for this phenomenon: in decidualized ES cells, the repressor ICER is titrated out by an excess of activator protein, such as CREM-{tau}2{alpha} or CREB. Within this concept, the presence of elevated ICER levels in cells constantly challenged by elevated cAMP might actually be viewed as an indicator for the presence of overriding amounts of transactivating CREM isoforms or CREB, phosphorylated by the elevated free catalytic subunits of PKA, which in turn contribute to the activation of decidualization markers such as the dPRL gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RT-PCR Analysis
Total RNA was extracted with RNA-clean (AGS, Heidelberg, Germany) and reverse transcribed with SuperScript RNase H- reverse transcriptase (GIBCO-BRL, Eggenstein, Germany). The following primers were based on the sequence of human CREM cDNA (41, 45); positions in brackets are given relative to the first ATG codon. Sense oligonucleotides were:

B (pos. -16 to +9; 5'-GACAAATCCAAGACAAATGA-CCATG-3'),

C (pos. 88–110; 5'-ACTGGGCAAATTTCAATCCCTGC-3'),

D (pos. 121–143; 5'-TGCAGTGAGCTGAGATCAGGCAC-3'). Antisense oligonucleotides were:

D' (pos. 177–201; 5'-ATGTATAGTTTGGCCCGAAGGT-AAC-3'),

E (pos. 296–318; 5'-AATTACACCTTCTGATTCTGCAG-3'),

F (pos. 537–561; 5'-ACCATCAGATCCTGGGTTAGAA-ATC-3'),

H (pos. 765–789; 5'-CAAACTTCCGGGCGATGCAGCC-ATC-3'),

J (pos. 1398–1423; 5'-CAGTTCATAGTTAAATATTTCTA-GTA-3'),

G (5'-GACTTGGGGCAAGTTCAGTTTCC-3'). Primer A is specific to the 5'-UTR of the ICER mRNA (45):

A (5'-TGGAACACTTTATGTTGAACTGTGG-3') Additional primers were designed to the novel exon {Psi} sequence:

{Psi}' (5'-CTATGAAAATCATCAGAGACCTAGC-3') (sense) and

{Psi}" (5'-CTCAATCCATCAGCAGAACAG-3') (antisense).

CREB primers were chosen upstream of the DBD of the human CREB cDNA (28):

CREB-1 (pos. 180–203 in exon B; 5'-GAAGCTGAAAACCAACAAATGACA-3', sense), CREB-2 (pos. 848–870 in exon H; 5'-CAATAGTGCTAGTGGGTGCTGTG-3', antisense), CREB-3 (pos. 406–427 in exon C; 5'-GTGAAGATTCACAGGAGTCAGT-3', sense).

Primers for amplification of the dPRL transcript were: 98 (pos. 43–72 in the decidual-specific exon 1a) (15), 173 (antisense to pos. 67–87 in exon 2 of the hPRL cDNA), and 630 (antisense to pos. 710–739 in the 3'-UTR) (46).

PCR on cDNA templates was performed with Taq polymerase (Promega, Madison, WI). Products were subjected to Southern blotting, hybridized with biotinylated oligonucleotide primers, and visualized with the Southern light detection system (Tropix, Bedford, MA). Genomic PCR was performed with the Expand long template PCR system on human genomic DNA (Boehringer, Mannheim, Germany). For sequencing and subcloning purposes, PCR products were isolated from a low melting agarose gel and ligated into the pGEM-T vector (Promega).

Constructs
To generate eukaryotic expression vectors, full-length CREM and ICER cDNAs were excised from pGEM-T by ApaI digestion, followed by polishing and NotI digestion. These fragments with a 5'-blunt and a 3'-NotI end were inserted into pRc/CMV (InVitrogen, San Diego, CA), which had been linearized with HindIII, polished, and further digested with NotI. Resultant plasmids were: pRc/CREM-10, pRc/CREM-17, pRc/CREM-20, pRc/CREM-23, pRc/CREM-24, pRc/ICER-I, and pRc/ICER-II. For immunological detection of CREM translation products, the FLAG sequence (DYKDDDDK) (47) was introduced 3' to the protein-coding region, replacing the stop codon after DBD I. CREM/FLAG fusion cDNAs were generated by PCR on the CREM cDNAs in pGEM-T with Pfu polymerase (Stratagene, Heidelberg, Germany) using oligonucleotide B (see above) as upstream primer. The following antisense oligonucleotide served as the downstream primer: 5'-GAATTGCGGCCGC TTA TTT GTC GTC ATC GTC TTT GTA GTC CTC TAC TTT ATG GCA ATA-3'; it contained a NotI site (underlined), the FLAG sequence terminated by a stop codon (in bold type), and CREM-specific sequence (pos. 979–996 in DBD I). After NotI digestion, the PCR products had a 5'-blunt and a 3'-NotI end and were inserted into pRc/CMV, which had been treated as described above to have a 5'-blunt and a 3'-NotI end. Thus, CREM-10/FLAG, CREM-17/FLAG, CREM-20/FLAG, CREM-22/FLAG, CREM-23/FLAG, and CREM-24/FLAG were created.

The expression vector for the catalytic subunit of PKA, pRSV-Cß, and an inactive mutant thereof, pRSV-Cßmutant, were kindly provided by Dr. R. Maurer (Oregon Health Sciences University, Portland, OR) (48). The generation of a reporter gene construct in pGL2-Basic (Promega) carrying 3000 bp of DNA flanking the dPRL promoter (dPRL-3000/luc) has been described previously (16). A fragment truncated to -332 bp was generated by PCR and inserted into pGL3-Basic (Promega) to yield dPRL-332/luc3. Plasmid CRE/-36rPRL/luc (kindly provided by Dr. M. G. Rosenfeld, Howard Hughes Medical Institute, San Diego, CA) (49) contains a composite CRE element 5'-TTGGCTGACGTCAGAGAGAGGCCGGCCCCTTACGTCAGAGGCGAG-3' linked to the minimal rPRL promoter fragment -36/+34. This fusion was isolated by PCR with the upstream primer 5'-CTTGG-CTGACGTCAGAGAGAG-3' and the downstream GL2 primer (Promega) antisense to the luciferase reporter gene. The product was restricted with HindIII at the 3'-end of the rPRL sequence and inserted into SmaI/HindIII-digested pGL3-Basic to give pCRE/-36rPRL/luc3.

Cell Culture
Purified cultures of human ES cells were prepared as previously described (16) and plated in basal media, a 1:1 mixture of DMEM and Ham’s F-12 containing 10% FCS that had been depleted of steroids by treatment with dextran-coated charcoal, 100 U/ml penicillin, and 100 µg/ml streptomycin, and supplemented with 10-9 M 17ß-estradiol and 1 µg/ml insulin. Cells kept under these conditions were referred to as -/- cells. Optionally basal medium was further supplemented with 2.5 x 10-7 M MPA (Sigma) (+/- cells) or with MPA and 100 ng/ml porcine relaxin (3000 U/mg protein; kindly supplied by S. Raiti, National Hormone and Pituitary Program, NIDDK, Baltimore, MD) (+/+ cells). Media were changed every 2–3 days. When they had reached confluence, monolayers were subcultured at a ratio of 1:3.

The human uterine sarcoma cell line SKUT-1B (HTB 115, American Type Culture Collection, Rockville, MD), obtained from the European Collection of Animal Cell Cultures (Salisbury, UK), was maintained in DMEM/Ham’s F-12 (1:1) with 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. COS-7 cells were cultured in DMEM with 10% FCS and antibiotics as above.

Transient Transfections, Nuclear Extract Preparations, and DNA-Binding Studies
Transfections were performed by the calcium phosphate precipitation method on SKUT-1B cells and on ES cells of the first passage plated in 12-well plates. SKUT-1B cells received 1.4 µg reporter plasmid, 0.6 µg pRSV-Cß (or pRSV-Cßmutant for controls), and 0.8 µg pRc/CREM or pRc/ICER constructs. For ES cells, 0.8 µg pRSV-Cß was used. The DNA precipitate was removed from the cells 16 h later and replaced by fresh medium, and cells were harvested after an additional 24 h for luciferase assay (Promega). All transfections were performed in triplicate wells on several independent cell preparations.

For preparation of nuclear extracts from COS-7 cells, transfections were performed in 6-cm dishes with a mixture of 5.5 µg CREM/FLAG constructs and 30 µl DOTAP (Boehringer) made up to a total volume of 150 µl with HEPES-buffered saline. The DNA/DOTAP mixture was added to the culture medium and left on the cells for 16 h. Medium was then replaced, and cells were harvested 24 h later in buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM dithiothreitol, and protease inhibitor cocktail (Complete; Boehringer)] and allowed to swell on ice for 5 min, after which NP-40 was added to 1%, and nuclei were spun down. The nuclear pellet was resuspended in buffer B (same as buffer A including 400 mM NaCl and 1% NP-40), and nuclear proteins were extracted for 15 min at 4 C.

EMSAs were performed using 10 µg nuclear extracts, 3 µl in vitro translation product, or 0.3 µg human CREB-1 bZIP domain (amino acids 254–327; Santa Cruz Biotechnology, Santa Cruz, CA). The CRE probe was a 32P-end-labeled double-stranded oligonucleotide with a consensus CRE (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'). Protein was incubated in bandshift buffer (40 mM HEPES, pH 7.9, 20 mM MgCl2, 240 mM KCl, 5 mM spermidine, 16% Ficoll) with 1 µg poly(deoxyinosinic acid-deoxycytidylic acid) for 15 min (competition was carried out in the presence of 100-fold molar excess of unlabeled CRE), followed by a 45-min incubation with probe (20,000 cpm) on ice. For supershift studies, incubation with probe was reduced to 30 min on ice, followed by addition of 1 µl FLAG antiserum and a 20-min incubation at room temperature. Reactions were then resolved on 6% polyacrylamide gels in Tris-borate-EDTA buffer.

Protein Analysis
CREM/FLAG constructs (0.5 µg) were subjected to in vitro transcription and translation using the TNT T7 coupled reticulocyte lysate transcription/translation system (Promega) in the presence of [35S]methionine in a final volume of 25 µl. Of this reaction, 20 µl were immunoprecipitated by incubation with FLAG antiserum (D-8; Santa Cruz Biotechnology) followed by protein A Sepharose (Pharmacia, Uppsala, Sweden) in Ripa buffer [PBS, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor set (Boehringer)]. Products were resolved on 3–27% or 10–20% polyacrylamide SDS gels (ICN, Meckenheim, Germany) and autoradiographed. For use of proteins in DNA-binding studies, in vitro transcription/translation was performed with unlabeled methionine.

Nuclear extracts from COS-7 cells transfected with CREM/FLAG constructs were immunoprecipitated with FLAG antiserum D-8 as above and fractionated on a 16.5% SDS polyacrylamide gel (50). Proteins were transferred to polyvinylidene fluoride membrane (Immobilon P; Millipore, Eschborn, Germany) in 0.025 M Tris, 0.192 M glycine, 20% methanol at 250 mA for 3 h. Immunodecoration was performed with FLAG antiserum D-8 followed by detection with the enhanced chemiluminescence (ECL) system (Amersham, Braunschweig, Germany) or with antibody against human CREM-1 (Santa Cruz) and the Western Light chemiluminescent detection kit (Tropix, Bedford, MA).

Immunofluorescence
SKUT-1B cells were plated in 24-well plates coated with poly-D-lysine (1 µg/cm2; Sigma Chemical Co., St. Louis, MO) and transiently transfected with 1 µg CREM/FLAG constructs as described above. Medium was replaced 16 h later, and cells were incubated for an additional 24 h. Cells were then washed in PBS, fixed in 2.5% paraformaldehyde in PBS for 10 min at 37 C, washed in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 10 min. To block nonspecific binding, 200 µl normal goat serum (Sigma) were added, left on the wells for 5 min and aspirated without subsequent wash. Monoclonal FLAG antibody M2 (Eastman Kodak, New Haven, CT) (20 µg/ml in PBS) or FLAG antiserum D-8 (Santa Cruz) (0.2 µg/ml in PBS) was added for 1 h at room temperature, followed by three washes in 0.001 M PBS. Fluorochrome-conjugated secondary antibody (anti-mouse Cy3 affinipure F(ab')2 goat IgG, or anti-rabbit Cy3 affinipure goat IgG; Dianova, Hamburg, Germany) was diluted 1:100 in 0.001 M PBS/2% normal goat serum and applied for 60 min, followed by three washes in 0.001 M PBS. Stained cells were visualized on an inverted microscope at 510–560 nm.


    ACKNOWLEDGMENTS
 
We wish to thank Ms. Katherine Bracken for help with the immunofluorescence, Dr. Richard Maurer for PKA expression plasmids, and Drs. Nicholas Hunt, Tom Krietsch, and Gabriel DiMattia for helpful discussions.


    FOOTNOTES
 
Address requests for reprints to: Birgit Gellersen, Institute for Hormone and Fertility Research, Division of Reproductive Sciences, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany.

This work was presented in part at the 10th International Congress of Endocrinology, San Francisco, CA, June 12–15, 1996.

This work is based in part on the doctoral study by R. T. performed at the Faculty of Biology, University of Hamburg, and was supported by Deutsche Forschungsgemeinschaft Grant Ge 748/1-2.

Received for publication August 26, 1996. Revision received October 21, 1996. Accepted for publication October 24, 1996.


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