Activation of Extracellular Signal-Regulated Kinases and CREB/ATF-1 Mediate the Expression of CCAAT/Enhancer Binding Proteins ß and -{delta} in Preadipocytes

Nathalie Belmonte, Blaine W. Phillips, Florence Massiera, Phi Villageois, Brigitte Wdziekonski, Perla Saint-Marc, Jennifer Nichols, Jérôme Aubert, Kumiko Saeki, Akira Yuo, Shuh Narumiya, Gérard Ailhaud and Christian Dani

Institute of Signaling, Development Biology and Cancer Research (N.B., B.W.P., F.M., P.V., B.W., P.S.-M., J.A., G.A., C.D.), UMR 6543 Centre Nationale de la Recherche Scientifique, Centre de Biochimie 06108 Nice Cedex 2, France; Centre for Genome Research (J.N.), University of Edinburgh, United Kingdom; Department of Hematology (K.S., A.Y.), Research Institute, International Medical Centre of Japan, Tokyo 162-8655, Japan; and Department of Pharmacology (S.N.), Kyoto University, Kyoto 606-8501, Japan

Address all correspondence and requests for reprints to: Dr. Christian Dani, Centre de Biochimie (UMR 6543 CNRS), UNSA, Faculté des Sciences, Parc Valrose, 06108 Nice cedex 2, France. E-mail: dani{at}unice.fr


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The essential role of CCAAT/enhancer binding proteins (C/EBPs) ß and {delta} for adipocyte differentiation has been clearly established. In preadipocytes, their expression is up-regulated by the activation of leukemia inhibitory factor receptor (LIF-R) and prostacyclin receptor (IP-R) via the extracellular signal-regulated kinase (ERK) pathway and cAMP production, respectively. However, the molecular mechanisms by which LIF and prostacyclin-induced signals are propagated to the nucleus and the transcription factors mediating ERK and cAMP-induced C/EBP gene expression were unknown. Here we report that both pathways share cAMP responsive element binding protein/activation transcription factor 1 (CREB/ATF-1) as common downstream effectors. LIF-R and IP-R activation induced binding of CREB and/or ATF-1 to C/EBP promoters and CREB-dependent transcription. Expression of dominant negative forms of CREB dramatically reduced the LIF- and prostacyclin-stimulated C/EBP ß and C/EBP {delta} expression. Upon stimulation of the IP-R, the ERK pathway was activated in a PKA-dependent manner. ERK activation by the PKA pathway was not required for CREB/ATF-1 phosphorylation but rather was necessary for CREB-dependent up-regulation of C/EBPs expression. Our findings suggest that ERK activation is required for CREB transcriptional activity, possibly by recruitment of a coactivator.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SEVERE OBESITY IS the result of an increase in fat cell size, i.e. adipocyte hypertrophy, in combination with increased fat cell number, i.e. adipose tissue hyperplasia (1). New fat cells arise from a preexisting pool of adipose precursor cells that are present irrespective of age (2, 3). Therefore, the formation of new fat cells remains an important issue from a physiological view point, and their enhanced number in obesity emphasizes the critical role of the proliferation of preadipose cells and their differentiation into adipose cells. The characterization of the molecular events regulating the adipose differentiation program (adipogenesis) has been the subject of numerous investigations in the past two decades. A wealth of information has been obtained both on the nature of the adipogenic factors, their cognate signaling pathways, and the identification of key transcriptional factors (4). So far, three families of transcriptional factors have been identified to be important in mature adipocyte development. Among members of the first family, PPAR{delta} (=PPARß=NUC-1) appears rapidly in confluent cells that express early markers of the adipogenic differentiation program but are still triacylglycerol free, termed preadipocytes (5). The fatty acid activation of PPAR{delta} ectopically expressing fibroblasts leads to the expression of PPAR{gamma}, which is critically required for terminal differentiation of preadipocytes to triacylglycerol-filled adipocytes (6). Both PPAR{delta} and PPAR{gamma} function as heterodimers with RXR{alpha} in a ligand-dependent fashion (7, 8). The second group of adipogenic factors is the basic-helix-loop-helix leucine zipper transcription factor family in which the /sterol regulatory element binding protein-c appears quite early during differentiation. Adipocyte determination and differentiation factor-1 stimulates the expression of fatty acid synthetase and lipoprotein lipase, which are responsible for fatty acid supply (9, 10). CCAAT/enhancer binding proteins (C/EBPs) are the third family of adipocyte-promoting transcription factors. They possess both leucine zipper and basic and acidic domains and are active as unliganded homodimers. It is now clear that transcription factors of all three families play a sequential role in the program of adipocyte differentiation and that the expression of C/EBPß and C/EBP{delta} is the earliest event to occur in preadipose cells. C/EBPß and C/EBP{delta} both promote adipogenesis of fibroblasts when they are ectopically expressed (11, 12) and up-regulate the subsequent expression of C/EBP{alpha} and PPAR{gamma}2. The essential role of these two C/EBPs for the differentiation of adipocytes both in vitro and in vivo has been clearly established using C/EBPß -/-. C/EBP{delta} -/- mice (13).

We have shown that activation of two cell surface receptors, i.e. the leukemia inhibitory factor-receptor (LIF-R) and the prostacyclin-receptor (IP-R), stimulates the expression of both C/EBPs and promotes adipogenesis. Preadipocytes but not adipocytes secrete functional LIF and simultaneously express LIF-R. An antagonist of LIF-R abolishes adipogenesis, strongly suggesting that secreted LIF acts via a paracrine/autocrine mechanism. The reduced capacity of lif-r-/- embryonic stem cells to undergo adipocyte differentiation demonstrates that this receptor plays a critical role in this process (14). Selective inhibitors of the extracellular signal-regulated kinase (ERK) cascade inhibit LIF-induced C/EBPß and C/EBP{delta} gene expression and prevent LIF-induced adipogenesis (14). Recently, we have delineated a second pathway, which up-regulates C/EBPß and C/EBP{delta} expression. Prostacyclin (PGI2), also secreted by preadipocytes, acts via IP-R, which is expressed in preadipocytes only. This pathway then up-regulates the expression of both C/EBPs by triggering cAMP production and also promotes adipogenesis (15). However, the molecular mechanisms by which LIF- and prostacyclin-induced signals are propagated to the nucleus and the transcriptional factor(s) mediating ERK- and cAMP-induced C/EBP gene expression in preadipocyte cells are not known. This issue has been addressed in this work.

In recent years, it has become clear that members of the leucine zipper class of transcriptional factor cAMP-responsive element binding proteins/activation transcription factor (CREB/ATF), respond to a variety of external signals and play roles in cell proliferation and differentiation (16, 17). CREB is the most extensively studied cAMP-responsive element (CRE)-binding protein. Phosphorylation of serine-133 is a critical event in CREB activation, leading to an increase in its trans-activation potential by allowing the recruitment and binding of coactivators such as CREB-binding protein (CBP). ATF-1, another CRE-binding protein, has significant sequence similarity to CREB, including in its phosphorylation domain. Initial studies identified PKA as the major kinase responsible for Ser-133 phosphorylation, but subsequent studies have identified additional pathways leading to CREB phosphorylation such as those that are regulated by ERKs. Recently CREB, which is expressed before the emergence of preadipocyte and adipocyte markers, has been shown to be required for adipocyte differentiation. Expression of a constitutively active form of CREB enhanced adipogenesis of 3T3-L1 cells while expression of a dominant-negative form of CREB blocked adipogenesis (18).

In this study, we used an antibody that specifically recognizes both phosphorylated CREB and phosphorylated ATF-1. Our results demonstrate that the regulation of C/EBPß and C/EBP{delta} gene expression via the LIF/LIF-R pathway is initially distinct from that of the prostacyclin/IP-R pathway but that both pathways converge on the activation of CREB/ATF-1. CREB/ATF-1 not only binds to a putative CRE in the promoters of C/EBPß and C/EBP{delta} genes but is also involved in the regulation of their transcription. Stably transfected 3T3-F442A and Ob1771 preadipose cells expressing dominant-negative forms of CREB exhibit a potent attenuation of C/EBPß and C/EBP{delta} gene expression upon stimulation by LIF-R and IP-R agonists. Activation of the ERK pathway upon LIF-R stimulation is sufficient for CREB phosphorylation and induction of C/EBPß and C/EBP{delta} transcription. While activation of the PKA pathway upon stimulation of the IP-R is sufficient to induce CREB phosphorylation, a PKA-dependent ERK activation is also required to induce CREB-dependent transcription and stimulation of C/EBP gene expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stimulation of C/EBPß and C/EBP{delta} Gene Expression by LIF and Prostacyclin in ip-r-/- or gp130-/- Mouse Embryo Fibroblasts
It has been reported that exposure of Ob1771 and 3T3-F442A preadipocytes to LIF stimulated C/EBPß and C/EBP{delta} expression and promoted adipogenesis. This response was transmitted via the cell surface LIF-R/gp130 receptor complex and the ERK pathway (14). Similarly, prostacyclin as well as its stable analog (carba)prostacyclin (cPGI2) also induced C/EBPß and C/EBP{delta} expression and appeared to act as a potent adipogenic hormone by signaling through its cell surface IP-R and the cAMP-dependent pathway in preadipocytes (15). As shown in Fig. 1Go (upper panel), C/EBP{delta} gene expression was stimulated 5.7 ± 0.2-fold by LIF and 4.8 ± 0.3-fold by cPGI2 whereas addition of both factors simultaneously led to an increase of 11.2 ± 1.6-fold. Similar data were obtained with induction of C/EBPß gene expression. These results suggest that the responses of the C/EBP genes to LIF and cPGI2 were additive. Because preadipocytes secrete basal levels of both LIF and prostacyclin, we were interested in determining whether each stimulus could independently trigger C/EBP gene expression. Stimulation by LIF and prostacyclin was examined in ip-r-/- mouse embryonic fibroblasts (MEFs) and gp130-/- MEFs. The gp130 subunit is known to be the common transducer of LIF and cytokines of the IL-6 family. Thus gp130-/- MEFs were deliberately used as both LIF and LIF-related cytokines appear to be secreted from MEFs (14). As shown in Fig. 1Go (left panel), ip-r-/- MEFs were unresponsive to the IP-R agonist with respect to the expression of C/EBP genes; however, the cells clearly remained responsive to LIF. Conversely, gp130-/- MEFs became unresponsive to LIF, as expected, but remained responsive to cPGI2 (Fig. 1Go, right panel). Taken together, these results indicate that each of the LIF-R/LIF and IP-R/prostacyclin systems can individually trigger the expression of C/EBPß and C/EBP, emphasizing a redundancy at this early stage of adipogenesis. The transcriptional factor(s) mediating ERK-and cAMP- induced C/EBPß and C/EBP{delta} gene expression and a possible convergence between these two pathways were then investigated.



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Figure 1. Stimulation of C/EBPß and C/EBP{delta} Expression in Preadipose Cells and in ip-r-/- and gp130-/- Mouse Embryo Fibroblasts

Upper panel, Ob1771 preadipose cells were maintained for 12 h in 1% FBS and then treated either with LIF (10 ng/ml) or cPGI2 (1 µM) or with LIF plus cPGI2 (10 ng/ml and 1 µM, respectively) for 1 h. Total RNA was extracted and 20 µg were subjected to Northern blot analysis. Quantification of the hybridization signal was performed as described in Materials and Methods, standardized to ß-actin RNA signal, and results are expressed setting the signal obtained in the absence of stimulation as 1. Results are the means of two independent experiments. Lower panel, MEF were maintained for 2 h in 1% FBS medium (C) and then treated with LIF (10 ng/ml) or cPGI2) (1 µM) for 1 h. Total RNA was extracted and 20 µg were subjected to Northern blot analysis.

 
Binding of Agonists to LIF-R and IP-R Induces CREB/ATF-1 Transcriptional Activity
Various lines of evidence now support the notion that CREB family members can be phosphorylated by several different kinases, which in turn are activated by a variety of exogenous signals. Therefore, the presence of Ser133 phosphorylated CREB and total CREB in preadipocytes was examined by Western blot analysis. Using a (Ser133)phospho-CREB (P-CREB) antibody which also detects the phosphorylated form of CREB-related proteins, we showed that CREB and ATF-1 were rapidly phosphorylated upon addition of LIF or cPGI2 to confluent Ob1771 and 3T3-F442A preadipocytes (Fig. 2AGo). In both cases, phosphorylated CREB/ATF-1 levels increased dramatically within 10 min of treatment (>30-fold) and remained elevated for 30 min while total CREB remained unchanged. Similar phosphorylation patterns have been reported in 3T3-L1 preadipocytes treated with cAMP-elevating agents (19). The ability of LIF and cPGI2 to regulate CREB/ATF-1 transcriptional activity was subsequently investigated. For this purpose, 3T3-F442A cells were transiently cotransfected with a firefly luciferase reporter gene under the control of 16 repeated CREs and a Renilla luciferase expression vector for normalization. As shown in Fig. 2BGo, addition of LIF or cPGI2 led to an increase in CRE-dependent transcription. These observations were extended by examining the capacity of nuclear extracts from Ob1771 preadipocytes stimulated by LIF, cPGI2, or both to recognize the putative CRE present in the C/EBPß and C/EBP{delta} promoters through the use of EMSAs. Potential CREs present in C/EBPß and C/EBP{delta} promoters and probes are shown in Fig. 3AGo. It has been previously reported that purified recombinant CREB was able to bind to these promoter sequences (18). The results of the EMSA in Fig. 3BGo show a strong enhancement of binding activity to C/EBPß and C/EBP{delta} probes in LIF- or cPGI2- treated nuclear extracts compared with that from unstimulated cells. These data suggest that phosphorylation of CREB/ATF-1 promotes binding to CRE sequences. This binding appeared specific as it was abolished by an excess of competitor consensus CRE but not by mutated CRE (Fig. 3CGo). Finally, supershift experiments performed with nuclear extracts from LIF-stimulated cells and a C/EBP{delta} probe demonstrated that the complex contained activated CREB/ATF1 (Fig. 3DGo). At present, the ratio of CREB and ATF-1 bound to the CRE has not been further analyzed.



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Figure 2. LIF-R and IP-R Activation Induces CREB/ATF-1 Phosphorylation and Transcriptional Activity

A, Ob1771 and 3T3-F442A cells were maintained for 15 h in 1% FBS and then treated with 10 ng/ml LIF or 1 µM cPGI2 for the indicated times. Protein lysates (25 µg) were used for Western blot analysis using antibodies against phospho-CREB and CREB. A representative result from three independent experiments is shown. B, 3T3-F442A cells were cotransfected with p{Delta}MC16-firefly Luc and pEF-renilla Luc constructs and were untreated (C) or treated for 16 h with LIF (10 ng/ml) or cPGI2 (1 µM). The figure plots normalized firefly luciferase activity. The values represent the mean ± SEM of three independent experiments performed in triplicate.

 


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Figure 3. LIF and cPGI2 Stimulate Binding Activity at the Putative CRE of C/EBPß and C/EBP{delta} Promoters

A, The potential CREs present in C/EBPß and C/EBP{delta} promoters are indicated by the box-enclosed regions. Nucleotide sequences of consensus CRE (cons CRE) and mutated CRE (mutCRE) used for competition are shown. B–D, Ob1771 cells were first maintained for 15 h in 1%FBS medium (cont) and then stimulated with 10 ng/ml LIF and/or 1 µM cPGI2. B, Nuclear protein extracts were harvested after 30 min and tested for protein-DNA interactions with polynucleotide kinase-labeled probes corresponding to the putative CRE of C/EBPß and C/EBP{delta} promoters. C, Same as in panel B, except that competition occurred by the inclusion of 100x molar excess of the consensus CRE (consCRE) or mutant CRE (mutCRE) oligonucleotides. D, Same as in panel B, except the inclusion of CREB antibody (dilution 1:40) plus phosphorylated CREB antibody (dilution 1:40) to produce a supershift.

 
Induction of C/EBPß and C/EBP{delta} in Stably Transfected Preadipocytes Expressing Dominant-Negative Forms of CREB and ATF-1
To directly assess the involvement of CREB/ATF-1 in the up-regulation of the C/EBPß and C/EBP{delta} genes by LIF or cPGI2, stable retroviral transfection was used to generate Ob1771 and 3T3-F442A preadipocytes expressing dominant-negative forms of CREB. Both dominant-negative forms, KCREB and ProCREB block gene activation by titrating endogenous CREB and ATF-1 and preventing their interaction with the CRE. KCREB has an Arg-to-Leu amino acid substitution at position 301 in the DNA-binding domain and can not bind to DNA while it still able to heterodimerize with endogenous CREB and ATF-1 (20). ProCREB contains a substitution of Arg at the same position by Pro, resulting in comparable dominant negative activity as KCREB (20). As shown in Fig. 4Go, treatment of control-Ob1771 preadipocytes by LIF or cPGI2 led to an increase in the levels of C/EBPß and C/EBP{delta} mRNA as expected. LIF- or cPGI2-induced C/EBP{delta} and C/EBPß gene expression was substantially reduced in KCREB-expressing preadipocytes. Similar results were obtained by stimulating the cells with the bisoxazole BMY45778, a potent agonist of the IP-R (21). These observations were extended to stably transfected 3T3-F442A preadipocytes expressing ProCREB. Similar to the results obtained with KCREB, expression of ProCREB in preadipocytes reduced C/EBP{delta} stimulation by LIF or cPGI2. Interestingly, 3T3-F442A preadipocytes did not respond to BMY45778, which will be discussed later. Altogether, these data indicate that activation of CREB/ATF-1 is necessary for the induction of C/EBPß and C/EBP{delta} genes, via the LIF-R and IP-R. Since our previous results demonstrated that ERK and cAMP-related events were implicated in the up-regulation of C/EBP genes, their involvement in CREB/ATF-1 activity was next investigated.



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Figure 4. Expression of Dominant-Negative Forms of CREB in Ob1771 and 3T3-F442A Cells Inhibits the LIF-R and IP-R Mediated Induction of C/EBPß and C/EBP{delta}

AKV- or AKV-KCREB-expressing Ob1771 cells (A) and AKV- or AKV-proCREB-expressing 3T3-F442A cells (B) were maintained for 2 h in 1% FBS and stimulated with LIF (10 ng/ml), cPGI2 (1 µM), or BMY45778 (1 µM). After 1 h, total RNA was prepared for Northern blot analysis. Blots were exposed overnight. Quantification of the hybridization signal was performed as described in Materials and Methods, standardized to ß-actin RNA signal, and results are expressed by setting at 1 the signal obtained in the absence of stimulation. Results are the means of two independent experiments.

 
A Pathway Linking PKA to ERK Is Required for the Activation of C/EBP Gene Expression by IP-R
We have previously reported that the selective MAPK kinase (MEK) inhibitor UO126 inhibits LIF-induced expression of C/EBP{delta} and C/EBPß, suggesting that the ERK pathway is involved. Stimulation of IP-R by BMY45778 or cPGI2 resulted in the activation of C/EBP{delta} (Fig. 5AGo). This activation was PKA dependent as pretreatment with 7–10 µM of the PKA inhibitor H89 led to the complete inhibition of IP-R agonist-induced C/EBP{delta} expression. Surprisingly, complete inhibition was also observed by prior treatment with 10 µM of the MEK inhibitor U0126 (Fig. 5AGo). This observation strongly suggested that IP-R induced the activation of ERK. As shown in Fig. 5BGo, both IP-R agonists induced ERK phosphorylation. This phosphorylation was inhibited by the MEK-inhibitor U0126, and the PKA inhibitor H89 (Fig. 5BGo). Altogether, these results demonstrated that activation of ERK by PKA was required for C/EBP gene expression.



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Figure 5. ERK Activation Induced by IP-R Is Required for C/EBP{delta} Gene Induction

A, Ob1771 cells were maintained for 2 h in 1% FBS, pretreated for 20 min with U0126 or H89 at the indicated concentrations, and then stimulated with BMY45778 (1 µM) or cPGI2 (1 µM) for 1 h. Total RNA was prepared and C/EBP{delta} expression was analyzed by Northern blot. B, Left panel, Ob1771 cells were stimulated with BMY45778 (1 µM), cPGI2 (1 µM), or LIF (10 ng/ml) for the indicated times. DMSO, the vehicle of cPGI2 and BMY45778, was also used. Right panel, Ob1771 cells were pretreated with the inhibitors U0126 (10 µM) or H89 (7 µM) for 20 min before stimulation by BMY45778 (1 µM) for the indicated times. Cells were lysed and analyzed by Western blot for ERKs expression and phosphorylation. The results are representative of three independent experiments.

 
Numerous studies have demonstrated that CREB phosphorylation and transcriptional activity can occur through either PKA- or ERK-dependent pathways (17). Therefore, we examined the contribution of both pathways for CREB activation via IP-R. As shown, the PKA-inhibitor H89 blocked CREB phosphorylation and transcriptional activity (Fig. 6Go, A and B, respectively). In contrast, the MEK-inhibitor U0126 did not affect CREB phosphorylation (Fig. 6AGo), but still inhibited the transcriptional activity of CREB induced by IP-R activation (Fig. 6BGo). This implied that an ERK-induced event was required in addition to CREB phosphorylation for CREB-dependent transcription of C/EBP. This requirement of ERK activation was confirmed by another approach. In contrast to Ob1771 cells, which respond to BMY45778 and cPGI2 through IP-R by up-regulating C/EBPß and C/EBP{delta} genes, 3T3-F442A cells appeared fully responsive to cPGI2 but unresponsive to BMY45778 (not shown). This lack of responsiveness was not due to the lack of coupling of IP-R as phosphorylation of CREB/ATF-1 was clearly observed under these conditions (Fig. 7AGo). One possible explanation is that the pathway linking PKA to ERK may not be functional in these cells when exposed to BMY45778. To gain insight into the difference(s) between the two cell lines and into the critical role of the ERK pathway, the status of ERKs and their phosphorylated forms was examined after exposure to different concentrations of BMY45778. Under these conditions, ERK1 and ERK2 phosphorylation occurred in Ob1771 preadipocytes at levels as low as 20 nM, but remained undetectable in 3T3-F442A preadipocytes even at 500 nM (Fig. 7BGo). The expression of B-Raf has been shown to be required for ERK activation by the PKA pathway in a number of cell lines (22, 23). As shown in Fig. 7CGo, B-Raf and Raf-1, the kinase upstream of MEK, were expressed in both preadipose cell lines. Altogether, these results strongly suggested that the lack of induction of C/EBP gene expression in 3T3-F442A cells upon stimulation with BMY45778 was due to the absence of ERK activation. These results confirmed that an ERK-induced event was required, in addition to CREB/ATF-1 phosphorylation, for CREB/ATF-1-mediated transcription of C/EBP genes.



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Figure 6. PKA and ERK Signaling Pathways Mediate CREB Transcriptional Activity Induced by IP-R Activation

A, Ob1771 cells were pretreated for 20 min with U0126 (10 µM) or H89 (7 µM) and then stimulated with cPGI2 (1 µM) for the indicated times. Phosphorylation of CREB/ATF1 was observed by Western blot analysis and normalized by total CREB expression. The results are representative of four independent experiments. B, 3T3-F442A cells stably transfected with p{Delta}MC16-Luc reporter gene were pretreated for 20 min with U0126 (10 µM) or H89 (7 µM) before stimulation by cPGI2 (1 µM). Luciferase activity was measured after 24 h.

 


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Figure 7. BMY45778 Induced Phosphorylation of CREB/ATF1 but not ERK in 3T3-F442A Preadipocytes

A, 3T3-F442A preadipocytes were stimulated with BMY45778 (1 µM) for the indicated times. Cells were lysed and analyzed by Western blot for CREB/ATF1 phosphorylation. B, Ob1771 or 3T3-F442A cells were stimulated either for 5 min with cPGI2 (1 µM) or BMY 45778 at the indicated concentrations or for 10 min with LIF (10 ng/ml). Cells were lysed and analyzed by Western blot for ERK phosphorylation. C, Unstimulated 3T3-F442A and Ob1771 preadipose cells were lysed and analyzed by Western blot for B-Raf and Raf-1 expression. Molecular weights are indicated. The presence of B-Raf isoforms have been previously reported (50 ).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have previously shown that the C/EBPß and C/EBP{delta} genes are up-regulated by both LIF/LIF-R and prostacyclin/IP-R, which activate the ERK and the PKA signaling pathways, respectively. However, the specific signaling molecules and transcription factors linking the receptor-mediated events to increased gene expression had not been yet identified. Our findings indicate that the LIF and prostacyclin-activated pathways share CREB/ATF1 as a common downstream effector, which in turn increases CRE-mediated expression of C/EBPß and C/EBP{delta} genes.

Several previous studies have demonstrated that the transcription factors CREB and ATF1 can be activated by both the ERK and PKA signaling pathways. This led us to examine whether CREB/ATF1 is involved in LIF or prostacyclin-induced up-regulation of C/EBPß and C/EBP{delta} genes. We observed for the first time that LIF stimulation results in the phosphorylation of CREB/ATF1. Similarly, cPGI2 also induced the phosphorylation of CREB/ATF1. The phosphorylation of CREB by both stimuli was rapid, occurring within 10 min, and preceded the up-regulation of C/EBPß and C/EBP{delta}, which takes place between 30 and 60 min. We next wanted to demonstrate that the CREB/ATF1 phosphorylation resulted in an active transcription factor, which could affect C/EBP ß and C/EBP {delta} induction. Stimulation of preadipocyte cells with either LIF or cPGI2 resulted in the interaction between CREB/ATF1 and the putative CRE present on the C/EBP genes as determined by EMSA. In some systems, a constitutive binding of CREB to CRE sequences in the absence of stimulus has been reported (24), where phosphorylation of CREB results in the enhancement of transcriptional activation by the recruitment of additional coactivators (24, 25, 26). Alternatively, phosphorylation of CREB might promote binding to the CRE (27, 28). The ability of LIF and cPGI2 to enhance its binding activity, without inducing any change in overall CREB levels, suggested that binding of CREB to C/EBP promoters could be regulated by phosphorylation in preadipocytes.

The use of dominant-negative forms of CREB and ATF-1 (KCREB or ProCREB) reduced the LIF and cPGI2-stimulated transcription of C/EBPß and C/EBP{delta} genes. These data collectively demonstrate that LIF and cPGI2-induced signaling pathways converge upon CREB/ATF1, and that the activation of this factor is associated with an increased expression of the C/EBPß and C/EBP{delta} genes. This conclusion is strongly supported by a previous study which demonstrates a role for CREB in adipogenesis (18). Reush et al. (18) showed that cAMP elevating agents induced CREB phosphorylation and subsequently adipogenesis in 3T3-L1 preadipocytes. However, this study did not examine the effect of phospho-CREB on the expression of the C/EBP genes. Taken together, these results highlight the importance of CREB in adipogenesis, possibly by up-regulating the C/EBPß and C/EBP{delta} genes.

After demonstrating the importance of CREB activation in the up-regulation of the C/EBP genes, we studied the mechanisms by which CREB is activated. Using a specific MEK inhibitor (UO126), we found that the ERK pathway is required for CREB phosphorylation and C/EBP expression induced by LIF. Similarly, the inhibition of the PKA pathway using a PKA inhibitor abolished CREB/ATF1 phosphorylation and C/EBP gene up-regulation in response to BMY45778 and cPGI2. These results were expected, as LIF and cPGI2 are known to signal through the ERK and PKA pathways, respectively. We were surprised, however, when the addition of the MEK inhibitor resulted in the suppression of C/EBP up-regulation after IP-R activation, as IP-R typically signals through PKA and does not directly require the ERK signaling cascade. This result demonstrated that ERK activation is an additional requirement for C/EBP gene up-regulation.

Previous results showed that preadipocytes secrete basal levels of LIF, which might then activate basal levels of ERK in an autocrine manner. We thus studied C/EBP gene activation using MEFs with targeted deletions in the gp130 and ip-r genes, enabling us to study the two signaling pathways in isolation. We clearly demonstrate that either ligand-receptor system alone is capable of inducing the C/EBPs in the absence of the other. Therefore, LIF-mediated ERK activation is not required for C/EBP expression induced via IP-R. It thus appears that the ERK pathway is directly activated by the IP-R ligands. A direct activation of the ERK cascade by the IP-R ligands was confirmed by the observation that there is a rapid phosphorylation of ERK after BMY45778 or cPGI2 treatment of Ob1771 preadipocytes. This phosphorylation was inhibited by the MEK-specific inhibitor, UO126, as well as by the PKA inhibitor, H89. This led us to propose a pathway linking PKA to ERK. While PKA is sufficient to phosphorylate CREB/ATF1, the activation of the ERK pathway is critical for the final transactivation of the C/EBP genes by IP-R activation.

We were able to confirm this result using another approach. Both cPGI2 and BMY45778 are considered to be high-affinity ligands for IP-R. The clonal preadipocyte cell line Ob1771 showed an up-regulation of C/EBP ß and C/EBP {delta} after stimulation by both ligands. However, while 3T3-F442A preadipocytes and mouse embryonic fibroblasts show a response to cPGI2, there is no change in C/EBP gene expression after BMY45778 stimulation. In 3T3-F442A cells we found that CREB is still phosphorylated, indicating that the PKA is still activated by BMY45778 but ERK phosphorylation was undetectable. Using this cell model, we confirmed that PKA-induced CREB phosphorylation is not sufficient for C/EBP expression and that ERK activation is additionally required.

A weaker effect of the agonist BMY45778 compared with cPGI2 has been previously described in the rat colon (29), which led the authors to propose that BMY45778 interacts with a novel subtype of IP-R. This proposal was supported by studies in the rat brain showing different binding properties of several prostacyclin analogs (30). Our results obtained in 3T3-F442A preadipocytes could thus be explained if BMY45778 induced a weak activation of IP-R leading to a PKA activation that was sufficient for CREB phosphorylation but not for ERK pathway activation. Future studies will address this hypothesis.

To date, the cAMP-induced stimulation of ERK activity has been reported in neuronal and PC12 cells (22, 23, 31, 32), lymphoma cells (33, 34), and melanocytes (35, 36). In preadipocytes the step in which PKA activity is required to promote MEK activity remains unclear. Because a MEK inhibitor blocked the IP-R-mediated activation of ERK, we can envision various regulatory points upstream of MEK linking PKA activity to the ERK signaling pathways. The different isotypes of Raf kinase, which is the major MEK activator, are known to be regulated by PKA (37). The expression of B-Raf is generally required for ERK activation by the PKA pathway (23). Raf-1 and B-Raf are expressed in 3T3-F442A and Ob1771 cell lines (see Fig. 7CGo). However, their role in PKA-dependent activation of ERK in preadipose cells remains to be investigated.

Our results show that to obtain CREB transcriptional activity, ERK activation is required in addition to CREB phosphorylation. We suggest that ERK activation is required for the regulation of CREB activity itself and does not activate other transcription factors for C/EBP induction as we show that the MEK inhibitor (UO126) blocked the cPGI2 up-regulation of a 16xCRE-luciferase reporter construct. This suggests that the ERK activation is required for CREB activity possibly by recruitment of a coactivator. The ability of MEK inhibitors to block CREB-mediated transcription downstream of CREB phosphorylation has been previously reported in PC12 cells (38) and in NIH 3T3 fibroblasts (39). Several transcriptional coactivators leading to CREB- mediated transcription have been identified (40, 41). The transcriptional coactivator CREB binding protein (CBP) is a putative candidate as it can associate with ERK (42), an ERK-mediated phosphorylation of CBP stimulates its histone acetyl transferase activity (43), and it has been proposed that the ERK pathway increases CBP activity (38).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Embryos from C57 BL/6J mice deficient for the prostacyclin receptor gene (ip-r-/-) at 14.5 d post coitus were used to prepare fibroblasts after removing head, heart, and legs. Embryos of strain (CD1) mice deficient for the gp130 gene (gp130-/-) were prepared similarly. Mouse embryo fibroblasts (MEF) thus obtained were termed ip-r-/- (44) and gp130-/- MEF, respectively.

Cell Cultures
ip-r-/- and gp130-/- MEF were used at passage 2 or 3 and grown to confluence at 37 C in H16 medium supplemented with 1x MEM nonessential amino acids, 2 mM glutamine, 1 mM pyruvate, and 10% heat-inactivated FBS (Dutcher, S. A., France). Cells from Ob1771 (45) and 3T3-F442A (46) preadipocyte clonal lines were grown to confluence in H16 medium containing 8% FBS. BOSC23 cells were grown in a 100-mm diameter culture dish in the presence of 10 ml of H16 medium containing 8% FBS.

Plasmids and Plasmid Constructions
The pCG expression vector containing KCREB or proCREB, two dominant-negative forms of CREB (20), was digested with BamHI and XbaI, and the resulting insert was ligated into the BlueScript vector. The resulting vector was then digested with SacI and HincII, and the insert was ligated in the retroviral AKV bipe 2 vector. KCREB was obtained as described previously (20), and ProCREB was obtained by substituting Arg by Pro on position 301. The p{Delta}M-C16-Luc expression vector (47) was a kind gift of J. C. Chambard (Nice, France).

Luciferase Assays
3T3-F442A Preadipose cells were seeded at 5 x 104 cells per 35-mm diameter well. After 24 h, transient transfections were performed using the FuGene 6 transfection system (Roche Molecular Biochemicals, Indianapolis, IN). Transfection mixes for each well contained 3 µg of p{Delta}M-C16-firefly Luc construct (47) and 60 ng of pEF-renilla Luc as internal control for normalizing firefly luciferase activities. The p{Delta}M-C16-firefly Luc construct contained 16 repeated CREs. Two days after transfection, cells reached confluence and were stimulated for 16 h with the appropriate effectors. Cells were lysed and both luciferase activities were determined using the Dual-Luciferase reporter Assay System following the manufacturer’s recommendations (Promega Corp., Madison, WI).

Retroviral Infection
For retroviral infection experiments, 8 x 105 BOSC23 cells were plated in a 100-mm diameter culture dish. Twenty four hours later, exponentially growing cells were transfected with 6 µg of AKV or AKV-KCREB/proCREB plasmids. The virus-containing media (10 ml) were collected and centrifuged for 5 min at 1,000 x g, and the supernatant was filtered through 0.45-µm pore size filters. 3T3-F442A and Ob1771 preadipose cells in 100-mm diameter culture dishes were infected at 50% confluence. After 24 h, infection media were removed and replaced every other day by fresh H16 medium containing 8% FBS and 400 µg/ml of G418. One week later, clones of cells were pooled and used for Northern blot analysis.

Nuclear Extracts and Gel Retardation Assays
Probes were generated by adding 60 ng of double-stranded oligonucleotide to T4-polynucleotide kinase buffer [0.5 M Tris-HCl, pH 7.6, 0.1 M MgCl2, 10 mM dithiothreitol (DTT), 10 mM EDTA, 25% polyethylene glycol (wt/vol)] with 1 µl {gamma}-32P-ATP and 5 U T4-polynucleotide kinase. The EMSA was performed by incubation of the radiolabeled probe (70,000 cpm) with 4 µg of nuclear extract in 1x binding buffer [10 mM Tris-HCl, pH 7.5, containing 50 mM NaCl, 0.5 mM DTT, 5 mM MgCl2, and 5% (vol/vol) glycerol] in the presence of a nonspecific competitor [0.5 µg poly(dI-dC)(dI-dC) (Sigma, St. Louis, MO)], in a final reaction volume of 20 µl. The binding reaction was electrophoresed on a 6% polyacrylamide gel in 1x TBE buffer (90 mM Tris-HCl, pH 8.0, 90 mM boric acid, 2 mM EDTA). The gels were run for 1.5 h at 10 V/cm2 at 4 C to retain the stability of the protein-DNA complexes.

RNA Analysis and DNA Probes
RNA was prepared and analyzed as previously described (48). Quantification of the hybridization signal was performed using a PhosphorImager screen (x-Bas1000, Fuji Photo Film Co., Ltd., Stamford, CT) coupled to the MacBas ver2.x bio-imaging analyzer. The C/EBPß cDNA and the C/EBP{delta} cDNA were isolated from MSV/C/EBPß and MSV/C/EBP{delta} plasmids (kindly provided by S. L. McKnight, Tularik Inc., San Francisco, CA).

Western Blot Analysis
Confluent Ob1771 and 3T3-F442A cells were first maintained for 12 h in H16 medium containing 1% FBS, and then treated with various effectors as indicated. Subsequently, cells were lysed with a mixture containing 50 mM Tris-HCl buffer, pH 7.5, 1% Triton X-100, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM ß-glycerophosphate, 2 µM sodium orthovanadate, 1 mM DTT, and 1x protease inhibitor cocktail (Roche, Paris, France). Proteins were separated on 10% SDS-polyacrylamide gels (30 µg of protein per lane) and transferred on polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Copenhagen, Denmark). Membranes were soaked for 1 h in Tris-HCl buffer, pH 7.5, containing 150 mM NaCl, 0.1% Tween-20, and 5% defatted milk. Total CREB and phosphorylated CREB were detected with primary antibodies raised in rabbits and a secondary peroxidase-conjugated antirabbit antibody according to the manufacturer’s instructions (New England Biolabs, Inc., Beverly, MA). Monoclonal antibody directed against the dually phosphorylated form of ERK1 and ERK2 was used according to the manufacturer’s procedure (Sigma). Polyclonal antibody against total ERKs and their use have been described (49). Polyclonal antibodies directed against B-Raf and Raf-1 were purchased from Santa Cruz Biotechnology, Inc. and used according to the manufacturer’s instructions.

Materials
Leukemia Inhibitory Factor (LIF) was obtained from Euromedex (France). cPGI2 was from Cayman Chemicals (Spibio, France). BMY45778 was a kind gift from N. Meanwell (Bristol-Myers Squibb Co., Princeton, NJ). The MEK inhibitor UO126 was purchased from Promega Corp. and the PKA inhibitor H89 was from FranceBiochem (France). Oligonucleotides were synthesized by Amersham Pharmacia Biotech (Denmark). All other chemicals were from Sigma-Aldrich (France).


    ACKNOWLEDGMENTS
 
The secretarial assistance of G. Oillaux is gratefully acknowledged. We are indebted to Drs. P. Lenormand (CNRS UMR 6543, Nice) and R. Busca (INSERM U385, Nice) for helpful discussion and to Drs. K. Yeow and R. Arkowitz for critical reading of the manuscript.


    FOOTNOTES
 
This work was supported by a special grant of the Bristol-Myers Squibb Co. Foundation to G. Ailhaud and a grant from Association pour la Recherche sur le Cancer (ARC) (to C.D.) (Grant 9982). B.W.P. was a recipient of an ARC fellowship.

Abbreviations: ATF-1, Activation transcription factor 1; CBP, CREB-binding protein; C/EBP, CCAAT/enhancer binding protein; cPGI2, (carba)prostacyclin; CRE, cAMP-responsive element; CREB, cAMP-responsive element binding protein; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; IP-R, prostacyclin receptor; LIF, leukemia-inhibitory factor; LIF-R, LIF receptor; MEFS, mouse embryonic fibroblasts; P-CREB, (Ser133)-phospho-CREB; PGI2, prostacyclin.

Received for publication March 2, 2001. Accepted for publication July 10, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. G Ailhaud, Amri E, Bertrand B, Barcellini-Couget S, Bardon S, Catalioto RM, Dani C, Deslex S, Djian P, Doglio A, Pradines-Figueres A, Forest C, Gaillard D, Grimaldi P, Negrel R, Vannier C 1990 Cellular and molecular aspects of adipose tissue growth. In: Bray G, Riquier D, Spiegelman B, eds. Obesity: towards a molecular approach. New York: Alan R. Liss, Inc; 219–236
  2. Ailhaud G, Hauner H 1998 Development of white adipose tissue. In: Bray G, Bouchard C, James PT, eds. Handbook of obesity. New York: M. Dekker Inc.; 359–378
  3. Hauner H, Entenmann G, Wabitsch M, Gaillard D, Ailhaud G, Negrel R, Pfeiffer EF 1989 Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically defined medium. J Clin Invest 84:1663–1670[Medline]
  4. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM 2000 Transcriptional regulation of adipogenesis. Genes Dev 14:1293–1307[Free Full Text]
  5. Amri EZ, Bonino F, Ailhaud G, Abumrad NA, Grimaldi PA 1995 Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. Homology to peroxisome proliferator-activated receptors. J Biol Chem 270:2367–2371[Abstract/Free Full Text]
  6. Bastie C, Holst D, Gaillard D, Jehl-Pietri C, Grimaldi PA 1999 Expression of peroxisome proliferator-activated receptor PPAR{delta} promotes induction of PPAR{gamma} and adipocyte differentiation in 3T3C2 fibroblasts. J Biol Chem 274:21920–21925[Abstract/Free Full Text]
  7. Ailhaud G 1999 Cell surface receptors, nuclear receptors and ligands that regulate adipose tissue development. Clin Chim Acta 286:181–190[CrossRef][Medline]
  8. Xu HE, Lambert MH, Montana VG, Parks DJ, Blanchard SG, Brown PJ, Sternbach DD, Lehmann JM, Wisely GB, Willson TM, Kliewer SA, Milburn MV 1999 Molecular recognition of fatty acids by peroxisome proliferator-activated receptors. Mol Cell 3:397–403[Medline]
  9. Kim JB, Spiegelman BM 1996 ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev 10:1096–1097[Abstract]
  10. Kim JB, Sarraf P, Wright M, Yao KM, Mueller E, Solanes G, Lowell BB, Spiegelman BM 1998 Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1. J Clin Invest 101:1–9[Abstract/Free Full Text]
  11. Wu Z, Xie Y, Bucher NL, Farmer SR 1995 Conditional ectopic expression of C/EBPß in NIH-3T3 cells induces PPAR{gamma} and stimulates adipogenesis. Genes Dev 9:2350–2363[Abstract]
  12. Lane MD, Tang QQ, Jiang MS 1999 Role of the CCAAT enhancer binding proteins (C/EBPs) in adipocyte differentiation. Biochem Biophys Res Commun 266:677–683[CrossRef][Medline]
  13. Tanaka T, Yoshida N, Kishimoto T, Akira S 1997 Defective adipocyte differentiation in mice lacking the C/EBPß and/or C/EBP{delta} gene. EMBO J 16:7432–7443[Abstract/Free Full Text]
  14. Aubert J, Dessolin S, Belmonte N, Li M, McKenzie FR, Staccini L, Villageois P, Barhanin B, Vernallis A, Smith AG, Ailhaud G, Dani C 1999 Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade. J Biol Chem 274:24965–24972[Abstract/Free Full Text]
  15. Aubert, J, Saint-Marc P, Belmonte N, Dani C, Negrel R, Ailhaud G 2000 Prostacyclin IP receptor up-regulates the early expression of C/EBPß and C/EBP{delta} in preadipose cells. Mol Cell Endocrinol 160:149–156[CrossRef][Medline]
  16. De Cesare D, Fimia GM, Sassone-Corsi P 1999 Signaling routes to CREM and CREB: plasticity in transcriptional activation. Trends Biochem Sci 24:281–285[CrossRef][Medline]
  17. Shaywitz AJ, Greenberg ME 1999 CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem 68:821–861[CrossRef][Medline]
  18. Reusch JE, Colton LA, Klemm DJ 2000 CREB activation induces adipogenesis in 3T3–L1 cells. Mol Cell Biol 20:1008–1020[Abstract/Free Full Text]
  19. Klemm DJ, Roesler WJ, Boras T, Colton LA, Felder K, Reusch JE 1998 Insulin stimulates cAMP-response element binding protein activity in HepG2 and 3T3–L1 cell lines. J Biol Chem 273:917–923[Abstract/Free Full Text]
  20. Saeki K, Yuo A, Suzuki E, Yazaki Y, Takaku F 1999 Aberrant expression of cAMP-response-element-binding protein (’CREB’) induces apoptosis. Biochem J 343:249–255[CrossRef][Medline]
  21. Wise H, Jones RL 1996 Focus on prostacyclin and its novel mimetics. Trends Pharmacol Sci 17:17–21[CrossRef][Medline]
  22. Dugan LL, Kim JS, Zhang Y, Bart RD, Sun Y, Holtzman DM, Gutmann DH 1999 Differential effects of cAMP in neurons and astrocytes. Role of B-raf. J Biol Chem 274:25842–25848[Abstract/Free Full Text]
  23. Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ 1997 cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 89:73–82[Medline]
  24. Quinn PG, Granner DK 1990 Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element. Mol Cell Biol 10:3357–3364[Medline]
  25. Quinn PG 1993 Distinct activation domains within cAMP response element-binding protein (CREB) mediate basal and cAMP-stimulated transcription. J Biol Chem 268:16999–17009[Abstract/Free Full Text]
  26. Felinski EA, Kim J, Lu J, Quinn PG 2001 Recruitment of an RNA polymerase II complex is mediated by the constitutive activation domain in CREB, independently of CREB phosphorylation. Mol Cell Biol 21:1001–1010[Abstract/Free Full Text]
  27. Yamamoto KK, Gonzalez GA, WH III Biggs, Montminy MR 1988 Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature 334:494–498[CrossRef][Medline]
  28. Nichols M, Weih F, Schmid W, DeVack C, Kowenz-Leutz E, Luckow B, Boshart M, Schutz G 1992 Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription. EMBO J 11:3337–3346[Abstract]
  29. Wise H, Qian YM, Jones RL 1995 A study of prostacyclin mimetics distinguishes neuronal from neutrophil IP receptors. Eur J Pharmacol 278:265–269[CrossRef][Medline]
  30. Takechi H, Matsumura K, Watanabe Y, Kato K, Noyori R, Suzuki M 1996 A novel subtype of the prostacyclin receptor expressed in the central nervous system. J Biol Chem 271:5901–5906[Abstract/Free Full Text]
  31. Qiu W, Zhuang S, von Lintig FC, Boss GR, Pilz RB 2000 Cell type-specific regulation of B-Raf kinase by cAMP and 14–3-3 proteins. J Biol Chem 275:31921–1929[Abstract/Free Full Text]
  32. Zanassi P, Paolillo M, Feliciello A, Avvedimento EV, Gallo V, Schinelli S 2001 cAMP-Dependent protein kinase induces cAMP-response element-binding protein phosphorylation via an intracellular calcium release/ERKdependent pathway in striatal neurons. J Biol Chem 276:11487–11495[Abstract/Free Full Text]
  33. Saxena M, Williams S, Tasken K, Mustelin T 1999 Crosstalk between cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase. Nat Cell Biol 1:305–311[CrossRef][Medline]
  34. Wan Y, Huang XY 1998 Analysis of the Gs/mitogen-activated protein kinase pathway in mutant S49 cells. J Biol Chem 273:14533–14537[Abstract/Free Full Text]
  35. Englaro W, Rezzonico R, Durand-Clement M, Lallemand D, Ortonne JP, Ballotti R 1995 Mitogen-activated protein kinase pathway and AP-1 are activated during cAMP-induced melanogenesis in B-16 melanoma cells. J Biol Chem 270:24315–24320[Abstract/Free Full Text]
  36. Busca R, Abbe P, Mantoux F, Aberdam E, Peyssonnaux C, Eychene A, Ortonne JP, Ballotti R 2000 Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes. EMBO J 19:2900–2910[Abstract/Free Full Text]
  37. Hagemann C, Rapp UR 1999 Isotype-specific functions of Raf kinases. Exp Cell Res 253:34–46[CrossRef][Medline]
  38. Grewal SS, Fass DM, Yao H, Ellig CL, Goodman RH, Stork PJ 2000 Calcium and cAMP signals differentially regulate cAMP-responsive element-binding protein function via a Rap1-extracellular signal-regulated kinase pathway. J Biol Chem 275:34433–34441[Abstract/Free Full Text]
  39. Seternes OM, Johansen B, Moens U 1999 A dominant role for the Raf-MEK pathway in forskolin, 12-O-tetradecanoyl-phorbol acetate, and platelet-derived growth factor-induced CREB (cAMP-responsive element-binding protein) activation, uncoupled from serine 133 phosphorylation in NIH 3T3 cells. Mol Endocrinol 13:1071–1083[Abstract/Free Full Text]
  40. Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859[CrossRef][Medline]
  41. Fimia GM, De Cesare D, Sassone-Corsi P 2000 A family of LIM-only transcriptional coactivators: tissue-specific expression and selective activation of CREB and CREM. Mol Cell Biol 20:8613–8622[Abstract/Free Full Text]
  42. Liu YZ, Thomas NS, Latchman DS 1999 CBP associates with the p42/p44 MAPK enzymes and is phosphorylated following NGF treatment. Neuroreport 10:1239–1243[Medline]
  43. Ait-Si-Ali S, Carlisi D, Ramirez S, Upegui-Gonzalez LC, Duquet A, Robin P, Rudkin B, Harel-Bellan A, Trouche D 1999 Phosphorylation by p44 MAP Kinase/ERK1 stimulates CBP histone acetyl transferase activity in vitro. Biochem Biophys Res Commun 262:157–162[CrossRef][Medline]
  44. Murata T, Ushikubi F, Matsuoka T, Hirata M, Yamasaki A, Sugimoto Y, Ichikawa A, Aze Y, Tanaka T, Yoshida N, Ueno A, Oh-ishi S, Narumiya S 1997 Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature 388:678–682[CrossRef][Medline]
  45. Doglio A, Dani C, Grimaldi P, Ailhaud G 1986 Growth hormone regulation of the expression of differentiation-dependent genes in preadipocyte Ob1771 cells. Biochem J 238:123–129[Medline]
  46. Green H, Kehinde O 1976 Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell 7:105–113[Medline]
  47. D Spengler, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L 1993 Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170–175[CrossRef][Medline]
  48. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef][Medline]
  49. Brondello JM, Brunet A, Pouyssegur J, McKenzie FR 1997 The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44MAPK cascade. J Biol Chem 272:1368–1376[Abstract/Free Full Text]
  50. Barnier JV, Papin C, Eychene A, Lecoq O, Calothy G 1995 The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. J Biol Chem 270:23381–23389[Abstract/Free Full Text]