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,
Martine Parren-van Amelsvoort,1
Andrea Kolbus,2,4
Kazuo Yamamoto,3
Peter Steinlein,2
Roel G. W. Verhaak,1
Tak W. Mak,3
Hartmut Beug,2
Bob Löwenberg,1 and
Marieke von Lindern1*
Department of Hematology, Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands,1 Research Institute of Molecular Pathology, A-1030 Vienna, Austria,2 The Campbell Family Institute for Breast Cancer Research, University Health Network, University of Toronto, Toronto, Ontario M5G 2C1, Canada,3 Department of Obstetrics and Gynecology, Medical University of Vienna, Vienna, Austria4
Received 6 September 2006/ Returned for modification 8 October 2006/ Accepted 20 February 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Erythroblasts can be expanded in vitro using serum-free medium supplemented with erythropoietin (Epo), stem cell factor (SCF), and glucocorticoids, which reflects the in vivo expansion of erythroblasts under stress conditions (7, 12, 25, 70). Immortal cultures of erythroblasts can reproducibly be established from p53–/– mice (60, 70). These cultures remain dependent on Epo, SCF, and glucocorticoids for their expansion and retain the ability to undergo complete differentiation into erythrocytes in the presence of Epo. The expansion of these cultures is dependent on Epo-induced activation of the tyrosine kinase receptor Ron/Stk (60), whereas differentiation relies on Epo-induced Stat5 phosphorylation (26). Both Epo and SCF activate the phosphatidylinositol 3-kinase (PI3K)-PKB pathway, although SCF induces phosphorylation of PKB more strongly (70). The inhibition of PI3K abrogates Epo/SCF-induced expansion of in vitro cultures, inducing differentiation instead, suggesting that pathways downstream of PI3K-PKB control the proliferation of erythroblasts (70). This was corroborated by in vivo experiments. Mice lacking the PI3K subunit p85 displayed transient fetal anemia with reduced numbers of burst-forming units-erythroid and CFU-erythroid (37). The lack of p85 did not increase apoptosis of erythroblasts and mast cells but decreased proliferation (29, 37, 48). Foxo3a–/– mice displayed compensated anemia with reticulocytosis, suggesting normal expansion but defects in erythrocyte maturation (18).
Both SCF and Epo were able to inhibit the expression of Foxo3a target genes Cdkn1b (p27KIP) and Btg1. Nevertheless, SCF delays erythroid differentiation, while Epo enables erythroid differentiation. By consequence, Foxo3a targets may be differentially regulated by Epo and SCF. Two lines of evidence support this. First, Foxo proteins integrate a variety of signaling pathways, and examples show cooperation with transforming growth factor ß signaling, I
B kinase, Wnt signaling, and the Jak-Stat pathway (27, 36, 44, 61). Furthermore, Foxo transcription factors can mediate gene expression independent of their DNA binding ability, underlining the importance of transcriptional coregulators (57, 68). Second, although Epo and SCF have overlapping functions (i.e., activation of the PI3K and Ras-mitogen-activated protein kinase pathways), there are also differences. Epo specifically activates the Jak-Stat pathway and the tyrosine kinase receptor Ron, which recruits the adaptor Gab1 (21, 67). Other targets are induced by both Epo and SCF but may have stimulus-specific effects. Epo and SCF activate Btk, but only SCF-induced Btk protects from Trail-induced apoptosis (60). Along similar lines, Epo and SCF may induce the differential regulation of Foxo3a target genes.
Finally, it is not known to what extent Foxo3a is involved in Epo/SCF-mediated repression of gene expression following factor deprivation (42), nor do we know to what extent all Foxo3a target genes are regulated by Epo/SCF-induced activation of the PI3K-PKB pathway. To analyze the relation between Epo- and SCF-controlled signal transduction and Foxo3a activity, we investigated Foxo3a-, Epo-, and SCF-induced gene expression on an expressed-sequence-tag (EST) microarray containing 17,000 cDNAs (17K EST cDNA array). We found that Foxo3a target genes are differentially affected by growth factor stimulation, and we analyzed two clusters of Foxo3a-upregulated genes in more detail. One cluster encompasses target genes that are repressed by Epo/SCF and upregulated in differentiation (Cdkn1b/p27Kip, Btg1, Ccng2/Cyclin G2, and Ulk1), while the other cluster contains genes that are less obviously inhibited by Epo/SCF and that are not upregulated during differentiation (Dcn, Sesn1, and Cited2). Interestingly, Cited2 appeared to be a Foxo3a target gene that was induced instead of repressed by Epo. Data presented demonstrate that the alleged growth-stimulatory transcription factor Stat5 cooperates with the alleged growth-inhibitory transcription factor Foxo3a to control the expression of Cited2. In contrast, the upregulation of Btg1 during differentiation appeared to be reinforced by the cooperation of Foxo3a with the cyclic AMP (cAMP)-responsive transcription factor CREB/ATF1. Our data imply that Foxo3a functions to integrate and transmit multiple signals that cooperate to regulate the gene expression program of erythroblasts.
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MATERIALS AND METHODS |
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cDNA array hybridizations and analysis. Total RNA was generated from cells treated with or without 4OHT. Dual labeling was used to hybridize the cDNAs pairwise to a custom-made 17K EST cDNA microarray. Profiles for factor-deprived and Epo- and SCF-restimulated cells were obtained in the same experiment but were reported previously (42). For a description of the procedures compliant with minimum information about a microarray experiment, see the supplemental material. Hierarchical clustering of data was performed and visualized with the Spotfire application using Euclidean distance and average linkage to assess distance.
Real-time Q-PCR. cDNA synthesis and quantitative reverse transcription-PCR were performed using TaqMan technology (PE Applied Biosystems model 7700 or 7900 sequence detector) and SYBR green detection (Applied Biosystems) of double-stranded DNA as described previously (42). For primers, see Table 1. The threshold cycle values of the RNase inhibitor were used for normalization. Melting curves were performed to determine the specificity of the quantitative PCR (Q-PCR) product.
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Constructs.
To construct the expression plasmids of Foxo3a deletion mutants, portions of Foxo3a were amplified by PCR using the following primers: 5'-AATGGATCCGGAAAAGCCCCCCGGCGGC-3' (forward) and 5'-AATCTCGAGTTCAGCCTGGCACCCAGCTCT-3' (reverse) for 
N (G244 to the end), 5'-AATGGATCCGCAGAGGCACCGGCTTCCCC-3' (forward) and 5'-AATCTCGAGTTCACTTGCTTACTGAAGGTGACAGG-3' (reverse) for the C mutant (the first Met to K360), and 5'-AATGGATCCGCGGCTGGGGGCTCCGGGCA-3' (forward) and 5'-AATCTCGAGTTCACTTGCTTACTGAAGGTGACAGG-3' (reverse) for the NC mutant. PCR products were digested with BamHI and XhoI (both sites are included in the forward and the reverse primers described above, respectively) and subcloned into the corresponding sites of the Myc-tagged fusion protein expression vector pcDNA3/Myc (53). The resultant plasmids were verified by sequencing for the amplified regions and the junctions with the vector. The dominant negative Stat5 construct was described previously (72).
The mouse Cited2 promoter was cloned into pGL3-basic (Promega) using primers 5'-CCTATTGCTCCACTGAACAAT-3' (forward) and 5'-CTCACCTTCCGTCTTTGCGATTTC-3' (reverse) and the Expand High Fidelity PCR system (Roche). For promoter alignment between human (GenBank accession number AF129290) and murine (accession number NT039491) Cited2, we used the DNAMAN program, version 5.2.9. The transcriptional start site has been adapted from the published human Cited2 promoter, and the positions of Foxo and Stat5 binding sites were numbered accordingly (47). Mutations in the Cited2 promoter were made using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. The Daf16 binding element (DBE) was mutated using forward primer 5'-GATCGCTGAGTTTAAATACAGAGCAGGGAC-3'; the Stat5 site in the Cited2 promoter was mutated using forward primer 5'-TGCTCCACTGAACAATTCAAGCTTCAAGGAAGACTAGTAGC-3'. The cAMP-responsive element (CRE) was mutated using forward primer 5'-AGCAGATTTGGTCAGCTCCTC-3'. The respective opposite strand was used as the reverse primer.
Luciferase reporter assays. Ba/F3 cells (10 x 106 cells) were electroporated (0.28 kV; capacitance, 960 µFD) with a maximum of 20 µg of DNA. After recovery for several hours in normal medium, cells were washed and grown overnight in the presence of SCF and stimulated for 7 h with IL-3 and/or LY294002 the next day. Luciferase activity was measured using the Steady-Glo system (Promega). Transfection efficiency was determined by cotransfecting lacZ and analyzing ß-galactosidase activity.
ChIP assay. Chromatin immunoprecipitation (ChIP) assays were performed by using an acetyl-histone H3 immunoprecipitation kit according to the manufacturer's instructions (catalog no. 17-245; Upstate). Approximately 75 x 106 erythroblasts (I/11) were used for a specific condition. DNA was purified by phenol-chloroform extraction and dissolved in 200 µl water. Two microliters was used for PCR. Primers used are listed in Table 1. Antibodies to precipitate complexes harboring Foxo3a (sc-11251), Stat5 (sc835), and c-myc (9E10) were obtained from Santa Cruz.
EMSA. Nuclear extracts were prepared and EMSA was performed as described previously by using the oligonucleotide probes shown in Table 1 (65).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All targets were induced two- to fourfold within 2 h in two independent Foxo3a(A3):ER clones (F17 and F18) but not in control cells (Fig. 2A). Quantitative expression analysis following factor deprivation and subsequent restimulation with Epo, SCF, or Epo/SCF of I/11 cells confirmed that the expression of cluster A targets Btg1, Ccng2, and Ulk1 was subject to PI3K-dependent repression by Epo, SCF, and Epo/SCF (Fig. 2B). In contrast, cluster D targets Dcn and Sesn1 were less-than-twofold downregulated by Epo/SCF, and inhibition of PI3K had no effect on their expression (Fig. 2B). Notably, the expression of Cited2 was repressed by SCF but upregulated by Epo and by Epo plus SCF. Inhibition of PI3K did not affect Epo-induced upregulation of Cited2 but further increased Epo/SCF-induced expression. Thus, not all Foxo3a-targeted genes showed the predictable response to PI3K activity during factor deprivation and restimulation. We next examined whether PI3K-dependent expression correlates with expression during differentiation. We have demonstrated increased Foxo3a expression during erythroid differentiation, with a concurrent decrease in PKB activity and Foxo3a phosphorylation (4). This decrease in PKB phosphorylation during differentiation correlates with EpoR activity. Epo-induced signaling occurs in early phases of differentiation but does not affect late differentiation (71). Consequently, the expression of Btg1 and Cdkn1b sharply increases from the moment unphosphorylated Foxo3a accumulates in differentiating erythroblasts (4). The expression of the cluster A genes Ccng2 and Ulk1 similarly increased 48 h after differentiation induction. However, none of the cluster D genes was significantly upregulated during differentiation (Fig. 2C).
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Epo induces a nuclear Foxo3a/Stat5 complex. To examine the differential regulation of Foxo3a targets in the presence of Epo, we investigated the expression of Cited2. Cited2 is a transcriptional cofactor that is able to bind CBP/p300, which results in the inhibition of Hif1. A Cited2 deficiency results in multiple developmental failures resulting in early embryonal lethality (5, 73, 74). Cited2 is a cytokine- and growth factor-inducible gene transcriptionally controlled by the Jak/Stat pathway (62). Because Epo robustly activates Stat5 in erythroblasts, we examined a possible association between endogenous Foxo3a and Stat5. Notably, Epo causes partial and SCF causes almost complete phosphorylation of Foxo3a, whereas Stat5 is phosphorylated exclusively by Epo (4, 70). Following factor deprivation and restimulation with Epo and/or SCF, Foxo3a appeared to coimmunoprecipitate with Stat5 from total cell lysate under all conditions (Fig. 3A). However, Foxo3a-Stat5 complexes in the nucleus were detected specifically in lysates of Epo-stimulated cells (Fig. 3A). With respect to gene regulation, the association of Foxo3a and Stat5 in the nucleus is important. Therefore, we examined the role of Epo and SCF in the cellular distribution of Foxo3a, Stat5, and the Foxo3a-Stat5 complex. Both Epo and SCF induced transient translocation of Foxo3a from the nucleus to the cytoplasm, but Epo induced partial phosphorylation of Foxo3a and consequently only partial exclusion of Foxo3a from the nucleus. In the presence of Epo plus SCF, phosphorylation and nuclear exclusion of Foxo3a were nearly complete (Fig. 3B). Foxo3a always coimmunoprecipitated with Stat5 in cytoplasmic fractions (exposure of cytoplasmic and nuclear fraction is not equal; phosphorylated and nonphosphorylated Foxo3a show as one band in Fig. 3C). However, the nuclear localization of Stat5 and nuclear Foxo3a/Stat5 association were induced by Epo (Fig. 3C). Nuclear extracts stained with the Foxo3a antibody show a slower-migrating, aspecific background band in serum-starved cells (t = 0 min), as shown before (Fig. 3C) (4).
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N), a C-terminal deletion mutant lacking amino acids 361 to 673 (C), and a mutant containing mainly the DNA-binding domain (NC) (Fig. 3D). The Foxo3a constructs were Myc tagged and expressed together with Stat5 in 293T cells (Fig. 3E to H). Expression of the N Foxo3a mutant was low compared to that of other constructs, probably due to a less stable protein product, because other N mutants tested were also expressed at lower levels (data not shown). Stat5 was efficiently immunoprecipitated from total lysates (Fig. 3F, lower panel), and no Foxo3a or Stat5 was precipitated in a nonspecific manner (Fig. 3F and G, lane 2). Full-length Foxo3a and the C and the NC Foxo3a mutants precipitated with Stat5 (Fig. 3F). In reverse, Stat5 immunoprecipitated with all Foxo3a constructs (Fig. 3G). This indicates that Stat5 associates with Foxo3a through a domain located within the overlapping C-terminal domain of amino acids 244 to 360. The fact that the N Foxo3a mutant could not be detected in a Stat5 immunoprecipitation (Fig. 3F), as well as the low levels of Stat5 coprecipitated by the N Foxo3a mutant (Fig. 3G), can be explained by the low expression levels of the N Foxo3a mutant (Fig. 3E). In conclusion, the allegedly proliferation-inhibiting factor Foxo3a and proliferation-promoting factor Stat5 associate, which may result in the regulation of common target genes such as Cited2. The nuclear localization of this complex is maximal in the presence of Epo, which seems to be due to the fact that only Epo induces the concurrent nuclear localization of Stat5 and Foxo3a.
Foxo3a- and Stat5-responsive elements in the Cited2 promoter. Next, we investigated the potential transcriptional control of the Cited2 promoter by the Foxo3a/Stat5 complex. Alignment of a mouse genomic DNA fragment from chromosome 10 (GenBank accession number NW_001030408) to a previously described fragment at positions –3300 to +18 encompassing the human CITED2 promoter (47) revealed one conserved, palindromic Stat5 response element (SRE) (at bp –1207 bp) and one conserved Foxo3a binding element (DBE at bp –872). Both sites are located in a highly conserved promoter region and are separated by 335 bp (Fig. 4A). We employed ChIP assays to test the binding of the Epo-induced Foxo3a/Stat5 complex to these sites (Fig. 4B and C). I/11 erythroblasts were factor deprived (4 h), which was followed by 20 min of Epo stimulation. PCR analysis revealed that immunoprecipitated Stat5-DNA complexes isolated from Epo-stimulated cells contain the SRE of Cis, an established Stat5 target gene, but not the DBE of Btg1 (Fig. 4B). In reverse, Foxo3a-DNA complexes isolated from factor-deprived cells contain the DBE of Btg1 but not the SRE of Cis (Fig. 4C). In addition, the binding of Foxo3a to the Btg1 promoter was reversed upon Epo stimulation (Fig. 4C). Importantly, both the DBE and the SRE of Cited2 were detected in Stat5-DNA complexes (Fig. 4B) and in Foxo3a-DNA complexes only in response to Epo stimulation (Fig. 4C), which is in accordance with the nuclear Foxo3a-Stat5 interaction following Epo stimulation. These data corroborate that the regulation of the two Foxo3a target genes Cited2 and Btg1 follows opposite kinetics, with Cited2 being upregulated and Btg1 being downregulated in response to Epo stimulation. Apparently, the Foxo3a-Stat5 complex is able to escape the repression by the PI3K-PKB pathway induced by Epo.
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750) (72), however, inhibited the activation of the Cited2 promoter (Fig. 5B). Mutation of either the SRE or the DBE was sufficient to abolish Foxo3a/Stat5-stimulated promoter activity (Fig. 5C), indicating that both sites are required for the Foxo3a-Stat5 complex to induce Cited2 expression. The fact that Foxo3a cannot activate the Cited2 promoter alone, but only in cooperation with Stat5, provides a mechanism for the differential regulation of Cited2 and Btg1 by Foxo3a. This was confirmed by comparing Cited2 and Btg1 promoter activities. Whereas IL-3 stimulation of Ba/F3 cells activated the Cited2 promoter, it repressed Btg1 promoter activity more than twofold (Fig. 5D). In addition, growth factor depletion induced the Btg1 promoter but did not affect the activity of the Cited2 promoter (Fig. 5E).
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CREB family members contribute to Btg1 expression. The Foxo3a-binding DBE sites in the Cited2 and Btg1 promoters are identical consensus sites. The presence of the SRE in the Cited2 promoter and the nuclear association between Foxo3a and Stat5 may explain why Cited2 and Btg1 are differentially regulated by Epo stimulation. Indeed, reanalysis of the Btg1 promoter did not reveal any potential SRE. Instead, we found a CRE at position –204, i.e., 15 bp downstream of the DBE in the Btg1 promoter (Fig. 6A) (4). Promoter activity was assayed in NIH 3T3 cells using luciferase activity as a reporter. Mutation of the CRE or the DBE repressed basal promoter activity to a similar extent, and repression was not enhanced when both elements were deleted (Fig. 6B). In addition, costimulation by Foxo3a and cAMP significantly enhanced Btg1 promoter activity (Fig. 6C).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Differential regulation of Foxo3a target genes. Foxo target genes are induced in response to various stress situations including oxidative stress, DNA damage, and growth factor depletion (17, 68). These stress conditions inhibit cell growth but regulate distinct target genes; Cdkn1b/p27Kip and Bim are upregulated in response to factor deprivation, while Gadd45 is induced in response to DNA damage and oxidative stress (15, 24, 30, 43, 50, 64). Oxidative stress induces the acetylation and phosphorylation of Foxo concomitant with nuclear localization and the association of Foxo proteins with the acetylase p300/CBP-associated factor and subsequently with the deacetylase Sirt1, which decreases the interaction of Foxo proteins with p300 (15, 68). The interaction of Foxo with Sirt-1 repressed the Foxo-induced expression of Bim, Cdkn1b, Peck, and IGFBP1 but induced the expression of Gadd45 (15, 52). As a result, cell cycle arrest and replicative senescence were enhanced, and apoptosis was suppressed (15). In addition to differential target gene regulation in response to stress (32), this study deals with differential target gene regulation in response to signaling.
Because we performed single hybridizations, the list of putative target genes may contain errors. However, expression data for Epo, SCF, Epo/SCF, Epo/SCF/Dex, and Epo/SCF/ZK profiles are highly consistent (42). Moreover, we validated selected target genes in distinct Foxo3a(A3):ER-expressing clones, which confirmed that Foxo3a target genes in cluster D (e.g., Sesn1, Dcn, and Cited2) are not involved in a cellular response to factor deprivation. Strikingly, genes in clusters A and D represent different cellular processes. For example, the cluster D representative Sestrin1 (Sesn1) regenerates the antioxidant potential of thioredoxin, decorin (Dcn) modulates growth factor signaling, and Cited2 modulates the interaction of p300/CBP with transcription factors (1, 11, 16, 38). In contrast, cluster A representative Cyclin G2 (Ccng2) as well as p27Kip/Cdkn1b and Rbl2 (p130Rb2) inhibit cell cycle progression (6), whereas other genes in cluster A have a functional role in mature erythrocytes. Our study showed that this diverse set of Foxo3a target genes enables us to understand how Foxo3a cooperates with various environmental signals to control the expression of the appropriate set of target genes in response to specific conditions.
Foxo3a integrates various signaling pathways. We showed that an Epo-induced Foxo3a/Stat5 complex regulates Cited2 expression. Epo-induced Cited2 expression was not affected by the PI3K inhibitor LY294002, which is not surprising, since Epo strongly induces Stat5 activation, while it causes only weak Foxo3a phosphorylation. Nevertheless, it is surprising that a ChIP with anti-Foxo3a antibodies precipitates the DBE of Cited2 only in presence of Epo, while the DBE of Btg1 is present predominantly in the absence of Epo. The two DBE sequences are both full consensus sites. This suggests that the affinity of Foxo3a for its recognition site is not very strong and may require stabilization through the interaction with other transcription factors. It is striking that Foxo-associated factors are controlled by signaling pathways that have such diverse functions. Whereas Stat5 signaling is associated with proliferation, the cAMP pathway is associated with differentiation and cell cycle arrest (33). Recently, Foxo was shown to interact with ß-catenin, the central effector of the Wnt-signaling pathway assumed to impose "stem-cellness" to progenitor cells (27, 58). Thus, Foxo3a functions to integrate various signaling pathways, and this may explain its versatile function. Complex formation with other transcription factors also explains how a mutant Foxo1, incapable of DNA binding, retains the ability to regulate the expression of part of its target genes (57). This mutant lost the ability to induce apoptosis but was still able to cause a G1 arrest, underscoring that different mechanisms of target gene activation are associated with distinct cellular responses.
Regulation of Cited2 expression by Foxo3a and Stat5. Cited2 is a modulator of transcription that has been implicated in developmental processes and cancer (5, 73). Its regulation of important regulatory processes such as Hif1 activity and the transforming growth factor ß pathway (74) suggests that Cited2 expression requires tight control. Foxo3a and Stat5 do not control expression per se, but they are important to adapt Cited2 expression to environmental conditions. This seems to be true for most of the identified target genes. For instance, Btg1, Cyclin G2, and Sestrin are known as p53 target genes, but they are expressed in p53-deficient I/11 cells in the absence of growth factor (i.e., activation of Foxo3a). Thus, in response to distinct stress factors, p53 and Foxo3a can activate expression independent of each other.
The phosphorylation of Stat5 does not seem to affect the association of Foxo3a with Stat5, because coimmunoprecipitation from cytoplasmic lysates is equally efficient in the presence or absence of Epo, and no phosphorylated Stat5 can be detected in the absence of Epo. Instead, it is colocalization in the nucleus that induces the transcriptional activity of a Stat5/Foxo3a complex in the presence of Epo.
The interaction of Foxo3a and Stat5 involves a domain immediately C terminal of the DNA binding domain. The Foxo3a domain at positions 244 to 360 is an important site for Foxo3a regulation. It contains the PKB phosphorylation sites S253, localized in the Forkhead domain, and S314, localized in the phosphorylation patch (68). Importantly, S253 phosphorylation of Foxo by PKB is regarded as being the triggering event leading to Foxo phosphorylation, resulting in nuclear export. Stat5 binding to the region at positions 244 to 360 may thus explain how Foxo3a escapes negative control by the PI3K pathway and is able to stimulate the transcription of Cited in response to Epo stimulation.
We did not observe a marked regulation of Cited2 during differentiation. This is not surprising because Cited2 expression in response to Epo (differentiation) or Epo plus SCF (renewal) is similar (Fig. 2). Notably, Stat5 is expressed early but not late in differentiation, while Foxo3a expression increases from the start of differentiation induction to reach maximal levels 48 h after differentiation induction, when Stat5 expression becomes undetectable (4, 26). The reduced expression of Stat5 most likely precludes the upregulation of Cited2 when Foxo3a expression in the nucleus increases.
Role of cAMP signaling in erythropoiesis.
cAMP levels rise late in differentiation. Erythroid cells express G
s-coupled receptors, among which are the adrenergic receptor and receptors for thrombin and PGE2 (56). Notably, the PGE2 receptor is upregulated late in differentiation (W. J. Bakker and M. von Lindern, unpublished data; 25), whereas prostaglandin dehydrogenase (Pgdh1) is induced by dexamethasone in expanding erythroblasts and rapidly downregulated during differentiation (42; Bakker and von Lindern, unpublished). As a consequence, PGE2 activity is restricted to the final phase of erythroid differentiation when it controls hemoglobin expression, particularly Ala-S, the rate-limiting enzyme in heme synthesis, and NF-E2, a transcription factor binding the ß-globin enhancer (2, 22, 31, 54, 55, 63).
The CRE site in the Btg1 promoter also recruits cJun and/or Atf-2. The cJun/Atf2 complex is not regulated by the cAMP-PKA pathway but is regulated through Jun kinase (JNK). JNK is a stress-activated kinase, and cJun/Atf2 transcriptional activity is strongly enhanced in response to genotoxic stress (23, 41, 66). Interestingly, Btg1 expression is induced in response to DNA damage (19). The binding of Creb/Atf1 to the CRE site in the Btg1 promoter accelerates Foxo3a-mediated expression late in differentiation, while the binding of cJun/Atf2 following the activation of JNK may enhance Foxo-mediated expression in response to oxidative or genotoxic stress.
In conclusion, we identified putative Foxo3a target genes that were assigned to different clusters based on their regulation by Epo and SCF signal transduction. Examples from each cluster show that Foxo3a integrates PI3K-PKB signals with other signaling pathways. On the Btg1 promoter, PI3K-PKB signaling is integrated with cAMP signaling. Surprisingly, we found that the cooperation of Foxo3a and Stat5, i.e., the cooperation of pathways generally regarded to inhibit and promote proliferation, respectively, induced the expression of Cited2. The data strengthen the notion that Foxo proteins function at the heart of signal-dependent gene regulation and in complexes that integrate multiple signals to decide between cell death or survival, senescence, and functional maturation.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Dutch Cancer Society (EUR 2000-2230), The Netherlands Organization for Scientific Research (050-10-051), the European Union (HPRN-CT-2000-00083), an Erasmus fellowship to T.B.V.D., and a fellowship of the Dutch Academy of Arts and Sciences to M.V.L.
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FOOTNOTES |
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Published ahead of print on 12 March 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands.
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DBE), a mutated SRE (
