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Am J Physiol Regul Integr Comp Physiol 282: R1219-R1226, 2002. First published December 21, 2001; doi:10.1152/ajpregu.00507.2001
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Vol. 282, Issue 4, R1219-R1226, April 2002

cAMP and in vivo hypoxia induce tob, ifr1, and fos expression in erythroid cells of the chick embryo

Stefanie Dragon1, Nina Offenhäuser2,3, and Rosemarie Baumann1

1 Physiologisches Institut, Universität Regensburg, 93053 Regensburg, Germany; 2 European Institute for Oncology, 20141 Milan; and 3 FIRC Institute for Molecular Oncology, 20139 Milan, Italy


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During avian embryonic development, terminal erythroid differentiation occurs in the circulation. Some of the key events, such as the induction of erythroid 2,3-bisphosphoglycerate (2,3-BPG), carbonic anhydrase (CAII), and pyrimidine 5'-nucleotidase (P5N) synthesis are oxygen dependent (Baumann R, Haller EA, Schöning U, and Weber M, Dev Biol 116: 548-551, 1986; Dragon S and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 280: R870-R878, 2001; Dragon S, Carey C, Martin K, and Baumann R, J Exp Biol 202: 2787-2795, 1999; Dragon S, Glombitza S, Götz R, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996; Dragon S, Hille R, Götz R, and Baumann R, Blood 91: 3052-3058, 1998; Million D, Zillner P, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 261: R1188-R1196, 1991) in an indirect way: hypoxia stimulates the release of norepinephrine (NE)/adenosine into the circulation (Dragon et al., J Exp Biol 202: 2787-2795, 1999; Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996). This leads via erythroid beta -adrenergic/adenosine A2 receptor activation to a cAMP signal inducing several proteins in a transcription-dependent manner (Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996; Dragon et al., Blood 91: 3052-3058, 1998; Glombitza S, Dragon S, Berghammer M, Pannermayr M, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R973-R981, 1996). To understand how the cAMP-dependent processes are initiated, we screened an erythroid cDNA library for cAMP-regulated genes. We detected three genes that were strongly upregulated (>5-fold) by cAMP in definitive and primitive red blood cells. They are homologous to the mammalian Tob, Ifr1, and Fos proteins. In addition, the genes are induced in the intact embryo during short-term hypoxia. Because the genes are regulators of proliferation and differentiation in other cell types, we suggest that cAMP might promote general differentiating processes in erythroid cells, thereby allowing adaptive modulation of the latest steps of erythroid differentiation during developmental hypoxia.

erythropoiesis; terminal differentiation; adaptation; gene expression


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

UNTIL THE LAST THIRD of embryonic development, circulating red blood cells (RBC) of the chick embryo are not fully differentiated cells but are mainly composed of polychromatic/orthochromatic erythroid cells (7). The latest steps of differentiation, such as cell cycle exit, nuclear shut-down, ribosomal and mitochondrial degradation, and induction of several erythroid genes, are accomplished in the embryonic circulation (7, 18, 28, 43). We previously showed that immature embryonic RBC (day 3 to day 17) possess a functional cAMP signaling system (3) coupled to beta -adrenergic and adenosine A2 receptors. In late embryonic development, progressive hypoxia stimulates the release of norepinephrine (NE) and adenosine into the blood (12, 13), which leads to an increase of cAMP in embryonic RBC via beta -adrenergic and adenosine A2 receptor activation (13, 17). As a result, the increased intracellular cAMP level activates the coordinate synthesis of carbonic anhydrase (CAII), erythroid 2,3-bisphosphoglycerate (2,3-BPG), the heat shock protein hsp70, and pyrimidine 5'-nucleotidase (P5N) in addition to a fall in RBC ATP concentration (11-13, 17). Although the induction of CAII and the change of the RBC organic phosphate pattern are adaptive processes leading to improved O2 and CO2 transport of erythroid cells (5), it could be shown that several other RBC proteins are also induced in a cAMP-dependent manner (11), suggesting that cAMP might have additional functions in terminal RBC differentiation. Thus P5N, which is also induced by cAMP (14), catalyzes the release of the diffusible nucleosides uridine and cytidine from pyrimidine monophosphates, which are the product of ribosomal RNA degradation during late erythroid differentiation.

To extend our knowledge about the role of cAMP signaling in terminal differentiation and to identify novel cAMP-regulated erythroid genes, we constructed a cDNA library from embryonic RBC after beta -adrenergic receptor activation and differentially screened the library for cAMP-induced genes. We isolated three genes (tob, ifr1, and fos), which are known to be involved in antiproliferative and differentiation-promoting processes. Their transcripts are strongly upregulated by cAMP-elevating agonists in vitro and, even more importantly, by hypoxia in vivo.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. If not otherwise stated, analytic grade reagents were purchased from Sigma Chemicals (Deisenhofen, Germany). NE, propranolol, and 5'-(N-cyclopropyl)carboxamidoadenosine (CPCA) were obtained from RBI Biotrend (Cologne, Germany).

Preparation of erythroid cells and RNA preparation. Fertilized eggs of White Leghorn chickens were incubated at 37.5°C and 60% relative humidity in a commercial forced-draft incubator for up to 11 days of development. All experimental procedures were carried out in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" recommended by the World Medical Association Declaration of Helsinki. Usually, the RBC of several eggs were pooled and washed three times with cold PBS (in mM: 137 NaCl, 2.7 KCl, 10 Na2HPO4, 1.8 mM KH2PO4, pH 7.4, at room temperature with HCl) before they were incubated for up to 12 h at 37°C in a gyratory water bath with varying agonists [cytokrit 4%, Ham's medium F10 (Seromed, Biochrom, Berlin, Germany) supplemented with 20 mmol/l HEPES, 10% FCS (Boehringer Mannheim, Germany), pH 7.4 at 37°C]. For in vivo hypoxia, after 11 days of normoxic incubation, one group of embryos was subjected to 13.0% O2 for 30 to 180 min while a control group was kept under normoxic conditions. In this case, the blood of individual eggs was collected for RNA preparation. Total RNA of embryonic RBC was extracted (10), and poly(A)+-RNA was prepared from total RNA with the ligotex mRNA kit (Quiagen, Hilden, Germany).

cDNA library construction and screening. The library was constructed with 5 µg poly(A)+-RNA, which originated from day 11 RBC treated for 4 h with 10 µM NE using the ZAP Express cDNA synthesis kit and the Gigapack III Gold cloning kit by Stratagene (La Jolla, CA). We obtained a cDNA library with 600,000 independent clones. Differential screening was performed with single-stranded cDNA probes synthesized from day 11 RBC treated with or without 10 µM NE for 4 h. The cDNA probes were prepared as follows: 500 ng oligo(dT)12-18 primer (Amersham Pharmacia Biotech, Freiburg, Germany) was annealed to 1 µg mRNA. Reverse transcription was performed using 200 U Superscript II (GIBCO-BRL Life Technologies, Karlsruhe, Germany) in (in mM) 50 Tris · HCl, pH 8.3; 75 KCl; 3 MgCl2; 10 DTT; 0.5 dATP, dGTP, and dCTP; 0.13 dTTP; and 0.07 dig-11-dUTP (Roche Molecular Biochemicals, Mannheim, Germany) in a total volume of 20 µl. The reaction proceeded for 1 h at 42°C. After 10 min at 70°C the RNA was degraded with 1 U RNase H (PeqLab Biotechnologies, Erlangen, Germany) at 37°C for 20 min. For differential screening, 25,000 plaques of the cDNA library were plated on five 80-mm plates. Plaques hybridizing preferentially to the NE probe were picked and subjected to a secondary and tertiary screening by a combined PCR and Southern blotting technique (32) with the cDNA probes used before. At the end the inserts of the interesting phages were PCR amplified with T3 and T7 primers of the flanking region of the vector, and the PCR products were sequenced by Taq cycle sequencing using fluorescent terminators with an ABI Prism 377-96 DNA-Sequencer (Perkin-Elmer Biosystems, Norwalk, CT). The sequences were translated with the help of the Virtual Genome Center (www.alces.med.umn.edu), and the amino acid sequences were compared with the European Molecular Biology Laboratory protein acid data base (Heidelberg, Germany).

RT and PCR. For gene expression studies, all RNA samples of an experiment were reverse transcribed into cDNA at the same time. Five micrograms of denatured total RNA was used as template in a 20-µl cDNA synthesis reaction. The RNA samples were incubated with 100 pmol random d(N)6 primers (Pharmacia, Freiburg, Germany) for 10 min at 60°C, chilled on ice, and incubated for 15 min at room temperature. With a master mix, 1× RT buffer (50 mM Tris · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 20 mM DTT, 0.5 mM each dNTP) and 200 U Superscript II were added per sample followed by incubation at 42°C for 60 min and 70°C for 15 min. Aliquots (usually 10 or 50 ng RNA) of the cDNA reactions were analyzed for gene expression with the appropriate primers in 50-µl PCR reactions (Table 1). Again, to reduce sample variability, all cDNAs to be analyzed on a given gel were amplified at the same time using a master mix containing (per sample) 1× reaction buffer (PAN systems, Nürnberg, Germany), 1.5 mM MgCl2, 200 µM each dNTPs, 10 pmol of each primer (MWG Biotech, Ebersberg, Germany, see Table 1), and 1.25 U PanScript DNA polymerase (PAN systems). PCR conditions were 94°C, 2.5 min followed by 18-30 cycles (depending on primers and expression level) of 50-60°C, 1 min; 72°C, 1 min; 94°C, 1 min. To ensure that the conditions used were within the linear range of PCR amplification, aliquots of the reaction were removed at four increasing cycles and analyzed. In addition, a control reaction was performed using fivefold of the initial template amount at time 0. The samples were analyzed on a 2% agarose gel stained with 0.5 µg/ml ethidium bromide (EtBr).

                              
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  		Table 1.   PCR primers

Northern blots. Per sample, 5 or 10 µg of total RNA was separated by electrophoresis through a 1% agarose formaldehyde gel. After being stained with EtBr, the RNA was transferred by capillary blotting with 10× SSC for 16-20 h onto a neutral nylon membrane (Porablot NY amp, Macherey-Nagel, Düren, Germany). The transferred RNA was ultraviolet crosslinked and fixed by baking for at least 30 min at 80°C. The probes were prepared by RT-PCR with dig-11-dUTP (Roche Molecular Biochemicals).

The nonradioactive hybridization and luminescence detection procedure (anti Dig-antibody, substrate CDP-Star from Roche Molecular Biochemicals) follows the manufacturer's instructions with some modifications (15). In some cases, the blots were quantified with a MultiImager (BioRad, Munich, Germany). For reprobing, the blots were washed for 5 min in distilled water at room temperature. After equilibrating the membranes in 5× SSC for 20 min, the probe was removed by a 2-min stripping in 0.1% SDS at 95°C.

Western blotting. Whole cell lysates were prepared as follows: 15 µl of packed cells were mixed in 200 µl of cold SDS sample buffer (62.5 mM Na-phosphate, pH 7.0, 10% glycerol, 2% SDS, 0.01% bromphenol blue, 5% beta -mercaptoethanol), and the lysate was sonicated for 10 s at 150 W with cooling. Until use, the samples were stored at -80°C for up to 2 wk. After being boiled for 5 min at 95°C, aliquots of 30-100 µg Hb (determined with the cyanmethemoglobin method) were subjected to SDS-PAGE (27). The proteins were transferred to reinforced nitrocellulose (Optitran BA-S 85, Schleicher & Schuell, Dassel, Germany) by semi-dry blotting (Trans-Blot SD, BioRad) according to the manufacturer's instructions. After being blocked for 20 min in PBS with 0.05% Tween 20 and 3% nonfat dry milk, the blot was incubated overnight at 4°C with rabbit anti-Fos antibody (K-25, #sc-253, Santa Cruz Biotechnology; www.scbt.com) at a 1:1,000 dilution. After two short washes with water, the blot was incubated with the secondary antibody (1:4,000 dilution of goat peroxidase-coupled anti-rabbit; Pierce, Rockford, IL) for 1.5 h at RT. The blot was washed two times in water, 3-5 min in PBS with 0.05% Tween 20, and rinsed in four or five changes of water. For enhanced chemiluminescent detection with a MultiImager (BioRad), the substrate SuperSignal West (#34095ZZ, Pierce) was used.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sequence analysis of chicken tob and ifr1. Differential screening of a chick erythroid cDNA library prepared from day 11 RBC identified two different full-length clones: one contained an open reading frame (ORF) of 1842 nt coding for a protein of 325 amino acids (accession no. AJ292766), which shows strong homology (up to 94%) to the Tob (APRO6) proteins of mammals. The homology of chick Tob to the mammalian counterparts extends through most of the chick sequence, except for the shorter glutamine-rich region (Q8 at position 236-243). Northern blot analysis of RNA obtained from day 11 RBC detects a single transcript of ~2.2 kb (Fig. 2B).

In mammals, the Tob protein family has only recently been established. Although it is broadly expressed (6, 16, 30, 45), the knowledge about a specific function is still scarce. It belongs to the anti-proliferative Tob/PC3/Tis21/Btg1 proteins (23, 30, 40) recently renamed APRO (anti-proliferative) gene family (31). The Tob gene had been initially identified in NIH3T3 cells as a molecule that interacts with the receptor-type protein tyrosine kinase ErbB-2 (30). In addition to the binding of Tob to ErbB-2, members of the family can associate with other cellular targets, like Btg1 (APRO2) with protein-arginine N-methyltransferase Prmt1 (31) and the homeobox transcription factor Hoxb9 (38), Pc3/Btg1/Tob2 (APRO1/2/5) with Caf1, a protein capable of binding cyclin-dependent kinases (CCR4-associated factor 1; 23), and Tob (APRO6) with the receptor-regulated Smad proteins, which are part of the BMP signaling pathway (46).

The second ORF with 2215 nt codes for a protein of 434 amino acids (accession no. AJ292765) that is homologous to the mammalian Ifr1 (PC4/TIS7) proteins. The homology (up to 93%) extends throughout the whole sequence, except for a 15-amino acid deletion in the chick sequence. The transcript size in RBC of day 11, detected by Northern blot analysis, is ~2.5 kb (Fig. 2B). The protein family is even less well characterized than the Tob family: the Ifr1 protein was initially isolated as an immediate-early response gene of PC12 cells after induction of differentiation by nerve growth factor (42). In addition, deprivation of Ifr1 causes a delay in muscle differentiation (19). In mouse embryonic development, a homologue of Ifr1 is expressed soon after gastrulation and later in the hepatic primordium, suggesting a possible involvement in early hematopoiesis (8).

cAMP-dependent gene expression in definitive RBC. To confirm the cAMP-dependent expression of the genes identified, we used embryonic RBC of day 11. These cells respond in vivo to hypoxia with induction of 2,3-BPG and CAII synthesis, and in vitro stimulation of these cells with cAMP-elevating agents can simulate these processes in a transcription-dependent manner (11-14, 17). Therefore, the cells are an excellent model to study physiological effects of cAMP on erythroid gene expression. We studied the erythroid gene expression by RT-PCR and Northern blot analysis: RBC that were incubated in the presence of the beta -adrenergic agonist NE (Fig. 1) for up to 4 h strongly upregulate their steady-state level of tob (within the first 15 min) and ifr1 (within the first hour, Fig. 1), whereas the level of the ribosomal protein s17 or beta -globin is not affected. The time course for the induction of the two genes is slightly different: the tob mRNA is elevated 30 min after stimulation and reached maximal levels after 2 h, whereas ifr1 mRNA induction is visible first at 60 min and increases up to 4 h. In addition to tob and ifr1, the transcription factor fos (GenBank M37000), a known cAMP-induced gene in other cellular systems (39, 44), is also upregulated by cAMP in embryonic RBC. The fos mRNA increases within 15 min after beta -adrenergic receptor activation, reaching maximal levels already at 30 min (Fig. 1). In addition, the expression of all three genes is also increased after a 4-h incubation with the adenosine A2 receptor agonist CPCA (Fig. 2, A and B). The NE-dependent induction of the three genes by beta -adrenergic receptor activation is completely abolished in the presence of the beta -blocker propranolol (P; Fig. 1), whereas a translational inhibition with cycloheximide could not suppress the induction (Fig. 4). This suggests that protein phosphorylation rather than a de novo protein synthesis step is required for upregulation of the three mRNAs species. In addition, the presence of cycloheximide amplifies the effects of NE at 30 min. Therefore, changes in mRNA and/or transcription factor stability might play a modulatory role in the profile of induced gene expression.


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  		Fig. 1.   Gene expression of tob, ifr1, and fos in definitive red blood cells (RBC) in response to beta -adrenergic receptor activation. Day 11 RBC were either processed without incubation (time 0; t0) or incubated for up to 4 h in the absence or presence of 1 µM norepinephrine (NE) ± 10 µM propranolol (P). Equal amounts (for time 0 also the 5-fold amount) of cDNA was amplified by PCR with specific primers (see METHODS). One of two independent experiments is shown.



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  		Fig. 2.   Adenosine A2 receptor activation induces tob, ifr1, and fos expression in definitive RBC. The RNA of day 11 RBC was analyzed by RT-PCR (n = 3) (A) or Northern blotting (B) (5 µg RNA per lane; n = 1) before (time 0) or after a 4-h incubation in the absence (-) or presence (CPCA) of 10 µM 5'-(N-cyclopropyl)carboxamidoadenosine (CPCA).

For fos, we also measured the protein expression by Western blotting. The Fos protein is not present in day 11 RBC unless they are stimulated with NE or the direct activator of adenylyl cyclases, forskolin (Fig. 3C). In the presence of NE, the time course of Fos protein (Fig. 1A) follows the course of fos mRNA expression (Fig. 3A). The protein level remains elevated at least during 12 h of beta -adrenergic receptor stimulation (Fig. 3B). Direct stimulation of the adenylyl cyclase with 100 µM forskolin results in a weaker production of cAMP than 1 µM NE (3) and, in consequence, a weaker induction of Fos protein expression (63.0 ± 15.8% of the NE response, n = 4; Fig. 3C).


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  		Fig. 3.   Fos protein expression in definitive (day 11) RBC before (time 0) and after stimulation with 1 µM NE ± 10 µM P for 4 h (A) and 12 h (B) and after (C) stimulation for 4 h with the adenylyl cyclase activator forskolin (F). Whole cell lysates of day 11 RBC (100 µg Hb per lane) were analyzed; a representative of 2-4 experiments is shown.

cAMP-dependent gene expression in primitive RBC. The first erythroid population of vertebrate embryos are the primitive RBC that originate from the yolk sac and are characterized by the presence of specific embryonic hemoglobins (5). In the chick, they are released into the circulation as immature cells with mitotic activity until day 5/6 of embryonic development. Around day 6, the first definitive RBC appear in the embryonic blood, and they become the main RBC population after day 8. We determined the relative mRNA levels of tob, ifr1, and fos in primitive day 5 RBC in response to beta -adrenergic receptor activation by RT-PCR (Fig. 6). As in definitive RBC, we could observe a prominent cAMP-dependent induction in gene expression of tob, ifr1, and fos after 4 h of incubation. To rule out that the expression we observe arises from contaminating definitive RBC, we determined the relative beta -globin expression level in the RNA preparations of day 5. Indeed, the very low level of beta -globin at day 5 compared with the high level in day 11 indicates that the induction of tob, ifr1, and fos takes place in primitive RBC. Parallel to the mRNA expression, the Fos protein expression of day 5, which is minimal in RBC before an induction, becomes strongly upregulated by cAMP (Fig. 6B). Likewise at day 4, when the erythroid cells are even more immature and show higher mitotic activity in the circulation, the Fos protein level can be upregulated by NE (Fig. 6C).

In vivo hypoxia induces gene expression of fos, tob, and ifr1. In the chick embryo, the decrease of the blood PO2 during normal development is a potent stimulus for the release of NE and adenosine into the embryonic circulation around day 13/14 (13). A rise in cAMP-important steps of terminal differentiation in erythroid cells are initiated, such as the induction of 2,3-BPG and CAII (protein and mRNA) synthesis and the fall in ATP (11, 12). The same erythroid processes can already be induced at day 8 by experimental hypoxia (chronic or acute) of chick embryos (4).

For CAII, a lasting induction of its mRNA has been demonstrated after day 13/14. In contrast, we could not observe a lasting induction for tob, ifr1, and fos after day 13/14 (data not shown). However, moderate acute hypoxia (13% O2) for 30-75 min of day 11 chick embryos leads to an upregulation of the mRNA expression of all three genes (Fig. 5A). For significant induction, a 30 min-period of hypoxia is sufficient to strongly upregulate the steady-state level of tob and fos (Fig. 5), whereas a less prominent induction is seen for ifr1. This is in agreement with the slower time course observed in the in vitro incubations (Figs. 1 and 4A). Additionally, after a 2- to 3-h period of hypoxic incubation, a strong induction of Fos protein was detected in the RBC of all embryos we examined (Fig. 5B). These data suggest that Tob, Ifr1, and Fos are part of the adaptation program of avian embryonic RBC in response to hypoxia.


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  		Fig. 4.   Upregulation of tob, ifr1, fos is independent of protein synthesis in definitive RBC. Day 11 RBC were preincubated 15 min with [norepinephrine + cycloheximide (NE+CY)] or without 35 µM CY (NE), and then 10 µM NE was added to the incubations. A: Northern blot analysis of tob and ifr1 with 10 µg RNA per lane, n = 1. The signals were standardized to s17 and the induction relative to the start level (= 1) was calculated. The results were confirmed by RT-PCR (n = 2), data not shown. B: RT-PCR results for fos expression, n = 2.



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  		Fig. 5.   Short-term hypoxia at day 11 induces erythroid expression of tob, ifr1, and fos in vivo. Eggs that had been incubated in air for 11 days were either kept under normoxia or subjected to 13.0% O2 for 30-75 min (A) to analyze gene expression by RT-PCR and for 120-180 min (B) to analyze protein expression by Western blotting (100 µg Hb per lane). The RBC of single embryos were analyzed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The precise signaling events that govern the penultimate steps of erythroid differentiation, i.e., the conversion from erythroblast to mature RBC, are unknown, not least of all because of the lack of suitable experimental systems allowing extensive biochemical analysis of late differentiation steps. The circulating, immature RBC of the avian (chick) embryo allows insight into these processes. The present data extend our previous findings, which already indicated that cAMP signaling has an important influence on the expression of erythroid genes like CAII and P5N. Using differential cDNA screening, we have now identified three factors involved in the regulation of the cAMP-dependent maturation and we obtained the following results: 1) the genes tob, ifr1, and fos are expressed in late erythroid differentiation, 2) the three genes are strongly and stably induced by cAMP in embryonic RBC of primitive and definitive lineage, 3) moderate experimental short-term hypoxia upregulates the expression of the three genes, and 4) based on the functional analysis of the homologous genes in other cellular systems, we suggest that in avian erythroid cells these genes could be involved in ongoing maturation processes, like exit from the cell cycle, promotion of differentiation, and organelle breakdown.

Potential functions of Tob, Ifr1, and Fos in the erythroid system. Although the precise role of the three cAMP-induced genes in late erythroid differentiation remains to be determined, their function in other cellular systems gives some idea of their potential role in the erythroid system. The protooncogene Fos builds up the transcription factor family composed of Fos, Fra-1, Fra-2, and FosB, which bind to Jun and Maf proteins (25, 26) and constitute the heterodimeric AP-1 proteins. The AP-1 protein binds to AP-1 sites that are widely distributed in regulatory regions of genes involved in division, differentiation, and transformation of cells (2). AP-1 sites have been identified in erythroid cells in the regulatory regions of genes expressed in terminally differentiating RBC, i.e., the globin (35), heme oxygenase 1 (24), anion exchanger 1 (41), and protoporphobilinogen deaminase (33) genes. In addition, the Fos protein has been detected in human and murine erythroblasts of fetal liver, indicating a potential involvement in fetal erythropoiesis (9). In the erythroid system of the chick embryo, the target genes of Fos are not yet determined. Although Fos is expressed before other cAMP-induced genes (Fig. 1; 11), we can certainly rule out an involvement of Fos in the induction of caII, tob, and ifr1, because a protein synthesis step is not required for their transcriptional activation (Fig. 4A; 11).

The Tob proteins belong to a family of APRO genes (31). They seem to be connected with several cellular signaling systems, e.g., tyrosine receptor kinases (30), receptor serine/theonine kinases (46), and cyclin-dependent protein kinases (23). In all cases investigated so far, the presence of Tob suppresses a mitogenic signal by binding to components of the signaling pathways (ErbB2, Smad1, 5, and 8, and Caf1). In the erythroid cells of the chick embryo, the signaling pathways remain to be investigated and all potential functions of Tob are therefore speculative. For example, the protein might promote the cell-cycle exit or it might be involved in one-way decisions like degradation of the ribosomes and mitochondria. The identification of Tob binding partners in the erythroid system will help to elucidate the role of Tob. However, finding a function for Tob (or Fos or Ifr1) in erythroid cells will also allocate a new function for cAMP in late erythropoiesis.

The Ifr1 protein seems to be a positive regulator of differentiation. The most extensive studies were done in the mouse myoblast cell line C2C12 (19). Sense and antisense cDNA transfection experiments showed that, although no effect was seen by overexpression of Ifr1, blocking the Ifr1 expression caused a delay in attaining the differentiated phenotype, with an impairment of myogenin and myosin expression. In addition, location studies in PC12 cells showed a cytoplasmic translocation to the plasma membrane on stimulation with NGF and subsequent accumulation of Ifr1 in the nucleus, which disappears again after nerve growth factor removal. The results suggest Ifr1 as a possible link between receptor signaling and transcriptional regulation, although no specific binding partners of Ifr1 have been identified so far. The putative role of Ifr1 as positive regulator of differentiation in other cells suggests a similar function in embryonic RBC.

Gene expression of tob, ifr1, and fos in definitive RBC. Although the expression of caII is stably induced in vitro and in vivo, we were not able to detect an accumulation of tob, ifr1, and fos mRNAs in the last third of embryonic development (unpublished observation). In contrast, induction of the genes by cAMP-elevating receptor agonists in vitro and by acute hypoxia in vivo occurs quickly and prominently (Figs. 1 and 5). During acute hypoxia and in vitro, the cells are exposed to a sharp rise of receptor agonists (13), affecting all cells in the circulation simultaneously and resulting in a coordinated response to all cells. On the other hand, developmental hypoxia leads to a more gradual increase of NE/adenosine in the embryonic plasma. In addition, there is a constant influx of erythroid cells into the circulation, which mix with other cells that are already induced. For this reason, we assume that tob, ifr1, and fos are transiently upregulated during erythroid differentiation. However, in vitro and during acute in vivo hypoxia the three erythroid genes are strongly induced parallel to other cAMP-dependent processes, like 2,3-BPG, CAII, and P5N synthesis and ATP degradation (4, 12-14, 33), which are characteristic for late erythroid differentiation, indicating a role of Tob, Ifr1, and Fos in vivo.

Gene expression in primitive RBC. Primitive day 5 RBC are able to upregulate in vitro the steady-state level of caII (11), tob, ifr1, and fos (Fig. 6) in response to the same hormonal agonists effective in day 11 RBC. Therefore, in primitive RBC the same cAMP signal transduction pathway exists (3). The presence of catecholamines in the early chick embryo and the yolk sac, the site of primitive erythropoiesis, has been documented (21, 22), indicating a potential role of the signaling pathway in advance of erythroid cell release to the circulation. Thereby, at the site of erythropoiesis, cAMP might control earlier differentiation steps as well, possibly with the participation of Tob, Ifr1, and Fos. With the isolation of erythroid cells from the yolk sac of the early chick embryo we can obtain further insight into an involvement of the three genes in earlier steps of erythropoiesis.


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  		Fig. 6.   Regulation of tob, ifr1, fos, and Fos protein expression in primitive RBC. The cells were incubated for 4 h in the presence of 1 µM NE ± 10 µM P. A: RT-PCR results of day 5 RBC; a representative of 4 experiments is shown. The preparation was essentially free of definitive RBC as the beta -globin expression was negligible compared with a preparation of day 11 RBC. B: Fos protein detection by Western blotting in day 5 RBC (70 µg Hb per lane); 1 of 2 experiments is shown. C: Fos protein detection in day 4 RBC (30 µg Hb per lane); 1 of 4 experiments is shown.

In conclusion, we identified through a differential screening procedure approach for NE-induced genes in chick RBC three proteins that are likely involved in terminal erythroid differentiation. In analogy to their mammalian counterparts we suggest that Tob, Ifr1, and Fos might regulate important steps in the exit from the cell cycle, differentiation and organelle breakdown.

Perspectives

The identification of the novel cAMP-induced genes in erythroid cells, especially tob, which regulates cell cycle progression and exit in other cell systems, as well as fos and ifr1, opens up a more general role for the signal molecule cAMP during terminal erythroid differentiation. We are still far from understanding how cAMP regulates erythroid gene expression, but in the future the functional characterization (i.e., localization studies, binding partners, Fos-regulated genes) of Tob, Ifr1, and Fos will help us understand the molecular mechanisms underlying RBC differentiation. Many steps of mammalian and nonmammalian erythropoiesis are similar. In addition, mammalian erythroid cells possess a functional cAMP signal transduction system (20, 36, 37) and are influenced by catecholamines via sympathetic innervation of the bone marrow (1, 29). Thus elucidating the function of cAMP in the avian erythroid cell differentiation might help the understanding of the potential role of cAMP in mammalian erythropoiesis.


    ACKNOWLEDGEMENTS

The authors thank R. Volkmann and F. Webinger for technical assistance. N. Offenhäuser acknowledges the generous support of P. P. Di Fiore.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Dragon, Physiologisches Institut, Universität Regensburg, 93053 Regensburg, Germany (E-mail: stefanie.dragon{at}vkl.uni-regensburg.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 21, 2001;10.1152/ajpregu.00507.2001

Received 17 August 2001; accepted in final form 26 November 2001.


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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 282(4):R1219-R1226
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