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Originally published In Press as doi:10.1074/jbc.M200131200 on April 9, 2002

J. Biol. Chem., Vol. 277, Issue 25, 22902-22908, June 21, 2002
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Mechanisms of FOXC2- and FOXD1-mediated Regulation of the RIalpha Subunit of cAMP-dependent Protein Kinase Include Release of Transcriptional Repression and Activation by Protein Kinase Balpha and cAMP*

Maria K. DahleDagger, Line M. GrønningDagger, Anna Cederberg§, Heidi Kiil Blomhoff, Naoyuki Miura, Sven Enerbäck§, Kristin A. Taskén||, and Kjetil Taskén**

From the Department of Medical Biochemistry, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, § Medical Genetics, Department of Medical Biochemistry, Göteborg University, SE 405 30 Göteborg, and  Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Hamamatsu 431-3192, Japan

Received for publication, January 5, 2002, and in revised form, March 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have reported recently that mice overexpressing the forkhead/winged helix transcription factor FOXC2 are lean and show increased responsiveness to insulin due to sensitization of the beta -adrenergic cAMP-PKA+ pathway and increased levels of the RIalpha subunit of cAMP-dependent protein kinase (PKA) (Cederberg, A., Grønning, L. M., Ahren, B., Taskén, K., Carlsson, P., and Enerbäck, S. (2001) Cell 106, 563-573). In this present study, we reveal that FOXC2 and a related factor, FOXD1, specifically activate the 1b promoter of the RIalpha gene in adipocytes and testicular Sertoli cells, respectively. By deletional mapping, we discovered two different mechanisms by which the Fox proteins activated expression from the RIalpha 1b promoter. In 3T3-L1 adipocytes, an upstream region represses promoter activity under basal conditions. Bandshift experiments indicate that overexpression of FOXC2 promotes the release of a potential repressor from this region. In Sertoli cells, sequences downstream of the transcription start sites mediate the activating effect of FOXD1, and protein kinase Balpha /Akt1 strongly induces this effect. Furthermore, we show that an inactive FOXD1 mutant lowers the cAMP-mediated induction of the RIalpha 1b reporter construct. In summary, winged helix transcription factors of the FOXC/FOXD families function as regulators of the RIalpha subunit of PKA and may integrate hormonal signals acting through protein kinase B and cAMP in a cell-specific manner.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The forkhead/winged helix family of transcription factors is characterized by a highly conserved monomeric DNA-binding domain called the winged helix (reviewed in Ref. 2). A number of forkhead and forkhead-related genes have been isolated to date (3-7), and the Fox1 nomenclature (Forkhead box) has now been adopted for all chordate forkhead genes (www.biology.pomona.edu/fox.html) (8). Among these are several forkhead related activators (FREACs) cloned from human (9-13) that all share the minimum requirement for a 7-bp core binding motif (RTAAAYA). One of these factors, FOXD1 (FREAC4, FKHL8), has expression restricted to kidney, the central nervous system testis, and is regulated by Ets-1 and p53 in kidney-derived cell lines (11, 14). FOXC2 (FREAC11, FKHL14, MFH-1) (15, 16) is restricted to adipocytes in adults (1), whereas the prenatal form is important for development. Mice lacking Foxc2 die during embryogenesis or perinatally and exhibit aortic arch and skeletal defects (17, 18). Instead, overexpression of FOXC2 in adipose tissue has been important in understanding its function in adult mice. FOXC2 transgenic mice develop a phenotype characterized by a high sensitivity to insulin and partial resistance to diet-induced obesity (1). This effect is partly due to up-regulation of beta -adrenergic receptors and PKA type Ialpha that lower the threshold for activation of PKA by cAMP, resulting in a hypersensitive beta -adrenergic pathway.

Activation of the cAMP-dependent protein kinase (PKA) proceeds by a concerted reaction in which binding of the intracellular second messenger cAMP to the regulatory subunit dimer (R2) in a positive, cooperative fashion results in dissociation and activation of two catalytic (C) subunits (reviewed in Ref. 19). Targeted disruption of the RIIbeta regulatory subunit gene in mice leads to a lean phenotype with elevated levels of uncoupling protein 1 and increased metabolic rate due to a shift in the PKA composition from PKA IIbeta (RIIbeta 2C2) to type Ialpha (RIalpha 2C2) holoenzyme (20-22). The effect of this regulatory subunit shift was shown to lower the threshold for PKA activation by cAMP and to modulate lipolysis (23). The RIIbeta knockout phenotype resembles that of the FOXC2 transgenic mice (1) and supports the notion that regulation of PKA isozyme composition, particularly RIIbeta versus RIalpha , is important for hormonal responsiveness and cAMP sensitivity.

The RIalpha gene is controlled by several promoters, giving rise to at least three mRNAs that differ in the first non-coding exon (24-26). Two of these mRNAs (RIalpha 1a and -1b) are expressed in most tissues (25), and we have recently reported cAMP-mediated post-transcriptional regulation of RIalpha 1b in Sertoli cells (27). We have shown that both FOXC2 and RIalpha are induced by cAMP in mouse 3T3-L1 adipocytes (28)2 and that FOXD1 and RIalpha are induced by cAMP in Sertoli cell primary cultures3 (27). We have also reported recently (28) that basal levels of Foxc2 mRNA are down-regulated during differentiation of 3T3-L1 adipocytes, with a simultaneous increase in Foxc2 responsiveness to insulin and TNFalpha .

In this study, we examine the effect of FOXD1 and FOXC2 on the RIalpha promoters, showing that the expression of RIalpha 1b but not RIalpha 1a is induced. Deletional mapping reveals that FOXC2 and FOXD1 regulate expression from the RIalpha 1b promoter through two different mechanisms, and EMSA and co-transfection experiments showed that in 3T3-L1 adipocytes FOXC2 induces a release of transcriptional repression. In Sertoli cells, PKBalpha strongly increases the effect of FOXD1, and a truncated FOXD1 inhibits cAMP-mediated induction of the RIalpha 1b promoter, indicating that FOXD1 may function as a mediator of signaling by both cAMP and PKB in a cell-specific manner.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- The RIalpha promoter constructs RIalpha 1a+1b (-882 to +77), RIalpha 1a (-882 to -310), RIalpha 1b (-406 to +77) (24), and the RIIbeta promoter construct (-4500 to -123) (29) were inserted into the pCAT basic reporter vector (Promega, Madison, WI). Five deletion constructs were made from the RIalpha 1b promoter region: A+ (-307 to +77), A (-307 to +4), B (-199 to +4), C (-92 to +4), and C+ (-92 to + 77) (27). Complementary DNAs encoding full-length forkhead genes were inserted in pEVRFO (FOXD1) or pCB6+ (FOXC2) expression vectors or C-terminal to the hemagglutinin epitope (HA) tag of the pEF-BOS vector (30). A cDNA fragment encoding the forkhead DNA binding domain (FOXD1-DBD, amino acids 127-221) was cloned into pEVRFO. Expression vectors for HA-tagged wild type or myristoylated PKBalpha /Akt1 were created in pCMV5 or pCMV6, respectively (31). All these expression vectors contain cytomegalovirus (CMV) promoters. In addition, the luciferase expression vector pGL3Control (Promega) was used as internal control in transfection experiments.

Cell Cultures-- Primary cultures of rat Sertoli cells were prepared from testes of 19 days old Sprague-Dawley rats (B&K Universal AS, Nittedal, Norway) according to the method of Dorrington et al. (32) with some modifications (33). The cells were plated in 6-well plates (35-mm/well) for transfections or in 10-cm culture dishes for preparation of nuclear extracts. Cells were grown in Eagle's minimal essential medium (Invitrogen) with addition of streptomycin (100 g/liter), penicillin (70 mg/liter), fungizone (0,25 mg/liter), L-glutamine (2 mM), and 10% fetal bovine serum at 32 °C in a humidified atmosphere with 5% CO2. After 3 days, the cells were incubated further in serum-free modified Eagle's minimal essential medium. After 2 days of culture in serum-free medium, LipofectAMINE-mediated transfections were performed as described elsewhere (34) using 2 µg of DNA (1.5 µg of CAT or luciferase reporter and 0.5 µg of internal luciferase or CAT control) with 5 µl of LipofectAMINE (Invitrogen) per 35-mm well for 3 h, after which media were changed. Mouse 3T3 L1 cells (American Type Culture Collection) were plated in 6-well plates for transfections or in 10-cm culture dishes for preparation of nuclear or whole cell extracts. Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) with 4.5 g/liter glucose with addition of streptomycin (100 mg/liter), penicillin (70 mg/liter), fungizone (0.25 mg/liter), anti-PPLO agent, and 10% fetal bovine serum at 37 °C, and transfected at ~80% confluency as described above.

Luciferase and CAT Assays-- All cells were harvested in reporter lysis buffer 48 h after transfection and assayed for luciferase activity (Promega, Madison, WI). CAT activities were measured using an organic phase extraction method (35) and normalized for expression of luciferase.

Immunoblotting-- Preconfluent 3T3-L1 cells (10-cm2 culture dish) were washed in 5 ml of cold phosphate-buffered saline and then scraped in 500 µl of a buffer containing 10 mM potassium phosphate, pH 6.8, 1 mM EDTA, 10 mM CHAPS (Sigma) and CompleteTM protease inhibitor mix (1 tablet/10 ml) (Roche Molecular Biochemicals). Cell suspensions were sonicated 3 times (Heat Systems Ultrasonics) and centrifuged for 5 min at 12,000 × g. Supernatants were stored at -70 °C until analysis. Protein samples were diluted in SDS sample buffer and denatured for 5 min at 100 °C before loading on a one-dimensional SDS-polyacrylamide gel (4.0% stacking gel, 10% separating gel). 20 µg of total protein was loaded in each lane, subjected to electrophoresis, and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) by electroblotting. The membranes were blocked in a solution containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 5% milk, followed by incubation with primary antibody for 1 h in blocking solution. The antibodies used are monoclonal antibodies against human RIalpha (1:500) (Transduction Laboratories) or human FOXC2 (1:250) (16), chicken polyclonal antibody against amino acids 1-12 in FOXD1 (1:200), or rabbit polyclonal antibody against PKA catalytic subunit (Calpha , 1:250) (Santa Cruz Biotechnology). Membranes were washed in a solution containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20. Immunoreactive proteins were visualized with enhanced chemiluminescence reagents (ECL, Amersham Biosciences) using a horseradish peroxidase-conjugated secondary antibody (1:20.000) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and subjected to autoradiography. Films were scanned, and band densities were quantitated using the Scion Image package (www.scioncorp.com).

Preparation of Nuclear Extracts-- Sertoli cells or preconfluent 3T3-L1 cells (10-cm2 culture dish) were scraped in Hanks' balanced salts solution containing 0.1% fatty acid-free bovine serum albumin, harvested by centrifugation at 320 × g for 5 min (4 °C), and washed once in cold phosphate-buffered saline. Cell pellets were resuspended in 450 µl of hypotonic buffer (10 mM Tris, pH 7.6, 10 mM NaCl, 3 mM MgCl2) and lysed by addition of 50 µl of 5% Nonidet P-40 in hypotonic buffer. The nuclei were pelleted by centrifugation (130 × g, 5 min, 4 °C); pellets were carefully washed in 1 ml of hypotonic buffer, centrifuged (130 × g), and then resuspended in 100 µl of a buffer containing 5 mM Hepes, pH 7.9, 26% glycerol, 1.5 mM dithiothreitol, and the protease inhibitors phenylmethylsulfonyl fluoride (0.5 mM), CompleteTM protease inhibitor mix (1 tablet/10 ml), and calpain inhibitor I (50 µM) (Roche Molecular Biochemicals). High salt extraction was accomplished by addition of NaCl to a final concentration of 400 mM while mixing for 30 min at 4 °C. Extracts were centrifuged (30,000 × g, 20 min, 4 °C), and the supernatants were stored at -70 °C until analysis.

DNA-Protein Complex Analysis-- Electrophoretic mobility shift assays (EMSAs) were performed using double-stranded 32P-end-labeled forkhead consensus oligonucleotide (5'-GATCCCTTAAGTAAACAGCATGAGATC-3') (9) or a 100-bp region of RIalpha exon 1a (-407 to -306). For each reaction, 5000 cpm of labeled probe were incubated with 2.5 (Fig. 3A) or 5 µg (Fig. 3B) of crude nuclear proteins from transfected 3T3-L1 preadipocytes and 0.5 (Fig. 3A) or 1 µg (Fig. 3B) of poly(dI·dC) in a buffer containing 5 mM Hepes, pH 7.9, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 25 (Fig. 3B) or 100 mM KCl (Fig. 3A) at room temperature for 15 min. Competition experiments were performed in the presence of 250-fold molar excess of unlabeled probe or with a nonspecific forkhead sequence (5'-GATCCAGGCCGTAAACAGCATGAGATC-3') (9). In addition, competitions were performed with oligonucleotides containing the RIalpha exon 1a (-407 to -306) or exon 1b (+1 to +68) sequences. In supershift experiments, a human monoclonal FOXC2 antibody (16) was added followed by incubation for 1 h at room temperature. Samples were run in 6% non-denaturing polyacrylamide gels at 150 V in Tris/glycine buffer (50 mM Tris pH 8.5, 380 mM glycine, 2 mM EDTA) at 4 °C. Subsequently, gels were dried and subjected to autoradiography.

Immunofluorescence-- Primary Sertoli cells were grown on polylysine-treated coverslips in 6-well plates and transfected with pEF-BOS/FOXC2, pEF-BOS/FOXD1, pCMV/PKBalpha , or pCMV/myristoylated PKBalpha (2 µg/well). Following incubation for 24 h, cells were washed twice in cold phosphate-buffered saline, and cells on coverslips were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and proteins blocked with 2% bovine serum albumin in phosphate-buffered saline, 0.01% Tween 20. Cells were then incubated with a mouse polyclonal antibody against the HA tag (C-20, 1:250) (Santa Cruz Biotechnology), followed by an anti-mouse fluorescein (FITC)-conjugated secondary antibody (1:1000). DNA was stained with 0.1 µg/ml Hoechst 33342. Observations were made and photographs taken as described previously (36).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

FOXC2 Induces RIalpha Protein in 3T3-L1 Cells-- In order to establish whether FOXC2 expression could induce RIalpha protein levels in 3T3-L1 cells as in the FOXC2 transgene, we harvested cells transfected with human FOXC2 expression vector or the corresponding empty vector and prepared whole cell extracts after 6, 12, and 24 h. RIalpha , the PKA catalytic subunit, Calpha , and FOXC2 protein levels were then examined by immunoblotting (Fig. 1). Basal levels of endogenous Foxc2 were observed in mouse 3T3-L1 cells, and a stronger band (2-fold) was observed in extracts of 3T3-L1 cells transfected for 6 h with the human FOXC2 construct. The levels of FOXC2 protein were strongly increased after 12 and 24 h of expression (6-fold), and the mobility (~62 kDa with the appearance of a faster migrating band) was similar to that observed previously (16). RIalpha levels were ~2-fold induced in the presence of FOXC2 at 6-24 h of expression as determined by densitometric scanning. In contrast, FOXC2 had no effect on the levels of Calpha .


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  		Fig. 1.   Expression of FOXC2 elevates levels of RIalpha protein in 3T3-L1 cells. Nuclear extracts from untransfected (-) or FOXC2-transfected (+) 3T3-L1 cells harvested after 6, 12, and 24 h were prepared and examined by immunoblotting using antibodies to human FOXC2, RIalpha , and Calpha . Mobility of the 66-kDa band of Rainbow molecular weight marker (Amersham Biosciences) is indicated. One representative of three experiments is shown.

FOXD1 and FOXC2 Induce Reporter Activity Driven by the RIalpha 1b Promoter in 3T3-L1 Cells-- To map the effect of FOXC2 on RIalpha promoter activity in adipocytes, we cotransfected 3T3-L1 cells at 80% confluency with reporter constructs containing the RIalpha 1a+1b promoter, the 1a or 1b promoters alone, and a construct containing 4500 bp of the RIIbeta promoter together with pCB6+/FOXC2 or the empty expression vector (Fig. 2A). We simultaneously tested whether FOXD1, which is not endogenously expressed in adipocytes, had a similar effect on the RIalpha promoter (Fig. 2B). FOXC2 expression induced a 4-5-fold increase in reporter activity directed from the RIalpha 1a+1b and the RIalpha 1b promoter constructs, and in contrast, the RIalpha 1a or RIIbeta promoters were slightly down-regulated by the presence of FOXC2. The same pattern of regulation was observed with FOXD1. We next analyzed expression from five 3' and/or 5' deletion constructs of RIalpha 1b in the pCAT basic reporter vector (Fig. 2C, left panel) (27). We observed a 7-fold induction of basal reporter expression when a 100-bp region in exon 1A was absent (RIalpha 1b A+). This deletion raised basal expression to the same levels as in the presence of FOXC2 and thereby abolished the induction by FOXC2 indicating that FOXC2 regulation may involve release of repression residing in this region. In construct RIalpha 1b B, however, basal levels were down to the level of the longest construct (RIalpha 1b), but the inductive effect of FOXC2 was not reconstituted. Interestingly, the elevated basal levels of the shortest constructs (RIalpha 1b C and RIalpha 1b C+) were again higher, which indicates that repressive effects may reside in RIalpha 1b B as well, although not regulated by FOXC2.


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  		Fig. 2.   FOXC2 and FOXD1 induce expression from the 1b promoter of the RIalpha gene in 3T3-L1 cells. RIalpha promoter constructs RIalpha 1a + 1b (-882 to +77), RIalpha 1a (-882 to -310), RIalpha 1b (-406 to +77), and RIIbeta promoter construct (-4500 to -123) inserted upstream of the CAT reporter gene in the pCATbasic (pCATb) reporter vector were transfected into preconfluent 3T3-L1 cells together with expression vectors for FOXC2 or FOXD1. Cell cultures were harvested after 48 h. Figure shows fold induction of reporter activity directed by the promoter in the presence of FOXC2 (A) or FOXD1 (B), relative to activity in the presence of the corresponding empty expression vectors. C, 3' and 5' deletion constructs of the RIalpha 1b promoter (depicted to the left) upstream of the CAT reporter gene in pCAT basic were transfected into 3T3-L1 cells together with expression vectors for FOXC2 (filled bars) or the corresponding empty vector (open bars). Data represent reporter activities (CAT/Luc) relative to transfection with the RIalpha 1b promoter and empty expression vector that was assigned the value of 1. Data are normalized for expression of luciferase from a cotransfected control vector (pGL3Control) and represent mean ± S.D. of three separate transfections performed in triplicate. The main responsive region is marked with a bold line below the figure.

Specific Binding of a Protein to an Upstream Region of the RIalpha 1b Promoter Is Abolished in the Presence of FOXC2 Expression-- Specific binding of nuclear proteins from 3T3-L1 cells transfected with FOXC2 expression vector or the corresponding empty vector (basal) to a 32P-labeled forkhead oligonucleotide or an oligonucleotide containing 100 bp of RIalpha exon 1A were examined (Fig. 3). Because FOXC1/FREAC3 and FOXC2 have identical DNA binding domains, we used the forkhead-binding site characterized for FOXC1 to detect FOXC2 by EMSA (9). Binding to the labeled forkhead consensus site was induced in FOXC2-transfected cells (Comp. I, lane 8 versus lane 2; Fig. 3A). However, this induction was not as profound as the increase in FOXC2 proteins levels observed by immunoblotting (Fig. 1), which can be explained by the fact that the labeled FOXC1 consensus probe is not specific for FOXC2 (9). An optimal DNA binding sequence for FOXC2 has not been determined. Furthermore, in extracts from FOXC2-transfected cells, we observed a small shift in complex I to a faster mobility compared with basal extracts. By supershift experiments using a human FOXC2 antibody, we identified FOXC2 as the protein forming complex I (lanes 7 and 13). Complex I binding to the labeled DNA fragment could only be displaced by the homologous unlabeled probe (lanes 3 and 9). Mutated oligos (lanes 4 and 10) or oligos from the FOXC2/FOXD1-responsive regions of the RIalpha 1b promoter containing 100 bp of RIalpha exon 1a (lanes 5 and 11) or 72 bp of RIalpha exon 1b (lanes 6 and 12) (see Figs. 2C and 4D for mapping of regions) did not compete for binding. These observations indicate that FOXC2 does not bind directly to the RIalpha 1b promoter. We next labeled oligos from the two responsive promoter regions located in exon 1a (100 bp) and exon 1b (72 bp). Although no changes in DNA-protein complex formation was observed with the 32P-labeled RIalpha exon 1b oligo (not shown), the exon 1a probe that corresponds to the FOXC2-responsive region formed a specific complex in 3T3-L1 nuclear extracts from untransfected cells (Fig. 3B, lane 2), which was strongly reduced by addition of the homologous unlabeled probe (lane 3). This specific DNA-protein complex was nearly abolished in extracts from 3T3-L1 cells transfected with FOXC2 for 24 and 48 h (lanes 4 and 5), which may indicate that FOXC2 is implicated in regulating release of a transcriptional repressor from the RIalpha promoter.


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  		Fig. 3.   A protein binding to the RIalpha 1b promoter is released following FOXC2 expression. EMSA was performed with a double-stranded forkhead oligonucleotide (5'-GATCCCTTAAGTAAACAGCATGAGATC-3') as the 32P-labeled probe (A). Complex (Comp.) formation was analyzed using 2.5 µg of nuclear extracts from 3T3-L1 cells transfected with FOXC2 expression vector (lanes 8-13) or empty vector (Basal, lanes 2-7). Lane 1 shows probe in the absence of nuclear extract. 250-Fold excess of homologous unlabeled probe (+, lanes 3 and 9), a forkhead oligo with altered flanking regions around the core sequence (5'-GATCCAGGCCGTAAACAGCATGAGATC-3' (mut, lanes 4 and 10), RIalpha exon 1a (lanes 5 and 11), and RIalpha exon 1b (lanes 6 and 12) were added as competitors. Supershifts with FOXC2 antibody (+, lanes 7 and 13) were obtained by incubation of antibody for 1 h at room temperature. B, EMSA experiment with a 32P-labeled 100-bp oligonucleotide from RIalpha exon 1a incubated with 2.5 µg of nuclear extracts from 3T3-L1 cells transfected with FOXC2 expression vector (lanes 4 and 5) or the empty vector (lanes 2 and 3). Lane 1 is probe in the absence of nuclear extract. 250-Fold excess of homologous unlabeled probe was added as competitor in lane 3. Results shown are representative of 3 observations using extracts made from separate cultures.

FOXD1 and FOXC2 Induce Reporter Activity Driven by the RIalpha 1b Promoter in Sertoli Cells-- We next wanted to investigate if FOXD1, which is expressed in Sertoli cells of the testis,3 regulated RIalpha levels through the same mechanism as in adipocytes. The level of transfection is much lower in Sertoli cell primary cultures (2-5% as opposed to 20-30% in 3T3-L1 cells), and we detected ectopically expressed HA-tagged FOXC2 and FOXD1 with FITC-conjugated anti-HA antibody (Fig. 4A), showing that expression and localization is restricted to the nucleus in Sertoli cells. To study the effect of FOXD1 on the RIalpha promoter region, we co-transfected reporter constructs containing the RIalpha 1a+1b promoter, the 1a or 1b promoters alone, or a construct containing 4500 bp of the RIIbeta promoter into rat Sertoli cell primary cultures together with expression vector for FOXD1 or the corresponding empty vector (Fig 4B). We also tested if expression of FOXC2 had the same effect on the RIalpha promoter (Fig. 4C). In Sertoli cell cultures, reporter activity from the RIalpha 1b promoter construct was increased 8-fold in the presence of FOXD1 and 5-fold with FOXC2. The RIalpha 1a+1b construct was not similarly induced. The RIalpha and RIIbeta constructs were also induced to a smaller extent by the presence of FOXD1 or FOXC2 (about 2-fold). Deletion of the exon 1a region (RIalpha 1b A+, Fig. 4D) elevated basal activity 2-fold, whereas a further 3' deletion of the region downstream of transcription start (RIalpha 1b A) again reduced basal activity. Some activating effect of FOXD1 (3-fold) was restored in constructs RIalpha 1b A and C+ indicating that a downstream region of the promoter mediated activation of RIalpha 1b by FOXD1. The presence of both upstream- and downstream-responsive regions appeared to have the maximal effect on activity of the 1b promoter. In Sertoli cells, the activation mediated by the downstream region appeared to be more profound than the cis-acting repressor activity residing in the upstream promoter region.


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  		Fig. 4.   FOXD1 and FOXC2 induce expression from the 1b promoter of the RIalpha gene in Sertoli cells. Sertoli cells were grown on coverslips and transfected with 2 µg of expression vectors for HA-tagged FOXD1 or FOXC2. Localization of the ectopically expressed forkheads was visualized by immunofluorescence using anti-HA primary antibody and FITC-conjugated secondary antibody (Ab) (A). Deletion constructs of the RIalpha promoter and a construct from the RIIbeta promoter were transfected into primary cultures of rat Sertoli cells together with expression vectors for FOXD1 (B) and FOXC2 (C) (filled bars) or the corresponding empty vector (open bars). Cell cultures were harvested after 48 h. The figure shows fold induction of reporter activity in the presence of the FOXD1/FOXC2 expression relative to the presence of corresponding empty expression vector. D, 3' and 5' deletion constructs of the RIalpha 1b promoter were inserted upstream of the CAT reporter gene in pCAT basic and transfected into Sertoli cells together with an expression vector for FOXD1 or the corresponding empty vector. Data represent reporter activities (CAT/Luc) relative to transfection with the RIalpha 1b promoter and empty vector that was assigned the value of 1. All data are normalized for expression of luciferase from a cotransfected luciferase control vector and represent mean ± S.D. of three separate transfections performed in triplicate. The responsive regions are marked with bold lines below the figure.

A Truncated FOXD1 Mutant Reduces cAMP-dependent Induction of the RIalpha Gene in Sertoli Cells-- Observing that the pattern of RIalpha regulation by FOXD1 in Sertoli cells mapped to the same downstream region that was identified as responsible for regulation by the cAMP pathway in our previous studies (27), we wished to examine if FOXD1 was implicated in cAMP-dependent regulation of expression from RIalpha 1b. We expressed full-length FOXD1 as well as a FOXD1 mutant containing only the DNA-binding region and no transactivating domains, which by overexpression would displace endogenous FOXD1 by occupying Fox-binding sites (Fig. 5). The RIalpha 1b reporter construct (1 µg/well) was transfected into primary rat Sertoli cells together with empty expression vector, the full-length FOXD1 expression vector, or the truncated FOXD1 vector (FOXD1-DBD) (0.5 µg/well) and left untreated (open bars) or stimulated by 8-(4-chlorophenyl)thio-cAMP (100 µM) for 28 h (filled bars). The induction of RIalpha by cAMP was ~6-fold in cells transfected with empty expression vector and 8-fold in the presence of full-length FOXD1. In the presence of FOXD1-DBD, however, cAMP-induced levels were reduced, which may indicate that FOXD1 is implicated in cAMP-mediated regulation of the RIalpha 1b promoter.


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  		Fig. 5.   Truncated FOXD1 abolishes cAMP-dependent induction of the RIalpha gene. The RIalpha 1b construct in the pCATbasic reporter vector was transfected into primary cultures of rat Sertoli cells together with expression vectors for the full-length FOXD1 vector (FOXD1), a FOXD1 mutant (FOXD1-DBD), or the corresponding empty vector (pEVRFO). The cell cultures were either left untreated (open bars) or stimulated with 8-CPT-cAMP (100 µM) for 28 h (filled bars). Data represent reporter activities (CAT/Luc) relative to unstimulated Sertoli cells transfected with the RIalpha 1b promoter and empty vector that was assigned the value of 1. Data are normalized for expression of luciferase from a cotransfected control vector (0.25 µg/culture dish) and represents mean ± S.D. of three separate transfections performed in triplicate.

Protein Kinase B (PKBalpha ) Induces the Effect of FOXD1 in Sertoli Cells-- The insulin-activated serine/threonine kinase PKB/Akt (reviewed in Ref. 37) has been reported to phosphorylate and negatively regulate forkhead/winged helix transcription factors of the FOXO-family (AFX, FKHR, and FKHRL1) by inducing transport out of the nucleus (38-42). Three highly homologous isoforms of PKB have been characterized and termed PKBalpha /Akt1, PKBbeta /Akt2, and PKBgamma /Akt3 (43-45). To test if PKB affected FOXD1 and FOXC2 in our system, we transfected primary cultures of Sertoli cells with pCMV expression vectors for wild type PKBalpha /Akt1 (0.5 µg/well) together with the FOXD1 expression vector (0.5 µg/well) and pCAT reporter constructs containing the RIalpha 1a or RIalpha 1b promoters (1.0 µg/well). We found that PKBalpha induced expression from the RIalpha 1b promoter 15-fold in the presence of FOXD1 but had no significant effect when FOXD1 was absent (Fig 6A). No effect was observed on the RIalpha 1a promoter. In contrast, overexpression of PKBalpha had no effect in the presence of FOXC2 or FOXD1 in preconfluent or fully differentiated 3T3-L1 adipocytes (not shown). Furthermore, expression of PKBalpha did not affect the level of FOXD1 as shown by immunoblotting. When we expressed a constitutively active and membrane-bound myristoylated PKBalpha mutant (Fig. 6B, dark gray bars), we observed a reduced activation of RIalpha 1b compared with cells expressing wild type PKBalpha (black bars). This might indicate that the activating mechanism of PKB partly depends on detachment of PKB from the membrane. Immunofluorescence data (Fig. 6C) show that HA-tagged wild type PKBalpha is located throughout the cell, whereas myristoylated PKBalpha mutant is restricted to the plasma membrane region as expected.


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  		Fig. 6.   PKB strongly induces the effect of FOXD1 on the RIalpha 1b promoter, whereas a membrane-bound PKB mutant is less effective. A, constructs containing the RIalpha 1a and RIalpha 1b promoters inserted in pCATbasic reporter vector and the corresponding empty reporter vector (1 µg/well) were cotransfected with 0.5 µg of expression vector for FOXD1 (hatched bars), 0.5 µg of expression vector for wild type (wt) protein kinase B/PKBalpha (gray bars), a combination of FOXD1/PKB (black bars) or corresponding empty vectors (open bars) in primary cultures of rat Sertoli cells. A pGL3 luciferase reporter vector (0.25 µg) was transfected as internal control. Data represent reporter activities (CAT/Luc) relative to transfection with the RIalpha 1b promoter and empty vector that was assigned the value of 1. Data are normalized for expression of luciferase from a cotransfected control vector (pGL3Control) and represent mean ± S.D. of three separate transfections performed in triplicate. A Western blot showing FOXD1 expression in nuclear extracts from cells transfected with expression vectors for FOXD1, FOXD1, in combination with PKBalpha , or the corresponding empty vector (pEVRFO)(-) is incorporated in A. B, a reporter construct containing the RIalpha 1b promoter region was cotransfected with 0.5 µg of FOXD1 expression vector (right 4 bars) or the corresponding empty vector (pEVRFO, left 4 bars) together with 0.5 µg of wild type PKBalpha expression vector (black bars), myristoylated (myr) PKBalpha mutant (dark gray bars), kinase dead PKBalpha (light gray bars), or the corresponding pCMV vector (open bars). C, Sertoli cells were grown on coverslips and transfected with 2 µg of expression vectors for HA-tagged wild type PKBalpha and myristoylated PKBalpha . Localization of the ectopically expressed proteins was visualized using anti-HA primary antibody and FITC-conjugated secondary antibody (Ab).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present work, we have performed detailed studies on regulation of the RIalpha subunit of PKA by FOXC2 and FOXD1 in 3T3-L1 adipocytes and Sertoli cell primary cultures, and we found that both transcription factors induce expression from the RIalpha 1b promoter in both Sertoli cells and adipocytes, whereas the RIalpha 1a and RIIbeta promoters were not similarly affected. The reasons for differentially regulated promoters and alternative splicing of leader exons in the RIalpha gene are not known, but one possible explanation is that the relative levels of RIalpha transcripts containing different 5'-UTRs create a fine-tuned control mechanism for rapid translation of RIalpha protein as a response to various signals. Sequences in the 5'-UTR may also affect subcellular localization.

Deletional mapping of the RIalpha 1b promoter in 3T3-L1 cells indicate that an upstream region of the 1b promoter (-400 to -300, part of exon 1a) represses basal transcription, and EMSA experiments show that FOXC2 mediates release of a factor from this region with subsequent activation of RIalpha expression. The responsive part of promoter 1b contains binding sites for the zinc finger transcriptional repressor Ikaros (IK1 and IK2) at positions -401, -392, and -315 (46, 47). We also found a binding site for GATA1 at position -382, another zinc finger protein known to interact with CBP/p300. Recently, an atypical GATA protein, TRPS1, was identified as a transcriptional repressor (48). The IK2 and GATA1 sites in the RIalpha promoter are thereby potential repressor binding elements.

In Sertoli cells, a region downstream of the transcription start site is essential for the inductive effect of FOXD1/FOXC2. We have reported recently (27) that the RIalpha 1b promoter is induced 8-fold by cAMP at the post- transcriptional level, and the cAMP responsiveness is mapped to the same downstream region. Here we suggest that FOXD1 may partly and indirectly be involved in cAMP-mediated regulation through this region, based on the observation that a truncated FOXD1 lacking the transactivating region and functioning as an inhibitor of FOXD1 activity lowers cAMP-induced expression from the RIalpha 1b promoter. FOXD1 and FOXC2 both contain putative PKA phosphorylation sites in the DNA binding domain, identically positioned 5 amino acids downstream of the 15-amino acid N-terminal forkhead signature. This indicates the possibility of a direct regulation of FOXD1 by PKA in addition to induction of the FOXD1 transcript.3 Another winged helix transcription factor is also reported to be regulated by the FSH signal in Sertoli cells (49).

In our study, we found that overexpression of the insulin-activated kinase PKBalpha /Akt1 induced the FOXD1-mediated activation of RIalpha 1b in Sertoli cells but did not affect regulation by FOXD1 or FOXC2 in preconfluent or fully differentiated 3T3-L1 cells. There is a possibility that different PKB isozymes mediate this effect in these cells. PKBalpha /Akt1 seems to regulate spermatogenesis with no effect on glucose tolerance and insulin sensitivity (50, 51), whereas PKBbeta /Akt2 was identified as the PKB isoform required for insulin to maintain normal glucose homeostasis (52, 53).

The FOXO family and several other transcription factors are regulated by direct phosphorylation by PKB/Akt. However, no consensus phosphorylation sites for PKB were found in the FOXC2 or FOXD1 sequences. Thus, we anticipate that a yet unknown factor mediates the effect of PKBalpha on FOXD1 or that FOXD1 and PKBalpha act in concert on downstream effectors that regulate RIalpha . Studies on the dynamics of PKB following activation in B-cells show that shortly after activation, PKB is distributed throughout the cytosol and nucleus (54). We found that ectopically expressed FOXD1 and FOXC2 are strictly localized to the nucleus, and a direct interaction between PKB and FOXD1 should thereby depend on the ability of PKB to dissociate from the membrane and travel to the nucleus. This is supported by the observation that a myristoylated, constitutively membrane-bound PKB mutant has a reduced effect on FOXD1-mediated induction of RIalpha 1b.

The effects of insulin and PKB on translation is well accounted for (55) and facilitates translational initiation of mRNAs containing strong cap-proximal secondary structures (56). We have reported recently (27) that the 5'-UTR of RIalpha 1b mRNA, in contrast to RIalpha 1a mRNA, contains a strong stem-loop and is regulated at the post-transcriptional level by cAMP in Sertoli cells, an effect that appeared to be cell-specific. The observation that both PKA and PKB appear to signal through FOXD1 and the RIalpha 1b 5'-UTR in regulation of RIalpha in Sertoli cells could indicate that a post-transcriptional regulatory mechanism may be involved.

A PKA-independent, FSH/cAMP-mediated activation of PKB, mimicking the insulin-like growth factor I response, has been reported in granulosa cells (57). This activation was found to involve cAMP-regulated guanine exchange factors that may activate phosphatidylinositol 3'-kinase. Other reports have suggested that PKA may activate PKB in a phosphatidylinositol 3'-kinase-independent manner (58). Our observations that activation of the RIalpha 1b promoter by FOXD2 is significantly elevated in the presence of both PKB and cAMP may implicate that cAMP activates PKB by PKA-dependent or PKA-independent mechanisms. Both PKA-dependent and PKA-independent cAMP-signaling pathways may stimulate proliferation in several cell types (59).

Sertoli cells and adipocytes are both highly responsive to hormonal stimuli and share many common features, such as glucose uptake, lipid storage, as well as hormone-sensitive lipase activity. Whereas adipocyte metabolism is regulated by catecholamines and sympatic tonus through the beta -adrenergic receptors, mature Sertoli cells support and control spermatogenesis in response to the pituitary follicle-stimulating hormone (FSH) testosterone, insulin, and insulin-like growth factors I/II (60, 61). Apparently, downstream pathways are parallel in the two cell types and involve cAMP, PKA, as well as Fox family members. This in turn tunes cAMP sensitivity by up-regulation of RIalpha levels (Fig. 7).


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  		Fig. 7.   Tuning of cAMP sensitivity, metabolism, and reproductive function by integration of signals in adipocytes and Sertoli cells. Stimulation of the beta -adrenergic receptor (beta -AR) by catecholamines (CAT) in adipocytes or the follicle-stimulating hormone receptor (FSHR) by FSH in Sertoli cells leads to increased levels of cAMP and activation of PKA. In both cells, PKA phosphorylates and activates hormone-sensitive lipase leading to enhanced lipolysis. In adipocytes, PKA signaling up-regulates levels of uncoupling protein-1 (UCP1) that contributes to increase energy expenditure. In Sertoli cells, PKA activity is essential for expression of several factors vital for spermatogenesis (e.g. androgen-binding protein, ABP). Furthermore, cAMP induces levels of the RIalpha regulatory subunit of PKA in both cell types and of the winged helix transcription factors Foxc2 in adipocytes and FoxD1 in Sertoli cells. Conversely, Fox proteins increase RIalpha levels by regulating the RIalpha 1b promoter. Insulin (Ins) and tumor necrosis factor alpha  (TNFalpha ) in adipocytes and various growth factors (GrF) in Sertoli cells signal through corresponding receptors (insulin receptor (IR), TNF receptor (TNFR), growth factor receptor (GrFR)) and phosphatidylinositol 3'-kinase (PI3K) to activate PKB and also to induce the levels of Foxc2 mRNA in adipocytes. In Sertoli cells, PKBalpha acts in concert with FoxD1 to further activate RIalpha levels, and possibly a similar function of PKBbeta may be found in adipocytes. In addition to this, stimulation of PKB activity by FSH has also been reported in Sertoli cells. Taken together, similar mechanisms regulate RIalpha expression and thereby increase the sensitivity of PKA to cAMP in both cell types (marked by bold arrow with *).

In conclusion, FOXD1 and FOXC2 both induce the RIalpha 1b promoter to elevate RIalpha /PKAI levels. In adipocytes the effect is mainly mediated through an upstream promoter region and the possible release of a repressor, whereas in Sertoli cells, FOXD1 primarily activates through a downstream region and mediates signals from the cAMP/PKA and PKB signaling pathways.

    ACKNOWLEDGEMENTS

We greatly appreciate the skillful technical assistance of Gladys Josefsen and Guri Opsahl, and we thank Dr. Naheed N. Ahmed for providing the PKBalpha /Akt1 expression vectors.

    FOOTNOTES

* This work was supported by the Norwegian Cancer Society, the Program for Advanced Studies in Medicine, the Norwegian Research Council, Anders Jahres Foundation, Novo Nordic Research Foundation Committee, the Swedish Medical Research Foundation, The Arne and IngaBritt Lundberg Foundation, the Juvenile Diabetes Foundation, the Wallenberg Foundation, Amersham Biosciences, The Upjohn Co., and a Junior Individual Grant from the Swedish Foundation for Strategic Research (to S. E.).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.

Dagger Both authors contributed equally to this work.

|| Present address: Oslo Urological University Clinic, Aker University Hospital, N-0514 Oslo, Norway.

** To whom correspondence should be addressed: Dept. of Medical Biochemistry, Inst. of Basic Medical Sciences, University of Oslo, P. O. Box 1112 Blindern, N-0317 Oslo, Norway. Tel.: 4722851454; Fax: 4722851497; E-mail: kjetil.tasken@basalmed.uio.no.

Published, JBC Papers in Press, April 9, 2002, DOI 10.1074/jbc.M200131200

2 M. K. Dahle, L. M. Grønning, A. Cederberg, H. K. Blomhoff, N. Miura, S. Enerbäck, K. A. Taskén, and K. Taskén, unpublished data.

3 K. A. Taskén, S. Ernstsson, H. K. Knutsen, L. M. Grønning, K. Taskén, and S. Enerbäck, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: Fox, forkhead box; C, catalytic subunit; CMV, cytomegalovirus; DBD, DNA binding domain; EMSA, electrophoretic mobility shift assay; FITC, fluorescein isothiocyanate; FSH, follicle-stimulating hormone; HA tag, hemagglutinin epitope tag; IK, ikaros; PKA, cAMP-dependent protein kinase; PKB, protein kinase B; R, regulatory subunit; TNF, tumor necrosis factor; 5'-UTR, 5'-untranslated region; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CAT, chloramphenicol acetyltransferase; Luc, luciferase; oligo, oligonucleotide.

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RESULTS
DISCUSSION
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