We have reported recently that mice overexpressing the forkhead/winged helix transcription factorFOXC2 are lean and show increased responsiveness to insulin due to sensitization of the β-adrenergic cAMP-PKA+pathway and increased levels of the RIα 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 RIα 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 RIα1b 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 Bα/Akt1 strongly induces this effect. Furthermore, we show that an inactive FOXD1 mutant lowers the cAMP-mediated induction of the RIα1b reporter construct. In summary, winged helix transcription factors of the FOXC/FOXD families function as regulators of the RIα subunit of PKA and may integrate hormonal signals acting through protein kinase B and cAMP in a cell-specific manner. We have reported recently that mice overexpressing the forkhead/winged helix transcription factorFOXC2 are lean and show increased responsiveness to insulin due to sensitization of the β-adrenergic cAMP-PKA+pathway and increased levels of the RIα 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 RIα 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 RIα1b 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 Bα/Akt1 strongly induces this effect. Furthermore, we show that an inactive FOXD1 mutant lowers the cAMP-mediated induction of the RIα1b reporter construct. In summary, winged helix transcription factors of the FOXC/FOXD families function as regulators of the RIα subunit of PKA and may integrate hormonal signals acting through protein kinase B and cAMP in a cell-specific manner. forkhead box catalytic subunit cytomegalovirus DNA binding domain electrophoretic mobility shift assay fluorescein isothiocyanate follicle-stimulating hormone hemagglutinin epitope tag ikaros cAMP-dependent protein kinase protein kinase B regulatory subunit tumor necrosis factor 5′-untranslated region 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid chloramphenicol acetyltransferase luciferase oligonucleotide 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. 2Gajiwala K.S. Burley S.K. Curr. Opin. Struct. Biol. 2000; 10: 110-116Crossref PubMed Scopus (461) Google Scholar). A number of forkhead and forkhead-related genes have been isolated to date (3Weigel D. Jackle H. Cell. 1990; 63: 455-456Abstract Full Text PDF PubMed Scopus (411) Google Scholar, 4Lai E. Clark K.L. Burley S.K. Darnell J.E., Jr. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10421-10423Crossref PubMed Scopus (306) Google Scholar, 5Lai E. Prezioso V.R. Tao W.F. Chen W.S. Darnell J.E., Jr. Genes Dev. 1991; 5: 416-427Crossref PubMed Scopus (448) Google Scholar, 6Dirksen M.L. Jamrich M. Dev. Genet. 1995; 17: 107-116Crossref PubMed Scopus (59) Google Scholar, 7Kaestner K.H. Lee K.H. Schlondorff J. Hiemisch H. Monaghan A.P. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7628-7631Crossref PubMed Scopus (129) Google Scholar), and the Fox1 nomenclature (Forkhead box) has now been adopted for all chordate forkhead genes (www.biology.pomona.edu/fox.html) (8Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar). Among these are several forkhead relatedactivators (FREACs) cloned from human (9Pierrou S. Hellqvist M. Samuelsson L. Enerbäck S. Carlsson P. EMBO J. 1994; 13: 5002-5012Crossref PubMed Scopus (379) Google Scholar, 10Cederberg A. Betz R. Lagercrantz S. Larsson C. Hulander M. Carlsson P. Enerbäck S. Genomics. 1997; 44: 344-346Crossref PubMed Scopus (12) Google Scholar, 11Ernstsson S. Pierrou S. Hulander M. Cederberg A. Hellqvist M. Carlsson P. Enerbäck S. J. Biol. Chem. 1996; 271: 21094-21099Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 12Ernstsson S. Betz R. Lagercrantz S. Larsson C. Ericksson S. Cederberg A. Carlsson P. Enerbäck S. Genomics. 1997; 46: 78-85Crossref PubMed Scopus (13) Google Scholar, 13Larsson C. Hellqvist M. Pierrou S. White I. Enerbäck S. Carlsson P. Genomics. 1995; 30: 464-469Crossref PubMed Scopus (62) Google Scholar) 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 (11Ernstsson S. Pierrou S. Hulander M. Cederberg A. Hellqvist M. Carlsson P. Enerbäck S. J. Biol. Chem. 1996; 271: 21094-21099Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 14Cederberg A. Hulander M. Carlsson P. Enerbäck S. J. Biol. Chem. 1999; 274: 165-169Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar).FOXC2 (FREAC11, FKHL14, MFH-1) (15van Dongen M.J. Cederberg A. Carlsson P. Enerbäck S. Wikstrom M. J. Mol. Biol. 2000; 296: 351-359Crossref PubMed Scopus (52) Google Scholar, 16Miura N. Iida K. Kakinuma H. Yang X.L. Sugiyama T. Genomics. 1997; 41: 489-492Crossref PubMed Scopus (29) Google Scholar) is restricted to adipocytes in adults (1Cederberg A. Grønning L.M. Ahren B. Taskén K. Carlsson P. Enerbäck S. Cell. 2001; 106: 563-573Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), whereas the prenatal form is important for development. Mice lacking Foxc2 die during embryogenesis or perinatally and exhibit aortic arch and skeletal defects (17Yang X.L. Matsuura H., Fu, Y. Sugiyama T. Miura N. FEBS Lett. 2000; 470: 29-34Crossref PubMed Scopus (20) Google Scholar, 18Iida K. Koseki H. Kakinuma H. Kato N. Mizutani-Koseki Y. Ohuchi H. Yoshioka H. Noji S. Kawamura K. Kataoka Y. Ueno F. Taniguchi M. Yoshida N. Sugiyama T. Miura N. Development. 1997; 124: 4627-4638Crossref PubMed Google Scholar). Instead, overexpression of FOXC2 in adipose tissue has been important in understanding its function in adult mice. FOXC2transgenic mice develop a phenotype characterized by a high sensitivity to insulin and partial resistance to diet-induced obesity (1Cederberg A. Grønning L.M. Ahren B. Taskén K. Carlsson P. Enerbäck S. Cell. 2001; 106: 563-573Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). This effect is partly due to up-regulation of β-adrenergic receptors and PKA type Iα that lower the threshold for activation of PKA by cAMP, resulting in a hypersensitive β-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. 19Taskén K. Skålhegg B.S. Taskén K.A. Solberg R. Knutsen H.K. Levy F.O. Sandberg M. Ørstavik S. Larsen T. Johansen A.K. Vang T. Schrader H.P. Reinton N.T. Torgersen K.M. Hansson V. Jahnsen T. Adv. Second Messenger Phosphoprotein Res. 1997; 31: 191-204Crossref PubMed Google Scholar). Targeted disruption of the RIIβ 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 IIβ (RIIβ2C2) to type Iα (RIα2C2) holoenzyme (20Cummings D.E. Brandon E.P. Planas J.V. Motamed K. Idzerda R.L. McKnight G.S. Nature. 1996; 382: 622-626Crossref PubMed Scopus (368) Google Scholar, 21Amieux P.S. Cummings D.E. Motamed K. Brandon E.P. Wailes L.A., Le, K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; 272: 3993-3998Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 22McKnight G.S. Cummings D.E. Amieux P.S. Sikorski M.A. Brandon E.P. Planas J.V. Motamed K. Idzerda R.L. Recent Prog. Horm. Res. 1998; 53: 139-159PubMed Google Scholar). The effect of this regulatory subunit shift was shown to lower the threshold for PKA activation by cAMP and to modulate lipolysis (23Planas J.V. Cummings D.E. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1999; 274: 36281-36287Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The RIIβ knockout phenotype resembles that of the FOXC2 transgenic mice (1Cederberg A. Grønning L.M. Ahren B. Taskén K. Carlsson P. Enerbäck S. Cell. 2001; 106: 563-573Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) and supports the notion that regulation of PKA isozyme composition, particularly RIIβ versus RIα, is important for hormonal responsiveness and cAMP sensitivity. The RIα gene is controlled by several promoters, giving rise to at least three mRNAs that differ in the first non-coding exon (24Solberg R. Sandberg M. Natarajan V. Torjesen P.A. Hansson V. Jahnsen T. Taskén K. Endocrinology. 1997; 138: 169-181Crossref PubMed Scopus (42) Google Scholar, 25Dahle M.K. Reinton N. Ørstavik S. Taskén K.A. Taskén K. Mol. Reprod. Dev. 2001; 59: 11-16Crossref PubMed Scopus (11) Google Scholar, 26Barradeau S. Imaizumi-Scherrer T. Weiss M.C. Faust D.M. FEBS Lett. 2000; 476: 272-276Crossref PubMed Scopus (18) Google Scholar). Two of these mRNAs (RIα1a and -1b) are expressed in most tissues (25Dahle M.K. Reinton N. Ørstavik S. Taskén K.A. Taskén K. Mol. Reprod. Dev. 2001; 59: 11-16Crossref PubMed Scopus (11) Google Scholar), and we have recently reported cAMP-mediated post-transcriptional regulation of RIα1b in Sertoli cells (27Dahle M.K. Knutsen H.K. Taskén K.A. Pilz R. Taskén K. Eur. J. Biochem. 2001; 268: 5920-5929Crossref PubMed Scopus (14) Google Scholar). We have shown that both FOXC2 and RIα are induced by cAMP in mouse 3T3-L1 adipocytes (28Grønning L.M. Cederberg A. Miura N. Enerbäck S. Taskén K. Mol. Endocrinol. 2002; 16: 873-883Crossref PubMed Scopus (54) Google Scholar) 2M. K. Dahle, L. M. Grønning, A. Cederberg, H. K. Blomhoff, N. Miura, S. Enerbäck, K. A. Taskén, and K. Taskén, unpublished data. and that FOXD1 and RIα are induced by cAMP in Sertoli cell primary cultures 3K. A. Taskén, S. Ernstsson, H. K. Knutsen, L. M. Grønning, K. Taskén, and S. Enerbäck, manuscript in preparation. (27Dahle M.K. Knutsen H.K. Taskén K.A. Pilz R. Taskén K. Eur. J. Biochem. 2001; 268: 5920-5929Crossref PubMed Scopus (14) Google Scholar). We have also reported recently (28Grønning L.M. Cederberg A. Miura N. Enerbäck S. Taskén K. Mol. Endocrinol. 2002; 16: 873-883Crossref PubMed Scopus (54) Google Scholar) 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 TNFα. In this study, we examine the effect of FOXD1 andFOXC2 on the RIα promoters, showing that the expression of RIα1b but not RIα1a is induced. Deletional mapping reveals thatFOXC2 and FOXD1 regulate expression from the RIα1b 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, PKBα strongly increases the effect of FOXD1, and a truncated FOXD1 inhibits cAMP-mediated induction of the RIα1b promoter, indicating that FOXD1 may function as a mediator of signaling by both cAMP and PKB in a cell-specific manner. The RIα promoter constructs RIα1a+1b (−882 to +77), RIα1a (−882 to −310), RIα1b (−406 to +77) (24Solberg R. Sandberg M. Natarajan V. Torjesen P.A. Hansson V. Jahnsen T. Taskén K. Endocrinology. 1997; 138: 169-181Crossref PubMed Scopus (42) Google Scholar), and the RIIβ promoter construct (−4500 to −123) (29Knutsen H.K. Taskén K. Eskild W. Richards J.S. Kurten R.C. Torjesen P.A. Jahnsen T. Hansson V. Guerin S. Taskén K.A. Mol. Cell. Endocrinol. 1997; 129: 101-114Crossref PubMed Scopus (14) Google Scholar) were inserted into the pCAT basic reporter vector (Promega, Madison, WI). Five deletion constructs were made from the RIα1b promoter region: A+ (−307 to +77), A (−307 to +4), B (−199 to +4), C (−92 to +4), and C+ (−92 to + 77) (27Dahle M.K. Knutsen H.K. Taskén K.A. Pilz R. Taskén K. Eur. J. Biochem. 2001; 268: 5920-5929Crossref PubMed Scopus (14) Google Scholar). 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 (30Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1521) Google Scholar). 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 PKBα/Akt1 were created in pCMV5 or pCMV6, respectively (31Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (490) Google Scholar). All these expression vectors contain cytomegalovirus (CMV) promoters. In addition, the luciferase expression vector pGL3Control (Promega) was used as internal control in transfection experiments. 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. (32Dorrington J.H. Roller N.F. Fritz I.B. Mol. Cell. Endocrinol. 1975; 3: 57-70Crossref PubMed Scopus (294) Google Scholar) with some modifications (33Øyen O. Frøysa A. Sandberg M. Eskild W. Joseph D. Hansson V. Jahnsen T. Biol. Reprod. 1987; 37: 947-956Crossref PubMed Scopus (68) Google Scholar). 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 (34Grønning L.M. Knutsen H.K. Eskild W. Hansson V. Taskén K. Taskén K.A. Eur. J. Endocrinol. 1999; 141: 75-82Crossref PubMed Scopus (3) Google Scholar) 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. 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 (35Pothier F. Ouellet M. Julien J.P. Guerin S.L. DNA Cell Biol. 1992; 11: 83-90Crossref PubMed Scopus (154) Google Scholar) and normalized for expression of luciferase. 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 Complete™ 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 RIα (1:500) (Transduction Laboratories) or humanFOXC2 (1:250) (16Miura N. Iida K. Kakinuma H. Yang X.L. Sugiyama T. Genomics. 1997; 41: 489-492Crossref PubMed Scopus (29) Google Scholar), chicken polyclonal antibody against amino acids 1–12 in FOXD1 (1:200), or rabbit polyclonal antibody against PKA catalytic subunit (Cα, 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). 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 mmNaCl, 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), Complete™ 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. Electrophoretic mobility shift assays (EMSAs) were performed using double-stranded32P-end-labeled forkhead consensus oligonucleotide (5′-GATCCCTTAAGTAAACAGCATGAGATC-3′) (9Pierrou S. Hellqvist M. Samuelsson L. Enerbäck S. Carlsson P. EMBO J. 1994; 13: 5002-5012Crossref PubMed Scopus (379) Google Scholar) or a 100-bp region of RIα exon 1a (−407 to −306). For each reaction, 5000 cpm of labeled probe were incubated with 2.5 (Fig. 3 A) or 5 μg (Fig.3 B) of crude nuclear proteins from transfected 3T3-L1 preadipocytes and 0.5 (Fig. 3 A) or 1 μg (Fig.3 B) of poly(dI·dC) in a buffer containing 5 mmHepes, 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.3 B) or 100 mm KCl (Fig. 3 A) 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′) (9Pierrou S. Hellqvist M. Samuelsson L. Enerbäck S. Carlsson P. EMBO J. 1994; 13: 5002-5012Crossref PubMed Scopus (379) Google Scholar). In addition, competitions were performed with oligonucleotides containing the RIα exon 1a (−407 to −306) or exon 1b (+1 to +68) sequences. In supershift experiments, a human monoclonal FOXC2 antibody (16Miura N. Iida K. Kakinuma H. Yang X.L. Sugiyama T. Genomics. 1997; 41: 489-492Crossref PubMed Scopus (29) Google Scholar) 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 mmglycine, 2 mm EDTA) at 4 °C. Subsequently, gels were dried and subjected to autoradiography. Primary Sertoli cells were grown on polylysine-treated coverslips in 6-well plates and transfected with pEF-BOS/FOXC2, pEF-BOS/FOXD1, pCMV/PKBα, or pCMV/myristoylated PKBα (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 (36Collas P. Liang M.R. Vincent M. Alestrom P. J. Cell Sci. 1999; 112: 1045-1054Crossref PubMed Google Scholar). In order to establish whether FOXC2 expression could induce RIα 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. RIα, the PKA catalytic subunit, Cα, 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 (16Miura N. Iida K. Kakinuma H. Yang X.L. Sugiyama T. Genomics. 1997; 41: 489-492Crossref PubMed Scopus (29) Google Scholar). RIα levels were ∼2-fold induced in the presence ofFOXC2 at 6–24 h of expression as determined by densitometric scanning. In contrast, FOXC2 had no effect on the levels of Cα. To map the effect of FOXC2 on RIα promoter activity in adipocytes, we cotransfected 3T3-L1 cells at 80% confluency with reporter constructs containing the RIα1a+1b promoter, the 1a or 1b promoters alone, and a construct containing 4500 bp of the RIIβ promoter together with pCB6+/FOXC2 or the empty expression vector (Fig.2 A). We simultaneously tested whether FOXD1, which is not endogenously expressed in adipocytes, had a similar effect on the RIα promoter (Fig.2 B). FOXC2 expression induced a 4–5-fold increase in reporter activity directed from the RIα1a+1b and the RIα1b promoter constructs, and in contrast, the RIα1a or RIIβ promoters were slightly down-regulated by the presence ofFOXC2. The same pattern of regulation was observed withFOXD1. We next analyzed expression from five 3′ and/or 5′ deletion constructs of RIα1b in the pCAT basic reporter vector (Fig.2 C, left panel) (27Dahle M.K. Knutsen H.K. Taskén K.A. Pilz R. Taskén K. Eur. J. Biochem. 2001; 268: 5920-5929Crossref PubMed Scopus (14) Google Scholar). We observed a 7-fold induction of basal reporter expression when a 100-bp region in exon 1A was absent (RIα1b 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 FOXC2regulation may involve release of repression residing in this region. In construct RIα1b B, however, basal levels were down to the level of the longest construct (RIα1b), but the inductive effect ofFOXC2 was not reconstituted. Interestingly, the elevated basal levels of the shortest constructs (RIα1b C and RIα1b C+) were again higher, which indicates that repressive effects may reside in RIα1b B as well, although not regulated by FOXC2. 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 RIα exon 1A were examined (Fig. 3). Because FOXC1/FREAC3 and FOXC2 have identical DNA binding domains, we used the forkhead-binding site characterized forFOXC1 to detect FOXC2 by EMSA (9Pierrou S. Hellqvist M. Samuelsson L. Enerbäck S. Carlsson P. EMBO J. 1994; 13: 5002-5012Crossref PubMed Scopus (379) Google Scholar). Binding to the labeled forkhead consensus site was induced inFOXC2-transfected cells (Comp. I, lane 8 versus lane 2; Fig. 3 A). However, this induction was not as profound as the increase in FOXC2proteins levels observed by immunoblotting (Fig. 1), which can be explained by the fact that the labeled FOXC1 consensus probe is not specific for FOXC2 (9Pierrou S. Hellqvist M. Samuelsson L. Enerbäck S. Carlsson P. EMBO J. 1994; 13: 5002-5012Crossref PubMed Scopus (379) Google Scholar). 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 theFOXC2/FOXD1-responsive regions of the RIα1b promoter containing 100 bp of RIα exon 1a (lanes 5 and11) or 72 bp of RIα exon 1b (lanes 6 and12) (see Figs. 2 C and 4 D for mapping of regions) did not compete for binding. These observations indicate that FOXC2 does not bind directly to the RIα1b 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 the32P-labeled RIα 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.3 B, 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 RIα promoter. We next wanted to investigate ifFOXD1, which is expressed in Sertoli cells of the testis,3 regulated RIα 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 andFOXD1 with FITC-conjugated anti-HA antibody (Fig.4 A), showing that expression and localization is restricted to the nucleus in Sertoli cells. To study the effect of FOXD1 on the RIα promoter region, we co-transfected reporter constructs containing the RIα1a+1b promoter, the 1a or 1b promoters alone, or a construct containing 4500 bp of the RIIβ promoter into rat Sertoli cell primary cultures together with expression vector for FOXD1 or the corresponding empty vector (Fig 4 B). We also tested if expression ofFOXC2 had the same effect on the RIα promoter (Fig.4 C). In Sertoli cell cultures, reporter activity from the RIα1b promoter construct was increased 8-fold in the presence ofFOXD1 and 5-fold with FOXC2. The RIα1a+1b construct was not similarly induced. The RIα and RIIβ constructs were also induced to a smaller extent by the presence ofFOXD1 or FOXC2 (about 2-fold). Deletion of the exon 1a region (RIα1b A+, Fig. 4 D) elevated basal activity 2-fold, whereas a further 3′ deletion of the region downstream of transcription start (RIα1b A) again reduced basal activity. Some activating effect of FOXD1 (3-fold) was restored in constructs RIα1b A and C+ indicating that a downstream region of the promoter mediated activation of RIα1b 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. Observing that the pattern of RIα 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 (27Dahle M.K. Knutsen H.K. Taskén K.A. Pilz R. Taskén K. Eur. J. Biochem. 2001; 268: 5920-5929Crossref PubMed Scopus (14) Google Scholar), we wished to examine if FOXD1 was implicated in cAMP-dependent regulation of expression from RIα1b. We expressed full-length FOXD1 as well as a FOXD1mutant containing only the DNA-binding region and no transactivating domains,
As a result of selecting triglycerides as the major vehicle for storing superfluous energy, evolution came up with a specialized cell type, the adipocyte, equipped to handle triglycerides and its potentially toxic metabolites - fatty acids. For the first time in history large human populations are subjected a wealth of cheap, accessible and palatable calories. This has created a situation, on a large scale not previously encountered, in which the capacity to store triglycerides in adipocytes is an important determinant of human health. Too few adipocytes (e.g. lipodystrophia) or a situation in which all adipocytes are filled, to their maximum capacity (e.g. severe obesity), will create very similar and unfavorable metabolic situations in which ectopic triglyceride stores will appear in tissues like liver and muscle. This review sets out to discuss the adipocyte and its role in metabolism as well as the consequences of a metabolic situation, in which the adipocyte has lost its fat storing monopoly. Keywords: Insulin Resistance, Adipocentric View, lipodystrophia, triglycerides
To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-beta-galactosidase reporter gene "knock-in" (Foxs1beta-gal/beta-gal) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a beta-galactosidase reporter gene. Staining for beta-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1beta-gal/beta-gal mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1beta-gal/lbeta-gal males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1beta-gal/beta-gal mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function.
We describe the cloning and sequence analysis of a nearly full-length cDNA as well as a corresponding 5.2-kilobase pair genomic fragment encoding FREAC-4, a member of the forkhead family of transcription factors. The cDNA is collinear with respect to the coding region of the intronless genomic clone. The conceptual translation product predicts a protein of 465 amino acids with a hyperacidic amino-terminal end, a DNA binding forkhead domain and a carboxyl-terminal part that is rich in homopolymeric runs of prolines and alanines. The transcription start is identified using an RNase protection assay. A 2.7-kilobase pair genomic DNA fragment, located immediately upstream of the translation start, was fused to a luciferase reporter gene. Significant levels of luciferase activity were detected when this construct was transfected into two kidney-derived cell lines, 293 and COS-7 cells, whereas only background reporter gene expression was observed in a cell line of nonkidney origin. Cotransfections with plasmids expressing WT-1, WTAR (a mutated form of WT-1), p53, and a mutated form of p53 revealed a complex pattern of regulation with a 3-fold induction with WT-1, a 7-fold induction with mutated p53, and a 4-fold repression with wild-type p53. A 5′-promoter deletion series delimits a DNA fragment necessary for WT-1 inducibility in cotransfection experiments. This fragment is shown to contain at least one cis-element that is capable of interacting with recombinant WT-1. We describe the cloning and sequence analysis of a nearly full-length cDNA as well as a corresponding 5.2-kilobase pair genomic fragment encoding FREAC-4, a member of the forkhead family of transcription factors. The cDNA is collinear with respect to the coding region of the intronless genomic clone. The conceptual translation product predicts a protein of 465 amino acids with a hyperacidic amino-terminal end, a DNA binding forkhead domain and a carboxyl-terminal part that is rich in homopolymeric runs of prolines and alanines. The transcription start is identified using an RNase protection assay. A 2.7-kilobase pair genomic DNA fragment, located immediately upstream of the translation start, was fused to a luciferase reporter gene. Significant levels of luciferase activity were detected when this construct was transfected into two kidney-derived cell lines, 293 and COS-7 cells, whereas only background reporter gene expression was observed in a cell line of nonkidney origin. Cotransfections with plasmids expressing WT-1, WTAR (a mutated form of WT-1), p53, and a mutated form of p53 revealed a complex pattern of regulation with a 3-fold induction with WT-1, a 7-fold induction with mutated p53, and a 4-fold repression with wild-type p53. A 5′-promoter deletion series delimits a DNA fragment necessary for WT-1 inducibility in cotransfection experiments. This fragment is shown to contain at least one cis-element that is capable of interacting with recombinant WT-1.
Insulin resistance plays a major role in the development of type 2 diabetes and may be causally associated with increased intracellular fat content. Transgenic mice with adipocyte-specific overexpression of FOXC2 (forkhead transcription factor) have been generated and shown to be protected against diet-induced obesity and glucose intolerance. To understand the underlying mechanism, we examined the effects of chronic high-fat feeding on tissue-specific insulin action and glucose metabolism in the FOXC2 transgenic (Tg) mice. Whole-body fat mass were significantly reduced in the FOXC2 Tg mice fed normal diet or high-fat diet compared with the wild-type mice. Diet-induced insulin resistance in skeletal muscle of the wild-type mice was associated with defects in insulin signaling and significant increases in intramuscular fatty acyl CoA levels. In contrast, FOXC2 Tg mice were completely protected from diet-induced insulin resistance and intramuscular accumulation of fatty acyl CoA. High-fat feeding also blunted insulin-mediated suppression of hepatic glucose production in the wild-type mice, whereas FOXC2 Tg mice were protected from diet-induced hepatic insulin resistance. These findings demonstrate an important role of adipocyte-expressed FOXC2 on whole-body glucose metabolism and further suggest FOXC2 as a novel therapeutic target for the treatment of insulin resistance and type 2 diabetes.
