| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Pediatrics (S.K.D., A.S., A.O.S., D.R.P.), Baylor College of Medicine, Houston, Texas 77030; Department of Medicine (D.Y., J.G.J.), University of Texas Health Science Center, San Antonio, Texas 78284; and Department of Pathology and Laboratory Medicine (F.G.B.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: David R. Powell, Texas Children’s Hospital, Feigin Center, MC 3–2482, 6621 Fannin, Houston, Texas 77030. E-mail: dpowell{at}bcm.tmc.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Insulin inhibits, and glucocorticoids stimulate, hepatic expression of phosphoenolpyruvate carboxykinase (PEPCK) and tyrosine aminotransferase (TAT). IREs mapped to the PEPCK and TAT gene promoters resemble the human and rat IGFBP-1 IREs by requiring the same T(G/A)TTT motif for maximal insulin inhibition and glucocorticoid stimulation of promoter activity (2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13). This suggests that the same protein(s) binds the T(G/A)TTT sequence to confer insulin and glucocorticoid responsiveness to these genes. Identifying this protein(s) is necessary to understand how insulin and glucocorticoids regulate hepatic expression of these, and probably many additional, genes.
The hepatic nuclear factor 3 (HNF3) family of proteins belong to the forkhead/winged helix superfamily of transcription factors that plays a role in embryogenesis and differentiation (14). HNF3 proteins bind the IGFBP-1, PEPCK, and TAT IREs (7, 11, 12, 15, 16). However, IRE-bound HNF-3 forms do not appear to confer insulin inhibition to these promoters (7, 15, 16), although they may confer glucocorticoid stimulation (17, 18).
In the nematode Caenorhabditis elegans, activity of the gene daf-2, an insulin receptor homolog, contributes to the rapid rate of aging. daf-2 activity also blocks entry into dauer phase; when food is scarce, young animals become dauers, which are long-lived forms that remain small and reproductively immature. Severe daf-2 mutations slow the rate of aging and allow entry into dauer phase even in the presence of food (19, 20). Life-span extension and dauer entry of these animals require the active product of the daf-16 gene. Thus, a key role for the insulin-receptor homolog daf-2 is to antagonize daf-16 action (21, 22).
daf-16 encodes a forkhead protein. The DNA binding domain of the DAF-16 protein resembles those in HNF3 forms but is most similar to those in FKHR, FKHRL1 and AFX proteins; indeed, DAF-16 and these three proteins comprise a distinct class of forkhead DNA binding domains (21, 22, 23, 24, 25). This suggests that, analogous to the ability of daf-2 to antagonize daf-16 activity in C. elegans, insulin acts through the insulin receptor to antagonize action of FKHR and related proteins in mammals. If so, then FKHR may bind the T(G/A)TTT IRE motif in the IGFBP-1, PEPCK and TAT promoters. The function of FKHR proteins is not clear since they were first identified as human oncogenic fusion proteins (25, 26); indeed, target DNA elements for FKHR proteins have not been identified.
Data presented in this manuscript show that FKHR binds to the IGFBP-1, PEPCK, and TAT promoter IREs, but not to a number of insulin-unresponsive IRE mutants. In addition, FKHR stimulated activity of the native IGFBP-1 promoter, but not IGFBP-1 promoter constructs containing insulin-unresponsive IRE mutants.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
), were
described previously (7).
|
To construct pGST-FKHR, which expresses GST-FKHR fusion protein in Escherichia coli, the 5' region of the FKHR cDNA was released from pcDNA3-FKHR with KpnI/SacI and subcloned into M13mp18 at SalI/SacI. A BamHI site was placed 5' to the FKHR translation start codon by site-directed mutagenesis using the Muta-Gene kit (Bio-Rad Laboratories, Inc., Hercules, CA) as described previously (7). The 3' region of FKHR cDNA, released from pcDNA3-FKHR with SacI/XbaI, was subcloned into pSP73 at SacI/XbaI. The 5' FKHR fragment was released from M13mp18 with BamHI/SacI and the 3' FKHR fragment was released from pSP73 with SacI/XhoI; these two fragments were annealed with GST expression vector pGEX-5X-3 (Pharmacia & Upjohn, Bridgewater, NJ), which had been linearized with BamHI/XhoI, creating pGST-FKHR.
A 5' FKHR DNA fragment released from pcDNA3-FKHR using HindIII/MluI, and a 3' FKHR DNA fragment released from pcDNA3-FKHR using MluI/XbaI, were ligated into the eukaryotic expression vector Rc/RSV (Invitrogen Corp., Carlsbad, CA) at the HindIII/XbaI sites to create pRSV-FKHR. To create the control plasmid pRSV, Rc/RSV was cleaved with HindIII/XbaI, Klenow-filled and then reannealed. Mutated sequences and construct orientations were confirmed by DNA sequencing using Sequenase (U.S. Biochemicals, Cleveland, OH) in the dideoxy chain termination method (27, 28).
Cell culture and DNA transfection
Maintenance and transfection of HEP G2 cells, and preparation of
HEP G2 whole cell extract, have been described (7, 27, 29). HEP G2
cells were cotransfected with 3 µg of CAT plasmid and either with 1
µg of pRSV, pRSV-FKHR, pcDNA3 or pcDNA3-FKHR; cotransfecting 1 µg
of pRSVL, which contains the RSV LTR 5' to the luciferase reporter gene
(30), controlled for transfection efficiency. Transfected cells were
washed in PBS and then incubated in serum-free medium as described (2).
Chloramphenicol acetyltransferase (CAT) and luciferase assays were
performed by standard methods (30, 31).
Northern analysis
After a 4-h incubation in serum-free medium, HEP G2 cells were
harvested and total RNA isolated (32). Integrity of total RNA from HEP
G2 and MCF-7 cells was confirmed by 1% formaldehyde-agarose gel
electrophoresis. Polyadenylated RNA was isolated from HEP G2 total RNA
using the Poly(A) Quik mRNA Isolation Kit (Stratagene, La
Jolla, CA) and then used for Northern analysis of FKHR transcripts
(33). The 1.5-kb SacI/XbaI fragment of the FKHR
DNA clone (23) was labeled with 32[P] and used as
probe.
RNase protection assay
A 410 bp BglII/ScaI fragment encoding
parts of FKHR exons 1 and 2 was subcloned into pSP72 (Promega Corp.) at BamHI/PvuII. After vector
linearization, the SP6 promoter was used to transcribe antisense
complementary FKHR RNA in vitro. This RNA was labeled with
32[P] and then hybridized with 25 µg of total HEP G2 or
MCF-7 RNA (25 µg). After hybridizing RNA with labeled probe,
single-stranded RNA was digested with RNase A, and protected fragments
then separated by 8 M urea 6% SDS-PAGE as described (34).
MspI-digested pBR322 fragments were end-labeled for use as
Mr markers.
Protein expression
GST-FKHR was expressed in Escherichia coli (BL21) and
purified on a glutathione sepharose 4B affinity column
(Pharmacia). For quantitation, affinity-purified GST-FKHR
ran in two lanes of a 10% SDS-PAGE gel; other lanes contained from 1
to 250 ng of BSA. One GST-FKHR lane was Southwestern blotted using
32[P]-labeled IGFBP-1 IRE as probe (see below). The other
lanes were silver stained to allow quantitation of GST-FKHR bands which
bound the 32[P]-labeled IGFBP-1 IRE probe.
FKHR was expressed in vitro in a reticulocyte lysate system. pcDNA3-FKHR was linearized with XbaI and transcribed with T7 RNA polymerase for 1 h at 37 C using the Riboprobe System (Promega Corp.). After treatment with DNaseI and extraction with phenol and chloroform, FKHR messenger RNA (mRNA) was precipitated with ethanol and then translated using the Rabbit Reticulocyte Lysate System (Promega Corp.).
Preparation of antisera to FKHR
Human FKHR peptide sequence was analyzed by Peptide Companion
(WindowChem Software, Fairfield, CA) to identify antigenic sequences.
BLAST analysis then selected the sequence with least homology to other
known proteins. This peptide, FKHR413–430 (SLNSP
SPNYQKYTYGQSS), was synthesized by the San Antonio Cancer Institute
Protein/Peptide Shared Resource. After conjugating
FKHR413–430 with keyhole limpet hemocyanin, 2 mg of
conjugated peptide were injected into each of two New Zealand white
rabbits on days 0, 14, 28 and 42 by standard protocol (Alpha Diagnostic
International, San Antonio, TX), creating antisera 2798 and
2799.
Immunoblotting
GST-FKHR, reticulocyte lysate, and HEP G2 whole cell extract
(WCE) were separated by SDS-PAGE on a 7.5–15% gradient gel under
reducing conditions [100 mM dithiothreitol (DTT)] and
then transfered to a nitrocellulose membrane (35). FKHR blots used
rabbit antihuman FKHR413–430 peptide antisera at
1:10,000–20,000 dilution; second antibody was a 1:15,000 dilution of
peroxidase Affinipure goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). GST blots used a
1:10,000 dilution of anti-GST antibody (Pharmacia); second
antibody was a 1:15,000 dilution of peroxidase Affinipure rabbit
antigoat IgG (Jackson ImmunoResearch Laboratories, Inc.).
Detection was by ECL (Amersham Life Science, Inc.) as
described previously (35).
Electromobility shift assay (EMSA)
Standard binding assay: complementary 33 bp oligonucleotides
(oligos) encoding either native or mutant IRE sequences within the
-124 to -96 bp region of the IGFBP-1 promoter, and 33 bp
complimentary oligos encoding the TAT IRE, the PEPCK IRE and a mutant
PEPCK IRE, were annealed and 32[P]-labeled (2, 7); Table 1
shows native and mutant IRE sequences. Labeled probe (
2–5 fmol)
was incubated with GST-FKHR, HEP G2 WCE or reticulocyte lysate in 20
mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol
(vol/vol), 2 mM DTT and 2 mg/ml BSA at 4 C in a final 20
µl volume; poly (dG-dC) served as nonspecific competitor (50 ng/lane
for reticulocyte lysate or WCE, 10 ng/lane for GST-FKHR). In some
studies, 1 µg of either anti-GST antibody or nonimmune goat IgG
(ChromPure goat IgG, Jackson ImmunoResearch Laboratories, Inc.) was added. After a 15 min incubation, the mixture was
separated at 4 C and 190 V over 3 h on a 5% nondenaturing
polyacrylamide gel (7).
Competition studies were performed as described (7) using the above
EMSA conditions. A graded excess of nonconcatamerized and unlabeled
competitor DNA was mixed with approximately 2 fmol of labeled IRE probe
before adding GST-FKHR (0.2 ng/lane). Binding was analyzed by EMSA.
After dried gels were exposed to a Storage Phosphor Screen for
approximately 1–24 h, relevant protein/DNA probe complexes were
quantified with a Storm 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) using ImageQuant software.
DNase I protection assays
Plasmids p1205CAT, pCCGG, pG/C-A/C, pHFH2–7, pTTR and pAm2Bm2
were digested with XhoI, labeled with
-32[P]dCTP and digested with PvuII to
release 293 bp IGFBP-1 promoter fragments labeled on the antisense
strand. The fragments were incubated with or without GST-FKHR and then
digested with DNase I (Worthington Biochemical Corp., Lakewood, NJ) as described previously (28).
Southwestern blotting
GST-FKHR was analyzed by SDS-PAGE on a 7.5–15% gradient gel
under reducing conditions (100 mM DTT). Separated proteins
were transfered to nitrocellulose membranes (35), which were blocked in
20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 5%
Carnation Nonfat Dry Milk for 1 h at room temperature. After
rinsing in 10 mM Tris HCl, pH 7.5, 50 mM NaCl,
1 mM DTT, membranes were incubated for 90 min at room
temperature in this same buffer containing 1 µg poly (dG-dC)/10 ml
buffer and 15 x 106 cpm of
32[P]-labeled IGFBP-1 IRE or Am2Bm2 probe/10 ml buffer;
these same probes were used in EMSA studies. Membranes were rinsed at
room temperature in 10 mM Tris-HCl, pH 7.5, 50
mM NaCl, and then autoradiographed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, total HEP G2 RNA protected an
approximately 410-bp fragment of labeled FKHR cRNA from RNase A
digestion.
|
, anti-FKHR413–430
antiserum 2799 recognized a protein of molecular mass (Mr)
approximately 75 kDa, the expected size of FKHR (23), in RL incubated
with FKHR mRNA (FKHR-RL) but not in RL incubated without FKHR mRNA
(Control-RL). In HEP G2 WCE, this same antiserum recognized a major
approximately 75-kDa band that comigrated with the putative FKHR band
in FKHR-RL. Preimmune serum did not recognize this approximately 75-kDa
protein in FKHR-RL or HEP G2 WCE.
|
) but not 32[P]-labeled
Am2Bm2 (data not shown). The largest (Mr 105 kDa),
intermediate (Mr 75 kDa), and smallest (Mr
60 kD) GST-FKHR forms bound the least, intermediate, and greatest
amount of IRE probe, respectively. When this membrane was immunoblotted
with anti-GST antibody, these same three IRE-binding FKHR forms were
seen. A further immunoblot with anti-FKHR413–430 antiserum
2798 identified the FKHR413–430 epitope in the two larger
GST-FKHR forms but not in the most abundant 60 kDa form (Fig. 3).
Because the Mr of GST is 26 kD, these data suggest that
the approximately 105 kDa form is intact GST-FKHR, while the smaller
forms consist of intact GST fused to C-terminal truncated FKHR that
either contains (75 kDa form) or does not contain (60 kDa form)
the FKHR413–430 epitope.
|
GST-FKHR binding to native and mutant IREs
By EMSA, the GST-FKHR preparation formed a major low mobility
protein/DNA complex with the the 33 bp probe encoding the native
IGFBP-1 IRE but not with the probe encoding the Am2Bm2 IRE mutant (Fig. 4). Anti-GST antibody prevented GST-FKHR
from complexing with labeled IRE probe, whereas nonimmune goat IgG did
not block GST-FKHR/IRE complex formation (data not shown). The GST-FKHR
preparation also formed a low-mobility complex with probes encoding the
TAT and PEPCK IREs, but not with a probe encoding the insulin
unresponsive M2 mutant of the PEPCK IRE (7, 8).
|
, GST-FKHR protects the
native IGFBP-1 IRE, but not the insulin unresponsive G/C-A/C IRE mutant
(7), from DNaseI digestion. GST-FKHR also protects the insulin
responsive CCGG mutant (7), but not the insulin unresponsive Am2Bm2
mutant (data not shown). These data were confirmed by EMSA using
competitive oligo binding; as shown in Fig. 6A, GST-FKHR was competed from labeled
IGFBP-1 IRE probe by unlabeled native IRE and CCGG oligos, but not by
G/C-A/C or Am2Bm2 oligos.
|
|
, GST-FKHR was competed from labeled IGFBP-1 IRE
probe by unlabeled oligos encoding native IRE and IRE mutants HFH2–7
and TTR, but not by IRE mutant Am2Bm2; the HFH2–7 mutant confers
insulin inhibition, but the TTR mutation does not (7). The relative
ability of a 25-fold excess of unlabeled oligo to compete with labeled
IRE for GST-FKHR binding was IRE = HFH2–7 > TTR. The
ability of GST-FKHR to bind the HFH2–7 and TTR mutant IREs was
confirmed by DNaseI protection assay (data not shown).
As shown in Fig. 6C, GST-FKHR was competed from labeled IGFBP-1 IRE
probe by unlabeled oligos encoding native IRE and IRE mutants ABm2 and
Am2B, but not by IRE mutant Am2Bm2. The relative ability of a 25-fold
excess of unlabeled oligo to compete with labeled IRE for GST-FKHR
binding was IRE > ABm2 > Am2B, similar to the ability of
these mutants to confer insulin inhibition to the IGFBP-1 promoter (3, 7).
Transfecting HEP G2 cells with pRSV-FKHR did not alter the pattern by which proteins in HEP G2 WCE bind labeled IGFBP-1 IRE probe during EMSA; similarly, expressing FKHR in RL did not change the pattern by which proteins in RL bind the IGFBP-1 IRE probe during EMSA (data not shown). Further, incubating anti-FKHR413–430 antisera with transfected or nontransfected HEP G2 WCE, with FKHR-RL or with purified GST-FKHR did not supershift any bands during EMSA. Thus, FKHR forms in HEP G2 WCE could not be identified with certainty by EMSA, precluding analysis of native FKHR binding to native and mutant IREs.
FKHR activates the IGFBP-1 promoter through the IRE
In p1205CAT, the CAT reporter gene is 3' to the first 1205 bp of
the IGFBP-1 promoter which contains the IRE; pAm2Bm2 and pG/C-A/C are
identical except for IRE mutations (Table 1) that do not allow FKHR
binding and are insulin-unresponsive. When p1205CAT was cotransfected
into HEP G2 cells with plasmids expressing FKHR (pRSV-FKHR or
pcDNA3-FKHR), promoter activity rose approximately 5- to 6-fold
relative to activity when FKHR nonexpressing plasmids pRSV or pcDNA3
were cotransfected (Fig. 7A). In
contrast, FKHR expression did not increase activity of pG/C-A/C (Fig. 7B) or pAm2Bm2 (data not shown) relative to control values.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The insulin-regulated factor must be present in insulin-responsive tissues. FKHR is clearly expressed in insulin-responsive HEP G2 cells. Also, FKHR and its close relative FKHRL1 were expressed in all 16 human tissues tested including liver, muscle, pancreas, and ovary (24). Thus, the tissue distribution of FKHR and FKHRL1 is compatible with a role for these proteins in confering insulin inhibition.
The insulin-regulated factor should bind specifically to the IGFBP-1 IRE. FKHR does so; by Southwestern blot, EMSA and DNase I protection assay, GST-FKHR binds the IGFBP-1 IRE but not the insulin-unresponsive Am2Bm2 mutant. Southwestern, immunoblot, and EMSA studies showed that both intact and C-terminal truncated FKHR forms bind the IRE, and DNase I footprints showed that FKHR binds the IRE in the proximal IGFBP-1 promoter. Although EMSA and DNAseI protection data were based mainly on C-terminal truncated GST-FKHR, FKHR expressed in vivo in HEP G2 cells activates the IGFBP-1 promoter construct containing the native IRE but not the Am2Bm2 mutant; this implies that, like GST-FKHR, biologically active FKHR binds specifically to native IRE. Unfortunately, binding of native HEP G2 FKHR to the IRE was never shown; this is likely due to very low levels of FKHR in HEP G2 cells.
The insulin-regulated factor should bind T(G/A)TTT-containing IREs from other genes, and binding to IRE mutants should parallel the insulin responsiveness of these mutants. By EMSA, FKHR does indeed bind TAT and PEPCK IREs, but not the insulin-unresponsive PEPCK-M2 mutant. Also, FKHR binding to 7 of 8 IGFBP-1 IRE mutants parallels the insulin responsiveness of these mutants. Of note, finding that FKHR neither binds the insulin-unresponsive G/C-A/C mutant, nor increases activity of an IGFBP-1 promoter construct containing this mutant, is important because HNF3 binding to this mutant in past studies strongly suggested that HNF3 forms do not confer insulin effect. Possible explanations for the one inconsistent finding, that FKHR binds the insulin-unresponsive TTR mutant by EMSA, are: 1) high affinity of abundant HNF3 forms for the TTR mutant (7) precludes FKHR binding to this mutant in vivo; or 2) another FKHR family member, which does not bind TTR, is the important insulin-regulated factor.
Although the insulin-regulated factor would not necessarily activate transcription by binding the IRE, FKHR clearly does so. The ability of FKHR to stimulate production of IGFBP-1, a protein which blocks IGF-mediated growth and substrate utilization in vivo (1), is consistent with the ability of the FKHR homologue DAF-16 to slow growth and shift metabolism away from energy utilization in C. elegans (21, 22).
Binding of insulin to the insulin receptor activates multiple kinase pathways. It is now clear that many metabolic actions of insulin are mediated through pathways, which include the activation of phosphatidylinositol 3-kinase (PI3K), and it is also likely that, in these pathways, the downstream target of PI3K is protein kinase B/Akt (PKB/Akt) (36). For example, PKB/Akt transduces the stimulatory effect of insulin from PI3K to glycogen synthase (36); also, PI3K and PKB/Akt mediate insulin inhibition of IGFBP-1 promoter activity through the IRE (38). Recent evidence now suggests that FKHR is a downstream target of PKB/Akt in these pathways. First, in C. elegans, the PKB/Akt homologs AKT-1 and AKT-2, when transducing insulin receptor-like signals from the PI3K homolog AGE-1, act primarily to antagonize action of the FKHR homolog DAF-16 (39). Second, FKHR sequence analysis reveals three copies of the consensus PKB/Akt phosphorylation motif, RXRXXS (39); these three sites and their location in the N terminus, DNA binding domain and C-terminus of FKHR are conserved in FKHRL1, AFX and DAF-16 proteins (40). These observations, combined with the ability of FKHR to bind the IGFBP-1 IRE, suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription. The additional observation that the forkhead protein TTF-2 mediates transcriptional activation of thyroid-specific genes by insulin (41), suggests a general role for forkhead proteins in confering insulin effects on transcription.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received January 22, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. Chahal, C.-C. Chen, M. J. Rane, J. P. Moore, M. T. Barati, Y. Song, and B. C. Villafuerte Regulation of Insulin-Response Element Binding Protein-1 in Obesity and Diabetes: Potential Role in Impaired Insulin-Induced Gene Transcription Endocrinology, October 1, 2008; 149(10): 4829 - 4836. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Logie, A. J. Ruiz-Alcaraz, M. Keane, Y. L. Woods, J. Bain, R. Marquez, D. R. Alessi, and C. Sutherland Characterization of a Protein Kinase B Inhibitor In Vitro and in Insulin-Treated Liver Cells Diabetes, September 1, 2007; 56(9): 2218 - 2227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Hall, X. L. Wang, L. George, S. R. Koch, and D. K. Granner Insulin Represses Phosphoenolpyruvate Carboxykinase Gene Transcription by Causing the Rapid Disruption of an Active Transcription Complex: A Potential Epigenetic Effect Mol. Endocrinol., February 1, 2007; 21(2): 550 - 563. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Qu, J. Altomonte, G. Perdomo, J. He, Y. Fan, A. Kamagate, M. Meseck, and H. H. Dong Aberrant Forkhead Box O1 Function Is Associated with Impaired Hepatic Metabolism Endocrinology, December 1, 2006; 147(12): 5641 - 5652. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Onuma, B. T. Vander Kooi, J. N. Boustead, J. K. Oeser, and R. M. O'Brien Correlation between FOXO1a (FKHR) and FOXO3a (FKHRL1) Binding and the Inhibition of Basal Glucose-6-Phosphatase Catalytic Subunit Gene Transcription by Insulin Mol. Endocrinol., November 1, 2006; 20(11): 2831 - 2847. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Finlay, A. J Ruiz-Alcaraz, C. Lipina, S. Perrier, and C. Sutherland A temporal switch in the insulin-signalling pathway that regulates hepatic IGF-binding protein-1 gene expression. J. Mol. Endocrinol., October 1, 2006; 37(2): 227 - 237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mounier, V. Dumas, and B. I. Posner Regulation of Hepatic Insulin-Like Growth Factor-Binding Protein-1 Gene Expression by Insulin: Central Role for Mammalian Target of Rapamycin Independent of Forkhead Box O Proteins Endocrinology, May 1, 2006; 147(5): 2383 - 2391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang, S. Patil, B. Chauhan, S. Guo, D. R. Powell, J. Le, A. Klotsas, R. Matika, X. Xiao, R. Franks, et al. FoxO1 Regulates Multiple Metabolic Pathways in the Liver: EFFECTS ON GLUCONEOGENIC, GLYCOLYTIC, AND LIPOGENIC GENE EXPRESSION J. Biol. Chem., April 14, 2006; 281(15): 10105 - 10117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. T. Vander Kooi, H. Onuma, J. K. Oeser, C. A. Svitek, S. R. Allen, C. W. Vander Kooi, W. J. Chazin, and R. M. O'Brien The Glucose-6-Phosphatase Catalytic Subunit Gene Promoter Contains Both Positive and Negative Glucocorticoid Response Elements Mol. Endocrinol., December 1, 2005; 19(12): 3001 - 3022. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Puig and R. Tjian Transcriptional feedback control of insulin receptor by dFOXO/FOXO1 Genes & Dev., October 15, 2005; 19(20): 2435 - 2446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.J. Kim, O.L. Buzzio, S. Li, and Z. Lu Role of FOXO1A in the Regulation of Insulin-Like Growth Factor-Binding Protein-1 in Human Endometrial Cells: Interaction with Progesterone Receptor Biol Reprod, October 1, 2005; 73(4): 833 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Perrot and M. M. Rechler The Coactivator p300 Directly Acetylates the Forkhead Transcription Factor Foxo1 and Stimulates Foxo1-Induced Transcription Mol. Endocrinol., September 1, 2005; 19(9): 2283 - 2298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. R. Stayrook, K. S. Bramlett, R. S. Savkur, J. Ficorilli, T. Cook, M. E. Christe, L. F. Michael, and T. P. Burris Regulation of Carbohydrate Metabolism by the Farnesoid X Receptor Endocrinology, March 1, 2005; 146(3): 984 - 991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Villafuerte, L. S. Phillips, M. J. Rane, and W. Zhao Insulin-response Element-binding Protein 1: A NOVEL Akt SUBSTRATE INVOLVED IN TRANSCRIPTIONAL ACTION OF INSULIN J. Biol. Chem., August 27, 2004; 279(35): 36650 - 36659. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Furuyama, K. Kitayama, Y. Shimoda, M. Ogawa, K. Sone, K. Yoshida-Araki, H. Hisatsune, S.-i. Nishikawa, K. Nakayama, K. Nakayama, et al. Abnormal Angiogenesis in Foxo1 (Fkhr)-deficient Mice J. Biol. Chem., August 13, 2004; 279(33): 34741 - 34749. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-S. Kwon, B. Huang, T. G. Unterman, and R. A. Harris Protein Kinase B-{alpha} Inhibits Human Pyruvate Dehydrogenase Kinase-4 Gene Induction by Dexamethasone Through Inactivation of FOXO Transcription Factors Diabetes, April 1, 2004; 53(4): 899 - 910. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-C. Tsai, N. Bhattacharyya, L.-Y. Han, J. A. Hanover, and M. M. Rechler Insulin Inhibition of Transcription Stimulated by the Forkhead Protein Foxo1 Is Not Solely due to Nuclear Exclusion Endocrinology, December 1, 2003; 144(12): 5615 - 5622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Altomonte, A. Richter, S. Harbaran, J. Suriawinata, J. Nakae, S. N. Thung, M. Meseck, D. Accili, and H. Dong Inhibition of Foxo1 function is associated with improved fasting glycemia in diabetic mice Am J Physiol Endocrinol Metab, October 1, 2003; 285(4): E718 - E728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Puig, M. T. Marr, M. L. Ruhf, and R. Tjian Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway Genes & Dev., August 15, 2003; 17(16): 2006 - 2020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Perrot and M. M. Rechler Characterization of Insulin Inhibition of Transactivation by a C-terminal Fragment of the Forkhead Transcription Factor Foxo1 in Rat Hepatoma Cells J. Biol. Chem., July 3, 2003; 278(28): 26111 - 26119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
|
