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Department of Pediatrics (S.K.D., A.O.S., R.M.W., A.S., D.R.P.), Baylor College of Medicine, Houston, Texas 77030; and Department of Clinical Genetics (S.V.A.), Karolinska Institute, S-171 76 Stockholm, Sweden
Address all correspondence and requests for reprints to: David R. Powell, Texas Children’s Hospital, Clinical Care Center, MC 3–2482, 6621 Fannin, Houston, Texas 77030. E-mail: dpowell{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hepatic nuclear factor 3 (HNF3) family of proteins play a role in hepatic development (17, 18). One class of HNF3-binding sites contains the sequence TGTTT (19), identical to the sequence shared in the above IREs. Early studies found that HNF3 forms do indeed bind to the IGFBP-1, PEPCK, and TAT IRE regions, and later studies suggested that HNF3 forms act through these IREs to confer glucocorticoid stimulation (13, 14, 20, 21, 22, 23). Although initial studies presented some indirect evidence to suggest that insulin does not work through HNF3 proteins to inhibit glucocorticoid-stimulated promoter activity (20, 21), a role for HNF3 proteins in this process must still be considered as they are the only known proteins to bind the IRE in each of these genes.
The high mobility group (HMG) I/Y family of proteins enhance transcription from some promoters by acting as accessory factors that enhance the DNA binding and activity of neighboring transcription factors (24). HMGI and the smaller HMGY proteins, which differ by only 11 amino acids due to alternative splicing of a single primary transcript (25), bind preferentially to A-T-rich sequences (26) similar to those present in the IGFBP-1 IRE, but binding of HMGI/Y forms to this IRE has not been evaluated.
The present study describes a search for proteins contained in hepatocyte extracts that bind specifically to the IGFBP-1 IRE. Additional studies identify these proteins and test their ability to bind: 1) an IRE from the Apo CIII promoter, which contains the T(G/A)TTT motif and confers insulin inhibition (12, 27); and 2) a series of IGFBP-1 IRE mutants that have variable insulin responsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(29), which expresses human glucocorticoid
receptor (hGR) under the control of the Rous Sarcoma Virus long
terminal repeat (RSV LTR), was kindly provided by Dr. Ronald M. Evans
(The Salk Institute for Biological Studies, La Jolla, CA). Plasmid
pGST-HNF3ß, used for expression of
glutathione-S-transferase (GST)-mHNF3ß fusion protein in
Escherichia coli, was constructed by digesting murine
HNF-3ß cDNA (kindly provided by Dr. Brigid L. M. Hogan,
Vanderbilt University School of Medicine, Nashville, TN) (30) and
pGEX2T (Pharmacia, Piscataway, NJ) with EcoRI, filling in
with Klenow polymerase, and then joining by blunt-end ligation.
Site-directed mutagenesis
The 1.3-kb hIGFBP-1 promoter fragment present in the M13-based
vector M13 mp18 was mutated by the Kunkel method using synthetic
oligonucleotides and the Muta-Gene kit (Bio-Rad, Hercules, CA). The
sequence of all mutations and orientation of all constructs was
confirmed by DNA sequence analysis using Sequenase (U.S. Biochemicals,
Cleveland, OH) in the dideoxy chain termination method (28, 31).
The putative HNF3-binding element AAACAAACTTAT spanning bp -116 to
-105 of the IGFBP-1 IRE was mutated as follows. Oligonucleotide
5'-GCACTAGCAAAAGAAACTTCTTTTGAACACTC-3' mutated the C and A
nucleotides at bp -113 and -106 to G and C, respectively, creating
pG/C-A/C. Oligonucleotide 5'-ACTAGCAAAACACCGGTATTTTGAACAC-3' mutated bp
-111 to -108 from AACT to CCGG, creating pCCGG. Oligonucleotide
5'-ACTAGCAAAACAGGATTATTTTGAACA-3' mutated bp -111 to -109 from AAC to
GGA, creating pGGA. Oligonucleotide
5'-TGCACTAGCAAAGTCAATA-ATCTTTGAACACTCA-3' mutated bp -114
to -105 from ACAAACTTAT to GTCAATAATC, creating pTTR. Oligonucleotide
5'-GCACTAG CAAAAACAACAAACTTTGAACACTCA-3' mutated bp -113 to -105 from
CAAACTTAT to ACAACAAAC, creating pHFH2–7. These mu-tant sequences
are presented in alignment with the native IRE as part of Fig. 6
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. One microgram
of pRSVL plasmid, which contains the RSV LTR upstream to the luciferase
reporter gene (32), was cotransfected to control for transfection
efficiency. Transfected cells were washed three times in PBS and then
incubated with serum-free medium (DMEM supplemented with 5
mM L-glutamine, 50 U/ml penicillin, and 50
µg/ml streptomycin) ± 100 nM dexamethasone (Sigma
Chemical Co., St. Louis, MO) and/or 100 nM insulin (kindly
provided by Eli Lilly Co., Indianapolis, IN).
Chloramphenicol acetyltransferase and luciferase assays
CAT and luciferase assays were performed by standard methods
(32, 33).
Preparation of cellular extracts
Whole cell extracts (WCE) were prepared as described previously
(34). HEP G2 cells (
5 x 107 cells) were washed
with PBS, harvested, and centrifuged. Cells were frozen at -80 C and
thawed by adding 3 volumes of lysis buffer [20 mM HEPES,
pH 7.9, 0.2 mM EDTA, 0.2 mM EGTA, 0.5
mM spermidine, 0.15 mM spermine, 1.0
mM dithiothreitol, 10% glycerol (vol/vol), 0.5
mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin A, 1
µg/ml leupeptin, 50 µM
L-1-tosylamide-2-phenylethyl/chloromethyl/ketone, 25
µM N--p-tosyl-L-lysine
chloromethylketone, 0.5 mM benzamidine, 10 mM
Na molybdate, 2 mM Na pyrophosphate, 2 mM
Na3VO4] and 1 volume of 2 M KCl.
The lysate was mixed for 60 min and centrifuged at 80,000 x
g for 30 min at 4 C. Supernatant was collected and diluted
with lysis buffer to a final KCl concentration of 0.15 M.
Precipitate was removed by centrifugation, and the extract was
aliquoted and stored at -80 C.
Perchloric acid extracts (PAE) were prepared from HEP G2 cells and from
human liver obtained with permission at autopsy; these extracts are
rich in HMG proteins (35). Briefly, HEP G2 cells (5 x
107 cells) were washed with PBS, harvested, and
centrifuged. Liver was frozen at -80 C, ground to fine pieces in a
cold mortar, and transferred to 100 ml hypotonic buffer (10
mM NaCl, 3 mM MgCl2, 10
mM Tris-HCl, pH 7.4). The cell mixture was passed through
cheese cloth and the hepatocytes were collected by centrifugation. Cell
pellets were resuspended in 2 volumes of ice-cold 5% perchloric acid.
After centrifugation, the supernatant was made 0.3 M in
HCl, mixed with 9 volumes of acetone, and then kept at -20 C
overnight. The precipitate was recovered by centrifugation at 4 C,
washed with acetone, aliquoted, air-dried at room temperature, and
stored at -80 C.
Protein expression
GST-mHNF3ß fusion protein expressed in E. coli
(BL21(DE3)pLysE) was purified on a glutathione Sepharose 4B affinity
column following the recommendations of the manufacturer (Pharmacia).
GST-mHNF3ß eluted from the column was concentrated 10-fold at 4 C
using a Centricon-30 Microconcentrator (Amicon, Beverly, MA).
Electrophoretic mobility shift assay (EMSA)
Proteins studied by EMSA included HEP G2 and liver WCE and PAE
described above; GST-mHNF3ß fusion protein described above; purified
recombinant human HMGI (hHMGI) kindly provided by Dr. Raymond Reeves,
Washington State University (Pullman, WA) (36); and recombinant
full-length rat His(6)-HNF3 (His(6)-rHNF3) kindly provided by Dr.
Kenneth Zaret, Brown University (Providence, RI) (37). These studies
also used anti- and preimmune sera to HMGI/Y, kindly provided by Dr.
Reeves (38), and antisera to rat HNF3 and rat HNF3ß, kindly
provided by Dr. James Darnell, Jr., Rockefeller University (New York,
NY) (17, 39).
Standard binding assay.
Complementary 33-bp oligonucleotides
encoding either native or mutant IRE sequences within the -124 to -96
bp region of the IGFBP-1 promoter were annealed and labeled as
described previously (5). In addition, six other pairs of
complementary 33-bp oligonucleotides were annealed and labeled as
above: 1) the Apo CIII probe, 5'-CTAGTGTGCCTTTACTCCAAACATCCCCCAGCC-3'
and 5'-CTAGGGCTGGGGGATGTTTGGAGTAAAGGCACA-3', spanning from -474 to
-446 bp of the human Apo CIII promoter and containing an IRE; 2) the
PEPCK probe, 5'-CTAGACCTCACAGCTGTGGTGTTTTGACAACCA-3' and
5'-CTAGTGGTTGTCAAAACACCACAGCTGTGAGGT-3', spanning from -428 to -400
bp of the PEPCK promoter and containing a well characterized IRE; 3)
the PEPCKm probe, 5'-CTAGACCTCACAGCTGTGGTGGGGGTACAACCA-3' and
5'-CTAGTGGTTGTACCCCCACCACAGCTGTGAGGT-3', which spans the identical
region of the PEPCK promoter but contains the M2 mutation, which blocks
the ability of the IRE to confer insulin effect (21); 4) the Am2B
probe, 5'-CTAGCACTAGCAACCATGACTTATTTTGAACAC-3' and
5'-CTAGGTGTTCAAAATAAGTCATGGTTGCTAGTG-3', which contains a mutation of
the IRE A element; 5) the ABm2 probe
5'-CTAGCACTAGCAAAACAAACCATGGTTGAACAC-3' and
5'-CTAGGTGTTCAACCATGGTTTGTTTTGCTAGTG-3', which contains a muta-tion
of the IRE B element; and 6) the Am2Bm2 probe
5'-CTAGCACTAGCAACCATGACCATGGTTGAACAC-3' and
5'-CTAGGTGTTCAACCATGGTCATGGTTGCTAGTG-3', which contains both the A and
B element mutations (6, 10, 12, 27). The Am2B, ABm2, and Am2Bm2
sequences are presented in alignment with the native IRE as part of
Fig. 4. Labeled probe (2–5 fmol) was incubated at 4 C with the
protein of interest in 10 mM Tris-HCl, pH 7.5, 50
mM NaCl, 5% glycerol (vol/vol) in a final volume of 20
µl; 1 µg poly (dG-dC) was also added as nonspecific competitor
except when purified hHMGI and GST-HNF3 fusion proteins were included.
After a 15-min incubation, the mixture was separated at 4 C and
190–210 V over 2–3.5 h on a 5% nondenaturing polyacrylamide gel
using a low ionic strength gel buffer system (5).
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2 fmol labeled IRE probe
before addition of the protein of interest. Binding was analyzed by
EMSA. Dried gels were exposed to a Storage Phosphor Screen for 1–24
h and then quantification of relevant protein/DNA probe complexes was
performed with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale,
CA) using ImageQuant software.
DNase I protection assays
The plasmid p207CCAAT (28), containing a hIGFBP-1 promoter
fragment spanning from -207 to +15 bp relative to the transcription
start site, was digested at the 5'-end with HindIII, labeled
with [-32P]deoxycytidine triphosphate (CTP), and then
digested at the 3'-end with EcoRI to release a 249-bp
fragment labeled on the antisense strand. This hIGFBP-1 promoter
fragment was incubated in the presence or absence of hHMGI and then
digested with DNase I (Worthington Biochemicals, Freehold, NJ) as
described previously (31). Plasmids p1205CAT, pTTR, and pHFH2–7 were
digested at the 3'-end with XhoI, labeled with
[-32P]dCTP, and then digested at the 5'-end with
PvuII to release 320-bp fragments, labeled on the sense
strand, which contained from -246 to +68 bp of the hIGFBP-1 promoter.
These hIGFBP-1 promoter fragments were incubated in the presence or
absence of His(6)-rHNF3 and then digested with DNase I (Worthington
Biochemicals). Specific nucleotides protected from DNase I digestion
were determined using sequence ladders derived from the appropriate
hIGFBP-1 promoter probes (40).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, multiple
proteins present in WCE from HEP G2 cells bind the native, but not the
AmBm mutant, IRE probe by EMSA. These proteins migrate in two groups,
one with low mobility (proteins 1 and 2) and the other with high
mobility (proteins 3–5). Additional studies using a variety of gel
shift conditions (heparin, salmon sperm DNA, poly (dA-dT), poly
(dI-dC), poly (dG-dC) acid, poly (dAG-dCT) as nonspecific competitors;
presence of ATP; presence of excess oligonucleotides that specifically
bind HNF3 with high affinity; absence of phosphatase inhibitors; high
ionic strength gels) failed to identify additional proteins that shift
the native IRE probe but not the AmBm mutant probe (data not
shown).
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and HNF3ß are IRE-binding proteins 1 and 2
or -ß, antisera to these HNF3 forms were incubated with HEP G2 WCE
and then separated by EMSA. Figure 1B
shows that HNF3 antiserum
blocked binding of protein 1 while HNF3ß antiserum blocked binding of
protein 2, suggesting that proteins 1 and 2 are probably HNF3 and
HNF3ß, respectively. Binding of HNF3 forms to the IGFBP-1 IRE was
confirmed by expressing mHNF3ß as a GST fusion and then incubating it
with native and AmBm mutant IRE probes. GST-mHNF3ß, purified on a
glutathione Sepharose 4B column, did not bind the AmBm probe (not
shown) but did bind the native IRE probe, and this binding was competed
most strongly by HNF3ß antiserum (Fig. 1C).
HMGI/Y proteins bind the IGFBP-1 IRE
HMGI/Y proteins are small basic proteins that migrate rapidly
during electrophoresis and preferentially bind AT-rich stretches of
double-stranded DNA (24, 26, 36, 41), suggesting that they might be
related to the high mobility proteins 3–5, which bind the IRE during
EMSA (Fig. 1A). Since PAE of cells are rich in HMG proteins, such
extracts were prepared from HEP G2 cells and from adult human liver. As
shown in Fig. 2, HEP G2 PAE contains the
same three high mobility proteins as WCE. The presence of HMGI
antiserum, but not preimmune antiserum, blocks binding of the two
fastest migrating species. Thus, proteins 4 and 5 are HMGI and -Y,
respectively, while protein 3, previously named IREBP (5), is not
recognized by HMGI antiserum. In contrast, PAE from adult human liver
contains HMGI and -Y but does not contain IREBP. Purified hHMGI protein
migrates as two bands recognized by HMGI antiserum; the slower
migrating band is probably intact HMGI, whereas the faster migrating
band is probably an HMGI fragment.
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and HMGI/Y binding within the IRE and hHMGI proteins footprinted the IRE. As
shown in Fig. 3A, 1 and 4 ng
His(6)-rHNF3 protected the A element (-118 to -112 bp) and the B
element (-107 to -101 bp) of the IGFBP-1 IRE from DNAse I digestion,
and a new hypersensitive site appeared in the A element at bp -114. As
shown in Fig. 3B, hHMGI protected the IGFBP-1 IRE from DNAse I
digestion. Interestingly, hHMGI also protected the AT-rich region from
-71 to -61 bp, which binds HNF-1 and is essential for basal activity
of the IGFBP-1 promoter (28).
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). HMGI/Y binds strongly only to the
native IRE probe; HMGI/Y binds weakly when the A element is mutated,
and binding is difficult to detect when either the B element is mutated
alone or the A and B elements are mutated together. This suggests that
HMGI binds more strongly to the B element than to the A element.
HNF3ß, but not HMGI/Y, binds to IREs from many genes
As shown in Fig. 5, GST-mHNF3ß
binds to the native IGFBP-1 IRE and to the Apo CIII and PEPCK IREs, but
not to the well characterized M2 mutant of the PEPCK IRE that is
unresponsive to insulin. In contrast, HMGI/Y proteins bind to the
native IGFBP-1 IRE, but not to the native PEPCK and Apo CIII IREs.
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, a series of mutations targeted 1) the
G and A nucleotides of the IRE A and B elements, respectively; 2) the
four nucleotides between the IRE A and B elements; or 3) the HNF3
sequence itself. Activity of the G/C-A/C mutant was increased by
dexamethasone, and this dexamethasone-mediated increase in activity was
not inhibited by insulin. Similar to the G/C-A/C mutant, the CCGG and
GGA mutants were responsive to dexamethasone; however, the
dexamethasone-mediated increase in activity of the CCGG and GGA mutants
was clearly inhibited by insulin, in contrast to the G/C-A/C mutant. In
the HFH2–7 plasmid, the HNF3 site in the IGFBP-1 IRE was mutated to a
sequence reported to bind HNF3/fork head 2 (HFH2) but not HNF3 forms
despite the presence of the TGTTT motif. In the TTR plasmid, the HNF3
site in the IGFBP-1 IRE was mutated to the sequence spanning -106 to
-96 bp of the TTR promoter, which binds HNF3 with high affinity but
does not contain the T(G/A)TTT motif (42). Although both plasmids were
responsive to dexamethasone, insulin inhibited the
dexamethasone-mediated increase in activity of only the HFH2–7
construct.
As shown in Fig. 7, insulin clearly
inhibited the basal activity of plasmids containing the native IRE and
the CCGG, GGA, and HFH2–7 mutants but not those containing the
G/C-A/C, TTR, and Am2Bm2 mutants, consistent with the effect of insulin
on dexamethasone-stimulated activity of these plasmids.
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and HNF3ß
bound to the native IRE with higher affinity than to the G/C-A/C and
CCGG IRE mutants (21). As shown in Fig. 8A, HNF3 and HNF3ß (proteins 1 and
2) present in HEP G2 WCE were competed from labeled IRE probe by
HFH2–7, TTR, and native IRE oligonucleotides, but not AmBm
oligonucleotide, during EMSA; TTR competed much more efficiently than
did IRE or HFH2–7 oligonucleotides. Labeled HFH2–7, TTR, and native
IRE probes, but not AmBm probe, also directly bound GST-mHNF3ß,
His(6)-rHNF3, and HNF3 forms in HEP G2 WCE (Fig. 8B); in these
studies, HNF3 and HNF3ß forms bound to the TTR probe as well as or
better than they bound to the IRE or HFH2–7 probes.
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at least as well as did the insulin-responsive HFH2–7
mutant and the native IRE, it is possible that His(6)-rHNF3 binds to
the TTR mutation in a different way than it binds to the HFH2–7
mutation or to the native IRE. As shown in Fig. 9, identical regions of the native IRE,
the TTR mutation, and the HFH2–7 mutation were protected from DNase I
digestion by His(6)-rHNF3. In addition, His(6)-rHNF3 induced the
appearance of the same hypersensitive site at -110 bp in the TTR and
HFH2–7 mutants; thus, if His(6)-rHNF3 binds these two IRE mutants
in a fundamentally different way, it could not be demonstrated by DNase
I digestion.
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, recombinant hHMGI, and also HMGI/Y
proteins present in liver PAE, bound to labeled G/C-A/C, CCGG, HFH2–7,
TTR, and native IRE probes, but not AmBm probe. HMGI/Y proteins bound
more tightly to the native IRE sequence than to the G/C-A/C, CCGG,
HFH2–7, and TTR mutant sequences, while hHMGI bound more uniformly to
the IRE and the mutant sequences; the reason for this is unclear. Of
greater significance, however, hHMGI bound comparably to CCGG and
G/C-A/C mutants, and all HMGI/Y proteins appeared to bind comparably to
TTR and HFH2–7 mutants.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the above evidence implicating HNF3 in insulin effect, initial
studies suggested that HNF3 is not involved. Unterman et al
(20) described an IRE mutation that disrupted HNF3 binding but
conferred a weak insulin effect (20). Also, the CCGG and G/C-A/C
mutants were used previously to explore this issue; the CCGG mutation
targets the HNF3-binding site between the A and B IRE elements, while
the G/C-A/C mutation targets the HNF3-binding site within the A and B
elements. These mutations demonstrated comparable affinity for HNF3
forms, but the CCGG mutation allowed insulin response while the G/C-A/C
mutation did not (Table 1 and 21 .
Nevertheless, the Unterman and G/C-A/C mutants show a decrease both in
insulin response and in affinity for HNF3. In the present study the IRE
was replaced with a high-affinity HNF3- binding site from the TTR
promoter. Despite the fact that HNF3 and HNF3ß forms bound to the
TTR mutation as well as or better than they bound to the native IRE or
the HFH2–7 mutation, the TTR mutation did not confer insulin
inhibition, in contrast to the native IRE and the HFH2–7 mutation.
Table 1 clearly shows that HNF3 binding to IRE mutants does not
correlate with the responsiveness of the mutants to insulin.
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). Thus the IGFBP-1, PEPCK, TAT, and Apo
CIII IREs, and all IGFBP-1 and PEPCK IRE mutants that confer insulin
inhibition, contain the T(G/A)TTT motif, whereas insulin-unresponsive
IRE mutants do not contain this motif (Refs. 5, 6, 10, 20, and 21 and
present study). At present, it is unclear what protein binds the
T(G/A)TTT motif to confer insulin effect. HNF3 proteins may be
responsible if they bind native IREs and IRE mutants that contain the
T(G/A)TTT motif (such as HFH2–7) in a different spatial orientation
than IRE mutants lacking the T(G/A)TTT motif (such as TTR); in this
scenario, insulin would activate only the HNF3 bound to
T(G/A)TTT-containing IREs. Although there is reason to suspect that
HNF3 forms may bind differently to T(G/A)TTT-containing elements than
to the TTR element (48), nevertheless HNF3 footprints the TTR and
HFH2–7 mutations over the same span of nucleotides, and HNF3
binding induces the same hypersensitive site in footprints of the TTR
and HFH2–7 IRE mutants. This suggests that HNF3 binds these two IRE
mutants in a very similar spatial orientation, making it unlikely that
insulin can selectively activate T(G/A)TTT-bound HNF3 alone.
Alternatively, HNF3 could participate in insulin inhibition by binding
to the IRE as a complex with other proteins; as part of the complex,
HNF3 would have an affinity for the IRE and IRE mutants that parallels
insulin response. Finally, a protein(s) other than HNF3 may confer
insulin effect to the IGFBP-1 IRE and related IREs. One candidate is
HFH2, a member of the fork head protein superfamily; HFH2 is expressed
in liver and other tissues during extrauterine life and should bind the
insulin-responsive HFH2–7 mutation (42, 49). To date, an exhaustive
search using HEP G2 extracts and a wide variety of EMSA conditions has
failed to identify additional bands that might represent IRE binding to
HFH2, other candidate proteins, or HNF3 as part of a protein
complex. This study identifies HMGI and -Y as proteins present in normal human liver that bind the IGFBP-1 IRE. HMGI/Y proteins bind in the minor groove of A-T-rich DNA and upon binding may bend the DNA helix; they may play a role in nucleosome positioning and also act as accessory factors that bend DNA to allow enhanced binding and activity of transcription factors (24, 26, 36, 41). Present and past (5) studies also identify a protein, designated IREBP, which 1) is expressed in HEP G2 cells but not normal liver; 2) binds the hIGFBP-1 IRE; and 3) shares many characteristics with HMGI/Y proteins. Based on electrophoretic mobility and expression in hepatoma cells rather than normal liver, IREBP is probably HMGI-C, which is highly expressed in HEP G2 cells and is a close relative of HMGI/Y (35). IREBP/HMGI-C were not studied further due to their lack of expression in normal liver tissue.
HMGI/Y proteins appeared initially to be candidate proteins for
conferring insulin effect. HMGI/Y proteins are likely targets of
insulin-regulated erks and cdc2 kinase in liver (43, 44, 45, 46). In fact, a
motif present in the DNA-binding domain of HMGI can be phosphorylated
by cdc2 kinase both in vitro and in vivo,
resulting in a 20-fold decrease in affinity of HMGI for DNA (36). Also,
IRE-ABP, which likely bends DNA upon binding to the 3'-region of the
upstream IRE present in the glyceraldehyde-3-phosphate dehydrogenase
promoter, is a member of the HMG superfamily (50, 51). Nevertheless,
the studies presented here suggest that HMGI/Y does not confer insulin
response: 1) HMGI/Y proteins do not bind the PEPCK or Apo CIII IREs,
which makes a role for HMGI/Y unlikely if it is assumed that these
structurally related IREs confer insulin effect by a common mechanism;
2) hHMGI bound as well to the insulin responsive CCGG mutant as to the
insulin unresponsive G/C-A/C mutant, and all HMGI/Y proteins bound as
well to the insulin-responsive HFH2–7 mutant as to the
insulin-unresponsive TTR mutant. Thus, there is no correlation between
ability of IRE mutants to confer insulin effect and their ability to
bind HMGI (Table 1); and 3) HMGI/Y proteins bind strongly to the
combined A and B elements of the native IRE, bind weakly to the B
element alone, and bind very weakly, if at all, to the A element alone;
in contrast, the IRE A element alone is more responsive to insulin than
is the B element and is almost as responsive to insulin as the combined
A and B elements (5, 6).
The IREs in the IGFBP-1, PEPCK, and TAT genes are also accessory sites necessary for glucocorticoids to exert their full stimulatory effect on gene transcription (6, 8, 11, 13, 14). Prior studies found that the ability of glucocorticoids to stimulate activity of IGFBP-1 promoter constructs containing either the native IRE or the CCGG and G/C-A/C IRE mutants correlated directly with ability of these sequences to bind HNF3 (21), suggesting an association between HNF3 binding and glucocorticoid responsiveness. Indeed, HNF3 proteins appear to augment the glucocorticoid effect on TAT and PEPCK gene transcription by binding to their respective IREs (22, 23). The present study was not designed to examine the role of HNF3 and HMGI/Y proteins in conferring glucocorticoid effect; glucocorticoids were used to amplify the inhibitory effect of insulin on IGFBP-1 promoter activity. Nevertheless, data from this study show that HNF3 forms bind the native IRE, the TTR mutant, and HFH2–7 mutant, which confer glucocorticoid effect, but do not bind the AmBm mutant, which does not confer glucocorticoid effect, suggesting that HNF3 forms may augment glucocorticoid stimulation of the IGFBP-1 promoter. However, these and past correlations do not prove an association; studies designed to show a direct interaction between HNF3 and GR pathways are needed and are underway in many laboratories. In contrast to HNF3 forms, HMGI/Y proteins are unlikely to confer glucocorticoid effect to the IGFBP-1 IRE. First, HMGI/Y proteins do not bind the A element in the ABm2 mutation of the IGFBP-1 IRE despite the fact that the A element in the ABm2 mutation is very responsive to glucocorticoids (6). Second, HMGI/Y proteins do not bind the PEPCK IRE, which makes a role for HMGI/Y unlikely if it is assumed that the structurally related IGFBP-1 and PEPCK IREs confer glucocorticoid effect by a common mechanism.
Most past studies examined the ability of IGFBP-1, PEPCK, and TAT IREs to confer insulin inhibition of glucocorticoid-stimulated promoter activity (6, 10, 15, 21), but it is unlikely that insulin inhibits IGFBP-1 transcription solely by inhibiting the IRE-binding protein that potentiates glucocorticoid effect: 1) the G/C-A/C and TTR mutants augment glucocorticoid stimulation but are totally unresponsive to insulin, suggesting that each hormone effect is conferred through a separate protein pathway; and 2) insulin inhibits basal activity of each IRE mutant examined in this study to roughly the same degree that it inhibits glucocorticoid-stimulated activity of the same mutant, suggesting that the mechanism of insulin inhibition is the same in the presence and absence of glucocorticoid stimulation. It is conceivable, then, that HNF3 confers the effects of glucocorticoids but not insulin on IGFBP-1 transcription.
The binding of HMGI/Y proteins to the IGFBP-1 IRE is similar to the binding of C/EBP proteins to the PEPCK IRE (12, 21, 52). HMGI/Y proteins bind only the IGFBP-1 IRE, while C/EBP proteins bind only the PEPCK IRE. Also, neither protein family appears to play a role in the insulin inhibition conferred through these IREs. Nevertheless, important and perhaps complementary roles for these proteins in the function of their respective IREs may ultimately be recognized; thus, a role for each protein family should be considered in any newly described IRE function.
In summary, both HNF3 and HMGI/Y proteins bind the IGFBP-1 IRE, but the role these proteins play while bound to the IRE is unclear. The inability of HMGI/Y proteins to bind other IREs suggests they do not play a role in insulin or glucocorticoid effects. The data presented here are compatible with, but not proof of, a role for HNF3 in conferring glucocorticoid stimulation of IGFBP-1 transcription. Although HNF3 forms are the only proteins found to specifically bind the related IGFBP-1, PEPCK, TAT, and Apo CIII IREs, any future hypothesis implicating HNF3 proteins in insulin inhibition must account for the dissociation of their binding to IRE mutants and the ability of those mutants to confer insulin effect. Ultimately, the protein(s) responsible for the insulin effect will be found to interact with the T(G/A)TTT sequence, and in particular with the G or A nucleotide, of the IRE motif to confer the inhibitory effect of insulin on IGFBP-1 gene transcription.
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Received February 12, 1997.
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belongs to a gene family in mammals
that is homologous to the Drosophila homeotic gene fork head. Genes Dev 5:416–427. Protein Express Purif 6:821–825[CrossRef][Medline]
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