(Received for publication, August 5, 1996, and in revised form, October 16, 1996)
From the Division of Endocrinology and Metabolism,
Department of Medicine, Emory University School of Medicine, Atlanta,
Georgia 30322, the ¶ School of Life Science, Queensland University
of Technology, Brisbane, Queensland, Australia 4001, and the
Murdoch Institute, Royal Children's Hospital,
Melbourne, Victoria, Australia 3052
The hepatic expression and serum levels of
insulin-like growth factor-binding protein-3 (IGFBP-3) are decreased in
insulin-dependent and insulin-resistant diabetes. Insulin
increases hepatic IGFBP-3 expression by enhancing gene transcription.
This report identifies sequences within the IGFBP-3 promoter that are
necessary and sufficient for the response to insulin in hepatic
nonparenchymal cells. By transient transfection, we mapped the insulin
response element to the 1150 to
1124 base pair (bp) region of the
rat IGFBP-3 promoter. Three tandem repeats of the
1150 to
1117 bp
region conferred insulin responses in a heterologous promoter. Gel
shift analyses revealed a 3-fold increase in DNA-protein complex
formation with nuclear extracts obtained from insulin-stimulated
nonparenchymal cells compared with cells incubated without insulin and
revealed 3-4-fold decrease in complex formation with nuclear extracts
obtained from the livers of streptozotocin-diabetic rats compared with control rats. Mutational analysis of this 34-bp region showed a core
sequence of 10 bp (
1148 to
1139) that is critical for interaction
with insulin-induced trans-acting factors. Southwestern blotting revealed a ~90-kDa protein that was increased 2-3-fold by
the addition of insulin. Thus, we have identified
cis-acting DNA sequences that are responsible for
regulation of IGFBP-3 transcription by insulin and essential for
binding of insulin-responsive nuclear factors.
Insulin-like growth factors I and II (IGF-I and -II)1 are peptides that have insulin-like metabolic and trophic effects and mediate some of the peripheral actions of growth hormone (1). The actions of IGFs are modulated by a family of six IGF-binding proteins (IGFBPs), which have different tissue distribution and production sites (2, 3). Most of the circulating plasma IGF-I and IGF-II is associated with IGFBP-3 and an acid-labile subunit, constituting a ~150-kDa complex, which serves as a reservoir for IGFs (4). Formation of this large molecular weight complex limits the access of IGFs to tissues and prevents the hypoglycemic effects of IGFs (5). Recent studies also suggest that the IGFBPs actively modulate the mitogenic and metabolic actions of IGFs in the cellular microenvironment (6-9).
The mechanisms by which IGFBP-3 is regulated are complex. IGFBP-3 may undergo post-translational processing to yield various proteolytically cleaved, phosphorylated, and glycosylated products (8-14). These processes have been shown to alter the binding of IGFBP-3 to the acid-labile subunit and cell surfaces and affect the affinity of IGFBP-3 for IGFs (15). IGFBP-3 can also associate with the cell surface and extracellular matrix; dissociation of cell-associated IGFBP-3 is one mechanism by which IGF-I increases release of IGFBP-3 into conditioned medium by fibroblasts and breast cancer cells (16, 17). While the post-translational regulation of IGFBP-3 appears to be important, IGFBP-3 is also regulated at the level of gene transcription.
The IGFBPs are structurally homologous, with strict conservation of the 18 cysteine residues clustered at the NH2 and COOH termini of the proteins (18). Despite their structural similarities, however, the IGFBPs differ in their pattern of developmental and hormonal regulation. IGFBP-1 and IGFBP-3 have been studied most extensively. While the serum protein and liver mRNA levels of IGFBP-1 reach the highest levels during fetal life and the neonatal period, the protein levels of IGFBP-3 in serum and liver mRNA levels are highest during puberty and adult life (19). Furthermore, IGFBP-1 levels decrease in the presence of anabolic hormones such as insulin and growth hormone, while IGFBP-3 levels increase in the presence of these hormones (20, 21). The IGFBP-1 and the IGFBP-3 genes are contiguously arranged in a tail-to-tail fashion within chromosome 7, separated by only 20 kilobase pairs of DNA (22). The juxtaposition of the genes and the high expression of both genes within the liver could theoretically allow cellular factors to regulate the genes similarly in a given physiological condition. To explain the markedly different responses of these structurally related proteins, there is interest in determining whether different sets of transcription factors regulate the promoter activity of these genes. At present, the cis-elements required for basal expression and insulin-mediated activity of the IGFBP-1 gene have been reported (23-26), while the cis-elements required for development- and hormone-mediated activity of the IGFBP-3 promoter have yet to be identified.
Our previous investigations showed that IGFBP-3 mRNA is an abundant
transcript in the liver and is secreted by the Kupffer and sinusoidal
endothelial cells (27). Through sequential collagenase/Pronase treatment, we were able to isolate nonparenchymal cells that express IGFBP-3 and maintain the phagocytic function of Kupffer cells, and we
have used this cultured cell model to study the mechanisms involved in
the hormonal regulation of IGFBP-3. We showed previously that insulin
increased IGFBP-3 expression by stimulating the rate of gene
transcription rather than through stabilization of mRNA transcripts
(28). In the present study, we define the cis-acting sequences that mediate the positive effects of insulin on IGFBP-3 transcription, and recognize insulin-responsive factors in nuclear extracts. By transient transfection, we mapped the insulin response element (IRE) in the rIGFBP-3 gene to the region spanning 1150 to
1124 bp. In gel mobility shift analyses, the IRE exhibited increased
DNA-protein complex formation with nuclear extracts obtained from cells
exposed to insulin compared with cells not exposed to insulin and
decreased complex formation with extracts from diabetic compared to
normal rat livers. Southwestern blotting revealed association of the
IGFBP-3 IRE region with 90- and 70-kDa nuclear factors; the 90-kDa
factor appears to be hormone-responsive and could contribute to
regulation of IGFBP-3 transcription by insulin.
The reagents listed were obtained from the
following sources: collagenase type 1 from Worthington; Pronase-E from
Calbiochem; type 1 rat tail collagen, human transferrin, fetal bovine
serum, and Williams' E medium from Sigma; Lipofectin
reagent, human recombinant insulin, medium 199, and deoxyribonuclease 1 (DNase I) from Life Technologies, Inc.; [-32P]ATP from
Amersham Corp. All restriction enzymes were from New England BioLabs
(Beverly, MA).
Livers from 180-200-g male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were perfused sequentially with 0.1% Pronase-E and 0.05% collagenase in situ and then incubated with 0.08% Pronase-E in 100 ml of Dulbecco's modified Eagle's medium/F12, pH 7.4, at 37 °C for 30 min. Cells were centrifuged, washed 3 times, and plated on 6-well collagen-coated culture plates at 1-2 × 106 cells/well. After overnight incubation, cultures were maintained with daily changes of medium, 20% fetal bovine serum containing medium 199 on day 2, and serum-free media thereafter.
Transient transfections of chimeric constructs containing various
deletions of rat IGFBP-3 promoter regions attached to the luciferase
reporter gene were undertaken in liver nonparenchymal cells on day 3 of
culture. Lipofectin reagent and DNA complexes were mixed at a 15 µg
to 2.5 µg ratio and incubated with the cells overnight. Medium was
replaced with serum-free medium 199 on day 4, with or without the
addition of 106 M insulin for 24 h, and
cell extracts were assayed on day 5 for gene activity using the
luciferase assay system (manufacturer's recommended protocol from
Promega) and measured by a Microsure 100 luminometer (LKB Wallac,
Turku, Finland). All readings were within the linear range of the
instrument when compared with known luciferase concentrations.
The rat IGFBP-3 promoter region in
pCAT basic vector (29) was subcloned to plasmids that carry the coding
region for firefly luciferase (pGL2-Basic from Promega, Madison, WI).
To obtain the 734/+34 pGL2 IGFBP-3, a
1047 to +34 AvaII
fragment of the IGFBP-3 promoter in pCAT Basic was blunt-ended, ligated
to PUC18, and cut with XbaI/SpeI and subsequently
cloned to the NheI site of pGL2-Basic vector. To obtain the
1600 pGL2 IGFBP-3, we ligated the XbaI-SpeI-cut
734 pGL2 IGFBP-3 to the NheI-XbaI fragment of
the
1.6-kilobase pair pCAT Basic vector. To obtain
1061 pGL2 IGFBP-3, we ligated the SalI-SpeI (
1061 to
734) fragment of the
1.06 kilobase pair IGFBP-3 in pCAT Basic to
XhoI-SpeI-digested
734 pGL2 IGFBP-3. To obtain
the
99 pGL2 IGFBP-3, we digested
734 pGL2 IGFBP-3 with
SmaI and relegated the construct. The
1201 pGL2 IGFBP-3
construct was obtained by 5
deletion of
KpnI/MluI-digested
1600 pGL2 IGFBP-3 with
exonuclease III.
To obtain the IGFBP-3 IRE (1150 to
1117 bp) concatemer,
double-stranded oligonucleotides corresponding to the first primer in
Table II were annealed, treated with T4 DNA ligase at 16 °C for 10 min, and then gel-purified and ligated upstream to a luciferase gene
reporter containing SV40 promoter (pGL3-Promoter from Promega). Orientation of the sequences and the number of tandem repeats were
confirmed by dideoxy sequencing.
|
To obtain the substitution mutants used in Fig. 4, oligonucleotides
corresponding to mutants 1-6 (Table II) were used as 5-primers for 30 cycles of PCR amplification with 3
-primer corresponding to the
503
to
522 bp region of the rat IGFBP-3 promoter. The amplified fragments
were subsequently gel-purified, cut with SpeI, and subcloned
into the SmaI-SpeI-digested
1201/+34 pGL2
IGFBP-3. The presence of mutated bases was confirmed by dideoxy
sequencing.
Nuclear Extracts from Hepatic Nonparenchymal Cells and Rat Liver
Cells were isolated and grown in vitro as
described above, and the nuclear extracts were prepared by pooling
cells from 10 100-mm plates, using the protocol described by Dignam
et al. (30) with slight modifications. Aprotinin and
leupeptin at 2 µg/ml, and pepstatin A at 1 µg/ml were added to 10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol. The cells were exposed to medium with or without 106 M
insulin for 48 h prior to nuclear protein extraction on day 5 of
culture. Liver nuclear extracts from normal and streptozotocin-diabetic rats were generous gifts of Dr. Ching-I Pao, and were obtained as
described previously (31).
For DNase I footprinting, end-labeled
140-bp SmaI/SalI fragments corresponding to the
1201 to
1061 bp region of rIGFBP-3 were incubated with 5-20 µg
of nuclear extract protein in 25 µl of binding buffer containing 20 mM Hepes, pH 7.9, 0.2 mM EDTA, 0.1 M KCl, 0.5 mM dithiothreitol, 1 µg of
poly(dI·dC) and 20% glycerol at 25 °C for 20 min. DNase I at 5 µg/ml in 25 mM NaCl, 10 mM Hepes, 5 mM MgCl2, and 1 mM
CaCl2 was then added, followed by incubation at 25 °C
for 2 min. The reaction was stopped with buffer containing 12.5 mM EDTA, 12.5 µg/ml proteinase K, 10 µg/ml yeast tRNA,
and 0.1% SDS at 37 °C for 10 min, followed by phenol/chloroform extraction, precipitation with ethanol, and electrophoresis on an 8 M urea, 6% polyacrylamide gel.
For gel mobility shift assay, [-32P]ATP-labeled
oligonucleotides (
1150 to
1117 bp fragment of rat IGFBP-3) was
incubated with different concentrations of nuclear extracts in 25 µl
of binding buffer containing 10 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 0.5 mM
dithiothreitol, 0.2% Nonidet P-40, 20 µg of bovine serum albumin, 36 µg of salmon sperm DNA, and 10% glycerol at 25 °C for 20 min.
Incubations were carried out with or without unlabeled competitors as
indicated. Protein-DNA complexes were separated from free probe on 6%
polyacrylamide gel in 0.25 × TBE at 12 V/cm for 2-3 h, and
visualized by autoradiography.
For Southwestern blotting, 20 µg/lane of nuclear extracts from
cultured cells treated or untreated with 106
M insulin and from pooled liver extracts from normal and
diabetic rats were subjected to SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose, and denatured by incubating in a solution containing 6 M guanidine HCl, 25 mM Hepes, pH
7.6, 12.5 mM MgCl2, 20% glycerol, 0.1%
Nonidet P-40, 0.1 M KCl, and 1 mM
dithiothreitol. This was followed by gradual renaturation using
decreasing concentrations of guanidine HCl and then incubation of the
filter with 32P-labeled oligonucleotide probe (
1150 to
1117 bp region of rat IGFBP-3), washing, and autoradiography.
Data were examined by analysis of variance and Duncan's multiple range test. Results were considered significant when p was <0.05.
To investigate the sequences required for insulin to
increase rat IGFBP-3 gene transcription, we developed a series of
5-deletions of the promoter region between nt
1600 and +34. All
plasmids were constructed with a common 3
-end (nt +34), attached to a luciferase reporter gene; this design preserves the TATA element (
28)
and a CpG island, allowing function as a basal promoter (29). Fig.
1 shows insulin-stimulated expression after transient transfection of hepatic nonparenchymal cells in primary culture. Using
plasmids with nested deletions from
1600 to
99 nt, insulin increased luciferase activity 168 ± 36% and 166 ± 15%
with IGFBP-3 plasmids that spanned
1600/+34 and
1201/+34 nt,
respectively, compared with cells not treated with insulin. Insulin had
no effect on luciferase activity with plasmids that spanned
1061/+34
and
734/+34 nt. However, insulin increased luciferase activity
58 ± 2% with plasmids containing the
99/+34 nt, presumably
representing effects on the basal promoter. These results indicate that
the deletion of bases
1201 to
1061 largely eliminated the ability of insulin to stimulate IGFBP-3 expression in liver cells, suggesting that an insulin-responsive element is located within this 140-bp region.
DNA-Protein Interactions Identified by DNase I Footprinting
DNase I footprinting was used to characterize the
pattern of nuclear factor binding to the 1201/
1061 nt region. In
the presence of nuclear extracts from hepatic nonparenchymal cells,
four regions were protected: sequences between
1185 and
1179 nt,
1150 and
1137 nt,
1135 and
1126 nt, and
1100 and
1096 nt,
as depicted in Fig. 2. Similar footprint patterns were
observed with both the antisense (Fig. 2) and the sense strands (not
shown). With this large fragment, nuclear extracts from unstimulated
and insulin-stimulated cells provided the same pattern of protection
(not shown).
IGFBP-3 Sequences Permit Insulin Action in a Heterologous Promoter
The sequences of the protected regions were compared
with known insulin regulatory sequences. Comparison was done by the
PILEUP Program from the Wisconsin Genetics Computer Group, which used progressive pairwise alignment. Comparing the sequences of the four
protected regions with known IREs, there is limited homology between
the second and third protected regions (1148 to
1117 bp) with the
IREs identified for glucagon, glyceraldehyde-3-phosphate dehydrogenase,
PEPCK, prolactin, and amylase genes, as shown in Table I
(32-36). Using the GAP comparison program of the Genetics Computer
Group, the homologous sequence is limited to 11 of 28 bases of the
glyceraldehyde-3-phosphate dehydrogenase IRE, 11 of 30 bases of the
glucagon IRE, 10 of 29 bases of the amylase IRE, and 8 of 22 bases of
the prolactin IRE.
|
To determine whether this IGFBP-3 sequence could confer insulin
responsiveness upon a heterologous promoter, we constructed an
oligonucleotide containing three tandem repeats of the IGFBP-3 promoter
region between 1150 and
1117 bp. This concatemer was inserted
upstream from the SV40 promoter in a luciferase reporter construct, and
transfected into hepatic nonparenchymal cells. As shown in Fig.
3, luciferase activity driven by the viral promoter alone was increased by 13 ± 5% in response to insulin, but the presence of the 34-bp IGFBP-3 sequence permitted a 92 ± 12%
increase in reporter activity in response to insulin. When the IGFBP
sequence was inserted in reverse orientation, insulin increased
luciferase expression 91 ± 12%. Thus, the insulin responsiveness
of the
1150 to
1117 bp region of the IGFBP-3 gene is portable to a
heterologous promoter, and the hormone responsiveness is independent of
orientation. In combination, these findings indicate that the IGFBP-3
region functions as an enhancer and is sufficient for
regulation by insulin.
Linker-scanning Mutations Identify an IRE in the IGFBP-3 Gene
To determine the minimal IGFBP-3 sequence required for
insulin responsiveness within the 34-bp region, a series of 5-bp
substitution block mutations scanning the 1150/
1117 region was
introduced into the wild type
1201/+34 promoter fragment, and
constructs were transfected into hepatic nonparenchymal cells. Table
II shows the wild type IGFBP-3 IRE sequence (
1150 to
1117 bp) and the internal substitution mutants used in these studies.
Fig. 4 shows the increase in expression in response to
insulin in cells transfected with these constructs. In the absence of
insulin, all of the constructs had basal activity that varied only
slightly above or below the level seen with the control plasmid (wild
type
1201/+34 bp). Substitutions of nt
1148/
1144 and
1143/
1139 (mutants 1 and 2) reduced the response to insulin to
3 ± 9% and 23 ± 14% of that of the wild type control
plasmid. Mutations of
1138/
1134,
1133/
1129, and
1128/
1124
bases (mutants 3, 4, and 5) reduced the insulin response to 60 ± 32, 61 ± 5, and 47 ± 18% of the control plasmid, respectively. In contrast, mutations of
1123/
1119 (mutant 6) and
1159 to
1150 bp (not shown) had no effects on insulin
responsiveness. Thus, mutation of the region from
1148 to
1139 bp
reduced the response to insulin by 80-90%, whereas mutation of the
1138 to
1124 bp region reduced the response to insulin by 50-60%,
while mutation in other neighboring regions had little effect. These data indicate that the region between
1148 and
1139 bp is
necessary for insulin responsiveness of IGFBP-3 gene
transcription.
To characterize
the interactions of nuclear factors with the IGFBP-3 IRE, we examined
binding activity by gel mobility shift analysis. End-labeled 1150 to
1117 bp oligonucleotides were incubated with nuclear extracts from
hepatic nonparenchymal cells and subjected to nondenaturing
polyacrylamide gel electrophoresis. As shown in Fig.
5A, one major DNA-protein complex was
observed consistently, with a second band of higher mobility that was
present intermittently. The binding activity of nuclear extracts
obtained from insulin-treated cells was about 3-fold higher than that
of extracts from control cells. Fig. 5B demonstrates the
specificity of complex formation. The formation of the major
DNA-protein complex was inhibited by a 50-100-fold excess of unlabeled
IGFBP-3 IRE, while an unrelated oligonucleotide (AP-3) had no effect.
Neither oligonucleotide affected formation of the higher mobility
complex, presumably representing nonspecific binding.
The physiologic relevance of insulin-induced DNA-protein complex
formation in hepatic nonparenchymal cells was assessed by determining
whether analogous regulation could be detected with hepatic nuclear
extracts prepared from normal and streptozotocin-diabetic rats. As
shown in Fig. 6, extracts from the livers of normal and diabetic rats formed complexes similar to those seen with cell extracts. The DNA-protein binding activity decreased by 3-4-fold with
extracts obtained from diabetic rat liver compared with normal rat
liver, indicating that decreased binding to this region may contribute
to the reduction in IGFBP-3 expression in conditions of diabetes
mellitus.
To identify the nucleotide sequences within the 34-bp region that were
necessary for complex formation, we generated a series of
oligonucleotides with 5-bp substitution mutations within the 1150 to
1117 bp fragment (as shown in Table II). Competition assays were
conducted using a wild type fragment as a probe, and an excess of
unlabeled mutant oligonucleotides as competitors. As shown in Fig.
7, the DNA-protein complexes were competed away in the
presence of a 100-fold excess of unlabeled wild type fragment. When the
mutants were used as competitors, the oligonucleotides with
substitution of sequences corresponding to nt
1148/
1144 (mutant 1)
and
1143/
1139 (mutant 2) could not compete away the DNA-protein
complexes (band intensity was comparable with that seen with a 100-fold
excess of unrelated oligonucleotides shown in Fig. 5B).
However, mutants with substitution of sequences from
1138 to
1119
(mutants 3-5) were able to compete for binding of nuclear protein(s)
to the IGFBP-3 IRE. These results suggest that the sequences between
1148 and
1139 are essential for binding of the nuclear factor(s) to
the IGFBP-3 IRE. Thus, the same 10-bp region that is critical for
insulin responsiveness in functional studies is also important for
binding of putative trans-acting factor(s).
To characterize the size and hormone responsiveness of proteins
associated with the IGFBP-3 IRE, nuclear extracts from hepatic nonparenchymal cells and rat livers were subjected to
SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose, and
probed with a labeled IGFBP-3 IRE oligonucleotide, as shown in Fig.
8. Proteins with apparent molecular mass of 90 and 70 kDa were present in both cell and liver extracts. Metabolic
responsiveness of the 70-kDa protein was not consistent with different
preparations of nuclear extracts, while the 90-kDa protein appeared to
be hormone-responsive. The abundance of the 90-kDa protein was
increased 2-2.5-fold with insulin treatment in cultured cells and was
increased 1.8-fold in hepatic extracts from normal compared with
diabetic animals.
Our previous studies demonstrated that the Kupffer and sinusoidal
endothelial cells of the liver are the locus of both high basal
expression of the IGFBP-3 gene and transcriptional regulation of
IGFBP-3 by insulin (27, 28). In this investigation, we used transient
transfection of these nonparenchymal cells to identify sequences within
the 1150 to
1124 bp region of the IGFBP promoter that are required
for the response to insulin. The 26-bp region appears to be both
sufficient and necessary for insulin responsiveness, since this region
can function as an IRE in the context of a heterologous promoter, and
mutations of these bases eliminate the insulin response of the parent
plasmid. The IGFBP-3 basal promoter (
99 to +34 bp) also exhibits
modest responsiveness to insulin, presumably reflecting involvement of
factors that are part of the basal transcriptional machinery, as has
been described previously (37). Our mutational analysis defines a 10-bp
core sequence between
1148 and
1139 bp that is critical for the
effects of insulin, but full responsiveness appears to require the
adjacent 16 bp.
The IGFBP-3 IRE appears to be composed of a dyad of AGG(A/G)A. This
sequence has a strong resemblance to the recognition sequence of
Ets-related transcription factors, AGGAA, which is contained within the
insulin response elements of both the prolactin and somatostatin genes
(38). The imperfect dyad suggests the possibility of a dimer binding
motif, and Ets-related proteins tend to function most effectively from
dyad recognition sites (39). In addition to the above cognate binding
sequence, 1134 to
1123 bp of the IGFBP-3 IRE (GGAAAGTCTCC on the
sense strand) strongly resembles the binding sites for HIV-
B
proteins, which recognize GGAAAGTCCC, and NF-
B proteins, which
recognize GGAAAGTCCCC (40). However, studies with antibodies to two
subunits of NF-
B (c-Rel, and P50) did not alter the DNA-protein
complexes as seen in Fig. 5 (not shown). The 10-bp core sequence of the
IGFBP-3 IRE that is most critical for insulin responses (
1148/
1139)
had no significant consensus sequence similarity to previously
identified transcription factor binding sites. We are currently in the
process of evaluating binding by these and other factors.
When the minimal sequence of the IGFBP-3 IRE is compared with the
sequence of other genes that are regulated by insulin, there are weak
homologies with the IREs of several genes, including prolactin,
glucagon, and amylase. As shown in Table I, the 5-end of the IGFBP-3
IRE exhibits somewhat more homology with IREs identified for glucagon
and prolactin, while the 3
-end of the IRE appears to have more
homology with the IREs from amylase and PEPCK. In addition, IGFBP-3
also contains a CAACAACAATTCC motif (
1159 to
1137 bp) which has
some homology to the hepatocyte nuclear factor 3
binding site of
IGFBP-1, a related protein which is inhibited by insulin in hepatocytes
(41, 42). However, our transfection studies indicate that such a region
is not required for regulation of IGFBP-3 transcription by insulin.
These limited homologies between the IGFBP-3 IRE and other known IREs
suggest the possibility that some common factor(s) may regulate the
expression of genes that are responsive to insulin.
Use of the IGFBP-3 IRE (1150 to
1117 bp) as a probe in gel mobility
shift analyses revealed a single insulin-inducible DNA-protein complex
with nuclear extracts from hepatic nonparenchymal cells. Similar
insulin-responsive up-regulation of protein binding has been reported
for upstream fatty acid synthase sequences in hepatocytes, and for the
prolactin gene in GH4 cells (38, 43). In contrast, the
presence of insulin does not affect DNA-protein interactions with the
PEPCK and IGFBP-1 genes. Such a difference may reflect the action of
insulin on gene transcription via direct or indirect mechanisms.
Insulin stimulates gene activity with increased binding of upstream
stimulatory factor 1 to fatty acid synthase (43) and Ets-related
proteins to the prolactin insulin response region (38) as opposed to
the negative effects of insulin on PEPCK and IGFBP-1 gene
transcription. Insulin is postulated to mediate such negative effects
indirectly, by inhibiting hepatocyte nuclear factor 3 binding that
supports glucocorticoid-induced transcription of the gene (41, 42). Our
findings are consistent with a model in which insulin induces factors
that bind to the IGFBP-3 IRE and increase IGFBP-3 gene transcription
directly. Since insulin deprivation in vivo in
streptozotocin-diabetic rats reduced DNA-protein complex formation with
the IGFBP-3 IRE in a manner similar to that seen with insulin
deprivation of hepatic nonparenchymal cells in primary culture, the
results in combination support the physiologic relevance of this
insulin-responsive DNA-protein complex in mediating the action of
insulin on the transcription of the IGFBP-3 gene.
Detailed analysis of the sequences critical to formation of DNA-protein
complexes with the IGFBP-3 IRE revealed that the retarded band
represents contact of factors with the 10-bp core sequence between
1148 and
1139 bp. (a) Oligonucleotides mutated in this region compete poorly with the labeled wild type probe. (b)
When such mutant oligonucleotides are used as probes, no DNA-protein complexes were found (data not shown). In addition, (c) use
of
1157 to
1139 bp oligonucleotides as probes (including the 10-bp core sequence but lacking the adjacent 16-bp region between
1138 and
1124 bp) revealed bands of mobility similar to those seen with the
1150 to
1117 bp probes; however, apparent affinity of DNA-protein
binding was considerably lower, suggesting that other proteins or DNA
contact of proteins with the
1138 to
1129 bp region may act
synergistically with a contiguous 5
-region to enhance transcription
factor binding. This indicates that there is no simple one-to-one
correspondence between the IRE DNA-binding motifs and the DNA/protein
interface; instead, the adjacent bases are critical for site-specific
recognition (44). This is consistent with the major DNA binding domain
requiring neighboring regions to make folding and docking of the
protein possible. Alternatively, multiple DNA binding domains may be
required for complete site-specific recognition, as could occur if the
binding protein is a homodimer or heterodimer or if the binding protein
contains different motifs in the same complex.
Our Southwestern blotting studies with IGFBP-3 IRE probes indicate the
presence of a 90-kDa insulin-responsive DNA-binding protein that can be
detected both in hepatic nonparenchymal cells in primary culture and in
rat liver. The 90-kDa band was consistently increased by the addition
of insulin to the cultured cell system and decreased by the induction
of insulin deficiency in streptozotocin-treated rats. In contrast, the
70-kDa protein did not exhibit consistent responsiveness to metabolic
status. Both proteins appear to bind directly to the core sequence of
the 1148 to
1139 bp core sequence, since similar bands were
observed with
1157 to
1139 bp and
1150 to
1117 bp probes. Thus,
both mutational analysis of IGFBP-3 function in cell transfection
models and studies of DNA-protein interactions in both gel shifts and
Southwestern blotting point to an alteration in the quantity and/or
activity of factors binding to the
1148 to
1139 bp region as a
critical mechanism for the control of IGFBP-3 transcription by insulin.
Our studies add to the understanding of the mechanism through which IGFBP-3 expression is regulated by insulin. We also demonstrate the feasibility of transient transfection in hepatic nonparenchymal cells in primary culture, which should be useful for other studies of genes that are uniquely expressed by these cells. Finally, our identification of an IRE in the IGFBP-3 gene and its association with hormone-responsive IRE binding proteins should provide insights important for future identification of the factors involved. Since IGFBP-3 is the major carrier protein for IGF-I, a critical growth factor, our observations may also shed light on the processes through which poor metabolic control of diabetes mellitus leads to impaired growth.
We thank Sharon Ann DePeaza and Mary Lou Mojonnier for assistance in preparing this manuscript. We thank Juan-li Zhu for helpful discussions and Weining Zhang for technical assistance.