(Received for publication, July 19, 1995; and in revised form, August 30, 1995)
From the
Insulin increases expression of somatostatin-chloramphenicol
acetyltransferase (CAT) constructs 10-fold and thymidine kinase-CAT
constructs 5-fold in GH4 cells. These responses are similar to our
previously reported data on insulin-increased prolactin-CAT expression.
They are also observed in HeLa cells and are thus not cell type
specific. The evidence suggests that the insulin responsiveness of
these genes is mediated by an Ets-related transcription factor. First,
linker-scanning mutations and/or deletions of the prolactin,
somatostatin, and thymidine kinase promoters suggest that their insulin
responsiveness is mediated by the sequence CGGA. This sequence is
identical with the response element of the Ets-related transcription
factors. Second, CGGA-containing sequences placed at -88 in the
MTV-CAT reporter plasmid conferred insulin responsiveness to the
mammary tumor virus promoter. Third, expression of the DNA-binding
domain of c-Ets-2, which acts by blocking effects mediated by
Ets-related transcription factors, inhibits the response of these
promoters to insulin. Finally, the Ets-related proteins Sap and Elk-1
bind to the prolactin, somatostatin, and thymidine kinase
insulin-response elements. An Ets-like element was found in all
insulin-sensitive promoters examined and may serve a similar function
in those promoters.
The mechanisms involved in the regulation of gene expression by insulin are not well characterized. Insulin-induced alterations in the steady-state levels of numerous mRNAs have been documented(1) . For several genes including phosphoenolpyruvate carboxykinase(2) , glyceraldehyde 3-phosphate dehydrogenase(3) , growth hormone(4) , and prolactin(5) , it has been established that these alterations are due to effects of insulin on the rate of transcription and not to effects on mRNA half-life or processing(1) .
The effects of hormones on transcription are mediated through response elements in the promoters of genes. Several response elements may exist for a particular hormone that differs slightly in sequence or orientation, perhaps to allow for fine tuning of the hormonal response or for interaction with different tissue-specific factors. However, these elements are sufficiently similar to be recognized by the hormone-activated transcription factors and are thus said to form a consensus response element. The consensus hormone response element for cAMP-activated genes is the sequence TGACGTCA(6) , while the thyroid-retinoid response element is a direct repeat of the sequence AGGTCA with varying numbers of intervening bases that determine hormone receptor specificity(7) . A specific sequence comprising an insulin response element has been identified only for a small proportion of insulin-responsive genes. Comparison of these insulin response elements has not revealed sequence homologies that could constitute a consensus insulin response element(8) .
The transcription factors that mediate responses to insulin have not been characterized. The putative insulin response element of the glyceraldehyde 3-phosphate dehydrogenase gene was identified by binding of an insulin-regulated protein to a specific sequence in the glyceraldehyde 3-phosphate dehydrogenase promoter. A protein that binds to this sequence has been cloned and is identical to the product of the testis determining gene, SRY(9) . However, its role in the activation of glyceraldehyde 3-phosphate dehydrogenase gene expression by insulin was not further established.
We previously identified an insulin response element in the prolactin promoter(8) . This insulin response element overlaps the cAMP response element of the prolactin gene TGACGGAA. However, mutagenesis and deletional analysis revealed that the insulin response element was separable from the cAMP response element and consisted of a direct repeat of the sequence CGGAAA. This sequence is identical to sequences that bind the Ets family of transcription factors. These studies report the identification of insulin response elements in the somatostatin and herpes simplex virus thymidine kinase genes that, together with the previously identified insulin response element of the prolactin gene, define a consensus insulin response element. The consensus sequence is identical to known binding sites for Ets-related transcription factors, and an Ets-factor inhibitor was found to inhibit insulin activation of all three promoters. The Ets-related proteins Sap-1 and Elk-1 specifically bind to these sequences.
The oligonucleotides used for these studies showing the putative
Ets-binding sites (underlined) and mutations (lower
case) include 1) an oligonucleotide containing the insulin
response element of the prolactin promoter (-106/-87)
5`-TCTTAATGACGGAAATAGATG-3`, 2) the putative insulin-responsive area of
the somatostatin promoter (-7/-47)
5`-GAAGGAGACGCTACTGGAGTCGTCTCTAGAGCCTGCGGACG-3`, 3) an 82-bp mutant of
this sequence (+1/+80 mutated)
5`-GGCGGCTGAAGcAGACGCTACTGcAGTCGTCTCTGCCGCCTGCGcACCTGCGTCTAGACTGACCCACCGCGCTCAAG-3`,
4) the sequence of the wild type thymidine kinase promoter from
-22/-2 5`-AAGGTGACGCGTGTGGCCTCG-3`, and 5) the
corresponding mutant sequence from the insulin-responsive
linker-scanning mutant of the thymidine kinase promoter
TK(ls-21/-12)CAT and MTV(TKb)CAT
(5`-ccGGatcCGCGTGTGGCCTCG-3`). These were purified on polyacrylamide
gels, annealed, and either end labeled with
[
P]dCTP or filled in with unlabeled
deoxyribonucleotides using the Klenow reaction. These labeled
oligonucleotides were then used in gel mobility shift experiments
performed as described(20) . 2 µg of nuclear extract or 2
µl of in vitro transcribed and translated Sap-1 or Elk-1
were incubated at 25 °C for 30 min with 10,000 cpm (10-20
fmol) of the
P-labeled oligonucleotide. The protein-DNA
complexes were then analyzed by electrophoresis on a 5% polyacrylamide
gel in Tris-acetate-EDTA buffer.
Figure 1:
Effect of insulin on cAMP-responsive
promoters in GH4 cells. GH4 cells were cotransfected with 10 µg of
the CAT construct indicated in the figure and 5 µg of pRT3HIR-2.
Following a 24-h incubation without hormone, insulin was added at 1
µg/ml. After an additional 24 h of incubation, the cells were
harvested, and CAT enzyme was assayed. The average % acetylation in the
control and insulin-incubated cells was determined, and the results
from the insulin-incubated cultures were compared with control levels
to determine the fold stimulation (Fold-Control). The results are from
three separate experiments done in duplicate. Basal CAT
expression/µg of protein was 0.052 ± 0.017% for
Pit-1(-738/+1)CAT, 0.23 ± 0.015% for
(-846/+44)CAT, 3.3 ± 0.27% for (AP1)3CAT, 0.66
± 0.078% for SS(-71/+80)CAT, and 0.23 ± 0.075
for TK(-95/+56)CAT.
Insulin also activates expression of
Prl(-173/+75)CAT, SS(-71/+80)CAT, and
TK(-95/+56)CAT in HeLa cells (Fig. 2). Low levels of
Prl(-173/+75)CAT expression are increased 16-fold in HeLa
cells in response to insulin. SS(-71/+80)CAT is increased
10-fold and TK(-95/+56)CAT is increased 4-fold in HeLa cells
in response to insulin. As in GH4 cells, the expression of CAT is not
increased by insulin using plasmids containing the Pit-1 promoter,
glycoprotein hormone -subunit promoter, or the repeated AP1
element. These data indicate that the effects of insulin to increase
transcription of these three genes is not unique to the GH cells.
Figure 2:
Effect of insulin on cAMP-responsive
promoters in HeLa cells. HeLa cells were cotransfected with 10 µg
of the CAT construct indicated in the figure and 5 µg of pRT3HIR-2.
RSV-Pit-1 was also included in cotransfections with the prolactin-CAT
reporter plasmid to achieve high level basal expression with this
construct(30) . Following a 24-h incubation without hormone,
insulin was added at 1 µg/ml. After an additional 24 h of
incubation, the cells were harvested, and CAT enzyme analysis was
performed as in Fig. 1. Basal CAT expression/µg of protein
was 0.022 ± 0.0058 for Pit-1(-738/+11)CAT, 0.27
± 0.067% for (-846/+44)CAT, 3.5 ± 0.023%
for (AP1)3CAT, 1.1 ± 0.04% for Prl(-173/+75)CAT, 0.26
± 0.016% for SS(-71/+80)CAT, and 0.23 ± 0.039%
for TK(-95/+56)CAT.
Figure 3:
The effect of insulin on different
thymidine kinase deletion and linker-scanning mutants in GH4 cells. GH4
cells were cotransfected with 10 µg of the thymidine kinase-CAT
construct indicated in the figure and 5 µg of pRT3HIR-2. Following
a 24-h incubation without hormone, insulin was added at 1 µg/ml.
After an additional 24 h of incubation, the cells were harvested, and
CAT enzyme analysis was performed as in Fig. 1. Basal CAT
expression/µg of protein was 0.16 ± 0.05% for
TK(-46/+56)CAT, 0.16 ± 0.028% for
TK(-725/-7)CAT, 5.1 10
±
0.99
10
% for TK(-725/-16)CAT,
0.8 ± 0.071% for TK(ls-46/-36)CAT, 0.46 ±
0.15% for TK(ls-42/-32)CAT, 0.28 ± 0.08% for
TK(ls-28/-18)CAT, 0.21 ± 0.04% for
TK(ls-21/-12)CAT, 0.19 ± 0.025 for
TK(ls-16/-6)CAT, and 0.057 ± 0.009% for
TK(-95/+56
BamHI)CAT.
To further confirm that the BamHI linker sequence used to make the linker-scanning
plasmids was sufficient to mediate the effects of insulin on thymidine
kinase-CAT expression, the linker sequence from the plasmid
TK(ls-21/-12)CAT between -22 and -2, containing
the CCGGAA motif, was cloned into MTV-CAT in both the normal and
inverted orientation to create
MTV(TKb)CAT and
MTV(TKbi)CAT (Fig. 4, top). Insulin did not affect CAT expression
from
MTV(TKa)CAT and
MTV(TKai)CAT that contain the
-22/-2 sequence from the wild type thymidine kinase gene in
both the normal and inverted orientation (Fig. 4, bottom). In contrast, insulin increases CAT expression 6-fold
in
MTV(TKb)CAT and
MTV(TKbi)CAT that contain the CGGA
sequence.
Figure 4:
The effect of insulin on MTV(TK)CAT
expression in GH4 cells. Top, the
MTV-CAT vector (7) was used to construct a hybrid promoter with sequences from
the thymidine kinase promoter or from a linker-scanning mutant of the
thymidine kinase promoter.
MTV-CAT contains 1200 bp of the mammary
tumor virus long terminal repeat linked to the CAT structural gene
terminated with an SV40 polyadenylation sequence. The glucocorticoid
enhancer region, -190/-88, was deleted, and a HindIII restriction sequence was inserted at -88.
Oligonucleotides were synthesized to the sequence -22/-2 of
the wild type thymidine kinase promoter and to the same location from
the linker-scanning mutant TK(ls-21/-12)CAT. These
oligonucleotides were then ligated into HindIII-digested
MTV-CAT. The resulting plasmids were sequenced to confirm the
presence of the proper sequence. The sequence of the final insert is
given in the figure. Bottom, the response of the
MTV(TK)-CAT plasmids to insulin was determined as above. GH4 cells
were transfected with 10 µg of the
MTV(TK)CAT plasmid
indicated and 5 µg of pRT3HIR-2. They were incubated with 1
µg/ml insulin as described in Fig. 1. The parental plasmid
MTV-CAT was unresponsive to insulin treatment (data not shown).
Basal CAT expression/µg of protein was 0.14 ± 0.065% for
MTV(TKa)CAT, 0.065 ± 0.011% for MTV(TKai)CAT, 0.1 ±
0.027% for MTV(TKb)CAT, and 0.31 ± 0.07% for
MTV(TKbi)CAT.
The deletion mutants shown in Fig. 5address the
possibility that the insulin response element of the native thymidine
kinase promoter is the CCGGAA sequence located at
-151/-146. The linker-scanning plasmid
TK(ls+5/+15)CAT was used to make 5`-deletions of the
thymidine kinase promoter since the 3`-deletion plasmids all have a
3`-CGGA sequence as a result of their construction. First, the BamHI site in the linker was removed with mung bean nuclease
to create the plasmid TK(ls+5/+15BamHI)CAT. The
remaining plasmid contains the thymidine kinase promoter between
-725 and +56. Insulin increases CAT expression 5-fold using
this plasmid. Deletion to -385 (TK(-385/+56)CAT, Fig. 5) did not reduce the increase in CAT expression due to
insulin. A further deletion to -218 (TK(-218/+15)CAT, Fig. 5) eliminates 5 potential Ets-binding sites including 2
CGGA sequences between -385 and -250, but this did not
reduce the insulin-mediated increase in CAT production with this
construct. Finally, a deletion to -128
(TK(-128/+56)CAT, Fig. 5), which eliminates the
CCGGAA at -151/-147, renders the thymidine kinase promoter
insensitive to insulin.
Figure 5:
The effect of insulin on deletion mutants
of the wild type thymidine kinase promoter in GH4 cells. GH4 cells were
cotransfected with 10 µg of the thymidine kinase-CAT construct
indicated in the figure and 5 µg of pRT3HIR-2. Following a 24-h
incubation without hormone, insulin was added at 1 µg/ml. After an
additional 24 h of incubation, the cells were harvested, and CAT enzyme
analysis was performed as in Fig. 1. Basal CAT expression/µg
of protein was 0.19 ± 0.09% for TK(ls+5/+15)CAT, 0.42
± 0.03% for TK(ls+5/+15BamHI)CAT, 1.97
± 0.65% for TK(-385/+56)CAT, 0.19 ± 0.088% for
TK(-218/+56)CAT, and 0.44 ± 0.083% for
TK(-128/+56)CAT.
Figure 6: Effect of insulin on 5`- and 3`-deletion mutants of somatostatin (-71/+80)CAT. GH4 cells were cotransfected with 10 µg of the somatostatin-CAT construct indicated in the figure and 5 µg of pRT3HIR-2. Following a 24-h incubation without hormone, insulin was added at 1 µg/ml. After an additional 24 h of incubation, the cells were harvested, and CAT enzyme analysis was performed as in Fig. 1. The results are from three separate experiments done in duplicate. A, somatostatin-CAT constructs. Basal CAT expression/µg of protein was 0.59 ± 0.012% for SS(-71/+80)CAT, 0.23 ± 0.09% for SS(-48/+80)CAT, 0.066 ± 0.022% for SS(-71/-1)CAT, and 0.04 ± 0.0008% for SS(-48/-1)CAT. B, mutated somatostatin-CAT constructs. Basal CAT expression/µg of protein was 0.18 ± 0.21% for SS(-71/+80)CAT, 0.24 ± 0.036% for SS(-71/+80EtsMut)CAT, 0.152 ± 0.046% for SS(-71/-1,+23/+80 mut)CAT, and 0.14 ± 0.0023% for SS(-71/-1,+7/+47)CAT.
Figure 7: Expression of high levels of the c-Ets-2 DNA binding domain inhibits the insulin response. GH4 cells were cotransfected with 10 µg of the promoter-CAT constructs indicated on the x axis and 5 µg of pRT3HIR-2 and with or without 30 µg of Ets-Z(25) . Following a 24-h incubation without hormone, 1 µg/ml insulin or 0.1 mM 8-(chlorophenylthio)-3`,5`-cyclic AMP was added to the indicated cultures. After an additional 24 h of incubation, the cells were harvested, and CAT enzyme analysis was performed as in Fig. 1. The results are from three separate experiments done in duplicate. Basal CAT expression/µg of protein was 0.034 ± 0.004% for Prl(-173/+75)CAT, 0.03 ± 0.007% for Prl(-173/+75)CAT + Ets-Z, 0.23 ± 0.03% for SS(-71/+80)CAT, 0.1 ± 0.04% for SS(-71/+80)CAT + Ets-Z, 0.048 ± 0.015% for TK(-95/+56)CAT, and 0.02 ± 0.004% for TK(-95/+56)CAT + Ets-Z.
Figure 8:
Ets-related proteins associate with
insulin-sensitive sequences but not with insulin-insensitive sequences.
Gel mobility shift experiments were conducted using P-labeled oligonucleotides to the prolactin (panel
A), somatostatin (panel B), and thymidine kinase
promoters (panel C). A,
P-labeled
prolactin -106/-87 containing a CGGAAA motif at
-97/-92 was incubated with various proteins indicated and
analyzed on a 5% polyacrylamide gel. Lane 1, GH4 cell nuclear
extract; lane 2, Elk-1; lane 3, Sap-1; lane
4, SRF; lane 5, Elk-1 + SRF; lane 6, Sap-1
+ SRF; lane 7, Elk-1 + Sap-1; lane 8, Elk-1
+ Sap-1 + SRF; and lane 9, unprogrammed lysate. B, lanes 1-3 and 7-9 are
P-labeled somatostatin +7/+47, and lanes
4-6 are
P-labeled somatostatin
+1/+80EtsMut with point mutations converting the three GGA
sequences in the somatostatin promoter to GCA. These oligonucleotides
were incubated with nuclear extract (lanes 1, 4, and 7), Sap-1 (lanes 2, 5, and 8), or
unprogrammed lysate (lanes 3, 6, and 9). Lanes 7-9 also had a 100-fold excess of unlabeled
somatostatin +7/+47. C, lanes 1-3 are
P-labeled thymidine kinase -22/-2, and lanes 4-9 are the
P-labeled
-22/-2 sequence from the linker-scanning mutant
TK(ls-21/-12)CAT (also found in MTV(TKb)CAT), in which the
addition of a BamHI linker introduces a CGGA motif. Lanes
7-9 also had a 100-fold excess of unlabeled TK-22/-2
mutant. These oligonucleotides were incubated with nuclear extract (lanes 1, 4, and 7), Sap-1 (lanes
2, 5, and 8), or unprogrammed lysate (lanes
3, 6, and 9).
Sap-1 incubation
with an oligonucleotide to the somatostatin promoter (Fig. 8B) produces a more slowly migrating protein-DNA
complex similar to that formed with Prl-106/-87 (Fig. 8B, lane 2). An excess of
non-radioactive somatostatin +7/+47 inhibits the formation of
this complex (Fig. 8B, lane 8). Nuclear
extract proteins also bind to the somatostatin promoter (Fig. 8B, lanes 1 and 7), but no
specific interactions of comparable migration with the Sap-1-DNA
complex were seen even on longer exposure (Fig. 8B, lane 1 versus lane 2). No specific binding of Sap-1 was seen
using an oligonucleotide whose Ets-binding sites were mutated by a
GC conversion (compare lane 5, Sap-1, with lane
6, unprogrammed lysate) (Fig. 8B).
Sap-1 binds
to the thymidine kinase promoter only when it has been mutated to
contain an Ets-binding site (Fig. 8C). The wild type
thymidine kinase promoter (-22/-2) shows no retarded bands
when labeled DNA is incubated with Sap-1 (Fig. 8C, lane 2) that are not also present with unprogrammed lysate (Fig. 8C, lane 3). The oligonucleotide TKb
corresponds to the this sequence (-22/-2) that is found in
the insulin-sensitive TK(ls-21/-12)CAT and MTVTKb-CAT.
Incubation of Sap-1 with P-labeled TKb results in two
retarded complexes (Fig. 8C, lane 5). These
bands are not seen with unprogrammed lysate (Fig. 8C, lane 6), and they are inhibited by an excess of unlabeled TKb (Fig. 8C, lane 8). Again, no specific bands
corresponding to the Sap-1 shifted DNA are seen using nuclear extract (Fig. 8C, lane 4).
Multiple lines of evidence presented here and previously (8) indicate that the sequence CGGA is a consensus response
element for insulin effects in GH and HeLa cells. First, deletion and
linker-scanning mutants of the prolactin promoter identified the
sequence CGGAAA as essential for the insulin effect on the prolactin
promoter, and this sequence could confer insulin responsiveness to
MTV-CAT(8) . Second, the expression of CAT from several
CGGA containing linker-scanning and deletion mutants of the thymidine
kinase promoter is increased by insulin. When this linker sequence is
inactivated, as in the plasmid
TK(-95/+56
BamHI)CAT, insulin responsiveness is
lost. The CGGA-containing sequence from one of the linker-scanning
mutants was shown to confer insulin sensitivity when inserted into
MTV-CAT. Finally, CAT expression from a somatostatin promoter
construct is also increased by insulin. Deletions that inactivate the
cAMP response element of this gene have no effect on insulin
regulation. However, deletion of sequences in the 5`-untranslated
region of the gene, containing three Ets-binding motifs, eliminates the
increased expression mediated by insulin. Point mutation of these
motifs reduces the effect of insulin 75%, and a 24-bp deletion that
removes the first 2 of these motifs completely eliminates the effect of
insulin. When the three motifs are added back to the
insulin-insensitive plasmid SS(-71/-1)CAT, the effect of
insulin is restored. These effects are seen both in GH cells and in
HeLa cells. Thus, the presence of one or more CGGA sites in the
proximal promoter region confers insulin responsiveness in these cell
lines.
The effect of insulin to increase gene expression can be
mediated by one copy of the insulin response element. The constructs
MTV(Prl-106/-77)CAT and
MTV(TKb)CAT are
stimulated 4- and 6-fold by insulin, and they have only one copy of
this sequence. The 5`-deletion mutants of the thymidine kinase promoter
are also insulin responsive with only one copy of this sequence.
However, the prolactin promoter has two Ets-related binding sequences,
and the response of the prolactin promoter is approximately twice that
of the thymidine kinase promoter constructs. Thus, multiple Ets motifs
may mediate an increased response.
This is not true of all cell
lines. Chinese hamster ovary cells transfected with the prolactin-CAT
constructs and an expression vector for Pit-1 show low levels of
prolactin-CAT expression. However, this activity is not inducible
either by insulin or cAMP. ()Thus, it appears that Chinese
hamster ovary cells lack transcription factors that are both important
for high basal expression of this construct and its regulated
expression. The sequence CGGA is able to confer insulin sensitivity
only in cells with a necessary complement of transcription factors.
The involvement of Ets-related proteins in insulin-increased gene transcription is suggested by the experiment with the dominant negative Ets plasmid and by the gel shift experiments. Cotransfection of cells with a plasmid that expresses the DNA-binding domain of c-Ets-2 causes a 75% reduction in the insulin sensitivity of the prolactin, somatostatin, and thymidine kinase promoters. The Ets-related proteins Elk-1 and Sap-1 were shown to bind sequences from these promoters that are insulin sensitive but not sequences that are insulin insensitive.
The location of this sequence may also be important for its ability to mediate responses to insulin. The three insulin-sensitive promoters that we have described have the insulin response element inserted close to the transcription start site, the farthest away being the putative insulin response element of the wild type thymidine kinase gene at -150. Since this sequence is not uncommon in the genome, it is likely that this sequence is only effective within the first few hundred base pairs of the transcription start site.
These data
allowed us to establish several criteria for screening
insulin-responsive genes for potential response elements. First, the
sequence GGA is key to the insulin response element. Second, preference
was given to sequences containing CGGA as in the prolactin and
thymidine kinase promoters, but (A/T)GGA sequences were also considered
(especially as a dimer with CGGA). Third, the limits
-250/+50 were established since the IRE in the somatostatin
gene is in the region +1/+50, and the upstream limit was
established in accord with other Ets-responsive promoters that contain
Ets-binding sites in the -200/-300 region. Finally, inverse
sequences were also considered since the insulin response is
transferred to MTV-CAT by both the normal and inverse prolactin
and thymidine kinase insulin response elements. These criteria allowed
us to identify potential insulin response elements in 22 genes
previously reported to be insulin sensitive.
The utility of this type of analysis is clear for promoters where extensive deletional analysis has defined a region that contains the insulin response sequence. For example, the insulin response element of gene 33 likely resides in the first 100 base pairs (26) of the promoter. Comparison of this region of the gene 33 sequence with the consensus insulin response element identifies the sequence -93 CCGGATTGGCTGCGCGGAGG -74 that contains a direct repeat of the insulin responsive sequence (underlined). The insulin response of the c-fos promoter was mapped to the serum response element(27) . This sequence contains the sequence -225 GCGGAAGGTCTAGGAGA -209 that binds Elk-1. Elk-1 is an Ets-related protein that was shown to be phosphorylated by insulin.
These studies do not rule out other insulin response elements, and it seems likely that there are other insulin-responsive sequences. Although all of the insulin-responsive promoters examined have sequences that are similar to the insulin response element of the prolactin promoter, the region of homology between our consensus insulin response element and the area of the gene known to be insulin sensitive does not correspond in all cases. For example, the negative insulin response element in the glucagon gene is apparently located at -268/-238, and this region does not contain a CGGAA sequence(28) . Similarly, AGGA sequences reside outside of the insulin-responsive region of the amylase gene -167/-137(29) .
In summary, we have defined a consensus insulin response element, CGGA, that can act in several different promoter contexts and in different cell types. The activity of this response element is most likely dependent on the presence of the proper insulin response pathway and insulin-responsive transcription factors in the cells.