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J Biol Chem, Vol. 274, Issue 26, 18625-18634, June 25, 1999
From the Several of the complications seen in patients
with both type I and type II diabetes mellitus are associated with
alterations in the expression of matrix metalloproteinases. To identify
the cis-acting elements that mediate the stimulatory effect
of insulin on collagenase-1 (matrix metalloproteinase-1) gene
transcription a series of collagenase-chloramphenicol acetyltransferase
(CAT) fusion genes were transiently transfected into HeLa cells.
Multiple promoter elements, including an Ets and activator protein-1
(AP-1) motif, were required for the effect of insulin. The AP-1 motif appears to be a target for insulin signaling because it is sufficient to mediate an effect of insulin on the expression of a heterologous fusion gene, whereas the data suggest that the Ets motif acts to
enhance the effect of insulin mediated through the AP-1 motif. Multiple
promoter elements were also required for the stimulatory effect of
phorbol esters on collagenase-CAT gene transcription, and the AP-1
motif was also a target for phorbol ester signaling. However, the
cis-acting elements required for the effects of insulin and
phorbol esters were not identical. Moreover, phorbol esters were a much
more potent inducer of collagenase-CAT gene transcription than insulin,
a difference that may be explained by selective effects of insulin and
phorbol esters on AP-1 expression.
The maintenance of the extracellular matrix is accomplished by a
balance of synthesis and degradation, the latter being determined by
the relative activities of a family of extracellular matrix proteinases, the matrix metalloproteinases
(MMPs),1 and the tissue
inhibitors of MMPs (1-3). Tissue inhibitors of MMPs block MMP action
by binding covalently to MMPs and preventing both activation of the MMP
by enzymatic modification and the ability of MMPs to bind substrate
(1-3).
The regulation of MMP gene expression has received considerable
attention primarily because of the involvement of increased MMP
activity in tumor progression, specifically the development of
malignant carcinomas (4). However, several of the complications associated with both type I, insulin-dependent diabetes
mellitus and type II, non-insulin-dependent diabetes
mellitus, including glomerulosclerosis of the kidney (5, 6),
retinopathy (7) periodontal disease (8), and some forms of cardiac
disease (9), are also characterized, in part, by alterations in the amount and composition of extracellular matrix protein. In the streptozotocin rat model of type I diabetes, collagenase-1 (MMP-1; referred to henceforth as collagenase) gene expression in the glomerulus is decreased (10), and this would be predicted to contribute
to an increase in the mesangial matrix and/or the glomerula basement
membrane, one of the characteristics of glomerulosclerosis (5, 6). This
decrease in collagenase gene expression is reversed by insulin
treatment (10), but it is unclear whether this represents a direct
effect of insulin as opposed to an indirect effect mediated through
changes in glucose concentration. The effect of insulin on collagenase
expression in glomerula-derived cell lines has not been studied;
however, in NIH 3T3 (11), Chinese hamster ovary (12), and HeLa cells
(13) insulin stimulates the expression of reporter genes when ligated
to the collagenase promoter. Interestingly, if insulin does directly
regulate collagenase gene expression in the glomerulus, then
collagenase expression would actually be predicted to be increased in
hyperinsulinemic individuals because the insulin resistance associated
with muscle, adipose tissue, and liver may not be manifest in the
kidney (14).
cis-Acting elements that mediate the action of insulin on
gene transcription are referred to as insulin response sequences (IRSs)
or elements (15). Several IRSs have been identified, but it is apparent
that a single consensus IRS does not exist (15). Instead, it is
predicted that multiple classes of consensus IRSs will be found, only
three of which have currently been identified (15). One of these has
the consensus sequence T(G/A)TTT(T/G)(T/G) and mediates the
insulin-dependent transcriptional inhibition of several
hepatic genes such as those encoding phosphoenolpyruvate carboxykinase
(PEPCK), insulin-like growth factor-binding protein-1, tyrosine
aminotransferase, apolipoprotein CIII, and glucose-6-phosphatase (16-20). The other consensus IRSs are the Ets motif (21) and the serum
response element (15, 22), which mediate stimulatory effects of insulin
on several genes. However, unlike the PEPCK-type IRS that confers a
selective effect of insulin (15), the Ets motif and serum response
element can mediate the effects of multiple other hormones on
gene transcription (23, 24).
The activator protein-1 (AP-1) motif, which binds members of the Fos
and Jun transcription factor families (25) and, like the Ets motif and
serum response element, mediates transcriptional changes in response to
multiple ligands (26), may represent a fourth class of consensus IRS.
Thus, Rutter et al. (12) have shown that in Chinese hamster
ovary cells, mutation of the AP-1 motif in the collagenase promoter
abolishes the stimulatory effect of insulin on the expression of a
collagenase-luciferase fusion gene. Similarly, in rat H4IIE hepatoma
cells, an AP-1 motif is required for the stimulatory effect of insulin
on the expression of a malic enzyme (ME)-chloramphenicol
acetyltransferase (CAT) fusion gene (13). However, paradoxically, in
HeLa cells insulin has almost no effect on ME gene transcription,
whereas it markedly stimulates collagenase gene transcription (13).
This observation raises the question as to why the AP-1 motif appears
to only mediate an insulin-dependent activation of gene
transcription in some cell contexts. To indirectly address this
question, we have analyzed the promoter elements required for the
stimulatory effect of insulin on collagenase gene transcription in HeLa
cells. The results demonstrate that multiple promoter elements are
required for the effect of insulin on collagenase gene transcription in
addition to the AP-1 motif. However, the collagenase AP-1 motif appears
to be a target of insulin signaling because it is sufficient to mediate
an effect of insulin on the expression of a heterologous fusion gene.
The additional collagenase promoter elements that are required for the
full stimulatory effect of insulin may bind accessory factors that act
to enhance the effect of insulin mediated through the AP-1 motif. The
data also suggest that the mechanism of insulin and phorbol ester
signaling through the collagenase-1 AP-1 motif are distinct.
Materials
[ Plasmid Construction
DNA manipulations were accomplished by standard techniques (27),
and DNA sequencing was performed using the USB Sequenase kit. All
plasmid constructs were purified by centrifugation twice through cesium
chloride gradients (27).
A BglII-HindIII fragment of the human
collagenase-1 promoter, spanning the sequence from A plasmid designated X(An) was generated by ligating a minimal
Xenopus 68-kDa albumin promoter (29) with the following
sequence (5'-AAGCTTGATCTCTCTGAGCAATAGTATAAAACTCGAG-3';
HindIII and XhoI restriction enzyme sites
underlined; TATA box in italics) into HindIII-XhoI cleaved pCAT(An). The plasmid XMB
was then generated by removing a BamHI site located just 5'
of the HindIII site by the use of mung bean nuclease.
Double-stranded complementary oligonucleotides, representing various
regions of the collagenase promoter (Table I), were synthesized with
HindIII compatible ends and ligated into
HindIII-cleaved XMB in multiple copies. The number of
inserts was determined by restriction enzyme analysis and confirmed by DNA sequencing.
Cell Culture and Transient Transfection
Human HeLa cervical carcinoma cells were grown to 90%
confluence in T150 flasks in DMEM containing 10% (v/v) calf serum and were replated the day before use into 55-cm2 culture dishes
(1 flask to 26 dishes). Attached cells were transfected by addition of
0.5 ml of a calcium phosphate-DNA co-precipitate (30), containing the
reporter gene construct (15 µg), an expression vector for
Hamster insulinoma tumor cells were grown to 70% confluence in T150
flasks in DMEM containing 2.5% (v/v) fetal bovine serum and 15% (v/v)
horse serum and were replated the day before use into
55-cm2 culture dishes (1 flask to 14 dishes). Attached
cells were then co-transfected as described previously (31) by addition
of 0.5 ml of a calcium phosphate-DNA co-precipitate containing 15 µg of reporter plasmid DNA and expression vectors encoding
Rat hepatoma H4IIE cells were grown in 12-well plates in DMEM
containing 10% (v/v) fetal bovine serum. Attached cells were transfected at ~60% confluence with the plasmid pCol·Luc (2 µg; Ref. 12) using 4 µl of the Tfx-50 transfection reagent (Promega) in a
final volume of 400 µl serum-free DMEM, as described previously (32).
After incubation for 2 h, the cells were then overlaid with 2 ml
of serum-containing DMEM. After an overnight incubation, the cells were
washed with serum-free DMEM for 2 h prior to incubation for
24 h in serum-free DMEM supplemented with or without various concentrations of insulin, as indicated in the Figure legends. Under
these growth and transfection conditions 100 nM insulin was
not toxic to these cells (see Fig. 2), in contrast to previous experiments in which the cells were grown in DMEM containing 2.5% (v/v) fetal calf serum and 2.5% (v/v) newborn calf serum and were transiently transfected in solution using calcium phosphate-DNA co-precipitation (13).
CAT, Cells were either harvested by trypsin digestion and sonicated
in 300 µl of 250 mM Tris (pH 7.8) containing 2 mM phenylmethylsulfonyl fluoride, or for luciferase assays,
cells were extracted by scraping into passive lysis buffer (Promega).
CAT, Gel Retardation Assay
Labeled Probes--
Oligonucleotides representing the sense and
antisense strands of the collagenase AP-1 and Ets motifs (Table I) were
synthesized with BamHI or HindIII compatible
ends, respectively, gel purified, annealed, and then labeled with
[ Nuclear Extract Preparation--
HeLa nuclear extracts were
prepared exactly as described previously (13).
AP-1 Binding Assay--
Labeled AP-1 oligonucleotide (~7.5
fmol, ~30,000 cpm) was incubated with HeLa (3 µg) nuclear extract
in a final reaction volume of 20 µl containing 20 mM
HEPES, pH 7.8, 100 mM NaCl, 0.38 mM spermidine,
0.08 mM spermine, 0.1 mM EDTA, 1 mM
EGTA, 2 mM dithiothreitol, 12.5% glycerol (v/v), and 1 µg of poly(dI-dC)·poly(dI-dC). After incubation for 10 min at room
temperature, the reactants were loaded onto a 6% polyacrylamide gel
and electrophoresed at room temperature for 90 min at 150 V in a buffer
containing 25 mM Tris·HCl at pH 7.8, 190 mM
glycine, and 1 mM EDTA. Following electrophoresis, the gels
were dried and exposed to Kodak XAR5 film, and binding was analyzed by autoradiography.
Ets Binding Assay--
When the Ets oligonucleotide was used as
the labeled probe, the binding conditions were identical to those
described for AP-1 except that the NaCl concentration was decreased to
50 mM and poly(dI-dC)·poly(dI-dC) was reduced to 0.5 µg. In addition, visualization of specific Ets binding (see Fig.
8B) required preincubation of HeLa nuclear extract with 10 ng of chymotrypsin for 2 min at room temperature prior to addition of
the labeled probe and binding buffer and a further 10 min of incubation
at room temperature. Binding was then analyzed by acrylamide gel
electrophoresis as described above.
Competition Experiments--
For competition experiments (see
Fig. 8), the indicated unlabeled double-stranded oligonucleotides
(100-fold molar excess) were mixed with the labeled oligomer prior to
addition of nuclear extract. Binding was then analyzed by acrylamide
gel electrophoresis as described above.
Gel Supershift--
Gel supershift assays (see Fig. 9) were
carried out by incubating HeLa nuclear extract (3 µg) with the
indicated antisera for 10 min at room temperature, prior to the
addition of the labeled AP-1 oligonucleotide probe and binding buffer
and incubation for an additional 10 min.
Insulin Stimulates Collagenase-CAT Fusion Gene Transcription in
HeLa Cells--
To begin to study the regulation of collagenase gene
transcription by insulin, a collagenase-CAT fusion gene construct,
containing collagenase promoter sequence from Multiple, Distinct cis-Acting Elements Are Required for the Actions
of Insulin and Phorbol Esters on Collagenase Gene
Transcription--
The result shown in Fig. 1 suggests that an IRS is
present in the collagenase promoter between
To delineate the collagenase IRS, a series of 5' deletion mutations of
the collagenase promoter was constructed in the CAT reporter plasmid
pCAT(An) (28), and the effect of insulin on CAT expression directed by
these constructs was analyzed following transient transfection into
HeLa cells. Fig. 4A shows that
insulin-stimulated collagenase-CAT gene transcription was unchanged
when the region of the promoter between
The progressive loss in insulin-stimulated collagenase-CAT gene
expression following deletion of the promoter sequence from The Collagenase Ets and AP-1 Motifs Are Both Required for the
Stimulatory Effects of Insulin and Phorbol Esters on Gene
Transcription--
To directly assess the relative importance of the
collagenase Ets and AP-1 motifs in mediating the induction of gene
transcription by insulin and phorbol esters, these
cis-acting elements were mutated within the context of the
The collagenase Ets and AP-1 motifs, particularly the latter, are also
important for basal collagenase gene transcription (Fig.
5C). Thus, although mutation of the Ets motif reduces basal collagenase-CAT gene expression, mutation of the AP-1 motif reduces basal gene expression to barely detectable levels, despite the presence
of an intact Ets motif (Fig. 5C). In addition, mutation of
the AP-1 motif within the context of the
Mutation of the Ets motif within the context of the The Collagenase AP-1 and Ets Motifs Confer a Stimulatory Effect of
Insulin and Phorbol Esters on the Expression of a Heterologous
Promoter--
The preceding experiments demonstrate that the AP-1
motif is required for the action of both insulin and phorbol esters on collagenase gene transcription and that the Ets motif enhances the
action of both agents, although it is inactive in the absence of the
AP-1 motif. However, these experiments do not prove that the
transcription factors binding the AP-1 and/or Ets motifs are the
targets of insulin and phorbol ester signaling, rather than acting as
accessory factors to enhance the effects of these agents mediated
through an unidentified response sequence. To determine whether the
AP-1 and/or Ets motifs are the targets of insulin and phorbol ester
signaling, multiple copies of an oligonucleotide representing the
collagenase promoter sequence between
We next investigated the ability of multimerized oligonucleotides
(Table I), containing mutations in either the Ets or AP-1 motifs, to
mediate an insulin or phorbol ester response in the context of the XMB
vector (Fig. 6). Mutation of the Ets motif reduced both basal fusion
gene expression (Fig. 6C) and the stimulatory effect of
phorbol esters (Fig. 6B), whereas mutation of the AP-1 motif
abolished the stimulatory effect of phorbol esters (Fig. 6B)
and greatly reduced basal gene expression, despite the presence of an
intact Ets motif (Fig. 6C). The effects of these mutations on both basal fusion gene expression and the response to phorbol esters
mimic the effects of these same mutations when made in the context of
the endogenous collagenase promoter (Fig. 5, B and
C). By contrast, mutation of the Ets motif in the
heterologous context of the XMB vector had only a minor effect on the
ability of insulin to stimulate expression of the fusion gene, whereas the same mutation in the context of the collagenase promoter markedly reduced the insulin response (Fig. 5A). The explanation for
this discrepancy is unclear but may indicate that in this context, the
presence of multiple AP-1 motifs reduces the requirement for the Ets
motif with respect to insulin, although not phorbol ester signaling.
This is consistent with the observation that the mechanisms of insulin
and phorbol esters signaling through the AP-1 motif are distinct (see
below). Mutation of the AP-1 motif in the heterologous context of the
XMB vector abolished the effect of insulin (Fig. 6A), as did
the same mutation in the context of the collagenase promoter (Fig.
5A).
Both Insulin and Phorbol Esters Signal through the Collagenase AP-1
Motif--
The preceding experiments demonstrate that the AP-1 motif
is required for the action of both insulin and phorbol esters on collagenase gene transcription (Fig. 5) as well as for mediating an
effect of these agents on the expression of a heterologous XMB fusion
gene through the Protein Binding to the Collagenase Ets and AP-1 Motifs--
The
specific binding of nuclear extract proteins to the collagenase AP-1
motif was analyzed using the gel retardation assay (Fig.
8). Two broad protein-DNA interactions
were detected when a double-stranded oligonucleotide representing
collagenase promoter sequence from
We also analyzed protein binding to an oligonucleotide representing the
wild-type collagenase Ets motif (Table I) but failed to detect a
protein-DNA interaction, the formation of which was selectively
competed for by an excess of the unlabeled wild-type oligonucleotide
and not by an oligonucleotide containing the same mutated Ets sequence
that abolished the effects of insulin and phorbol esters mediated
through this element in the context of the collagenase promoter (Fig.
8B, left panel). This result contrasts with those of White
et al. (42), who were able to detect specific binding to the
collagenase Ets motif. We would speculate that this discrepancy may
reflect a difference in the amount or nature of the Ets family
transcription factors expressed in HeLa cell nuclear extracts, as used
here, and fibroblast nuclear extracts, as used by White et
al. (42).
A number of investigators have previously shown that DNA binding by Ets
proteins is repressed by an intramolecular mechanism (43-45) that can
be relieved through partial proteolysis (45). To determine whether a
similar phenomenon was preventing the detection of specific protein
binding to the wild-type Ets oligonucleotide, HeLa nuclear extract was
preincubated with 10 ng of chymotrypsin prior to analysis of
protein-DNA binding using the gel retardation assay. Using this
strategy, a protein-DNA interaction was now detected, the formation of
which was selectively competed for by an excess of the unlabeled
wild-type oligonucleotide and not by an oligonucleotide containing the
same mutated Ets sequence that abolished the effects of insulin and
phorbol esters mediated through this element in the context of the
collagenase promoter (Fig. 8B, right panel, arrow).
Additional gel retardation experiments with an oligonucleotide
containing both the collagenase AP-1 and Ets motifs, using HeLa nuclear
extract without chymotrypsin treatment, failed to reveal the presence
of a protein-DNA interaction that might be indicative of the formation
of a ternary complex, although specific binding to the AP-1 motif was
detected (data not shown). Thus, the presence of AP-1 did not appear to
be sufficient to relieve the repression of Ets protein binding.
The Mechanisms of Insulin and Phorbol Ester Signaling through the
Collagenase AP-1 Motif Are Distinct--
The magnitude of insulin- and
phorbol ester-stimulated collagenase-CAT fusion gene transcription are
markedly different, with phorbol ester being the more potent activator
(Figs. 2-5). This difference in potency was maintained when the
effects of insulin and phorbol esters were compared on the expression
of heterologous constructs containing multimerized oligonucleotides
representing either the collagenase promoter sequence between
Because AP-1 is composed of homodimers and heterodimers of various
members of the Fos and Jun transcription factor families (25), we
hypothesized that the difference in potency of insulin and phorbol
ester signaling might be due to differences in the ability of these
factors to selectively induce the binding of specific members of the
Fos/Jun families. To test this hypothesis, nuclear extracts were first
prepared from HeLa cells incubated for 5 h in the presence or
absence of insulin and phorbol esters, and then polyclonal antisera to
specific Fos/Jun family members were assayed for their ability to
supershift the specific protein-DNA complex detected using the
collagenase AP-1 motif in gel retardation assays (Fig.
9). These experiments showed that
although both insulin and phorbol esters induced AP-1 binding activity,
several differences were apparent in the composition of the induced
complex. Most notably, phorbol esters stimulated a marked increase in
c-Fos binding and a slight increase in Fra-1 binding (Fig.
9B), whereas insulin was without effect on these proteins
(Fig. 9A). By contrast, insulin induced the expression of
Fra-2 and Jun D (Fig. 9A), whereas phorbol esters only had a
minor effect on the binding of these factors (Fig. 9B).
Because phosphatase inhibitors were not present during the preparation
of nuclear extracts, it is likely that these changes in AP-1 binding
reflect changes in AP-1 expression rather than phosphorylation.
Although insulin induces c-Fos mRNA in multiple cell types, this
effect is transient (13), which may thus explain the lack of
insulin-stimulated c-Fos binding (Fig. 9A).
This analysis of the cis-acting elements that mediate
the stimulatory effects of insulin and phorbol esters on collagenase gene transcription reveals that multiple elements are required for the
action of both agents. The results obtained with respect to phorbol
ester signaling are similar to those reported by Brinckerhoff and
colleagues (reviews in Refs. 1 and 37) in their studies on the
regulation of the rabbit collagenase We first became interested in the regulation of collagenase gene
transcription by insulin as a result of earlier studies on the
regulation of ME gene transcription (13). Those experiments demonstrated that whereas insulin clearly stimulated ME-CAT gene transcription in H4IIE cells, it had little effect on ME-CAT gene transcription in HeLa cells (13). The action of insulin on ME gene
transcription in H4IIE cells was shown to be mediated, in part, through
an AP-1 motif in the ME promoter (13). Because insulin stimulates
collagenase-CAT gene transcription in HeLa cells (13) (Fig. 1) and
because the stimulatory effect of insulin on collagenase gene
transcription requires an intact AP-1 motif in the collagenase promoter
(12) two questions arose, namely (i) why does insulin stimulate
collagenase-CAT expression, but not ME-CAT expression, in HeLa cells
given that both promoters contain AP-1 motifs and (ii) why does insulin
stimulate ME-CAT gene transcription in H4IIE cells but not in HeLa
cells? We postulated that the latter may reflect an inherent difference
in insulin-signaling through AP-1 motifs in the H4IIE and HeLa cell
lines, whereas the answer to the first question could reflect some
inherent difference between the AP-1 motifs in the ME and collagenase
promoter (13). Alternatively, we proposed that both observations might
be explained by a difference in the promoter context in which the AP-1
motif was located; in other words, the nature of the transcription
factors associated with the promoter other than AP-1 (13). The data presented in this paper demonstrates that the latter explanation is at
least part of the answer. Thus, whereas the AP-1 motif is required, the
marked stimulation of collagenase-CAT gene transcription by insulin in
HeLa cells is mediated through the interaction of multiple collagenase
promoter elements and their associated factors. By analogy, the
presence of an AP-1 motif in the ME promoter would be predicted to be
insufficient by itself for the induction of ME-CAT gene transcription
by insulin in HeLa cells. The question of why insulin stimulates ME-CAT
gene transcription in H4IIE cells but not in HeLa cells remains to be
answered. We have previously shown that phorbol esters also fail to
induce ME-CAT fusion gene expression in HeLa cells and that there is an
inherent difference in the ability of the collagenase and ME AP-1
motifs to mediate a phorbol ester response in a heterologous context
(13). However, it is apparent that the marked stimulation of
collagenase-CAT gene expression by phorbol esters is also dependent on
multiple elements in addition to the AP-1 motif (Figs. 4 and 5).
Based on the data presented in Figs. 5 and 6, we propose that the
transcription factor binding the collagenase Ets motif acts as
accessory factor to enhance the effect of insulin mediated through the
AP-1 motif. Such an arrangement would be similar to that found in the
glucose-6-phosphatase promoter in which HNF-1 Although the multimerized collagenase Ets motif fails to mediate an
insulin response when ligated to the heterologous XMB vector (Fig. 7),
the possibility that insulin directly signals through the Ets binding
factor as well as through AP-1 cannot be entirely ruled out. Thus,
Blackshear and colleagues (22) have demonstrated that at least in some
cell lines, the stimulatory effect of insulin on c-fos gene
transcription is mediated by MAP kinase through the phosphorylation of
the Ets motif-binding protein, p62TCF. p62TCF
only binds the c-fos serum response element as a ternary
complex with p67SRF (23). By analogy, the possibility
therefore exists that insulin does directly signal through the
collagenase Ets motif-binding protein, but that this cannot be
demonstrated in a heterologous context because AP-1 is required to
stabilize binding of the Ets factor (22). MAP kinase also
phosphorylates the Ets protein GABP In summary, the experiments described in this report demonstrate that
multiple promoter elements are required for the stimulatory effect of
insulin on collagenase gene transcription in HeLa cells. The AP-1 motif
in the collagenase promoter appears to be the target of both insulin
and phorbol ester signaling; however, the mechanisms of insulin and
phorbol ester action are distinct. Future studies will examine whether
insulin also regulates collagenase gene expression in glomerula-derived
cell lines. Although insulin receptor levels are low in the mesangial
cells of the glomerulus (55), they increase in the diabetic state (56).
Furthermore, it is interesting to note that in the streptozotocin rat
model of type I diabetes, collagenase gene expression in the glomerulus
decreases (10). This may indicate that the regulation of collagenase
gene expression in this tissue by phorbol esters differs from that
reported in HeLa cells because the hyperglycemia in these animals has
been postulated to lead to activation of protein kinase C (57), which would be anticipated to lead to an increase, rather than a decrease, in
collagenase gene expression.
We thank Rob Hall for useful suggestions
during the course of this project and for insightful comments on the
manuscript. We thank John Hassell and Howard Crawford for assistance
with the analysis of Ets-binding proteins and Howard Towle and Jonathan Whittaker for the pCAT(An) and insulin receptor expression vector plasmids, respectively. We also thank Julia Breyer, Ray Harris, and R. Brooks Robey for interesting discussions on the potential role of
decreased collagenase-1 gene expression in the pathophysiology of
glomerulosclerosis. HeLa and hamster insulinoma tumor cells were kindly
provided by Roland Stein and Eva Henderson. Data analysis was performed
in part through the use of the Vanderbilt University Medical Center
Cell Imaging Resource (CA68485 and DK20593).
*
This research was supported by a grant from the Mark Collie
Foundation and Grant RO1 DK52820 from the National Institutes of Health
(to R. M. O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
A British Diabetic Association Senior Research Fellow.
The abbreviations used are:
MMP, matrix
metalloproteinase;
PEPCK, phosphoenolpyruvate carboxykinase;
PMA, phorbol 12-myristate 13-acetate;
CAT, chloramphenicol
acetyltransferase;
DMEM, Dulbecco's modified Eagle's medium;
ME, malic enzyme;
IRS, insulin response sequence.
Multiple Promoter Elements Are Required for the Stimulatory
Effect of Insulin on Human Collagenase-1 Gene Transcription
SELECTIVE EFFECTS ON ACTIVATOR PROTEIN-1 EXPRESSION MAY EXPLAIN
THE QUANTITATIVE DIFFERENCE IN INSULIN AND PHORBOL ESTER ACTION*
,,,,,,
Department of Molecular Physiology and
Biophysics, Vanderbilt University Medical School,
Nashville, Tennessee 37232 and the § Department of
Biochemistry, School of Medical Sciences, University of Bristol,
Bristol BS8 1TD, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP (>3000 Ci·mmol
1)
and [3H] acetic acid, sodium salt (>10
Ci·mmol1) were purchased from Amersham Pharmacia
Biotech and ICN, respectively. Insulin was obtained from Collaborative
Bioproducts, and phorbol 12-myristate 13-acetate (PMA) and
-chymotrypsin were from Sigma. Specific antisera to c-Fos (sc-52),
Fra-1 (sc-183), Fra-2 (sc-604), c-Jun (sc-45), Jun B (sc-46), and Jun D
(sc-74) were all obtained from Santa Cruz Biotechnology, Inc.
518 to +64, was
isolated from the plasmid pCol·Luc (12) and ligated, in the same
orientation as that of the endogenous gene, into the
BglII-digested polylinker of the pCAT(An) expression vector,
a generous gift from Dr. Howard Towle (28). The noncompatible 3'
HindIII-BglII junction was filled in using the
Klenow fragment of Escherichia coli DNA polymerase I prior
to blunt-end ligation. The pCAT(An) vector has polyadenylation signals
located 5' of the polylinker to prevent read-through transcription (28). Control experiments demonstrated that there was no basal CAT
expression and no effect of insulin or PMA when the pCAT(An) vector,
minus the collagenase promoter, was transiently transfected into HeLa
cells (data not shown). A series of truncated collagenase-CAT fusion
genes was then generated, with the 5' end points shown in Fig. 4, using
the 518/+64 pCAT(An) construct as a template, by either restriction
enzyme digestion or polymerase chain reaction, using standard
techniques (27). The 3' polymerase chain reaction primer was designed
to conserve the junction between the collagenase promoter and CAT
reporter gene to be the same as that in all other collagenase-CAT
fusion gene constructs. All promoter fragments generated by polymerase
chain reaction were completely sequenced to ensure the absence of
polymerase errors, whereas promoter fragments generated by restriction
enzyme digestion were only sequenced to confirm the 5' end points.
Site-directed mutants of the collagenase Ets and AP-1 motifs in the
context of the 97 to +64 promoter fragment, as shown in Fig. 5, were
generated by polymerase chain reaction in conjunction with 5' primers
that incorporated the mutated sequence (Table I).
-galactosidase (2.5 µg), and an expression vector encoding the
insulin receptor (5 µg), courtesy of Dr. Jonathan Whittaker, to the
10 ml of culture medium. After an overnight incubation the medium was
removed and the cells incubated in 10 ml PBS for 10 min at room
temperature. The cells were then incubated for a further 8-24 h in 10 ml serum-free DMEM supplemented with or without various concentrations
of PMA or insulin, as indicated in the Figure legends, prior to harvesting.
-galactosidase (2.5 µg) and the insulin receptor (5 µg) to the
10 ml of culture medium. After incubation for between 4 and 6 h,
the cells were treated for 2 min with 20% glycerol in serum-free DMEM
(5 ml/dish). The cells were then rinsed for 5 min with serum-free DMEM
(5 ml/dish) prior to incubation for 24 h in serum-free DMEM (10 ml/dish) supplemented with or without various concentrations of
insulin, as indicated in the Figure legends.
-Galactosidase, and Luciferase Assays
-galactosidase, and luciferase assays were performed exactly as
described previously (20, 30, 32). To compare the relative basal CAT
expression obtained with the various reporter gene constructs
described, CAT activity in samples from control cells was corrected for
the -galactosidase activity in the same sample. Because phorbol
esters and insulin affect Rous sarcoma virus- galactosidase
expression in HeLa cells (data not shown), CAT activity in these
samples was corrected for the protein concentration in the cell lysate, as measured by the Pierce BCA assay, and each plasmid construct was
analyzed in duplicate in multiple transfections, as specified in the
Figure legends.
-32P]dATP using the Klenow fragment of
Escherichia coli DNA polymerase I to a specific activity of
approximately 2.5 µCi/pmol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
518 to +64, relative to
the transcription start site at +1 (33), was transiently transfected into HeLa cells (Fig. 1). An effect of
insulin on reporter gene expression was only detected when the
collagenase-CAT fusion gene was co-transfected with an expression
vector encoding the insulin receptor (Fig. 1A). In the
absence of insulin, co-transfection with the insulin receptor alone was
insufficient to activate collagenase-CAT fusion gene expression,
suggesting a low level of signaling through the basal receptor (Fig.
1B). Stanley (34) has previously shown that co-transfection
with an insulin receptor expression vector is also required to observe
an effect of insulin on the expression of a transiently transfected
prolactin-CAT fusion gene in rat pituitary tumor GH4 cells.
Interestingly, insulin stimulates the expression of the endogenous
prolactin gene in these cells in the absence of receptor
co-transfection (34). The maximal effect of insulin on collagenase-CAT
gene expression was seen at 100 nM (Fig.
2A). This concentration of
insulin is 10-fold higher than that required to see a maximal
repression of glucocorticoid-stimulated PEPCK-CAT fusion gene
expression in rat H4IIE hepatoma cells (35, 36). This requirement for
100 nM insulin to manifest the maximal effect of the
hormone is not explained by the degradation of insulin by HeLa cells
because a similar EC50 for this effect is obtained following an 8 or 24 h incubation with insulin (Fig.
2B). In addition, the maximal effect of insulin on
collagenase-CAT gene expression was also seen at 100 nM in
hamster insulinoma tumor cells (Fig. 2C), H4IIE cells (Fig.
2D), and Chinese hamster ovary-T cells (data not shown).
Although cell line-dependent variations in insulin sensitivity could arise due to differences in the expression of molecules in the insulin signaling pathway, our results suggest that
different signaling pathways are used by insulin to regulate PEPCK and
collagenase gene transcription.
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Fig. 1.
Insulin stimulates collagenase-CAT fusion
gene transcription in HeLa cells. HeLa cells were transiently
co-transfected, as described under "Experimental Procedures," with
a collagenase-CAT fusion gene (15 µg) containing promoter sequence
from 518 to +64, an expression vector encoding -galactosidase (2.5 µg), and either with (+IREV) or without
(IREV) an expression vector encoding the insulin receptor
(5 µg). Following transfection, cells were incubated for 24 h in
serum-free medium in the presence or absence of 10 nM
insulin. The cells were then harvested, and both CAT and
-galactosidase activity were assayed as described previously (20,
30). In A, results are presented as the ratio of CAT
activity, corrected for protein concentration in the cell lysate, in
insulin-treated versus control cells and are expressed as
fold induction. In B, results are presented as the ratio of
CAT to -galactosidase activity in control cells and are expressed as
arbitrary units. Results represent the mean of ± S.E. of three
experiments, in which each condition was assayed in duplicate.
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Fig. 2.
Concentration dependence of
insulin-stimulated collagenase-CAT fusion gene transcription. HeLa
and hamster insulinoma tumor (HIT) cells were transiently
co-transfected using calcium phosphate co-precipitation, as described
under "Experimental Procedures," with a collagenase-CAT fusion gene
(15 µg), containing promoter sequence from 518 to +64, and
expression vectors encoding -galactosidase (2.5 µg) and the
insulin receptor (5 µg). H4IIE cells were transiently transfected
using the Tfx-50 transfection reagent, as described under
"Experimental Procedures," with a collagenase-luciferase fusion
gene (2 µg), containing promoter sequence from 518 to +64.
Following transfection, cells were incubated for either 24 h
(A, C, and D) or 8 h (B) in
serum-free medium in the presence or absence of various concentrations
of insulin. The cells were then harvested and CAT or luciferase
activity was assayed as described previously (20, 30, 32). Results are
presented as the ratio of CAT or luciferase activity (corrected for
protein concentration in the cell lysate in A-C although
not in D) in insulin-treated versus control cells
and are expressed as fold induction. Results represent the mean of ± S.E. of three to eight experiments, in which each condition was
assayed in duplicate.
518 and +64. Because the
cis-acting elements that mediate the stimulatory effect of
phorbol esters on collagenase gene transcription have been studied in
detail (1, 2, 33, 37), and given the long running controversy concerning the potential role of protein kinase C in insulin action (38), it was of interest to determine whether insulin and phorbol esters both mediate their stimulatory action on collagenase gene transcription through the same elements. The phorbol ester PMA has a
biphasic effect on collagenase-CAT gene transcription, with a
maximal stimulation of ~150-fold seen at 10 nM (Fig.
3). The reduction in PMA-stimulated CAT
expression at higher PMA concentrations probably reflects the
down-regulation of protein kinase C (39). By contrast, the maximal
stimulation obtained with insulin was ~30-fold (Fig. 2A),
and the stimulation of collagenase-CAT fusion gene expression by
insulin and PMA was not additive (data not shown).
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Fig. 3.
Concentration dependence of PMA-stimulated
collagenase-CAT fusion gene transcription in HeLa cells. HeLa
cells were transiently co-transfected, as described under
"Experimental Procedures," with a collagenase-CAT fusion gene (15 µg), containing promoter sequence from 518 to +64, and expression
vectors encoding -galactosidase (2.5 µg) and the insulin receptor
(5 µg). The insulin receptor expression vector was included to be
consistent with all other transfection experiments. Following
transfection, cells were incubated for 24 h in serum-free medium
in the presence or absence of various concentrations of PMA. The cells
were then harvested, and CAT activity was assayed as described
previously (20, 30). Results are presented as the ratio of CAT
activity, corrected for protein concentration in the cell lysate, in
PMA-treated versus control cells and are expressed as fold
induction. Results represent the mean of ± S.E. of four
experiments, in which each condition was assayed in duplicate.
518 and 159 was deleted. By
contrast, the effect of PMA was reduced more than 50% (Fig.
4B), indicating that these two agents do not have an
identical mechanism of action on collagenase gene transcription.
Further deletion of the collagenase promoter sequence between 158 and
124 resulted in a slight decrease in insulin-stimulated
collagenase-CAT gene transcription (Fig. 4A) and a slight
increase in phorbol ester-stimulated collagenase-CAT gene transcription
(Fig. 4B), as previously reported in a study by Auble and
Brinckerhoff (40) on the rabbit collagenase-1 promoter in fibroblasts.
Two additional collagenase-CAT fusion genes, in which the collagenase
promoter sequence from 123 to 98 and then from 97 to 80 were
deleted, both directed decreased phorbol ester- and insulin-stimulated
CAT expression (Fig. 4, A and B).
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Fig. 4.
Progressive deletion of the collagenase
promoter sequence between 518 and 80 reduces both basal
collagenase-CAT fusion gene transcription and the stimulatory effects
of insulin and PMA. HeLa cells were transiently co-transfected, as
described under "Experimental Procedures," with a series of
collagenase-CAT fusion genes, with 5' deletion end points as shown on
the abscissa, and expression vectors encoding
-galactosidase and the insulin receptor. Following transfection,
cells were incubated for 24 h in serum-free medium in the presence
or absence of 10 nM insulin or 100 nM PMA. The
cells were then harvested, and both CAT and -galactosidase activity
were assayed as described previously (20, 30). Results are presented as
the ratio of CAT activity, corrected for protein concentration in the
cell lysate, in insulin-treated (A) or PMA-treated
(B) versus control cells, and are expressed as
fold induction. In C, results are presented as the ratio of
CAT to -galactosidase activity in control cells and are expressed as
arbitrary units. Results represent the mean of ± S.E. of 4-13
experiments, in which each construct was assayed in duplicate.
158 to
80 is indicative of the presence of either multiple insulin response
sequences in this region or multiple binding sites for accessory
factors that enhance the action of a more proximal IRS. These same
deletions result in decreased basal collagenase-CAT expression (Fig.
4C). Whether the transcription factors that confer high
basal collagenase gene expression through the 158 to 98 promoter
region are the same as those that mediate/enhance the insulin effect
remains to be determined. Instead, we decided to focus this study on
the more proximal portion of the promoter, between 97 and +64,
because this region contains the Ets and AP-1 motifs that have
previously been shown to be important in mediating the induction of
collagenase gene transcription by phorbol esters (1, 2, 24, 37, 41).
Moreover, this proximal region of the promoter confers a response to
both insulin and phorbol esters that is 50% of that seen with the
full-length promoter (Fig. 4, A and B).
97 to +64 promoter fragment (Fig. 5).
Mutation of the Ets motif reduced the stimulatory effects of both
insulin and phorbol esters on collagenase-CAT fusion gene transcription, whereas mutation of the AP-1 motif abolished the response to both agents (Fig. 5, A and B). Thus,
an intact AP-1 motif is essential for the induction of collagenase-CAT
gene expression by insulin and phorbol esters, whereas the Ets motif is
not.
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Fig. 5.
Site-directed mutation of the Ets and AP-1
motifs in the collagenase promoter reduces both basal collagenase-CAT
fusion gene transcription and the stimulatory effects of insulin and
PMA. HeLa cells were transiently co-transfected, as described
under "Experimental Procedures," with a series of collagenase-CAT
fusion genes, with 5' deletion end points and mutations as shown on the
abscissa, and expression vectors encoding -galactosidase
and the insulin receptor. Following transfection, cells were incubated
for 24 h in serum-free medium in the presence or absence of 10 nM insulin or 100 nM PMA. The cells were then
harvested, and both CAT and -galactosidase activity were assayed as
described previously (20, 30). Results are presented as the ratio of
CAT activity, corrected for protein concentration in the cell lysate,
in insulin-treated (A) or PMA-treated (B)
versus control cells and are expressed as fold induction. In
C, results are presented as the ratio of CAT to
-galactosidase activity in control cells and are expressed as
arbitrary units. Results represent the mean of ± S.E. of 4-13
(insulin) or 3 (PMA) experiments, in which each construct was assayed
in duplicate.
79 to +64 promoter fragment
reduces basal fusion gene expression to the same level as that seen
when the AP-1 motif is mutated in the context of the 97 to +64
promoter fragment, despite the presence of the intact Ets motif (Fig.
5C).
97 to +64
promoter fragment has the same quantitative effect, on both basal
fusion gene expression and the response to insulin, as deletion of the
97 to 80 sequence (Fig. 5, A and C). By
contrast, deletion of the 97 to 80 sequence is more deleterious to
the phorbol ester response than a mutation of the Ets motif in the
context of the 97 to +64 promoter fragment (Fig. 5B). This
suggests that the transcription factor(s) binding the Ets motif
mediates both basal gene expression and the response to insulin
conferred through this region but that an additional element is
important for the action of phorbol esters. Finally, mutation of the
AP-1 motif in the context of the 79 to +64 promoter fragment almost
completely abolishes the small effect of insulin and phorbol esters on
the expression of this truncated fusion gene, just as it does in the context of the 97 to +64 promoter fragment (Fig. 5, A and
B).
97 and 64, which contains
both the Ets and AP-1 motifs (Table
I), were ligated into
the polylinker of a heterologous vector, designated XMB, which contains
a minimal Xenopus 68-kDa albumin promoter (29) ligated to
the CAT reporter gene, and the resulting construct was transiently
transfected into HeLa cells (Fig. 6).
Neither basal CAT expression nor insulin/PMA-induced CAT expression
were detected with the basic XMB vector (Fig. 6) or with a fusion gene construct in which a single copy of the 97/64 oligonucleotide had
been ligated into the polylinker of the XMB vector (data not shown). By
contrast, the multimerized 97/64 oligonucleotide was able to
mediate a stimulatory effect of both insulin and phorbol esters on
fusion gene expression, although the effect of insulin was small
relative to that of phorbol esters (Fig. 6).
Sequence of oligonucleotides used in these studies
97 and 64 was
synthesized with either BamHI (GATC)- or HindIII
(AGCT)-compatible ends. Both the BamHI- and
HindIII-compatible ends contribute to the collagenase
sequence such that the oligonucleotide with BamHI ends
actually represents the collagenase sequence between 98 and 64,
whereas the oligonucleotide with HindIII ends actually
represents the collagenase sequence between 97 and
61.
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Fig. 6.
The collagenase promoter sequence between
97 and 64 can confer a stimulatory effect of both phorbol esters
and insulin on the expression of a heterologous
Xenopus-CAT fusion gene. HeLa cells were
transiently co-transfected, as described under "Experimental
Procedures," with expression vectors encoding -galactosidase and
the insulin receptor and the basic XMB vector or constructs in which
oligonucleotides representing the wild-type (WT) or
individually mutated (MUT) Ets (E) and AP-1
(A) motifs in the collagenase promoter sequence from 97 to
64 had been ligated into the HindIII site of the
Xenopus promoter in multiple (4-5) copies. Following
transfection, cells were incubated for 24 h in serum-free medium
in the presence or absence of 10 nM insulin or 100 nM PMA. The cells were then harvested, and both CAT and
-galactosidase activity were assayed as described previously (20,
30). Results are presented as the ratio of CAT activity, corrected for
protein concentration in the cell lysate, in insulin-treated
(A) or PMA-treated (B) versus control
cells, and are expressed as fold induction. In C, results
are presented as the ratio of CAT to -galactosidase activity in
control cells and are expressed as arbitrary units. Results represent
the mean of ± S.E. of three experiments, in which each construct
was assayed in duplicate.
97/64 collagenase sequence (Fig. 6). In the
collagenase promoter the Ets motif enhances the action of both agents,
although it is inactive in the absence of the AP-1 motif (Fig. 5).
However, it is unclear from these experiments whether the Ets
motif-binding protein can also directly mediate an insulin/phorbol
ester response or whether it just acts as an accessory factor to
enhance the effects of these agents mediated through the AP-1 motif. To
address this question, oligonucleotides representing just the
individual collagenase Ets or AP-1 motifs (Table I) were ligated into
the polylinker of the XMB vector (Fig.
7). Neither basal CAT expression nor
insulin/PMA-induced CAT expression were detected with fusion gene
constructs containing single copies of the Ets and AP-1
oligonucleotides (data not shown). However, the multimerized AP-1 motif
was able to mediate an induction of fusion gene expression by both
insulin and phorbol esters, whereas the multimerized Ets motif was
unable to confer a response to either agent (Fig. 7). Mutation of the
AP-1 motif in this context abolished the induction of XMB fusion gene
expression (Fig. 7), just as it did in the context of the
collagenase-CAT fusion gene (Fig. 5).
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Fig. 7.
The collagenase AP-1 motif, in contrast to
the Ets motif, can confer a stimulatory effect of both phorbol esters
and insulin on the expression of a heterologous
Xenopus-CAT fusion gene. HeLa cells were
transiently co-transfected, as described under "Experimental
Procedures," with expression vectors encoding -galactosidase and
the insulin receptor and XMB vector constructs in which
oligonucleotides representing the wild-type collagenase Ets motif
(E(WT)) or the wild-type or mutated collagenase AP-1 motif
(A(WT) and A(MUT), respectively) had been ligated
into the HindIII site of the Xenopus promoter in
multiple (3-5) copies. Following transfection, cells were incubated
for 24 h in serum-free medium in the presence or absence of 10 nM insulin or 100 nM PMA. The cells were then
harvested and CAT activity was assayed as described previously (20,
30). Results are presented as the ratio of CAT activity, corrected for
protein concentration in the cell lysate, in insulin-treated
(A) or PMA-treated (B) versus control
cells, and are expressed as fold induction. Results represent the mean
of ± S.E. of three or four experiments, in which each construct
was assayed in duplicate.
78 to 63 (Table I), which
contains the AP-1 motif, was used as the labeled probe (Fig.
8A). A 100-fold molar excess of the unlabeled 78/63
oligonucleotide competed effectively against the labeled probe for
binding of the upper complex, indicating that this protein-DNA complex
represents a specific interaction (Fig. 8A). By contrast, an
oligonucleotide representing the same 78 to 63 collagenase
sequence, but with a mutation in the AP-1 core sequence (Table I) that
abolishes insulin and phorbol ester signaling through this element
(Fig. 7), failed to compete for protein binding with the labeled probe,
even in a 100-fold molar excess (Fig. 8A). Thus, the
formation of this specific protein-DNA complex correlates with the
effect of insulin and phorbol ester mediated through this sequence in
the context of the collagenase promoter and the heterologous XMB
vector. This specific protein-DNA complex detected in the gel
retardation assay contains members of the AP-1 transcription factor
family (Ref. 13 and see below).
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Fig. 8.
Specific protein-DNA complexes are detected
in gel retardation assays using the collagenase AP-1 and Ets motifs as
the labeled probes. Protein binding to labeled oligonucleotide
probes, representing either the collagenase AP-1 motif sequence between
78 and 63 (A) or the Ets motif sequence between 97 and
76 (B), was analyzed using HeLa cell nuclear extracts and
the gel retardation assay, as described under "Experimental
Procedures." The labeled probes were incubated in the absence () or
presence of a 100-fold molar excess of unlabeled oligonucleotides
representing either the wild-type (WT) or mutant
(MUT) collagenase 78/63 AP-1 sequence (A) or
97/76 Ets sequence (B) prior to the addition of nuclear
extract. In B, nuclear extract was pretreated with or
without 10 ng of chymotrypsin. Representative autoradiographs are
shown. In A, the AP-1 complex and a nonspecific
(NS) protein-DNA interaction are indicated by the
arrows, whereas in B, the specific protein-DNA
interaction revealed by chymotrypsin treatment is indicated by the
arrow. FP, free probe.
97 and
64, which contains both the Ets and AP-1 motifs (Fig. 6), or
collagenase promoter sequence between 78 to 63, which contains just
the AP-1 motif (Fig. 7). This suggests that the difference in potency of insulin and phorbol esters on collagenase gene transcription can be
at least partly ascribed to a difference in signaling through the AP-1 motif.
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Fig. 9.
Insulin and phorbol esters stimulate
selective protein binding to the collagenase AP-1 motif.
A, HeLa cells were transiently transfected, as described
under "Experimental Procedures," with an expression vector encoding
the insulin receptor (5 µg). Nuclear extracts were then prepared from
HeLa cells incubated for 5 h in serum-free medium (C)
or serum-free medium supplemented with 100 nM insulin
(I). B, nuclear extracts were prepared from
nontransfected HeLa cells incubated for 5 h in serum-free medium
(C) or serum-free medium supplemented with 1 µM PMA (P). Nuclear extract was preincubated
in the absence of antibody (None) or in the presence of 1 µg of the indicated antisera for 10 min at room temperature, prior to
the addition of a labeled oligonucleotide probe, representing the
collagenase AP-1 motif sequence between 78 and 63, and binding
buffer and incubation for an additional 10 min at room temperature.
Protein binding was then analyzed using the gel retardation assay as
described under "Experimental Procedures." In the representative
autoradiograph shown, only the retarded complexes are visible and not
the free probe, which was present in excess. The AP-1 complex and a
nonspecific (NS) protein-DNA interaction are indicated by
the arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 gene in fibroblasts. Although
several of these elements are required for the effects of both insulin
and phorbol esters, it is apparent that there are also unique features
to their mechanisms of action. Thus, phorbol ester-stimulated
collagenase-CAT gene transcription was reduced when the region of the
promoter between 518 to 159 was deleted, whereas the effect of
insulin was unchanged (Fig. 4). In addition, deletion of the 97 to
80 sequence was more deleterious to the phorbol ester response than a
site-directed mutation of the Ets motif in the context of the 97 to
+64 promoter fragment, whereas the insulin response was equally reduced
by both manipulations (Fig. 5). Perhaps the most striking difference,
though, was the observation that the mechanisms of insulin and phorbol
ester signaling through the AP-1 motif appear to be distinct (Fig. 9).
Thus, these agents induce selective increases in the binding of
specific members of the Fos/Jun transcription factor families; most
notably, phorbol esters stimulated a marked increase in c-Fos binding
(Fig. 9B), whereas insulin was without effect (Fig.
9A). We hypothesize that these selective effects on AP-1
binding may explain the greater potency of phorbol esters in
stimulating collagenase gene transcription, although the existence of
additional differences in insulin- and phorbol ester-stimulated changes
in AP-1 phosphorylation cannot be excluded.
acts as an accessory
factor to enhance the effect of insulin mediated through a more
proximal IRS (46). Interestingly, Ets and AP-1 motifs are found
juxtaposed in several promoters, including those encoding collagenase
(41, 47), matrilysin (48), and tissue inhibitor of MMP-1 (49) genes and
the polyoma virus enhancer (50). In these genes, the Ets and AP-1
motifs mediate a synergistic activation of transcription in response to
various agonists (for review, see Ref. 2). In addition, it has been
shown that Ets proteins are capable of directly interacting with
components of the AP-1 complex in vitro (49, 51, 52) and
in vivo (51, 52), thus providing a molecular model to
explain the ability of Ets motif-binding proteins to enhance signaling
through AP-1 motifs.
, which mediates the stimulatory
effect of insulin on prolactin gene transcription (53), although the
significance of this phosphorylation is unclear because recent data has
shown that PD98059 does not block this effect of insulin (54).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Molecular Physiology and Biophysics, 761 MRB II, Vanderbilt
University Medical School, Nashville, TN 37232-0615. Tel.:
615-936-1503; Fax: 615-322-7236.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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