1 Division of Endocrinology, Diabetes, and Metabolism, Washington University School Of Medicine, St. Louis, Missouri 63110; and 2 Department of Cell and Developmental Biology, Oregon Health Services University, Portland, Oregon 97201
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ABSTRACT |
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Elk-1, a member of the ternary
complex factor family of Ets domain proteins that bind serum response
elements, is activated by phosphorylation in a cell-specific manner in
response to growth factors and other agents. The purpose of the current
study was to determine whether Elk-1 activation contributes to
glucose-/depolarization-induced Ca2+-dependent induction of
immediate early response genes in pancreatic islet -cells. The
results of experiments in insulinoma (MIN6) cells demonstrated that
Elk-1-binding sites (Ets elements) in the Egr-1 gene promoter
contribute to transcriptional activation of the gene. Treatment with
either epidermal growth factor (EGF), a known inducer of
-cell
hyperplasia, glucose, or KCl-induced depolarization resulted in
Ser383 phosphorylation and transcriptional activation of
Elk-1 (4 ± 0.3-, P = 0.003, 2.3 ± 0.19-, P = 0.002, and 2.2 ± 0.1- fold, P = 0.001 respectively). The depolarization response was inhibited by the
Ca2+ channel blocker verapamil and by the MEK inhibitor
PD98059 (53 ± 6 and 55 ± 0.5%, respectively). EGF-induced
activation of Elk-1 was also inhibited by PD98059 (60 ± 5%). A
dominant negative Ras produced partial inhibition (42%) of the
depolarization-induced Elk-1 transcriptional activation. Transfection
with a constitutively active Ca2+/calmodulin kinase IV
plasmid also resulted in Elk-1 transcriptional activation. Experiments
with p38, phosphatidylinositol 3-kinase, and protein kinase A
inhibitors indicated that these pathways are not involved. We conclude
that Elk-1 activation contributes to glucose-/depolarization-induced
Ca2+-dependent induction of immediate early growth response
genes in pancreatic islet
-cells. Furthermore, the results
demonstrated a convergence of nutrient- and growth factor-mediated
signaling pathways on Elk-1 activation through induction of
Ras/mitogen-activated protein kinase ERK-1 and -2. The role of these
pathways in the glucose-induced proliferation of islet
-cells can
now be assessed.
depolarization; growth factors; Egr-1; epidermal growth factor
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INTRODUCTION |
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RECENT
SUCCESS with human islet transplantation in diabetic patients
underscores the need for adequate numbers of islet -cells for this
therapy. A compensatory increase in pancreatic islet
-cell mass
occurs with insulin resistance states, yet the mechanisms involved in
this response are little understood (14). Plasma glucose
appears to be a major component in this response, as prolonged hyperglycemia in experimental animal models leads to increase in
-cell mass (4). Glucose induces pleiotropic effects in islet
-cells that are depolarization and Ca2+ dependent,
and these effects are potentially mediated by activation of multiple
intracellular signaling pathways. Depolarization-induced Ca2+ influx has been implicated in the activation of
Ca2+/calmodulin (CaM) kinases II (CaMKII) and IV
(CaMKIV), protein kinases A (PKA) and C (PKC), extracellular
signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)
(MEK) (16, 32), and phosphatidylinositol 3-kinase (PI
3-kinase) in islet
-cells (16, 23, 27, 32). For
glucose-induced depolarization and insulin-like growth factor (IGF)
treatment of insulinoma cells, studies with pharmacological inhibitors
have implicated the MAPK/ERK and PI 3-kinase pathways in proliferative
responses (19). The relationships between these
activated pathways, their downstream targets, and glucose-regulated
growth responses have not been defined (35).
A prominent component of the cellular response to mitogenic stimuli is
the rapid and transient transcriptional activation of a group of genes
termed immediate early genes (IEGs) (38), most notably
c-fos and Egr-1 (17). This rapid response of
IEG transcription to mitogenic stimuli suggests that the products of
these genes likely have a regulatory role in the cellular responses to
growth factors (10). Like growth factors, glucose, peptide hormones (glucoincretins), and other nutrients have been shown to
activate transcription of several IEGs including Egr-1 in insulinoma cell lines and pancreatic islets (22, 39). Egr-1 is a
zinc-finger DNA-binding transcription factor that is expressed in
multiple tissues and is induced by diverse stimuli, of which the best
studied examples have been the treatment with peptide mitogens
(7, 22, 25, 30). Recently, in an effort to define the
signal transduction pathways involved in glucose regulation of Egr-1 transcription, we found that glucose-induced -cell depolarization triggers a cascade of events in which Ca2+ influx leads to
the activation of PKA and CaM pathways (3). The initiation
of these signaling pathways leads to activation of cAMP response
element (CRE)-binding protein (CREB) and serum response factor (SRF),
transcription factors involved in growth responses. The Egr-1 induction
after depolarization was shown to result predominantly through serum
response element (SRE)-dependent transcription. These studies defined a
role of SRF activation as a mechanism for the glucose effect on
pancreatic
-cell gene expression.
SRF is a transcription factor early defined as being one of many that mediate mitogenic responses and regulate fibroblast proliferation in response to growth factors (42). Activated SRF by phosphorylation cascades induces transcription by binding to SREs and by recruiting ternary complex factors (TCFs). TCFs are members of the Ets family of transcription factors that are involved in the formation of the ternary complex with the SRF and SRE. Elk-1 is the most prominent member of the TCF family of transcription factors (33, 41). Whether Elk-1 activation contributes to SRE-dependent transcriptional responses appears to depend on the cell type and the stimulus (15).
In the present studies, we found that glucose-induced depolarization of
pancreatic islet -cells triggers a cascade of events in which
Ca2+ influx leads to Elk-1 activation through the
Ras/MEK/ERK pathway. In addition, activation of the CaMKIV pathway also
results in Elk-1-dependent transcription by an ERK-dependent mechanism.
Epidermal growth factor (EGF) treatment, like depolarization, also
results in Elk-1-dependent transcriptional activation by a similar
pathway. These studies demonstrate convergence of depolarization- and
growth factor-activated mitogenic responses on Elk-1 transcriptional activation in islet
-cells.
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EXPERIMENTAL PROCEDURES |
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Chemicals. Phorbol 12-myristate 13-acetate, KCl, verapamil, EGF, growth hormone, prolactin, and IGF-II were obtained from Sigma (St. Louis, MO). PD98059, H89, SB203580, and wortmannin were obtained from Biomol (Plymouth Meeting, PA).
Cell culture conditions. The MIN6 insulinoma cell line was obtained from Y. Oka (Yamaguchi University, Yamaguchi, Japan) (20) and was maintained in Dulbecco's modified Eagle's medium (DMEM) as previously described (3). All of the experiments were performed in cells between passages 20 and 30.
Plasmids.
Plasmids containing the murine Egr-1 promoter linked to the luciferase
vector pXP2 constructs have been described in detail previously
(9). The cis-reporter plasmid 5XSRF+Ets-Luc
contains the luciferase reporter gene driven by a basic promoter
element (TATA box) joined to five tandem repeats of the
c-fos SRE containing the SRF and Ets-binding elements
(Stratagene). The cis-reporter plasmid 5XSRF-Luc contains
the luciferase reporter gene driven by a basic promoter element (TATA
box) joined to five tandem repeats of the c-fos SRF
and lacking the Ets-binding elements (Stratagene). The Elk-GAL4
luciferase system includes a trans-activator plasmid that
expresses a fusion protein containing the activator domain of Elk-1
fused to the DNA-binding domain of GAL4 (residues 1-147; Stratagene) and was cotransfected with a reporter gene containing a
synthetic promoter with five tandem repeats of the yeast GAL4-binding sites that control expression of the luciferase gene pRC-Luc
(Stratagene). The constitutively active forms of CaMKIV (CaMKIVCT) and
CaMKII (CaMKIIBC) and the dominant negative forms of CaMKIV
(CaMKIVK75E) and CaMKII (CaMKII
BI) were gifts from Dr. T. Chatila
(Washington University, St. Louis, MO) and have been described
previously (31). The plasmid containing the catalytic unit
of PKA (pFC-PKA) was purchased from Stratagene. The plasmids pZIP-ras
Ras wild-type (WT) and pZIP-ras 15A (Ras 15A) and the vector pZIP-NeoSV
were provided by Dr. C. Der (University of North Carolina) (6,
11). The plasmids encoding the MAP kinase phosphatase 1 (MKP-1)
and Elk-1 (CMV-Elk-1) were kindly provided by Dr. J. Pessin (University of Iowa) and Dr. J. Schwartz (University of Michigan)
(26). The pRL-TK control vector contains the herpes
simplex virus thymidine kinase promoter upstream of Renilla
luciferase (Promega).
Transient transfections.
MIN6 cells were transfected by lipofectamine and Plus Reagent
(GIBCO-BRL) by using the suggested amounts of DNA according to the
manufacturer's protocol. Briefly, 1 × 105 cells were
plated in 12-well plates 3 days before transfection. Cells at
~60% confluence were transfected by mixing the indicated amount of
DNA described in the figure legends and a lipid mixture containing a
1:2 ratio of lipofectamine and Plus Reagent in 1 ml of OPTI-MEM media
(GIBCO-BRL). Afer 3 h of incubation, 0.5 ml of DMEM media
containing 5 mM glucose and 2% serum was added to the cells. After
12 h, the medium-DNA complexes were replaced by preincubation
media containing DMEM with 5 mM glucose and 2% FBS, and the cells were
left for 24 h. At the end of the 24 h, the specific
stimulating agent was added to the media, and the cells were harvested
6 h later. For the overexpression experiments with pFC-PKA,
CaMKIVCT, CaMKIIBC, MEK, and MEKK, MIN6 cells were transfected as
described, followed by incubation in DMEM containing 5 mM glucose and
2% FBS for 30 h until harvesting. For the experiments that
include the dominant negative forms of CaMKII and IV, and pZIP-ras
(WT), pZIP-ras (15A), MIN6 cells were cultured as described, but the
last 6 h of the experiment were in the presence of the stimulating
agent. Total DNA was maintained constant in all of the transfection
experiments by using the empty vector of the respective cDNA to be
overexpressed. To correct for differences in transfection efficiencies,
2 ng of pRL-TK Renilla luciferase plasmid were
simultaneously transfected. All results are normalized for transfection
efficiency and expressed as the ratio of firefly to Renilla luciferase.
Electrophoretic mobility shift assay.
The sense strand sequences of the oligonucleotides used were
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Luciferase assay. Cell lysis was performed using 200 µl of passive lysis buffer (Promega). Firefly and Renilla luciferase were measured by the Dual-Luciferase Reporter Assay System (Promega) with the use of 20 µl of cell lysate. Luciferase activity was measured in a Monolight 3010 luminometer.
Immunoblotting. MIN6 cells were plated in six-well plates and transfected by lipofectamine and Plus Reagent (GIBCO-BRL) with 1 µg of CMV-Elk-1 as described. Thirty-six hours later, cells were preincubated in Krebs-Ringer bicarbonate-HEPES buffer (KRBH) and 2% albumin for 1 h followed by stimulation with the indicated agents. Cells were lysed with buffer containing 1× PBS, 0.1% SDS, 0.01 M dithiothreitol, and one-half of a tablet of "Complete" protease inhibitor cocktail (Boehringer Mannheim). After boiling, proteins were separated by electrophoresis through 10% polyacrylamide, 0.1% SDS gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated overnight at room temperature in blocking buffer containing 0.2% I-Block (Tropix) and 1:1,000 Tween 20. Subsequently, the membranes were hybridized at 4°C overnight in blocking buffer containing the Elk-1 and the phospho-Elk-1 antibody with the dilutions recommended by the manufacturer (New England Biolabs). After three washes at room temperature, the membranes were incubated in secondary horseradish peroxidase antibody for 1 h. After a 1-h washing, immunodetection was performed with an ECL Western blotting detection system (Amersham) following the manufacturer's protocol.
Kinase assay. MAPK assays were performed using the P44/42 MAP Kinase Assay Kit (Cell Signaling Technology) according to the manufacturer's protocol. MIN6 cells were precultured in KRBH-2% albumin and stimulated as indicated in the figure legends. Briefly, cell lysates were immunoprecipitated overnight at 4°C with an anti-phospho-p44/42 MAPK (Thr202 and Tyr204) antibody. The resulting immunoprecipitate was then incubated with an Elk-1 fusion protein in the presence of 200 µM ATP and kinase buffer for 30 min. Proteins were separated through 10% SDS-PAGE and transferred to nitrocellulose membranes. ERK activity was measured by phosphorylation of Elk-1 at Ser383 in Western blotting by use of a phospho-Elk-1 (Ser383) antibody. The total Elk-1 level was assessed by blotting the same membranes with Elk-1 antibody (Santa Cruz, CA).
Statistical analysis. Triplicate samples were analyzed for each experiment, and experiments were replicated to ensure consistency of the responses. Significant differences between control and the indicated treatments were determined using unpaired Student's t-test.
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RESULTS |
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Ets elements are necessary for depolarization induction of
SRE-dependent transcription in MIN6 insulinoma cell lines.
Previous studies showed that the proximal 530 bp of the Egr-1 promoter,
containing five SREs, are sufficient for depolarization activation of
the promoter (3). The purpose of the present studies was
to determine the role of Ets-dependent transcription in this process.
SREs are comprised of SRF-binding sites and adjacent Ets motifs (GGA)
that have been implicated in mediating a component of their enhancer
activity in other cells (41). To define a minimal promoter
suitable to investigate the contribution of the Ets elements in
depolarization induction of Egr-1 in pancreatic -cells, several
deleted constructs of the Egr-1 promoter were assessed. As shown in
Fig. 1A, although the 1.2-kb
promoter was induced ninefold (P < 0.003), deletion of
the two activating protein 1 (AP-1) elements, the two proximal SRE/Ets
elements, and the CRE (construct C) still retained sixfold induction
(P = 0.03) by depolarization. This suggested that the
distal cluster of SRE/Ets elements in construct C could be used to
refine the contribution of these binding sites in the depolarization
response. Parenthetically, basal activity for construct C was increased
relative to the full promoter construct A, suggesting perhaps the
presence of inhibitory elements in the proximal promoter.
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Nuclear extracts from MIN6 cells exhibit Elk-1-binding to Ets
motifs.
The SRE is continuously occupied in vivo by SRF and Ets proteins of the
ternary complex subfamily (15, 36, 40). In most cell types
examined, formation of this complex is constitutive rather than
inducible (15, 48). Previous studies have shown that the
sequence encompassing SRE-3 to -5 in the Egr-1 promoter can interact
with SRF and Elk-1 proteins at the SRF and Ets-binding domains,
respectively (43, 44). Furthermore, Elk-1 in combination with SRF participates in the initiation of Egr-1 gene transcription under other experimental conditions (41). To determine
whether Elk-1-binding activity in MIN6 cells is involved in
SRE-dependent transcription by depolarization, nuclear extracts from
MIN6 cells were incubated with a c-fos SRE. This SRE was
chosen as consensus sequence on the basis of initial gelshift
experiments with a probe containing the c-fos SRE and
unlabeled Egr-1 SRE-1, -2, -3, -4, and -5. Binding of nuclear protein
was competed for by each of the five Egr-1 SREs (data not
shown). As observed in other systems, two DNA:protein complexes
were observed (Fig. 2A). They
included one slower migrating band (band A) and a
diffuse faster migrating complex (band B) containing four
bands (lane 1). Band A migrated similarly to
bands identified by others as the ternary complex composed of the SRE,
SRF, and Elk-1 or another TCF (18, 26, 37, 41). The
identity of complex B is not known, and the intensity varies with cell
type (26). To ensure that some of these complexes contained an Ets family member, competitions with an unlabeled oligonucleotide containing the Drosophila E74A-binding site
was performed (34). This site has been shown to bind Elk-1
and compete for TCF binding, thereby inhibiting TCF at the
c-fos SRE (21, 28). Bands A
and B did not disappear when a nonspecific cold competitor in 5-, 25-, and 50-fold molar excess was used (lanes 2-4). In contrast, complexes A and B were progressively
competed for by addition of unlabeled E74A oligonucleotide (lanes
5-7). These results suggested that band
A is due to binding of SRF and an Ets family member. The
disappearance of some of the components of band B
suggested that some of these bands most likely contain homo- or
heterodimers of Ets family members. Multiple DNA-protein complexes have
also been observed when recombinant Elk-1 was incubated with E74
oligonucleotide (34). To recognize the identity of the
proteins present in the SRE complex (bands A and
B), nuclear extracts from MIN6 cells were preincubated with
anti-SRF and anti-Elk-1 antibody (SRF and
Elk-1, respectively).
Addition of
SRF to nuclear extracts resulted in disappearance of
band A and appearance of a more slowly migrating
band C (Fig. 2B, lane 2).
The presence of
Elk-1 reduced complex A by 80% as assessed by
densitometric analysis (n = 3; Fig. 2B,
lane 3), suggesting that Elk-1 is part of the SRE complex.
Incubation of the nuclear extracts with a nonspecific antibody
(anti-CREB) did not disrupt the protein complex (data not
shown). Disruption by
Elk-1 of the protein complexes containing
Elk-1 has been observed in other systems (1, 8). When
nuclear extracts were incubated with an oligonucleotide containing the
c-fos SRE with mutated SRF-binding site, no supershift was obtained with
SRF antibody, and a similar decrease in intensity of
the bands was observed with the
Elk-1 antibody (data not shown). These findings suggest that Elk-1 and the SRF are part of the ternary
complex in MIN6 cells.
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Depolarization and growth factors induce phosphorylation of Elk-1
in pancreatic -cells.
In other cells, it has been shown that EGF can regulate Elk-1
transcriptional activity by inducing phosphorylation of
Ser383 (33). Having shown that depolarization
activates Egr-1 transcription via SRE/Ets-dependent interaction and
that Elk-1 and SRF are part of the ternary complex in MIN6 cells, we
next sought to determine whether Elk-1 is phosphorylated on
Ser383 in MIN6 cells after depolarization. Cells were
transfected with a plasmid encoding Elk-1 and then subjected to Western
blotting with the use of anti-phospho-Ser383 antibodies.
Although the effects on phosphorylation of endogenous Elk-1 to
depolarization and EGF could be observed, the signals with transfected
Elk-1 were more readily evaluated. As shown in Fig.
3A, a rapid Ser383
phosphorylation of Elk-1 by glucose was observed as early as 5 min,
which appeared to be diminishing by 30 min. The total amount of Elk-1
protein did not differ with either of the agents tested, as indicated
by use of an antibody to nonphosphorylated Elk-1. KCl-induced
depolarization resulted in a similar time course of Elk-1
phosphorylation (Fig. 3B).
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Depolarization induces Ca2+-dependent
transcriptional activation of Elk-1.
To determine whether depolarization-induced Ser383
phosphorylation of Elk-1 is associated with transcriptional activation,
a transactivator plasmid Elk1-GAL4 encoding a fusion protein containing Elk-1 transactivation domain (amino acids 307-427) ligated
downstream of the sequence encoding the GAL4 DNA-binding and
dimerization domain was used. The Elk1-GAL4 plasmid was cotransfected
with a reporter gene containing a synthetic promoter with five tandem repeats of the yeast GAL4-binding site upstream of the luciferase gene.
As shown in Fig. 4A,
KCl-induced depolarization activated Elk-1-dependent transcription in a
dose-dependent manner. Addition of 25 mM glucose to cells preincubated
with 2 mM glucose, a treatment previously shown to result in
depolarization-dependent induction of Egr-1 transcription
(17), also induced Elk-1-dependent transcriptional activation 2.3-fold (P = 0.002, Fig. 4B).
Stimulation with EGF (100 ng/ml) resulted in similar transcriptional
activation of Elk-1. The results of these experiments indicated that
both a nutrient and a growth factor could lead to transcriptional
activation of Elk-1.
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Elk-1 transcriptional activation is mediated by
Ca2+-activated Ras/MAPK signaling.
Calcium regulation of gene transcription in various cell types involves
multiple signaling pathways (15). These include the
Ser/Thr kinases PKA, MAPK, and CaMKs. As shown in Fig.
5A, the PKA inhibitor H89 (10 µM) did not inhibit KCl-induced Elk-1-mediated transcriptional
activation. Consistent with this result, no response was observed when
a plasmid encoding the catalytic unit of PKA was cotransfected.
Evidence of the biological activity of this plasmid was previously
demonstrated in a CREB-GAL4 transactivation assay (3).
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CaMKIV activates Elk-1-dependent transcription.
Previous evidence obtained with Ca2+/CaM inhibitors
suggested that activation of Egr-1 transcription in insulinoma cells by depolarization involved this pathway (3). To determine
whether activation of CaMKIV and CaMKII, in addition to the Ras/MAPK
pathways, modulate Elk-1-dependent transcription, plasmids encoding
constitutively active forms of these kinases were assessed in the
GAL4-Elk-1 transcriptional activation assay. Transfection with CaMKIVCT
or CaMKIIBC, respectively, activated Elk-1-dependent transcription 5- (P = 0.01) and 2.3-fold (P = 0.002), respectively (Fig.
7A). Neither dominant negative
forms of CaMKIV (CaMKIVKT) nor CaMKII (CaMKIIBI) inhibited the
depolarization induction of Elk-1-dependent transcription (data not
shown). These results may be explained by the alternative activation
through the Ras pathway. That both pathways do contribute to
depolarization activation of Elk-1 was suggested by an additional 25%
inhibition of Elk-1 activation with cotransfection of a dominant
negative CaMKIV together with the dominant negative Ras (data not
shown).
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DISCUSSION |
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A common cellular response to a variety of growth stimuli is the
activation of various kinase-dependent signaling pathways. Several of
these kinase-mediated pathways have been shown to be triggered by
glucose treatment of pancreatic -cells. Although it is recognized
that hyperglycemia serves as a growth factor for these cells, the
proximal mechanisms whereby glucose-induced depolarization and
Ca2+ influx alter gene expression are still undetermined.
To appreciate the mechanisms concerned in Ca2+ regulation
of islet
-cell transcription, we have focused on the identification
of signal transduction pathways by which Ca2+ leads to the
phosphorylation and activation of transcription factors regulating the
Egr-1 promoter. In the current studies, we demonstrated 1)
that the Ets elements are important components in
depolarization-activated SRE-dependent Egr-1 transcription, 2) that Elk-1 is one of the TCFs that bind the SRE in
pancreatic
-cells, 3) that Elk-1 is phosphorylated and
transcriptionally activated by glucose and other depolarizing agents,
as well as by growth factors, and 4) that the MEK/ERK
pathway is involved in Elk-1-dependent transcription activated by both
glucose and growth factors. These studies also confirm the tissue
specificity of Ca2+-dependent TCF-mediated transcription,
since similar findings were described in cortical neurons and the
pituitary cell line AtT20, but not in hippocampal neurons or PC12 cells
(2). Most important, the results of these studies are the
first to show the convergence of glucose-induced depolarization
and growth factor treatment on activation of a transcription factor in
insulinoma cells.
It was noted that KCl-induced depolarization increased both phosphorylation and transactivation of Elk-1 to a greater extent than glucose treatment. The differences in the responses between glucose and depolarization can be explained by differences in the magnitude of the stimulus, which we have previously shown is extracellular Ca2+. This conclusion is based on the observation that inhibition of both glucose and KCl induction of Egr-1 transcription was blocked by the hyperpolarizing agent diazoxide and by Ca2+ channel blockers (3). Because it was previously shown that KCl-induced depolarization elicited a higher magnitude of Ca2+ influx, we decided to use this stimulus to study the signaling pathways induced by depolarization.
Efforts to define the proximal pathways whereby Ca2+ influx triggers the MEK/ERK pathway involved transfections with Ras and CaMK plasmids. Although expression of wild-type Ras resulted in robust Elk-1 transcriptional activation, expression of a dominant negative form of Ras, under conditions where others have observed marked inhibition (6), resulted in only partial inhibition. These results suggested that other pathways besides Ca2+ activation of Ras could be involved. In the present study, we demonstrated that activation of the CaMKIV pathway resulted in Elk-1-dependent transcriptional activation. This activation was ERK dependent as suggested by the complete inhibition of CaMKIV transactivation of Elk-1 by the ERK-1-specific MAP kinase phosphatase (Fig. 7B). Similar activation of the ERK pathway by CaMKIV has been shown in NG108 neuronal cells (13). Although the present study showed that activation of the CaMKIV pathway resulted in Elk-1-dependent transcriptional activation, the lack of inhibition by a dominant negative form of CaMKIV raised the concern about the role of endogenous CaMKIV. An additional inhibition was observed when a combination of dominant negative forms of Ras and CaMKIV was used, however (data not shown). The results of these experiments suggested that the CaMKIV pathway contributes to, but is not essential for, depolarization induction of Elk-1 in insulinoma cells.
EGF has been shown to modulate islet growth in animal models, and
overexpression of EGF in -cells in transgenic mice results in
pancreatic
-cell hyperplasia (24). Recently, EGF
receptor-deficient mice were shown to have generalized defects in islet
-cell proliferation (29). EGF activation of Elk-1
transcription has been demonstrated in several cell types. In the
present studies, we showed that EGF treatment of MIN6 insulinoma cells
results in Elk-1 activation. As demonstrated in other systems, this
activation occurs via a Ras/MAPK pathway, as this effect was inhibited
by PD98059. The results of the present studies also indicated a
convergence of glucose/depolarization/Ca2+ activation
and EGF-mediated signaling pathways through activation of
Ras/MAPK/ERK-1/2. Thus both mitogenic stimuli result in transcriptional activation of Elk-1 via similar mechanisms. In this regard, in preliminary experiments we have demonstrated that, in insulinoma cells,
inhibition of the MEK MAPK pathway by PD98059 decreases thymidine
incorporation induced by both EGF and glucose (data not shown). The
present studies thus provide an in vitro experimental model to begin to
assess the molecular mechanisms involved in EGF responses in transgenic animals.
On the basis of the results of previous (3) and the
present studies, a schematic respresentation of the signal transduction pathways involved in depolarization/Ca2+ regulation of
SRE-dependent transcription in pancreatic -cells is suggested in
Fig. 8. Using the Egr-1 gene as a model,
we have shown that the Egr-1 promoter contains five SREs and
that the transcriptional response to depolarization is SRE dependent.
In response to increased glucose metabolism, depolarization and
Ca2+ influx result in CaMKIV activation of SRF
(3). The present studies now demonstrate
Ca2+/CaM/CaMK activation of another transcription factor,
Elk-1, involved in SRE-mediated transcription. In contrast to the
activation of SRF by CaMKIV, Elk-1-dependent transcription requires
activation of the Ras/MEK pathway. EGF activation of Elk-1-dependent
transcription also requires the Ras/MEK pathway. Thus the results of
the present experiments show that both glucose/depolarization and EGF
treatment converge on a common pathway leading to Elk-1 activation. How this pathway contributes to the proliferation of
-cells to prolonged hyperglycemia and insulin resistance can now be tested in different experimental models.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Michael D. Shornick, Cris M. Welling, and Jon Wasson for technical assistance, and Burton Wice for careful comments and suggestions. We also thank Gary Skolnick for preparation of the manuscript.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16746 (M. A. Permutt) and the Diabetes Research and Training Center for technical support.
Address for reprint requests and other correspondence: E. Bernal-Mizrachi, Division of Endocrinology, Diabetes and Metabolism, Washington Univ. School Of Medicine, Campus Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: ebernal{at}im.wustl.edu).
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.
Received 13 June 2001; accepted in final form 26 July 2001.
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