Regulation of Insulin Gene Transcription by a Ca2+-Responsive Pathway Involving Calcineurin and Nuclear Factor of Activated T Cells
Michael C. Lawrence,
Harshika S. Bhatt,
Jeannette M. Watterson and
Richard A. Easom
Department of Molecular Biology and Immunology, University of North
Texas Health Science Center at Fort Worth, Fort Worth, Texas
76107-2699
Address all correspondence and requests for reprints to: Dr. Richard A. Easom, Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699. E-mail: reasom{at}hsc.unt.edu
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ABSTRACT
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Immunosuppressants such as FK506 (tacrolimus), the primary cellular
target of which is calcineurin, decrease ß-cell insulin content and
preproinsulin mRNA expression. This study offers an explanation for
this effect by establishing that calcineurin is an important regulator
of insulin gene expression through the activation of a transcription
factor, nuclear factor of activated T cells. Three putative nuclear
factor of activated T cells binding sites were located within the
proximal region of the rat insulin I gene promoter (-410 to +1 bp).
Expression of nuclear factor of activated T cells in both clonal
(INS-1) and primary (islet) ß-cells was confirmed by immunoblot and
immunocytochemical analyses. Moreover, nuclear factor of activated T
cells DNA-binding activity was detected in INS-1 and islet nuclear
extracts by EMSAs. Activation of the insulin gene promoter by glucose
or elevated extracellular K+ (to depolarize the ß-cell)
was totally prevented by FK506 (510 µM).
K+-induced promoter activation was suppressed (>65%) by a
2-bp mutation of a single nuclear factor of activated T cells binding
site in -410 rInsI. Both stimulants also activated a minimal
promoter-reporter construct containing tandem nuclear factor of
activated T cells consensus sequences. The effects of FK506 on
K+-induced nuclear factor of activated T cells reporter or
insulin gene promoter activity were not mimicked by rapamycin,
indicating specificity toward calcineurin. These findings suggest that
the activation of calcineurin by ß-cell secretagogues that elevate
cytosolic Ca2+ plays a fundamental role in maintenance of
insulin gene expression via the activation of nuclear factor of
activated T cells.
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INTRODUCTION
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POSTTRANSPLANT DIABETES MELLITUS is among
the most serious adverse effects of immunosuppressive therapy using
FK506 (tacrolimus) and is manifested by hyperglycemia, insulin
resistance, and the appearance of islet cell antibodies
(1). The incidence of diabetes in recipients of kidney
transplants has been reported to be as high as 20% (2).
The onset of diabetes may be even more widespread since tacrolimus is
not only established for primary immunosuppression in liver and kidney
transplantation, but has also been considered for therapies after solid
organ transplantation of heart, lung, and pancreas
(1).
The diabetogenic action of FK506 is not understood, but a direct effect
on ß-cell function is invoked. The chronic administration of FK506
in vivo results in a marked but reversible reduction in
insulin content of endocrine islets (3). This precedes
morphological changes that are partially characterized by a loss of
dense core secretory granules (4). Similar results are
observed with the use of another immunosuppressant, cyclosporin A
(CsA), although often to a lesser extent. FK506, or the structural
analog L-683,590, also reduces insulin content and ultimately insulin
secretion in isolated islets or cultured ß-cells in vitro
(5, 6). Since FK506 does not acutely affect insulin
secretion (7), these effects are presumed to be explained
by a reduced capacity to synthesize insulin. At the molecular level,
FK506 has been shown to reduce ß-cell preproinsulin mRNA expression
(3, 5, 6) and dampen glucose activation of the insulin
promoter (6). It is reasoned, therefore, that a primary
effect of FK-506 is to interfere with the transcriptional activation of
the insulin gene.
This mechanistic scenario is similar to the activated T cell in
which the immunosuppressant properties of FK506 and CsA are accounted
for by their common action to inhibit the
Ca2+/calmodulin-dependent phosphatase 2B,
calcineurin (8, 9, 10). Both compounds target calcineurin via
their interaction with immunophilins, FK506 binding proteins (FKBPs)
and cyclophilin for FK506 and CsA, respectively. Calcineurin is
critically required for the induced expression of cytokine genes
necessary for the initiation and coordination of an immune response
(10). Under normal conditions, the action of calcineurin
in the cell cytosol results in the dephosphorylation (on multiple
serines) of NFAT (nuclear factor of activated T cells) (11, 12). The resultant exposure of a nuclear localization
sequence promotes the rapid translocation of NFAT to the nucleus
(13) where it binds, generally in cooperation with other
trans-acting factors such as fos/jun components of activator
protein-1 (14, 15), to cis-elements
located in the promoters of several cytokine genes (9, 16). To date, most of the known therapeutic and toxic effects of
FK506 and cyclosporin A are attributable to the inhibition of
calcineurin (10).
Calcineurin is widely distributed among tissues (17), and
several reports have documented its expression in islet cells of the
endocrine pancreas (3, 6, 7, 18). It is now apparent that
NFAT expression is also diverse in that it is detected in nonimmune
tissues and cell types such as skeletal muscle, heart, neurons,
adipocytes, and the pancreas (12, 19). The current study
was therefore initiated to assess the involvement of NFAT in ß-cell
gene expression. The evidence generated suggests that calcineurin, via
NFAT, is an important regulator of insulin gene transcription and that
the disruption of this pathway may contribute to the diabetogenic
effects of FK506.
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RESULTS
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Identification of NFAT Consensus Sites within the Insulin Gene
Promoter
Examination of the first 410 bp of the rat I insulin gene promoter
(-410 rInsI), a region that controls >90% of the transcriptional
regulation of preproinsulin gene (23), revealed the
presence of three putative NFAT binding sequences [consensus
(T/A)GGAAA(A/N)(A/T/C), where N = any base] (Fig. 1
). These sequences are located at
positions -139 to -131 (1NFAT), -299 to -291 (2NFAT), and -308 to
-316 (3NFAT) on -410 rInsI relative to the transcription start site
(+1). Two of these binding sequences (1NFAT and 3NFAT) are positionally
conserved in other mammalian insulin gene promoters, such as in human,
mouse, and dog. The insulin gene promoter resembles other known
NFAT-dependent promoters in that it displays, in common, a multiplicity
of NFAT-binding sites (12), implying that higher-order
interactions among NFAT-containing complexes are required for effective
transcription.

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Figure 1. Multiple NFAT Consensus Sequences Are Present
within the Insulin Gene Promoter
Shown is the promoter region (-410 bp to +1) for the rat insulin 1
(-410 rInsI) gene. The boxes represent select sequence
elements previously identified as regulatory sites. The three
conceptualized NFAT sites (13NFAT) are indicated above the promoter
along with the DNA sequence conforming to the NFAT consensus motif.
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NFAT Is Expressed in Pancreatic ß-Cells
Three independent pieces of evidence were acquired to support a
role of NFAT in ß-cell function. First, the expression of NFAT in
ß-cells was ascertained based on immunochemical analyses in rat
pancreatic slices. Using an antibody raised against a peptide common to
all known NFAT family members, NFAT immunoreactivity was primarily
associated with islets of Langerhans with minimal reactivity in
surrounding exocrine tissue (Fig. 2A
).
Within the islet, NFAT labeling was detected in cells also expressing
insulin, supporting its association with the ß-cell. By this
analysis, it was apparent that NFAT was also expressed in peripheral
cells of the islet, but no further attempt was made to ascertain their
identity as
,
, or pp-cells. In addition, by immunoblot, NFAT was
identified in isolated rat islets, as well as in the cultured clonal
ß-cell line (INS-1) (Fig. 2B
) and calculated to have an approximate
Mr of 70,000. Furthermore, by EMSA, nuclear
extracts of islets and INS-1 cells displayed specific binding activity
toward DNA probes harboring NFAT consensus sequences from -410 rInsI.
The NFAT DNA-binding complex supershifts in the presence of anti-NFAT
antibody raised against a peptide sequence common to all known NFAT
family members (Fig. 2C
). The figure shown (2NFAT) is representative of
what is exhibited from EMSAs of all three identified -410 rInsI
promoter NFAT sites. Although two complexes were routinely resolved,
only the upper band was found to represent a specific binding event
under these experimental conditions based on its competition by excess
NFAT DNA probe. These observations confirm a functional expression of
NFAT in the ß-cell.

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Figure 2. NFAT Is Expressed in Pancreatic Cells
A, Immunocytochemistry: Cryosections of a rat pancreas
double-stained (same section) with antiinsulin (left,
Cy-2) and anti-NFAT796 (right, Texas-red) antibodies. B,
Western analysis: Whole cell extracts from INS-1, pancreatic islets,
and Jurkat cells were probed with an anti-NFATp antibody. C, EMSA: An
NFAT probe (2NFAT) from the rat insulin I promoter was used to detect
NFAT-DNA binding activity in INS-1 and pancreatic islet cells. The
NFAT-DNA binding complex was competed with excess nonlabeled probe (2X,
20X, or 200X) incubated before (pre) or after (post) the addition of
the radiolabeled NFAT probe of the insulin gene promoter. The
lower band was not competed by the unlabeled probe,
indicative of nonspecific binding. The NFAT-DNA binding complex was
supershifted (complex indicated by arrow) in the
presence of anti-NFAT796 antibody; lane1: no nuclear extract; lanes
24: INS-1 nuclear extract; lanes 57: islet nuclear extract; lanes 3
and 6: anti-NFAT796 Ab (Ab+); lanes 4 and 7: nonimmunized
rabbit serum (Ab-).
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Glucose and Potassium-Induced Insulin Gene Transcription
To permit an assessment of the involvement of calcineurin/NFAT
signaling in insulin gene transcription, INS-1 ß-cells were
transfected with an insulin promoter-reporter construct (pGL2-rInsI).
The activity of this promoter was initially monitored in cells
stimulated by glucose (11 mM) or by high extracellular
concentrations (30 mM) of K+,
conditions reasoned to induce the elevation of intracellular
Ca2+
([Ca2+]i) and the
activation of calcineurin (24). Stimulatory concentrations
of either glucose (11 mM) or K+ (30
mM) induced a similar (
7-fold) elevation in reporter
enzyme activity (luciferase) within 6 h of stimulation relative to
basal conditions (2 mM glucose, 5 mM
K+) (Fig. 3A
).
Since high K+ induces cell depolarization without
the influence of glucose metabolism, these observations support the
conclusion that an elevation in
[Ca2+]i is capable of
enhancing insulin gene promoter activity.

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Figure 3. Glucose (Glc)- and
K+-Induced Insulin Gene Transcription Is
Inhibited by FK506
INS-1 cells were transfected with pGL2-rInsI and then incubated in
basal (2 mM glucose) or stimulatory (11 mM
glucose or 30 mM K+) media for 6
h. Inhibitors were added 2 h before cell stimulation. Luciferase
activity in cell lysates was normalized with respect to CAT activity
and expressed as fold over basal. A, FK506 (5 µM) or
control vehicle (DMSO) was added. B, Increasing concentrations of FK506
(110 µM) were added. C, Increasing concentrations of
rapamycin (110 µM) were added. Data are expressed as a
fold increase in luciferase activity (normalized to CAT activity) over
controls in the presence of 2 mM glucose. Data are
means ± SE for three or more independent
determinations.
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Calcineurin Is Required for Up-Regulating Insulin Gene Promoter
Activity
Significantly, the effect of glucose or K+
to enhance insulin gene promoter activity was antagonized by the
presence of FK506, a selective inhibitor of calcineurin (Fig. 3
). FK506
dose-dependently inhibited insulin promoter activity; complete
inhibition was observed at a concentration of 510 µM
FK506 (Fig. 3B
). This effect appeared to be specific since it had no
effect on the expression of a control vector (pSV-CAT) cotransfected
with pGL2-rInsI. Furthermore, K+-induced insulin
promoter activity was not inhibited by rapamycin (Fig. 3C
), an analog
of FK506 that binds the same intracellular receptor as FK506 (FKBP-12)
but does not affect calcineurin activity (25, 26). In
contrast, rapamycin inhibited insulin promoter activity driven by
glucose nearly as efficiently as FK506. This latter observation
suggests other signaling mechanisms independent of calcineurin are also
necessary for glucose activation of insulin gene transcription.
The involvement of changes in
[Ca2+]I in the activation
of the insulin promoter by K+ was investigated
using two different Ca2+ inhibitors.
First, BAPTA, an intracellular Ca2+ chelator,
completely blocked the effect of K+ to activate
the insulin promoter (Fig. 4A
). Second,
verapamil, a selective inhibitor of L-type Ca2+
channels, also profoundly decreased this response to
K+ (Fig. 4A
), providing evidence that the
activation of the insulin promoter was, at least in part, the result of
Ca2+ influx. A direct effect of calcineurin to
modulate insulin gene transcription was further demonstrated in INS-1
cells overexpressing a constitutively active form of calcineurin A
(CaN
CaM-AI) lacking the autoinhibitory domain and a functional
calmodulin binding domain. In cells cotransfected with pGL2-rInsI and
constitutive calcineurin (CaN
CaM-AI) in basal glucose
concentrations, reporter activity was increased 6-fold relative to
cells transfected with the control vector (no calcineurin). This
stimulation approximated reporter activation achieved in the presence
of 30 mM K+ (Fig. 4B
, cf.
Fig. 3A
). This effect was further heightened by the addition of 11
mM glucose, which enhanced reporter activities
14-fold over those observed under basal conditions (Fig. 4A
). These
observations demonstrate that calcineurin can directly up-regulate
insulin gene transcription and enhance the ability of glucose to
modulate the activity of the insulin promoter.

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Figure 4. Calcium and Calcineurin Can Modulate Insulin Gene
Transcription
A, INS-1 cells were transfected with pGL2-rInsI and then incubated in
basal conditions (2 mM glucose, 3 mM
K+) or stimulatory K+ (30 mM) for
6 h. The cells were treated with calcium inhibitors verapamil or
BAPTA for 2 h and then stimulated with 30 mM
K+ for 6 h. B, INS-1 cells were cotransfected with
pGL2-rInsI and constitutively active calcineurin A
(pSR CaN CaM-AI) or empty vector. Data are expressed as a fold
increase in luciferase activity (normalized to CAT activity) over
controls in the presence of 2 mM glucose. Data are
means ± SE for three or more independent
determinations.
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Activation of NFAT in ß-Cells
To evaluate whether glucose and cell depolarization by
K+ activate NFAT in ß-cells, INS-1 cells were
transfected with an NFAT-reporter construct (NFAT-Luc) in which
multiple NFAT-consensus sites were inserted upstream of a minimal
promoter (IL-2) (8). Stimulatory concentrations of glucose
(11 mM) and K+ (30 mM)
increased NFAT-Luc reporter activity by 6-fold and 8-fold,
respectively, over basal conditions (Fig. 5
). In both cases, reporter activity was
completely blocked by 5 µM FK506 as observed in cells
transfected with pGL2-rInsI (cf. Fig. 3C
). In contrast,
rapamycin up to a concentration of 10 µM, had
no significant effect on NFAT-mediated transcription induced by either
glucose or K+. Thus, insulin secretagogues
activate NFAT in pancreatic ß-cells by a calcineurin-dependent
mechanism.

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Figure 5. Glucose and K+ Induce NFAT Activation
in ß-Cells
INS-1 cells were transfected with a NFAT-Luc reporter construct and
incubated with basal (2 mM glucose) or stimulatory (11
mM glucose, Glc, or 30 mM K+)
conditions for 6 h. A, Cells were incubated in the presence of 10
µM FK506 (gray bar) or 10 µM
rapamycin (striped bar). Control cells were supplemented
with vehicle alone (white and black bars). B, Cells were
incubated in increasing concentrations of FK506 (solid
symbols) or rapamycin (open symbols). Data are
expressed as a fold increase in luciferase activity (normalized to CAT
activity) over controls in the presence of 2 mM glucose.
Data are means ± SE for three or more independent
determinations.
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Effect of an NFAT-Mutated Insulin Gene Promoter on
Transcription
To confirm the importance of calcineurin/NFAT in the activation of
insulin gene transcription, site-directed mutagenesis was employed to
eliminate an NFAT element from the insulin gene promoter. In light of
the poorly defined binding of PDX-1 to A-box and the potential
overlap with NFAT binding sequences, an NFAT site was chosen (2NFAT)
that did not exist within either of the A and E element enhancers
(Far-FLAT and Nir-P1) (Fig. 1
). The double-point-mutated 2NFAT site
does not disrupt any known elemental binding sites, such as that for
pancreas duodenal homeobox-1 (PDX-1) or insulin enhancer
factor-1, which are essential transcription factors for insulin
gene promoter activity. There was little difference between luciferase
reporter activities in cells transfected with wild-type (pGL2-rInsI) or
mutant (pGL22NFATm) promoter constructs in the presence of
stimulatory concentrations of glucose (Fig. 6A
). In contrast, the mutation of 2NFAT
resulted in a marked suppression (
68%) of luciferase reporter
expression induced by depolarizing concentrations of extracellular
K+. This mutation also resulted in the dramatic
loss (
60%) of the insulin promoter to stimulation by the
over-expression of constitutively active calcineurin (Fig. 6B
).

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Figure 6. Elimination of 2NFAT Binding Element Reduces
Insulin Promoter Activity in Response to K+
INS-1 cells were transfected with either rInsI-Luc (WT) or INS-2NFATm
(2NFATm). A, Cells were incubated with 11 mM glucose or 30
mM K+ in the absence and presence of FK506 or
rapamycin (5 µM each). B, rInsI-Luc or INS-2NFATm was
cotransfected with a plasmid expressing constitutively active
calcineurin (CaN; pSR CaN CaM-AI). Data are expressed as fold
increase in luciferase activity (normalized to CAT activity) over
controls in the presence of 2 mM glucose. Data are
means ± SE for three or more independent
determinations. *, P < 0.05 vs.
rInsI-Luc, 2 mM glc).
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DISCUSSION
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In addition to its central role in the coordination of cytokine
expression in the activated T cell, calcineurin is now known to
influence transcriptional regulation in a variety of nonimmune cells
(27). Most dramatic perhaps is its role in the
transcriptional regulation of genes associated with hypertrophic growth
in cardiac and skeletal muscles (28). Calcineurin has also
been implicated in the transcriptional regulation of a noninsulin gene
(i.e. glucagon) in the ß-cell (24). This
study has now established that calcineurin has the capacity to directly
modulate the insulin gene promoter. This is exhibited directly by the
effect of overexpression of constitutively active calcineurin (CaN A)
to up-regulate rInsI and is further supported by the attenuation of
promoter activity by FK506. These data suggest that calcineurin may be
required for physiological regulation of insulin gene expression by
ß-cell stimuli.
Most significantly, the modulation of insulin gene transcription by
calcineurin was found to be mediated via NFAT and thus is similar to
that of the activated T cell. The observation that both primary (islet)
and clonal ß-cells express NFAT was not unexpected based on the
widened scope of detection of this transcription factor in nonimmune
system cells. However, it is not yet determined which of the isoforms
of this large multigene family of proteins (16) are
represented in ß-cells. Nevertheless, the application of an NFAT
promoter-reporter system demonstrates that the insulin secretagogues,
glucose and K+, both activate NFAT in the
ß-cell. Despite the fact that NFAT can be dephosphorylated by a
number of phosphatases, the specific involvement of calcineurin is
supported by the complete inhibition achieved in the presence of FK506,
but not rapamycin. The same discriminatory sensitivity was observed
with the rInsI insulin gene promoter, at least in response to
K+, arguing that NFAT activation by calcineurin
is also required for insulin gene expression under these conditions.
This link is strengthened by the observation that the influence of
calcineurin and K+ on insulin gene promoter
activity were both significantly dampened by the 2-bp mutation of a
single NFAT site (2NFAT) in this promoter. Collectively, these data
establish a functional pathway by which calcineurin can modulate
insulin promoter activity through the interaction of NFAT with specific
sites and argue that NFAT should be added to the already large
repertoire of transcription factors capable of influencing insulin gene
transcription.
The lack of effect of rapamycin on K+-induced
insulin promoter activity is in contrast to a previous study, which
emphasized an autocrine effect of insulin secreted, in response to cell
depolarization, on insulin gene transcription (29). This
autocrine effect may be an important contributor to the regulation of
insulin biosynthesis (30), but the identification of a
calcineurin/NFAT pathway in the ß-cell forwards a direct
mechanism by which Ca2+-responsiveness may
be conferred on the insulin promoter (31, 32). Numerous
other studies have established that cell depolarization-induced
regulation of gene expression in the ß-cell is dependent on
Ca2+ influx (33, 34). In the case of
glucagon gene promoter, activation in HIT cells (ß-cells) is mediated
by calcineurin modulation of cAMP response element binding protein
interaction with a cAMP response element in this promoter (34, 35). Despite the presence of a cAMP response element within the
-410 rInsI promoter, the context of this site does not appear to
permit it to be responsive to FK506 (36). Consideration of
these studies suggests that the prevention of NFAT activation is a
primary mechanism by which FK506 perturbs insulin gene
transcription.
The involvement of calcineurin in glucose regulation of the insulin
promoter is less clear and confused by the observation that rapamycin
mimics the effects of FK506 on glucose activation of the insulin
promoter. This action of rapamycin has been observed previously
(29). However, in the current study, glucose-induced
activation of NFAT-reporter construct was refractory to rapamycin
treatment, suggesting that the early cellular events of calcineurin and
NFAT activation are not disrupted. Rather than inhibiting calcineurin,
the rapamycin/FKBP12 complex targets mammalian target of
rapamycin (also named FRAP, RAFT1, and RAPT1) (37)
and then PHAS1 (38) and p70S6K
(39), which are involved in the regulation of protein
translation. The effect of rapamycin is unlikely to be a nonspecific,
global inhibition of transcription because it has no effect on the
insulin promoter activation induced by K+ or on
the constitutive chloramphenicol transferase (CAT) expression from the
control vector, pSV-CAT (data not shown). A potential suggestion,
therefore, is that rapamycin affects the biosynthesis of insulin
promoter-specific factors required to sustain insulin gene
transcription induced by glucose. Alternatively, the inhibition of
p70S6K, an integral component of the insulin
signaling pathway, may interfere with an autocrine effect of secreted
insulin to regulate its own transcription in the ß-cell
(29), but this suggestion is minimized by the lack of
effect of rapamycin on K+-induced activation of
-410 rInsI (see above). Considering the increased complexity of
glucose signaling relative to cell depolarization (23), it
is more likely that rapamycin interferes indirectly with some aspect of
transcriptional regulation by glucose. In any case, it is evident that
there are at least two distinct pathways arising from glucose
metabolism that effect insulin gene transcription: a
rapamycin-sensitive pathway and a calcium-dependent (FK506-sensitive)
pathway (Fig. 7
). The rapamycin-sensitive
pathway, which provides factors that are responsible for determining
the full effect of glucose-stimulated insulin gene transcription,
requires the glucose-induced calcium-dependent pathway.

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Figure 7. Schematic of the Activation of the Insulin Gene
Promoter by Glucose
High glucose (11 mM) activates the insulin gene
transcription by at least two distinct pathways. The calcium-dependent
pathway involves calcineurin and NFAT, whereas the rapamycin-sensitive
pathway involves factors derived or activated by glucose metabolism
which target the insulin gene promoter.
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A more extensive interaction of trans-acting factors on
-410 rInsI, relative to K+, may account for the
lack of effect of 2NFATm mutation on glucose-induced activation of the
insulin promoter. This site may have greater significance in the
context of the action of incretins, such as GLP-1, which
can heighten intracellular calcium and up-regulate insulin gene
transcription to a higher level than induced by glucose alone
(40). In fact, our preliminary data (not shown) suggest
that 2NFAT is important for maximal promoter activity in the combined
presence of glucose and GLP-1. Divergent heterologous
partnering between transcription factors interacting among the
distinctly arranged NFAT sites may account for differences in response
to a complex combination of integrated signals to which the ß-cell is
exposed. Furthermore, preliminary experiments, in which we tested the
effect of mutations of the other NFAT sites, suggest that 1NFAT may be
most important to glucose signaling, and may therefore form the primary
target of FK506 in experimental conditions involving glucose alone
(data not shown).
Curiously, of the two NFAT sites that are conserved among mammalian
insulin promoters, the site most proximal to the transcriptional start
site is in close proximity to the A2 binding for the homeodomain
protein PDX1 that is acutely activated by glucose (41, 42). This site also overlaps with a CAAT/enhancer box
binding site for CAAT/enhancer-binding protein ß, a known repressor
of insulin gene transcription but only in conditions of persisting
hyperglycemia (45). Although deciphering the significance
of this NFAT site may prove challenging, trans-acting
factors to these sites may represent intricate mechanisms by which the
ß-cell fine tunes the activity of the insulin gene promoter in
response to various signals. Extensive studies in immune-system cells
have shown that NFAT commonly, but not always, binds to DNA in concert
with a partner, e.g. AP-1 activator protein, from the
bZIP family of transcription factors (12, 43). A full
understanding of how NFAT regulates the -410 rInsI promoter in the
ß-cell thus hinges on the identification of other factors with which
it cooperates and the DNA sequences with which they interact.
In summary, this study has demonstrated that calcineurin regulates
insulin gene transcription via a mechanism involving NFAT interaction
with specific elements within the insulin promoter. It is suggested
that the disruption of this pathway in vivo under chronic
FK506 treatment contributes to the diabetogenic effect of
immunosuppressant therapy involving FK506. It is worth noting that
immunosuppressant therapies involving low-dose FK506 treatment result
in long-term survival of islet transplants (44). The
further study of this mechanism is necessary to permit the development
of new pharmacological approaches that clinically prevent tissue
rejection with reduced risk of posttransplant diabetes.
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MATERIALS AND METHODS
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Cell Culture
INS-1 and Jurkat cells were cultured in RPMI 1640 medium
supplemented with 10% FBS, 2 mM L-glutamine,
streptomycin (100 µg/ml), and penicillin (100 U/ml) at 37 C under an
atmosphere of 95% air/5% CO2.
Plasmids and Mutagenesis
A vector construct (pSYNT) harboring the promoter region (-410
to +1) of the rat insulin 1 gene (-410 rInsI) (20) was
kindly provided by Dr. M. German (San Francisco, CA). The -410
rInsI fragment was amplified by PCR with primers incorporating a
5'-XhoI linker and directionally cloned into the pGL2-Basic
luciferase promoter-reporter mammalian expression vector (Promega Corp., Madison, WI). The resultant construct is designated
pGL2-rInsI. Mutagenesis of the second NFAT site within the insulin gene
promoter (2NFAT) (Fig. 1
) was achieved by a four-primer mutagenesis
method designed to create two-point mutations and thus the disruption
of the core NFAT sequence (5'-GGAAA to 5'-TCAAA). The PCR fragment was
cloned into the pCR2.1 TA cloning vector (Invitrogen,
Carlsbad, CA) and sequenced for verification of the site-directed point
mutations. The mutated fragment was then amplified to incorporate
5'-XhoI linker and cloned into pGL2-Basic as described
above. The expression vector, pSR
CaN
CaM-AI, harboring
constitutively active calcineurin A (CaN-A) in which the calmodulin
binding and autoinhibitory domains were deleted (21), was
a generous gift from Dr. Stephen OKeefe (Merck Research Laboratories,
Whitehouse Station, NJ). For calcineurin overexpression
experiments, a control vector was generated by religation of pSR
after restriction enzyme digestion to eliminate the CaN-A insert. The
NFAT-luciferase (NFAT-Luc) reporter plasmid was a generous gift of Dr.
Gerald Crabtree (Stanford, CA).
Isolation of Islets
Pancreati were isolated from male Wistar rats by collagenase P
(Roche Molecular Biochemicals, Indianapolis, IN) digestion
followed by centrifugation on a discontinuous Ficoll gradient. Islets
were cultured in CMRL-1066 containing 5.5 mM glucose and
supplemented with 2 mM L-glutamine, 10%
heat-inactivated FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin
overnight at 24 C under an atmosphere of 95% air/5%
CO2. Immediately before experimentation, the
islets were incubated at 37 C for a minimum of 60 min.
Western Blot Analysis
Cell extracts from INS-1, pancreatic islets, and Jurkat cells
were prepared by lysis in Laemmli buffer. Samples were boiled for 5 min
and loaded (30 µg protein per lane) on an SDS-6% polyacrylamide gel.
The proteins were electrotransferred to a nitrocellulose membrane
(Osmonics, Westborough, MA) and blotted with affinity-purified
NFATp antibody (a gift from Dr. Karen L. Leach, Pharmacia & Upjohn, Inc., Kalamazoo, MI). Washes were done in PBS with 0.1%
polyoxyethylene sorbitan monolaurate (Tween-20). The enhanced
chemiluminescence system was used as the method of detection by a
secondary goat antirabbit IgG antibody conjugated to horseradish
peroxidase (Amersham Pharmacia Biotech, Piscataway,
NJ).
Immunocytochemistry
Rat pancreati were excised from Wistar rats and fixed on ice for
46 h by immersion in PBS (137 mM NaCl, 2.7 mM
KCl, 1.5 mM
KH2PO4, 100 mM
Na2HPO4, pH 7.2)
supplemented with 4% paraformaldehyde. After overnight equilibration
at 4 C in PBS containing 30% sucrose, the pancreati were embedded in
tissue freezing medium (OCT compound) and cryosectioned (
70 nm) as
previously described (22). On the day of
immunocytochemistry, frozen pancreatic sections were rehydrated and
permeabilized with PBS containing 0.2% Triton X-100 and blocked with
PBS containing 4% BSA and 5% serum from the host animal species in
which the secondary antibody was raised. Incubations with primary
antibodies anti-NFAT796 (a generous gift of Dr. Nancy Rice, ABL-Basic
Research Program, Frederick, MD) or antiinsulin (1:200 dilution)
(Linco Research, Inc., St. Charles, MO) were continued
overnight at 4 C and followed by incubation with
fluorochrome-conjugated secondary antibodies (1:200) for 1 h at 37
C. All washes were done in PBS containing 0.1% Triton X-100.
Visualization of slides was conducted on a Nikon
Microphot FXA microscope.
EMSAs
Complementary oligonucleotides
(5'-ATGAGGTGGAAAATGCTCAG) containing a -410 rInsI NFAT
consensus site (2NFAT) were synthesized (Genosys, MO), hybridized, and
end-labeled by T4 polynucleotide kinase (Amersham Pharmacia Biotech) in the presence of
[
-32P]-ATP. INS-1 cells (
4 x
106 cells) were lysed in 400 µl of buffer A
[10 mM Tris (pH 8.0), 1.5 mM
MgCl2, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 10 mM NaF, 0.6% NP-40, 1
mM dithiothreitol (DTT), 0.5 phenylmethylsulfonyl fluoride,
and 10 µg/ml leupeptin]. Nuclear pellets were spun down and
resuspended in 50 µl buffer B [10 mM Tris (pH 8.0), 1.5
mM MgCl2, 400 mM NaCl, 1
mM EDTA, 1 mM EGTA, 10 mM NaF, 1
mM DTT, 0.5 mM phenylmethylsulfonyl fluoride,
and 10 µg/ml leupeptin] to harvest extracts. Equal amounts of
nuclear extract (20 µg) were incubated for 30 min with
double-stranded 32P-labeled NFAT probe (20,000
cpm) in reaction buffer (10 mM Tris, pH 8.0, 50
mM KCl, 1 mM EDTA, 1 mM DTT, 6%
glycerol). Increasing amounts (2-, 20-, or 200-fold after; 2- or
20-fold before) excess of cold probe were added to competition
reactions either 15 min before or after the labeled probe. Anti-NFAT
antibody was added 15 min after labeled probe in supershift
experiments. The reactions were subjected to electrophoresis on 6%
polyacrylamide gels, and bands were detected using a Packard Instant
Imager Electronic Autoradiography System (Packard, CT).
Cell Transfections and Reporter Assays
INS-1 cells were cultured in 12-well plates in RPMI medium as
described, and then brought to 2 mM glucose 6 h before
transfection. INS-1 cell transfection was achieved using FuGene-6
(Roche Molecular Biochemicals) according to the
manufacturers directions. All cells were cotransfected with a control
vector (pSV-CAT) for the normalization of transfection efficiency.
Eighteen hours after transfection, the cells were stimulated with
either 11 mM glucose or 30 mM KCl. In the
latter case, the cell incubations (post 18 h) were performed using
a modified Krebs Ringer bicarbonate medium (25 mM HEPES, pH
7.4, 115 mM NaCl, 24 mM
NaHCO3, 5 mM KCl, 2.5 mM
CaCl2, 1 mM
MgCl2) with 0.1% BSA; a 30 mM KCl
isotonic Krebs Ringer bicarbonate solution was generated by adjusting
the relative concentrations of KCl and NaCl to 30 mM and 90
mM, respectively. For inhibitor studies, FK506 (1, 5, 10
µM) or rapamycin (1, 5, 10 µM) was added to
the media 2 h before cell stimulation. The cells were harvested
24 h after transfection by lysis in Reporter Lysis Buffer,
(Promega Corp., Madison, WI). After brief centrifugation
(
16,000 x g, 5 min) to remove cell debris, the
supernatant was assayed for luciferase activity based on Luciferase
Assay System (Promega Corp.) using a TD-20/20
bioluminometer (Turner Designs) or CAT activity by the CAT-ELISA method
(Roche Molecular Biochemicals).
Statistical Analysis
Statistical significance was calculated by one-tailed
t test.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Anne Marie Brun for generating
pancreas cryosections.
 |
FOOTNOTES
|
---|
This work was supported by a grant (009768-022 to R.A.E.) from the
Advanced Research Program of the Texas Higher Education Coordinating
Board.
Abbreviations: [Ca2+]i, Intracellular
Ca2+; CAT, chloramphenicol acetyltransferase; CsA,
cyclosporin A; DTT, dithiothreitol; NFAT, nuclear factor of activated T
cells; PDX-1, pancreas duodenal homeobox-1; rInsI, rat I insulin gene
promoter.
Received for publication November 10, 2000.
Accepted for publication June 8, 2001.
 |
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