(Received for publication, December 17, 1996, and in revised form, February 4, 1997)
From the Department of Molecular Sciences, Central
Research Division, Pfizer, Inc., Groton, Connecticut 06340 and the
§ Department of Molecular Physiology and Biophysics,
Vanderbilt University Medical Center, Nashville, Tennessee 37232
Islet amyloid polypeptide is expressed almost
exclusively in pancreatic - and
-cells. Here we report that
cell-specific expression of the human islet amyloid polypeptide gene is
principally regulated by promoter proximal sequences. The sequences
that control tissue-specific expression were mapped between nucleotides
2798 and +450 of the human islet amyloid polypeptide (IAPP) gene
using transgenic mice. To localize the cis-acting elements
involved in this response, we examined the effects of mutations within these sequences using transfected islet amyloid polypeptide promoter expression constructs in pancreatic
cell lines. The sequences between
222 and +450 bp were found to be necessary for
cell-specific expression. Linker-scanning mutations of the 5
-promoter
proximal region defined several key distinct control elements,
including a negative-acting element at
111/
102 base pairs (bp),
positive-acting elements like the basic helix-loop-helix-like binding
site at
138/
131 bp, and the three A/T-rich, homeobox-like sites at
172/
163,
154/
142, and
91/
84 bp. Mutations within any one of
these elements eliminated transcriptional expression by the promoter.
Gel mobility shift assays revealed that the PDX-1 homeobox factor,
which is required for insulin gene transcription in
cells,
interacted specifically at the
154/
142- and
91/
84-bp sites.
Since PDX-1 is highly enriched in
and
cells, these results
suggest that this factor plays a principal role in defining islet
cell- and
cell-specific expression of the IAPP gene.
Amyloid deposits are a common feature in individuals with
non-insulin-dependent diabetes mellitus
(NIDDM)1 (1). Islet amyloid polypeptide
(IAPP) or amylin, which is a member of the calcitonin gene family (2),
is the most abundant component of pancreatic amyloid. This 37-amino
acid peptide is normally co-secreted with insulin from cells (3)
and is also expressed in a subset of islet
cells (4).
The physiological role IAPP plays normally or in NIDDM is unclear. As a consequence of its abilities to inhibit insulin secretion in isolated islets (5, 6) and to counteract insulin action in peripheral tissues (7), IAPP has been proposed to play an important role in regulating plasma glucose levels in mammals. The formation of diabetes-associated amyloid deposits appears to be related to the primary sequence of IAPP, as only certain species, which include primates and cats, encode a hydrophobic amyloidogenic core of amino acids (amino acids 20-29) that allow fibril formation (8). However, since amyloid deposits are not normally found in nondiabetic individuals, other factors must also contribute to disease formation. It has been proposed that elevated expression of IAPP may be one such factor (9).
Experiments conducted by German et al. (10) indicate that
transcription of the IAPP gene is controlled by a factor that is also
involved in cell-specific expression of the insulin gene. Thus, it
was demonstrated that the
176 to
117-bp region of the human IAPP
gene could direct cell-specific transcription from a heterologous
promoter in transient transfection assays with characteristics similar
to the
247 to
197-bp region of rat insulin I gene. This region of
the insulin gene I is regulated by A/T-rich elements and a
basic helix-loop-helix (B-HLH) factor binding site (10, 11). The
regulatory factors that bind to and activate the A/T-rich elements
appear to be shared between the IAPP and insulin genes (10).
The PDX-1 homeoprotein, which is selectively expressed in islet and
cells as well as specific enteroendocrine cell-types in the
duodenum (12), activates expression from A/T-rich elements of the
insulin gene (13-16). Interestingly, homozygous PDX-1 mutant mice fail
to form a pancreas (17, 18), and the enteroendocrine cells in the
duodenum are also affected (18). Recently, two laboratories have
identified factors that appear to regulate expression from the promoter
proximal A/T-rich elements in the IAPP gene. However, the conclusions
drawn from these studies are quite different. Bretherton-Watt et
al. (19) concluded that PDX-1 interacts with these A/T-rich
elements in
cells, whereas Wang and Drucker (20) demonstrated that
a distinct factor, the LIM homeodomain protein Isl-1, can activate IAPP
expression.
In the present study, we have examined the effects of mutations
throughout the human IAPP gene on cell-type-specific expression. This resulted in the identification of a minimal control region spanning sequences from
222 to +450 bp, which was regulated by a
number of distinct cis-acting elements. The positive control elements included the A/T-rich elements at
172/
163,
154/
142, and
91/
84 bp and a B-HLH-like binding site at
138/
131. In addition, this analysis identified a negative-acting element at
111/
102 bp. Using the gel shift assay, we found that PDX-1 composed the major
-cell binding activity with the human IAPP elements at
154/
142 and
91/
84 bp. In contrast, Isl-1 did not appear to bind
to these elements. These results indicate that there are several
distinct factors acting upon the sequences within the 5
-flanking
region of the IAPP gene to control islet
cell-specific expression.
Furthermore, one of these factors, PDX-1, is common to the insulin
gene.
The IAPP-GH fusion gene was constructed from human IAPP
and growth hormone (GH) sequences using standard cloning techniques. IAPP sequences were isolated from the luciferase reporter plasmid pSLA12 (21) and spanned 2798 to +450 bp (exon 1 (102 bp) + intron 1 (333 bp) + exon 2 (15 bp)) of the human 5
-flanking and -untranslated
leader region; human growth hormone sequences were obtained from the
plck-hGH (22) plasmid, a gift of Dr. Roger Perlmutter. IAPP-GH DNA was
injected into the male pronuclei of fertilized FVB/N mouse oocytes.
Founder animals were identified using Southern blotting and polymerase
chain reaction (PCR) methods. IAPP-GH RNA was detected by Northern
analysis in pancreas samples in one (strain HGC) of six germline
positive founders. IAPP-GH expression was examined
immunohistochemically in nontransgenic and the HGC transgenic strain
using growth hormone (1:250 dilution, Peninsula Laboratories, Belmont,
CA) and insulin (1:400 dilution, Dako Laboratories, Carpinteria, CA)
antiserum in 3-4-µm paraformaldehyde-fixed, paraffin-embedded
sections by the peroxidase-anti-peroxidase method as described
previously (23).
All of the IAPP promoter mutants were
cloned into the chloramphenicol acetyltransferase (CAT) expression
vector, pTC, which is a Bluescript II SK plasmid
(Stratagene) containing the CAT reporter gene fused to
-globin
poly(A) sequences. Each of the clones described below were verified by
DNA sequencing. The 5
deletion constructs were made with the human
IAPP sequences in pSL (
2798 to +450; Ref. 21) using an exonuclease
III/mung bean digestion kit (Pharmacia Biotech Inc.). The constructs
were named according to their 5
-flanking end point. The exon I
internal deletion mutants were generated in pAm-391 by standard
molecular techniques. The intron I deletion mutant, pAm-241 (
+104 to
+434), was constructed in pAm-241 by deleting the amylin intron 1. The
oligonucleotide PCR primers spanned human IAPP sequences from
214/
231 (5
-AGATCTGATGGCAAATTC-3
) and bridged exon 1 and exon 2 (5
-CATTAAAAGAAAATTTGAGAAGCCATGG-3
). The IAPP-human elongation
factor-1 (EF-1) intron mutants were generated by cloning EF-1 intron 2 (+1726 to +2091) and EF-1 intron 4 (+2674 to +2756) fragments generated
by the PCR reaction between the IAPP +103 (exon 1) and +436 (exon 2)
sequences in pAm-391 to yield pAm-391EF-1Int2 and pAm-391EF-1Int4. The
IAPP promoter linker-scanning mutants were constructed in pAm-222 by a
modification of the procedure of Gustin and Burke (24). The IAPP
promoter mutant oligos used in the PCR reactions are listed in Fig.
5.
Cell Culture and Transient Transfections
TC3 (kindly
provided by Shimon Efrat, Albert Einstein College of Medicine, Bronx,
NY), RINm5F (from ATCC), and HeLa S3 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 units/ml penicillin, and 100 µg/ml streptomycin. All expression
constructs were introduced into cells by electroporation with the
Cell-porator (Life Technologies, Inc., Gaithersburg, MD) using
conditions described previously (25). Routinely, 10 µg of IAPP-CAT
chimera was co-transfected along with 2 µg of RSV-long terminal
repeat promoter luciferase (LUC) expression plasmid, RSV-LUC; the LUC
activity from RSV-LUC was used to control for transfection efficiency.
Expression from the IAPP-CAT constructs was compared with a herpes
simplex virus thymidine kinase promoter-driven CAT reporter construct,
pBLCAT2 (26), transfected in parallel. LUC and CAT activities were
measured from 10 µg of crude extract 40-48 h after transfection. The
protein concentration of the crude lysate was determined by the method of Bradford (27). CAT and LUC activities were determined using the
methods described by Kingston and Sheen (28). Each experiment was
repeated a minimum of 3 times.
Nuclear extracts from TC3 cells
and human adult islets were prepared as described (29).
TC3 (0.5-10
µg) and islet (10 and 20 µg) extract protein were resolved on a
12% SDS-PAGE and electrotransferred to an Immobilon polyvinylidene
difluoride membrane (Millipore, Bedford, MA). The membranes were either
probed with N-terminal PDX-1 or Isl-1 K5 (31) polyclonal antisera
(1:500 dilution in a buffer composed of 5% nonfat dry milk in
Tris-buffered saline (140 mM NaCl, 10 mM
Tris-HCL, pH 7.5) + 0.5% Tween 20). The N-terminal XlHbox8 antisera
was developed against the first 75 amino acids of the
Xenopus XlHbox8 protein and cross-reacts with the mouse
PDX-1 protein (14); and the Isl-1 K-5 antisera was generated against
C-terminal residues 178-349 of rat Isl-1 (31). The blot was washed and
probed with peroxidase-conjugated protein A as described previously
(14). The positions of the bound antibodies were detected by
autoluminography with the ECL detection kit (Amersham Life Science
Inc., Arlington Heights, IL).
Nuclear extract (5 µg) from HeLa and TC3 cell lines and human islet extracts were
used in gel shift assays with double-stranded 32P-labeled
human IAPP oligonucleotides probes (A1,
96GATGGAAATTAATGACAGAGG-76
; A2,
163ACTGATGAGTTAATGTAATAATGACC-138; A3,
183TATTTGCTACGTTAATATTTACT-161) using the
conditions described previously (14). The sequence of the wild-type and
mutant PDX-1 binding oligonucleotides in the rat insulin II gene
(
213/
192 bp) have been described (14). The antibody addition
experiments contained 3 µl of either N-terminal XlHbox 8, Isl-1 (A8
and K5), or Mox-2 antibodies. The Mox-2 antibody was raised to amino
acids 2-139 of the mouse Mox-2 protein (30); and Isl-1 A8 antisera was
generated against residues 86-175 of chick Isl-1 (31). Preincubation
reactions with antisera and
TC3 extract were conducted for 10 min at
room temperature prior to initiation of the DNA binding reactions. The
competition analyses were conducted with 100-fold excess of competitor
DNA, the radiolabeled oligonucleotide probe, and extract. The samples
were analyzed on a 6% nondenaturing polyacrylamide gel run at 4 °C
in TGE buffer (50 mM Tris, 380 mM glycine, and
2 mM EDTA, pH 8.5). After electrophoresis, the gel was
dried and subjected to autoradiography.
To localize the
sequences of the human IAPP gene that were sufficient for
islet-specific expression, a 3.3-kilobase region of the genomic clone
h201 (32) was ligated to the human GH gene and expression examined
in transgenic mice. The IAPP sequences contained within the IAPP-GH
reporter included 2798 bp of 5
-flanking sequence, exon 1 (102 bp),
intron 1 (333 bp), and a portion of exon 2 (15 bp). There were no IAPP
protein coding sequences present in the GH reporter construct. IAPP-GH
was subjected to pronuclear microinjection to generate six founder
lines, one of which (line HGC) was shown by Northern analysis to
express the transgene. To determine whether the transgene was expressed
within islets, we prepared serial sections from the pancreas of
transgenic and nontransgenic littermates and examined them for GH and
insulin expression immunohistochemically (Fig. 1). We
detected GH staining within the islets of transgenic mice but not their
nontransgenic littermates. In contrast, insulin expression was observed
in the islets of both sets of animals. The co-incidence of GH and
insulin staining indicates that the majority of human GH-containing
cells were
cells. These results are consistent with the expression pattern for the endogenous IAPP protein (4). In contrast, GH staining
was not detected in any other tissues tested, including kidney, liver,
spleen, stomach, skin, muscle, and brain.2
These results indicated that the region from
2798 to +450 bp was
sufficient to direct islet-specific expression of the IAPP gene.
Identification of 5
As a first step toward identifying the
IAPP sequences within the 2798/+450 bp control region that were
necessary for islet
cell expression, we subcloned a series of IAPP
control region mutants into a bacterial CAT expression plasmid. The CAT
activity from these chimeras was assayed in two well established,
rodent, pancreatic islet
cell lines,
TC3 (mouse) and RIN-m5F
(rat), and a non-IAPP producing cell line, HeLa S3 (human). The insulin and IAPP genes are expressed in both of these
cell lines (10, 15,
21, 42). IAPP-CAT activity was compared with pTC, the parent CAT
expression vector, and a ubiquitously expressed herpes simplex
thymidine kinase promoter-driven CAT reporter construct, pBLCAT2. CAT
activity from each transfected construct was normalized to the activity
obtained from a cotransfected Rous sarcoma virus-luciferase expression
plasmid.
To determine the exon 1 and intron 1 sequences that were important in
expression, internal deletion mutants within these regions were
constructed in the context of human IAPP sequences spanning 391/+450
(Fig. 2) or
241/+450 (Fig. 3). There
was only a small change in IAPP-CAT reporter gene activity as a
consequence of this 5
deletion (compare pAm-391 with pAm-222 in Fig.
4). All of the exon 1 deletion mutants were active in
the transfected
cell lines and inactive in HeLa cells (Figs. 2 and
3; data not shown). The activity of the largest exon 1 deletion mutant,
which spanned sequences from +5 to +47 bp, was reduced approximately 2.8-fold when compared with pAm-391 (Fig. 2, B and
C). In contrast, precise deletion of intron 1 from pAm-241
resulted in a 20-fold drop in activity (Fig. 3A). To
determine whether the loss in pAm-241
104-434 activity resulted from
the removal of essential cis-active transcriptional control
sequences, we replaced the IAPP intron with nonrelated intron sequences
from the generally expressed elongation factor 1 (EF-1) gene (33). The
activity of the constructs containing either EF-1 intron 2 or EF-1
intron 4 sequences were very similar to the IAPP wild-type construct
(compare pAm-391 with either pAm-391EF-1Int2 or pAm-391EF-1Int4 in Fig.
3B). Together, these results indicate that IAPP transgene
transcription within
cells was not profoundly affected by the human
IAPP noncoding sequences present in the construct. However, our results
suggest that a post-transcriptional processing event may have been very
important in the expression of IAPP-GH. We therefore have retained the
IAPP noncoding sequences to +450bp within each of the 5
-flanking
control region mutant constructs in the studies described below.
To identify the 5-flanking region sequences within the human IAPP
promoter that were important in
-cell expression, we generated a
series of 5
-flanking IAPP-CAT deletion constructs. The expression pattern obtained with these mutants was similar between the
cell
lines,
TC3 and RIN-m5F (Fig. 4, B and C).
Thus, the chimera with only 222 bp of upstream sequence, pAm-222, had
approximately the same activity as the
2798 construct, pAm-2798. In
contrast, deletion of the IAPP sequences between
222 to
138
resulted in a 6-10-fold drop in activity in both
cell lines. This
region contains the A/T-rich motifs that appear to be functionally
similar to those of the insulin gene (10). The generally lower activity of the IAPP-CAT construct in RIN-m5F cells, when compared with
TC-3,
is also reflected in their IAPP and insulin levels (21, 42). All of the
5
-deletion constructs were inactive in HeLa cells (Fig.
4D). These studies indicate that the sequences between
222
to +450 bp are responsible for directing IAPP gene expression to
cells.
To identify the IAPP control elements
within the 5 proximal region that were important in
cell-type-specific activity, we constructed a series of 10-bp
linker-scanner mutants between positions
211 and
32 bp in pAm-222
(Fig. 5). This strategy allowed us to analyze the
importance of small regions of the IAPP gene in transcriptional control
without altering the spacing between putative promoter elements. The
activity of each construct was assayed in
TC3 and RINm5f cells.
There were several IAPP promoter sequences sensitive to mutation in
transfected cells (Fig. 5). In some instances, we observed that
mutant IAPP-CAT activity was reduced primarily in only one of the
cell lines. In this category were the mutants at
201/
192, and
81/
71 bp. We presume that their activators were only limiting for
IAPP-CAT expression in the sensitive
cell line. We believe that the
more interesting mutants were those that influenced IAPP-CAT activity
in both RIN-m5F and
TC-3 cells as these probably represent the
action of regulators that are essential for transcription. The
phenotype of these mutants indicates that IAPP gene expression is
regulated by both positive- and negative-acting cellular activities. Thus mutations within activator sites, like the A/T-rich elements at
172/
163,
154/
142, and
91/
84 bp or the B-HLH-like site at
138/
131, reduced activity by 65-75% (compare the activity of the
C, D, E, F, G, and L mutants with pAm-222 in Fig. 5A). In
accordance with the nomenclature used in naming the A/T-rich and B-HLH
sites in the insulin gene (34), the A/T-rich elements at
172/
163,
154/
142, and
91/
84 bp will be referred to as A3, A2, and A1,
respectively, and the B-HLH-like site as E1. Each of the A and E
element mutants profoundly affected pAm-222 activity, implying that the
activators functioning at these sites act cooperatively to mediate IAPP
expression. In contrast to the A/T-rich and B-HLH-like site mutants,
the properties of
111/
102 mutant indicates that it is a repressor
binding site. This result does not appear to be caused by artifactual
generation of an activator binding site since the 2-8-fold increase in
activity observed with the
111/
102 site mutant was also obtained
with two other linker-scanner mutants over this
region.3
The gel mobility shift assay was used to determine the
distribution of the cellular factors interacting with the A1, A2, and A3 elements. Nuclear extracts were prepared from TC3 and HeLa S3
cells. Binding reactions were conducted in the presence of extract and
32P-labeled human IAPP A-element probes spanning sequences
at
183/
161 (A3),
163/
138 (A2), and
96/
76 (A1) bp. Binding
to these IAPP elements was compared with the insulin A2 element binding
site at
213 to
192 bp, which interacts with the PDX-1 transcription factor (14). Several protein-DNA complexes were detected with these
IAPP probes in these extracts (Fig. 6). Specificity of
binding was determined in competition assays with wild-type IAPP and
with wild-type and mutant alleles of the insulin
213/
192 element as
competitors. The competition pattern indicates that there was at least
one common, specific protein-IAPP element complex detected using
TC3
cell extract with the A1 and A2 probes (Fig. 6, A and B). This complex composes the major
cell binding
activity and co-migrates with the PDX-1-insulin complex (Fig.
6D); it was not detected in HeLa extracts (Fig. 6). The
presence of PDX-1 in this complex was also confirmed by
antibody-supershift analysis. Thus, the addition of an antibody that
recognizes PDX-1 specifically super-shifted this complex with the IAPP
A1 and A2 probes, whereas there was no effect upon incubation with a
polyclonal antisera to the unrelated murine mesoderm-specific Mox-2
homeoprotein (Fig. 6). In contrast, the IAPP A3 element does not bind
to this factor (Fig. 6C).
Our gel-shift results indicated that the homeoprotein PDX-1 was the
principal A element binding protein of the IAPP gene. Recent studies by
Bretherton-Watt et al. (19) have also come to a similar
conclusion. However, Wang and Drucker (20) proposed that Isl-1, a LIM
homeodomain protein, binds to and activates IAPP expression through an
IAPP A/T-rich element. Their results indicate that Isl-1 actions are
primarily through the A2 element. To compare the relative contribution
of Isl-1 and PDX-1 with A2 element binding activity, we analyzed the
effect of PDX-1 and Isl-1 polyclonal antisera on protein-DNA complex
formation in extracts prepared from TC-3 cells and human islets.
Isl-1 antisera raised to either amino acids 86-175 or 178-349 were
used in this analysis. Western blot analysis indicated that there was
approximately 80-fold more PDX-1 than Isl-1 in
TC-3 cells and
1.5-fold more in islets (Fig. 7). The PDX-1 binding
complex formed with the A2 element probe was quantitively removed from
these extracts upon addition of the PDX-1 antisera (Fig.
8). In contrast, the Isl-1 and preimmune antisera did
not have any specific influence on A2 element complex levels.
Increasing the amount of Isl-1 antisera had no effect on this result
nor did changing the gel-shift reaction conditions to those of Wang and
Drucker.4 These results strongly suggest
that PDX-1 is the major IAPP A2 element binding factor in
cells,
with little or no contribution from Isl-1. We infer from these results
that PDX-1 is an activator of IAPP A/T-element driven activity in islet
cells.
IAPP is a major component of islet amyloid plaques found in NIDDM
patients (1). Although the physiological and pathophysiological role of
IAPP is unclear, this factor appears to inhibit insulin action by
affecting both its release from the pancreas (5, 6) and its activity in
target tissues such as skeletal muscle (7). It has been proposed that
overexpression of IAPP could contribute to the development of disease
(9). Recent studies with transgenic mice that overexpress human IAPP
support this proposal (35). Thus, inhibiting IAPP transcription by
targeting the action of factors uniquely required for expression could
be a viable therapeutic initiative. In the present study, we have identified the cis-acting DNA elements of the human IAPP
gene that are required for islet cell expression. Our analyses
revealed that human IAPP sequences, which reside between nucleotides
222 to +450, contain the control elements that impart
cell-specific expression. Furthermore, we observed that transcription
appears to be mediated through the interaction of both positive- and
negative-acting cellular factors. These results also indicate that the
PDX-1 transcription factor contributes to the correct,
cell-type-specific expression of both the IAPP and insulin genes.
The transgenic reporter gene IAPP-GH, which contained human IAPP gene
sequences from 2798 to +450 bp driving growth hormone expression, was
expressed in the same islet-specific manner as the endogenous gene and
therefore served to define the region of the IAPP gene required for
transcription. To determine more precisely the regions of the IAPP gene
that were important in
cell expression, we constructed a series of
5
- and 3
-flanking deletion mutants spanning the sequences from
2798
to +450 and examined their activity in IAPP expressing and
nonexpressing cell lines. Two regions appear to be important in islet
-cells, a promoter proximal region between
222 and
91 bp and an
intron region between +104 and +434 bp. The promoter region contains the cis-acting elements required for islet-specific
transcriptional activity. In contrast, the intron region appears to be
important in post-transcriptional control. This conclusion is based on
our observation that replacement of IAPP first intron sequences with non-related sequences from the ubiquitously expressed EF-1 gene restored IAPP-CAT expression to a level comparable with the wild-type gene. Although the exact regulatory mechanism has not been elucidated, these results suggest that splicing is required for maximal IAPP expression. Studies conducted with other mammalian genes (36, 37) and
in plants (38) indicate that this may be an important regulatory
process. Detailed experiments conducted by Huang and Gorman (39)
suggest that splicing is coupled to efficient polyadenylation and
transport of the mRNA to the cytoplasm.
To systematically identify the elements within the human IAPP proximal
promoter region that were important in cell expression, linker-scanner mutants were constructed spanning the sequences between
211 and
32. Our results suggest that activation of IAPP transcription is primarily mediated by an interplay between several distinct regulators acting at the A elements at
172/
163,
154/
142, and
91/
84 bp and the E-like site at
138/
131 bp.
The activator of the A1 and A2 elements appears to be PDX-1, a factor
which plays a key role in pancreatic determination and insulin
transcription in
cells (14-18). This conclusion is based upon
several observations. First, antibodies to PDX-1 recognize the
principal binding complex associated with each of these elements in gel
shift experiments performed with
cell line and islet extracts. The
recent binding and antibody supershift studies conducted with the human
IAPP gene by Bretherton-Watt et al. (19) also support this
conclusion. Second, the endogenous expression pattern of PDX-1 and IAPP
appear to be identical (4, 12, 14, 16). Finally, Serup et
al. (40) have recently demonstrated that PDX-1 stably transfected into the islet
cell line AN 697 coordinately induces IAPP and insulin gene expression. Interestingly, PDX-1 did not affect the expression of other genes selectively transcribed in islet
cells, like the glucose transporter 2,
glucokinase, or glucagon-like peptide 1 receptor genes in their study. In contrast, co-transfection experiments conducted by Wang and Drucker (20) indicates that Isl-1 can
activate A2 element-mediated expression. These studies were conducted
in the islet
cell line, InR1 G9. Although we cannot detect any
binding of Isl-1 to this element in either
TC-3 or islet extracts,
it is still possible that there are circumstances where this generally
distributed islet factor (41) is important for A2 element activity.
However, we believe that the islet cell types used in the various
investigations may explain the discrepancies between studies. Wang and
Drucker (20) used an islet
cell line. In contrast, our results and
those of Bretherton-Watt et al. (19) were obtained with IAPP
producing
cell lines (10, 15, 21, 42). As a consequence, we believe
that PDX-1 is the IAPP activator of A1- and A2-mediated expression in
islet
and
cells.
Given the ability of PDX-1 to induce both IAPP and insulin expression,
there must be other regulators that control their distinct expression
patterns. Transcription between insulin and IAPP may be distinguished
by factors that bind to and activate expression at the A3 or E1
elements. Insulin transcription is also regulated by proteins in the
B-HLH family (43). However, previous studies have demonstrated that the
B-HLH-like site of the IAPP gene will not functionally substitute for
the insulin B-HLH site (10), arguing that a distinct factor is
important in control. Alternatively, the negative regulator that acts
at the 111/
102-bp element or another of the cis-active
elements identified by our linker-scanner analysis may be key to
selective expression. Further understanding of the importance of each
of these factors in IAPP transcription may lead to better therapeutic
strategies for preserving
cell function in NIDDM.
We thank Drs. Yi Qiu and Arun Sharma for constructive criticism of the manuscript, Drs. Jeff Hanke and Roger Perlmutter for plasmid plck-hGH, Dr. Sam Pfaff for providing the Isl-1 A8 and K5 antisera, and Dr. Christopher Wright for the polyclonal N-terminal XlHbox8 antisera. We also thank the Diabetes Research and Training Center, Islet Isolation Core Facility (supported by U. S. Public Health Service Grant DK20579 from the National Institutes of Health) at Washington University School of Medicine for providing human adult islets; Jeff Stock, Jane Bennett, and Dr. John McNeish for generation of the HAPhGh founder animals; and Drs. Patrick Roche and Peter Butler of the Mayo Clinic, Rochester, MN for immunostaining of the mouse tissue sections.
During review of this manuscript, another
report was published that describes the importance of PDX-1 in IAPP
transcription in the cell (Watada, H., Kajimoto, Y., Kaneto, H.,
Matsuoka, T., Fujitani, Y., Miyazaki, J., and Yamasaki, Y. (1996)
Biochem. Biophys. Res. Commun. 229, 746-751).