(Received for publication, August 10, 1995; and in revised form, October 18, 1995)
From the
Syntaxin 1/HPC-1 is an integral membrane protein, which is
thought to be implicated in the regulation of synaptic neurotransmitter
release. We investigated syntaxin 1 expression in pancreatic
cells and the functional role of syntaxin 1 in the insulin release
mechanism. Expression of syntaxin 1A, but not 1B, was detected in mouse
isolated islets by the reverse transcriptase-polymerase chain reaction
procedure. An immunoprecipitation study of metabolically labeled islets
with an anti-rat syntaxin 1/HPC-1 antibody demonstrated syntaxin 1A
protein with an apparent molecular mass of
35 kDa.
Immunohistochemistry of the mouse pancreas demonstrated that syntaxin
1/HPC-1 was present in the plasma membranes of the islets of
Langerhans. In order to determine the functional role of syntaxin 1 in
pancreatic
-cells, rat syntaxin 1A or 1B was overexpressed in
mouse
TC3 cells using the transient transfection procedure.
Transfection of
TC3 cells with either syntaxin 1 resulted in
approximately 7-fold increases in their immunodetectable protein
levels. Glucose-stimulated insulin release by syntaxin
1A-overexpressing cells was suppressed to about 50% of the level in
control cells, whereas insulin release by syntaxin 1B-overexpressing
and control cells did not differ. Next, we established stable
TC3
cell lines that overexpressed syntaxin 1A and used them to evaluate the
effect of syntaxin 1A on the regulatory insulin release pathway. Two
insulin secretogogues, 4-
-phorbol 12-myristate 13-acetate or
forskolin, increased insulin release by untransfected
TC3 cells
markedly, but their effects were diminished in syntaxin
1A-overexpressing
TC3 cells. Glucose-unstimulated insulin release
and the proinsulin biosynthetic rate were not affected by syntaxin 1A
overexpression, indicating a specific role of syntaxin 1A in the
regulatory insulin release pathway. Finally, in vitro binding
assays showed that syntaxin 1A binds to insulin secretory granules,
indicating an inhibitory role of syntaxin 1A in insulin exocytosis via
its interaction with vesicular proteins. These results demonstrate that
syntaxin 1A is expressed in the islets of Langerhans and functions as a
negative regulator in the regulatory insulin release pathway.
There are two types of secretory pathway in eukaryotic cells.
One is the constitutive secretory pathway which is involved in
continuous exocytosis and the other is the regulated secretory pathway,
in which soluble proteins and other substances are stored in secretory
vesicles for later release. Neurons and endocrine cells exhibit
regulated release, which mediates chemical signaling in these
systems(1) . Regulated secretion occurs by docking and/or
fusion of the secretory vesicles with the plasma membrane when chemical
signals reach the targeted cells, which arises when membrane
depolarization or a secretagogue stimulates a second messenger system.
In the case of pancreatic cells, the membrane depolarization
caused by high glucose stimulant activates voltage-dependent
Ca
channels (2, 3, 4) and
triggers intracellular Ca
release(5, 6) , initiating insulin secretion as
a result of docking and/or fusion of insulin secretory granules with
the plasma membrane. Although the molecular basis of vesicle fusion is
poorly understood, a number of proteins involved in the targeted
movement and fusion reactions of the vesicle membrane have recently
been identified in neurons(7, 8) . The molecular
components of secretion by pancreatic endocrine cells may share many
attributes with those identified in neurons. In this study, we focused
on the role of syntaxin 1/HPC-1 in the mechanism of insulin secretion
by pancreatic
cells. Syntaxin 1/HPC-1, originally isolated from
rat hippocampus, is an integral membrane protein (9, 10) that has been postulated to act upon a family
of vesicular transport receptors(11) , which suggests it plays
a general role in protein traffic to the plasma membrane. Here, we
demonstrated the syntaxin 1A expression in mouse pancreatic islets and
determined its functional role in the insulin secretory pathway.
In order to produce stable TC3 cell lines
that overexpressed syntaxin 1A protein, the construct described above
was introduced into
TC3 cells by lipofectAMINE(TM), cultured in
DMEM for 48 h, after which they were plated (10
cells/plate) on a 10-cm dishes and grown in the presence of 800
mg/ml G418 (Life Technologies, Inc.). Multiple, single stable clones of
each of the constructs were isolated and expanded. In order to
determine their syntaxin 1A contents, the cells were labeled with
[
S]Met-Cys, lysed, and immunoprecipitated with
the anti-syntaxin 1/HPC-1 antibody, as described above. The amount of
syntaxin 1A protein immunoprecipitated was estimated by densitometric
analysis of the autoradiographic band. The stable transformants were
selected and maintained in DMEM containing 10% FBS without G418.
As the regulated secretory pathways of neurons and endocrine
cells show similarities(20) , and there are several
similarities between secretory mechanisms in yeast and mammalian
cells(21, 22) , the molecular components involved in
secretion from pancreatic cells may share common features with
those identified in neurons. In neuronal cells, syntaxin 1/HPC-1 binds
to many exocytosis-related proteins, such as VAMP, NSF,
-SNAP,
SNAP-25(7) , synaptotagmins, and N-type-Ca
channels(23, 24) , suggesting that it is an
essential part of the fusion apparatus involved in forming a molecular
machinery complex(25) . The results of our immunohistochemical
study showed clearly that syntaxin 1/HPC-1 is localized in the plasma
membranes of the islets of Langerhans (Fig. 1), indicating that
it may function in insulin exocytosis as a part of the molecular
machinery complex for docking and/or fusion apparatus. This led us to
investigate the expression and functional role of syntaxin 1/HPC-1 in
pancreatic
cells.
Figure 1: Immunofluorescence photomicrograph of the mouse pancreas stained for syntaxin 1/HPC-1. Sections of the mouse pancreas were incubated with anti-syntaxin 1 antiserum followed by swine anti-rabbit immunoglobulin coupled to fluorescein. The islets of Langerhans and exocrine tissues were immunostained. Immunoreactivity of syntaxin 1 can be seen in the plasma membranes of many cells in the islets of Langerhans, but little is evident in the exocrine tissues.
In order to determine whether mouse
pancreatic islets express syntaxin 1A and/or 1B, we utilized the
reverse transcriptase-PCR technique. Total islet RNA was subjected to
the reverse transcriptase-PCR using degenerate oligonucleotide primers
and syntaxin 1A, but no 1B mRNA, was detected in mouse isolated islets (Fig. 2). Northern blot analysis of the total RNA (15 µg)
from mouse isolated islets detected no syntaxin 1A transcripts (data
not shown), indicating that the expression level of syntaxin 1A mRNA in
cells was quite low. Therefore, we attempted to detect syntaxin
1A protein in mouse isolated islets. For this purpose, about 400 islets
were labeled metabolically with [
S]Met-Cys for 3
h in RPMI 1640 medium containing 11 mM glucose.
Autoradiography of the protein immunoprecipitated from the labeled cell
extract with rabbit anti-rat syntaxin 1/HPC-1 antiserum after SDS-PAGE
revealed a protein band with a molecular mass of approximately 35 kDa (Fig. 2). This protein was identified as syntaxin 1A on the
basis of the following criteria: 1) it migrated as a 35-kDa protein, as
expected on the basis of the predicted molecular mass; 2) it was not
immunoprecipitated from a nonimmune control serum; 3) the addition of
50 µg of unlabeled rat syntaxin 1A displaced this band (data not
shown); and 4) reverse transcriptase-PCR analysis of mouse isolated
islets detected only syntaxin 1A, not 1B mRNA. It is of interest that
only syntaxin 1A was expressed in mouse isolated islets. As
immunohistochemical and in situ hybridization studies of the
rat brain have shown that the localization of their proteins is not
identical, (
)the biological role of syntaxin 1A may be
different from that of syntaxin 1B. Our findings suggest that syntaxin
1A expressed in pancreatic
cells plays an important role in the
docking and/or fusion of insulin secretory granules with the plasma
membrane.
Figure 2:
Expression of syntaxin 1/HPC-1 mRNA and
protein in mouse isolated islets. Upper panel, autoradiogram
of PCR-amplified syntaxin cDNA fragments from mouse isolated islets.
cDNAs, reverse-transcribed from total RNAs (100 ng) of islets,
were subjected to PCR, as described under ``Experimental
Procedures.'' The PCR products were separated on a 1.2% agarose
gel, blotted onto a nitrocellulose filter, and hybridized with syntaxin
1A or 1B cDNA probe. The predicted size of the amplified fragment is
340 bp. Lower panel, identification of the syntaxin 1/HPC-1
protein band in mouse isolated islets. Mouse islets (300
400)
isolated by collagenase digestion were labeled for 3 h with
[
S]Met-Cys in RPMI 1640 (10% (v/v) dialyzed FBS)
containing 11 mM glucose. The cells were disrupted and
immunoprecipitated with rabbit anti-rat syntaxin 1/HPC-1 antiserum (lane 1), or rabbit normal serum (lane 2), as
described under ``Experimental Procedures.'' The
immunoprecipitates were analyzed using 10% SDS-PAGE, followed by
fluorography with Amplify(TM) and
autoradiography.
In order to investigate the functional role of syntaxin 1A
in pancreatic cells, we induced overexpression of rat syntaxin 1A
or 1B in mouse
TC3 cells, which possess both regulated and
constitutive insulin secretion pathways(26, 27) ,
because manipulation of protein expression levels has proved to be a
powerful tool for studying biological activities of
proteins(28, 29) . Mouse
TC3 cells were
transfected transiently with rat syntaxin 1A or 1B using
lipofectAMINE(TM) under the control of the Rous sarcoma virus long
terminal repeat promoter. As shown in Fig. 3, the transfection
of
TC3 cells with either syntaxin 1 resulted in approximately
7-fold increases in their respective immunoprecipitable proteins, which
were quantified by densitometric scanning of the blots, relative to
untreated
TC3 cell levels. The impact of syntaxin 1 overexpression
was evaluated by incubating the cells under glucose-stimulated
conditions (11 mM glucose) for 2 days posttransfection and
measuring insulin secretion into the medium. As shown in Fig. 4A, glucose-stimulated insulin release by syntaxin
1A-overexpressing
TC3 cells was reduced to about half the level in
control cells. Although the data from the independent transfection
experiments are not shown, the inhibitory effect of syntaxin 1A
overexpression on insulin secretion varied among the independent
transfection experiments. This is probably due to the different
transfection efficiency in each experiment (see Fig. 3), because
the levels of syntaxin 1A overexpression were compatible with the
degrees of insulin release inhibition (data not shown). In contrast,
insulin release by syntaxin 1B-overexpressing cells did not differ from
that by control cells. The biosynthetic rate of immunopurified
proinsulin was also measured by labeling the cells with
[
S]Met-Cys for 30 min. There were no difference
in the glucose-stimulated proinsulin biosynthetic rates between the
syntaxin 1-overexpressing and control cells (Fig. 4B).
In agreement with the above results, insulin content in syntaxin
1A-overexpressing cells was elevated about 2-fold relative to control
cells (Table 1). Thus, syntaxin 1A overexpression in
TC3
cells inhibited glucose-stimulated insulin release, and this inhibitory
effect was specific for the insulin release pathway.
Figure 3:
Immunoprecipitation analysis of syntaxin 1
expression in transiently transfected mouse TC3 cells.
TC3
cells were transfected transiently with rat syntaxin 1A or 1B
constructed expression vectors using lipofectAMINE(TM) under the
control of the Rous sarcoma virus long terminal repeat promoter. Two
days later, the cells were labeled with
[
S]Met-Cys and processed for immunoprecipitation
analysis, as described in the legend to Fig. 2. The figure
represents four independent transfection experiments for each syntaxin
1 isoform. The syntaxin 1A and 1B expression levels were determined by
densitometric scanning of the blots.
Figure 4:
A, inhibitory effect of syntaxin 1A
overexpression in mouse TC3 cells on glucose-stimulated insulin
release.
TC3 cells transfected with the indicated vector, or left
untreated, were incubated in RPMI 1640 (10% (v/v) FBS) containing 11
mM glucose for 2 days, after which the media were collected
and insulin secretion was analyzed by IRI. The insulin secretion data
were calculated after pooling the data obtained from all of
transfection experiments, as described in the legend to Fig. 3. B, proinsulin biosynthesis stimulated by 11 mM glucose in mouse
TC3 cells. Transfected and untransfected
TC3 cells were labeled with [
S]Met-Cys for
30 min and then processed for measurement of the proinsulin
biosynthetic rate as described under ``Experimental
Procedures.''
To address the
issue of the effect of syntaxin 1A on the regulatory insulin release
pathway in detail, we established the stable TC3 cell lines that
overexpressed syntaxin 1A and examined the effect of syntaxin 1A in the
regulatory insulin secretion pathway by utilizing two insulin
secretogogues, TPA (4-
-phorbol 12-myristate 13-acetate) and
forskolin, which activate protein kinase C and protein kinase A,
respectively(30, 31, 32, 33) . We
obtained three different clones that overexpressed syntaxin 1A protein,
in all of which glucose-stimulated insulin release was inhibited (data
not shown). We investigated further by subjecting a stable clone
designated
TC3-hpc1, which expressed approximately 20 times more
syntaxin 1A protein than control
TC3 cells, shown by
immunoprecipitation analysis, and immunoblot analysis showed that the
protein was present in the plasma membrane fraction (Fig. 5),
indicating that the overproduced syntaxin 1A was processed correctly in
the cells. In agreement with the result of the transient transfection
experiments, glucose-stimulated insulin release by
TC3-hpc1 cells
was lower than that by control cells (Fig. 6). The inhibitory
effect of syntaxin 1A on insulin secretion was only observed when
TC3-hpc1 cells were incubated under glucose-stimulated conditions
(11 mM glucose); it was not apparent under
glucose-unstimulated conditions (0 mM glucose), indicating
that syntaxin 1A affects only the regulatory insulin release pathway.
We further examined the effect of syntaxin 1A overproduction on the
regulatory insulin release pathway by performing experiments using
potent secretogogues. As described previously(27) , the
incubation of
TC3 cells with TPA for 1 h under either the absense
or presence of glucose increased insulin release markedly (Fig. 7). Forskolin increased insulin release only in the
presence of glucose, in agreement with the previous
report(34, 35) . On the other hand, the insulin
release by
TC3-hpc1 cells was completely abolished when cells were
incubated with TPA in the absence of glucose or in the presence of
glucose with forskolin. However, when
TC3-hpc1 cells were
incubated with TPA in the presence of glucose, insulin release was
stimulated even by syntaxin 1A-overproducing
TC3-hpc1 cells,
although the levels of insulin release by
TC3-hpc1 were only half
of the levels of control
TC3 cells. The reason why only the
combination of both glucose and TPA stimulated the insulin release by
TC3-hpc1 cells is presently unknown. A possible explanation is
that this combination increases the efficiency of
-SNAP
biosynthesis or its stabilization in the cells, which is known to
stimulate the Ca
-dependent exocytosis(36) ,
thereby overcoming the inhibitory effect of overproducing syntaxin 1A
protein. Taken together with these results, the insulin release
stimulated by three different insulin secretogogues, such as glucose,
TPA, and forskolin, was inhibited by syntaxin 1A protein overproduction
in
TC3 cells, suggesting that syntaxin 1A plays an essential role
as a negative regulator in the regulatory insulin release pathway,
which is probably associated with the docking and/or fusion of insulin
secretory granules, as suggested for neurotransmitter
release(8, 37) .
Figure 5:
Comparison of the amounts of syntaxin 1A
protein in TC3-hpc1 and
TC3 cells and its presence in the
plasma membranes. Stable
TC3 cells that overexpressed syntaxin 1A
protein were produced by transfecting
TC3 cells with a rat
syntaxin 1A expression vector, as described under ``Experimental
Procedures.'' Stable transformants were cloned by selection in 800
mg/ml G418 and designated
TC3-hpc.
TC3-hpc1 and control
TC3 cells were labeled with [
S]Met-Cys for
3 h, and immunoprecipitation analysis with an anti-syntaxin 1/HPC-1
antibody was performed as described. The intensity of the syntaxin 1A
protein band was estimated by densitometric scanning. In order to
investigate whether syntaxin 1A protein was present in plasma
membranes, the plasma membrane fractions were isolated from cells.
Samples of protein (10 µg) were analyzed by SDS-PAGE in 10%
polyacrylamide gel and electrotransferred to a nitrocellulose filter,
immunoblotted with the anti-syntaxin 1/HPC-1 antibody, and detected by
alkaline-phosphatase method.
Figure 6:
Insulin release from TC3-hpc1 cells.
TC3-hpc1 cells were incubated in RPMI 1640 (10% (v/v) dialyzed
FBS) with and without 11 mM glucose for 2 days, after which
the media were collected and the amount of insulin secreted into each
medium was analyzed by IRI. *, p < 0.0001 (versus
TC3 cells)
Figure 7:
Effect of TPA and forskolin on insulin
release by syntaxin 1A-overexpressed cells (TC3-hpc1 cells).
TC3-hpc1 and
TC3 cells were cultured in DMEM on 60-mm plates
to
80% confluence, then incubated with 50 nM TPA or 10
uM forskolin under either the absence or presence of glucose
for 1 h at 37 °C, after which the media were collected and insulin
release was measured by IRI. *, p < 0.0001 (versus
TC3 cells)
Although the biochemical mechanism
underlying the inhibitory effect of syntaxin 1A on the regulatory
insulin release pathway is unknown, we hypothesize that syntaxin 1A
acts as a suppressor of exocytosis via interaction with synaptotagmins,
which are expressed not only in brain but also in pancreatic
cells(38) , and have been suggested to inhibit neurotransmitter
exocytosis in neurons by unknown mechanism (39, 40) .
Indeed, syntaxin 1 is known to bind to synaptotagmins(41) ,
which are major components of vesicular proteins(37) , probably
regulating the Ca
-stimulated exocytosis process by
undergoing Ca
-dependent conformational
changes(42) . Therefore, in the present study, we characterized
the interaction of syntaxin 1A with insulin secretory granules, which
may possess vesicular proteins, such as synaptotagmins,
synaptotagmin-like molecules, or VAMP, by in vitro binding
assays. After purified syntaxin 1A fused to maltose-binding protein
(MBP-syntaxin) were coupled to agarose beads, they were incubated with
TC3 cell homogenates. The bound proteins were eluted,
electrophoresed, and immunoblotted with anti-insulin or anti-PC2
antibodies. As shown in Fig. 8, the eluted proteins were
proinsulin, insulin, and PC2 (a converting enzyme present in the
insulin secretory granules) (43, 44) , indicating that
syntaxin 1A may interact with insulin secretory granules via vesicular
proteins. Thus, in pancreatic
cells as well as in neurons,
syntaxin 1A is thought to bind to secretory vesicles, thereby
overproduced syntaxin 1A may suppress insulin exocytosis via the
interaction with vesicular proteins, probably by mechanisms similar to
those observed in neurons(39, 40) .
Figure 8:
Binding of syntaxin 1A to insulin
secretory granules. The MBP fusion syntaxin 1A bound to amirose
resin-agarose beads were incubated with the total TC3 cell extract
at 4 °C for 24 h. Proteins bound to washed beads were analyzed by
SDS-PAGE, following by immunoblotted with antibodies recognizing 9-kDa
precursor and 6-kDa mature insulin and 65-kDa
PC2.
Binding of
syntaxin 1 to munc 18 homologues(45, 46) , which is
also expressed in MIN6 insulinoma cell line(46) , was reported
to inhibit its interaction with SNAP-25(47) , resulting in
inhibition of the formation of the core complex (syntaxin
1-synaptotagmin-SNAP-25), which serves as a receptor for SNAP and NSF
(called SNARE, SNAP/NSF receptor) (48) . Since the overproduced
syntaxin 1A protein binds tightly not only to vesicular proteins, such
as synaptotagmins and VAMP, but also to munc 18 homologues and SNAP-25,
the ``core complex'' may not be assembled effectively during
the final steps between vesicle docking and fusion, and eventually the
fusion reaction may be disturbed. An inhibitory role of syntaxin 1A in
the exocytosis process was also observed in our recent studies, which
showed that 1) inhibition of the function of syntaxin 1 protein as a
result of microinjection of an anti-rat syntaxin 1 antibody into PC12 h
cells stimulated norepinephrine release in the regulatory
pathway(49) ; 2) syntaxin 1A proteins translated from rat
syntaxin 1A capped cRNAs injected into the embryonic cells of Japanese
newt inhibited secretion of the extracellular matrix(50) . In
contrast, insulin release under glucose-unstimulated conditions was
unaffected by syntaxin 1A protein overproduction, suggesting that
syntaxin 1A is not obligatory for the constitutive pathway in
pancreatic cells. Taken together with these results, our data
indicate that syntaxin 1A contributes to the pathway involved in the
regulation of insulin release, probably as a negative regulator of
regulatory exocytosis.
In conclusion, syntaxin 1A, but not 1B, is
expressed in pancreatic cells and plays an essential role in the
regulatory insulin release pathway as a negative regulator. Therefore,
any dysfunction in the expression, function, or regulation of syntaxin
1A may result in impaired insulin release and may be involved in the
pathogenesis of non-insulin-dependent diabetes mellitus.