Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555-0536
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ABSTRACT |
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Neurotensin (NT) plays an
important role in gastrointestinal secretion, motility, and growth. The
mechanisms regulating NT secretion are not entirely known. Our purpose
was to define the role of the PKC signaling pathway in secretion of NT
from BON cells, a human pancreatic carcinoid cell line that produces
and secretes NT peptide. We demonstrated expression of all 11 PKC isoforms at varying levels in untreated BON cells. Expression of
PKC-, -
2, -
, and -µ isoforms was most pronounced.
Immunofluorescent staining showed PKC-
and -µ expression
throughout the cytoplasm and in the membrane. Also, significant
fluorescence of PKC-
was noted in the nucleus and cytoplasm.
Treatment with PMA induced translocation of PKC-
, -
, and -µ
from cytosol to membrane. Activation of PKC-
, -
, and -µ was
further confirmed by kinase assays. Addition of PKC-
inhibitor
Gö-6976 at a nanomolar concentration, other PKC inhibitors
Gö-6983 and GF-109203X, or PKC-
-specific inhibitor rottlerin
significantly inhibited PMA-mediated NT release. Overexpression of
either PKC-
or -
increased PMA-mediated NT secretion compared with control cells. We demonstrated that PMA-mediated NT secretion in
BON cells is associated with translocation and activation of PKC-
,
-
, and -µ. Furthermore, inhibition of PKC-
and -
blocked PMA-stimulated NT secretion, suggesting a critical role for these isoforms in NT release.
protein kinase C; phorbol ester; BON cell line; gut endocrine cells
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INTRODUCTION |
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THE ALIMENTARY TRACT POSSESSES a sophisticated regulatory system that controls secretion of digestive juices, gastrointestinal (GI) motility, blood flow, and the growth and differentiation of GI tissues (21). Regulatory hormones, localized to specialized endocrine cells of the GI mucosa, are of central importance in coordinating and controlling these events (21, 53). Endocrine cells of the GI tract are regulated by ingested nutrients, neurotransmitters, and other hormones (21). Although the PKC pathway appears to play an important role in the signal transduction of regulated peptide secretion in certain endocrine cells, the exact molecular mechanisms regulating stimuli-induced gut hormone release are not entirely understood.
The PKC isoform family represents a group of widely distributed
serine-threonine kinases that play important roles in signal transduction of various physiological stimuli, including growth factors, hormones, and transmitters (39, 41). Eleven PKC
isoforms grouped into three major classes have been identified to date: the conventional PKCs (,
1,
2, and
), the novel PKCs (
,
,
, and
), and the atypical PKCs (
,
, and µ)
(39, 41). PKC-µ, also known as protein kinase D (PKD),
may be considered an additional family (25, 48). The
conventional isoforms require both Ca2+ and diacylglycerol
(DAG), novel isoforms are Ca2+ independent, and atypical
isoforms are Ca2+ independent and DAG or phorbol ester
resistant (39, 41). In endocrine cells, particularly in
the pancreas and thyroid, PKC isoforms have been detected and linked to
the regulation of hormone secretion (9, 16, 20, 22, 23, 35, 42,
49, 50, 57). On activation, PKC isoforms translocate to new
cellular sites, including the plasma membrane, cytoskeletal elements,
and the nucleus, as well as other subcellular compartments (39, 41). Although the secretory mechanisms responsible for hormone release from endocrine cells of the pancreas and thyroid have been
extensively studied, the mechanisms leading to release in gut
enteroendocrine cells have not been well defined, largely due to the
lack of appropriate intestinal endocrine cell models.
Although attempts have been made to isolate enteroendocrine cells from the canine GI mucosa (4), these primary cultures are insufficiently pure and difficult to maintain for more than a few days, thus rendering these cells unsuitable for many molecular approaches. Our laboratory has been particularly interested in the expression and cellular effects of the gut hormone neurotensin (NT), a tridecapeptide localized predominantly to specialized enteroendocrine cells (N cells) of the adult small bowel (10) that facilitates fatty acid translocation (10), affects gut motility and secretion (2, 10, 51), and stimulates growth of normal gut mucosa and certain cancers (8, 10, 13, 15). For our studies of NT expression, the BON cell line, which was established and characterized in our laboratory from a human pancreatic carcinoid tumor (12), has served as an invaluable in vitro model. Similar to the terminally differentiated N cell of the small bowel, BON cells express high levels of NT/neuromedin N mRNA (11, 14), synthesize and secrete NT peptide, and process the NT/neuromedin N precursor protein in a fashion identical to that of the normal intestine (6). BON cells exhibit morphological and biochemical characteristics consistent with the enteroendocrine cell phenotype, including the presence of numerous dense core granules and the expression and secretion of chromogranin A and other peptides (e.g., pancreastatin) (12, 45, 57). Unlike primary cultures of enteroendocrine cells from canine GI mucosa, BON cells can be maintained in long-term culture without noticeable changes in their secretory activity. Thus the BON cell line provides an excellent model to discern the mechanisms underlying NT peptide secretion.
In initial studies (57), we demonstrated stimulation of NT
secretion in BON cells after treatment with PMA. In another study (23), the potential role of PKC was suggested in bombesin
(BBS)-mediated peptide secretion in BON cells stably transfected
with the human gastrin-releasing peptide (GRP) receptor (GRP-R).
However, the specific PKC isoforms involved in NT secretion are
not known. The purpose of the present study was to better delineate the
PKC isoforms involved in NT secretion by using the BON and BON/GRP-R cell lines. Specifically, we wished to determine which PKC isoforms are
expressed in untreated BON cells, to assess whether PKC translocation occurs in these cells with stimulation, and to delineate the PKC isoforms associated with NT secretion. Importantly, we demonstrate activation of PKC-, -
, and -µ in BON cells stimulated with PMA and inhibition of NT release in BON and BON/GRP-R cells by PKC-
and
-
inhibitors. Our results indicate that PKC-
, -
, and/or -µ
are involved in NT secretion.
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MATERIALS AND METHODS |
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Materials.
PMA, aprotinin, and leupeptin were purchased from Sigma (St. Louis,
MO). BBS was obtained from Bachem (Torrance, CA). The anti-PKC isoform
polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz,
CA). The rat monoclonal anti-hemagglutinin (HA) antibody (clone 3F10)
was from Roche Molecular Biochemicals (Indianapolis, IN). The PKC
inhibitors Gö-6976, GF-109203X, and rottlerin were from BIOMOL
(Plymouth Meeting, PA). The PKC inhibitor Gö-6983 was purchased
from Calbiochem (La Jolla, CA). Secondary antibodies were from Pierce
(Rockford, IL). [-32P]ATP was obtained from New
England Nuclear (Boston, MA). The PKC-
and -
expression plasmids
(pTB701-HA-PKC
and pTB701-HA-PKC
, respectively) and the control
plasmid (pTB701-HA) were kindly provided by Dr. Yoshitaka Ono (Kobe
University, Japan). LipofectAMINE regent for transfections was obtained
from Invitrogen (Carlsbad, CA). X-ray film was purchased from Eastman
Kodak (Rochester, NY). The enhanced chemiluminescence system for
Western immunoblot analysis was from Amersham (Arlington Heights, IL).
Protein A-Sepharose was from Pharmacia Biotech (Piscataway, NJ). The
concentrated protein assay dye reagent was purchased from Bio-Rad
(Hercules, CA). Tissue culture media and reagents were obtained from
GIBCO-BRL (Grand Island, NY). All other reagents were of molecular
biology grade and purchased from either Sigma or Amresco (Solon, OH).
Cell culture and transfections.
The BON cell line was developed and characterized in our laboratory
from a human pancreatic carcinoid tumor (12). The
BON/GRP-R cell line was stably transfected with a human GRP-R cDNA and
described previously (23). BON and BON/GRP-R cells were
maintained in a 1:1 mixture of DMEM and nutrient mixture, F12K,
supplemented with 5% or 10% FBS in 5% CO2 at 37°C.
Geneticin (G418; 400 µg/ml) was added in the medium for the BON/GRP-R
cells. For the experiments involving PKC- and -
overexpression,
cells were transiently transfected with the PKC-
, PKC-
, or
control (empty) plasmid using LipofectAMINE according to the
manufacturer's instructions.
Cell treatments. Before each experiment, the cells were detached by using trypsin and seeded in six-well plates or 150-mm dishes at a density of 5 × 104/cm2 for 24-36 h. Cells were washed with secretion medium (DMEM-F12K containing 1% dialyzed BSA) and preincubated for 30 min in secretion medium before the reagents were added. For NT release experiments, cells were treated with PMA or BBS at various concentrations for 30 or 15 min, respectively. Alternatively, cells were pretreated with PKC inhibitors Gö-6976, Gö-6983, GF-109203X, and rottlerin for 30 min at various dosages, followed by combined treatments with PMA (10 nM) or BBS (10 nM) and the PKC inhibitors. In translocation experiments, cells were treated with PMA (10 nM) over a time course.
Confocal microscopy and immunofluorescence. BON cells were cultured in coverslips in six-well plates. After treatment, cells were washed with PBS three times and fixed with methanol for 20 min at 4°C. After three washes with PBS, the cells were blocked with 1% BSA-PBS for 20 min. The cells were incubated with rabbit or goat polyclonal anti-PKC antibody diluted 1:100 with 1% BSA-PBS for 1 h at room temperature or overnight at 4°C. Cells were washed three times with PBS and incubated with secondary anti-rabbit or Alexa 488-conjugated goat antibody diluted 1:200 in 1% BSA-PBS. The fluorescence of PKC isoform immunoreactivity was observed under a confocal laser scanning fluorescent microscope with excitation at 488 nm (green).
Cytosol and membrane fractionation.
BON cells were harvested on ice; all procedures were performed at
4°C. Cells were washed with ice-cold PBS and scraped into homogenization buffer containing 25 mM Tris · HCl, pH 7.4, 2 mM EDTA, 1 mM PMSF, 10 mM -mercaptoethanol, 10% glycerol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Cells were allowed to swell for 10 min and then homogenized with 30 strokes of a Kontes Tenbroech tissue
grinder. Cell lysates were centrifuged at 2,000 g for 5 min
at 4°C, supernatants (collected as postnuclear cell lysates) were
centrifuged at 100,000 g for 30-60 min, and the
resulting supernatant was used as the cytosolic fraction. The pellet
was rinsed with PBS three times and extracted with homogenization buffer containing 1% Triton X-100 for 60 min. The Triton-soluble component (particulate fraction) was centrifuged at 14,000 g
for 20 min at 4°C. Protein concentrations were determined by the
Bio-Rad protein assay using BSA as a standard.
Protein preparation and Western blotting. For total protein, BON cells were washed with cold PBS, scraped into RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40 detergent, 1 mM PMSF, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and 0.25% sodium deoxycholate and proteinase inhibitors, and incubated on ice for 30-60 min. The protein concentration of the supernatants was determined by a Bio-Rad assay. For PKC translocation studies, cytosolic and membrane fractions were separated as described above and protein concentration was determined in both fractions. Equal amounts of protein (15 µg) were resolved on 10% Novex Tris-Glycine gels (Invitrogen) and electrophoretically transferred to polyvinylidene difluoride membranes. After the nonspecific binding sites were blocked with 5% dried skimmed milk in TBST (120 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature or overnight at 4°C, PKC isoforms were detected by using rabbit or goat anti-human antibodies (1:500 dilution) for 3 h at room temperature or overnight at 4°C. The membranes were incubated with secondary antibodies (1:10,000 dilution) conjugated with horseradish peroxidase. Membranes were developed by using the enhanced chemiluminescence detection system.
In vitro kinase assays.
To examine the effect of PMA on the enzymatic activity of PKC-,
-
, and -µ, we treated BON cells with either vehicle control (DMSO)
or various concentrations of PMA (0.1-1,000 nM). Cells were
collected 10 min after treatment, and the endogenous PKC-
, -
, and
-µ activity was determined by immunocomplex kinase assays. Proteins
(50 µg) from particulate fractions were incubated with anti-PKC-
(C-20), -
(C-20) or -µ (C-20) antibodies (1:50) on a shaker for
2 h or overnight at 4°C followed by another 2 h incubation with 30 µl of protein A-Sepharose beads at 4°C. The immunocomplexes were washed six times with the homogenization buffer and three times
with the kinase buffer. The immunocomplexes were suspended in 20 µl
kinase buffer {35 mM Tris · HCl, pH 7.4, 10 mM
MgCl2, 0.5 mM EGTA, 0.1 mM CaCl2 containing 2 mg histone type III-SS (Sigma), and 5 µCi
[
-32P]ATP} and were incubated for 10 min at 30°C.
Reactions were stopped by the addition of 2× SDS-PAGE sample buffer.
Samples were denatured by boiling for 5 min and separated by 10% Novex
Tris-Glycine gels. Gels were incubated in Gel-Dry drying solution
(Invitrogen) for 5 min and dried at 80°C for 50 min followed
by exposure to X-ray film. Radioactive-labeled phosphorylated histone
signals were quantified by densitometry.
NT radioimmunoassay.
After 24-36 h, BON cells were washed with secretion medium
(DMEM-F12K containing 1% dialyzed BSA) and preincubated for 30 min in
secretion medium. Cells were pretreated with PKC inhibitors in fresh
secretion medium for 30 min and treated with PMA (10 nM) and PKC
inhibitors for another 30 min. BON/GRP-R cells were washed three times
with PBS and treated with various concentrations of BBS or PKC
inhibitors (diluted in Krebs-Henseleit buffer) plus 25 mM HEPES, 2 mM
CaCl2, and 0.1% BSA at room temperature. Medium was
collected and stored at 20°C until RIA for NT. Cells were removed
from culture plates by scraping and then sonicated in 1 M acetic acid
containing 5 mM EDTA, 1 mM PMSF, and 100 U/ml aprotinin. After being
boiled for 5 min, cell extracts were centrifuged at 20,000 g
for 20 min at 4°C. Supernatants were saved for NT assays. RIA for NT
was performed in duplicate samples as described previously
(26).
Statistical analysis. All experiments were repeated at least three times, and data are reported as means ± SE. Data were analyzed by the Friedman test (Fig. 3), the Kruskal-Wallis test (Figs. 5 and 7) or one-way classification analysis of variance Fisher's least-significant difference procedure for multiple comparisons with Bonferroni's adjustment for number of comparisons (Figs. 4 and 6). A P value <0.05 was considered significant.
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RESULTS |
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Predominance of PKC-, -
2, -
, and -µ in BON cells.
To confirm the effects of PMA and establish the optimal dosages for NT
release, BON cells were treated with PMA at various concentrations
(10-400 nM) for 30 min and NT release was measured by RIA. PMA
treatment induced a dose-dependent release of NT from BON cells, with
the most marked increase in NT peptide secretion noted with a dosage of
100 nM; a corresponding decrease in NT cellular content was noted (data
not shown). The induction of NT release by PMA suggested a role for the
PKC signal-transduction pathway in stimulated NT release.
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PMA induces translocation of PKC-, -
, and -µ isoforms in
BON cells.
We next determined whether PMA treatment induced translocation of the
PKC-
, -
2, -
, and -µ isoforms by subcellular fractionation and Western blotting (Fig. 2,
left). PMA (10 nM) induced translocation of the PKC-
(Fig. 2A, left), -
(Fig. 2B,
left), and -µ (Fig. 2C, left)
isoforms to the membrane fraction 1 min after treatment. Each blot was
reprobed with actin as a control for contamination from the cytosolic
fraction; the membrane fractions contained a small amount of cytosolic
proteins, but this does not affect the overall interpretation of the
results. In contrast, no change in the translocation of PKC-
2 was
detected (data not shown).
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Increased kinase activity of PKC-, -
, and -µ with PMA
treatment.
To further establish a role for the PKC-
, -
, and -µ isoforms in
PMA-mediated NT release, BON cells were treated with PMA and in vitro
kinase assays were performed (Fig.
3). Cells were treated with various doses
of PMA for 10 min; the membrane extracts were immunoprecipitated
by PKC-
, -
, and -µ antisera and collected by protein
A-Sepharose beads; and catalytic activity was then assayed by using
histone type III as an exogenous substrate. Extracts immunoprecipitated
with PKC-
antisera contained a basal level of histone type III
kinase activity (without stimulation); activity was markedly enhanced
by PMA in a dose-dependent fashion (Fig. 3A). Induction of
catalytic activity was noted at a concentration of 0.1 nM compared with
untreated control cells. The membrane extracts were also
immunoprecipitated by PKC-
antibody; the catalytic activity of
PKC-
, similar to PKC-
, was increased by PMA in dose-dependent pattern (Fig. 3B). Dose-dependent induction of PKC-µ
activity was also detected (Fig. 3C). Therefore, consistent
with our translocation experiments, PMA treatment stimulates PKC-
-,
-
-, and -µ-associated kinase activities.
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Inhibitors of PKC- and -
decreased PMA-mediated NT secretion.
To better ascertain the PKC isoforms that contribute to NT secretion,
BON cells were pretreated with various PKC inhibitors for 30 min before
treatment with PMA. After 30 min of PMA (10 nM) treatment, the medium
was collected and NT release was measured by RIA (Fig.
4). Treatment with PMA alone
significantly increased NT secretion. In contrast, 4
-PMA, the
inactive isomer of PMA, did not affect NT secretion. For these
studies, we used four well-characterized PKC inhibitors: Gö-6976,
Gö-6983, GF-109203X, and rottlerin, which are structurally
unrelated and have different activity profiles against the conventional
and novel PKC isoforms. Rottlerin, a PKC-
-specific inhibitor at
5-10 µM (17), significantly inhibited PMA-stimulated NT release, thus demonstrating a role for PKC-
in
PMA-stimulated NT secretion. The indolocarbazole compound, Gö-6976, has been shown to inhibit conventional PKC isoforms exclusively (IC50
2 nM against PKC-
in vitro)
(31), with no demonstrable in vitro inhibitory activity
against novel Ca2+-independent or atypical PKC isoforms
even at high micromolar concentrations. Pretreatment with Gö-6976
resulted in a dose-dependent inhibition of PMA-stimulated NT release.
Similarly, the bisindolmaleimide compound GF-109023X and Gö-6983,
which are known to inhibit both conventional and novel PKC isoforms
(IC50
8 nM for PKC-
) (55), significantly inhibited NT secretion.
|
Overexpression of PKC- and -
increased PMA-stimulated NT
secretion.
We examined the role of PKC-
and -
isoforms by overexpression of
wild-type PKC-
and -
in BON cells. Cells were transiently transfected with a plasmid containing either wild-type PKC-
or PKC-
linked to HA or, as a control, the empty vector (pTB701-HA) and
then treated with PMA (10 nM) (Fig. 5).
These plasmids have been used previously to assess the overexpression
of these isoforms on insulin-stimulated translocation of the glucose
transporter GLUT4 in rat adipocytes (3).
|
Inhibition of PKC- and -
decreases BBS-mediated NT secretion
in BON/GRP-R cells.
The hormone GRP (the human equivalent of BBS) stimulates the release of
all intestinal hormones, including NT (19). We next assessed the potential role of the PKC-
and -
isoforms on the release of NT mediated by BBS by using BON cells containing a stably
transfected human GRP-R plasmid (BON/GRP-R). BBS treatment induced a
dose-dependent release of NT from BON/GRP-R cells with a significant
effect identified with a dose of BBS as low as 10 nM (Fig.
6A). In addition, the cellular
content of NT decreased concomitant with increased NT release
(data not shown). Similar to BON cells treated with PMA,
pretreatment of BON/GRP-R cells with the PKC inhibitors
Gö-6976, Gö-6983, and rottlerin significantly decreased
BBS-mediated NT secretion (Fig. 6B). Therefore, these findings establish a role for the PKC-
and -
isoforms in NT secretion stimulated by BBS.
|
Overexpression of PKC- and -
increased BBS-stimulated NT
secretion.
To further confirm the role of PKC-
and -
isoforms in
BBS-mediated NT release in BON/GRP-R cells, the cells were transiently transfected with wild-type PKC-
(Fig.
7A) and -
(Fig.
7B) plasmids or, as control, the empty vector. The cells
were then treated with BBS (10 nM for 15 min) 48 h after
transfection as described previously. Expression of transfected and
endogenous PKC-
was confirmed by Western blotting by using the
anti-HA antibody (Fig. 7A) or anti-PKC-
antibody (Fig.
7A; bottom bands represent endogenous PKC-
and
top bands, denoted by the asterisk, represent PKC-
from
the PKC-
plasmid). NT secretion was significantly increased by BBS
(10 nM) treatment of BON/GRP-R cells transfected with wild-type PKC-
compared with BBS treatment of BON/GRP-R cells transfected with the
empty vector. Similar results were obtained from BON/GRP-R cells
transfected with PKC-
(Fig. 7B). Therefore, these
findings further support a role for the PKC-
and -
isoforms in
BBS-mediated NT release.
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DISCUSSION |
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The PKC isoforms are widely distributed at varying levels in
mammalian tissues; however, some of the isoforms appear to be more
specific for certain tissues (40, 41). For example,
PKC- is predominantly expressed in the brain and spinal cord
(41), PKC-
is expressed in T cells (37)
and skeletal muscle (44), and PKC-
is highly expressed
in the respiratory tract, digestive tract, and epithelia of the skin
(43). In our present study, we identified expression of
all 11 PKC isoforms, including PKC-
, -
, and -
, in the BON
endocrine cell line, with predominant expression of PKC-
, -
2,
-
, and -µ. An assessment of PKC isoforms in other endocrine cells
has been performed with varying results depending on cell type. The PKC
isoforms
,
2,
,
, and
are expressed in whole pancreatic
islets of mouse and rat and in the insulinoma-derived
-cells
(24, 28). PKC-
was identified in the insulin-secreting cell line RINm5F (49), and recently the presence of
PKC-µ has been demonstrated in the MIN6
-cell line
(52). PKC-
, -
, -
, and -
predominate in the
FRTL-5 thyroid cell line (32, 54), and PKC-
, -
,
-
, and -
are found in AtT-20 cells, a mouse anterior pituitary
tumor cell line (36). In primary cultures of human antral
G cells, Moore et al. (38) demonstrated expression of the
PKC isoforms
,
,
,
,
, and µ. Therefore, most
endocrine cells, including the BON cell line, express the
,
, and
isoforms but differ with respect to
,
,
,
,
, and
µ.
In BON cells, PKC- was observed throughout the cytoplasm as well as
in the membrane, as demonstrated by either confocal imaging or Western
blotting. This is consistent with a previous study in FRTL-5 thyroid
cells (32). In addition, Moore et al. (38) found PKC-
associated with small vesicles distributed throughout the
cytoplasm in human antral G cells by using confocal imaging. We
demonstrate that PKC-
expression was found in the nucleus and
cytoplasm in BON cells, which is consistent with other endocrine cells
(28, 29). It is generally thought that the PKC isoforms, when quiescent, are located in the cytoplasm and, on activation, they
translocate to the plasma membrane, cytoskeleton, perinuclear and
nuclear areas, or other intracellular organelles (30).
However, information regarding PKC translocation in endocrine cells is relatively limited. In our study, PMA-mediated NT secretion was associated with translocation of the PKC-
and -
isoforms from the
cytosol to the membrane, and in vitro kinase assays further confirmed
the activation of these isoforms. Therefore, these studies would
suggest that the PKC-
and -
isoforms play a role in stimulated NT
secretion in the BON endocrine cell line. Consistent with our results
in BON cells, stimulation of the thyroid cell line FRTL-5 by PMA
results in the translocation of PKC-
and -
from the cytosol to
the membrane (54). Stimulation of mouse or rat islet cells or the RINm5F cell line results in insulin release and translocation of
the
-,
-, and
-isoforms (27, 29). In contrast,
stimulation of gastrin release from antral G cells results in
translocation of PKC-
and -
(38). Therefore,
although PKC isoforms are ubiquitously expressed in a number of cell
types, the isoforms that contribute to stimulated release of hormones
from endocrine cells appears limited to a subset of isoforms that are
specific to the particular cell type.
To further delineate a role for the PKC- and -
isoforms in
stimulated NT secretion, BON cells were pretreated with different PKC
inhibitors of varying specificity. NT release was effectively blocked
by pretreating BON cells with the PKC-
-specific inhibitor rottlerin
before PMA stimulation. In contrast, transfection of the PKC-
plasmid, which overexpresses PKC-
, significantly enhanced PMA-mediated stimulation. Therefore, these results, together with our
findings of PKC-
translocation and induction of in vitro kinase
activity, strongly suggest a role for this isoform in the stimulated
release of NT. Although the PKC-
isoform has been most extensively
characterized for its role in the regulation of proliferation and
differentiation of various types of cells, this isoform is important
for thyroid hormone release (32, 54) and insulin secretion
(27, 29). In addition to rottlerin, BON cells were
pretreated with PKC inhibitors that, at nanomolar concentrations, are
relatively effective in blocking the conventional PKCs and, in
particular, PKC-
(55). Inhibition of NT release was
noted with each of three inhibitors (Gö-6976, Gö-6983, and GF-109203X); these data, in combination with the translocation results,
suggest a role for PKC-
as well in stimulated NT release. These
findings are further supported by our demonstration that overexpression
of PKC-
elicited significant PMA-stimulated NT release. Similar to
our findings, PKC-
appears to be involved in thyroid hormone
secretion (32, 54) and contributes to insulin release from
-cells stimulated by GRP and in cholinergic potentiation of insulin
secretion (28, 52).
In our present study, PKC-µ, a ubiquitous PKC isoform (25,
48), was observed in the cytoplasm and membrane in the BON cell
line. PKC-µ translocation from the cytosol to the membrane occurred
rapidly in BON cells after PMA stimulation, suggesting a role for the
PKC-µ isoform, as well as the PKC- and -
isoforms, in
NT-stimulated release. PKC-µ (human) or PKD (mouse) is a
serine/threonine protein kinase with structural, enzymologic, and
regulatory properties distinctly different from other PKC family
members (25, 48). In nonendocrine cells, PKC-µ/PKD can
be activated by pharmacological agents, including phorbol esters or
bryostatin 1 (1, 33), and physiological agents such as G
protein-coupled receptor (GPCR) agonists (7, 58), growth
factors (1, 58), and antigen-receptor engagement
(34). We found that phosphorylation of histone type III by
PKC-µ was enhanced by PMA in a dose-dependent fashion, although less
than that of PKC-
and PKC-
. PKC-µ/PKD activation is mediated by
a PKC-dependent signal transduction pathway, suggesting that
PKC-µ/PKD can function downstream of PKC by a novel signaling pathway
(5, 46, 56), but the precise role of this isoform has not
been conclusively demonstrated. Interestingly, a recent study by Guha
et al. (18) found that NT induced a rapid activation of
PKC-µ in the pancreatic cancer cell line PANC-1, which may be
responsible for the mitogenic effect of NT in these cells. Future
studies will better elucidate the role of PKC-µ in NT release.
The amphibian peptide BBS, and its mammalian counterpart GRP, potently
stimulate the release of all known GI hormones (19). BBS-related peptides interact with GPCR, especially of the BBS/GRP subtype, to activate phospholipase C and the generation of the two
secondary messengers, inositol-1,4,5-trisphosphate and DAG, which
results in mobilization of intracellular Ca2+ and PKC
activation (47). Moore et al. (38) reported
that stimulation of G cells with BBS resulted in the translocation of
PKC- from a predominantly intracellular location to the plasma membrane. Another study, using the STC-1 cell line, demonstrated that
CCK secretion in response to BBS involves MAPK activation through a
PKC-dependent mechanism (50). We found that BBS-mediated NT secretion in the BON/GRP-R cell line, similar to PMA stimulation, was inhibited by using the PKC inhibitors, including the specific PKC-
inhibitor rottlerin. The role of PKC-
and -
isoforms in BBS-mediated NT release was also supported by the overexpression of
wild-type PKC-
and -
. Consistent with our current findings, Hellmich et al. (23) found that pretreatment of BON/GRP-R
cells with the PKC inhibitor GF-109203X completely blocked
BBS-stimulated release of chromogranin A, indicating that BBS-mediated
peptide release from endocrine cells requires activation of PKC. In the case of BBS-mediated secretion from BON/GRP-R cells, an increase in
Ca2+ is necessary (23), thus further
supporting a role for PKC-
in BBS-stimulated hormone release.
In conclusion, we demonstrate expression of all 11 PKC isoforms with
prominent expression of the PKC-, -
2, -
, and -µ isoforms. Stimulation of BON cells with PMA resulted in the release of NT peptide, translocation of the PKC-
, -
, and -µ isoforms, and induction of in vitro kinase activity. The role for the PKC-
and
-
isoforms was further confirmed by using a battery of PKC inhibitors that blocked PMA-mediated NT release and overexpression of
PKC-
and -
, which enhanced PMA-mediated NT release. In addition, the PKC-
and -
isoforms appear to also be involved in the
stimulation of NT release by BBS. Therefore, our findings, using the
novel endocrine cell lines BON and BON/GRP-R, identify a role for the PKC isoforms
and
in both PMA- and BBS-mediated NT release.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jason Reed, Michael P. Cooper, and Leoncio A. Vergara for technical assistance, Tatsuo Uchida for statistical analysis, and Eileen Figueroa and Karen Martin for manuscript preparation.
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FOOTNOTES |
---|
This paper was presented, in part, at the annual meeting of the American Gastroenterological Association (May 19-22, 2002, San Francisco, CA) and was previously published in abstract form (30a).
This work was supported by Grants 2R37-AG-10885, RO1-DK-48489, and PO1-DK-35608 from the National Institutes of Health.
Address for reprint requests and other correspondence: B. M. Evers, Dept. of Surgery, The Univ. of Texas Medical Branch, 301 Univ. Boulevard, Galveston, TX 77555-0536 (E-mail: mevers{at}utmb.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.
August 14, 2002;10.1152/ajpgi.00177.2002
Received 13 May 2002; accepted in final form 1 August 2002.
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