Phorbol ester-mediated neurotensin secretion is dependent on the PKC-alpha and -delta isoforms

Jing Li, Mark R. Hellmich, George H. Greeley Jr., Courtney M. Townsend Jr., and B. Mark Evers

Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555-0536


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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-alpha , -beta 2, -delta , and -µ isoforms was most pronounced. Immunofluorescent staining showed PKC-alpha and -µ expression throughout the cytoplasm and in the membrane. Also, significant fluorescence of PKC-delta was noted in the nucleus and cytoplasm. Treatment with PMA induced translocation of PKC-alpha , -delta , and -µ from cytosol to membrane. Activation of PKC-alpha , -delta , and -µ was further confirmed by kinase assays. Addition of PKC-alpha inhibitor Gö-6976 at a nanomolar concentration, other PKC inhibitors Gö-6983 and GF-109203X, or PKC-delta -specific inhibitor rottlerin significantly inhibited PMA-mediated NT release. Overexpression of either PKC-alpha or -delta 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-alpha , -delta , and -µ. Furthermore, inhibition of PKC-alpha and -delta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 (alpha , beta 1, beta 2, and gamma ), the novel PKCs (delta , epsilon , eta , and theta ), and the atypical PKCs (zeta , iota , 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-alpha , -delta , and -µ in BON cells stimulated with PMA and inhibition of NT release in BON and BON/GRP-R cells by PKC-alpha and -delta inhibitors. Our results indicate that PKC-alpha , -delta , 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). [gamma -32P]ATP was obtained from New England Nuclear (Boston, MA). The PKC-alpha and -delta expression plasmids (pTB701-HA-PKCalpha and pTB701-HA-PKCdelta , 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-alpha and -delta overexpression, cells were transiently transfected with the PKC-alpha , PKC-delta , 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 beta -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-alpha , -delta , 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-alpha , -delta , and -µ activity was determined by immunocomplex kinase assays. Proteins (50 µg) from particulate fractions were incubated with anti-PKC-alpha (C-20), -delta (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 [gamma -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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ABSTRACT
INTRODUCTION
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Predominance of PKC-alpha , -beta 2, -delta , 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.

To better define the role of the PKC pathway in NT release, we first performed Western immunoblots to identify the PKC isoforms present in the BON endocrine cells by using whole cell extracts. Untreated BON cells contained proteins immunoreactive with antibodies to all 11 PKC isoforms. PKC-alpha , -beta 2, -delta , and -µ are predominantly expressed (Fig. 1A), whereas the others are only weakly detected (data not shown). To further confirm the results by Western immunoblot, cells were stained for immunofluorescence and assessed by confocal microscopy (Fig. 1B). The fluorescence of PKC-alpha and -µ was observed throughout the cytoplasm as well as in the membrane. In contrast, PKC-delta was noted in the nucleus and cytoplasm. PKC-beta 2 was detected in particulate structures in the cytoplasm.


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Fig. 1.   Expression of PKC isoforms in BON cells. A: BON cells were incubated for 30 min with secretion medium. Denatured proteins from total lysates of BON cells or rat brain (positive control) were separated on 10% Novex Tris-Glycine gels, transferred to polyvinylidene difluoride membranes, and immunoblotted for PKC-alpha , -beta 2, -delta , and -µ. The specificity of each isoform-specific signal was confirmed by preabsorption of the primary antibodies with corresponding blocking peptide before cell labeling. Each blot is representative of 3 different experiments. B: confocal micrographs of untreated BON cells incubated for 30 min with secretion medium. Cells were then fixed with methanol and incubated with anti-PKC-alpha , -beta 2, -delta , and -µ antibodies, followed by incubation with Alexa 488-conjugated secondary anti-rabbit or anti-goat antibody. The specificity of each isoform-specific signal was confirmed by preabsorption of the primary antibodies with corresponding blocking peptide before cell labeling. In these experiments, the fluorescence signal was of the same magnitude as the background signal obtained when the primary antibody was omitted. The images shown are representative of at least 3 separate experiments.

PMA induces translocation of PKC-alpha , -delta , and -µ isoforms in BON cells. We next determined whether PMA treatment induced translocation of the PKC-alpha , -beta 2, -delta , and -µ isoforms by subcellular fractionation and Western blotting (Fig. 2, left). PMA (10 nM) induced translocation of the PKC-alpha (Fig. 2A, left), -delta (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-beta 2 was detected (data not shown).


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Fig. 2.   Translocation of PKC-alpha , -delta , and -µ with PMA treatment. Left: BON cells were preincubated for 30 min with secretion medium and then incubated with 0.1% DMSO (-) or PMA (10 nM) over a time course (given in minutes). Denatured proteins from cytosolic and membrane fractions were separated on 10% Novex Tris-Glycine gels, transferred to polyvinylidene difluoride membranes, and immunoblotted for PKC-alpha (80 kDa; A), -delta (80 kDa; B), and PKC-µ (115 kDa; C). Each blot was reprobed with actin. Representative blots from at least 3 separate experiments are shown. Right: BON cell monolayers were treated with PMA, fixed with methanol, and incubated with anti-PKC-alpha (A), -delta (B), or -µ (C) antibodies followed by incubation with Alexa-conjugated secondary antibody. Confocal micrographs demonstrate translocation to the membrane of all 3 isoforms in BON cells treated with PMA (10 nM) for 10 min (arrows).

To verify the results obtained by subcellular fractionation, we performed immunofluorescence studies to assess the effects of PMA on PKC translocation (Fig. 2, right). Consistent with our Western blot results, PKC-alpha (Fig. 2A, right), -delta (Fig. 2B, right), and -µ (Fig. 2C, right) translocated to the membrane after treatment with PMA (10 nM). Together, these results demonstrate translocation of the PKC-alpha , -delta , and -µ isoforms following PMA treatment, suggesting a role for these isoforms in PMA-mediated NT release in BON cells.

Increased kinase activity of PKC-alpha , -delta , and -µ with PMA treatment. To further establish a role for the PKC-alpha , -delta , 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-alpha , -delta , 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-alpha 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-delta antibody; the catalytic activity of PKC-delta , similar to PKC-alpha , 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-alpha -, -delta -, and -µ-associated kinase activities.


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Fig. 3.   PMA stimulates PKC-alpha -, -delta -, and -µ-associated kinase activities in BON cells. A: cells were treated with various doses of PMA for 10 min, and membrane extracts were immunoprecipitated (IP) with polyclonal anti-PKC-alpha antibody for 2 h or overnight at 4°C and incubated for another 2 h with protein A-Sepharose beads. The activity was assayed by phosphorylation of histone type III as an exogenous substrate in vitro. The radioactive bands were separated by 10% Novex Tris-Glycine gels. Densitometric analysis of the kinase activation of PKC-alpha is presented relative to basal activity (open bar). B and C: catalytic activity of PKC-delta and -µ was also detected as described above. Results are expressed as means ± SE and are representative of 3 separate experiments. *P < 0.05 vs. control (-).

Inhibitors of PKC-alpha and -delta 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, 4alpha -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-delta -specific inhibitor at 5-10 µM (17), significantly inhibited PMA-stimulated NT release, thus demonstrating a role for PKC-delta in PMA-stimulated NT secretion. The indolocarbazole compound, Gö-6976, has been shown to inhibit conventional PKC isoforms exclusively (IC50 approx  2 nM against PKC-alpha 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 approx  8 nM for PKC-alpha ) (55), significantly inhibited NT secretion.


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Fig. 4.   Effect of PKC inhibition on neurotensin (NT) secretion. BON cell monolayers were incubated with secretion medium and then preincubated with vehicle or the PKC inhibitors Gö-6976, Gö-6983, GF-109203X (GFX), or rottlerin for 30 min before addition of PMA (10 nM). After another 30 min, the medium was collected and centrifuged to remove particulate matter, and the level of NT secreted into the medium was measured by RIA using duplicate samples. Two control groups were utilized: one group treated with vehicle alone (-) and another group treated with 4alpha -PMA, the inactive isomer of PMA. Experiments were performed in triplicate; results are expressed as means ± SE (n = 6); **P < 0.05 vs. vehicle or 4alpha -PMA; *P < 0.05 vs. 10 nM PMA; dagger P < 0.05 vs. 10 nM Gö-6983; Dagger P < 0.05 vs. 10 nM GFX.

Overexpression of PKC-alpha and -delta increased PMA-stimulated NT secretion. We examined the role of PKC-alpha and -delta isoforms by overexpression of wild-type PKC-alpha and -delta in BON cells. Cells were transiently transfected with a plasmid containing either wild-type PKC-alpha or PKC-delta 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).


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Fig. 5.   Overexpression of PKC-alpha and -delta significantly increases PMA-mediated NT secretion. BON cells were transiently transfected with the empty vector (pTB701-HA) or the corresponding PKC-alpha and -delta vectors and harvested 48 h after transfection. Protein expression was analyzed by Western blotting. NT release was measured by RIA. A: overexpression of PKC-alpha was shown by using either anti-HA antibody or anti-PKC-alpha antibody (endogenous PKC-alpha expression is indicated by bottom band, and expression of PKC-alpha from the transfected plasmid is shown by top band, denoted with an asterisk). Anti-beta -actin antibody was used as loading control. BON cells were transiently transfected with the empty vector or the PKC-alpha plasmid (pTB701-HA-PKCalpha ), and 48 h later cells were preincubated with secretion medium for 30 min and then treated with vehicle (0.1%, DMSO) (open bar denotes cells transfected with empty vector and treated with vehicle) or 10 nM PMA (hatched bar denotes cells transfected with empty vector, and closed bar denotes cells transfected with the PKC-alpha plasmid). The supernatants were collected for measurement of NT secretion by RIA. B: overexpression of PKC-delta was shown using either anti-hemagglutinin (HA) antibody or anti-PKC-delta antibody (endogenous PKC-delta expression is indicated by bottom band and expression of PKC-delta from transfected plasmid is shown by top band, denoted with asterisk). Anti-beta -actin antibody was used as a loading control. NT secretion was measured by RIA as described above. Experiments were performed in triplicate. Results are expressed as means ± SE (n = 6). *P < 0.05 vs. empty vector (pTB701-HA); dagger P < 0.05 vs. PMA and empty vector.

First, BON cells were transfected with the PKC-alpha plasmid (pTB701-HA-PKCalpha ) or the empty vector; overexpression of PKC-alpha was confirmed by Western blotting and probing for the HA tag in cells transfected with the PKC-alpha plasmid (Fig. 5A, top). HA expression was not detected in the cells transfected with the empty vector. In addition, an increase in PKC-alpha expression was identified in cells transfected with the PKC-alpha plasmid (bottom band represents endogenous PKC-alpha , and top band, denoted by the asterisk, represents PKC-alpha from the PKC-alpha plasmid; Fig. 5A, middle). NT secretion was significantly increased by PMA (10 nM) treatment of BON cells transfected with the empty vector (Fig. 5A, bottom). Importantly, PMA treatment of cells transfected with the PKC-alpha plasmid resulted in a significantly enhanced NT secretion compared with PMA treatment of BON cells transfected with the empty vector.

In a similar fashion, BON cells were transfected with the PKC-delta plasmid (pTB701-HA-PKCdelta ) or the empty vector (pTB701-HA) (Fig. 5B). Western blotting demonstrated overexpression of PKC-delta in transfected cells using the anti-HA antibody (Fig. 5B, top). Moreover, blotting with the anti-PKC-delta antibody further confirmed increased PKC-delta expression in cells transfected with the PKC-delta plasmid (similar to PKC-alpha , bottom band represents endogenous PKC-delta expression and top band, denoted by the asterisk, represents PKC-delta expression from the transfected PKC-delta plasmid; Fig. 5B, middle). Treatment of the PKC-delta -transfected BON cells with PMA (10 nM) for 30 min resulted in a significantly greater increase of NT release compared with PMA treatment of cells transfected with the empty vector (Fig. 5B, bottom).

Inhibition of PKC-alpha and -delta 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-alpha and -delta 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-alpha and -delta isoforms in NT secretion stimulated by BBS.


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Fig. 6.   Bombesin (BBS)-mediated NT secretion from BON/gastrin-releasing peptide (GRP) receptor (GRP-R) cells. A: BON/GRP-R cells were treated with 0.1% DMSO (-) or BBS at the indicated doses for 15 min. NT release was measured by RIA. Results are expressed as means ± SE (n = 6). *P < 0.05 vs. vehicle control (0.1% DMSO). B: BON/GRP-R cells were preincubated with vehicle or PKC inhibitors (Gö-6976, Gö-6983, or rottlerin) for 30 min before addition of BBS (10 nM). After another 15 min, medium was collected and centrifuged to remove particulate matter, and the level of NT secreted into the medium was measured by RIA using duplicate samples. Two control groups were used: an untreated group (-) and a group treated with vehicle alone (0.1% DMSO). Experiments were performed in triplicate. Results are expressed as means ± SE (n = 6). *P < 0.05 vs. BBS alone; dagger P < 0.05 vs. no treatment (-) and DMSO (0.1%).

Overexpression of PKC-alpha and -delta increased BBS-stimulated NT secretion. To further confirm the role of PKC-alpha and -delta isoforms in BBS-mediated NT release in BON/GRP-R cells, the cells were transiently transfected with wild-type PKC-alpha (Fig. 7A) and -delta (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-alpha was confirmed by Western blotting by using the anti-HA antibody (Fig. 7A) or anti-PKC-alpha antibody (Fig. 7A; bottom bands represent endogenous PKC-alpha and top bands, denoted by the asterisk, represent PKC-alpha from the PKC-alpha plasmid). NT secretion was significantly increased by BBS (10 nM) treatment of BON/GRP-R cells transfected with wild-type PKC-alpha 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-delta (Fig. 7B). Therefore, these findings further support a role for the PKC-alpha and -delta isoforms in BBS-mediated NT release.


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Fig. 7.   Overexpression of PKC-alpha and -delta significantly increases BBS-mediated NT secretion in BON/GRP-R cells. BON/GRP-R cells were transiently transfected with the empty vector (pTB701-HA) or the corresponding PKC-alpha and -delta vectors and harvested 48 h after transfection. NT release was measured by RIA. Protein expression was analyzed by Western blotting. A: overexpression of PKC-alpha was demonstrated by probing with anti-HA antibody and anti-PKC-alpha antibody (endogenous PKC-alpha expression is indicated by bottom band and expression of PKC-alpha from the transfected plasmid is shown by top band, denoted with an asterisk). Anti-beta -actin antibody was used as loading control. BON/GRP-R cells were transiently transfected with the empty vector or the PKC-alpha plasmid (pTB701-HA-PKCalpha ), and 48 h later treated with vehicle or BBS (10 nM). The supernatants were collected for measurement of NT secretion by RIA. B: overexpression of PKC-delta was demonstrated by probing with anti-HA antibody or anti-PKC-delta antibody (endogenous PKC-delta expression is indicated by bottom band and expression of PKC-delta from transfected plasmid is shown by top band, denoted with the asterisk). Anti-beta -actin antibody was used as a loading control. NT secretion was measured by RIA as described above. Experiments were performed in triplicate. Results are expressed as means ± SE (n = 6). *P < 0.05 vs. empty vector (pTB701-HA); dagger P < 0.05 vs. BBS and empty vector.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma is predominantly expressed in the brain and spinal cord (41), PKC-theta is expressed in T cells (37) and skeletal muscle (44), and PKC-eta 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-gamma , -theta , and -eta , in the BON endocrine cell line, with predominant expression of PKC-alpha , -beta 2, -delta , and -µ. An assessment of PKC isoforms in other endocrine cells has been performed with varying results depending on cell type. The PKC isoforms alpha , beta 2, delta , epsilon , and zeta  are expressed in whole pancreatic islets of mouse and rat and in the insulinoma-derived beta -cells (24, 28). PKC-iota was identified in the insulin-secreting cell line RINm5F (49), and recently the presence of PKC-µ has been demonstrated in the MIN6 beta -cell line (52). PKC-alpha , -delta , -epsilon , and -zeta predominate in the FRTL-5 thyroid cell line (32, 54), and PKC-alpha , -beta , -epsilon , and -zeta 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 alpha , gamma , theta , epsilon , zeta , and µ. Therefore, most endocrine cells, including the BON cell line, express the alpha , epsilon , and zeta  isoforms but differ with respect to beta , gamma , delta , eta , theta , and µ.

In BON cells, PKC-alpha 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-alpha associated with small vesicles distributed throughout the cytoplasm in human antral G cells by using confocal imaging. We demonstrate that PKC-delta 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-alpha and -delta 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-alpha and -delta 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-alpha and -delta 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 alpha -, beta -, and zeta -isoforms (27, 29). In contrast, stimulation of gastrin release from antral G cells results in translocation of PKC-gamma and -theta (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-delta and -alpha 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-delta -specific inhibitor rottlerin before PMA stimulation. In contrast, transfection of the PKC-delta plasmid, which overexpresses PKC-delta , significantly enhanced PMA-mediated stimulation. Therefore, these results, together with our findings of PKC-delta translocation and induction of in vitro kinase activity, strongly suggest a role for this isoform in the stimulated release of NT. Although the PKC-delta 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-alpha (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-alpha as well in stimulated NT release. These findings are further supported by our demonstration that overexpression of PKC-alpha elicited significant PMA-stimulated NT release. Similar to our findings, PKC-alpha appears to be involved in thyroid hormone secretion (32, 54) and contributes to insulin release from beta -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-alpha and -delta 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-alpha and PKC-delta . 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-gamma 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-delta inhibitor rottlerin. The role of PKC-alpha and -delta isoforms in BBS-mediated NT release was also supported by the overexpression of wild-type PKC-alpha and -delta . 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-alpha in BBS-stimulated hormone release.

In conclusion, we demonstrate expression of all 11 PKC isoforms with prominent expression of the PKC-alpha , -beta 2, -delta , and -µ isoforms. Stimulation of BON cells with PMA resulted in the release of NT peptide, translocation of the PKC-alpha , -delta , and -µ isoforms, and induction of in vitro kinase activity. The role for the PKC-alpha and -delta isoforms was further confirmed by using a battery of PKC inhibitors that blocked PMA-mediated NT release and overexpression of PKC-alpha and -delta , which enhanced PMA-mediated NT release. In addition, the PKC-alpha and -delta 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 alpha  and delta  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.


    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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