help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takekoshi, K.
Right arrow Articles by Nakai, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takekoshi, K.
Right arrow Articles by Nakai, T.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Endocrinology Vol. 142, No. 11 4861-4871
Copyright © 2001 by The Endocrine Society

ARTICLES

Leptin Stimulates Catecholamine Synthesis in a PKC-Dependent Manner in Cultured Porcine Adrenal Medullary Chromaffin Cells

Kazuhiro Takekoshi, Kiyoaki Ishii, Toru Nanmoku, Shunsuke Shibuya, Yasushi Kawakami, Kazumasa Isobe and Toshiaki Nakai

Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, 305-8575, Japan

Address all correspondence and requests for reprints to: Dr. Kazuhiro Takekoshi, Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305-8575, Japan. E-mail: k-takemd{at}md.tsukuba.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that murine recombinant leptin directly stimulates catecholamine synthesis through the long form of the leptin receptor (Ob-Rb) expressed in cultured porcine chromaffin cells. Additionally, we found that leptin activates IP3 production after PLC activation. It is well established that activation of PLC elicits IP3 production as well as an increase in diacylglycerol, a compound that stimulates PKC. Therefore, we investigated the involvement of PKC in leptin-induced catecholamine synthesis. Leptin was found to induce significant increases in PKC activity in a dose-dependent manner (1, 10, and 100 nM); chelation of extracellular Ca2+ by EDTA abolished this PKC stimulatory activity. We also confirmed by Western blot analysis that leptin (at 100 nM) induced significant increases in Ca2+-dependent PKC{alpha}, -ßI, and -{gamma} expression. The activity of the rate-limiting enzyme tyrosine hydroxylase (TH) in the biosynthesis of catecholamine is regulated at the transcriptional and posttranscriptional levels. TH enzyme activity and TH mRNA levels induced by 100 nM leptin were significantly inhibited by the PKC inhibitor Ro 32-0432 as well as by EDTA. In addition, increases in TH protein and intracellular catecholamine content stimulated by leptin were completely inhibited by Ro 32-0432. Leptin markedly activated ERKs and, to a lesser extent, JNK; these stimulatory effects on ERKs and JNK were completely inhibited by Ro 32-0432 as well as EDTA. In contrast, leptin did not activate P38 MAPK. Similar to leptin, PMA activated ERK and JNK. Nicardipine and {omega}-conotoxin GVIA, each at 1 µM, were effective at inhibiting leptin-induced TH enzyme activity, TH mRNA accumulation, PKC activity, and ERK activity. Leptin increased activating protein-1 DNA-binding activity, and this was diminished by Ro 32-0432 as well as EDTA, similar to the reduction of TH mRNA levels. In addition, using supershift analysis, we documented the involvement of c-Fos and, to a lesser extent, c-Jun in leptin-induced activating protein-1 activity. These results indicate that leptin stimulates Ca2+-dependent PKC isoform-dependent catecholamine synthesis in porcine chromaffin cells. Previously, we had shown that leptin stimulated cAMP. The present study also showed that H89 (a PKA inhibitor) moderately, but significantly, inhibited leptin-induced ERK and TH mRNA. Consistent with this finding, leptin is shown here to activate novel PKC{epsilon}, which is assumed to stimulate Raf, upstream of ERKs, via cAMP, supporting the suggestion that Ca2+-independent novel PKC may also play some physiological role in regulating catecholamine synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE ob gene product, is secreted mainly by mature adipocytes (1). Leptin acts not only as a satiety factor, but also affects energy expenditure through hypothalamic effects (1, 2, 3, 4, 5). The long form of the leptin receptor (Ob-Rb) is expressed at high levels in the hypothalamus and associates with the Janus family of tyrosine kinase-signal transducers and activators of transcription (JAK-STAT) (6, 7, 8, 9, 10, 11, 12, 13).

Leptin, however, is a member of the cytokine family, and its signal transduction system may be similar to that of other cytokines, suggesting that pathways other than JAK-STAT may be involved. Earlier studies had shown that MAPKs are involved in leptin-stimulated signal transduction (14, 15, 16). MAPKs form a family of serine/threonine kinases that are activated by phosphorylation in response to a variety of extracellular stimuli. Recently, the MAPKs family has been divided into three subgroups (17): 1) the ERKs, including P44 and P42; 2) the stress-activated protein kinase/c-Jun Nterminal kinase (JNK), and 3) the p38 MAP kinase (p38 MAPK).

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamine (18). TH activity can be regulated by both short- and long-term mechanisms. Short-term regulation of enzyme activity occurs at the posttranscriptional level. Central to this regulation is the phosphorylation of TH, which results in activation of the enzyme (19, 20). Indeed, TH is phosphorylated and activated by a variety of protein kinases, including PKC (21, 22). A long-term regulation has been shown to be exerted at the TH protein synthesis level after TH gene transcription (23). Similar to their effect on TH enzyme activity, several protein kinases, including PKC, also induce an increase in levels of TH mRNA (24).

Several transcription factors were reported to bind to the 5'-flanking sequences of the TH gene, including activating protein-1 (AP-1), AP-2, POU/OCT, SP-1, and cAMP response element sites. In particular, the AP-1 site is highly conserved in rat, murine, bovine, and human TH genes, suggesting that it may be important for transcriptional regulation of the TH gene (25, 26). Indeed, binding of the AP-1 (Fos/Jun) heterodimer to the TH-tetradecanoyl phorbol 13-acetate-responsive element (TH-TRE) site is a prerequisite for TH gene activation in rat pheochromocytoma PC12 cells (27).

Recently, novel peripheral roles for leptin have been identified. Indeed, we have previously shown that leptin directly induces catecholamine synthesis through Ob-Rb expressed in cultured porcine chromaffin cells (28, 29). These results indicated that 1) leptin (>=1 nM) stimulates TH enzyme activity, which is completely inhibited by depletion of extracellular Ca2+; 2) murine recombinant leptin (>=1 nM) stimulates TH mRNA accumulation associated with TH protein increases; 3) leptin causes an increase in the activity of MAPKs, accompanied by increased phosphorylation of STAT-3 and -5, but not STAT-1; 4) TH mRNA increases stimulated by leptin are inhibited by the MAPK kinase-1-inhibitor PD-98059; 5) leptin increases intracellular Ca2+ ([Ca2+]i) through activation of voltage-dependent Ca2+ channels (VDCC); and 6) leptin increases [Ca2+]i by mobilizing Ca2+ from intracellular pools accompanied by IP3 production.

It is established that activation of PLC elicits IP3 production as well as increased diacylglycerol (DAG), a compound that stimulates PKC. The PKC family consists of three isoforms: classical Ca2+-dependent PKC [(c)PKC] ({alpha}, ßI, ßII, and {gamma}), which are sensitive to Ca2+, DAG, or phorbol esters; novel PKC [(n)PKC] ({delta}, {epsilon}, {eta}, and {theta}), which are regulated by DAG or phorbol esters but are independent of Ca2+; and atypical PKC [(a)PKC] ({zeta}, {lambda}, µ), which are insensitive to Ca2+, DAG, and phorbol esters (30, 31). As described above, growing evidence suggests that activation of PKC plays an important role in regulating catecholamine synthesis in chromaffin cells. However, the involvement of PKC in leptin-induced TH activity is not yet determined.

The major aim of the present study was to delineate the role of PKC in leptin-induced catecholamine synthesis in cultured porcine adrenal medullary cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Unless otherwise noted, all reagents were purchased from Wako Seiyaku (Tokyo, Japan). Recombinant murine leptin was purchased from PeproTech (London, UK). H89 was purchased from Sigma (St. Louis, MO). The MAPK kinase-1 inhibitor, PD-98059, was purchased from New England Biolabs, Inc. (Beverly, MA). Ro 32-0432, a PKC inhibitor, was purchased from Calbiochem (La Jolla, CA).

Cell culture
Primary dissociated cells from porcine adrenal medulla were prepared and purified by the differential plating method as previously described (28, 29). In brief, the cells (1 x 106) were plated into 35-mm polystyrene dishes and maintained as a monolayer culture in DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% FBS (Life Technologies, Inc.), 100 U/ml penicillin, 100 µg/ml streptomycin, and 1.3 µg/ml fungizone in a humidified atmosphere of 5% CO2/95% O2 at 37 C for 2–3 d, then used for experiments.

TH enzyme activity
TH enzyme activity was measured using a method previously published by Kumai et al. (32). Experiments were initiated by replacing the medium with HEPES-buffered Krebs buffer containing various test substances, and the cells were incubated at 37 C for 10 min. The cells were then homogenized in 0.25 M sucrose (50 vol) using a glass tissue grinder. The standard incubation medium consisted of the following components in a total volume of 250 µl: 100 µl tissue homogenate, 40 µl 1 M sodium acetate buffer (pH 6.0), 40 µl 1 mM L-tyrosine or D-tyrosine, 20 µl 1 M 6-methyl-5,6,7,8-tetra-hydropterine in 1 M 2-mercaptoethanol, 20 µl 20 mM catalase, and 30 µl water. The medium was incubated at 37 C for 30 min, and the reaction was stopped with 1 M perchloric acid containing dihydroxybenzylamine as an internal standard and then 0.2 M EDTA in an ice bath. Finally, 1 M potassium carbonate and 0.2 M Tris-HCl (pH 8.5) containing 1% EDTA were added. The 3-(3,4-di-dihydroxyphenyl)alanine was extracted using the aluminum oxide method. Forty microliters of extracted medium were mixed with 0.1 N NaOH and TSK-GEL ODS-120T (TOSOH, Tokyo, Japan) and analyzed by HPLC. The mobile phase consisted of the following components: 50 mM sodium acetate, 20 mM citric acid, 12.5 mM sodium octyl sulfate, 1 mM di-n-butylamine, and 0.134 mM EDTA. All separations were performed isocratically at a flow rate of 0.6 ml/min at 28 C. The detector potential was maintained at +0.65 V. The TH enzyme activity was calculated as the amount of 3-(3,4-di-dihydroxyphenyl)-alanine formed from tyrosine per mg protein/min.

Northern blot analysis
For Northern blot analysis, total RNA was extracted from the samples using an ISOGEN kit (Nippon Gene, Tokyo, Japan). The RNA concentration was determined spectrophotometrically (at 260 nm). RNA (10 µg sample) was fractionated by electrophoresis on 1% agarose/5% formaldehyde gels (80 V, 2 h). After staining with ethidium bromide and visual inspection of the UV fluorescence to confirm the presence of equal amounts of 18S and 28S ribosomal RNA in each lane, the RNA was transferred to a nitrocellulose membrane and hybridized to 32P-labeled probes. The probe used for TH was a 1.9-kbp EcoRI fragment of pTHT1. Plasmid pTHT1 contained the full-length cDNA for human TH type 1 cDNA. This plasmid was developed by T. Nagatsu and was provided by the RIKEN Gene Bank (Ibaraki, Japan). The probe was labeled using a random primer extension labeling kit (NEN Life Science Products, Boston, MA). Rat glyceraldehyde3-phosphate dehydrogenase cDNA was used as an internal standard (CLONTECH Laboratories, Inc., Palo Alto, CA). Hybridization signals were scanned in an image analyzer (BAS2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).

Western blot analysis for TH
Western blot analyses were performed as previously described (28, 29). In brief, the cells were solubilized with 0.1% SDS containing 1% Triton X-100, 1% sodium deoxycholate, and 20 mM Tris-HCl, pH 7.4. The supernatant containing 10 mg protein was separated on 10% SDS-polyacrylamide gels, then transferred to nitrocellulose using a Transblot apparatus (Bio-Rad Laboratories, Inc.). After transfer, the nitrocellulose sheets were incubated for 1 h with BLOTTO buffer (5% skimmed milk, 0.05% Triton X-100, 100 mM NaCl, and 200 mM Tris-HCl, pH 7.4). The nitrocellulose membranes were then washed three times for 10 min each time with 0.05% Triton X-100, 20 mM Tris-HCl (pH 7.4), and 150 mM NaCl, then for 1 h with 1 mg/ml of a monoclonal antibody to TH (Roche, Mannheim, Germany). The nitrocellulose membranes were then washed three times for 10 min each time with 0.05% Triton X-100, 20 mM Tris-HCl (pH 7.4), and 150 mM NaCl and incubated for 1 h with horseradish peroxidase-labeled protein A (Amersham Pharmacia Biotech, UK). Finally, the blots were washed three times, incubated with ECL reagent (Amersham Pharmacia Biotech, Little Chalfont, UK) for 1 min, and photographed on Polaroid (Fuji, Tokyo) films (ISO 3000).

Western blot analysis for ERKs
Phosphorylation of ERKs proteins from porcine adrenal medullary cells was measured using an antibody kit (New England Biolabs, Inc.) according to the manufacturer’s instructions. Very briefly, cells were starved for 4 h, and then the medium was replaced with HEPES-buffered Krebs buffer including various test substances. Lysates were immunoprecipitated with anti-ERK antibody. Immunoprecipitates were subjected to 7.5% SDS-PAGE and analyzed by immunoblotting using antiphosphotyrosine antibody.

Western blot analysis for PKC isoforms
In brief, cells were starved for 4 h, and then the medium was replaced with HEPES-buffered Krebs buffer containing leptin (100 nM). The incubation time was 10 min. The cells were washed with PBS, then lysed in lysis buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue]. Lysates were immunoprecipitated with PKC antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1 µg/ml, in blocking solution). Immunoprecipitates were subjected to 7.5% SDS-PAGE and analyzed by immunoblotting using goat antirabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc.).

PKC activity
PKC activity was measured in cultured confluent cells as previously described (33). Cell cultures (in 90-mm dishes) were incubated in control medium (5% glucose) or with test substance for 10 min. The cells were then harvested, homogenized by sonification in 20 mM Tris-HCl (pH 7.5), and incubated for 30 min with 100 µl reaction buffer solution containing a pseudosubstrate and various phospholipids from a commercially available kit (Pep Taq-Nonradioactive PKC activity kit, Promega Corp., Madison, WI). The reaction was terminated by heating, and the reaction mixture was separated into phosphorylated and nonphosphorylated substrates on a 0.9% agarose gel. For quantification, the gel bands were visualized by UV light, then excised, and the absorbance was measured at 570 nm. The results are expressed in OD units.

Measurement of JNK activity
JNK activity was measured by solid phase kinase assay as previously described (34). Cell lysates were incubated with glutathione-S-transferase-c-Jun-(1–79) fusion protein bound to glutathione-Sepharose beads for 8 h at 4 C. Beads were recovered by centrifugation (10,000 x g for 30 sec), washed twice with buffer A [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µl/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride], and then ß-glycerophosphate washed twice with a kinase buffer [25 mM Tris-HCl (pH 7.5), 5 mM, 2 mM dithiothreitol, 0.1 mM Na3VO4, and 10 mM MgCl2]. The beads were then incubated with 50 µl kinase buffer containing 100 µM ATP (at 37 C for 30 min). Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. To detect phosphorylated c-Jun, the membranes were incubated with polyclonal anti-phospho-c-Jun (1 h at 25 C). The blots were then incubated with peroxidase-conjugated goat anti-IgG (1 h at 25 C), and the proteins were detected using the ECL system.

Measurement of p38 MAPK activity
p38 MAPK activity was measured by solid phase kinase assays as previously described (34). Cell lysates were incubated with 4 µl anti-p38 MAPK (overnight at 4 C). The immunocomplexes were preincubated with protein G-Sepharose (2 h at 4 C). The beads were recovered by centrifugation (1000 x g for 30 sec) and then washed twice with buffer A and twice with a kinase buffer. p38 MAPK activity in immunoprecipitates was measured using the p38 MAPK assay kit (New England Biolabs, Inc.) according to the manufacturer’s instructions. Briefly, the beads were incubated with 50 µl kinase buffer containing 200 µM ATP and 2 µg activating transcription factor-2 as a substrate (30 min at 30 C). SDS-PAGE and Western blot were performed as described above. Phosphorylated activating transcription factor-2 was detected using polyclonal phospho- activating transcription factor-2-specific antibody.

Determination of intracellular catecholamine levels
Cells were treated with test substances for 24 h. The catecholamine levels in the cells were determined as described previously (35), using a catecholamine autoanalyzer (TOSOH, H8030) with a built-in high performance liquid chromatograph and a spectrofluorometer.

Gel mobility shift assay
Nuclear extracts from cells were isolated as previously described (36). Synthetic oligonucleotide (TRE: 5'-CAG GAT GAT TCA GAG GCA GG -3') was used as a probe and competitor. Sense and antisense strands of oligonucleotide were annealed and then labeled using a T4 polynucleotide kinase kit with [{alpha}-32P]ATP (3000 Ci/mmol). Nuclear extracts (5 µg) were preincubated for 15 min at 0 C in a buffer [10 mM HEPES (pH 7.8), 50 mM KCl, 1 mM EDTA, 5 mM Mg Cl2, 1 mM dithiothreitol, 0.7 mM phenylmethylsulfonylfluoride, 5% glycerol, and 5 µg poly(dI-dC)] with or without antisera or competitor oligonucleotide. End-labeled oligonucleotide probes (0.5 ng) were then added to each mixture, and incubation was carried out for another 30 min at room temperature. DNA-protein complexes were resolved on 5% polyacrylamide gels and then visualized using a BAS 2000 image analyzer (Fuji Photo Film Co., Ltd.).

Statistical analysis
Data groups were analyzed by one-way ANOVA by means of the StatView computer software program (Abacus Concepts, Inc., Berkeley, CA). When ANOVA showed significant differences, post-hoc analysis was performed by Tukey’s test. P < 0.05 was considered significant. All data are expressed as the mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin stimulates TH enzyme activity in a PKC-dependent manner in porcine adrenal medullary cells
We previously reported that leptin (>=1 nM) significantly stimulated TH enzyme activity (29). To determine whether PKC was involved in leptin-induced TH enzyme activity, PKC was blocked by the PKC inhibitor, Ro 32-0432 (100 nM) (37). This resulted in complete inhibition of the TH enzyme activity induced by leptin (Fig. 1Go). Additionally, we examined whether ERKs were involved in leptin-induced TH enzyme activity. Pretreatment of cells with the MAPK kinase-1 inhibitor PD-98059 (50 µM) inhibited leptin (100 nM)-induced TH enzyme activity significantly (Fig. 1Go). We previously showed that chelation of extracellular Ca2+ by EDTA completely suppressed 100 nM leptin-induced TH enzyme activity (29). To establish whether L-type and/or N-type Ca2+ channels were functionally involved in TH enzyme activity induced by 100 nM leptin, we examined the effects of nicardipine or {omega}-conotoxin GVIA on this process. As shown in Fig. 1Go, nicardipine and {omega}-conotoxin GVIA significantly inhibited leptin-induced TH enzyme activity by 67% and 28%, respectively. In contrast, xestospongin C (a membrane-permeable blocker of IP3-mediated Ca2+ release) did not alter TH enzyme activity induced by leptin at this concentration, indicating that internal Ca2+ may have a minimal role in regulating TH enzyme activity (29). In agreement with this previous study, the intracellular Ca2+ chelator BAPTA-AM (at 30 µM) did influence 100 nM leptin-induced TH enzyme activity (Fig. 1Go).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Leptin stimulates TH enzyme activity in a PKC-dependent manner. Cells were incubated for 10 min with leptin alone (100 nM), leptin plus Ro 32-0432 (Ro; 100 nM, 30-min pretreatment), leptin and PD-98059 (PD; 50 µM, 1-h pretreatment), leptin and BAPTA (30 µM, 30-min pretreatment), or leptin and nicardipine or {omega}-conotoxin (1 µM each, 30-min pretreatment). TH enzyme activity was then measured as described in Materials and Methods. The values represent the mean ± SD (n = 6). *, Significantly different (P < 0.05) from the basal level. #, Significantly different (P < 0.05) from the value induced by leptin (100 nM) alone.

 
Leptin stimulates TH mRNA in a PKC-dependent manner
We previously reported that leptin (>=1 nM) significantly stimulated increased TH mRNA levels (28). This stimulatory effect was completely abolished by Ro 32-0432 (at 100 nM). Comparable to TH enzyme activity, EDTA, but not 1-2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), abolished leptin-induced TH mRNA accumulation (Fig. 2Go, A and B). Nicardipine and {omega}-conotoxin GVIA, each at 1 µM, were effective at decreasing leptin-induced TH mRNA levels by 53% and 21%, respectively (Fig. 2Go, C and D). Previously, we had shown that leptin stimulated cAMP (28). Further, to confirm that cAMP-PKA is indeed involved in TH mRNA accumulation caused by leptin, we examined the effect of a PKA inhibitor, H89, on the level of leptin-induced TH mRNA. H89 (10 µM) significantly inhibited leptin-induced TH mRNA accumulation by 37% (Fig. 2Go, C and D). The concentration of H89 was set at 10 µM according to Marley et al. (38) and Hwang et al. (39).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 2. Leptin stimulates TH mRNA in a PKC-dependent manner. Cells were incubated for 8 h with A) leptin alone (100 nM), leptin plus Ro 32-0432 (Ro; 100 nM, 30-min pretreatment), leptin plus EDTA (0.5 mM, 10-min pretreatment), or leptin plus BAPTA (30 µM, 30-min pretreatment); or B) leptin and PD-98059 (PD; 50 µM, 1-h pretreatment), leptin and nicardipine or {omega}-conotoxin (1 µM each, 30-min pretreatment), or leptin and H89 (10 µM, 30-min pretreatment). RNA (10 µg/lane) was then isolated and used for Northern blot analysis as described in Materials and Methods. Representative data are shown. B and D, The values represent the mean ± SD (n = 4–6). *, Significantly different (P < 0.05) from basal level. #, Significantly different (P < 0.05) from the value induced by leptin (100 nM) alone.

 
Effect of leptin on PKC activity
We examined the effect of leptin on PKC activity. As shown in Fig. 3AGo, leptin (1, 10, and 100 nM) significantly induced increases in PKC activity by approximately 1.3-, 1.9-, and 2.4-fold, respectively. PKC has been shown to phosphorylate and activate Raf, and this may lead to ERK activation (40, 41). In agreement with these reports, the stimulatory effect of leptin on PKC activity was completely abolished by Ro 32-0432, whereas PD-98059 (50 µM) did not affect PKC activity (Fig. 3BGo), suggesting that PKC could lie upstream from ERKs in the signaling cascade in our experimental system. Depletion of external Ca2+ by EDTA significantly suppressed, but did not completely abrogate, leptin-induced PKC activity (Fig. 3BGo). Nicardipine and {omega}-conotoxin GVIA, each at 1 µM, attenuated leptin-induced PKC activity by 48% and 24%, respectively. In contrast, BAPTA-AM (30 µM) did not alter leptin-induced PKC activity. These findings indicate that mobilization of Ca2+ from extracellular pools, but not from intracellular pools, may contribute to the PKC activity induced by leptin.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. Effects of leptin on PKC activity in porcine adrenal medullary cells. A, Cells were incubated for 10 min with various concentrations of leptin (1–100 nM) alone. B, Cells were incubated for 10 min with leptin alone (100 nM), leptin and Ro 32-0432 (Ro; 100 nM, 30-min pretreatment), leptin and PD-98059 (PD; 50 µM, 1-h pretreatment), leptin and EDTA (0.5 mM, 10-min pretreatment), leptin and BAPTA (30 µM, 30-min pretreatment), or leptin and nicardipine or {omega}-conotoxin (1 µM each, 30-min pretreatment) as indicated. PKC activity was then assayed as described in Materials and Methods. *, Significantly different (P < 0.05) from the basal value. #, Significantly different (P < 0.05) from the value induced by leptin (100 nM) alone.

 
Effect of leptin on PKC isoforms
We determined that porcine chromaffin cells contain {alpha}, ßI, ßII, {gamma}, {theta}, {epsilon}, and {zeta} PKC isoforms (data not shown). To confirm whether (c)PKC isoforms are indeed involved in leptininduced PKC activity, we initially tested (c)PKC isoforms, {alpha}, ßI, ßII, ß, and {gamma} by Western blot analysis. As shown in Fig. 4Go, leptin (100 nM) significantly induced increases in PKC-{alpha}, -ßI, and -{gamma} expression by approximately 2.1-, 1.6-, and 3.2-fold, respectively. Conversely, the expression of PKC ßII caused by leptin was not altered under these experimental conditions. Furthermore, we tested the effect of leptin on (n)PKC and (a)PKC. Leptin (100 nM) significantly induced increases in (n)PKC-{epsilon} by approximately 1.6-fold. However, neither (n)PKC-{theta} nor (a)PKC-{zeta} was induced by leptin.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. Effects of leptin on PKC isoforms in porcine adrenal medullary cells. Cells were incubated for 10 min with leptin (100 nM). PKC isoforms in chromaffin cells were then assayed by Western blot as described in Materials and Methods. Representative data are shown.

 
Effects of PKC inhibitors on leptin-induced TH-protein levels and intracellular catecholamine levels
To confirm the involvement of PKC in increased catecholamine synthesis after TH mRNA induction, the effects of leptin on TH protein levels were examined. Cells were treated with leptin (100 nM) alone or in the presence of Ro 32-0432 for 24 h. TH protein was then assessed by Western blot analysis. Ro 32-0432 completely inhibited leptininduced TH protein production, as it did TH mRNA (Fig. 5Go). To further confirm this finding, we examined intracellular catecholamine levels. Cells were treated with either leptin alone or leptin together with Ro 32-0432 for 24 h, followed by HPLC assay of intracellular catecholamine levels. As shown in Table 1Go, Ro 32-0432 significantly attenuated leptin-induced intracellular catecholamine levels.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 5. Effect of leptin on the TH protein level in cultured porcine adrenal medullary cells. A, Cells were incubated for 24 h with either leptin (100 nM) or leptin and Ro 32-0432 (Ro; 100 nM). TH protein levels were then measured by Western blot as described in Materials and Methods. Representative data are shown. B, The values represent the mean ± SD (n = 6). *, Significantly different (P < 0.05) from basal level. #, Significantly different (P < 0.05) from the value induced by leptin (100 nM) alone.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Effect of Ro 32-0432 on leptin-induced intracellular catecholamine content

 
Effect of leptin on ERKs
We had previously shown that leptin (1, 10, and 100 nM) induced significant increases in MAPK activity in a dose-dependent manner (29). In addition, PD-98059 (50 µM) completely inhibited leptin-induced MAPK activity. To elaborate on this finding, we used a specific antibody directed against the phosphorylated form of ERK. Similar to our previous findings (29), leptin (100 nM) significantly increased the activity of ERKs by about 5.9-fold, and this was completely inhibited by PD-98059 (50 µM; Fig. 6Go). We also examined the involvement of (c)PKC in leptin-induced ERK activity. Leptin-induced ERK phosphorylation was inhibited by Ro 32-0432. Also, depletion of external Ca2+ by EDTA significantly, but incompletely, suppressed leptin (100 nM)induced ERK activity. In the presence of nicardipine (1 µM) or {omega}-conotoxin GVIA (1 µM), leptin-induced ERK levels were inhibited by approximately 52% and 21%, respectively. H89 (10 µM) significantly inhibited leptin-induced ERK activity by 42%. The stimulatory effect of leptin on ERKs was mimicked by treatment with the PKC activator PMA (100 nM) for 10 min. These findings indicate that leptin may activate ERKs through a (c)PKC-dependent pathway, in agreement with our PKC profile (Fig. 4Go).



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Effect of leptin on ERK activity in porcine chromaffin cells. A, Cells were incubated for 10 min with leptin alone (100 nM), leptin plus Ro 32-0432 (Ro; 100 nM, 30-min pretreatment), leptin plus PD-98059 (PD; 50 µM, 1-h pretreatment), leptin plus EDTA (0.5 mM, 10-min pretreatment), PMA alone (100 nM, 15 min), leptin plus nicardipine or {omega}-conotoxin (1 µM each, 30-min pretreatment), or leptin plus H89 (10 µM, 30-min pretreatment). ERK activity was determined as described in Materials and Methods. Representative data are shown. B, The values represent the mean ± SD (n = 4) of the densitometric measurement of the phospho-ERK (pERK1+ pERK2). *, Significantly different (P < 0.05) from basal value. #, Significantly different (P < 0.05) from value induced by leptin (100 nM).

 
Effect of leptin on JNK activity
We examined whether leptin may affect JNK activity. As shown in Fig. 7Go, leptin (100 nM) moderately, but significantly, induced increases in JNK activity by about 2.7-fold. Similar to ERKs, EDTA significantly, but incompletely, suppressed leptin-induced ERK activity. The stimulatory effect of leptin on JNK was also abolished by Ro 32-0432, and a short exposure to PMA mimicked the effects of leptin-induced JNK activity. PD-98059 (50 µM) did not affect JNK activity.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 7. Effect of leptin on JNK activity in porcine chromaffin cells. A, Cells were incubated for 10 min with leptin alone (100 nM), leptin and Ro 32-0432 (Ro; 100 nM, 30-min pretreatment), leptin and PD-98059 (PD; 50 µM, 1-h pretreatment), leptin and EDTA (0.5 mM; 10-min pretreatment), or short-term PMA alone (100 nM, 15 min). JNK activity was determined as described in Materials and Methods. Representative data are shown. B, The values represent the mean ± SD (n = 4) of the densitometric measurement of phospho-c-Jun. *, Significantly different (P < 0.05) from basal value. #, Significantly different (P < 0.05) from value induced by leptin (100 nM).

 
Effect of leptin on p38 MAPK activity
We tested the effect of leptin on P38 MAPK activity. However, leptin had no effect on P38 MAPK activity (data not shown).

Effect of leptin on AP-1 DNA-binding activity
It has been suggested that MAPKs stimulate activation of Fos and Jun, resulting in activation of AP-1, which is a prerequisite for TH gene activation mechanisms in chromaffin cells (27). To determine whether leptin could induce AP-1 DNA-binding activity, nuclear extracts from leptin-stimulated chromaffin cells were incubated with 32P-labeled AP-1 consensus sequences. As shown in Fig. 8AGo, 100 nM leptin increased the DNA-binding activity of AP-1. The specificity of this DNA-binding activity was confirmed by its marked reduction in the presence of an excess of unlabeled AP-1 probe (36). To further determine whether PKC may be involved in leptin-induced AP-1 DNA-binding activity, we examined the effect of Ro 32-0432 on this. Pretreatment of cells with Ro 32–0432 abolished the stimulatory effect of leptin on AP-1 activity. In addition, the effect of leptin on AP-1 formation was blocked by chelation of extracellular Ca2+ by EDTA as well as PD-98059. Further to investigate the involvement of c-Fos and c-Jun in leptin-induced AP-1 activity, we added antibodies against c-Fos and c-Jun to the binding reaction (supershift analysis). As shown in Fig. 8BGo, leptin-induced AP-1 consisted mainly of c-Fos and, to a smaller extent, c-Jun proteins.



View larger version (70K):
[in this window]
[in a new window]
  		Figure 8. Effect of leptin on AP-1 DNA-binding activity in porcine chromaffin cells. A, Cells were exposed to 100 nM leptin for 2 h, then nuclear extracts were subjected to a gel mobility shift assay. Cells were pretreated with Ro 32-0432, PD-98059, or EDTA for 60 min, followed by stimulation with leptin (100 nM). Nuclear extracts were then incubated with a radiolabeled oligonucleotide containing AP-1-binding sites in the absence or presence of unlabeled AP-1. A representative autoradiogram is shown (n = 4). B, Nuclear extracts were incubated with a radiolabeled oligonucleotide in the presence or absence of antibodies against c-Jun or c-Fos. Supershifted DNA-binding complexes containing c-Jun or c-Fos protein are indicated by the arrowhead. A representative autoradiogram is shown (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrated that leptin significantly increased PKC activity in a dose-dependent manner in cultured porcine chromaffin cells. It is of note that the stimulatory effect of leptin on PKC was observed at a physiological concentration (1 nM). The increase in PKC activity stimulated by leptin (100 nM) was completely inhibited by Ro 32-0432, confirming that Ro 32-0432 is indeed a PKC inhibitor in our experimental system (37). It should be noted that the increases in both TH enzyme activity and mRNA levels evoked by leptin stimulation were inhibited by Ro 32-0432. In addition, we showed that Ro 32-0432 markedly inhibited leptin-induced TH protein levels as well as intracellular catecholamine levels. These findings support the idea that leptin stimulates catecholamine synthesis in a PKC-dependent manner.

We have previously reported that leptin increases [Ca2+]i mainly through activation of L- and N-type Ca2+ channels, resulting in catecholamine secretion (29). The present study showed that TH mRNA induction by leptin occurred in an extracellular Ca2+-dependent manner. Furthermore, leptin-induced PKC activity also occurred in an extracellular Ca2+-dependent manner, and the leptininduced PKC isoforms were of the Ca2+-dependent type, such as {alpha}, ßI, and {gamma}. Thus, it is likely that (c)PKC could play a major role in catecholamine biosynthesis evoked by leptin in chromaffin cells. Also, both L- and N-type Ca2+ channels could be involved in leptin-induced PKC activity and TH mRNA accumulation.

In contrast, BAPTA did not alter either TH enzyme activity or TH mRNA levels induced by leptin, indicating that internal Ca2+ may have a minimal role in regulating leptin-induced catecholamine synthesis. In agreement with these findings, pretreatment of cells with BAPTA did not affect PKC activity induced by leptin, supporting the suggestion that internal Ca2+ may have a minimal role in regulating PKC activity.

We previously reported that leptin significantly stimulated IP3 production after PLC activation (29). Thus, it can be suggested that leptin stimulates PLC breakdown, which leads to stimulation of the PKC pathway. This, in turn, results in the stimulation of TH enzyme activity and TH synthesis. However, the precise relationships and/or interactions between leptin receptors (JAK/STAT pathway) and PLC activation remain unclear. Further studies will be needed to clarify this point.

We demonstrated that leptin stimulated these kinases, with the strongest activation of ERKs (5.9-fold), somewhat less for JNK (2.8-fold), and not at all for p38 in our experimental system. In agreement with our findings, Bouloumie et al. (42) reported that leptin markedly induced JNK activity in human umbilical vascular epithelial cells. In addition, Harvey et al. (43) reported that leptin did not alter p38 activity in the CRI-G1 insulinoma cell line. However, the physiological relevance of the differences among the three MAPK families observed in the present study remains unclear.

Mounting evidence suggests that PKC, in particular (c)PKC, is capable of phosphorylating and activating Raf, which, in turn, results in the activation of ERKs in several systems involving PC12 cells (40, 41, 42). Indeed, short-term PMA treatment caused an activation of ERKs similar to that of leptin, confirming that PKC can trigger activation of the ERKs pathway in chromaffin cells. In addition, Ro 32-0432 inhibited ERK activity induced by leptin. These results indicate that PKC can stimulate ERKs in response to leptin in chromaffin cells. Consistent with this idea, Ro 32-0432 inhibited ERK activity induced by leptin, whereas PD-098059 did not affect PKC activity, suggesting that PKC could lie upstream of ERKs (Fig. 9Go).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 9. Outline of proposed model for the effects of leptin on TH transcription in chromaffin cells. Results were suggested by the current experiments together with data that were the basis of our previous reports (28 29 ).

 
It is also of note that leptin-induced ERK activity is dependent on extracellular Ca2+, mainly through L- and N-type Ca2+ channels. Taken together with the finding that leptin is capable of inducing (c)PKC isoforms, we speculate that induction of ERKs by leptin could be mediated by (c)PKC isoforms. Similar to the situation with ERKs, leptin also seems to activate JNK through a (c)PKC in porcine chromaffin cells.

Growing evidence suggests that transcriptional regulation of TH is controlled by the expression of immediate early genes. Immediate early genes appear to play a key role in the transduction of short-lived environmental signals into long-term changes in cell functions through binding to specific sites on their target genes. Immediate early genes are basic leucine zipper proteins that bind to DNA as dimers. Jun family proteins can bind to the TRE as homo- or heterodimers, whereas Fos family members form heterodimers with Jun family members (45, 46). Transcriptional activation of TRE-containing promoters of genes such as TH is mediated by the AP-1 complex composed of different Fos/Jun families (27).

The transcriptional activity of c-Fos and c-Jun is regulated by phosphorylation (47). It has been shown that several kinases are responsible for phosphorylation of c-Fos and c-Jun. Indeed, c-Fos protein can be phosphorylated at Ser374 by ERK (48). In addition, other kinases, including Fos-regulating kinase, PKA, and ribosomal S-6 kinase, have been shown to phosphorylate c-Fos protein (49, 50). JNK phosphorylates c-Jun at Ser63 and Ser73 (51). Recently, it has also been shown that JNK can activate c-Fos via activation of Elk-1, the target of ERK (52). We demonstrated that leptin induced AP-1 DNA-binding activity. The specificity of this DNA-binding activity was confirmed by its marked reduction in the presence of an excess of unlabeled AP-1 probe. The stimulatory effect of leptin was prevented by Ro 32-0432 as well as EDTA, consistent with the idea that leptin induction of ERKs and JNK could be mediated by (c)PKC.

It was shown by supershift analysis that leptin-induced AP-1 consisted mainly of c-Fos and, to a smaller extent, c-Jun proteins. This is consistent with our present findings that leptin much more strongly activates ERK activity compared with JNK. Thus, it can be suggested that ERK activation induced by leptin appears to be especially important in regulating TH transcription through AP-1 formation. Consistent with this idea, leptin-induced AP-1 formation was abolished by pretreatment with PD-98059. This finding was also in agreement with our previous data that leptin-induced TH mRNA was significantly inhibited by PD-98059 (29).

We previously showed that depletion of external Ca2+ completely suppressed TH enzyme activity induced by 100 nM leptin (29). The present study also showed that nicardipine and {omega}-conotoxin GVIA were effective at inhibiting leptin-induced TH enzyme activity. These findings indicate that external Ca2+ plays a critical role in regulating TH enzyme activity mainly via L- and N-type VDCC. To support these previous findings the present study showed that leptin can induce PKC activity and that these leptin-induced PKC isoforms were indeed Ca2+ dependent. In addition, TH enzyme activity induced by leptin was abolished by PKC inhibitors. It has been reported that the phosphorylation sites on TH can be identified as Ser8, Ser19, Ser31, and Ser40 in bovine chromaffin cells (53, 54). In particular, it was demonstrated that Ser31 may be phosphorylated by ERKs through PKC. In agreement with this previous finding, PD-98059 inhibited leptin-induced TH enzyme activity. Further studies (i.e. to determine which phosphorylations cause TH enzyme activation after leptin stimulation) will be required to clarify detailed mechanisms.

Another interesting point made in this study was that leptin significantly induced increases in (n)PKC isoform {epsilon}, which is assumed to be independent of Ca2+. This is in agreement with the PKC data that depletion of extracellular Ca2+ significantly, but incompletely, inhibited leptininduced PKC activity. Recently, PKC isoform {epsilon} was reported to stimulate Raf, upstream of ERKs (55). It was also shown that bradykinin-induced activation of PKC-{epsilon} was mediated via the stimulation of cAMP in PC12 cells (56). We previously reported that leptin induced increases in cAMP production (28). Thus, it is possible that the cAMP production induced by leptin may converge to the PKC-{epsilon} (Fig. 9Go). Similar to PKC activity, it is of note that depletion of extracellular Ca2+ did not completely inhibit leptin-induced TH mRNA accumulation or ERKs and JNK activity. Furthermore, a PKA inhibitor moderately, but significantly, inhibited leptin-induced ERK and TH mRNA accumulation. Although we have no evidence that the increase in cAMP is associated with increased PKC-{epsilon} in our experimental system, it can be suggested that this cAMP-PKC-{epsilon}-mediated pathway may play some physiological role in regulating leptin-induced catecholamine synthesis.

In conclusion, we can summarize our findings on leptin-induced TH regulation and propose the following model as follows (Fig. 9Go). Leptin increases [Ca2+]i through activation of VDCC mainly via L- and N-type Ca2+ channels. Calcium activates PKC, thereby triggering the MAPKs cascade, involving ERKs and, to a lesser extent, JNK, which, in turn, causes the sequential phosphorylation and activation of c-Fos and c-Jun, respectively. Consequently, c-Fos/c-Jun dimers (AP-1 transcription factor) bind to TH-TRE sites and subsequently initiate transcription. In addition, a probable cAMP-PKC-{epsilon}-mediated pathway needs to be considered.


   Acknowledgments
 


   Footnotes
 
This work was supported by in part by a Grant 11770624 from the Ministry of Education and the University of Tsukuba Research Project.

Abbreviations: (a)PKC, Atypical PKC; [Ca2+]i, intracellular Ca2+; BAPTA-AM, 1-2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester; (c)PKC, Ca2+-dependent PKC; DAG, diacylglycerol; JAK, Janus family of tyrosine kinase; JNK, c-Jun N-terminal kinase; (n)PKC, novel PKC; STAT, signal transducer and activator of transcription; TH, tyrosine hydroxylase; TRE, tetradecanoyl phorbol 13-acetate-responsive element; VDCC, voltage-dependent Ca2+ channels.

Received February 13, 2001.

Accepted for publication July 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432[CrossRef][Medline]
  2. Collins S, Kuhn CM, Petro AE, Swick AG, Chrunyk BA, Surwit RS 1996 Role of leptin in fat regulation. Nature 380:677[CrossRef][Medline]
  3. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, WintersD, Boone T, Collins F 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–543[Abstract/Free Full Text]
  4. Levin N, Nelson C, Gurney A, Vandlen R, de Sauvage F 1996 Decreased food intake does not completely account for adiposity reduction after ob protein infusion. Proc Nat Acad Sci USA 93:1726–1730[Abstract/Free Full Text]
  5. Scarpace PJ, Matheny M, Pollock BH, Tumer N 1997 Leptin increases uncoupling protein expression and energy expenditure. Am J Physiol 273:226–230
  6. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, et al 1995 Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271[CrossRef][Medline]
  7. Lee GH, Proenca R, Montez JM, Carroll KM, Darvishzadeh JG, Lee JI, Friedman JM 1996 Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632–635[CrossRef][Medline]
  8. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG 1996 Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101–1106[Medline]
  9. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP 1996 Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491–495[CrossRef][Medline]
  10. Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC 1996 Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA 93:6231–6235[Abstract/Free Full Text]
  11. Baumann H, Morella KK, White DW, Dembski M, Bailon PS, Kim H, Lai CF, Tartaglia LA 1996 The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci USA 93:8374–8378[Abstract/Free Full Text]
  12. Vaisse C, Halaas JL, Horvath CM, Darnell Jr JE, Stoffel M, Friedman JM 1996 Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet 14:95–97[CrossRef][Medline]
  13. Rosenblum CI, Tota M, Cully D, Smith T, Collum R, Qureshi S, Hess JF, Phillips MS, Hey PJ, Vongs A, Fong TM, Xu L, Chen HY, Smith RG, Schindler C, Van der Ploeg LH 1996 Functional STAT 1 and 3 signaling by the leptin receptor (OB-R); reduced expression of the rat fatty leptin receptor in transfected cells. Endocrinology 137:5178–5181[Abstract]
  14. Yamashita T, Murakami T, Otani S, Kuwajima M, Shima K 1998 Leptin receptor signal transduction: OBRa and OBRb of fa type. Biochem Biophys Res Commun 246:752–759[CrossRef][Medline]
  15. Takahashi Y, Okimura Y, Mizuno I, Iida K, Takahashi T, Kaji H, Abe H, Chihara K 1997 Leptin induces mitogen-activated protein kinase-dependent proliferation of C3H10T1/2 cells. J Biol Chem 272:12897–900[Abstract/Free Full Text]
  16. Bjorbaek C, Uotani S, da Silva B, Flier JS 1997 Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 272:32686–32695[Abstract/Free Full Text]
  17. Lewis TS, Shapiro PS, Ahn NG 1998 Signal transduction through MAP kinase cascades. Adv Cancer Res 74:49–139[Medline]
  18. Stachowiak MK, Jiang HK, Poisner AM, Tuominen RK, Hong JS 1990 Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems. J Biol Chem 265:4694–4702[Abstract/Free Full Text]
  19. Haycock JW, Wakade AR 1992 Activation and multiple-site phosphorylation of tyrosine hydroxylase in perfused rat adrenal glands. J Neurochem 58:57–64[CrossRef][Medline]
  20. Zigmond RE, Schwarzschild MA, Rittenhouse AR 1989 Acute regulation of tyrosine hydroxylase by nerve activity and by neurotransmitters via phosphorylation. Annu Rev Neurosci 12:415–461[CrossRef][Medline]
  21. Haycock JW 1990 Phosphorylation of tyrosine hydroxylase in situ at serine 8, 19, 31, and 40. J Biol Chem 265:11682–11691[Abstract/Free Full Text]
  22. Albert KA, Helmer-Matyjek E, Nairn AC, Muller TH, Haycock JW, Greene LA, Goldstein M, Greengard P 1984 Calcium/phospholipid-dependent protein kinase (protein kinase C) phosphorylates and activates tyrosine hydroxylase. Proc Natl Acad Sci USA 81:7713–7717[Abstract/Free Full Text]
  23. Campbell DG, Hardie DG, Vulliet PR 1986 Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase. J Biol Chem 261:10489–10492[Abstract/Free Full Text]
  24. Hwang O, Kim ML, Lee JD 1994 Differential induction of gene expression of catecholamine biosynthetic enzymes and preferential increase in norepinephrine by forskolin. Biochem Pharmacol 48:1927–1934[CrossRef][Medline]
  25. Le Bourdelles B, Boularand S, Boni C, Horellou P, Dumas S, Grima B, Mallet J 1988 Analysis of the 5' region of the human tyrosine hydroxylase gene: combinatorial patterns of exon splicing generate multiple regulated tyrosine hydroxylase isoforms. J Neurochem 50:988–991[CrossRef][Medline]
  26. Cambi F, Fung B, Chikaraishi D 1989 5' flanking DNA sequences direct cell-specific expression of rat tyrosine hydroxylase. J Neurochem 53:1656–1659[CrossRef][Medline]
  27. Gizang-Ginsberg E, Ziff EB 1994 Fos family members successively occupy the tyrosine hydroxylase gene AP-1 site after nerve growth factor or epidermal growth factor stimulation and can repress transcription. Mol Endocrinol 8:249–262[Abstract]
  28. Takekoshi K, Motooka M, Isobe K, Nomura F, Manmoku T, Ishii K, Nakai T 1999 Leptin directly stimulates catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells. Biochem Biophys Res Commun 261:426–431[CrossRef][Medline]
  29. Takekoshi K, Ishii K, Kawakami Y, Isobe K, Nakai T 2001 Ca2+ mobilization, tyrosine hydroxylase (TH) activity and signaling mechanisms in cultured porcine adrenal medullary chromaffin cells: effects of leptin. Endocrinology 142:290–298[Abstract/Free Full Text]
  30. Nishizuka Y 1995 Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9:484–496[Abstract]
  31. Newton AC 1995 : Protein kinase C: structure, function, and regulation. J Biol Chem 270:28495–28498[Free Full Text]
  32. Kumai T, Tanaka M, Tateishi T, Watanabe M, Nakura H, Asoh M, Kobayashi S 1998 Effects of anti-androgen treatment on the catecholamine synthetic pathway in the adrenal medulla of spontaneously hypertensive rats. Naunyn Schmiedebergs Arch Pharmacol 357:620–624[CrossRef][Medline]
  33. Wellner M, Maasch C, Kupprion C, Lindschau C, Luft FC, Haller H 1999 The proliferative effect of vascular endothelial growth factor requires protein kinase C-{alpha} and protein kinase C-{zeta}. Arterioscler Thromb Vasc Bio 19:178–185[Abstract/Free Full Text]
  34. Viedt C, Soto U, Krieger-Brauer HI, Fei J, Elsing C, Kubler W, Kreuzer J 2000 Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species. Arterioscler Thromb Vasc Biol 20:940–948[Abstract/Free Full Text]
  35. Takekoshi K, Ishii K, Kawakami Y, Isobe K, Nakai T 2000 {kappa}-Opioid inhibits catecholamine biosynthesis in PC12 rat pheochromocytoma cell. FEBS Lett 477:273–277[CrossRef][Medline]
  36. Yukimasa N, Isobe K, Nagai H, Takuwa Y, Nakai T 1999 Successive occupancy by immediate early transcriptional factors of the tyrosine hydroxylase gene TRE and CRE sites in PACAP-stimulated PC12 pheochromocytoma cells. Neuropeptides 33:475–482[CrossRef][Medline]
  37. Chabot-Fletcher M, Breton JJ 1998 Effect of staurosporine on transcription factor NF-{kappa}B in human keratinocytes. Biochem Pharmacol 56:71–78[CrossRef][Medline]
  38. Marley PD, Thomson KA, Bralow RA 1995 Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase. Br J Pharmacol 114:1687–1693[Medline]
  39. Hwang O, Park SY, Kim KS 1997 Protein kinase A coordinately regulates both basal expression and cyclic AMP-mediated induction of three catecholamine-synthesizing enzyme genes. J Neurochem 68:2241–2247[Medline]
  40. Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H, Finkenzeller G, Marme D, Rapp UR 1993 Protein kinase C{alpha} activates RAF-1 by direct phosphorylation. Nature 364:249–252[CrossRef][Medline]
  41. Sozeri O, Vollmer K, Liyanage M, Frith D, Kour G, Mark III GE, Stabel S 1992 Activation of the c-Raf protein kinase by protein kinase C phosphorylation. Oncogene 7:2259–2262[Medline]
  42. Bouloumie A, Marumo T, Lafontan M, Busse R 1999 Leptin induces oxidative stress in human endothelial cells. FASEB J 13:1231–1238[Abstract/Free Full Text]
  43. Harvey J, McKay NG, Walker KS, Van-der-Kaay J, Downes CP, Ashford ML 2000 Essential role of phosphoinositide 3-kinase in leptin-induced K(ATP) channel activation in the rat CRI-G1 insulinoma cell line. J Biol Chem 275:4660–4669[Abstract/Free Full Text]
  44. Deleted in proof
  45. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M 1987 Phorbol ester-inducible genes contain a common cis element recognized by a by a TPA-modulated trans-acting factor. Cell 49:729–739[CrossRef][Medline]
  46. Angel P, Karin M 1991 The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:129–157[Medline]
  47. Abate C, Baker SJ, Lees-Miller SP, Anderson CW, Marshak DR, Curran T 1993 Dimerization and DNA binding alter phosphorylation of Fos and Jun. Proc Natl Acad Sci USA 90:6766–6770[Abstract/Free Full Text]
  48. Chen RH, Abate C, Blenis J 1993 Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase. Proc Natl Acad Sci USA 90:10952–10956[Abstract/Free Full Text]
  49. Deng T, Karin M 1994 c-Fos transcriptional activity stimulated by H-Ras-activated protein kinase distinct from JNK and ERK. Nature 371:171–175[CrossRef][Medline]
  50. Taylor LK, Marshak DR, Landreth GE 1993 Identification of a nerve growth factor- and epidermal growth factor-regulated protein kinase that phosphorylates the protooncogene product c-Fos. Proc Natl Acad Sci USA 90:368–372
  51. Hibi M, Lin A, Smeal T, Minden A, Karin MI 1993 Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev 7:2135–2148[Abstract/Free Full Text]
  52. Whitmarsh AJ, Davis RJ 2000 A central control for cell growth. Nature 403:255–256[CrossRef][Medline]
  53. Haycock JW 1990 Phosphorylation of tyrosine hydroxylase in situ at serine 8, 19, 31, and 40. J Biol Chem 265:11682–11689
  54. Haycock JW 1993 Multiple signaling pathways in bovine chromaffin cells regulate tyrosine hydroxylase phosphorylation at Ser19, Ser31, and Ser40. Neurochem Res 18:15–26[CrossRef][Medline]
  55. Cai H, Smola U, Wixler V, Eisenmann-Tappe I, Diaz-Meco MT, Moscat J, Rapp U, Cooper GM 1997 Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase. Mol Cell Biol 17:732–741[Abstract]
  56. Graness A, Adomeit A, Ludwig B, Muller WD, Kaufmann R, Liebmann C 1997 Novel bradykinin signalling events in PC-12 cells: stimulation of the cAMP pathway leads to cAMP-mediated translocation of protein kinase Cepsilon. Biochem J 327:147–154



This article has been cited by other articles:


Home page
 J. Physiol.Home page
I. H. Trevenzoli, M. M. R. Valle, F. B. Machado, R. M. G. Garcia, M. C. F. Passos, P. C. Lisboa, and E. G. Moura
Neonatal hyperleptinaemia programmes adrenal medullary function in adult rats: effects on cardiovascular parameters
J. Physiol., April 15, 2007; 580(2): 629 - 637.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
H. Cui, F. Cai, and D. D. Belsham
Leptin signaling in neurotensin neurons involves STAT, MAP kinases ERK1/2, and p38 through c-Fos and ATF1
FASEB J, December 1, 2006; 20(14): 2654 - 2656.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Valerio, V. Ghisi, M. Dossena, C. Tonello, A. Giordano, A. Frontini, M. Ferrario, M. Pizzi, P. Spano, M. O. Carruba, et al.
Leptin Increases Axonal Growth Cone Size in Developing Mouse Cortical Neurons by Convergent Signals Inactivating Glycogen Synthase Kinase-3beta
J. Biol. Chem., May 5, 2006; 281(18): 12950 - 12958.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Lappas, M. Permezel, and G. E. Rice
Leptin and Adiponectin Stimulate the Release of Proinflammatory Cytokines and Prostaglandins from Human Placenta and Maternal Adipose Tissue via Nuclear Factor-{kappa}B, Peroxisomal Proliferator-Activated Receptor-{gamma} and Extracellularly Regulated Kinase 1/2
Endocrinology, August 1, 2005; 146(8): 3334 - 3342.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
T Nanmoku, K Takekoshi, T Fukuda, K Ishii, K Isobe, and Y Kawakami
Stimulation of catecholamine biosynthesis via the PKC pathway by prolactin-releasing peptide in PC12 rat pheochromocytoma cells
J. Endocrinol.,