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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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ßI, and -
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 
-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
, 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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] (,
ßI, ßII, and ),
which are sensitive to Ca2+, DAG, or phorbol
esters; novel PKC [(n)PKC] (
, , 
, and 
), which are
regulated by DAG or phorbol esters but are independent of
Ca2+; and atypical PKC [(a)PKC] (
, 
,
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
[-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). 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. 1). 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 -conotoxin GVIA on this process. As
shown in Fig. 1, nicardipine and -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. 1).
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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. 2, A and
B). Nicardipine and -conotoxin GVIA, each at 1
µM, were effective at decreasing leptin-induced TH mRNA
levels by 53% and 21%, respectively (Fig. 2, 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. 2, C and
D). The concentration of H89 was set at 10 µM according
to Marley et al. (38) and Hwang et
al. (39).
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, 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. 3B), 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. 3B).
Nicardipine and -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.
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,
ßI, ßII, , , ,
and PKC isoforms (data not shown). To confirm whether (c)PKC
isoforms are indeed involved in leptininduced PKC activity, we
initially tested (c)PKC isoforms, , ßI,
ßII, ß, and by Western blot analysis. As
shown in Fig. 4, leptin (100
nM) significantly induced increases in PKC-,
-ßI, and - 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- by approximately 1.6-fold.
However, neither (n)PKC- nor (a)PKC- was induced by
leptin.
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). 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 1, Ro 32-0432 significantly attenuated
leptin-induced intracellular catecholamine levels.
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). 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
-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. 4
).
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, 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.
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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. 8A, 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. 8B, leptin-induced
AP-1 consisted mainly of c-Fos and, to a smaller extent, c-Jun
proteins.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 , ßI, and . 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. 9).
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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 -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 , 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 was reported
to stimulate Raf, upstream of ERKs (55). It was also shown
that bradykinin-induced activation of PKC- 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- (Fig. 9). 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- in our
experimental system, it can be suggested that this
cAMP-PKC--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. 9). 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--mediated pathway needs to be
considered.
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Acknowledgments |
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Footnotes |
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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.
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