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(Received for publication, June 15, 1995) From the
In mammalian melanocytes, melanin synthesis is controlled by
tyrosinase, the critical enzyme in the melanogenic pathway. We and
others showed that the stimulation of melanogenesis by cAMP is due to
an increased tyrosinase expression at protein and mRNA levels. However,
the molecular events connecting the rise of intracellular cAMP and the
increase in tyrosinase activity remain to be elucidated. In this study,
using B16 melanoma cells, we showed that cAMP-elevating agents
stimulated mitogen-activated protein (MAP) kinase,
p44
In melanocytes and melanoma cells, melanin synthesis is
controlled by a cascade of enzymatic reactions regulated at the level
of tyrosinase. This enzyme synthesizes dopaquinone from tyrosine and
appears to control the rate-limiting step of melanogenesis. Melanin
synthesis is stimulated by a large array of effectors including
1-oleyl-2-acetyl-glycerol(1) , ultraviolet B
radiations(2) , and cAMP-elevating agents (forskolin, IBMX, ( The proline-directed serine/threonine kinases of the MAP kinase
family (p44
Figure 1:
cAMP-elevating agents stimulate
p44
The kinase activity
of MEK was monitored in a cell-free system, after immunoprecipitation
with specific antibody, using as substrate p44
Figure 2:
[Nle
Since Raf-1 was described as operating immediately upstream
of MEK, similar experiments were performed to examine the effect of M
+ I on Raf-1 activity. Raf-1 was isolated from B-16 melanoma cells
stimulated as described above, and its kinase activity was evaluated
using as substrate MEK immunoprecipitated from unstimulated cells (Fig. 3). In the first lane, we observed a band at 45 kDa
corresponding to the basal autophosphorylation of MEK. The other bands
appeared to be nonspecific, since they were precipitated by preimmune
serum (not shown). With Raf-1 incubated alone, no autophosphorylation
was observed (lanes2-4). When MEK was added to
Raf-1, no significant increase in the basal phosphorylation of MEK was
observed with Raf-1 precipitated from control or M + I-treated
cells (lanes5 and 7). In contrast, MEK
phosphorylation was markedly increased in the presence of Raf-1
extracted from TPA-treated cells (lane6). These
results indicate that cAMP-elevating agents did not stimulate Raf-1
activity in B-16 cells. Thus, the effect of [Nle
Figure 3:
TPA but not M + I stimulates Raf-1 in
B-16 cells. Raf-1 was immunoprecipitated from unstimulated cells (lanes2 and 5), cells treated with 16
nM TPA for 15 min (lanes3 and 6),
or cells treated with 1 µM [Nle
The localization of
p44
Figure 4:
Immunolocalization of the p44
Figure 5:
[Nle
Figure 6:
Characterization of the AP-1 complex
induced by TPA and [Nle
The molecular mechanisms by which cAMP stimulates melanin
synthesis in melanocytes and melanoma cells remain to be identified. In
this aim, we characterized the molecular events triggered by cAMP in
B-16 melanoma cells. Our results demonstrate that cAMP activated
p44 Following
its activation by cAMP, we observed a transient translocation of
p44 In this study we showed that melanin synthesis,
tyrosinase activity, and amount were simultaneously increased by
[Nle Dendritogenesis, another feature of melanocyte differentiation is
stimulated during cAMP-induced melanogenesis in B-16 melanoma cells.
Interestingly, cAMP-elevating agents induce in PC12 a differentiated
phenotype characterized by neurite outgrowth and an activation of
p44 In summary, the data gathered in this study demonstrate that the MAP
kinase pathway and AP-1 are activated during cAMP-induced
melanogenesis. The role of p44
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24315-24320
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. This effect was mediated by the activation
of MAP kinase kinase. cAMP-elevating agents induced a translocation of
p44
to the nucleus and an activation of the
transcription factor AP-1. cAMP-induced AP-1 contained FOS-related
antigen-2 in association with JunD, while after phorbol ester
stimulation AP-1 complexes consist mainly of JunD/c-Fos heterodimers.
In an attempt to connect these molecular events to the control of
tyrosinase expression that appears to be the pivotal point of
melanogenesis regulation, we hypothesized that following its activation
by cAMP, p44
activates AP-1. Then AP-1 could
stimulate tyrosinase expression through the interaction with specific
DNA sequences present in the mouse tyrosinase promoter.
)
-MSH)(3, 4, 5) . Few data
are available concerning the molecular mechanisms triggered by these
melanogenic agents. Protein kinase C was thought to be involved in the
induction of melanogenesis by 1-oleyl-2-acetyl-glycerol and ultraviolet
B radiations(6, 7) . However, a recent report of
Carsberg et al.(8) has shown that the stimulation of
melanogenesis by these agents was not affected by RO485, a potent
inhibitor of protein kinase C. While the role of protein kinase C in
the induction of melanogenesis remains controversial, compelling data
have shown that cAMP-elevating agents stimulate melanogenesis in both
melanocytes and melanoma cells, indicating that the cAMP pathway plays
a key role in the regulation of melanogenesis (3, 4, 5) . The effect of cAMP on
melanogenesis is due to a stimulation of tyrosinase activity. This
appears to be the consequence of an augmentation of enzymatic activity
of preexisting tyrosinase(4, 9) following
post-translational modifications such as (i) phosphorylation or
glycosylation(10) , (ii) association with an
activator(11, 12) , and (iii) dissociation from an
inhibitor(13) . Alternatively, cAMP was shown to increase
tyrosinase mRNA(14, 15) , resulting in an augmentation
of tyrosinase amount, suggesting that cAMP stimulates tyrosinase
transcription(16) . However, the molecular events connecting
the stimulation of tyrosinase activity or the activation of tyrosinase
gene expression to the rise of cellular cAMP remain to be identified. and p42
)
are activated upon phosphorylation on both threonine 192 and tyrosine
194 by the dual specificity MAP kinase kinase (MEK)(20) . MEK
is itself phosphorylated and activated by Raf-1 kinase (20) or
by a recently identified MAP kinase kinase kinase (MEK
kinase)(21) . MAP kinases were shown to be involved in the
control of cell growth(17) , in the regulation of some
metabolic processes such as glycogen
synthesis(18, 19) , and more recently in the
regulation of pheochromocytoma and adipocytes
differentiation(22, 23) . In melanocytes and melanoma
cells the induction of melanogenesis is associated with cell
differentiation. Thus, we hypothesized that MAP kinases could be
activated during cAMP-induced melanogenesis. Using the well
characterized mouse melanoma cells B-16, we demonstrated that
cAMP-elevating agents such as forskolin, IBMX, and 4-norleucine
7-D-phenylalanine-
-melanocyte stimulating hormone
([Nle
,D-Phe
]-MSH), a
potent analog of -MSH, stimulated p44 through the activation of the MEK. Further investigations
demonstrated a translocation of p44
to the
nucleus and an activation of the transcription factor AP-1 by
cAMP-elevating agents. In this condition the AP-1 complex contained
predominantly JunD and Fra-2. Our results provide meaningful clues
concerning the molecular mechanisms triggered by cAMP in B-16 melanoma
cells and suggest that the MAP kinase pathway and AP-1 could play a
role in melanogenesis regulation by cAMP.
Materials
Dulbecco's modified
Eagle's medium, 4-norleucine
7-D-phenylalanine--melanocyte stimulating hormone
([Nle,D-Phe]-MSH),
IBMX, TPA, forskolin, BSA, myelin basic protein from bovine brain,
protein A-Sepharose, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF),
aprotinin, and leupeptin were purchased from Sigma. Polyclonal rabbit
antisera to human tyrosinase (PEP-7) was provided by Dr. V. Hearing
(Bethesda, MD). Antisera to p44 and MEK were generous
gifts from Dr. E. Van Obberghen (Nice, France). Antibodies to MEK
kinase, Raf-1, c-Fos, c-Jun, JunB, JunD, and the AP-1 and AP-2
synthetic oligonucleotides are from Santa Cruz Biotechnology. Rabbit
polyclonal anti-Fra-2 antibody was prepared using recombinant GST
fusion protein of Fra-2. L-[ring-3`,5`-
H]tyrosine
(40-60 Ci/mmol; 1 mCi/ml); L-3,4-dihydroxyphenyl
3-[
C]alanine (7-12 mCi/mol, 50
µCi/ml); [-P]dCTP; and
[
P]ATP (3000 Ci/mmol) were from Amersham
(Buckinghamshire, United Kingdom).
Cell Cultures
B-16/F10 murine melanoma cells (from
Dr. V. Hearing) were cultured in Dulbecco's modified
Eagle's medium with 10% fetal calf serum and
penicillin/streptomycin (100 IU/50 µg/ml) in a humidified
atmosphere containing 5% CO
in air at 37 °C.Determination of Tyrosinase Activity and Melanin
Synthesis
Tyrosinase activity in living cells was estimated by
the amount of HO released into the culture
medium during the hydroxylation of
[3,5-H]tyrosine to hydroxyphenylalanine,
according to Oikawa et al.(24) . The melanin formation
assay uses L-3,4-dihydroxyphenyl
3-[C]alanine (7-12 mCi/mmol) as
precursor(25) .Western Blot Analysis
B-16 cell lysates were
separated by SDS-PAGE (10% acrylamide gels) and transferred to Hybond-C
extra membranes. The blots were probed with PEP-7 antibody directed to
the C terminus part of tyrosinase(26) , and then proteins were
visualized by the Amersham ECL system and quantified by image analysis. Northern Blot Analysis
Total cellular RNAs were
purified using the method described by Chomczynski and
Sacchi(27) . For Northern blot analysis, total cellular RNA (25
µg/lane) were fractionated on 1% agarose, 0.66 M formaldehyde gels and transferred onto nylon membranes. Mouse
tyrosinase cDNA probe (generously provided by Dr. B. Bouchard) and
human glyceraldehyde-3-phosphate deshydrogenase cDNA probe were labeled
with [-P]dCTP using a random priming kit
(Stratagene). Membranes were autoradiographed and quantified by image
analysis.
Image Analysis
Morphometric measurements were
performed using a Biocom 500 (BIOCOM SA, Les Ulis, France) image
analysis system coupled to a CCD video camera and a Nikon TMS inverted
light microscope. After treatment, cells were viewed using a 20 
phase contrast objective and projected onto the video screen. Outlines
of 100 cells of each experimental condition were acquired manually,
quantitative measurements of area (A) and perimeter (P) being performed using the Mima software. Then the
dendricity factor was evaluated as P (4A)
according to Lacour et
al.(28) .
p44
Following a 2-h serum
starvation period, B-16 cells were incubated with effectors. Cells were
washed with cold phosphate-buffered saline and lysed in a
solubilization buffer (1% Triton X-100, 50 mM Hepes, pH 7.4,
150 mM NaCl, 10 mM EDTA, 10 mM
NaAssay
PO, 2 mM sodium
orthovanadate, 100 mM NaF with 100 IU/ml aprotinin, 20 mM leupeptin, 1 mM AEBSF). Solubilized proteins were
incubated for 2 h at 4 °C with antibody to p44 preadsorbed to protein A-Sepharose. Immune complexes were washed
twice with solubilization buffer and twice with HNTG buffer (50 mM Hepes, 150 mM NaCl, 0.1% (v/v) Triton X-100, 10% (v/v)
glycerol with 0.2 mM sodium orthovanadate) and resuspended in
50 µl of the same buffer supplemented with 30 mM magnesium
acetate, 0.2 mg/ml myelin basic protein, and
[
-
P]ATP (15 µM; specific
activity 30 Ci/mmol). The phosphorylation reaction was allowed to
proceed for 45 min at room temperature and was stopped by spotting the
sample onto 3MM Chr Whatman papers, which were then dropped into 10%
(v/v) trichloroacetic acid containing 5 mM pyrophosphate.
After three washes the radioactivity was determined by Cerenkov
counting.
MEK and Raf-1 Assay
MEK activity was measured in a
reconstitution assay as the ability of immunopurified MEK to
phosphorylate in vitro p44 immunoprecipitated
from nonstimulated cells. Pellets containing immunoprecipitated MEK and
p44
were washed as described above, mixed, and
resuspended in 50 µl of HNTG buffer supplemented with 10 mM MnCl
. Raf-1 activity was measured with the same
procedure using MEK immunoprecipitated from nonstimulated cells as
substrate for Raf-1. The phosphorylation reaction was performed in 20
mM Hepes, pH 7.4, 10 mM MgCl, 1 mM MnCl, 1 mM dithiothreitol, 10 mMp-nitrophenyl phosphate. Reactions were initiated by the
addition of [-
P]ATP (10 µM, 60
Ci/mmol). After incubation at room temperature for 20 min, assays were
stopped by the addition of Laemmli sample buffer. The samples were
analyzed by SDS-PAGE (10% acrylamide) and autoradiography.
Immunofluorescence Studies
After stimulation, B-16
cells were washed with PBS and fixed at -20 °C for 10 min
with methanol/acetone (3:7, v/v). After a 10-min rehydration at 25
°C in PBS containing 3% BSA (PBS/BSA), fixed cells were incubated
with the primary antibody directed to the C terminus part of
p44 (1:500) for 60 min at 25 °C. Cells were then
washed five times with PBS and incubated in PBS/BSA for 60 min at 25
°C with fluorescein isothiocyanate-conjugated secondary antibody
(anti-rabbit, 1:100). Finally, cells were washed five times with PBS
and examined under confocal laser scanning microscopy.
Nuclear Extracts and Gel Mobility Shift
Assay
Serum-starved B-16 cells were stimulated for 2 h with the
different effectors, and the nuclear extracts were prepared essentially
as described by Dignam (29) . In vitro binding
reaction of AP-1 in a total volume of 25 µl was performed by
incubation of 10 µg of nuclear extract in a binding buffer
containing 10 mM Hepes, pH 7.8, 50 mM KCl, 2
mM dithiothreitol, 1 mM EDTA, 5 mM MgCl, 10% glycerol, 3 mM AEBSF, 2 µg of
poly(dI-dC), 1 mg/ml BSA. After 10 min of preincubation on ice,
50,000-100,000 cpm of P end-labeled oligonucleotide
probe was added and incubated at 25 °C for 20 min. Then DNA-protein
complexes were resolved by electrophoresis on 4% polyacrylamide gels in
TAE buffer (10 mM Tris, 9 mM sodium acetate/acetic
acid, 275 µM EDTA) for 8 h at 120 volts, dried, and
subjected to autoradiography. When indicated, an excess of cold
competitor oligonucleotides was added during preincubation. For
antibody supershift assays, 1 µl of the corresponding antisera were
preincubated with the nuclear extracts for 1 h on ice in the binding
reaction buffer before adding the labeled probe.
Stimulation of Melanogenesis in B-16 Melanoma
Cells
We first studied the melanogenic activity and the
morphological changes of B-16 melanoma cells in response to
cAMP-elevating agents. In preliminary experiments, various
cAMP-elevating agents including forskolin, cholera toxin, -MSH,
[Nle, D-Phe]-MSH, and
IBMX were found to increase melanin synthesis in B-16 melanoma cells.
The most important stimulation was obtained with [Nle, D-Phe]-MSH plus IBMX.
[Nle, D-Phe]-MSH plus
IBMX (M + I) stimulated tyrosinase activity, melanin synthesis,
tyrosinase expression at the protein, and RNA messenger levels (Table 1). These results suggest that the stimulation of
tyrosinase gene expression plays a key role in the control of
melanogenesis by cAMP-elevating agents. Simultaneously, we observed
morphological differentiation characterized by numerous and arborescent
dendrite outgrowths. These changes were quantified by image analysis as
described under ``Experimental Procedures.'' The results
presented in Table 1show that the dendricity factor is markedly
increased after 24 and 48 h of incubation with M + I.
Activation of MAP Kinase by cAMP-elevating Agents in B-16
Melanoma Cells
We focused our attention on the activation of
p44 by cAMP-elevating agents. In this aim, B-16 melanoma
cells were stimulated for 15 min by forskolin, [Nle
, D-Phe]-MSH, IBMX, or
[Nle, D-Phe]-MSH plus
IBMX. Solubilized proteins were submitted to immunoprecipitation with a
specific antibody raised against the C terminus domain of
p44. Kinase activity was then measured using myelin
basic protein as substrate and quantified by filter paper assay (Fig. 1). With forskolin, [Nle
, D-Phe]-MSH, or IBMX we observed a
2.5-fold stimulation of p44 activity. A 4-fold
stimulation was achieved with [Nle
, D-Phe]-MSH, and IBMX in association (M
+ I). The most efficient stimulation of p44 activity (8-fold) was obtained with TPA (not shown). Longer
stimulation time either with TPA or cAMP-elevating agents did not
result in higher stimulation.
activity in B-16 cells. B-16 cells were
incubated either with 10 µM forskolin (FORSK), 1
µM [Nle
, D-Phe]-MSH (MSH), 0.1
mM IBMX, or M + I for 10 min. Then cells were
solubilized, p44 was immunoprecipitated, and
its kinase activity was measured using myelin basic protein as
substrate. Results are expressed as -fold stimulation of the basal
p44
activity from unstimulated cells (CONT). Data are means ± S.E. of five experiments
performed in triplicate.
Effect of cAMP-elevating Agent on MEK and on Raf-1
Kinase
In an attempt to understand the mechanism by which cAMP
stimulated p44, we studied the effect of M + I on
MAP kinase kinase and Raf-1 kinase activities.
extracted
from unstimulated cells (Fig. 2). Lane1 shows
the basal autophosphorylation of unstimulated p44
at 44
kDa. A faint band at 45 kDa in lanes3 and 4 indicates that MEK autophosphorylation was stimulated by TPA and M
+ I compared with the basal autophosphorylation (lane2). When phosphorylation was performed in the presence of
both MEK and p44
, we observed a strong phosphorylation
of a protein at 44 kDa, indicating that MEK phosphorylated p44
(lane5). This phosphorylation was increased
when MEK was extracted from TPA or M + I-treated cells (lanes6 and 7), demonstrating that MEK was stimulated
by TPA and [Nle
, D-Phe]-MSH plus IBMX in B-16 melanoma
cells.
, D-Phe]-MSH plus IBMX activates MEK in
B-16 cells. MEK was immunoprecipitated from unstimulated cells (lanes2 and 5), cells treated with 16
nM TPA for 15 min (lanes3 and 6),
or cells treated with 1 µM [Nle, D-Phe]-MSH plus 0.1 IBMX (M + I)
for 10 min (lanes4 and 7). Then MEK was
phosphorylated alone (lanes2-4) or in the
presence of p44 (lanes5-7). p44
, isolated from
unstimulated cells, was also phosphorylated alone (lane1). Phosphorylated proteins were analyzed by SDS-PAGE and
autoradiography. Molecular masses, indicated on the left, are
expressed in kilodaltons.
, D-Phe]-MSH plus IBMX on p44 is mediated by MEK that is activated by an unknown mechanism
independently on Raf-1 kinase stimulation.
, D-Phe]-MSH plus 0.1 mM IBMX (M
+ I) for 10 min (lanes4 and 7). Then
Raf-1 was phosphorylated alone (lanes2-4) or
in the presence of MEK (lanes5-7). MEK,
isolated from unstimulated cells, was also phosphorylated alone (lane1). Phosphorylated proteins were analyzed by
SDS-PAGE and autoradiography. Molecular masses, indicated on the left, are expressed in
kilodaltons.
Translocation of p44
Stimulation of tyrosinase gene expression plays a key
role in the regulation of melanogenesis by cAMP. Thus we hypothesized
that the cAMP signal should be transmitted to transcription factors
present in the nucleus. To verify this hypothesis, we studied the
effect of [Nle to the
Nucleus
, D-Phe]-MSH plus IBMX on p44 localization in B-16 melanoma cells.
in cells treated or not treated with
[Nle
, D-Phe]-MSH plus
IBMX was studied by immunofluorescence and confocal laser scanning
microscopy (Fig. 4). Using an antipeptide to the C terminus part
of p44, we observed, in the absence of M + I, a
strong perinuclear and a weak nuclear labeling. After a 60-min exposure
to M + I, the cytoplasm and the nucleus appeared equally labeled,
indicating that p44
translocated to the nucleus. This
phenomenon was transient, since after 150 min in presence of M +
I, nucleus labeling decreased, suggesting that p44
returned to the cytoplasm.
in B-16 cells stimulated with [Nle
, D-Phe]-MSH plus IBMX. Immunofluorescence
labeling was performed using anti-p44 antibody
and analyzed by a Leica confocal laser microscope. Optical sections,
through the center of the nuclei, at 8 µm from cell bottom are
shown. A, unstimulated B-16 cells; B, cells
stimulated with 1 µM [Nle
, D-Phe]-MSH plus 0.1 mM IBMX for
60 min; C, as in B, but for 150 min. Bar in A represents 10 µm.
Activation of Transcription Factors by cAMP-elevating
Agents
Since p44 is activated and translocated to
the nucleus by M + I, it is tempting to propose that these
phenomena are followed by an activation of transcription factors. Among
the transcription factors activated by p44
, AP-1 was
reported to stimulate gene expression through the binding to TRE
sequences. The presence of TRE-like sequences in the mouse tyrosinase
promoter prompted us to study the activation of AP-1 by
[Nle
, D-Phe]-MSH plus
IBMX. B-16 melanoma cells were stimulated either with TPA as positive
control or with M + I. Then nuclear extracts were prepared, and
the gel retardation assay was performed with a labeled oligonucleotide
containing the TRE consensus sequence (TGACTCA) (Fig. 5A). In control, TPA, and M + I conditions,
two strong bands with fast electrophoretic mobility were observed.
These bands were totally displaced by a 10-fold excess of unlabeled
cAMP-responsive element (CRE) (TGACGTCA), indicating that these bands
corresponded to a nonspecific binding of CRE binding protein (CREB) to
the TRE probe. With TPA or M + I, a third band with a slower
electrophoretic mobility appeared. This band was identified as a
classical AP-1 complex, since it was displaced more efficiently by
unlabeled TRE than by unlabeled CRE. These data demonstrate that
[Nle, D-Phe]-MSH plus
IBMX activated the transcription factor AP-1 in B-16 melanoma cells.
Additionally, an activation of the transcription factor AP-2 by cAMP
but not by TPA (Fig. 5B) was observed in B-16 melanoma
cells.
, D-Phe]-MSH plus IBMX activates AP-1 and
AP-2 in B-16 cells. DNA-binding activity of 10 µg of proteins from
the various cell extracts was measured by gel mobility shift assay
using a labeled oligonucleotide presenting the AP-1 consensus binding
sequence (TRE) (A). B-16 cells were stimulated with 16 nM TPA or 1 µM [Nle, D-Phe]-MSH plus 0.1 mM IBMX
(M+I) for 2 h at 37 °C. C, unstimulated cells.
Specificity of the complexes was tested by competition with increasing
molar excess (10 , 50 ) of cold oligonucleotides
containing the TRE or CRE consensus sequence. DNA binding activity was
measured in the same condition using a labeled oligonucleotide
presenting the AP-2 consensus binding site (B).
Characterization of AP-1 Complexes Induced by cAMP and
TPA
Interestingly, the AP-1 complex induced by M + I
migrated more slowly than that observed with TPA, suggesting that these
AP-1 complexes contain different components. AP-1 consists either in
homodimers of Jun family proteins or in heterodimers of Jun/Fos family
proteins. Three Jun proteins (c-Jun, JunB, JunD) and at least four Fos
proteins (c-Fos, FosB, Fra-1, Fra-2) were found in AP-1 complexes. The
composition of AP-1 complexes in both TPA and M + I conditions was
investigated by supershift experiments using specific antibodies to
c-Jun, JunB, JunD, c-Fos, and Fra-2 (Fig. 6). With a nuclear
extract from TPA-treated cells, AP-1 complexes were almost totally
shifted by anti-JunD (lane10) and anti c-Fos (lane11). AP-1 complexes were also shifted, but to a
lesser extent, by antibodies to JunB (lane8), c-Jun (lane9), and Fra-2 (lane12). AP-1
complexes induced by M + I were completely shifted by anti-JunD (lane16) and anti-Fra-2 (lane18).
We also detected a small amount of JunB (lane14),
but neither c-Jun (lane15) nor c-Fos antibodies (lane17) interacted with these AP-1 complexes. Thus
cAMP-induced AP-1 complexes contain mainly JunD and Fra-2.
, D-Phe]-MSH plus IBMX. Nuclear extracts
from unstimulated cells (BASAL), TPA-stimulated cells (TPA),
or M + I were incubated for 1 h with preimmune serum (lanes1, 7, and 13) or specific antibodies:
JunB (lanes2, 8, and 14), c-Jun (lanes3, 9, and 15), JunD (lanes4, 10, and 16), c-Fos (lanes5, 11, and 17), Fra-2 (lanes6, 12, and 18). Then samples
were incubated with labeled oligonucleotide containing a TRE sequence
and analyzed by gel mobility shift assay.
through the stimulation of MEK, the enzyme
immediately upstream from MAP kinases. The molecular mechanisms of MEK
activation by cAMP in B-16 melanoma cells differ from those already
reported in other cell types(30) . Indeed, neither Raf-1, which
is not activated by cAMP, nor MEK kinase, which is not detected in B-16
melanoma cells, is apparently involved in MEK activation by cAMP. The
involvement of another member of the Raf kinase family, i.e. A-Raf or B-Raf that is mainly expressed in neuronal cells (31) may be suggested. However, the inhibition of B-Raf kinase
activity by cAMP observed in PC12 cells(32) , makes this
hypothesis unlikely. It remains possible that in B-16 melanoma cells
cAMP activates an isoform of MEK kinase, different from that previously
described by Lange-Carter(21) . Alternatively, inhibition by
cAMP of phosphatase 2A activity, which was reported to dephosphorylate
and deactivate MEK(33) , can be also suggested.
to the nucleus. Similar observations were reported
in serum-treated fibroblast (34) or in NGF-stimulated PC12
cells(35) . In the nucleus, p44
is thought to
phosphorylate and activate numerous transcription factors such as
p62
(36) , c-Myc(37) , and
AP-1(38) . In B-16 melanoma cells, we showed that cAMP
stimulated AP-1 binding to an oligonucleotide containing a TRE
sequence. cAMP-induced AP-1 contained mainly JunD and Fra-2 components,
while in TPA-induced AP-1, we found JunB, c-Jun, JunD, c-Fos, and
Fra-2, JunD and c-Fos being the major components of these AP-1
complexes. Recently Tamir et al.(39) reported the
activation of AP-1 by cAMP in lymphocyte and ascribed the activation of
AP-1 by cAMP to the inactivation of the AP-1 inhibitory protein, IP-1,
upon phosphorylation by cAMP-dependent kinase (protein kinase
A)(40) . However, AP-1 can be also activated following the
phosphorylation of serines 63 and 73 of the N terminus domain of Jun
proteins. These sites are phosphorylated by Jun N-terminal kinases (41, 42) and by MAP kinases (38) , suggesting
that MAP kinases are involved in AP-1 activation. Additionally, a
recent report indicates that MAP kinases are involved in the regulation
of the expression of Fos family proteins, leading thereby to the
stimulation of AP-1 activity(43) . Thus, it is tempting to
propose that p44
through JunD phosphorylation or Fra-2
up-regulation is accountable for AP-1 activation by cAMP in B-16
melanoma cells.
, D-Phe]-MSH plus
IBMX. These effects appear to be the consequence of the augmentation of
tyrosinase mRNA. These observations confirmed previous
reports(14, 15) suggesting that the control of
tyrosinase mRNA expression is a key step in the cAMP-mediated
stimulation of melanogenesis in B-16 melanoma cells. Usually,
regulation of gene expression by cAMP is mediated by CRE through the
binding of CREB family transcription factors that are phosphorylated
and activated by protein kinase A(44) . However, no canonical
CRE was found in the mouse tyrosinase promoter. The presence of two
TRE-like sequences (2.1- and 0.18-kilobase upstream transcription start
site) in the mouse tyrosinase promoter suggests that the stimulation of
AP-1 by cAMP could lead to an increased tyrosinase gene expression.
AP-2, another transcription factor, was also shown to mediate the
effect of cAMP on gene expression(45) . The presence of a
putative AP-2 binding site in the mouse tyrosinase promoter and its
activation by cAMP suggest that AP-2 could participate, in coordination
with AP-1, in the regulation of mouse tyrosinase gene expression.
Interestingly, TPA and cAMP display a common set of cellular responses, i.e. activation of p44 and of AP-1, but they
promote opposite effects on melanogenesis(14, 46) .
This could be explained by the respective nature of TPA and
cAMP-induced AP-1 complexes, suggesting that JunD/Fra-2 would
transactivate tyrosinase gene expression while JunD/c-Fos would
inhibit, directly or indirectly, tyrosinase gene transcription.
(47, 48, 49) . Further, the
transfection of these cells with a constitutively active MEK leads to
spontaneous neuritogenesis (22) , demonstrating that the MAP
kinase pathway plays a pivotal role in the regulation of PC12
differentiation. Since dendritogenesis and neuritogenesis are closely
related processes, we hypothesize that MAP kinase could play a critical
role in the control of differentiation in neural crest-derived cells.
and that of AP-1 in the
regulation of melanogenesis remain to be proved. Nevertheless, we would
like to suggest that p44
, possibly through the
regulation of AP-1, plays a pivotal role in the control of tyrosinase
gene expression and thereby in the regulation of melanogenesis by cAMP
in B-16 melanoma cells.
)-MSH, -melanocyte stimulating
hormone; Fra-2, Fos-related antigen-2; MEK,
mitogen-activated/extracellular signal-regulated protein kinase; BSA,
bovine serum albumin; CRE, cAMP-responsive element; PAGE,
polyacrylamide gel electrophoresis; TPA,
12-O-tetradecanoylphorbol-13-acetate; TRE, TPA-responsive
element; M + I, [Nle, DPhe]-MSH plus IBMX; AEBSF,
4-(2-aminoethyl)benzenesulfonyl fluoride; CREB, CRE binding protein.
We are grateful to Pr. E. Van Obberghen for
anti-p44 and anti-MEK sera and to Dr. M. Yaniv
for antibodies to Fra-2. We thank Dr C. Rouvière
for confocal laser microscopy observations. We also thank A. Grima and
C. Minghelli for illustration work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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