(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. 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.
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, ()
-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.
The proline-directed serine/threonine kinases of the MAP kinase
family (p44 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.
Figure 1:
cAMP-elevating agents stimulate
p44 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.
The kinase activity
of MEK was monitored in a cell-free system, after immunoprecipitation
with specific antibody, using as substrate p44 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.
Figure 2:
[Nle, 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.
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, D-Phe
]
-MSH plus IBMX on p44
is mediated by MEK that is activated by an unknown mechanism
independently on Raf-1 kinase stimulation.
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, 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.
The localization of
p44 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.
Figure 4:
Immunolocalization of the p44 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.
Figure 5:
[Nle, 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).
Figure 6:
Characterization of the AP-1 complex
induced by TPA and [Nle, 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.
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 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.
Following
its activation by cAMP, we observed a transient translocation of
p44 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.
In this study we showed that melanin synthesis,
tyrosinase activity, and amount were simultaneously increased by
[Nle, 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.
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(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.
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 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.