(Received for publication, August 12, 1996, and in revised form, October 18, 1996)
From the Department of Biochemistry and Molecular
Biology, School of Medicine, University of Murcia, 30100 Espinardo,
Murcia, Spain, and the ¶ Swiss Institute for Experimental Cancer
Research (ISREC), Chemin des Boveresses 155, 1066 Epalinges, Switzerland
Current evidence suggests that melanogenesis is
controlled by epidermal paracrine modulators. We have analyzed the
effects of transforming growth factor-1 (TGF-
1) on the basal
melanogenic activities of B16/F10 mouse melanoma cells. TGF-
1
treatment (48 h) elicited a concentration-dependent
decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine
(Dopa) oxidase activities, to less than 30% of the control values but
had no effect on dopachrome tautomerase activity (TRP-2). The
inhibition affected to similar extents the Dopa oxidase activity
associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This
inhibition was noticeable between 1 and 3 h after the addition of
the cytokine, and maximal after 6 h of treatment. The decrease in
the enzymatic activity was paralleled by a decrease in the abundance of
the TRP-1 and tyrosinase proteins. TGF-
1 mediated this effect by
increasing the rate of degradation of tyrosinase and TRP-1. Conversely,
after 48 h of treatment, the expression of the tyrosinase gene
decreased only slightly, while TRP-1 and TRP-2 gene expression was not
affected. An increased rate of proteolytic degradation of TRP-1 and
tyrosinase seems the main mechanism accounting for the inhibitory
effect of TGF-
1 on the melanogenic activity of B16/F10 cells.
Melanin pigmentation in mammals is a highly regulated and complex event occurring within specialized cells, the melanocytes (1-3). The pathway starts by two reactions catalyzed by tyrosinase (monophenol monooxygenase, EC 1.14.18.1), the hydroxylation of L-tyrosine to L-Dopa,1 and the oxidation of L-Dopa to dopaquinone. In the absence of thiolic compounds, dopaquinone evolves spontaneously to L-dopachrome, and then to melanin. Two other enzymes, TRP-1 and TRP-2, have been shown to participate in the process. TRP-2, also called DCT (EC 5.3.2.3.), catalyzes the non-decarboxylative rearrangement of L-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (4-6). This stable diphenol is oxidized by TRP-1 to an unstable o-quinone that undergoes further polymerization reactions to yield melanin (7, 8). Moreover, other melanosomal proteins such as pmel 17 could also be involved in the control of the distal melanogenic reactions (9, 10). Tyrosinase itself could also participate in other oxidative steps, thus enhancing the polymerization rate of melanogenic intermediates (11, 12).
Due to the high reactivity of the melanogenic intermediates,
melanogenesis is confined in specialized organelles called melanosomes. Epidermal melanization involves not only the synthesis of the pigment
but also the transfer of melanized melanosomes to the keratinocytes
surrounding the dendritic processes of melanocytes. Thus, melanocytes
are in intimate physical and functional contact with other epidermal
cells, which could contribute to the control of the melanogenic status
of melanocytes through the secretion of several cytokines and paracrine
factors. In keeping with this view, the skin contains a variety of
chemical messengers with the potential to affect the proliferation and
differentiation of melanocytes. These include several cytokines (13)
and proopiomelanocortin-derived peptides (14-17). Interestingly, the
production of these factors by epidermal cells is modulated by
environmental conditions, including contact irritants, epidermal
wounds, and UV light. It has been shown that some interleukins normally
present in the skin are able to inhibit melanization and stimulate
proliferation of mouse melanoma cells (18). IL-1, IL-6, and TNF-
have also been shown to inhibit both melanization and proliferation in
normal human melanocytes (19). The mechanisms involved in the
inhibition of melanin synthesis by these chemical agents are still
unknown.
Here we report a study on TGF-1 inhibition of melanogenesis, using
B16/F10 mouse melanoma cells as a model. Our results show that this
cytokine inhibits in a concentration-dependent way the activities of tyrosinase and TRP-1, with no significant effect on TRP-2
activity. At the concentrations tested, TGF-
1 was not cytotoxic and
the possibility of induction of an enzymatic inhibitor was ruled out.
The inhibition was instead explained by a decreased half-life of the
melanogenic proteins, and for tyrosinase, by a slight but significant
decrease in mRNA levels. These results provide the first
description, at the molecular level, of the hypopigmenting effect of
TGF-
1. Moreover, they might provide a framework for the study of
more complex regulatory events, like the relationship between
hyperpigmenting stimuli, such as MSH and related peptides, and
hypopigmenting signals such as TGF-
1 and other cytokines.
B16/F10 mouse melanoma cells were originally a kind gift from Dr. V. Hearing (National Institutes of Health, Bethesda, MD). Cells were cultured in MEM with 10% fetal calf serum and 1% penicillin/streptomycin. When necessary, cytokines diluted in MEM were added to the culture medium 24 h after seeding. Cells were grown to semiconfluence, harvested with 0.1 mg/ml trypsin and 0.2 mg/ml EDTA in Hank's balanced salt solution, and counted before detergent solubilization.
ReagentsHuman recombinant TGF-1 (1 µg/ml, 4 × 10
8 M), TNF-
(10 µg/ml, 6 × 10
7 M), IL-1
(1 µg/ml, 6 × 10
8 M), and IL-6 (2 µg/ml,
10
7 M) were from Boehringer Mannheim.
TGF-
1, IL-6, and IL-1
were aliquoted and stored at
20 °C
without dilution. TNF-
was diluted to 1.25 × 10
8
M in MEM and aliquoted before storage at
20 °C.
Aliquots were thawed and used immediately. Refreezing was avoided. The
radioactive substrate L-[3,5-3H]tyrosine,
specific activity 50 Ci/mmol, was from DuPont NEN. The
-MSH analogue
[Nle4,D-Phe7]-
-MSH,
L-tyrosine, bovine serum albumin, L-Dopa,
phenylmethylsulfonyl fluoride, MBTH, SDS, and 3,3
-diaminobenzidine
were from Sigma. Trichloroacetic acid, glycine, Tris,
EDTA, and monosodium and disodium phosphate were from Merck (Darmstadt,
Germany). Acrylamide, bisacrylamide, TEMED, Tween 20, and ammonium
persulfate were obtained from Bio-Rad. 5,6-Dihydroxyindole-2-carboxylic
acid was prepared according to Wakamatsu and Ito (20).
Peroxidase-labeled mouse anti-rabbit IgG was from Chemicon (Temecula,
CA). All other reagents were of the highest purity available from
Probus (Barcelona, Spain).
Cells were washed with Hank's balanced salt solution and solubilized in 1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 10 mM phosphate buffer, pH 6.8. Tyrosine hydroxylase activity was measured by the method of Pomerantz (21) modified as described elsewhere (22). Dopa oxidase activity determinations were performed according to Winder and Harris (23), with minor modifications. DCT activity was quantitated by high performance liquid chromatography as described previously (7). All activity units were defined as the amount of enzyme that catalyzes the transformation of 1 µmol of substrate/min.
Electrophoretic ProceduresAnalytical SDS-PAGE was performed as described by Laemmli (24) in 12% acrylamide gels. Samples were mixed with sample buffer yielding a final SDS concentration of 3%, but omitting 2-mercaptoethanol and heating, in order to preserve enzymatic activity. Electrophoresis was run at 4 °C at a constant current of 25 mA in a Mini-PROTEAN cuvette (Bio-Rad). After electrophoresis, the gels were equilibrated in 50 mM sodium phosphate buffer, pH 6.0, for 15 min and incubated for 15 min at 37 °C in a phosphate-buffered solution of 2 mM L-Dopa and 4 mM MBTH (25).
Immunochemical TechniquesWestern blotting was performed
after reducing SDS-PAGE in 12% polyacrylamide gels, as described
previously (7). The antibodies used for the detection of TRP-1,
tyrosinase, and TRP-2 were PEP1,
PEP7, and
PEP8, respectively,
a kind gift from Dr. V. Hearing; the specificities and characteristics
of these antibodies have been described (26, 27). Visualization of
immunoreactive bands was performed after incubation in a solution of
3,3
-diaminobenzidine (0.6 mg/ml), cobalt(II) chloride (0.03%), and
0.001% H2O2 for 5 min (28), or in
chemiluminescence blotting substrate (Boehringer Mannheim) for 1 min,
followed by drying, and autoradiography on x-ray film.
75-cm2
culture flasks were used for a single RNA preparation. Total RNA was
isolated using guanidinium isothiocyanate, separated in a
formaldehyde-containing agarose gel, and blotted onto nylon membrane
(Hybond, Amersham). Prehybridization was performed for 2 h at
65 °C in 5 × Denhardt's solution, 1% SDS, 5 × SSC, 0.1 mg/ml fragmented salmon sperm DNA, previously denatured for 5 min at
95 °C. The filters were hybridized overnight at 65 °C to different probes,32P-labeled by random priming (29), in
2 × Denhardt's solution, 1% SDS, 5 × SSC, 0.1 mg/ml
denatured and fragmented salmon sperm DNA. The following probes were
used: mouse tyrosinase, pTy1.1 (1.1-kb EcoRI fragment) (30);
mouse TRP-1, pMT4 (1.6-kb HindIII fragment) (31); mouse
TRP-2, clone 5A (1.2-kb EcoRI fragment) (31, 32).
Glyceraldehyde-3-phosphate dehydrogenase was used as control for
comparable loading and transfer. In this case, the probe was a 300-base
pair PCR fragment obtained with primers: CGTCTTCACCACCATGGAGA and
CGGCCATCACGCCACAGTTT (nucleotides 294-313 and 593-574 of the human
glyceraldehyde-3-phosphate dehydrogenase sequence, respectively) (33).
Following hybridization, the filters were washed at 65 °C for 10 min, once in 2 × SSC, 1% SDS, and twice in 0.1 × SSC, 1%
SDS. They were then exposed for appropriate times at 70 °C with
XAR-5 autoradiography film. Before rehybridization, the membranes were
boiled for up to 60 min in 1 mM EDTA, 0.1% SDS, 10 mM Tris-HCl, pH 8.0, until no remaining probe could be detected by autoradiography.
Protein was determined using the BCA kit (Pierce) and bovine serum albumin as standard. Quantitation of gels, filters, and autoradiograms was performed by laser scanning densitometry, using an Ultroscan XL densitometer from Pharmacia Biotech Inc., and by digital image processing performed with an IMCO 10 image computer from Kontron (Zurich, Switzerland), with comparable results. Cell viability was measured by the MTT assay (34) and by trypan blue dye exclusion.
The effect of several cytokines normally present in the
epidermis on the melanogenic activities of B16 melanoma cells was tested. Treatment of cells with -MSH markedly increased the Dopa oxidase and tyrosine hydroxylase activities (Fig. 1).
This treatment was included as a positive control for cell
responsiveness. Conversely, IL-1
had no detectable effect, whereas
TGF-
1 and TNF-
mediated a noticeable decrease of these
activities. A similar inhibitory effect of TNF-
on the melanogenic
activity of normal human melanocytes has been reported recently (19).
The effect of the most potent inhibitor identified, TGF-
1, was
further analyzed.
Differential Regulation of the Melanogenic Enzymes by TGF-
After 48 h of culture in the presence of TGF-1,
both the tyrosine hydroxylase and the Dopa oxidase activities of B16
melanocytes were inhibited in a concentration-dependent
way, but DCT activity remained stable (Fig. 2). The
effect was apparently mediated by high affinity receptors, since the
inhibition was evident at concentrations as low as 10
12
M and 50% inhibitions were observed at 10
11
M. The cytokine inhibited the Dopa oxidase (and probably
the tyrosine hydroxylase) activity of the two tyrosinase isoenzymes, tyrosinase and TRP-1, as shown by electrophoretic separation followed by activity stain (25, 35) (Fig. 3).
The doses of cytokine used were not significantly cytotoxic for B16
cells, as determined by the MTT assay (Fig. 4). The cell number increased during a 72-h incubation period, even at high concentrations of TGF-1. However, TGF-
1 appeared to have a small cytostatic effect on B16 cells after 72 h of treatment. To avoid possible artifacts arising from this effect, all further experiments were performed at shorter incubation times, never exceeding 48 h.
The Inhibition of Tyrosinase Activities Is Not Related to the Induction of a Melanogenic Inhibitor
The possible induction by
TGF-1 of a tyrosinase inhibitor was ruled out based on the following
observations. First, the activity detected was linear with the amount
of extract in the assay mixture (data not shown), for control and
TGF-
1-treated cells. This constitutes a kinetic criterion for the
absence of effectors in an enzymatic preparation (36). Second, the
activity of mixtures of extracts from control and TGF-
1-treated
cells was identical to the theoretical sum of activities from both
components (data not shown). Moreover, the residual activity at
different TGF-
1 concentrations was very similar as measured
spectrophotometrically or by densitometric quantitation of
activity-stained SDS-PAGE gels (Fig. 5). Finally, the
Km for tyrosine was identical in extracts from
control and TGF-
1-treated cells (Table I), and the
decrease in Vmax was consistent with the
observed decreases in enzyme abundance (see below).
|
The inhibitory
effect of TGF-1 (10
10 M) was rapid, since
decreases in tyrosine hydroxylase and Dopa oxidase activities were detected as soon as 1 h after addition of the cytokine to the culture medium. The kinetics of inhibition were similar for both activities (Fig. 6). Maximal inhibition was reached
around 6 h after addition of TGF-
1, and then the activity
levels remained constant at around 20% of control values.
The inhibition was most likely due to decreased intracellular enzyme
levels. This was shown by Western blot experiments using specific
antibodies directed against the tyrosinase isoenzymes (Fig.
7). Decreased levels of TRP-1 were detected in
TGF-1-treated cells, as soon as 30 min after addition of the
cytokine. Consistent with previous reports (37), the levels of
tyrosinase in control cells, as detected with
PEP7, appeared lower
than those of TRP-1. Therefore, we used chemiluminescent substrate to
enhance the signal. Under these conditions, a decrease in the amount of
enzyme could be detected. The decrease in the amount of proteins
estimated by quantitation of the blots approximately matched the
inhibition of the melanogenic activities, further suggesting that
inhibition is the result of a lowered enzyme abundance. Conversely, no
changes in the intracellular levels of TRP-2 could be detected with the specific antibody
PEP8, even after a 48-h treatment at the highest concentration of TGF-
1 tested throughout this study.
The Decrease in Tyrosinase and TRP-1 Abundance Does Not Result from Reduced Levels of Their mRNA
As shown in Fig.
8, the levels of TRP-2 mRNA were similar in control
and TGF-1-treated cells (10
10 M, 48 h). No significant changes were also observed for TRP-1 mRNA.
Conversely, a moderate decrease (30%) in the abundance of the
tyrosinase message was detected. However, this decrease was lower than
the one observed for the enzymatic activity and for the protein levels
(80%, approximately).
TGF-
The effect of TGF-1 on the half-lives of tyrosinase
and TRP-1 was then considered. Cells were pretreated with TGF-
1
(10
10 M, 24 h). After addition of
cycloheximide (10 µg/ml), cells were collected at different times and
their residual tyrosine hydroxylase and Dopa oxidase activities were
measured (Fig. 9). Control cells yielded half-lives of
3.2 h and 3.8 h for the Dopa oxidase and tyrosine hydroxylase
activities, respectively. Upon treatment with the cytokine, the
half-life decreased to values near 1.7 h for Dopa oxidase, and 2.2 for tyrosine hydroxylase. Therefore, in both cases the half-life
decreased to values around 50% of the controls.
The occurrence in normal skin of a variety of cytokines and
proopiomelanocortin-derived peptides is well documented. Keratinocytes synthesize and secrete IL-1 and TNF- (38), IL-6 (39), and TGF-
1
(40). Melanoma cells secrete IL-1
and
, IL-6, IL-8, TGF-
and
-
(41, 42), and normal human melanocytes are able to synthesize
IL-1
and -
(43). Moreover, epidermal cells are able to synthesize
and process of proopiomelanocortin, thus yielding potentially
melanogenic peptides (14-17). These observations suggest that
cutaneous melanogenesis might be regulated, at least partially, by
paracrine or autocrine factors. However, although it is increasingly evident that some interleukins could act as paracrine inhibitors of
melanogenesis, as well as regulators of melanocyte proliferation, the
mechanism(s) by which they mediate their inhibitory effect, as well as
their interactions and cross-talk with melanogenic stimuli, are largely
unknown.
Among the cytokines tested in this study, both TNF- and TGF-
1
induced a potent inhibition of tyrosine hydroxylation, the rate-limiting step in melanin synthesis. The inhibition mediated by
TGF-
1 was selected for further study, since TGF-
1 appears to
fulfill several important functions in normal and perturbed mammalian
skin. TGF-
1 supports the growth of skin fibroblasts (44) and
inhibits the proliferation of keratinocytes (45, 46), although it could
also act as a survival factor for these cells by blocking their entry
in apoptosis (40). Through the control of the expression of integrins
and matrix metalloproteases, it may play a major role in tissue
remodeling, angiogenesis, and re-epithelization after cutaneous wounds
(47-50). It has been suggested that wound healing might indeed be a
major physiological role of this growth factor (reviewed in Ref. 51),
and, accordingly, it is strongly expressed in healing wounds (52).
Our data suggest that TGF-1 inhibition of melanogenesis is a
specific effect, rather than the result of extensive cellular damage or
a nonspecific event following the induction of intracellular proteases.
Indeed, although TGF-
1 inhibits the growth of a variety of cells,
this effect does not result from cytotoxicity (reviewed in Ref. 51).
B16/F10 cells continued to proliferate, under the conditions used in
this study, although at a rate somewhat slower than control cells.
Similar results have been reported by others (53). Moreover, most of
the experiments performed throughout this study were conducted at
TGF-
1 concentrations and incubation times such that the cytostatic
effect of the cytokine was not detectable. On the other hand, the
observation that DCT activity is not affected in TGF-
1-treated
melanocytes lends further support to the view that the inhibition of
tyrosine hydroxylase and Dopa oxidase activities is a specific process.
DCT is much more sensitive than tyrosinase to proteolysis (5), and
should be the melanogenic enzyme more potently inhibited by nonspecific
proteolysis.
Several possibilities were considered to account for the specific
inhibition of the rate-limiting melanogenic enzymes. A variety of
tyrosinase inhibitors of different chemical structure have been
described (3). Moreover, modulations of the intracellular levels of
such inhibitors have been postulated as a mechanism for tyrosinase
regulation by extracellular messengers (54). Therefore we tested
whether the inhibition mediated by TGF-1 could be accounted for by
the induction of a tyrosinase inhibitor. This possibility was ruled out
based on a variety of kinetic criteria, and on the observation that the
degree of inhibition of tyrosinase activity in extracts from
TGF-
1-treated cells was the same as measured under conditions
allowing for intermolecular interactions (radiometric and
spectrophotometric assays), or after SDS-PAGE separation of the
extract. A lower activity in the spectrophotometric assay, as opposed
to the electrophoretic method, would be indicative of the presence of
an inhibitor in the crude extracts. However, comparable results were
obtained with the electrophoretic and spectrophotometric assays.
Therefore, it is very unlikely that the inhibitory effect of TGF-
1
might be mediated by induction of a tyrosinase inhibitor.
On the other hand, the decrease of tyrosine hydroxylase and Dopa
oxidase activities could not be explained by a decreased rate of
transcription of the tyrosinase and TRP-1 genes, or by an increased
rate of degradation of the corresponding mRNA. The levels of
mRNA for TRP-1 remained unchanged, whereas those of tyrosinase
mRNA only displayed 30% decrease. As opposed to this minor effect
on mRNA levels, the abundance of tyrosinase and TRP-1 proteins
rapidly dropped following TGF-1 treatment. This effect was
particularly dramatic for TRP-1, which was barely detectable 6 h
after challenging the melanocytes with the cytokine. Therefore, under
these experimental conditions, the levels of the tyrosinase and TRP-1
proteins did not match those of their corresponding mRNA. Similar
observations have been reported by others for melanocytes treated with
a variety of agents able to modulate melanogenesis. For instance,
Abdel-Malek et al. (55) have shown that normal human
melanocytes respond to
-MSH by increasing the intracellular protein
levels of tyrosinase, TRP-1, and TRP-2 without any noticeable effect on
their mRNA, even after prolonged treatments.
It appears, therefore, that the inhibitory effect of TGF-1 on
tyrosinase and TRP-1 results from decreased levels of the proteins mainly due to post-translational events, and that this
post-translational control of the melanogenic enzymes might be a rather
general mechanism of control of pigmentation, not only by
hypopigmenting agents, but also by hyperpigmenting stimuli. Our data
showing decreased half-lives for tyrosinase and TRP-1 suggest that, at
least for TGF-
1, the post-translational control on these enzymes
would be exerted through an increased rate of degradation. This lowered enzyme stability might, in turn, be explained by several mechanisms. On
one hand, the "de novo" induction of a specific protease
might be envisaged, although the kinetics of TGF-
1 inhibition appear too rapid, at least for TRP-1, to be dependent on the de
novo synthesis of a protein. On the other hand, the melanosomes
themselves are a rich source of proteases and other hydrolytic enzymes
(56). TGF-
1 might therefore activate a preexisting protease or,
alternatively, mediate an unstabilization of the tyrosinase isoenzymes,
thus making them sensitive to such an enzyme. Work is under way to test
these possibilities.
In summary, we have shown for the first time that TGF-1 is a potent
inhibitor of melanin synthesis, through post-translational events
leading to decreased half-lives and protein levels of tyrosinase and
TRP-1. These data might provide a model for the study of the post-translational control of melanin synthesis in mammals. Moreover, they might provide a framework to understand a variety of pigmentary disorders associated to several physiological and pathological conditions, where an imbalance of paracrine and possibly autocrine melanogenic regulators might result in altered proliferation or differentiation of epidermal melanocytes. Finally, it should be noted
that the effects of TGF-
1 are exerted at low concentrations of the
cytokine. Since melanoma cells are able to synthesize and secrete
TGF-
1, our results suggest that an autocrine regulatory loop may
contribute to the control of melanogenesis, at least in melanocytes.
This possibility will be the subject of further studies.