First published online 4 March 2003
doi: 10.1242/jcs.00366
Sphingosine-1-phosphate decreases melanin synthesis via sustained ERK activation and subsequent MITF degradation
Dong-Seok Kim1,
Eui-Soo Hwang2,
Jai-Eun Lee2,
Sook-Young Kim2,
Sun-Bang Kwon2 and
Kyoung-Chan Park2,*
1 Research Division for Human Life Sciences, Seoul National University, 28
Yongon-Dong, Chongno-Gu, Seoul 110-744, Korea
2 Department of Dermatology, Seoul National University College of Medicine, 28
Yongon-Dong, Chongno-Gu, Seoul 110-744, Korea
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Fig. 2. Effects of S1P and kojic acid on melanogenesis in Mel-Ab cells. Cells were
cultured with 1-10 µM S1P or 1-100 µM kojic acid for 5 days, and the
tyrosinase activity (A) or melanin content (B,C) was measured, as described in
Materials and Methods. (D) To test the direct effect on tyrosinase, tyrosinase
activity was measured in a cell-free system, also as described in Materials
and Methods. Results are the average of three independent experiments ±
s.d. **P<0.01, *P<0.05 compared with control.
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Fig. 3. S1P induces the degradation of MITF and stimulates the ERK signaling
pathway. (A) After serum starvation, Mel-Ab cells were stimulated with 10
µM of S1P at the times indicated. Whole cell lysates were then subjected to
western blot analysis with antibodies against MITF, phospho-specific ERK, and
phospho-specific MEK. Equal protein loading was checked by reaction with
actin, phosphorylation-independent ERK, and MEK antibodies. (B) RT-PCR
analysis of MITF mRNA levels in S1P-stimulated cells. Cells were treated with
S1P as in A. Total RNA was isolated from the cells and cDNA prepared.
Equivalent amounts of cDNA were amplified with primers specific for MITF, and
actin primers were used as a control to ensure the even loading of target
cDNA. The resulting PCR products were analyzed by agarose gel electrophoresis.
The lane on the left shows markers of the indicated size. NC, negative
control.
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Fig. 4. Effects of S1P and PD98059 on melanogenesis in Mel-Ab cells. (A) Cells were
cultured with 10 µM S1P and/or 20 µM PD98059 for 5 days, and phase
contrast pictures were taken using a color video camera. The cells were
pretreated with 20 µM of PD98059 for 1 hour and then cultured with 10 µM
of S1P for 5 days, and tyrosinase activity (B) and melanin content (C) were
measured, as described in Materials and Methods. Values indicate the mean of
three independent experiments ± s.d.
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Fig. 5. S1P stimulation induces MITF degradation via the ERK signaling pathway. (A)
After serum starvation, Mel-Ab cells were stimulated with 10 µM of S1P at
the times indicated. Cell lysates were then immunoprecipitated with antibody
against ERK1/2, and the MITF level was measured by immunoblotting. Cells were
stimulated with 10 µM S1P for 10 minutes (B) or for 180 minutes (C) in the
absence or presence of 20 µM PD98059. Whole cell lysates were then
subjected to western blot analysis with antibodies against MITF,
phospho-specific ERK, tyrosinase and TRP-1. Actin antibody was used a loading
control.
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© The Company of Biologists Ltd 2003
),
sphingosine (•) or N,N-dimethylsphingosine (
). After serum
starvation, cells were incubated for 24 hours in serum-free media with various
concentrations (1-10 µM) of sphingolipids. The viability of the cells was
determined by the crystal violet assay. Each determination was made in
triplicate and the data represent means ± s.d.