I. Melanosomal pH controls multiple stages of melanin production, with near neutral pH being optimal for human tyrosinase activity and melanogenesis. II. Neutralizing melanosomal pH in Caucasian melanocytes and melanoma cells leads to increased melanin production and a preferential increase in eumelanin over phaeomelanin. III. Melanosomal pH also regulates the maturation rate of melanosomes, with neutral pH resulting in more mature melanosomes.

1. Melanosomal pH Controls Rate of
Melanogenesis, Eumelanin/Phaeomelanin
Ratio and Melanosome Maturation in
Melanocytes and Melanoma Cells
Janis Ancans,* Desmond J. Tobin,* Martin J. Hoogduijn,* Nico P. Smit,†
Kazumasa Wakamatsu,‡ and Anthony J. Thody*,
<13. 04. 27> 김준섭
- contents-
1. ABSTRACTS
2. INTRODUCTION
3. MATERIALS AND METHODS
4. RESULTS
5. DISCUSSION
▶OBJECTIVE : melanosomal pH

2. INHA University - Bioengineering
ABSTRACT
The skin pigment melanin is produced in melanocytes in highly specialized organelles known
as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal
pathway and can have a low internal pH. In the present study we have shown that melanin
synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the
role of intramelanosomal pH as a possible control mechanism for melaznogenesis. To do this
we examined the effect of neutralizing melanosomal pH on tyrosinase activity and
melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte
cultures (9 of 9) from Caucasian skin as well as two melanoma cell lines with comparable
melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to
neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential
increase in eumelanin production. Electron microscopy revealed an accumulation of melanin
and increased maturation of melanosomes in response to pH neutralization. In summary, our
findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity
and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low
melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of
melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is
an essential factor which regulates multiple stages of melanin production. Furthermore, since
we have recently identified that pink locus product (P protein) mediates neutralization of
melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We
would further propose that the wide variations in both constitutive and facultative skin
pigmentation seen in the human population could be associated with the high degree of P-
locus polymorphism.

3. INHA University - Bioengineering
ABSTRACT
<melanocyte>
melanosome
endosome
lysosome
pH ↓↓
pH ↓↓
melanin synthesis in human pigment cell lysates is maximal at pH 6.8.
We therefore investigated the role of intramelanosomal pH as a
possible control mechanism for melanogenesis.
However,

4. INHA University - Bioengineering
ABSTRACT
I. near neutral melanosomal pH is optimal for human tyrosinase activity and
melanogenesis
II. melanin production in Caucasian melanocytes is suppressed by low
melanosomal pH
III. the ratio of eumelanin/phaeomelanin production and maturation rate of
melanosomes can be regulated by melanosomal pH
Finding in experiments
melanosomal pH is an essential factor which regulates multiple stages of
melanin production.
※Caucasian : 백인

5. INHA University - Bioengineering
INTRODUCTION
This variation does not depend upon differences in the numbers of
epidermal melanocyte since these are similar in all ethnic groups.
However, there are inherited differences in the melanogenic activity of the
melanocytes and the production rate of melanin as well as the relative
amounts of the two main types of melanin, eumelanin (dark brown to
black) and phaeomelanin (yellow to reddish brown).
•races
•same ethnic group
skin color varies
Why??
melanosome의 pH가 다르다!

6. INHA University - Bioengineering
MATERIALS AND METHODS
Cells and Cell Cultures
RT-PCR
Tyrosinase Activity
Melanin Assay
Cellular homogenates
Whole cells
Labeling of Cells with Acridine Orange (AO)
Measurement of Eumelanin and Phaeomelanin
Light and Transmission Electron Microscopy
Human melanoma cell line :
FM3, FM55, FM81, FM94

7. INHA University - Bioengineering
RESULTS
Characterization of Melanogenic System in Human Pigment Cells
FM94 melanoma cell
optimal pH
 near neutral pH

8. INHA University - Bioengineering
RESULTS
Effect of Neutralizing Melanosomal pH on Melanogenesis and
Eumelanin/Phaeomelanin Ratio
FIG. 2. Visualization of acidic organelles.
Red fluorescence indicates the
presence of acidic vesicles
(A) and (B) : C803 cells
(C) and (D) : FM55 cells
(E) and (F) : FM94 cells
Treatment with ConA (10 nM)
(B), (D), and (F)
neutral pH
※ConA : proton pump inhibitor

9. INHA University - Bioengineering
RESULTS
Effect of Neutralizing Melanosomal pH on Melanogenesis and
Eumelanin/Phaeomelanin Ratio
<tyrosinase assay>
FIG. 3. Effects of 24 h incubation with proton pump inhibitor ConA (10 nM) on
tyrosinase activity (A and B)
control (striped bars) > presence of proton
pump inhibitors (closed bars)

10. INHA University - Bioengineering
RESULTS
Effect of Neutralizing Melanosomal pH on Melanogenesis and
Eumelanin/Phaeomelanin Ratio
FIG. 3. Effects of 24 h incubation with proton pump inhibitor ConA (10 nM) on melanin content
and type (C and D)
Cell
ConA treat •increase of melanin
•increase of PTCA/AHP ratio
※PTCA : eumelanin
※AHP : phaeomelanin

11. INHA University - Bioengineering
RESULTS
Effects of Neutralizing Melanosomal pH on Melanosome Maturation
FIG. 4. Effects of ConA (10 nM) on the numbers of mature melanosomes in C36 melanocytes as
revealed using thin-section light microscopy (A, B) and EM (C, D). After 24 h of treatment with
proton pump inhibitor increases in pigment granule numbers were observed (B and D) compared
with controls (A and C). Arrows point to mature melanosomes; scale bars 5 5 mm (A, B) and 1
mm (C, D).

12. INHA University - Bioengineering
DISCUISSION
melanosomal pH
FIG. 5. Melanosomal pH is controlled and maintained by the v-type proton
pump and P protein.
the activity of tyrosinase
the rate of melanin production
the ratio of eumelanin/phaeomelanin
the maturation of melanosomes